IL322837A

IL322837A - Peptidoglycan hydrolases with bactericidal activity

Info

Publication number: IL322837A
Application number: IL322837A
Authority: IL
Inventors: Lorenzo Corsini; Robert Kluj; Anna-Margarita Sch?Tta; Benedikt Bauer; Zehra Visram; Adriana Badarau; Anna Spier; Johannes Kraml; Olaf Othersen; Maren Lang; Leon Bichmann; Ilya Kizhvatov
Original assignee: BioNTech SE; Lorenzo Corsini; Robert Kluj; Sch?Tta Anna Margarita; Benedikt Bauer; Zehra Visram; Adriana Badarau; Anna Spier; Johannes Kraml; Olaf Othersen; Maren Lang; Leon Bichmann; Ilya Kizhvatov
Priority date: 2023-03-09
Filing date: 2024-03-08
Publication date: 2025-10-01
Also published as: EP4676598A1; JP2026507918A; CN121127584A; WO2024184533A1; AU2024234022A1

Description

New PCT application BioNTech SEVossius ref.: AF3920 PCT S3 Peptidoglycan hydrolases with bactericidal activity Field of the invention The present invention is generally in the fields of pharmaceuticals, in particular antibacterials, and protein engineering. In particular, the present invention relates to peptidoglycan hydrolases such as endolysins and nucleic acids, e.g., RNAs, encoding the peptidoglycan hydrolases of the invention, as well as medical uses thereof, for example, for treating diseases caused by and/or associated with a Staphylococcus (e.g., S. aureus) infection. Furthermore, the present invention relates to solidified yeast culture media for screening yeast cells for the secretion of peptidoglycan hydrolases with bactericidal activity and corresponding screening methods.

Background Staphylococci, in particular Staphylococcus aureus strains, are major human pathogens responsible for a vast array of pathologies, both acute and chronic, varying from mild to life threatening, including skin and soft tissue infections, bone-related infections, pneumonia, and sepsis. For example, S. aureus is a leading cause of mortality among antibiotic resistant bacterial pathogens, with ~ 700.000 deaths per year due to antibiotic resistant S. aureus globally; Antimicrobial Resistance Collaborators (2022), The Lancet, 399. The pathogenesis of S. aureus infection involves several critical steps: invasion of host tissues, evasion of the immune system, adhesion to surfaces, and biofilm formation. For example, by persisting in biofilm, bacteria evade neutrophil killing and display decreased susceptibility to antibiotics. Despite decades of research and promising preclinical data, there is no available vaccine against S. aureus.

Peptidoglycan hydrolases (belonging to the class of "enzybiotics") such as bacteriophage-encoded endolysins, are a promising alternative to antibiotics; Fischetti (2010), International Journal of Medical Microbiology, 300(6); Schmelcher (2012), Future Microbiology, 7; Hojckova (2013), BMC Microbiol. 13. Bacteriophages produce these enzymes, in particular endolysins, towards the end of the lytic cycle. The enzymes cleave peptidoglycans (PG) in the bacterial cell wall, thus lysing the cells and releasing the progeny phages. Peptidoglycan hydrolases, in particular endolysins (also abbreviated as "lysins"), have several advantages over antibiotics; especially, their narrow host specificity, which is often limited to a single genus or even a single species (Fischetti (2010), International Journal of Medical Microbiology, 300(6)), and their rather low propensity for generating resistance in their hosts (Schuch (2014), The Journal of Infectious Diseases 209(9)). Bacteriophages that invade Gram-positive bacteria encode a variety of highly diverse endolysins. Typically, endolysins have a modular structure consisting of one or more enzymatically active domains (EADs) connected by a flexible interdomain linker to at least one cell wall-binding domain (CBD). Both domains may contribute to the specificity for a given genus or species of bacteria; Oliveira (2013), J. Virol. 87. However, bacteriophage derived endolysins containing solely an enzymatically active domain, e.g., a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain (but no separate CBD), also exist. Such lysins break down peptidoglycan, in particular, from outside the bacterial cell.

A native (i.e. natural) phage lysin targeting S. aureus, CF-301 (ContraFect, also known as exebacase), delivered intravenously, showed therapeutic benefit in methicillin-resistant Staphylococcus aureus (MRSA) blood stream infections in a phase II clinical study (Fowler (2020), J. Clin. Invest., 130(7)), but the results could not be recapitulated in a phase III clinical study. Another native endolysin, SAL200, administrated intravenously to patients with persistent S. aureus bacteremia in a Phase II clinical study, resulted in serious adverse effects including pneumonia and respiratory failure (NCT03089697; Danis-Wlodarczyk (2021), Antibiotics, 10(12)); moreover, a very short half-life and an immune response against the enzyme was believed to limit its usefulness (WHO technical document, January 15, 2022: 2019 antibacterial agents in clinical development: an analysis of the antibacterial clinical development pipeline, https://www.who.int/publications/i/item/9789240000193).

On the other hand, certain peptidoglycan hydrolases delivered locally (intranasally and/or topically) have shown some success in S. aureus decolonization, e.g. ectolysin P128 and endolysin SA.100 (Danis-Wlodarczyk (2021), Antibiotics, 10(12)) as well as lysostaphin (Jayakumar (2020), J. Appl. Microbiol.)). Exposure of S. aureus to lysostaphin, a glycylglycine endopeptidase, was however shown to generate rapid resistance both in vitro anti in vivo, due to loss of function mutations in femA, which is required for the incorporation of the second and third glycine in the cross-bridges (Climo (2001), Antimicrobial Agents and Chemotherapy, 45 (5).

In view of the caveats of native phage endolysins, it has been tried to improve drug-like properties of endolysins by protein engineering; De Maesschalck V (2020) Crit Rev Microbiol, 46(5). The modular architecture of endolysins has served as a basis for lysin engineering, via domain shuffling, truncation, as well as random and/or site-directed mutagenesis; Gerstmans (2020), Sci Adv. 6(23). So far, lysin optimization focused on optimizing either functional activity (Gerstmans (2020), Sci Adv. 6(23)) or stability (Ritter (2019), Appl Environ Microbiol., 85(10)). Moreover, for the latter, only marginal improvements have been achieved, i.e., 4°C increase in melting temperatures for the best engineered variants.

Hence, there is a need for improved antibacterials, in particular peptidoglycan hydrolases with improved pharmaceutical properties, as well as improved means and methods for generating the same.

Summary of the invention Accordingly, the present invention relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain that has (i) a sequence identity of at least 60% to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions as compared to the amino acid sequence from position to position 215 in SEQ ID NO: 1.

As further described below, said CHAP domain is considered herein and in context of the present invention as a variant of the CHAP domain of LO482, i.e., a variant of the sequence from position 72 to position 215 in SEQ ID NO: 1.

Preferably, the peptidoglycan hydrolase of the present invention has a killing activity against a Staphylococcus species or strain, preferably Staphylococcus aureus. Furthermore, the peptidoglycan hydrolase of the invention has preferably the ability of being secreted from a eukaryotic cell. Furthermore, the peptidoglycan hydrolase of the invention is, preferably, stable up to a temperature of at least about 40°C, e.g., at least 37°C. Furthermore, the peptidoglycan hydrolase of the present invention is, preferably, an endolysin.

In context of the present invention, the peptidoglycan hydrolase of the invention is, preferably, contained in a pharmaceutical composition and/or, preferably, used for the treatment of a disease. Herein and in context of the present invention, the disease is, in particular, a bacterial disease, preferably, a disease caused by and/or associated with a Staphylococcus infection, e.g., a S. aureus infection.

Selection of the LYSM-CHAP domain architecture as starting point for further protein engineering and directed evolution The invention is, partly, based on the surprising finding, as illustrated in the appended Examples, that lysins with a LYSM-CHAP domain architecture such as LO482 (SEQ ID NO: 1) reliably have a good killing activity against Staphylococci, in particular Staphylococcus aureus (S. aureus) including methicillin-resistant Staphylococcus aureus (MRSA) strains such as ATCC43300. Therefore, lysins with a LYSM-CHAP domain architecture such as LO482 provide a particularly good starting point for protein engineering and directed evolution approaches. In particular, it has been surprisingly found in context of the present invention that 12 out of 13 lysins with a LYSM-CHAP domain architecture (i.e., >92%) effectively killed S. aureusceWs, whereas lysins with other domain architectures did often not show considerable killing activity against S. aureus, see, e.g., Example 1 and Figure 1. Moreover, it has been found that a representative of these LYSM-CHAP lysins, i.e., LO482 (SEQ ID NO: 1), had a good killing activity against many different S. aureus strains and also against other Staphylococcus species such as S. warned and S. capitis anti coagulate-negative Staphylococci such as S. epidermidis; see, e.g. Example 1 and Figure 2. Surprisingly, the killing activity against the tested Staphylococcus species and strains was even increased compared to one of the clinically most developed lysins, i.e., exebacase (also known as CF-301, ContraFect or described as "LO466" herein) which has a CHAP-SH3 domain architecture; see Figure 2. Of note, the CHAP domain of LYSM-CHAP lysins such as LO482 is very different to the CHAP domain of exebacase. In particular, the sequence identity of the CHAP domain of exebacase (i.e. the sequence from position 19 to position 164 in SEQ ID NO: 304) to the CHAP domain of LO482 (i.e. the sequence from position 72 to position 215 in SEQ ID NO: 1) is merely about 20%.

Furthermore, it has been found in context of the present invention that LysM-CHAP lysins such as LO482 (SEQ ID NO: 1) can be secreted in active form by eukaryotic cells in order to kill S. aureus, see, e.g., Example 2 and Figure 3. Nevertheless, it has been also observed by the present inventors that the stability and solubility of the wild-type (WT) LO482 (SEQ ID NO: 1) and its secretion from eukaryotic cells as well as its killing activity upon secretion from eukaryotic cells is still not optimal; see, e.g., Example 4 and Figure 5, Example 5 and Figure 6, and Example 12 and Figure 10. Yet, a high killing efficiency against the target bacterium (e.g., S. aureus), a good solubility in aqueous solutions, a high stability (including a sufficient thermostability and a low tendency for aggregation) are important properties of anti-bacterial compounds, in particular, enzybiotics such as peptidoglycan hydrolases. These pharmaceutical properties are particularly important for the treatment of bacterial infections in mammals, e.g., humans, as described herein. Furthermore, administration of a peptidoglycan hydrolase in form of a nucleic acid (e.g., an mRNA) encoding the peptidoglycan hydrolase to cells in a subject (e.g., a human) has, inter alia, the advantage of continuous production, i.e., secretion, of the peptidoglycan hydrolase from the cells of the subject at the site of a bacterial infection. Hence, the ability of being efficiently secreted from eukaryotic cells, e.g., human cells, is another beneficial property of peptidoglycan hydrolases.

In brief, the inventors surprisingly found that LO482 (SEQ ID NO: 1) is an optimal starting point for further protein engineering and directed evolution.

Accordingly, the invention further relates to a peptidoglycan hydrolase having bactericidal activity which has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1 and which comprises one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 1. This sequence identity is, in particular, calculated over the full length of the sequence of SEQ ID NO: 1 (and not over the full length of the sequence of the peptidoglycan hydrolase of the invention). Thus, any additional domains, peptides or tags that may be comprised in (or fused to) the peptidoglycan hydrolase of the invention, e.g., a signal peptide or a PKtag, should not be considered when determining the sequence identity of the peptidoglycan hydrolase of the invention to the sequence of SEQ ID NO: 1. Hence, the peptidoglycan hydrolase of the invention may comprise an amino acid sequence having a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, and, optionally, one or more further domains, peptides or tags, e.g. a signal peptide, a PK tag, a further peptide linker etc., as described herein.

Preferably, the peptidoglycan hydrolase of the invention comprises a CHAP domain according to the present invention, i.e., a CHAP domain that has (i) a sequence identity of at least 60% to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions as compared to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1.

LO482 variants with improved pharmaceutical properties (brief summary) By protein engineering, as illustrated in Example 4, and subsequent directed evolution, as illustrated in Example 5, the inventors surprisingly found LO482 variants which had several improved pharmaceutical properties at the same time. In particular, the inventors found LO482 variants which had an enhanced killing activity against S. aureus, an enhanced protein stability and an enhanced ability of being secreted from human cells, as illustrated e.g., in Example 6 and Figure 8.

Enhanced killing activity As described in more detail herein below and as also illustrated in the appended Examples, an enhanced killing activity against a target bacterium such as 5. az/rez/s corresponds to a lower minimal inhibitory concentration (MIC). In particular, herein and in context of the present invention, the MIC is defined as the minimal concentration at which the optical density at 620 nm (OD620) of a bacterial (e.g. 5. aureus) liquid culture comprising 5xl05 cfu/ml of bacterial (e.g. 5. aureus) cells is kept below 0.1 for at least 24h at 37°C. Thus, the inventive peptidoglycan hydrolases provided herein may be used at a lower concentration for treating a bacterial disease than comparable peptidoglycan hydrolases, which may increase the efficiency and safety of the treatment. For example, the hit variants G1 (SEQ ID NO: 3) and H5 (SEQ ID NO: 11) found in context of the present invention have a 4- or 8-fold lower MIC than WT LO482 (SEQ ID NO: 1), respectively, i.e. a MIC of 1 pg/ml for G1 and a MIC of 0.5 pg/ml for H5, compared to a MIC of 4 pg/ml for the WT L0482; see, e.g., Figure 8. Notably, the lower MICs have been observed with the methicillin-resistant Staphylococcus aureus ^AWSK) strain "ATCC43300" further highlighting that the peptidoglycan hydrolases of the invention may be particularly suitable as antibacterials, in particular, for medical uses.

The enhancement of the killing activity of peptidoglycan hydrolases according to invention was further confirmed with an OD reduction assay showing that, for example, H5 (SEQ ID NO: 11) killed S. aureuszeWs with faster kinetics than wild-type LO482 (SEQ ID NO: 1); see, e.g., Figure II.

Enhanced protein stability An enhanced protein stability, e.g., an enhanced thermostability and/or a reduced propensity for aggregation, further improves the pharmaceutical properties of a peptidoglycan hydrolase. For example, a sufficient thermostability, in particular, the ability of being stable of up to a temperature of at least about 40°C, e.g., at least 37°C, is critical for the use as a pharmaceutical in many mammals including humans (in view of the body temperatures). Furthermore, an enhanced thermostability facilitates storage and distribution of a pharmaceutical. Thus, the inventive peptidoglycan hydrolases provided herein may be particularly suitable as pharmaceuticals. Furthermore, a decreased propensity for aggregation enhances the manufacturability of a peptidoglycan hydrolase. Thus, the inventive peptidoglycan hydrolases provided herein may be more efficiently manufactured than comparable peptidoglycan hydrolases. Moreover, a decreased propensity for aggregation further reduces the immunogenicity of a peptidoglycan hydrolase in a mammalian subject, e.g., a human, which, in turn, may further increase the efficiency and/or safety of a treatment.

Enhanced ability of being secreted from human cells An enhanced ability of being secreted from human cells, is particularly important for the expression of a peptidoglycan hydrolase from a nucleic acid, e.g., a RNA. As described herein, when a suitable nucleic acid construct encoding a peptidoglycan hydrolase of the invention is introduced into cells in a subject (e.g. a human patient), said cells can continuously produce and secrete the peptidoglycan hydrolase protein. This may provide a more efficient treatment of the bacterial infection and, for example, provide a higher efficacy in treating difficult to treat bacterial infections such as bacterial biofilms. Therefore, the peptidoglycan hydrolases of the invention may be particularly suitable for delivery as a nucleic acid, e.g., a RNA. Moreover, the present invention provides more efficient means, e.g., nucleic acids encoding a peptidoglycan hydrolase of the invention, for treating bacterial infections.

Enhanced solubility An enhanced stability (e.g. a reduced propensity for aggregation) and an enhanced ability of being secreted from human cells may be associated with each other as well as with an enhanced solubility in an aqueous solution such as PBS. Indeed, it has been observed in context of the present invention that many of the generated LO482 variants which were found to have an enhanced stability and an enhanced ability of being secreted from human cells also showed an enhanced solubility in PBS. Thus, the inventive peptidoglycan hydrolases provided herein may have a better solubility in aqueous solutions than comparable peptidoglycan hydrolases. This is highly beneficial, in particular, for the manufacturability and useability of the peptidoglycan hydrolases. Moreover, an enhanced solubility in aqueous solutions may increase the efficiency and/or safety of a peptidoglycan hydrolases in the treatment of a disease.

Reduced propensity of generating resistance A CHAP domain (contained in LO482 and LO482 variants as described herein) may have a dual enzymatic activity, i.e., an amidase activity and a peptidase activity; Frankel (2012), J Biol Chern. 23;287(13). Both, the amidase activity and the peptidase activity, may contribute to the hydrolysis/cleavage of peptidoglycan in the cell wall of bacteria, as described herein. Therefore, a peptidoglycan hydrolase of the present invention comprising a CHAP domain of the invention may have a reduced propensity of generating resistance in target bacteria, e.g., S. aureus, compared to other peptidoglycan hydrolases such as lysostaphin. Thus, the peptidoglycan hydrolases of the invention may be particularly effective for treating bacterial infections for this additional reason.

Consensus mutations / the variant H3 The inventors unexpectedly found a LO482 variant, i.e., H3 (SEQ ID NO: 9), which (i) had all of the beneficial properties assayed (i.e. an enhanced killing activity against S. aureus, an enhanced stability and an enhanced ability of being secreted from mammalian cells) compared to the parental LO482 variant L0482ag (SEQ ID NO: 2), and which (ii) contained exclusively amino acid substitutions that were deemed to be among the most beneficial ones as they were consistently observed (in slightly different combinations) among the best hits obtained by the three rounds of directed evolution, i.e., G1-G4, H1-H10 and 11-130; see, e.g., Examples 6 and 7 and Figures 8 and 9.

H3 (SEQ ID NO: 9) has been obtained by removing two glycosylation sites in LO482 (SEQ ID NO: 1) to yield the aglycosylated variant L0482ag (SEQ ID NO: 2) (see Example 4), followed by two rounds of directed evolution with L0482ag as a starting point; see Example 5. H3 (SEQ ID NO: 9) contains in addition to the two aglycosylation mutations of L0482ag, i.e. N68K, N73G, exclusively the following mutations, i.e., amino acid substitutions: T82S, N85G, R86K, S130N, H136K, D169N, N185Y and N186G, in reference to the wild-type LO482 sequence SEQ ID NO: 1.

Furthermore, it has been found that several of the hit variants identified upon directed evolution of L0482ag (SEQ ID NO: 2) contained the amino acid substitution H136R instead of H136K. Hence, H136R is considered herein and in context of the present invention as a very good alternative to H136K.

Thus, H3 (SEQ ID NO: 9) reflects the consensus sequence for particularly improved LO482 variants, and the corresponding amino acid substitutions, i.e. T82S, N85G, R86K, S130N, H136K/R, D169N, N185Y and N186G, in reference to the wild-type LO482 sequence SEQ ID NO: 1, are considered herein as consensus mutations, in particular, consensus amino acid substitutions.

The most beneficial amino acid substitutions / the variant H5 Furthermore, it has been surprisingly found in context of the present invention that the LO482 variant H5 (SEQ ID NO: 11), obtained in the same way as H3, had an even more enhanced stability, killing activity against S. aureus, and ability of being secreted from human cells compared to H3 (SEQ ID NO: 9); see, e.g., Example 6. Surprisingly, the only difference between H5 (SEQ ID NO: 11) and H3 (SEQ ID NO: 9) was an additional amino acid substitution in H5, namely F155Y. This further demonstrates that a single amino acid substitution found in context of the present invention, e.g., F155Y, can improve several pharmaceutical properties of LO482, as described herein, at the same time.

Hence, the amino acid substitutions T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1 are considered herein and in context of the present invention as the most beneficial amino acid substitutions for enhancing the killing activity against a target bacterium, e.g., S. aureus, and/or other pharmaceutical properties (e.g., the stability, solubility and secretion from human cells) of LO482 derived peptidoglycan hydrolases (LO482 variants).

Moreover, it needs to be emphasized that it has been surprisingly found in context of the present invention that all of the most beneficial amino acid substitutions occurred in the CHAP domain of LO482, i.e., positions 72 to 215 in SEQ ID NO: 1. It is therefore considered herein and in context of the present invention that the CHAP domain is the most important domain of LO482 derived peptidoglycan hydrolases. ר Hence, the present invention further relates, in particular, to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1 and that has (ii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

Preferably, said CHAP domain further comprises an aglycosylation mutation as described herein, i.e., an amino acid substitution or deletion, preferably an amino acid substitution, at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is, preferably, substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine.

The surprising finding of LO482 variants (e.g., H3 and H5), wherein several pharmaceutical properties were improved at the same time, is partly based on the improved means and method for protein engineering and directed evolution developed in context of the present invention and described herein.

Means and methods for screening peptidoglycan hydrolases for bactericidal activity As illustrated in the appended Examples, the inventors surprisingly found that solidified yeast culture media suchas agar plates containing autoclaved (i.e. dead) target bacteria, e.g. dead S. aureus cells, are particularly suitablefor screening yeast cells for the secretion of an active peptidoglycan hydrolase, i.e., for identifying peptidoglycan hydrolases with a good killing activity against target bacteria, e.g., S. aureus. The corresponding screening method called "Yeast on dead aureus" (YODA) is based on the inventive concept that only an active (but not an inactive) peptidoglycan hydrolase secreted from a yeast colony cultured on the solidified yeast culture medium of the invention is able to break down the peptidoglycan of the dead bacterial cells in vicinity of the colony. Hence, only a secreted and active peptidoglycan hydrolase renders the previously turbid culture medium locally translucent and thereby generates a halo around the colony secreting said peptidoglycan hydrolase; see Example 3 and Figure 4. YODA-derived methods are extremely simple and efficient methods which allows to easily distinguish yeast cells/colonies expressing peptidoglycan hydrolases with a good killing activity against a target bacterium from yeast cells/colonies expressing inactive peptidoglycan hydrolases. Furthermore, YODA-derived methods are very sensitive since the lysins are constantly secreted from the cells. Thus, small secretion rates may be sufficient to see a halo (when the peptidoglycan hydrolase is active). Moreover, as described herein, e.g., in Example 3, the YODA method or derivatives thereof as described herein (e.g., YODB) have additional advantages over prior art screening methods, e.g., the double agar layer (DAL) assay described in Zhao (2014). Appl Environ Microbiol 80(9).

Accordingly, the present invention further relates to a solidified yeast culture medium comprising substrate particles intermixed with said medium, wherein said substrate particles comprise dead bacterial cells and/or fragments thereof, and/or peptidoglycan particles.

Furthermore, the invention relates to a method of screening yeast cells for the secretion of an active peptidoglycan hydrolase (i.e., a peptidoglycan hydrolase with bactericidal activity against a target bacterium, e.g., S. aureus), said method comprising the steps of: a) providing a solidified yeast culture medium comprising substrate particles intermixed with said medium, wherein said substrate particles comprise dead bacterial cells and/or fragments thereof, and/or peptidoglycan particles; b) culturing yeast cells expressing a peptidoglycan hydrolase on a surface of said solidified medium until at least one yeast colony is detectable, in particular wherein the yeast cells are able to secrete the peptidoglycan hydrolase; c) evaluating whether a halo is apparent around a yeast colony, in particular wherein the halo corresponds to a locally reduced optical density of said solidified medium around said colony, for example, in a radius of about 0.1 to 1 cm from the colony, especially compared to a region of the solidified medium that is free of yeast colonies; d) determining that a yeast colony secretes an active peptidoglycan hydrolase when a halo around the colony is apparent, or determining that a yeast colony does not secrete an active peptidoglycan hydrolase when no halo around the colony is apparent.

As illustrated in the appended Examples, the inventive YODA-derived screening method provided herein has been proven very useful for identifying improved peptidoglycan hydrolases with bactericidal activity, e.g., against 5. aureus, see, e.g., Examples 4 to 6.

Aglycosylation mutations enhance bactericidal activity upon expression in eukaryotic cells As shown in Example 4 and Figure 5A, it has been found in context of the present invention that WT LO482 (SEQ ID NO: 1) has a good killing activity against 5. aureus, i.e., a minimum inhibitory concentration (MIC) of 4 pg/ml, when produced in Escherichia coii (E coih but only a moderate killing activity (i.e. a MIC of 352 pg/ml when secreted from Pichia pastoris^P. pastorisp i.e., yeast.

The inventors reasoned that N-and O-glycosylation of WT L0482 during its maturation in the secretory pathway in eukaryotic cells might have negatively affected its bactericidal activity. In particular, the inventors speculated that transgenes such as lysins may be glycosylated at sites essential for folding and activity, which could potentially lead to the secretion of a less active protein (while secretory proteins are normally evolutionary adapted to these modifications).

It has been found that LO482 (SEQ ID NO: 1) contains two motifs of N-glycosylation at residues N68 (which is in the linker sequence) and N73 (which is in the CHAP domain). Therefore, the inventors removed the two N- glycosylation motifs in LO482 in a degenerate codon screen by employing the YODA method as described in Example and Figure 5B.

The inventors surprisingly found that the most frequent amino acid substitution pairs contained in active LO4mutants were N68K/N73G (i.e. N68N73 to KG), N68K/N73Y (i.e. N68N73 to KY), and N68A/N73H (i.e. N68N73 to AH). It was confirmed that all three LO482 mutants (i.e., having the N68K/N73G, N68K/N73Y and N68A/N73H substitutions, respectively) were aglycosylated; see, e.g., Example 4 and Figure 5C. In addition, the further mutation pairs N68N73 to ML, RE, KL and AA were also found in active LO482 mutants by YODA.

Moreover, it has been surprisingly found by the inventors that the aglycosylated N68K/N73G (i.e., KG) mutant, also called L0482ag herein, had a killing activity against S. aureus which was almost as good as for the WT LO482 upon production in E. coir, see, e.g., Example 4 and Figure 5C.

Accordingly, the present invention further relates to aglycosylated variants of LO482 (SEQ ID NO: 1) as illustrated by but not limited to the N68K/N73G (i.e., L0482ag; SEQ ID NO: 2) mutant described herein. Herein and in context of the invention, e.g., in the context of a peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, an aglycosylation mutation refers to an amino acid substitution or deletion (preferably to an amino acid substitution) at position 68 or 73 in SEQ ID NO: 1 or at any position corresponding to these positions. In context of the CHAP domain of the invention, an aglycosylation mutation refers to an amino acid substitution or deletion (preferably to an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position.

In particular, an amino acid substitution at position 68 in SEQ ID NO: 1 or at a position corresponding to this position means that the asparagine ("N") at position 68 in SEQ ID NO: 1 is substituted with another amino acid, as described herein.

Following the same logic, an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position means that the asparagine ("N") at position 73 in SEQ ID NO: 1 or the asparagine corresponding to position 73 in SEQ ID NO: 1 is substituted with another amino acid, as described herein.

The following amino acid substitutions are considered herein and in context of the present invention as particularly effective aglycosylation mutations or aglycosylation amino acid substitutions: N68K, N68A, N68M, N68R, N73G, N73Y, N73H, N73L, N73E, and N73A in SEQ ID NO: 1 or at positions corresponding to these positions. Since the CHAP domain is of most relevance herein and in context of the present invention, an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, e.g. N73G (as contained in L0482ag; SEQ ID NO: 2), is of particular relevance herein and in context of the present invention.

LO482 variants having at least one aglycosylation mutation (e.g., an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position), as described herein and as illustrated by L0482ag (SEQ ID NO: 2), may be a particularly good starting point for further protein engineering and/or directed evolution, as described herein. As illustrated in the appended Examples, and as further described herein, additional permissive, beneficial, particularly beneficial or most beneficial amino acid substitutions may be introduced into LO482 variants having at least one aglycosylation mutation, in particular in the CHAP domain thereof, to further enhance the pharmaceutical properties of the LO482 variants (e.g. the stability solubility and ability of being secreted from a human cell but also the killing activity against a target bacterium, e.g. S. aureus), as described herein.

Thus, an aglycosylation mutation (preferably an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position (more preferably N73G), is, preferably, combined with at least one of the amino acid substitutions described herein in context of the L0482ag variants obtained upon directed evolution, in particular with at least one permissive or beneficial amino acid substitution, preferably with at least one particularly beneficial amino acid substitution, more preferably with at least one of the most beneficial amino acid substitutions, as further described herein, i.e., more preferably with at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in reference to SEQ ID NO: 1.

Hence, the present invention further relates, in particular, to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1 and that has (ii) an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than phenylalanine or lysine such as glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine.

In addition, the present invention relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase has (i) a sequence identity of at least 60% to the sequence of SEQ ID NO: 1 and has (11) at least one amino acid substitution at positions 68 and 73 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably at least at position 73 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the residue at position 68 (i.e. the "N") in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than threonine or serine such as lysine, methionine, arginine or alanine, preferably lysine. Furthermore, the residue at position 73 (i.e. the "N") in SEQ ID NO: 1 or at a position corresponding to this position is preferably substituted with another amino acid residue than phenylalanine or lysine such as glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine. Preferably, said peptidoglycan hydrolase comprises a CHAP domain according to the present invention.

Methods for screening peptidoglycan hydrolases having several improved pharmaceutical properties / directed evolution As already indicated herein above, the inventors evaluated in context of the present invention whether it would be possible to not only improve the bactericidal activity of lysins but also further pharmaceutical properties of the lysins at the same time, (e.g., the solubility, secretion from eukaryotic cells and/or stability). As already indicated above and as further described in the following, the inventors surprisingly found that several pharmaceutical properties (including the bactericidal activity) of LO482 variants, in particular L0482ag, could be further improved.

To this end, the inventors developed a combinatorial screening method based on the inventive YODA-derived method which combines eukaryotic cell display, e.g. yeast display, with YODA; see, e.g., Example 5. As illustrated in the appended Examples, it has been surprisingly found that this inventive combinatorial screening method, allows to simultaneously improve, inter alia, the solubility, bactericidal activity and eukaryotic secretion of peptidoglycan hydrolases, e.g., endolysins such as LO482 (SEQ ID NO: 1) or derivatives thereof such as L0482ag (SEQ ID NO: 2). The inventors reasoned that the display (e.g. yeast display) step allows, in particular, to screen for peptidoglycan hydrolase variants which are adapted to the eukaryotic secretory pathway, and, hence, have an improved expression and secretion profile in eukaryotic cells. Moreover, the ability of being efficiently secreted is often associated with a good stability and good solubility of the protein, as described herein. Hence these biophysical and pharmaceutically relevant properties can be also improved by the inventive method. The YODA step, in particular, allows to screen for secreted peptidoglycan hydrolase variants which have good bactericidal activity, in particular against the desired target bacterium, as described herein. Hence, the combinatorial screening method of the invention allows to screen for improved peptidoglycan hydrolase variants which are adapted to the eukaryotic secretory pathway and effectively kill a target bacterium, e.g. 5. aureus, and which may have further beneficial pharmaceutical properties such as an enhanced stability and an enhanced solubility as described herein and as illustrated in the appended Examples.

As already mentioned above, the inventive YODA-based method refers to a method of screening yeast cells for the secretion of an active peptidoglycan hydrolase, said method comprising the steps of: a) providing a solidified yeast culture medium comprising substrate particles intermixed with said medium, wherein said substrate particles comprise dead bacterial cells and/or fragments thereof, and/or peptidoglycan particles; b) culturing yeast cells expressing a peptidoglycan hydrolase on a surface of said solidified medium until at least one yeast colony is detectable; c) evaluating whether a halo is apparent around a yeast colony; d) determining that a yeast colony secretes an active peptidoglycan hydrolase when a halo around the colony is apparent, or determining that a yeast colony does not secrete an active peptidoglycan hydrolase when no halo around the colony is apparent.

Herein and in context of the present invention, said YODA-based method can be combined with a eukaryotic cell display method. In particular, said method of screening yeast cells for the secretion of an active peptidoglycan hydrolase may be performed in step III) of the method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell according to the invention, in particular, as described in the following: Hence, the present invention further relates to a method of identifying an active peptidoglycan hydrolase variant (i.e., a peptidoglycan hydrolase variant with bactericidal activity against a target bacterium, e.g., 5. aureu^ that is optimized for secretion by a eukaryotic cell, said method comprising the steps of: 1) preparing a library of eukaryotic cells, preferably yeast cells, expressing peptidoglycan hydrolase variants on the cell surface; II) selecting eukaryotic cells, e.g. yeast cells, based on a high level of peptidoglycan hydrolase on the cell surface relative to other cells in the library; for example, selecting the 10%, 5%, 1% or 0.5% of cells in the library with the highest peptidoglycan level on the cell surface; III) performing the inventive method of screening yeast cells for the secretion of an active peptidoglycan hydrolase provided herein (i.e. a YODA-derived method), wherein yeast cells that are able to secrete the peptidoglycan hydrolase variants expressed in the eukaryotic cells selected in step II) are cultured in stepb) of said method of screening yeast cells for the secretion of an active peptidoglycan hydrolase; and IV) determining that a yeast colony that has been determined in step d) of said method of screening yeast cells for the secretion of an active peptidoglycan hydrolase to secrete an active peptidoglycan hydrolase produces an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell.

Moreover, said method, i.e., the combinatorial screening method of the present invention, can be used for directed evolution of peptidoglycan hydrolases, e.g. endolysins, such as LO482 (SEQ ID NO: 1) or derivatives thereof, e.g., L0482ag (SEQ ID NO: 2), as illustrated in the appended Examples.

Therefore, in certain embodiments, at least two rounds of steps I) to IV) of the inventive method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell are performed, wherein in step I) of each subsequent round, a further library of eukaryotic cells is prepared, and wherein the cells in the library express a different set of peptidoglycan variants compared to the library employed in the preceding round(s).

Summary of beneficial and permissive amino acid substitutions found by the aglycosylation screen or directed evolution As already indicated above and as further described herein and as illustrated in the appended Examples, the inventive screening methods provided herein contributed to finding the inventive peptidoglycan hydrolases, in particular, LO482 variants, which surprisingly had several improved pharmaceutical properties at the same time. In particular, 252 LO482 variants, especially, aglycosylated LO482 variants derived from L0482ag (SEQ ID NO: 2), have been generated in three rounds of directed evolution; see, e.g., Example 5 to 7. Importantly, all these LO4variants had the ability of being secreted by eukaryotic cells, in particular yeast cells, and are deemed to have a killing activity against 5. aureus as determined by the YODA method described in Example 3. Based on this large and very informative data set the inventors not only were able to find the most beneficial mutations described herein but many further beneficial, or at least permissive, amino acid substitutions at many positions in SEQ ID NO: (or SEQ ID NO: 2) that were contained in the active and secreted L0482ag variants; see, e.g., Example 7 and Table 3. It is credible that essentially all amino acid substitutions found in active and secreted L0482ag variants are beneficial or at least permissive for the desired pharmaceutical properties of LO482 variants. In particular, it is credible that essentially all, or at least the vast majority, of these amino acid substitutions alone or in combination confer to the LYSM-CHAP lysins, in particular LO482 variants, a sufficient killing activity against Staphylococcus species or strains, in particular 5. aureus, a sufficient ability of being secreted from eukaryotic cells, a sufficient stability and/or a sufficient solubility.

As illustrated in the appended Examples, e.g., in Examples 4 to 7, amino acid substitutions in the CHAP domain of LO482 which may confer, enhance, or, at least, maintain (i) a killing activity against Staphylococcus species or strains, in particular S. aureus, and (ii) the ability of being secreted from eukaryotic cells, and preferably (iii) a sufficient solubility and/or stability, have been identified to occur, in particular, at positions 72 to 76, 78, 81, 82, 85, 86, 93, 96,104,107,108, 111, 113, 115, 117, 121, 124, 125, 129, 130, 133 to 136,138,140 to 142,144,145, 148, 149, 152, 153, 155, 157, 159, 169, 173, 175 to 178, 185,186, 190 to 194,197,198, 199, 201, 203, 204, 207, and 212 to 215 in SEQ ID NO: 1, or at positions corresponding to these positions.

Accordingly, the present invention relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1 and that has (ii) one or more amino acid substitutions at positions to 76, 78, 81, 82, 85, 86, 93, 96, 104,107, 108, 111, 113, 115, 117, 121, 124, 125, 129, 130, 133 to 136, 138, 140 to 142, 144, 145, 148, 149, 152, 153, 155, 157, 159, 169, 173, 175 to 178, 185, 186, 190 to 194, 197, 198, 199, 201, 203, 204, 207, and 212 to 215 in SEQ ID NO: 1, or at positions corresponding to these positions, wherein the amino acid residue at position 72 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine or serine, more preferably glycine, the amino acid residue at position 74 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or proline, the amino acid residue at position 75 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine or proline, the amino acid residue at position 76 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 78 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or tyrosine, the amino acid residue at position 81 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, arginine or serine, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or methionine, preferably lysine, the amino acid residue at position 93 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or leucine, the amino acid residue at position 96 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 104 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 107 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or tyrosine, the amino acid residue at position 108 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position ill in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 113 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 115 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, the amino acid residue at position 121 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 124 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine or histidine, the amino acid residue at position 125 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 129 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or serine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, isoleucine or asparagine, preferably asparagine, the amino acid residue at position 133 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or threonine, the amino acid residue at position 134 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine or valine, the amino acid residue at position 135 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine or histidine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue at position 138 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 140 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or aspartic acid, the amino acid residue at position 141 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 142 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine, the amino acid residue at position 144 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 145 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or leucine, the amino acid residue at position 148 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine or valine, the amino acid residue at position 149 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or threonine, the amino acid residue at position 152 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 153 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or serine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 157 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 159 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, asparagine or arginine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, glutamine or threonine, the amino acid residue at position 176 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine, the amino acid residue at position 177 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 178 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine or arginine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, histidine, serine or tyrosine, preferably tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 190 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 191 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine, the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine, the amino acid residue at position 193 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 194 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 197 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 198 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 199 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 201 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or serine, the amino acid residue at position 203 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or valine, the amino acid residue at position 204 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, serine or tyrosine, the amino acid residue at position 207 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine or valine, the amino acid residue at position 212 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 213 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or threonine. the amino acid residue at position 214 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine, and/or the amino acid residue at position 215 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, valine or serine.

Furthermore, it is preferred that said CHAP domain has an aglycosylation substitution, i.e., an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, and in addition, at least one amino acid substitution at the other positions described herein above, i.e., at positions 72, to 76, 78, 81, 82, 85, 86, 93, 96, 104,107, 108, 111, 113, 115, 117, 121, 124, 125, 129, 130, 133 to 136, 138, 140 to 142, 144, 145, 148, 149, 152, 153, 155, 157, 159, 169, 173, 175 to 178, 185, 186, 190 to 194, 197, 198, 199, 201, 203, 204, 207, and 212 to 215 in SEQ ID NO: 1, or at positions corresponding to these positions, preferably at least one amino acid substitution at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions.

Accordingly, the present invention further relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1, and that has (ii) one or more amino acid substitutions at positions 72, 74 to 76, 78, 81, 82, 85, 86, 93, 96, 104, 107,108, 111, 113, 115, 117, 121, 124, 125, 129, 130, 133 to 136, 140 to 142,144,145, 148, 149,152,153,155, 157, 159, 169, 173, 175 to 178, 185, 186, 190 to 192, 194, 197, 198, 199, 201, 203, 204, 207, and 212 to 215 in SEQ ID NO: 1, or at positions corresponding to these positions, preferably at positions 82, 85, 86, 130, 136, 155, 169,185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein. Preferably, said CHAP domain further has an aglycosylation mutation, i.e. an amino acid substitution or deletion, preferably a substitution, at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

Particularly beneficial amino acid substitutions / selected hit variants As already indicated herein above, the present inventors found several hit variants of L0482ag (SEQ ID NO: 2) upon each round of directed evolution, i.e., G1 to G4 (SEQ ID NO: 3 to 6) upon round 1, Hl to H10 (SEQ ID NO: to 16) upon round 2 and II to 130 (SEQ ID NO: 17 to 46) upon round 3. The various amino acid substitutions contained in these hit variants (in addition to the two aglycosylation substitutions N68K and N73G) are considered herein and in context of the present invention as particularly beneficial mutations, i.e., amino acid substitutions; see, e.g., Examples 5 to 7.

As illustrated in the appended Examples, particularly beneficial amino acid substitutions in the CHAP domain of LO482 which may confer, enhance, or, at least, maintain (i) a killing activity against Staphylococcus species or strains, in particular S. aureus, and (11) the ability of being secreted from eukaryotic cells, and (ill) preferably a sufficient solubility and stability, have been identified to occur, in particular, at positions 73, 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191 to 194, 198, 204, 212 and 215 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein.

Accordingly, the present invention further relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1, and that has (ii) one or more amino acid substitutions at positions 73, 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191, 192, 194, 198, 204, 212 and 215 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine or serine, preferably glycine, the amino acid residue at position 75 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 78 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 81 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 104 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 107 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 115 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 124 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 125 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 133 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 135 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue at position 140 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 141 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 178 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 191 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 194 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 198 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 204 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 212 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue at position 215 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine.

Furthermore, it is preferred that said CHAP domain has an aglycosylation mutation, preferably, an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, more preferably N73G, and in addition, at least one particularly beneficial amino acid substitution at the other positions described herein above, i.e., at positions 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191, 192, 194, 198, 204, 212 and 215 in SEQ ID NO: 1, or at positions corresponding to these positions, preferably at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions.

Accordingly, the present invention further relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 2, and that has (ii) one or more amino acid substitutions at positions 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191, 192, 194, 198, 204, 212 and 215 in SEQ ID NO: 1, or at positions corresponding to these positions, as described herein, preferably one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein.

Most beneficial amino acid substitutions As already indicated above, based on the characterizations of the hit variants of LO482 (see, e.g., Example 6 and Figure 8) found in context of the present invention and comparative analyses of the sequences of these hits (see, e.g., Example 7), the inventors were able to find the most beneficial amino acid substitutions, i.e., T82S, N85G, R86K, S130N, H136K/R (preferably H136K), F155Y, D169N, N185Y and N186G in SEQ ID NO: 1.

As mentioned above, the most beneficial amino acid substitutions refer to the only amino acid substitutions contained in the hit variant H5 (SEQ ID NO: 11) vis a vis the parental L0482ag variant (SEQ ID NO: 2); see, e.g., Figure 9A. H5 is considered herein as the variant obtained by two rounds of directed evolution with the best pharmaceutical properties. As mentioned above, the most beneficial amino acid substitutions in H5 strongly enhanced the killing activity against S. aureus including S. aureus biofilms, the protein stability and the ability of being secreted from humans cells; see, e.g., Figures 8, 9,11 and 12.

Therefore, as already mentioned above, the present invention further relates, in particular, to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1, and that has (ii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

Preferably, said CHAP domain has a plurality of said most beneficial amino acid substitutions (e.g., 2, 3, 4, 5, 6, 7, or 9), preferably at least 6 or 7, more preferably at least 8, most preferably all of said most beneficial amino acid substitutions. Furthermore, said CHAP domain has preferably further an aglycosylation mutation, preferably an amino acid substitution, at position 73 in SEQ ID NO: 1 or at a position corresponding to this mutation as described herein, more preferably N73G.

The most recurrent one of the most beneficial amino acid substitutions: R86K Furthermore, it has been surprisingly found in context of the present invention that the amino acid substitution R86K was contained in all 44 identified hit variants, i.e., G1-G4, H1-H10 and 11-130, and in 96.08% of all LO4variants that were secreted from eukaryotic cells and that were determined to have a killing activity against S. aureus, as described herein and as illustrated in the appended Examples; see, e.g., Example 7 and Figure 9.

Therefore, R86K in SEQ ID NO: 1 is, herein and in context of the present invention, a particularly preferred amino acid substitution among the most beneficial amino acid substitutions identified. Accordingly, in particularly preferred embodiments, the CHAP domain of the present invention has an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with lysine.

Recurrent pairs of amino acid substitutions among the most beneficial amino acid substitutions It has been further surprisingly found in context of the present invention that certain amino acid substitutions occurred together in pairs in the identified hit variants, i.e., G1-G4, H1-H10 and 11-130; see, e.g., Example 7 and Figure 9. These mutation pairs are specifically: (i) T82S and N85G, (11) S130N and H136K/R and (ill) N185Y and N186G. Each of said 44 hit variants contained at least one of said mutation pairs. Of note, it is beneficial but not necessary that these amino acid substitutions, i.e., T82S and N85G, S130N and H136K/R, and N185Y and N186G, occur in pairs since among the 252 active LO482 variants secreted from yeast cells found in context of the present invention, variants having only one of these paired mutations (but not the other) were found as well.

Therefore, a CHAP domain of the invention having an amino acid substitution at position 82 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 85 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Similarly, a CHAP domain of the invention having an amino acid substitution at position 85 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

Furthermore, a CHAP domain of the invention having an amino acid substitution at position 130 in SEQ ID NO: or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 136 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Similarly, a CHAP domain of the invention having an amino acid substitution at position 136 of SEQ ID NO: 1 or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 130 of SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

Furthermore, a CHAP domain of the invention having an amino acid substitution at position 185 in SEQ ID NO: or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 186 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Similarly, a CHAP domain of the invention having an amino acid substitution at position 186 of SEQ ID NO: 1 or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 185 of SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

Consensus mutation units As already mentioned herein above, the hit variant H3 (SEQ ID NO: 9) reflects the consensus sequence for particularly improved LO482 variants. H3 contains exclusively, the following amino acid substitutions, also referred to herein as "consensus mutations": T82S, N85G, R86K, S130N, H136K/R (esp. H136K), D169N, N185Y and N186G in reference to SEQ ID NO: 1.

Furthermore, the inventors found five consensus mutation units for particularly improved LO482 variants based on the 44 L0482ag hit variants and the consensus sequence reflected by H3 (SEQ ID NO: 9) found in context of the present invention. These consensus mutation units consist of 1 or 2 amino acid substitutions, i.e.: (i) R86K (which is particularly preferred), (ii) T82S and N85G, (iii) S130N and H136K/R (preferably H136K), (iv) D169N, and (v) N185Y and N186G.

Accordingly, in preferred embodiments, the CHAP domain of the present invention has at least one amino acid substitution or substitution pair ( i.e. at least one consensus mutation unit) selected from the group consisting of the following (i) to (v):(i) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with lysine; (ii) an amino acid substitution at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine;(iii) an amino acid substitution at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 in SEQ ID NO: or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine;(iv) an amino acid substitution at position 169 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with asparagine; and(v) an amino acid substitution at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

Preferably, said CHAP domain has at least two, preferably at least three, more preferably at least four, most preferably all of said consensus mutation units.

Preferably, said CHAP domain has further an aglycosylation mutation as described herein, preferably an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein, more preferably the substitution N73G.

Furthermore, said CHAP domain has preferably the amino acid substitution F155Y.

In certain preferred embodiments, the CHAP domain of the present invention has a sequence identity of at least 94% (in particular, at least 93.9%), at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 9 (H3). Preferably, said CHAP domain has a) (i) at least one of the consensus mutations or consensus mutation units described herein and (ii) at least one aglycosylation mutation, preferably an amino acid substitution at position 73 as described herein, and/or b) at least two of the consensus mutations, e.g., at least one amino acid substitution pair, as described herein. Furthermore, in context of these preferred embodiments or in similar preferred embodiments, the CHAP domain of the present invention has at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, at most 1 or no amino acid substitutions in the sequence from position 72 to position 215 in SEQ ID NO: 9 (H3) or in the sequence from a position corresponding to position 72 in SEQ ID NO: 9 to a position corresponding to position 215 in SEQ ID NO: 9.

Furthermore, said CHAP domain has preferably the amino acid substitution F155Y.

In certain preferred embodiments, the peptidoglycan hydrolase of the present invention has a sequence identity of at least 95% (in particular at least 95.4%), at least 96%, at least 97%, at least 98% or at least 99% to the sequence of SEQ ID NO: 9 (H3). Furthermore, in context of these preferred embodiments or in similar preferred embodiments, the peptidoglycan hydrolase of the present invention has at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, at most 1 or no amino acid substitutions in SEQ ID NO: 9.

Preferably, said peptidoglycan hydrolase comprises a CHAP domain according to the present invention.

A single amino acid substitution improving several pharmaceutical properties at once: F155Y As further indicated herein above, the best performing LO482 variant found and characterized in context of the present invention, i.e. H5 (SEQ ID NO: 11), surprisingly contained exclusively one additional amino acid substitution vis a vis H3 (SEQ ID NO: 9), i.e., F155Y. As further described herein, said amino acid substitution improved several pharmaceutical properties compared to H3 (SEQ ID NO: 9) at the same time, i.e. the killing activity against S. aureus, the protein stability and the ability of being secreted from human cells; see, e.g, Figure 8.

Therefore, F155Y in SEQ ID NO: 1 is considered herein and in context of the present invention as a further particularly preferred amino acid substitution among the most beneficial amino acid substitutions identified. Accordingly, in particularly preferred embodiments, the CHAP domain of the present invention has an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with tyrosine.

Preferably, said CHAP domain further has at least one consensus mutation, as described herein, (i.e., T82S, N85G, R86K, S130N, H136K/R (preferably H136K), D169N, N185Y and/or N186G), or, more preferably, at least one consensus mutation unit, as described herein, i.e., (i) R86K (particularly preferred), (ii)T82S and N85G, (iii) S130N and H136K/R (preferably H136K), (iv) D169N, and/or (v) N185Y and N186G; and/or an aglycosylation substitution as described herein, i.e., an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein, preferably N73G.

In further preferred embodiments, the CHAP domain of the present invention has a sequence identity of at least 93% (in particular, at least 93.2%), at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 11 (H5). Preferably, said CHAP domain has a) (i) at least one of the most beneficial amino acid substitutions and/or at least one consensus mutation unit, as described herein and (ii) at least one aglycosylation mutation, preferably an amino acid substitution at position 73, as described herein, and/or b) at least two of the most beneficial amino acid substitutions, e.g., at least one amino acid substitution pair, as described herein. Furthermore, in context of these preferred embodiments or in similar preferred embodiments, the CHAP domain of the present invention has at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, at most 1 or no amino acid substitutions in the sequence from position 72 to position 215 in SEQ ID NO: 11 (H5) or in the sequence from a position corresponding to position in SEQ ID NO: 11 to a position corresponding to position 215 in SEQ ID NO: 11.

In certain preferred embodiments, the peptidoglycan hydrolase of the present invention has a sequence identity of at least 95% (in particular at least 95.0%), at least 96%, at least 97%, at least 98% or at least 99% to the sequence of SEQ ID NO: 11 (H5). Furthermore, in context of these preferred embodiments or in similar preferred embodiments, the peptidoglycan hydrolase of the present invention has at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, at most 1 or no amino acid substitutions in SEQ ID NO: 11.

Furthermore, the peptidoglycan hydrolase of the present invention is, in particular, a single polypeptide, i.e., a single amino acid chain.

Administration in form of a nucleic acid As already indicated above, administration of a peptidoglycan hydrolase of the invention in form of a nucleic acid (e.g., an mRNA) encoding said peptidoglycan hydrolase to a subject (e.g., a human), in particular introducing the nucleic acid into cells in a subject, has certain advantages. For example, when a nucleic acid (e.g., an mRNA) encoding a peptidoglycan hydrolase of the invention is introduced and expressed in cells in a subject, e.g., in a patient that has a bacterial, e.g. Staphylococcus, infection, the cells can continuously produce and secrete the peptidoglycan hydrolase protein. This may provide a more efficient treatment of the bacterial infection and, for example, provide a higher efficacy in treating difficult to treat bacterial infections such as bacterial biofilms, e.g., Staphylococcus'oioYiVms. Furthermore, the nucleic acid may be introduced into cells at a particular location, e.g. the site of a bacterial infection, and/or specific cell types which may further improve the efficiency and/or safety of the treatment. Furthermore, nucleic acids, in particular RNAs, have further certain practical advantages over proteins with respect to their manufacturing, safety profile and/or adaptability.

It has been further found by the inventors that all hit variants found in context of the invention and further tested, i.e., G1 to G4 and Hl to H6, were efficiently secreted from human cells when expressed from an RNA in these cells. In particular, it has been found that the secretion from human cells was highly enhanced compared to a control RNA expressing WT L0482 (SEQ ID NO: 1); see Examples 11 and 12 and Figure 10.

Therefore, the present invention further relates to a nucleic acid encoding the peptidoglycan hydrolase of the invention, preferably comprising a CHAP domain that has at least one of the most beneficial mutations (i.e. T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1) and/or at least one of the consensus mutation units, as described herein. Preferably, the nucleic acid of the invention is a RNA, preferably a mRNA. Preferably, the RNA, e.g., the mRNA, of the present invention comprises at least one modified nucleoside such as pseudouridine (w), Nl-methyl-pseudouridine (mly) or 5-methyl-uridine (m5U), preferably Nl-methyl- pseudouridine (mly), in place of at least one uridine, preferably in place of multiple, more preferably all uridines.

Detailed description of the invention Peptidoglycan hydrolases The term "peptidoglycan hydrolase", as used herein and in context of the present invention, refers to a polypeptide (i.e., a single amino acid chain) which is capable of hydrolyzing peptidoglycan (also called "murein") of at least one bacterial species or strain, preferably at least one Staphylococcus spec ies or strain, more preferably Staphylococcus aureus. A peptidoglycan hydrolase of the invention may comprise naturally occurring amino acids and/or non- naturally occurring amino acids as well as modifications such as, but not limited to, glycosylation (in particular 0- glycosylation and/or N-glycosylation), acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, or a protective group. Preferably, the peptidoglycan hydrolase of the invention is comprised of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% naturally occurring amino acids (which may comprise modifications or not), as described herein. The term "peptidoglycan hydrolase" encompasses, for example, endolysins, lysozymes, tail-spike depolymerases, Virion- associated peptidoglycan hydrolases (VAPGH), bacteriocins and autolysins. In preferred embodiments, the term "peptidoglycan hydrolase" refers to an endolysin. The terms "endolysin" and "lysin" are used interchangeably herein and in context of the present invention.

Isolated peptidoglycan hydrolases The peptidoglycan hydrolase of the present invention may be an isolated peptidoglycan hydrolase, e.g., an isolated endolysin. In particular, herein, "isolated" means removed (e.g., purified) from the natural state. For example, a nucleic acid, peptide or polypeptide naturally present in a living animal is not "isolated", but the same nucleic acid, peptide or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid, peptide or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. Preferably, an isolated peptidoglycan hydrolase, as used herein, refers to a peptidoglycan hydrolase which is isolated (e.g. purified) from its natural context, e.g., from a bacterial cell where it occurs in nature.

Engineered and natural peptidoglycan hydrolases In context of the present invention, a "peptidoglycan hydrolase" or an "endolysin", is, preferably, a modified, i.e., an engineered, "peptidoglycan hydrolase" or "endolysin", respectively. In particular, a "modified" (i.e., an "engineered", synthetic", "recombinant", or "artificial") peptidoglycan hydrolase or endolysin does not occur in nature and thus may be also considered herein and in context of the present invention as a "non-natural" (i.e., "non-native") peptidoglycan hydrolase or endolysin, respectively.

Thus, in certain embodiments, the peptidoglycan hydrolase of the invention is an engineered peptidoglycan hydrolase.

Furthermore, in certain embodiments, the peptidoglycan hydrolase of the invention is a non-natural peptidoglycan hydrolase.

However, in certain embodiments of the invention, the peptidoglycan hydrolases may also include natural (i.e. native) peptidoglycan hydrolases, in particular, natural endolysins, for example, in context of pharmaceutical compositions, medical uses, fusion proteins of a peptidoglycan hydrolase and another (poly)peptide (e.g. a PKtag), RNA constructs, and/or modified nucleic acids, e.g. RNAs containing a modified nucleoside such as N1-methyl- pseudouridine (mly) in place of at least one uridine, as described herein.

Endolysins Herein, and in context of the present invention, the term "endolysin" refers, in particular, to natural peptidoglycan hydrolases encoded by bacteriophages (or bacterial viruses), or engineered peptidoglycan hydrolases, in particular modified endolysins, derived from such natural peptidoglycan hydrolases (e.g., from LO482; SEQ ID NO: 1). Natural endolysins act, in particular, by hydrolyzing the host cell wall and subsequently allow the release of bacteriophage progenies. The peptidoglycan hydrolases, e.g., the endolysins, of the present invention are also capable of hydrolyzing peptidoglycan in the cell wall of a bacterial species or strain (e.g., S. aureus) and, thus, have a killing activity against said bacterial species or strain (e.g., S. aureus), as described herein.

The term "cell wall" as used herein refers to all components that form the outer cell enclosure of bacteria (containing, in particular, peptidoglycan), as commonly understood in the art.

Natural endolysins usually have a molecular weight ranging from about 15 to about 60 kDa, which is also a preferred range for the peptidoglycan hydrolases (e.g. modified endolysins) of the present invention. However, the peptidoglycan hydrolases of the invention may also have a different, e.g., higher, molecular weight, for example when they further comprise additional domains or tags such as a PK tag, as described herein. Moreover, natural endolysins typically have a modular configuration. Herein and in context of the present invention, an endolysin refers, in particular, to a modular endolysin, i.e., a polypeptide (i.e., a single amino acid chain) comprising one or more enzymatically active domains (EADs) and, preferably, additionally one or more cell wall-binding domains (CBDs). Moreover, it is possible, also in context of a modular endolysin, that the endolysin only comprises one module, namely an EAD, e.g. a CHAP domain of the invention, but not a separate cell wall-binding domain. Nevertheless, it is preferred that the peptidoglycan hydrolase of the invention comprises at least one EAD, in particular the CHAP domain of the invention, and additionally at least one CBD. Furthermore, the various domains in a modular endolysin, e.g., an EAD and a CBD, may be separated by linker regions, in particular, by short and flexible linkers, as described herein. An EAD may be N-terminally or C-terminally of a CBD. In LO482 (SEQ ID NO: 1), a single cell wall-binding domain (CBD), i.e. the LYSM domain, is N-terminally of a single enzymatically active domain (EAD), i.e. the CHAP domain. Furthermore, in LO482, the LYSM and CHAP domains are separated by a linker region.

LO482 Herein, and in context of the present invention, LO482 (i.e., wild-type L0482; also called "lytN") has the following sequence, wherein the LYSM domain (positions 1 to 51) is underlined, the linker (positions 52 to 71) is in italics, and the CHAP domain (positions 72 to 215) is in bold : REAPKTQIYTVKKGDTLSAIALKYKTTVSNIONTNNIANPNLIFIGOKLKVPMTPLVEPKPKTVSSNNKSNSNSSTLNYLKILE Egwdfdgsygwqcfdlvnvywnhlyghglkgygakdipyannfn EeakiyEntptfkaepgdlvvfsgrGgg GYGHTAIVLNGDYDGKLMKFOSLDONWNIGGWRKAEVAHKVVHNYENDMIFIRPFKKA(SEQ ID NO: 1) In the above sequence, the two glycosylation positions in SEQ ID NO: 1, i.e., positions 68 and 73, which may be deleted or, preferably, substituted with another amino acid (e.g., lysine and glycine, respectively) in order to obtain "aglycosylated" LO482 variants (e.g., L0482ag; SEQ ID NO: 2), as described herein, are further highlighted in grey. Furthermore, the positions which may be substituted with the most beneficial amino acid residues contained in the best performing characterized variant, i.e., H5 (SEQ ID NO: 11), as described herein, i.e., positions 82, 85, 86,130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1, are shown in white on black background.

The CHAP domain of LO482 (SEQ ID NO: 1) is further shown in SEQ ID NO: 301; the LYSM domain of of LO4(SEQ ID NO: 1) is further shown in SEQ ID NO: 302; and the linker region of LO482 (SEQ ID NO: 1) is further shown in SEQ ID NO: 303.

In context of the present invention, a natural peptidoglycan hydrolase, in particular the endolysin LO482 (SEQ ID NO: 1), is usually modified by at least one amino acid substitution, as described herein. Furthermore, one or more deletions, insertions and/or additions of amino acid residues may also occur. A modified LO482 protein is also considered herein as an LO482 variant. Furthermore, the peptidoglycan hydrolase according to the present invention, in particular a modified LO482 variant, may lack the LYSM domain or the CHAP domain of LO482, as well as the linker region, as described herein. Alternatively, the LYSM domain or the CHAP domain, as well as the linker region, may be replaced by another cell-wall binding domain or enzymatically active domain, or linker region, respectively, as described herein.

Accordingly, the peptidoglycan hydrolase according to the present invention comprises (I) a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1, as described herein; and/or (II) a LYSM domain that has (i) a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1 and (ii) one or more amino acid substitutions as compared to the sequence from position 1 to position 51 in SEQ ID NO: 1, as described herein.

Domains of peptidoglycan hydrolases, in particular, of modular endolvsins As already indicated above, the enzymatically active domains (EAD) of modular endolysins function, in particular, to cleave certain peptidoglycan bonds in the murein (i.e. peptidoglycan) layer of a host bacterium. Cell wall-binding domains (CBD) are typically enzymatically inactive, and, in particular, recognize and bind to certain epitopes in the cell wall of the host bacterium for proper fixation of the catalytic effect of the EAD. Preferably, herein and in context of the present invention, the cell wall binding domain is a peptidoglycan binding domain which binds, in particular, to the peptidoglycan structure of a target bacterium. The different domains of an endolysin can be connected by a peptide linker, also called "domain linker". Moreover, as described herein, an EAD (e.g. a CHAP domain) may hydrolyse the peptidoglycan of a target bacterium e.g., the host bacterium, by itself, i.e., without the need for interacting with a separate CBD. For example, an EAD such as a CHAP domain may have (in addition to the peptidoglycan hydrolase activity) some intrinsic cell wall-binding activity and, therefore, have bactericidal activity by itself. This has been already demonstrated, e.g., for some peptidoglycan hydrolases such as lysozyme. In particular, binding of a substrate in the cell wall of a target bacterium to or close to the catalytic pocket of an EAD may be sufficient to initiate docking and subsequent lysis of the bacterium. Therefore, a CHAP domain of the invention may be also considered herein and in context of the present invention as a peptidoglycan hydrolase which may have a bactericidal activity by itself.

Accordingly, a peptidoglycan hydrolase according to the present invention comprises at least one enzymatically active domain (EAD) which is also called "catalytic domain" herein. Preferably, said at least one enzymatically active domain comprises at least a CHAP domain of the invention, as described herein, i.e., a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1.

Enzymatic peotidoalvcan cleavage mechanisms As used herein, the term "peptidoglycan hydrolase" is, in general, not restricted to a specific enzymatic cleavage mechanism. In particular, an enzymatically active domain of a peptidoglycan hydrolase may function as a glycosidase, as an amidase (i.e. an amidohydrolase) and/or as a peptidase. Hence, a peptidoglycan hydrolase comprising one or more EADs as described herein and in context of the present invention, e.g., an endolysin, may function as a glycosidase, an amidase and/or a peptidase. As used herein, glycosidases such as acetylmuramidases, lytic transglycosylases or glucosaminidases generally cleave the backbone of glycan. In particular glycosidases may cleave the 81,4־ glycosidic bonds linking alternating polymeric structures of N-acetylmuramic acids (MurNAc) and N-acetylglucosamines (GIcNAc) in a peptidoglycan layer. Amidases (i.e. amidohydrolases) generally cleave the side- chain peptide, in particular they may catalyze the cleavage of amide bonds between the MurNAc and the first amino acid in the peptide stem moiety, i.e., L-alanine. Peptidases (in particular, endopeptidases and carboxypeptidases) generally cleave within the peptide side-chain, in particular they may cleave bonds between two amino acids of the stem peptide of peptidoglycan, whereby bond cleavage can either occur within interpeptide bridge or stem peptide- interpeptide bridge.

Herein and in context of the present invention, the term "CHAP domain" refers to a cysteine, histidine-dependent amidohydrolase/peptidase domain which is an enzymatically active domain (EAD) of a peptidoglycan hydrolase (in particular of a LO482 variant), as described herein. A CHAP domain may be further considered herein and in context of the present invention as a peptidoglycan hydrolase.

Moreover, the CHAP domain of LO482 (SEQ ID NO: 1) may function as an amidase and as a peptidase in order to hydrolyse peptidoglycan. In particular, it has been reported that the CHAP domain has an N-acetylmuramyl L-Ala amidase activity and a D-Ala-Gly endopeptidase activity; Frankel (2012), J Biol Chern. 23;287(13). Hence, the CHAP domain of the invention may also function, in particular, as an amidase and/or as a peptidase, preferably as an amidase and as a peptidase. Accordingly, the peptidoglycan hydrolase of the present invention functions, preferably, as an amidase (i.e. it has, preferably, an amidase activity) and/or as a peptidase (i.e. it has, preferably, a peptidase activity). In particular, the peptidoglycan hydrolase of the present invention may have an N- acetylmuramyl L-Ala amidase activity and/or a D-Ala-Gly endopeptidase activity. More preferably the peptidoglycan hydrolase of the present invention functions as an amidase and as a peptidase, in particular, wherein it may have an N-acetylmuramyl L-Ala amidase activity and a D-Ala-Gly endopeptidase activity.

Moreover, a CHAP domain which functions as an amidase and/or as a peptidase in order to hydrolyse peptidoglycan, may be also considered herein and in context of the present invention as a peptidoglycan hydrolase with amidase and/or peptidase activity, respectively. Thus, the CHAP domain of the invention may be a peptidoglycan hydrolase with amidase (e.g. N-acetylmuramyl L-Ala amidase activity) and/or peptidase activity (e.g. D-Ala-Gly endopeptidase activity). Accordingly, a peptidoglycan hydrolase of the present invention comprising the CHAP domain of the invention has, preferably, an amidase and/or peptidase activity, as described herein.

Herein, and in context of the present invention, hydrolyzing peptidoglycan in the cell wall of a bacterium may refer also to breaking down and/or cleaving said peptidoglycan.

Bactericidal activity Hydrolyzing, in particular breaking down and/or cleaving, peptidoglycan in the cell wall of a bacterium, usually kills the bacterium. Therefore, the peptidoglycan hydrolase of the present invention has, in particular, a bactericidal activity. Herein, and in context of the present invention, the term "bactericidal activity" refers to the ability of a peptidoglycan hydrolase to kill at least one bacterium, i.e., at least one bacterial species or strain, in particular at least one target bacterium (i.e., a bacterial species or strain to be killed). Preferably, a peptidoglycan hydrolase of the invention has the ability to kill at least one gram-positive bacterium, more preferably a Staphylococcus species or strain, most preferably at least Staphylococcus aureus, preferably, including methicillin-resistant Staphylococcus aureus strains. A peptidoglycan hydrolase which has the ability to kill a certain bacterium, e.g., S. aureus, is also referred to herein as a peptidoglycan hydrolase which has a "killing activity" against said bacterium, e.g., S. aureus.

Herein and in context of the present invention, the killing activity of a peptidoglycan hydrolase against a certain bacterium, e.g., S. aureus, is preferably measured by determining the minimum concentration at which the peptidoglycan hydrolase growth-inhibits a liquid culture of said bacterium, e.g., S. aureus. Said minimum concentration is also referred to herein as "minimal inhibitory concentration" (MIC). Herein and in context of the present invention, the "minimal inhibitory concentration" (MIC) is, in particular, defined as the minimum concentration which keeps the optical density at 620 nm (OD620) of a liquid culture comprising 5x105 cfu/ml of a target bacterium (e.g., S. aureus) below 0.1 for at least 24h at 37OC incubation. Preferably, the culture medium of said liquid culture is cation adjusted Muller-Hinton broth (caMHB) medium supplemented with 25% horse serum, in particular, when the target bacterium is a Staphylococcus species or strain such as S. aureus. When the killing activity against S. aureus is measured, the S. aureus cells in the liquid culture correspond, preferably, to 5xlcfu/ml of ATCC43300 which is a methicillin-resistant Staphylococcus aureus strain. The term "cfu" is the abbreviation of "colony forming units", and refers to the estimated number of viable bacterial cells as commonly understood in the art.

A detailed assay for measuring the killing activity against a target bacterium, e.g., a Staphylococcus species or strain such as S. aureus, is provided in Example 6: First, a peptidoglycan hydrolase (e.g., a LO482 variant of the present invention) is produced in E. coli, as described in Example 6 under the heading "Production of LO482 variants in E. coif. Then, the peptidoglycan hydrolase (e.g., the LO482 variant of the present invention) is purified from E. coli, as described under the heading "Purification of L0482 variants from E. coli' in Example 6. Finally, the bactericidal activity of the peptidoglycan hydrolase (e.g., the LO482 variant of the present invention) against a target bacterium (e.g. S. aureus) is determined as described in Example 6 under the heading "Determination of bactericidal activity of LO482 variants".

Detailed assays for measuring the killing activity of a peptidoglycan hydrolase against a biofilm or free-floating aggregate of a target bacterium, e.g., a biofilm or free-floating aggregate of Staphylococcus species or strain such as S. aureus, are provided in Example 6 under the heading "Determination of anti-biofilm activity of LO482 variants" which may be preferably employed. One of these assays makes use of a peg biofilm in plasma (PBA) and is particularly well suitable for determining the killing activity of a peptidoglycan hydrolase against a classical biofilm that is attached to a surface. The other assay makes use of a free-fioating aggregate in synovial fluid (FBA) and is particularly well suitable for determining the killing activity of a peptidoglycan hydrolase against a free-floating (biofilm-like) aggregate.

As used herein and in context of the present invention, the term "activity" of a peptidoglycan hydrolase refers, in particular, to the bactericidal activity of the peptidoglycan hydrolase, as described herein. Furthermore, because the bactericidal activity of a peptidoglycan hydrolase against a certain bacterial species or strain, e.g. S. aureusas described herein, is tightly correlated to its capability of hydrolyzing (and breaking down and/or cleaving) peptidoglycan in the cell wall of said bacterial species or strain, it is not necessary to further measure or determine the ability of the peptidoglycan hydrolase according to the present invention to hydrolyze, breakdown and/or cleave peptidoglycan by an enzymatic assay; it is sufficient to determine the bactericidal activity of the peptidoglycan hydrolase, i.e., its killing activity against the bacterial species or strain, e.g. S. aureus, as described herein. In other words, a peptidoglycan hydrolase (e.g. an endolysin) according to the present invention is considered to implicitly have the ability to hydrolase peptidoglycan in a target bacterium when it is able to kill said target bacterium, as described herein. However, to further corroborate that a peptidoglycan hydrolase functions indeed as a "peptidoglycan hydrolase", it is also possible to perform additional assays. For example, the reduction of purified peptidoglycan at OD620 nm in the present of a peptidoglycan hydrolase of the invention may be easily measured.

Furthermore, purified peptidoglycan may be incubated with a peptidoglycan hydrolase of the invention followed by analysis and identification of cleavage products by mass spectrometry. Furthermore, the YODA-derived method of screening yeast cells for the secretion of an active peptidoglycan hydrolase according to the invention, as described herein may be employed. As described herein, in said screening method, the "activity" of a peptidoglycan hydrolase, i.e., its killing activity against a certain bacterium, is determined by measuring its ability to break down peptidoglycan contained in dead bacterial cells of said bacterium and/or fragments thereof or corresponding peptidoglycan particles (in particular via hydrolysis of the peptidoglycan).

Gram-positive and gram-negative bacteria In gram-positive bacteria, the cytoplasmic membrane is surrounded by a peptidoglycan layer. A main purpose of the cell wall of Gram-positive bacteria is to maintain the shape of the bacteria and counteract the pressure inside the bacterial cells. Peptidoglycan or murein is a polymer composed of sugar and amino acid. The sugar component is composed of N-acetylglucosamine residuesand a N-acetyl mu ramie acid residues that are p-(l,4) linked. A peptide chain consisting of 3 to 5 amino acids is bound to N-acetylmu ramie acid. Peptide chains can be cross-linked to peptide chains of other chains to form a 3D mesh-like layer. The peptide chain can contain D- and L-amino acid residues, and its composition can vary depending on the type of bacteria.

In contrast to gram-positive bacteria, gram-negative bacteria have an outer membrane with a characteristic asymmetric bilayer. The outer membrane bilayer consists of an inner monolayer containing phospholipids (primarily phosphatidylethanolamine) and an outer monolayer composed primarily of lipopolysaccharide (LPS). This outer membrane overlays a peptidoglycan layer which is normally much thinner than in gram-positive bacteria.

As described herein, the peptidoglycan hydrolase of the invention has, in particular, a killing activity against at least one gram-positive bacterium, preferably a Staphylococcus species or strain, more preferably at least Staphylococcus aureus, as described herein.

Variants and sequence identity to a reference sequence Herein and in context of the present invention, a polypeptide (e.g., a peptidoglycan hydrolase of the invention) or a part thereof (e.g., a CHAP domain of the invention) which is derived from a certain polypeptide (e.g. LO482) or a part thereof (e.g. the CHAP domain of LO482) is considered herein and in context of the present invention as a "variant" of said polypeptide or part thereof, respectively, (e.g. a LO482 variant or CHAP domain variant, respectively). Furthermore, a peptidoglycan hydrolase of the invention comprising a CHAP domain of the invention, i.e., a CHAP domain derived from the CHAP domain of LO482, is also considered herein and in context of the present invention as an LO482 variant.

Herein, and in context of the present invention, the terms "variant" and "mutant" may be used interchangeably.

A variant (e.g. the CHAP domain of the invention) has, in particular, a sequence identity of at least n % (i.e. at least 60%) to a corresponding reference sequence (e.g. the sequence from position 72 to position 215 in SEQ ID NO: 1). Herein, a reference sequence, usually, refers to the sequence of the (poly)peptide or part thereof from which the variant is derived (e.g. the CHAP domain of LO482). Moreover, it is possible that a variant itself is employed as a reference sequence (e.g. H5; SEQ ID NO: 11) for describing another variant.

Furthermore, a variant, i.e. a polypeptide (e.g., a peptidoglycan hydrolase of the invention) ora part thereof (e.g., a CHAP domain of the invention) which is derived from a certain polypeptide (e.g. LO482) or a part thereof (e.g. the CHAP domain of LO482) that is used as a reference sequence, has at least one mutation, i.e., at least one amino acid substitution, deletion, insertion and/or addition, preferably at least one amino acid substitution, relative to the reference sequence (e.g. at least one mutation in SEQ ID NO: 1, or in the sequence from position 72 to position 215 in SEQ ID NO: 1, respectively).

As commonly understood in the art and as also used herein and in context of the present invention, the term "sequence identity", refers to the extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment. Typically, the "sequence identity" is expressed as a percentage. Moreover, one of the two sequences may be considered as a reference sequence to which the other sequence has a sequence identity of at least n %.

Thus, herein and in context of the present invention, a certain (poly)peptide (e.g., a peptidoglycan hydrolase of the invention) or a part thereof (e.g., a CHAP domain of the invention) can be structurally defined by having a sequence identity of at least n % to a corresponding reference sequence, with n being an integer between 60 and 99, in particular 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. As just mentioned above, said reference sequence refers to a (poly)peptide (e.g. SEQ ID NO: 1) or a part thereof (e.g. the sequence from position 72 to position 2in SEQ ID NO: 1) from which said (poly)peptide or part thereof is derived, respectively.

Furthermore, it may be specifically indicated herein and in context of the present invention that a certain polypeptide or part thereof has a higher minimal sequence identity to a corresponding reference sequence than "at least 60%", i.e., a sequence identity of at least n %, with n being an integer between 61 and 99. For example, when the sequence identity is at least 80%, n is an integer between 80 and 99.

Furthermore, when it is indicated herein that a certain polypeptide or part thereof has a minimal sequence identity of at least 90% to a corresponding reference sequence, i.e., a sequence identity of at least n %, with n being an integer between 90 and 99, n may also refer to a decimal number with one decimal place between 89.5 and 99.9, in particular, 89.5,89.6, 89.7, 89.9,90.0,90.1,90.2 etc. or 99.9. In particular, when the minimal sequence identity is indicated by an integer, i.e., at least n % with n being an integer (e.g. at least 95%), n may also refer to a decimal number with one decimal place that can be rounded by conventional rounding rules to said integer. For example, a sequence identity of at least 95% may also refer to a sequence identity of at least 94.5%, at least 94.6%, at least 94.7%, at least 94.8%, at least 94.9%, at least 95.0%, at least 95.1%, at least 95.2%, at least 95.3% or at least 95.4%.

Generally, herein and in context of the present invention, the higher the sequence identity to a reference sequence, the more preferred it is. Furthermore, certain minimal sequence identities (other than "at least 60%") are indicated herein in specific contexts and thus are considered as preferred minimal sequence identities in these contexts. However, other minimal sequence identities to a reference sequence, in particular, a sequence identity of at least n %, with n being an integer between 60 and 99, or n % with n being a decimal number with one decimal place between 89.5 and 99.9, are considered herein as well in context of any (poly)peptide or part thereof described herein. Of note, the term "(poly)peptide", as used herein, can refer to a polypeptide or a peptide, as commonly understood in the art.

The degree of sequence identity can be determined according to methods well known in the art using, preferably, suitable computer algorithms such as BLAST and/or CLUSTAL Omega. In particular, BLAST may be used in combination with CLUSTAL Omega.

Furthermore, such computer algorithms such as BLAST allow to identify and compare peptidoglycan hydrolase variants having at certain sequence coverage (e.g. at least 80%) and a sequence identity (e.g. at least 60%) to a reference sequence. The coverage is a filter that selects sequences with the same architecture as the reference sequence, e.g., LYSM-CHAP endolysins.

For example, in context of the present invention and as illustrated in the appended Examples, endolysin sequences were isolated from the NCBI nucleotide database using BLAST, wherein the sequences were first sorted with a cutoff of 80% sequence coverage and then with a cutoff of 60% identity to LO482 (SEQ ID NO: 1) or a certain variant thereof. CLUSTAL Omega was then employed to align the sequences.

When using the Clustal Omega analysis method (e.g. in combination with BLAST) to determine whether a particular sequence is, for instance, at least 60% identical to a reference sequence default settings may be used.

Preferably, Clustal Omega (Madeira F, Park YM, Lee J, et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Research. 2019 Jul;47(Wl):W636-W641. DOI: 10.1093/nar/gkz268. PMID: 30976793; PMCID: PMC6602479) is used for the comparison of amino acid sequences in context of the present invention. In the case of pairwise comparisons/alignments, the following default settings are preferably chosen: Program : clustalo; Version : 1.2.4; Input Parameters: Output guide tree: true; Output distance matrix: false; Dealign input sequences: false; mBed-like clustering guide tree: true; mBed-like clustering iteration: true; Number of iterations: 0; Maximum guide tree iterations: -1; Maximum HMM iterations: -1; Output alignment format: clustal_num; Output order: aligned; Sequence Type: protein. Preferably, the degree of sequence identity is calculated over the complete length of the reference sequence.

Amino acid residues located at a position corresponding to a position in a reference sequence, e.g., the amino acid sequence shown in SEQ ID NO:1, can be easily identified by the skilled person by methods known in the art. For example, such amino acid residues can be identified by aligning the sequence in question with the reference sequence, e.g., the sequence shown in SEQ ID NO:1, and by identifying the positions which correspond to the indicated positions in the reference sequence, e.g. in SEQ ID NO:1. The alignment can be done with means and methods known to the skilled person, e.g. by using a known computer algorithm such as the Lipman-Pearson method (Science 227 (1985), 1435) or the CLUSTAL algorithm. It is preferred that in such an alignment maximum homology is assigned to conserved amino acid residues present in the amino acid sequences.

Preferably, Clustal Omega is used for the comparison of amino acid sequences in context of the present invention, and hence for determining positions corresponding to positions in a reference sequence. In the case of pairwise comparisons/alignments, the following default settings are preferably chosen: Program : clustalo; Version : 1.2.4; Input Parameters: Output guide tree: true; Output distance matrix: false; Dealign input sequences: false; mBed- like clustering guide tree: true; mBed-like clustering iteration: true; Number of iterations: 0; Maximum guide tree iterations: -1; Maximum HMM iterations: -1; Output alignment format: clustal_num; Output order: aligned; Sequence Type: protein. When the amino acid sequences (e.g. of LO482 variants) are aligned by means of such a method, regardless of insertions, deletions or additions that occur in the amino acid sequences, the positions of the corresponding amino acid residues can be determined (e.g., in each of the LO482 variants).

For example, SEQ ID NO: 1 (WT L0482) has a length of 215 amino acids and an arginine ("R") at position 86. The LO482 hit variant H3 (SEQ ID NO: 9), for example, does not show any deletions, insertions or additions compared to the sequence of SEQ ID NO: 1 but has a lysine ("K") at position 86. Thus, H3 (SEQ ID NO: 9) has an amino acid substitution at position 86 in reference to SEQ ID NO: 1, wherein the residue at said position (i.e., the arginine) is substituted with lysine. In short, H3 has the R86K mutation in reference to SEQ ID NO: 1, as described herein. In the LO482 hit variant "Hl" (SEQ ID NO: 7), however, five amino acid residues are deleted at the N-terminus. Thus, Hl only has a length of 210 amino acid residues. However, by aligning SEQ ID NO: 1 and SEQ ID NO: 7 by standard means as described herein and as illustrated in the appended Examples, it is immediately evident that Hl (SEQ ID NO: 7) has a lysine ("K") at a position corresponding to position 86 in SEQ ID NO: 1, similarly as H3; see, e.g., Figure 9A. Thus, HI (SEQ ID NO: 7) is considered herein and in context to the present invention to have an amino acid substitution at a position corresponding to position 86 in SEQ ID NO: 1, wherein the residue at said position (i.e., the arginine) is substituted with lysine. In short, Hl has the same R86K mutation in reference to SEQ ID NO: as other hit variants such as H3.

A similar logic also applies to any other mutations, e.g., amino acid substitutions, described herein and in context of the present invention.

Herein and in the context of the present invention, an "amino acid substitution" or short "substitution" at a certain position in a reference amino acid sequence or at a position corresponding to a certain position in a reference sequence (e.g. in SEQ ID NO: 1) means that the amino acid residue at said position is substituted with another amino acid residue, as described herein. In particular, the terms "amino acid substitution" or "substituted with another amino acid residue" mean that the respective amino acid residue at the indicated position can be substituted with any other possible amino acid residue, e.g. a naturally occurring amino acid or a non-naturally occurring amino acid (Brustad and Arnold, Curr. Opin. Chern. Biol. 15 (2011), 201-210), preferably with a naturally occurring amino acid, i.e., an amino acid residue selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Herein and in the context of the present invention, in particular with respect to mutations within amino acid sequences, "deleted" or "deletion" means that the amino acid at the indicated position is deleted.

Herein and in the context of the present invention, in particular with respect to mutations within amino acid sequences, "inserted" or "insertion" means that at the respective position at least one amino acid residue, e.g., one or two, preferably one residue, is inserted after the indicated position.

Herein and in the context of the present invention, in particular with respect to mutations within amino acid sequences, "added" or "addition" means that at least one amino acid residue is added at the N-terminus and/or the C-terminus of the reference sequence.

Furthermore, a standard tool, preferably Clustal Omega, is used for determining the sequence identity of a nucleic acid sequence to a corresponding reference nucleic acid sequence in context of the present invention, preferably by using default settings.

CHAP domain of the invention and peptidoglycan hydrolases comprising a CHAP domain of the invention As described herein above, the present invention relates, in some aspects, to a peptidoglycan hydrolase comprising a CHAP domain according to the present invention. In particular, said peptidoglycan hydrolase has bactericidal activity, preferably a killing activity against a gram-positive bacterium, more preferably against Staphylococcus species or strain, most preferably against Staphylococcus aureus, preferably including methicillin-resistant Staphylococcus aureus (MRSA) strains, as described herein. The peptidoglycan hydrolase of the invention may have a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, as described herein.

In particular, herein and in context of the present invention, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1, and has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1, as described herein. Furthermore, the CHAP domain of the invention may have one or more amino acid deletions, insertions or additions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1. However, the extent of the amino acid substitutions, deletions, insertions and/or additions may not be such that the sequence identity of the CHAP domain of the invention to the sequence from position 72 to position 215 in SEQ ID NO: 1 is below 60%.

In particular, the CHAP domain according to the invention functions as a peptidoglycan hydrolase and, preferably, has bactericidal activity, as described herein.

As already mentioned above, the sequence from position 72 to position 215 in SEQ ID NO: 1 is also shown in SEQ ID NO: 301.

Thus, the CHAP domain according to the invention has, in other words, a sequence identity of at least 60% to the sequence of SEQ ID NO: 301 and one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 301.

As described herein, the comparison to a reference sequence comprises, in particular, performing a sequence alignment. Thus, regardless of the occurrence of any amino acid deletions, insertions or additions, positions in the CHAP domain of the invention corresponding to positions in SEQ ID NO: I can be readily identified. In particular, in the absence of any deletions, insertions or additions, the CHAP domain of the invention has one or more amino acid substitutions in the sequence from position 72 to position 215 in SEQ ID NO: 1. Otherwise, the CHAP domain of the invention may have one or more amino acid substitutions in the sequence from a position corresponding to position 72 in SEQ ID NO: 1 to a position corresponding to position 215 in SEQ ID NO: 1, i.e., in the sequence which corresponds to the sequence from position 72 to position 215 in SEQ ID NO: 1 in a sequence alignment (and which may have deletions, insertions and/or additions relative to the sequence from position 72 to position 215 in SEQ ID NO: 1). In this context, it is not necessary that positions corresponding to positions 72 and 215 are present in the CHAP domain of the invention because other positions between these positions are sufficient to perform a sequence alignment.

In certain embodiments, the peptidoglycan hydrolase of the invention consists of the CHAP domain of the invention, in particular, a sequence that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1. In particular, said CHAP domain (and hence said peptidoglycan hydrolase) has bactericidal activity, preferably a killing activity against a Staphylococcus species or strain, more preferably against Staphylococcus aureus, as described herein.

Thus, in some aspects, the invention relates to the CHAP domain of the invention, i.e. a CHAP domain which has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1, and which has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 2in SEQ ID NO: 1, as described herein. In particular, the CHAP domain of the invention may be advantageously employed in a peptidoglycan hydrolase of the invention (e.g. in combination with a cell wall binding domain) or considered itself as a peptidoglycan hydrolase of the invention.

In certain embodiments, the peptidoglycan hydrolase of the invention (i.e., a peptidoglycan hydrolase comprising or consisting of the CHAP domain of the invention), has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. This sequence identity is, in particular, calculated over the full length of the sequence of SEQ ID NO: 1 (and not over the full length of the sequence of the peptidoglycan hydrolase of the invention). Thus, any additional domains, peptides or tags that may be comprised in (or fused to) the peptidoglycan hydrolase of the invention, e.g., a signal peptide or a PKtag, should not be considered when determining the sequence identity of the peptidoglycan hydrolase of the invention to the sequence of SEQ ID NO: 1. Hence, the peptidoglycan hydrolase of the invention may comprise (I) an amino acid sequence having a sequence identity of at least 60% to the sequence of SEQ ID NO: 1 (comprising the CHAP domain of the invention, and optionally the LYSM domain and/or peptide linker of the invention), and, optionally, (II) one or more further domains, peptides or tags, e.g. a signal peptide, a PK tag, a further peptide linker etc., as described herein. In particular, said CHAP domain has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1, and has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1, as described herein.

Aglycosylation mutations As described herein and as illustrated in the appended Examples, the removal of glycosylation sites in LO482 (i.e. at positions 68 and 73 in SEQ ID NO: 1) resulting in so-called "aglycosylated" LO482 variants strongly enhanced the bactericidal activity upon expression in eukaryotic cells; see, e.g., Example 4.

Position 68 in SEQ ID NO: 1 is in the linker sequence of LO482, whereas position 73 in SEQ ID NO: 1 is in the CHAP domain of LO482. As already mentioned above, the CHAP domain is considered herein and in context of the invention as particularly important for the function of the peptidoglycan hydrolases of the invention, especially more important than the linker. Thus, an aglycosylation mutation at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is more preferred in context of the present invention than an aglycosylation mutation at position 68 in SEQ ID NO: 1 or at a position corresponding to this position.

Thus, in some embodiments, e.g., in context of aglycosylation mutations and aglycosylated LO482 variants, the inventive CHAP domain of the invention has a mutation, in particular, an amino acid substitution or a deletion, at position 73 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, e.g., as specified in the subsequent embodiments. In other words, in the CHAP domain of the invention, the arginine ("N") at position in SEQ ID NO: 1 or the arginine at a position corresponding to position 73 in SEQ ID NO: I may be deleted or, preferably, substituted with another amino acid residue, as described herein. As further described herein, said position 73 may be considered herein and in context of the present invention as a glycosylation position which is, preferably, aglycosylated. Therefore, the inventive CHAP domain of the invention has preferably an aglycosylation mutation, preferably an amino acid substitution, at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

Thus, in some preferred embodiments, the inventive CHAP domain of the invention has an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, in particular, an aglycosylation substitution as described herein.

In certain embodiments, e.g., in context of a mutation or substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, the peptidoglycan hydrolase of the invention does not have a sequence as shown in any one of SEQ ID NO: 276 to 278.

Herein and in context of the present invention, in particular in context of the inventive CHAP domain, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position may be, for example, substituted with another amino acid residue than methionine, phenylalanine or lysine.

In certain embodiments, e.g., with respect to peptidoglycan hydrolases consisting of the inventive CHAP domain and/or lacking a cell wall binding domain as described herein, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than methionine.

In further embodiments of the inventive peptidoglycan hydrolase or CHAP domain, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than phenylalanine or lysine.

Preferably herein and in context of the present invention, in particular in context of the inventive CHAP domain and/or aglycosylation substitutions, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, for example, glycine, tyrosine, leucine, glutamic acid, alanine, or histidine.

In some embodiments, in particular in context of the inventive peptidoglycan hydrolase and/or the inventive CHAP domain, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than phenylalanine, lysine or serine.

Preferably herein and in context of the present invention, in particular in context of the inventive CHAP domain and/or aglycosylation substitutions, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine. For example, the CHAP domain of the invention may have a sequence as shown in positions 72 to 215 in SEQ ID NO: 2.

In certain embodiments, in particular in context of a peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the peptidoglycan hydrolase of the invention further comprises a deletion or an amino acid substitution (preferably an amino acid substitution) at position 68 in SEQ ID NO: 1 or at a position corresponding to this position. In other words, the arginine at position 68 in SEQ ID NO: 1 or at a position corresponding to this position may be deleted or, preferably, substituted with another amino acid residue, as described herein. As already indicated above, a mutation at position 68 in SEQ ID NO: 1 or at a position corresponding to this position may be considered herein and in context of the present invention as an aglycosylation mutation, in particular in context of a peptidoglycan hydrolase having a sequence identity of at least 60% to the sequence of SEQ ID NO: 1.

In certain embodiments, in particular in context of the inventive peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted, for example, with another amino acid residue than threonine, serine or lysine, e.g., another amino acid residue than threonine or serine.

In certain embodiments, in particular in context of the inventive peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than threonine, serine or lysine, e.g. another amino acid residue than threonine or serine, and/or the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than phenylalanine, lysine or serine, e.g., another amino acid residue than phenylalanine or lysine.

In certain preferred embodiments, in particular in context of the inventive peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine, arginine or alanine, preferably lysine. In addition, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position may be substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine.

In certain preferred embodiments, in particular in context of the inventive peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the peptidoglycan hydrolase has a pair of amino acid substitutions at positions 68 and 73 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein a) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, serine, tyrosine or leucine, b) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with methionine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, c) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, or d) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or histidine.

In certain particularly preferred embodiments, in particular in context of the inventive peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the residue at position 68 in SEQ ID NO: or at a position corresponding to this position is substituted with lysine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

As already mentioned above, a peptidoglycan hydrolase of the invention having at least one aglycosylation mutation, in particular in the CHAP domain, (e.g. an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position such as K73G, for example as show in SEQ ID NO: 2) is, inter alia, a particularly good starting point for further protein engineering and/or directed evolution.

Thus, the peptidoglycan hydrolase of the invention may have instead or in addition (preferably in addition) to the mutations at positions 68 and/or 73 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein, for example in the embodiments just above, at least one amino acid substitution described herein in context of beneficial/permissive, particularly beneficial or most beneficial amino acid substitutions found in LO482 variants obtained by the directed evolution (see, e.g., Examples 5-7) or found in context of deimmunization analyses or experiments (see, e.g., Example 10). Beneficial/permissive, particularly beneficial, most beneficial amino acid or deimmunizing amino acid substitutions are described herein, for example, in the following.

Beneficial and permissive amino acid substitutions found by the aglycosylation screen, directed evolution or the in s/Z/co deimmunization screen As described herein and as illustrated in the appended Examples, the inventors found 252 LO482 variants that were active and secreted from eukaryotic cells. These variants contained various amino acid substitutions which are considered herein as beneficial or at least permissive with respect to the pharmaceutical properties of the peptidoglycan hydrolases of the invention; see, e.g., Example 7 and Table 3. In particular, said amino acid substitutions may be beneficial (or at least permissive) for the bactericidal activity (e.g. against S. aureu^ and the ability of being secreted from eukaryotic cells (e.g. human cells) of LO482 variants, as described herein.

Furthermore, the deimmunizing amino acid substitutions identified in siiico may be considered as beneficial for the safety and/or efficacy of the LO482 variants in context of medical uses and at least permissive for other pharmaceutical properties such as the bactericidal activity; see, e.g., Example 10 and Table 5.

Furthermore, the inventors found amino acid substitutions at position 73 in SEQ ID NO: 1, i.e. aglycosylation substitutions in the CHAP domain of LO482, which enhance the bactericidal activity upon expression in eukaryotic cells; see, e.g., Example 4.

The term "at least permissive" means that introducing a corresponding amino acid substitution does not abrogate the functionality of an LO482 variant as a pharmaceutical. In other words, a permissive amino acid substitution, as used herein, does not worsen the pharmaceutical properties of an LO482 variant to extent that it no longer can be used as a pharmaceutical. As a minimal requirement for being useful as a pharmaceutical, a LO482 variant must have a bactericidal activity as described herein. Furthermore, the LO482 variant should, ideally, have a sufficient stability as described herein when being used as a pharmaceutical. When the LO482 variant is provided in form of a nucleic acid (e.g. an RNA) for medical uses, the LO482 variant should, ideally, further have the ability of being secreted from a mammalian cell. However, it is not necessary that a permissive amino acid substitution renders a LO482 variant suitable for a pharmaceutical use by itself or improves the pharmaceutical properties of a LO4variant. Thus, permissive amino acid substitutions may be rather employed in combination with at least one particularly beneficial amino acid substitution, or, preferably, with at least one of the most beneficial amino acid substitutions, as described herein.

A "beneficial" amino acid substitution, as used herein, may improve or contribute to the improvement of at least one pharmaceutical property of a LO482 variant, e.g., the bactericidal activity, the ability of being secreted from mammalian cells, the stability and/or the reduction of the immunogenicity. Although beneficial amino acid substitution may be employed alone, they are, preferably, employed in combination with at least one particularly beneficial amino acid substitution, or, more preferably, with at least one of the most beneficial amino acid substitutions, as described herein.

Thus, in some embodiments, in particular in context of permissive/beneficial and/or deimmunizing mutations, as well as active and secreted LO482 variants obtained by directed evolution, the CHAP domain of the invention has one or more amino acid substitutions at positions 72 to 83, 85, 86, 93, 96, 99, 102 to 104, 107, 108, 111, 113 to 115, 117, 121 to 125, 129 to 131,133 to 145, 148, 149, 152, 153, 155, 157, 159, 166, 169, 170, 173 to 178, 185, 186, 190 to 194, 196 to 199, 201, 203 to 207, and 212 to 215 in SEQ ID NO: 1, or at positions corresponding to these positions. Preferably, in these embodiments, the amino acid residue at position 72 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, aspartic acid, histidine or threonine, the amino acid residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine, serine, aspartic acid or threonine, the amino acid residue at position 74 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or proline, the amino acid residue at position 75 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, proline, aspartic acid or glutamine, the amino acid residue at position 76 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, aspartic acid or asparagine, the amino acid residue at position 77 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, aspartic acid, glycine, histidine, lysin, asparagine, glutamine, serine, or threonine, the amino acid residue at position 78 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or tyrosine, the amino acid residue at position 79 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine or tryptophan, the amino acid residue at position 80 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, aspartic acid, methionine or asparagine, the amino acid residue at position 81 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, arginine, serine, alanine or glycine, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or glycine, the amino acid residue at position 83 in SEQ ID NO: 1 or at a position corresponding to this position is substitutedwith glutamine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine or tyrosine, the amino acid residue at position 93 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or leucine, the amino acid residue at position 96 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 99 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine, glutamine, threonine or valine, the amino acid residue at position 102 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, glycine, serine or threonine, the amino acid residue at position 103 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or glycine, the amino acid residue at position 104 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, aspartic acid, methionine, glutamine or tyrosine, the amino acid residue at position 107 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, tyrosine or alanine, the amino acid residue at position 108 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, glycine, lysine or tyrosine, the amino acid residue at position ill in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 113 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 114 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine, the amino acid residue at position 115 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, aspartic acid, glycine or serine, the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, aspartic acid or asparagine, the amino acid residue at position 121 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 122 in SEQ ID NO: 1 or at a position corresponding to this position is substitutedwith leucine, the amino acid residue at position 123 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, serine, threonine or tryptophan, the amino acid residue at position 124 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, histidine, glutamine or threonine, the amino acid residue at position 125 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 129 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or serine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, isoleucine, asparagine or tyrosine, the amino acid residue at position 131 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, glutamine or tyrosine, the amino acid residue at position 133 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, threonine or glutamine, the amino acid residue at position 134 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, valine, lysine or glutamine, the amino acid residue at position 135 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine, histidine or serine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, arginine, glutamic acid or threonine, the amino acid residue at position 137 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 138 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 139 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, aspartic acid, glutamine or serine, the amino acid residue at position 140 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or aspartic acid, the amino acid residue at position 141 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, aspartic acid, histidine or threonine, the amino acid residue at position 142 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine, the amino acid residue at position 143 in SEQ ID NO: 1 or at a position corresponding to this position is substitutedwith proline, the amino acid residue at position 144 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glutamine, the amino acid residue at position 145 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or leucine, the amino acid residue at position 148 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine or valine, the amino acid residue at position 149 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or threonine, the amino acid residue at position 152 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 153 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or serine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 157 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 159 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 166 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, glutamic acid or threonine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or aspartic acid, the amino acid residue at position 170 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, histidine or serine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, asparagine or arginine, the amino acid residue at position 174 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, histidine, lysine, arginine, serine or threonine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, glutamine or threonine, the amino acid residue at position 176 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, arginine, alanine, glutamine, serine or threonine, the amino acid residue at position 177 in SEQ ID NO: 1 or at a position corresponding to this position is substitutedwith leucine, the amino acid residue at position 178 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, arginine, serine or threonine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, histidine, serine, tyrosine or aspartic acid or asparagine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 190 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, alanine, cysteine, aspartic acid, histidine, asparagine, serine or threonine, the amino acid residue at position 191 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, arginine, aspartic acid or asparagine, the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, threonine, glutamic acid, glycine or lysine, the amino acid residue at position 193 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 194 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, glycine, proline or threonine, the amino acid residue at position 196 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine, the amino acid residue at position 197 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or proline, the amino acid residue at position 198 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 199 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, asparagine, serine or threonine, the amino acid residue at position 201 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, serine or lysine, the amino acid residue at position 203 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or valine, the amino acid residue at position 204 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, serine or tyrosine, the amino acid residue at position 205 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or valine, the amino acid residue at position 206 in SEQ ID NO: 1 or at a position corresponding to this position is substitutedwith glutamine, the amino acid residue at position 207 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, valine, histidine or tryptophan, the amino acid residue at position 212 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, aspartic acid, glutamic acid, histidine, lysin or asparagine, the amino acid residue at position 213 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, tyrosine or threonine. the amino acid residue at position 214 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine, and/or the amino acid residue at position 215 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, valine or serine.

The amino acid substitutions just listed herein above correspond to the amino acid residues shown in Example 7 in Table 3 in the column "beneficial/permissive residues" which are not surrounded by parentheses, i.e. not marked by "()", as well as the amino acid residues shown in Example 10 in Table 5 in the column "deimm". In particular, the positions (short: "pos") in Tables 3 and 5 corresponds to the positions in SEQ ID NO: 1. Thus, an amino acid residue listed at a certain position in these Tables means that the amino acid at said position or at a position corresponding to said position in SEQ ID NO: 1 is substituted with an amino acid residue indicated at said position in the column "beneficial/permissive residues" in Table 3 and/or in the column "deimm" in Table 5. As an illustrative example in this context, "ER(H)" in the column "beneficial/permissive residues" in Table 3 means that the amino acid residue (i.e. the lysine; "K") at position 176 in SEQ ID NO: 1 or at a position corresponding to position 1SEQ ID NO: I is substituted with glutamic acid ("E") or arginine ("R") in the L0482ag variants that were secreted from yeast cells and found to be active by YODA, as described herein and as illustrated in the appended Examples. Thus, said mutations, i.e. amino acid substitutions, are considered herein and in context of the present invention as "beneficial" or at least "permissive" for the desired pharmaceutical properties of peptidoglycan hydrolases, in particular, a sufficient killing activity (e.g., against S. aureus), a sufficient stability and a sufficient ability of being secreted from eukaryotic cells. Furthermore, the amino acid residues shown within parentheses in the column "beneficial/permissive residues" in Table 3 (e.g. the histidine "(H)" at position 176 in the above illustrative example) may be also at least permissive for said pharmaceutical properties.

Table 3 shows positions in SEQ ID NO: 1 where beneficial or permissive amino acid substitutions have been found. These are the positions for which the column "beneficial/permissive residues" in Table 3 shows at least one amino acid residue, regardless of whether said residue is shown without parentheses or in parentheses. These positions are also called "permissive positions" herein. Furthermore, positions 72 to 215 in Table 3 for which the column "beneficial/permissive residues" shows at least one amino acid residue, regardless of whether said residue is shown without parentheses or in parentheses are considered as "permissive positions" in the CHAP domain herein. Herein and in context of the present invention, an amino acid residue in SEQ ID NO: 1 corresponding to a beneficial or permissive position may be substituted with an amino acid residue shown in the column "beneficial/permissive residues" in Table 3 for said position, i.e. a residue shown without parentheses or in parentheses in said column in Table 3, preferably with a residue that is shown without parentheses in said column in Table 3.

In some embodiments, in particular in context of permissive/beneficial mutations and Table 3, the CHAP domain of the present invention has at least one amino acid substitution at the permissive positions in the CHAP domain or at positions corresponding to said permissive positions in the CHAP domain. Preferably, in these embodiments, at least one amino acid residue at said permissive positions in the CHAP domain or at positions corresponding to said permissive positions in the CHAP domain is substituted with an amino acid residue shown in the column "beneficial/permissive residues" in Table 3 for said positions, more preferably with a residue that is shown without parentheses in said column in Table 3.

In some embodiments, in particular in context of a peptidoglycan hydrolase having a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, permissive/beneficial mutations and Table 3, the peptidoglycan hydrolase of the present invention has at least one amino acid substitution at the permissive positions or at positions corresponding to said permissive positions. Preferably, in these embodiments, at least one amino acid residue at said permissive positions or at positions corresponding to said permissive positions is substituted with an amino acid residue shown in the column "beneficial/permissive residues" in Table 3 for said positions, more preferably with a residue that is shown without parentheses in said column in Table 3.

Thus, in certain embodiments, in particular in context of permissive/beneficial mutations, as well as active and secreted LO482 variants obtained by directed evolution, the CHAP domain of the invention has one or more amino acid substitutions at positions 72 to 76, 78, 81, 82, 85, 86, 93, 96, 104, 107, 108, 111, 113,115, 117, 121, 124, 125, 129, 130, 133 to 136, 138, 140 to 142, 144, 145, 148, 149, 152, 153, 155, 157, 159, 169, 173, 175 to 178, 185, 186, 190 to 194, 197, 198, 199, 201, 203, 204, 207, and 212 to 215 in SEQ ID NO: 1, or at positions corresponding to these positions. Preferably, in these embodiments, the amino acid residue at position 72 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine or serine, the amino acid residue at position 74 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or proline, the amino acid residue at position 75 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine or proline, the amino acid residue at position 76 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 78 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or tyrosine, the amino acid residue at position 81 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, arginine or serine, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substitutedwith glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or methionine, the amino acid residue at position 93 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or leucine, the amino acid residue at position 96 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 104 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 107 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or tyrosine, the amino acid residue at position 108 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position ill in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 113 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 115 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, the amino acid residue at position 121 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 124 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine or histidine, the amino acid residue at position 125 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 129 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or serine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, isoleucine or asparagine, the amino acid residue at position 133 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or threonine, the amino acid residue at position 134 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine or valine, the amino acid residue at position 135 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine or histidine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 138 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 140 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or aspartic acid, the amino acid residue at position 141 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 142 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine, the amino acid residue at position 144 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 145 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or leucine, the amino acid residue at position 148 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine or valine, the amino acid residue at position 149 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or threonine, the amino acid residue at position 152 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 153 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or serine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 157 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 159 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, asparagine or arginine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, glutamine or threonine, the amino acid residue at position 176 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine, the amino acid residue at position 177 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 178 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine or arginine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, histidine, serine or tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 190 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 191 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine, the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine, the amino acid residue at position 193 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 194 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 197 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 198 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 199 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 201 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or serine, the amino acid residue at position 203 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or valine, the amino acid residue at position 204 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, serine or tyrosine, the amino acid residue at position 207 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine or valine, the amino acid residue at position 212 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 213 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or threonine. the amino acid residue at position 214 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine, and/or the amino acid residue at position 215 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, valine or serine.

The amino acid substitutions described just herein above refer to all the substitutions found in the sequenced LO4variants that were secreted from yeast cells and determined to be active by the YODA method as described herein and as illustrated in the appended Examples; see, e.g., Example 7 and Table 3, in particular the "beneficial/permissive residues" shown without parentheses in Table 3.

Particularly beneficial amino acid substitutions Furthermore, the inventors determined hit variants among all these LO482 variants, which are particularly well adapted for the production in eukaryotic cells and which may have particularly beneficial pharmaceutical properties, as described herein; see, e.g., Examples 5-7, Table 4 and Figures 8 and 9. As described herein, the amino acid substitutions found in these hit variants, i.e. G1-G4, H1-H10 and 11-130, are considered herein and in context of the present invention as "particularly beneficial mutations" or "particularly beneficial amino acid substitutions" which may be particularly beneficial for maintaining or enhancing the desired pharmaceutical properties of peptidoglycan hydrolases, in particular, the bactericidal activity (e.g., against S. aureus), the stability and/or the ability of being secreted from eukaryotic cells, e.g. human cells.

Herein, and in context of the present invention, the particularly beneficial amino acid substitutions are preferred over amino acid substitutions that are described as "beneficial" or "permissive" herein.

Accordingly, in some preferred embodiments, the CHAP domain of the invention has one or more amino acid substitutions at positions 73, 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169,173,175,178,185,186,191,192,194,198, 204, 212 and 215 in SEQ ID NO: 1 or at positions corresponding to these positions. Preferably, in these embodiments, the amino acid residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine or serine, the amino acid residue at position 75 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 78 in SEQ ID NO: 1 or at a position corresponding to this position is substitutedwith tyrosine, the amino acid residue at position 81 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 104 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 107 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 115 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 124 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 125 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 133 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 135 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 140 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 141 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substitutedwith asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 178 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 191 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 194 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 198 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 204 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 212 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue at position 215 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine.

In further preferred embodiments, the CHAP domain of the invention has an aglycosylation mutation at position or at a position corresponding to this positions, as described herein, and additionally one or more amino acid substitutions at positions 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191 to 194,198, 204, 212 and 215 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein, for example, as just described herein above.

G1 as an exemplary first-round hit variant comprising particularly beneficial amino acid substitutionsG1 (SEQ ID NO: 3) is a hit variant obtained in the first round of the directed evolution, which showed an increased thermostability, an enhanced bactericidal activity and an enhanced secretion from eukaryotic cells as compared to the starting for the directed evolution (L0482ag); see Figure 8.

G1 (SEQ ID NO: 3) contains the following amino acid substitutions in the CHAP domain as compared to L0482ag (SEQ ID NO: 2): T82S, N85G, R86K, D169N, K173N, M175Q and A192Q.

Thus, in some embodiments, the CHAP domain of the invention has one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. threonine) is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. asparagine) is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. arginine) is substituted with lysine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. aspartic acid) is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. lysin) is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. methionine) is substituted with glutamine, and/or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. alanine) is substituted with glutamine.

Furthermore, the CHAP domain may have a sequence identity of at least 95% to the sequence from position 72 to position 215 in SEQ ID NO: 3.

In some embodiments, the CHAP domain has (a) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; (b) an amino acid substitution pair at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; and/or (c) an amino acid substitution at position 169 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine.

Furthermore, said CHAP domain may comprise one or more amino acid substitutions at positions 173, 175 and 1in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and/or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

Preferably, the CHAP domain further has an aglycosylation mutation at position 73 or at a position corresponding to this position, as described herein, preferably wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, more preferably with glycine.

In some embodiments of the present invention, the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 80% to the sequence from position 72 to position 215 in SEQ ID NO: 1; (ii) an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and (iii) one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and/or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine; with the proviso that said CHAP domain does not have a sequence as set forth in SEQ ID NO: 381, 382, 383, 3or 385.

In some embodiments of the present invention, the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 91% to the sequence from position 72 to position 215 in SEQ ID NO: 1; (ii) an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and (iii) one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and/or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine; with the proviso that said CHAP domain does not have a sequence as set forth in SEQ ID NO: 381 or 382.

In some further embodiments of the invention, the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 80% to the sequence from position 72 to position 215 in SEQ ID NO: 1; (ii) an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and (iii) at least two amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and/or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

In some embodiments of the invention, the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; (ii) an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; and (iii) one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and/or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

Most beneficial amino acid substitutions As described herein and as illustrated in the appended Examples, the inventors found a set of most beneficial mutations, in particular a set of 9 most beneficial amino acid substitutions, i.e.,T82S, N85G, R86K, S130N, H136K/R (preferably H136K), F155Y, D169N, N185Y and N186G in reference to SEQ ID NO: 1. As described herein, the variant H5 (SEQ ID NO: 11) which has been found to have overall the best pharmaceutical properties among the LO482 variants obtained by two rounds of directed evolution, contains exclusively these 9 most beneficial amino acid substitutions; see, e.g., Figures 8 and 9. In particular, H5 (SEQ ID NO: 11) has a strongly enhanced ability of being secreted from human cells, a strongly enhanced killing activity against S. aureus, and an improved (thermo)stability as compared to the parental L0482ag (SEQ ID NO: 2) and also as compared to WT LO482 (SEQ ID NO: 1); see, e.g., Examples 4-6 and 12 and Figures 8, 10 and 6. In particular, H5 (SEQ ID NO: 11) showed a 8-fold lower MIC than WT LO482 (SEQ ID NO: 1), i.e. WT LO482 had a MIC of 4 pg/ml, whereas H5 had a MIC of 0.5 pg/ml. Moreover, H5 (SEQ ID NO: 11) showed the strongest killing activity against S. aureus (together with H7; SEQ ID NO: 13), the best stability (in particular a melting temperature of 47°C), and a good secretion from human cells; see Figure 8. H5 also showed an enhanced ability of being secreted from human cells, an enhanced killing activity against S. aureus, and an improved (thermo)stability as compared to G1 (SEQ ID NO: 3) which is an improved LO482 variant obtained in the first round of the directed evolution; see Figure 8. In particular, G1 had a MIC of 1 pg/ml, whereas H5 had a MIC of 0.5 pg/ml.

H5 (SEQ ID NO: 11) further showed enhanced (i.e. faster) killing kinetics against S. aureus in an OD reduction assay than WT LO482 (SEQ ID NO: 1); see Example 6 and Figure 11. In addition, H5 (SEQ ID NO: 11) effectively killed S. aureus biofilms and showed an even greater anti-biofilm activity than G1 (SEQ ID NO: 3); see Example and Figure 12. This is in line with the particularly low MIC observed for H5, which is lower than the MIC of G1.

Thus, the most beneficial amino acid substitutions (i.e the "H5" mutations) are particularly beneficial for enhancing the desired pharmaceutical properties of peptidoglycan hydrolases, in particular, the bactericidal activity (e.g., against S. aureus including S. aureus biofilms), the stability and/or the ability of being secreted from eukaryotic cells, e.g. human cells.

In context of the present invention, the most beneficial amino acid substitutions are preferred over amino acid substitutions described as "particularly beneficial" and even more preferred over amino acid substitutions described as "beneficial" or "permissive".

As already mentioned above, it has been further found in context of the present invention that the LO482 variant H3 (SEQ ID NO: 9) obtained by the directed evolution contained 8 out of the 9 most beneficial amino acid substitutions and exclusively these 8 mutations. Since the 8 "H3" amino acid substitutions occurred recurrently in slightly different combinations among the 44 hit variants obtained by three rounds of directed evolution, they are considered herein and in context of the present invention as the consensus mutations. Furthermore, H3 reflects the consensus sequence. In particular, the 8 consensus mutations refer to T82S, N85G, R86K, S130N, H136K/R, D169N, N185Y and N186G, in reference to the sequence of SEQ ID NO: 1.

Furthermore, as described herein and as illustrated in the appended Examples, H3 (SEQ ID NO: 9) has improved pharmaceutical properties, e.g., an improved ability of being secreted from eukaryotic cells compared to WT LO4and L0482ag as well as a good killing activity against S. aureus, see, e.g., Figures 8 and 10. This further demonstrates the advantageous effects of the consensus mutations with respect to the pharmaceutical properties of LO482 variants, as described herein.

Thus, in some particularly preferred embodiments, e.g., in context of the consensus mutations, the CHAP domain of the present invention has one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions. Preferably, in these embodiments, the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In other words, the CHAP domain of the invention has, in some particularly preferred embodiments, at least one consensus mutation, i.e. at least one amino acid substitution selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, D169N, N185Y and N186G in SEQ ID NO: 1.

Preferably, said CHAP domain (i.e. a CHAP domain comprising at least one of the consensus mutations described herein) further comprises (i)an aglycosylation mutation at position 73 or at a position corresponding to this position, as described herein, for example, wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine or serine, more preferably glycine; and/or (ii) an amino acid substitution at position 155 or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

As also already mentioned above, it has been surprisingly found in context of the invention that the LO482 variant H5 (SEQ ID NO: 11) which had an enhanced killing activity against 5. aureus, an enhanced stability (e.g thermostability/melting temperature) and an enhanced ability of being secreted from human cells (Figure 8) as compared to H3, differed from H3 by only one amino acid, i.e., it had, in addition to the consensus mutations, the amino acid substitution F155Y in reference to SEQ ID NO: 1 (Figure 9).

Furthermore, it has been found that the amino acid substitution H136R occurred in many of the hit variants (i.e. G1-G4, H1-H10 and 11 to 130) instead of H136K and thus is a very good alternative to H136K.

In particular, the LO482 variant H3 has the following sequence, wherein the LYSM domain (positions 1 to 51) is underlined, the linker (positions 52 to 71) is in italics, and the CHAP domain (positions 72 to 215) is in bold : REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGOKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKELEE CGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNnEAKIYCNTPTFKAEPGDLVVFSGRFGG GYGHTAIVLNGIYDGKLMKFQSLDQNWZGGWRKAEVAHKVVHNYENDMIFIRPFKKA(SEQ ID NO: 9).

Furthermore, the LO482 variant H5 has the following sequence, wherein the LYSM domain (positions 1 to 51) is underlined, the linker (positions 52 to 71) is in italics, and the CHAP domain (positions 72 to 215) is in bold : REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGOKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKELEE [Sgwdfdgsygwqcfdlvnvywnhlyghglkgygakdipyannfn QeakiyQntptfkaepgdlwfsgrQgg GYGHTAIVLNGYDGKLMKFQSLDQNWZGGWRKAEVAHKVVHNYENDMIFIRPFKKA(SEQ ID NO: 11).

In the above H3 and H5 sequences (i.e SEQ ID NO: 9 and SEQ ID NO 11, respectively), the two glycosylation positions, i.e., positions 68 and 73, wherein aglycosylation amino acid residues have been introduced in H3 and H(i.e. lysine at position 68 and glycine at position 73) are highlighted in grey.

The amino acid residues in SEQ ID NO: 9 shown in white on black background refer to the consensus mutations, and the amino acid residues in SEQ ID NO: 11 shown in white on black background refer to the most beneficial amino acid substitutions, as described herein and in context of the present invention. Of note, H3 (SEQ ID NO: 9) contains all of the most beneficial amino acid substitutions except F155Y. Also see Figure 9A for the alignments.

Notably, all of the most beneficial amino acid substitutions, i.e. T82S, N85G, R86K, S130N, H136K/R (preferably H136K), F155Y, D169N, N185Y and N186G in reference to SEQ ID NO: 1, occurred within the CHAP domain of LO482, i.e. within the sequence from position 72 to position 215 in SEQ ID NO: 1. Herein and in context of the present invention any of these most beneficial mutations or any combination thereof is, preferably, combined with an aglycosylation mutation at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein, e.g., with the amino acid substitution N73G in SEQ ID NO: 1. In certain embodiments, e.g. in context of a peptidoglycan hydrolase which has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, any of these most beneficial mutations or any combination thereof is further combined with at least one aglycosylation mutation at positions 68 and 73 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein, e.g., with the amino acid substitutions N68K and/or N73G in SEQ ID NO: 1.

However, it is by no means necessary that the peptidoglycan hydrolase or CHAP domain of the invention contains all of the 9 most beneficial mutations described herein at once. For example, it has been found that the LO4variant Hl (SEQ ID NO: 7) did not show any mutations at positions corresponding to positions 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 and, nevertheless, had enhanced pharmaceutical properties compared to the parental L0482ag lysin (SEQ ID NO: 2), in particular, an enhanced killing activity against S. aureus, an enhanced stability (e.g. thermostability/melting temperature), and an enhanced ability of being secreted from human cells. As a further example, it has been found that the LO482 variant H9 (SEQ ID NO: 15) did not show any mutations at positions corresponding to positions 82, 85, 155, 185 and 185 in SEQ ID NO: 1, and also showed enhanced pharmaceutical properties compared to the parental L0482ag lysin (SEQ ID NO: 2); see, e.g., Figures 8 and 9. In fact, the amino acid substitution R86K is the only one among the most beneficial substitutions which Hl (SEQ ID NO: 7) and H(SEQ ID NO: 15) have in common. This clearly demonstrates that the most beneficial amino acid substitutions found in context of the present invention may be employed in various combinations and reliably enhance the pharmaceutical properties of LO482-derived peptidoglycan hydrolases, as described herein. However, this finding does, in no way, contradict the notion that the group of the most beneficial amino acid substitutions described herein has been purposefully and carefully selected by the present inventors in order to improve the pharmaceutical properties of LO482-derived peptidoglycan hydrolases. For example, when the additional amino acid substitutions N185Y and N186G are introduced in H9 (SEQ ID NO: 15) resulting, e.g., in G4 (SEQ ID NO: 6), H2 (SEQ ID NO: 8), H8 (SEQ ID NO: 14) or H10 (SEQ ID NO: 16), some or all of the pharmaceutical properties assayed can be further improved; see Figure 8 and 9. Furthermore, as already mentioned above, the single amino acid substitution F155Y (occurring, e.g., in H5) improved all pharmaceutical properties compared to H3, further demonstrating that individual most beneficial amino acid substitutions can already have highly beneficial effects. In fact, the LO4variant H5 (SEQ ID NO 11) which had all of the most beneficial mutations described herein and exclusively these mutations, showed the best pharmaceutical properties among all hit variants analyzed, as described herein.

Thus, in some particularly preferred embodiments, e.g., in context of the most beneficial amino acid substitutions, the CHAP domain of the present invention has one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions. Preferably, in these embodiments, the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In other words, the CHAP domain of the invention has, in some particularly preferred embodiments, at least one amino acid substitution selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1.

Preferably, said CHAP domain (i.e. a CHAP domain comprising at least one of the most beneficial amino acid substitutions described herein) further comprises an aglycosylation mutation at position 73 or at a position corresponding to this position, as described herein, for example, wherein the residue at position 73 in SEQ ID NO: or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine or serine, more preferably glycine.

Amino acid substitution pairs and consensus mutation unitsAs further described herein above, the inventors further surprisingly found that certain amino acid substitutions, in particular some of the consensus mutations (i.e. T82S, N85G, R86K, S130N, H136K/R (esp. H136K), D169N, N185Y and N186G in reference to SEQ ID NO: 1) occurred very often as substitution pairs in the active and secreted LO482 variants obtained by the directed evolution, in particular in the hits variants, i.e., G1 to G4, Hl to H10 and to 130. Thus, the consensus mutations (which are particularly preferred amino acid substitutions in context of the invention) may be further grouped as consensus mutation units herein and in context of the present invention.

As mentioned above, these consensus mutation units consist of 1 or 2 amino acid substitutions and refer to: (i) R86K (which is particularly preferred), (ii) T82S and N85G, (iii) S130N and H136K/R (preferably H136K), (iv) D169N, and (v) N185Y and N186G.

Thus, in some preferred embodiments, e.g., in context of the most beneficial amino acid substitutions and/or consensus mutation units, the CHAP domain of the invention has at least one pair of amino acid substitutions a) at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, b) at positions 130 and 1in SEQ ID NO: 1 or at positions corresponding to these positions, and/or c) at positions 185 and 186 in SEQ ID NO: or at positions corresponding to these positions.

In context of these embodiments, in a), preferably, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; in b), preferably, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine; and/or in c), preferably, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In other words, the CHAP domain of the invention has, in some preferred embodiments, at least one substitution pair selected from the group consisting of: a) T82S and N85G, b) S130N and H136K/R (preferably H136K), and c) N185Y and N186G.

Furthermore, said CHAP domain (i.e. a CHAP domain having at least one substitution pair) further has, preferably, an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

Furthermore, said CHAP domain (i.e. a CHAP domain having at least one substitution pair, and preferably an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position) further has, preferably, an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

Furthermore, said CHAP domain, has, preferably, an aglycosylation mutation at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

In some further preferred embodiments, the CHAP domain of the present invention has at least one consensus mutation unit, i.e., at least one amino acid substitution or substitution pair selected from the group consisting of the following (i) to (v): (i) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at said position is substituted with lysine (i.e. R86K); (ii) an amino acid substitution at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine (i.e. T82S), and the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine (i.e. N85G); (iii) an amino acid substitution at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine (i.e. S130N), and the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine (i.e. H136K/R), preferably lysine (H136K); (iv) an amino acid substitution at position 169 (i.e. the aspartic acid) in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with asparagine (i.e. D169N); and (v) an amino acid substitution at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine (i.e. N185Y), and the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine (i.e. N186G).

Preferably, in context of these embodiments, the CHAP domain of the invention has at least 2, preferably at least 3, more preferably at least 4 of the amino acid substitutions or substitution pairs (i) to (v) (i.e. consensus mutation units), as just described herein above.

In some particularly preferred embodiments, the CHAP domain of the invention has (i) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at said position is substituted with lysine; and has at least one, preferably at least two, more preferably at leastthree of the following amino acid substitutions or substitution pairs (ii) to (v) (i.e. consensus mutation units): (ii) an amino acid substitution at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; (iii) an amino acid substitution at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, and the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine; (iv) an amino acid substitution at position 169 (i.e. the aspartic acid) in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with asparagine; and/or (v) an amino acid substitution at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

Generally, herein and in context of the present invention, a consensus mutation or any combination thereof (i.e. at least one substitution selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, D169N, N185Y and N186G, in SEQ ID NO: 1) or a consensus mutation unit or any combination thereof (i.e. at least one consensus mutation unit selected from the group consisting of: (i) R86K, (ii) T82S and N85G, (iii) S130N and H136K/R, (iv) D169N, (V) N185Y and N186G) is, preferably, combined with an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Preferably, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine (i.e. F155Y), as described herein.

Furthermore, generally herein and in context of the present invention, a consensus mutation or consensus mutation unit or any combination thereof (which may be further combined with F155Y as just described) is, preferably, combined with at least one aglycosylation mutation (preferably at least one aglycosylation substitution) as described herein, preferably an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, more preferably, with glycine, as described herein.

What has been just described herein above in context of an amino acid substitution at position 155 in SEQ ID NO: or at a position corresponding to this position, an aglycosylation mutation, and an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is, in particular, also the case for the following embodiments: In some preferred embodiments, the CHAP domain of the invention has the amino acid substitution R86K, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S and N85G, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N and H136K/R, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K and D169N, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K and N185Y and N186, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, S130N and H136K/R, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, and D169N, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, and N185Y and N186G, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, D169N, N185Y and N186G, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N, H136K/R, N185Y and N186G, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N, H136K/R, and D169N, as described herein.

In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, N185Y and N186G, as described herein.

In some particularly preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, S130N, H136K/R and D169N, as described herein.

In some particularly preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, S130N, H136K/R, N185Y and N186, as described herein.

In some particularly preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N, H136K/R, D169N, N185Y and N186G, as described herein.

In some particularly preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, D169N, N185Y and N186G, as described herein.

In some of the most preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, S130N, H136K/R, D169N, N185Y and N186G, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S and N85G, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N and H136K/R, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G and D169N, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G, N185Y and N186G, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N, H136K/R and D169N, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N, H136K/R, N185Y and N186G, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N, H136K/R, D169N, N185Y and N186G, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions S130N and H136K/R, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions S130N, H136K/R and D169N, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions S130N, H136K/R, N185Y and N186G, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions S130N, H136K/R, D169N, N185Y and N186G, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitution D169N, as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions D169N, N185Y and N186G as described herein.

In some embodiments, the CHAP domain of the invention has the amino acid substitutions N185Y and N186G, as described herein.

Furthermore, herein and context of the present invention, and hence, also in the above embodiments, the substitution H136K is preferred over the substitution H136R.

Particularly important individual amino acid substitutions: R86K and F155YAs already mentioned above and as illustrated in the appended Examples, all hit variants, i.e. G1-G4, H1-H10 and 11-130, had the amino acid substitution R86K in reference to SEQ ID NO: 1. Thus, this amino acid substitution is considered herein and in context of the present invention as a particularly important substitution among the most beneficial amino acid substitutions, as described herein.

Thus, in some of the most preferred embodiments, e.g., in context of the most beneficial amino acid substitutions, the CHAP domain of the present invention has an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position. For example, in context of these embodiments, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position may be substituted with another amino acid residue than serine. Preferably, in context of these embodiments, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or methionine. More preferably, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine.

Furthermore, said CHAP domain, has, preferably, an aglycosylation mutation at position 73 in SEQ ID NO 1 or at a position corresponding to this position, as described herein.

As also already mentioned above, the amino acid substitution F155Y further improved several pharmaceutical properties (e.g. the killing activity against S. aureus, the stability and the ability of being secreted from human cells) in H5 (SEQ ID NO: 11) compared to H3 (SEQ ID NO: 9) containing only the consensus mutations.

Therefore, in some preferred embodiments, the CHAP domain of the invention has an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

Generally, herein and in context of the present invention, the CHAP domain, has, preferably, an aglycosylation mutation at position 73 in SEQ ID NO 1 or at a position corresponding to this position, as described herein. This is also true, inter alia, for all the following embodiments: Further embodimentsIn some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 91% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) one or more amino acid substitutions at positions 82, 85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 84% to the sequence of SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) one or more amino acid substitutions at positions 82,85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, CT the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 78% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) at least two amino acid substitutions at positions 82, 85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 73% to the sequence in SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) at least two amino acid substitutions at positions 82,85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 78% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) one or more amino acid substitutions at positions 82, 85, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 73% to the sequence in SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) one or more amino acid substitutions at positions 82, 85,130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) at least six amino acid substitutions at positions 82, 85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; has (iii) an amino acid substitution at position 82 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine; and has (iv) an amino acid substitution at position 85 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: or at a position corresponding to this position is substituted with glycine. In other words, said CHAP domain has the R86K substitution and a substitution pair consisting of T82S and N85G in reference to SEQ ID NO: 1.

In some particular embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 60% to the sequence of SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; has (iii) an amino acid substitution at position 82 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine; and has (iv) an amino acid substitution at position 85 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine. In other words, said peptidoglycan hydrolase has the R86K substitution and the substitution pair consisting of T82S and N85G in reference to SEQ ID NO: 1.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 83% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution or a deletion (preferably, an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and has (iii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 73% to the sequence of SEQ ID NO: 1; has (II) an amino acid substitution ora deletion (preferably, an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and has (iii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and (ii) an amino acid substitution at position in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the asparagine) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine. Preferably, said CHAP domain further has (iii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169,185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution or a deletion (preferably, an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and has (iii) an amino acid substitution at position 82 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution or a deletion (preferably, an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and has (iii) an amino acid substitution at position 130 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position or a deletion (preferably, an amino acid substitution) in SEQ ID NO: 1 or at a position corresponding to this position; and has (iii) an amino acid substitution at position 185 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 1in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine; and has (iii) one or more amino acid substitutions at positions 82, 85, 86, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 78% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 1in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and has (iii) at least two amino acid substitutions at positions 82, 85, 86, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 73% to the sequence of SEQ ID NO: 1; has (II) an amino acid substitution at position 155 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and has (iii) at least two amino acid substitutions at positions 82, 85, 86, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 78% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 155 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and has (iii) one or more amino acid substitutions at positions 82, 85, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 73% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 1in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and has (iii) one or more amino acid substitutions at positions 82, 85, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 1in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and has (iii) at least six amino acid substitutions at positions 82, 85, 86, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

In some embodiments, e.g., in context of LO482 variants obtained by the directed evolution, permissive or beneficial mutations, particularly beneficial mutations, most beneficial mutations, consensus mutations or consensus mutation units, the peptidoglycan hydrolase of the invention does not have a sequence as shown in any one of SEQ ID NO: 279 to 293.

In some embodiments, the CHAP domain of the present invention has a sequence identity of at least 90%, preferably at least 95%, more preferably at least 97% or at least 98%, e.g. at least 97.3%, to the CHAP domain of a hit variant as described herein, i.e., to the CHAP domain of any one of G1 to G4, Hl to H10 and 11 to 130 (SEQ ID NO: 3 to 46, respectively), as described herein. In particular, the CHAP domain of a hit variant (e.g. HI) refers to the sequence from a position in the sequence of said hit variant (e.g. SEQ ID NO: 7) which corresponds to position 72 in SEQ ID NO: 1 to a position in the sequence of said hit variant (e.g. SEQ ID NO: 7) which corresponds to position 215 in SEQ ID NO: 1. In particular, said CHAP domain has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to the sequence from position 72 to position 215 in SEQ ID NO: 1 as a reference sequence, as described herein.

In some further embodiments, the peptidoglycan hydrolase of the present invention has a sequence identity of at least 90%, preferably at least 95%, more preferably at least 97%, e.g. at least 96.8%, to a hit variant as described herein, i.e., to any one of SEQ ID NO: 3 to 46. In particular, said peptidoglycan hydrolase has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to SEQ ID NO: 1 as a reference sequence, as described herein.

In some preferred embodiments, the CHAP domain of the invention has a sequence identity of at least 93.2% or at least 94% to the sequence from position 72 to position 215 in SEQ ID NO: 11. In particular, said CHAP domain has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to the sequence from position to position 215 in SEQ ID NO: 1 as a reference sequence, as described herein.

In some preferred embodiments, the peptidoglycan hydrolase of the invention has a sequence identity of at least 95.0% or at least 96% to the sequence of SEQ ID NO: 11. In particular, said peptidoglycan hydrolase has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to SEQ ID NO: 1 as a reference sequence, as described herein.

In some preferred embodiments, the CHAP domain of the invention has a sequence identity of at least 93.9% or at least 94% to the sequence from position 72 to position 215 in SEQ ID NO: 9. In particular, said CHAP domain has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to the sequence from position to position 215 in SEQ ID NO: 1 as a reference sequence, as described herein.

In some preferred embodiments, the peptidoglycan hydrolase of the invention has a sequence identity of at least 95.4% or at least 96% to the sequence of SEQ ID NO: 9. In particular, said peptidoglycan hydrolase has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to SEQ ID NO: 1 as a reference sequence, as described herein.

Conserved positions and conserved segment In some embodiments, a segment of the inventive CHAP domain has a sequency identity of at least 80%, preferably at least 90%, to the sequence from position 87 to position 128 in SEQ ID NO: 1. In particular, said segment is contained in the inventive CHAP domain at positions corresponding to positions 87 to 128 in SEQ ID NO: 1, i.e., it is a corresponding segment. As shown, e.g., in Example 7, said segment may be considered herein and in context of the present invention as a particularly conserved CHAP segment.

Furthermore, in some of these embodiments or in other embodiments, the CHAP domain of the invention has at most six, five, four, three or two, more preferably at most one, most preferably no amino acid substitutions or deletions at positions 80, 87, 88,98,99,103,106,110,114,122,126,128,137,182, 202, and 208 of SEQ ID NO: or at positions corresponding to these positions. As shown, e.g., in Example 7, said positions may be considered herein and in context of the present invention as conserved positions within the CHAP domain.

In context of the present invention, the CHAP domain of the invention has, preferably, one or more amino acid substitutions at other positions than at the conserved positions described herein. Nevertheless, the CHAP domain may also comprise one or more mutations, e.g. amino acid substitutions, at the conserved positions. For example, the in-silico de-immunizing screen illustrated in Example 10 revealed possible amino acid substitutions at many different positions including "conserved positions". Of note, this de-immunizing screen has been designed such that advantageous pharmaceutical properties, in particular the bactericidal activity, stability and ability of being secreted from eukaryotic cells, are maintained upon the de-immunization. Thus, mutations such as the de-immunization mutations found in context of the invention may be also introduced at positions which are considered as "conserved" herein.

Moreover, a conserved CHAP segment has in context of the present invention, preferably, less mutations than other segments of the CHAP domain of the invention. Thus, a corresponding segment of the CHAP domain of the invention has, preferably, a higher sequency identity (e.g. about 90%) to the sequence from position 87 to position 128 in SEQ ID NO: 1, as compared to the sequence identity of the CHAP domain of the invention to the sequence from position 72 to position 215 in SEQ ID NO: 1 (which may be in this example, e.g., about 60% to 80%).

Hence, the present invention further relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1, and wherein (a) a corresponding segment of said CHAP domain has a sequency identity of at least 80%, preferably at least 90%, to the sequence from position 87 to position 128 in SEQ ID NO: 1, and/or (b) said CHAP domain has at most six, five, four, three or two, preferably at most one, more preferably no amino acid substitutions or deletions at positions 80, 87, 88, 98, 99, 103, 106,110, 114, 122, 126, 128, 137, 182, 202, and 208 of SEQ ID NO: 1 or at positions corresponding to these positions.

Preferably, said CHAP domain comprises at least one aglycosylation mutation at position 73 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, preferably the substitution N73G, and/or at least one amino acid substitution described herein in context of the L0482ag variants obtained upon directed evolution, preferably at least one of the most beneficial mutations, as described herein, i.e., at least amino acid substitution selected from the group consisting of: T82S, N85G, R86K, S130N, H136K/R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1.

Additional cell wall binding domain(s) Preferably, herein and in context of the present invention, the peptidoglycan hydrolase of the invention comprises, in addition to the CHAP domain of the invention, at least one cell wall binding domain.

As mentioned above, endolysins often have at least one cell wall-binding domain (CBD) which recognize and bind to certain epitopes in the cell wall of the host bacterium for proper fixation of the catalytic effect of an enzymatically active domain (EAD). Normally, CBDs are enzymatically inactive by themselves. Thus, a domain which has catalytic activity and is able to recognize and bind to certain epitopes in the cell wall of a bacterium is rather considered herein as a an enzymatically active domain (EAD). Preferably, herein and in context of the present invention, the cell wall binding domain is a peptidoglycan binding domain which binds, in particular, to the peptidoglycan structure of a target bacterium.

Suitable cell wall binding domains to be used in context of the present invention, include, inter alia״ , a LYSM domain, a SH3 domain and a choline binding domain.

Preferably, in context of the present invention, the at least one cell wall binding domain comprises a LYSM domain and/or a SH3 domain, more preferably, a LYSM domain, as described herein.

Preferably, herein and in context of the present invention, the cell wall binding domain is derived from an endolysin, in particular a cell wall binding domain thereof. Preferably, said endolysin has a killing activity against a Staphylococcus species or strain, more preferably against Staphylococcus aureus.

In context of the present invention, the cell wall binding domain is, preferably, derived from an endolysin comprising a LYSM domain or a SH3 domain, more preferably an endolysin comprising a LYSM domain. Preferably, the LYSM domain is derived from an endolysin comprising a LYSM domain and a CHAP domain, wherein the LYSM domain is, preferably, N-terminally of the CHAP domain. In other words, the LYSM domain of the invention is, preferably, derived from an endolysin having a LYSM-CHAP architecture, as described herein, e.g. LO482 (SEQ ID NO: 1) or LO499 (SEQ ID NO: 47).

Hence, in preferred embodiments, the peptidoglycan of the invention comprises, in addition, to the CHAP domain of the invention, a LYSM domain, as described herein.

Preferably, in context of the invention, the cell wall binding domain, e.g. the LYSM domain, is N-terminally of the CHAP domain.

Preferably herein, the LYSM domain according to the invention is defined by the sequence from position 1 to position in SEQ ID NO: 1 or it has a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1. In other words, the LYSM domain according to the invention is, preferably, defined by the sequence of SEQ ID NO: 302 or has a sequence identity of at least 60% to the sequence of SEQ ID NO: 302.

In some embodiments, the LYSM domain is defined by the sequence from position 1 to position 47 in SEQ ID NO: or it has a sequence identity of at least 60% to the sequence from position X to position Y in SEQ ID NO: 47.

In some embodiments, the SH3 domain is defined by the sequence of SEQ ID NO: 376 or has a sequence identity of at least 60% to the sequence of SEQ ID NO: 376.

In some embodiments, the SH3 domain is defined by the sequence of SEQ ID NO: 377 or has a sequence identity of at least 60% to the sequence of SEQ ID NO: 377.

Furthermore, the cell wall binding domain of the invention, e.g. the LYSM domain of the invention, has, preferably, the ability to bind to the cell wall of a Staphylococcus species or strain, more preferably to Staphylococcus aureus, as described herein.

Beneficial and permissive amino acid substitutions in the LYSM domain found by directed evolution or in siiico deimmunization screensAs described herein and as illustrated in the appended Example, the inventors found a variety of amino acid substitutions which were contained in the LYSM domain of L0482ag variants obtained by directed evolution; see, e.g., Table 3, the column "beneficial/permissive residues". Of note, the L0482ag variants containing these amino acid substitutions were all well secreted from eukaryotic cells and determined to be active by the YODA method.

Furthermore, as also described herein and as illustrated in the appended Examples, the inventors found deimmunizing substitutions in the LYSM domain of LO482 variants which may decrease the immunogenicity of the LYSM domain of the invention and peptidoglycan hydrolases containing the LYSM domain of the invention; see, e.g., Table 5, column "deimm".

Thus, the LYSM domain of the invention (which preferably has a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1) may have one or more amino acid substitutions at positions 1 to 6, 8, 10 to 13, 16 to 20, 22 to 30, 32 to 45 and 47 to 51 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably, wherein the amino acid residue at position 1 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tryptophan, glutamine or asparagine, the amino acid residue at position 2 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 3 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 4 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 5 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 6 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine or proline, the amino acid residue at position 8 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine or alanine, the amino acid residue at position 11 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or isoleucine, the amino acid residue at position 12 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glycine, glutamine or serine, the amino acid residue at position 13 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, aspartic acid, asparagine, proline or arginine, the amino acid residue at position 16 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with methionine, the amino acid residue at position 17 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, isoleucine, methionine or phenylalanine, the amino acid residue at position 18 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine or proline, the amino acid residue at position 19 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, histidine, lysine, asparagine, glutamine, serine or valine, the amino acid residue at position 20 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with methionine or valine, the amino acid residue at position 22 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, aspartic acid, glycine, histidine, lysine, glutamine, arginine or tryptophan, the amino acid residue at position 23 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, asparagine, glutamine or tryptophan, the amino acid residue at position 24 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine, histidine, threonine or asparagine, the amino acid residue at position 25 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, aspartic acid, glycine, histidine, glutamine, serine or arginine, the amino acid residue at position 26 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 27 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 28 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glutamic acid, glutamine, serine, threonine or alanine, the amino acid residue at position 29 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 30 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, alanine, glutamic acid, glutamine or serine, the amino acid residue at position 32 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, lysine, methionine or arginine, the amino acid residue at position 33 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, alanine, glutamic acid, glycine, lysine, glutamine or serine, the amino acid residue at position 34 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, tryptophan or isoleucine, the amino acid residue at position 36 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glycine, glutamine isoleucine or serine, the amino acid residue at position 37 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, aspartic acid, lysine, methionine, asparagine, glutamine or valine, the amino acid residue at position 38 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, serine or glycine, the amino acid residue at position 39 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, alanine, aspartic acid, glycine or isoleucine, the amino acid residue at position 40 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, aspatic acid, glycine, asparagine, serine, leucine or glutamine, the amino acid residue at position 41 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, aspartic acid or threonine, the amino acid residue at position 42 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, histidine, lysine, methionine, asparagine, glutamine or serine, the amino acid residue at position 43 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, valine or threonine, the amino acid residue at position 44 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glutamic acid, histidine, serine, tryptophan, or leucine. the amino acid residue at position 45 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, aspartic acid, glutamic acid, lysine, proline, glutamine or threonine, the amino acid residue at position 47 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 48 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glutamic acid, asparagine, glutamine, isoleucine or arginine, the amino acid residue at position 49 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, methionine or asparagine, the amino acid residue at position 50 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, histidine, asparagine, threonine or isoleucine, and/or the amino acid residue at position 51 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine.

The above amino acid substitutions may be considered as beneficial or, at least, permissive for the desired pharmaceutical properties of the peptidoglycan hydrolases of the invention, as described herein, e.g., as described in context of beneficial or permissive amino acid substitutions in the CHAP domain.

Furthermore, the LYSM domain of the invention (which preferably has a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1) may have one or more amino acid substitutions at positions 1, 8, 10, 12, 13, 17, 19, 22 to 25, 28 to 30, 32 to 45 and 48 to 51 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably, wherein the amino acid residue at position 1 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tryptophan or glutamine, the amino acid residue at position 8 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 12 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glycine, glutamine or serine, the amino acid residue at position 13 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, aspartic acid, asparagine or proline, the amino acid residue at position 17 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, isoleucine or methionine, the amino acid residue at position 19 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, histidine, lysine, asparagine or glutamine, the amino acid residue at position 22 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, aspartic acid, glycine, histidine, lysine, glutamine, arginine or tryptophan, the amino acid residue at position 23 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, asparagine, glutamine or tryptophan, the amino acid residue at position 24 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine, histidine or threonine, the amino acid residue at position 25 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, aspartic acid, glycine, histidine, glutamine or serine, the amino acid residue at position 28 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glutamic acid, glutamine, serine or threonine, the amino acid residue at position 29 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 30 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, alanine, glutamic acid or glutamine, the amino acid residue at position 32 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, lysine or methionine, the amino acid residue at position 33 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, alanine, glutamic acid, glycine, lysine or glutamine, the amino acid residue at position 34 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or tryptophan, the amino acid residue at position 36 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glycine or glutamine, the amino acid residue at position 37 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, aspartic acid, lysine, methionine, asparagine or glutamine, the amino acid residue at position 38 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or serine, the amino acid residue at position 39 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, alanine, aspartic acid or glycine, the amino acid residue at position 40 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, aspartic acid, glycine, asparagine, or serine, the amino acid residue at position 41 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or aspartic acid, the amino acid residue at position 42 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, histidine, lysine, methionine, asparagine, glutamine or serine, the amino acid residue at position 43 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine or valine, the amino acid residue at position 44 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glutamic acid, histidine, serine or tryptophan, the amino acid residue at position 45 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, aspartic acid, glutamic acid, lysine, proline, glutamine or threonine, the amino acid residue at position 48 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glutamic acid, asparagine or glutamine, the amino acid residue at position 49 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, methionine or asparagine, the amino acid residue at position 50 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, histidine, asparagine or threonine, and/or the amino acid residue at position 51 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine.

The above amino acid substitutions may be considered as beneficial or, at least, permissive for the desired pharmaceutical properties of the peptidoglycan hydrolases of the invention, as described herein.

As already mentioned above, e.g. in context of the CHAP domain of the invention, Table 3 shows positions in SEQ ID NO: 1 where beneficial or permissive amino acid substitutions have been found. These are the positions for which the column "beneficial/permissive residues" in Table 3 shows at least one amino acid residue, regardless of whether said residue is shown without parentheses or in parentheses. These positions are also called "permissive positions" herein. Furthermore, positions 1 to 51 in Table 3 for which the column "beneficial/permissive residues" shows at least one amino acid residue, regardless of whether said residue is shown without parentheses or in parentheses are considered as "permissive positions" in the LYSM domain herein.

As already mentioned above, herein and in context of the present invention, an amino acid residue in SEQ ID NO: corresponding to a beneficial or permissive position may be substituted with an amino acid residue shown in the column "beneficial/permissive residues" in Table 3 for said position, i.e. a residue shown without parentheses or in parentheses in said column in Table 3, preferably with a residue that is shown without parentheses in said column in Table 3.

Thus, in some embodiments, in particular in context of permissive/beneficial mutations and Table 3, the LYSM domain has at least one amino acid substitution at the permissive positions in the LSYM domain or at positions corresponding to said permissive positions in the LYSM domain. Preferably, in these embodiments, at least one amino acid residue at said permissive positions in the LSYM domain or at positions corresponding to said permissive positions in the LYSM domain is substituted with an amino acid residue shown in the column "beneficial/permissive residues" in Table 3 for said positions, more preferably with a residue that is shown without parentheses in said column in Table 3.

Particularly beneficial amino acid substitutions in the L YSM domainFurthermore, as described herein, the inventors found hits variants, i.e. G1 to G4, Hl to H10 and 11 to 130, which contained certain amino acid substitutions in the LYSM domain; see, e.g., Table 4. These amino acid substitutions are thus considered in context of the invention as particularly beneficial mutations, i.e., amino acid substitutions, in the LYSM domain. As described herein, e.g., in context of the CHAP domain of the invention, "particularly beneficial amino acid substitutions" may be particularly beneficial for maintaining or enhancing the desired pharmaceutical properties of peptidoglycan hydrolases, in particular, the bactericidal activity (e.g., against S. aureus), the stability and/or the ability of being secreted from eukaryotic cells, e.g. human cells. This is also true for the particularly beneficial amino acid substitutions in the LYSM domain.

Hence, the LYSM domain of the invention (which preferably has a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1), has, preferably, one or more amino acid substitutions at positions 1, 8, 10, 13, 23 to 25, 30, 33, 37, and 39 to 41 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably, wherein the amino acid residue at position 1 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tryptophan, the amino acid residue at position 8 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 13 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 23 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 24 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine, the amino acid residue at position 25 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 30 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 33 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 37 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 39 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 40 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, and/or the amino acid residue at position 41 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine.

As mentioned above, the cell wall binding domain of the invention, e.g. the LYSM domain of the invention, has, preferably, the ability to bind to the cell wall of a Staphylococcus species or strain, more preferably to Staphylococcus aureus.

Furthermore, a peptidoglycan hydrolase comprising a CHAP domain and/or a LYSM domain as described herein has, preferably, a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, as described herein.

Additional linker Preferably, herein and in context of the present invention, in particular in context of peptidoglycan hydrolase comprising a CHAP domain and a CBD, the peptidoglycan hydrolase comprises a peptide linker between the CHAP domain and the cell wall binding domain.

The peptide linker is not limited to any specific linkers and any linkers used in the art for connecting different domains or parts of proteins such as fusion proteins may be used herein and in context of the present invention.

As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence which connects two or more domains (e.g., the CHAP domain or the invention and a cell wall binding domain, and/or the CHAP of the invention and an extended pharmacokinetic (PK) peptide) in a linear amino acid sequence of a polypeptide chain. Preferably, the linker is a flexible linker. Exemplary linkers include glycine-serine-polypeptide linkers, glycine-proline- polypeptide linkers, and proline-alanine polypeptide linkers. An exemplary linker with a furin cleavage site (which may be also used in context of the present invention) is shown in SEQ ID NO: 370.

In some embodiments, the linker is a glycine-serine linker, i.e., a peptide that predominantly, essentially or exclusively consists of glycine and serine residues. Herein and in context of the present invention, a glycine-serine linker may comprise, for example, one or multiple copies (e.g. 2 to 5 copies) of the sequence shown in SEQ ID NO: 297 (i.e. GGGGS). Preferably, said copies are directly adjacent to each other, for example, as shown in SEQ ID NO: 298 or 299 (i.e. GGGGSGGGGS (GS2: 2x GGGGS), or GGGGSGGGGSGGGGSGGGGS (GS4: 4x GGGGS), respectively. Moreover, GS3 (3x GGGGS) or G5 (5x GGGGS) may be equally used. Further suitable glycine-serine linkers are shown in SEQ ID NO: 363 to 369. Another suitable glycine-serine linker has the sequence "GGS".

In certain embodiments, the peptide linker is derived from an endolysin, in particular, a linker sequence thereof. Preferably, said endolysin has a killing activity against a Staphylococcus species or strain, preferably Staphylococcus aureus.

In some preferred embodiments, the peptide linker is a LO482-derived linker, i.e. a linker which has a sequence identity of at least 60% to the sequence from position 52 to position 71 in SEQ ID NO: 1.

Beneficial and permissive amino acid substitutions in the LO482 linker found by agiycosyiation screen, directed evolution, or in siiico deimmunization screenAs described herein and as illustrated in the appended Examples, the inventors found a variety of amino acid substitutions which were contained in the linker sequence of LO482 variants obtained by directed evolution; see, e.g., Table 3, the column "beneficial/permissive residues". Of note, the LO482 variants containing these amino acid substitutions were all well secreted from eukaryotic cells and determined to be active by the YODA method. Furthermore, as also described herein and as illustrated in the appended Examples, the inventors found deimmunizing substitutions in the linker sequence of LO482 variants which may decrease the immunogenicity of the linker sequence and peptidoglycan hydrolases containing such a linker; see, e.g., Table 5, column "deimm".

Furthermore, the inventors found amino acid substitutions at position 68 in SEQ ID NO: 1, i.e. agiycosyiation substitutions in the linker of LO482, which enhance the bactericidal activity upon expression in eukaryotic cells, as described herein; see, e.g., Example 4.

Thus, the L0482-derived linker (which may have a sequence identity of at least 60% to the sequence from position to position 71 in SEQ ID NO: 1), may have one or more amino acid substitutions at positions 52 to 56, 58, to 66, and 68, to 71 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably wherein the amino acid residue at position 52 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 53 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, alanine, glycine, lysine, glutamine or serine, the amino acid residue at position 54 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glutamic acid, histidine, asparagine, alanine or isoleucine, the amino acid residue at position 55 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, aspartic acid or serine, the amino acid residue at position 56 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or isoleucine, the amino acid residue at position 58 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 60 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or isoleucine, the amino acid residue at position 61 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine or glutamine, the amino acid residue at position 62 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or asparagine, the amino acid residue at position 63 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, alanine or methionine, the amino acid residue at position 64 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or leucine, the amino acid residue at position 65 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, aspartic acid, asparagine or arginine, the amino acid residue at position 66 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with , lysine, methionine, arginine, alanine or serine, the amino acid residue at position 69 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, histidine, asparagine or glutamine, the amino acid residue at position 70 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and/or the amino acid residue at position 71 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or aspartic acid.

Furthermore, the peptide linker (which may have a sequence identity of at least 60% to the sequence from position to position 71 in SEQ ID NO: 1) may have one or more amino acid substitutions at positions 52 to 56, 58, 63, 65, 68, 69 and 71 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably wherein the amino acid residue at position 52 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 53 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, alanine, glycine, lysine, glutamine or serine, the amino acid residue at position 54 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, glutamic acid, histidine or asparagine, the amino acid residue at position 55 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine or aspartic acid, the amino acid residue at position 56 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 58 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 63 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, the amino acid residue at position 65 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine or aspartic acid, the amino acid residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine, arginine, alanine or serine, the amino acid residue at position 69 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, histidine, asparagine or glutamine, and/or the amino acid residue at position 71 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine.

As already mentioned above, e.g. in context of the CHAP domain of the invention, Table 3 shows positions in SEQ ID NO: 1 where beneficial or permissive amino acid substitutions have been found. These are the positions for which the column "beneficial/permissive residues" in Table 3 shows at least one amino acid residue, regardless of whether said residue is shown without parentheses or in parentheses. These positions are also called "permissive positions" herein. Furthermore, positions 52 to 71 in Table 3 for which the column "beneficial/permissive residues" shows at least one amino acid residue, regardless of whether said residue is shown without parentheses or in parentheses are considered as "permissive positions" in the linker of LO482 herein.

Herein and in context of the present invention, an amino acid residue in SEQ ID NO: 1 corresponding to a beneficial or permissive position may be substituted with an amino acid residue shown in the column "beneficial/permissive residues" in Table 3 for said position, i.e. a residue shown without parentheses or in parentheses in said column in Table 3, preferably with a residue that is shown without parentheses in said column in Table 3.

In some embodiments, in particular in context of permissive/beneficial mutations and Table 3, the LO482-derived peptide linker has at least one amino acid substitution at the permissive positions in the linker region of LO482 or at positions corresponding to said permissive positions in the linker region of LO482. Preferably, in these embodiments, at least one amino acid residue at said permissive positions the linker region of LO482 or at positions corresponding to said permissive positions in the linker region of LO482 is substituted with an amino acid residue shown in the column "beneficial/permissive residues" in Table 3 for said positions, more preferably with a residue that is shown without parentheses in said column in Table 3.

Particularly beneficial amino acid substitutions in the LO482 linkerFurthermore, as described herein, the inventors found hits variants, i.e. G1 to G4, Hl to H10 and 11 to 130, which contained certain amino acid substitutions in the linker region of LO482 variants; see, e.g., Table 4. These amino acid substitutions are thus considered in context of the invention as particularly beneficial mutations, i.e., amino acid substitutions, in the LO482-derived peptide linker. As described herein, e.g., in context of the CHAP domain of the invention, "particularly beneficial amino acid substitutions" may be particularly beneficial for maintaining or enhancing the desired pharmaceutical properties of peptidoglycan hydrolases, in particular, the bactericidal activity (e.g., against S. aureus), the stability and/or the ability of being secreted from eukaryotic cells, e.g. human cells. This is also true for the particularly beneficial amino acid substitutions in the linker of LO482.

Thus, in some preferred embodiments, in particular in context of LO482-derived peptide linkers, the peptide linker has a sequence identity of at least 60% to the sequence from position 52 to position 71 in SEQ ID NO: 1, and has one or more amino acid substitutions at positions 53, 55, 56, 58, 63, 65 and 68 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably wherein the amino acid residue at position 53 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, the amino acid residue at position 55 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, the amino acid residue at position 56 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 58 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 63 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, the amino acid residue at position 65 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and/or the amino acid residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine.

Preferably, said peptide linker has an aglycosylation mutation, e.g., a deletion (preferably and amino acid substitution) at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Preferably, the LO482-derived peptide linker has an amino acid substitution at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with lysine, methionine, arginine or alanine, preferably lysine. Furthermore, said peptide linker has (in addition to said aglycosylation mutation), preferably, at least one amino acid substitution at positions 53, 55, 56, 58, 63 and 65 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein, e.g., as just described above.

Furthermore, a peptidoglycan hydrolase comprising a CHAP domain, a LYSM domain and/or an L0482-derived peptide linker, as described herein, has, preferably, a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, as described herein.

LYSM domain of the invention and peptidoglycan hydrolases comprising a LYSM domain of the invention As described herein and as illustrated in the appended Examples, the inventors found particularly beneficial amino acid substitutions in the LYSM domain of LO482.

Thus, the present invention relates, in some aspects, to a LYSM domain that has (i) a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions at positions 1, 8, 10, 13, 23 to 25, 30, 33, 37, and 39 to 41 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 1 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tryptophan, the amino acid residue at position 8 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 13 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 23 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 24 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine, the amino acid residue at position 25 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 30 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 33 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 37 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 39 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 40 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, and/or the amino acid residue at position 41 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine.

In particular, said LYSM domain has the ability to bind to the cell wall of a Staphylococcus species or strain, more preferably to Staphylococcus aureus, as described herein.

Moreover, said LYSM domain is particularly advantageous for use in a peptidoglycan hydrolase having bactericidal activity, as described herein and in context of the present invention.

Thus, the present invention further relates, e.g. in context of the LYSM domain of the invention, to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises (a) an enzymatically active domain and (b) a LYSM domain that has (i) a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions at positions 1, 8,10,13, 23 to 25, 30, 33, 37, and 39 to 41 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 1 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tryptophan, the amino acid residue at position 8 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 13 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 23 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 24 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine, the amino acid residue at position 25 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 30 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 33 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 37 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 39 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 40 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, and/or the amino acid residue at position 41 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine.

Furthermore, said enzymatically active domain is, preferably, a CHAP domain, more preferably a CHAP domain of the invention as described herein.

Furthermore, said peptidoglycan hydrolase may have a peptide linker, preferably an L0482-derived peptide linker as described herein.

Furthermore, said peptidoglycan hydrolase (comprising an enzymatic domain and a LYSM domain of the invention) has, preferably, a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, as described herein.

L0482-derived peptide linker of the invention and peptidoglycan hydrolases comprising an L0482- derived peptide linker of the invention As described herein and as illustrated in the appended Examples, the inventors found particularly beneficial amino acid substitutions in the LO482-derived peptide linker of LO482.

Thus, the present invention relates, in some aspects, to a peptide linker that has (i) a sequence identity of at least 60% to the sequence from position 52 to position 71 in SEQ ID NO: 1, and that has (ii) one or more amino acid substitutions at positions 53, 55, 56, 58, 63, 65 and 68 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 53 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, the amino acid residue at position 55 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, the amino acid residue at position 56 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 58 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 63 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, the amino acid residue at position 65 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and/or the amino acid residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine, arginine or alanine, preferably lysine.

Preferably, said peptide linker has an aglycosylation mutation, e.g., a deletion (preferably and amino acid substitution) at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Preferably, the L0482-derived peptide linker has an amino acid substitution at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with lysine, methionine, arginine or alanine, preferably lysine. Furthermore, said peptide linker has (in addition to said aglycosylation mutation), preferably, at least one amino acid substitution at positions 53, 55, 56, 58, 63 and 65 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein, e.g., as just described above.

Moreover, said L0482-derived peptide linker is particularly advantageous for use in a peptidoglycan hydrolase having bactericidal activity, as described herein and in context of the present invention.

Thus, the present invention further relates, e.g. in context of the LO482-derived peptide linker of the invention, to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises (a) an enzymatically active domain, (b) a cell wall binding domain and (c) a linker that has (i) a sequence identity of at least 60% to the sequence from position 52 to position 71 in SEQ ID NO: 1, and that has (ii) one or more amino acid substitutions at positions 53, 55, 56, 58, 63, 65 and 68 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 53 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, the amino acid residue at position 55 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, the amino acid residue at position 56 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 58 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 63 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, the amino acid residue at position 65 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and/or the amino acid residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine, arginine or alanine, preferably lysine.

Preferably, said peptide linker has an aglycosylation mutation, e.g., a deletion (preferably and amino acid substitution) at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. More preferably, the L0482-derived peptide linker has an amino acid substitution at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with lysine, methionine, arginine or alanine, preferably lysine. Furthermore, said peptide linker has (in addition to said aglycosylation mutation), preferably, at least one amino acid substitution at positions 53, 55, 56, 58, 63 and 65 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein, e.g., as just described above.

Furthermore, said cell wall binding domain is, preferably, a LYSM domain, more preferably a LYSM domain of the invention as described herein.

Furthermore, said peptidoglycan hydrolase (comprising an enzymatic domain, a LYSM domain of the invention and a LO482 derived peptide linker of the invention) has, preferably, a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, as described herein.

Pharmaceutical properties of the peptidoglycan hydrolase of the invention Bactericidal activity, in particular against Staphylococci As already mentioned above, the peptidoglycan hydrolase of the invention (or the CHAP domain of the invention) has, preferably, a killing activity against at least one gram-positive bacterium, more preferably against at least one Staphylococcus species or strain, most preferably against S. aureus which, preferably, includes methicillin-resistant Staphylococcus aureus (MRSA) strains.

For example, the peptidoglycan hydrolase of the invention may have a killing activity, inter alia, against Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Staphylococcus warned (S. warned), Staphylococcus capitis (S. capitis), and/or Staphylococcus simuians (S. simulans). Moreover, the peptidoglycan hydrolase of the invention has, in particular, the ability to lyse the cell wall of a Staphylococcus species or strain, preferably Staphylococcus aureus, as described herein. More specifically, the peptidoglycan hydrolase of the invention may have the ability to break down, cleave and/or hydrolyze peptidoglycan in the cell wall of a Staphylococcus species or strain, preferably Staphylococcus aureus, as described herein.

In some embodiments, the peptidoglycan hydrolase of the invention may have a killing against a Streptococcus species or strain, for example, in addition to a killing activity against a Staphylococcus species or strain.

Preferably herein, the peptidoglycan hydrolase of the invention has a killing activity against S. aureus, as described herein.

Furthermore, it has been found that WT LO482 has a killing activity against methicillin-resistant Staphylococcus aureus {M9SK) strains, e.g., ATCC43300, A57, B94, Al; see Figure 2. Moreover, as described herein and as illustrated in the appended Examples, the MRSA S. aureus strain ATCC43300 has been employed in context of the present invention to assess the killing activity of LO482 variants against S. aureus.

Thus, the peptidoglycan hydrolase of the invention may further have a killing activity against at least one Staphylococcus aureus strain that is resistant to at least one antibiotic, e.g., methicillin, vancomycin, daptomycin and/or linezolid. For example, the peptidoglycan hydrolase of the invention may have a killing activity against at least one methicillin-resistant Staphylococcus aureus (MRSA) strain, at least one vancomycin-intermediate Staphylococcus aureus (yilSK} strain, at least one vancomycin-resistant Staphylococcus aureus (VRSA) strain, at least one daptomycin-resistant Staphylococcus aureus (DRSA) strain and/or at least one linezolid-resistant Staphylococcus aureus {tRSh} strain. Preferably, the peptidoglycan hydrolase of the invention has a killing activity against at least one methicillin-resistant Staphylococcus aureus (MRSP) strain.

Furthermore, the peptidoglycan hydrolase of the invention may have a killing activity against at least one coagulate- negative Staphylococcus species or strain, e.g. S. epidermidis.

Furthermore, the peptidoglycan hydrolase of the invention (or the CHAP domain of the invention) may, preferably, have a killing activity against a biofilm and/or a (biofilm-like) free-floating aggregate, in particular, a biofilm/free- floating aggregate of at least one gram-positive bacterium, more preferably against a biofilm/free-floating aggregate of at least one Staphylococcus species or strain, most preferably against a biofilm/free-floating aggregate of S. aureus. Said biofilm/free-floating aggregate of S. aureus may comprise or consist of at least one Staphylococcus aureus strain that is resistant to at least one antibiotic, e.g., methicillin and/or vancomycin, as described herein. In particular, the peptidoglycan hydrolase of the invention (or the CHAP domain of the invention) may have a killing activity against a biofilm of such a gram-positive bacterium which is attached to a surface and/or against a free-floating aggregate of such a gram-positive bacterium; see also the section "Pharmaceutical compositions and medical uses of the peptidoglycan hydrolase of the invention", infra, Example 6 and Figure 12. Detailed assays for determining the killing activity against biofilms and free-floating aggregates (e.g. S. aureus biofilms or free-floating aggregates) are described in Example 6.

The term "bactericidal activity", as used herein and in context of the present invention refers, preferably, to a killing activity against at least one Staphylococcus species or strain, more preferably, to a killing activity against Staphylococcus aureus, as described herein.

As already mentioned above, herein and in context of the present invention, the killing activity of a peptidoglycan hydrolase against a certain bacterium, e.g., S. aureus, is, preferably, measured by determining the minimum concentration at which the peptidoglycan hydrolase growth-inhibits a liquid culture of said bacterium, e.g., S. aureus. Said minimum concentration is also referred to herein as "minimal inhibitory concentration" (MIC). Herein and in context of the present invention, the "minimal inhibitory concentration" (MIC) is, in particular, defined as the minimum concentration which keeps the optical density at 620 nm (OD620) of a liquid culture comprising 5x1cfu/ml of a target bacterium (e.g., S. aureus^ below 0.1 for at least 24h at 37OC incubation. Preferably, the culture medium of said liquid culture is cation adjusted Muller-Hinton broth (caMHB) medium supplemented with 25% horse serum, in particular, when the target bacterium is a Staphylococcus species or strain such as S. aureus. When the killing activity against S. aureus is measured, the S. aureus cells in the liquid culture correspond, preferably, to 5xl05 cfu/ml of ATCC43300.

A detailed assay for measuring the killing activity against a target bacterium, e.g., a Staphylococcus species or strain such as S. aureus, is provided in Example 6: First, a peptidoglycan hydrolase (e.g., a LO482 variant of the present invention) is produced in E. coli, as described in Example 6 under the heading "Production of LO482 variants in E. coll'. Then, the peptidoglycan hydrolase (e.g., the LO482 variant of the present invention) is purified from E. coii, as described under the heading "Purification of L0482 variants from E. coil' in Example 6. Finally, the bactericidal activity of the peptidoglycan hydrolase (e.g., the LO482 variant of the present invention) against a target bacterium (e.g. S. aureus) is determined as described in Example 6 under the heading "Determination of bactericidal activity of LO482 variants".

Preferably, herein and in context of the present invention, the peptidoglycan hydrolase of the invention is able to growth-inhibit a Staphylococcus aureus liquid culture at a concentration of about 40 pg/ml or less, about 20 pg/ml or less or about 10 pg/ml or less, preferably at a concentration of about 4 pg/ml or less, more preferably at a concentration of about 2 pg/ml, about 1 pg/ml or less, or about 0.5 pg/ml or less. As just indicated, in context of the present invention, growth-inhibiting a Staphylococcus aureus qu6 culture is, in particular, defined as keeping the optical density at 620 nm (OD620) of a Staphylococcus aureus liquid culture comprising 5xl05 cfu/ml of Staphylococcus aureus, preferably ATCC43300, below 0.1 for at least 24h at 37°C, preferably wherein the culture medium of said liquid culture is cation adjusted Muller-Hinton broth (caMHB) medium supplemented with 25% horse serum, as described herein, and as illustrated in the appended Examples. Said "growth-inhibition" of a bacterial liquid culture may also refer, herein and in context of the present invention, as sterilizing said bacterial liquid culture.

Ability of being secreted from eukaryotic cells, in particular, from human cells Furthermore, the peptidoglycan hydrolase of the invention has, preferably, the ability of being secreted from a eukaryotic cell, in particular, when expressed in said cell. Said cell may be, for example, a yeast cell or a mammalian cell. A peptidoglycan hydrolase of the invention which has the ability of being secreted from a eukaryotic cell, preferably, further comprises a signal peptide, as described herein. Moreover, a peptidoglycan hydrolase of the invention which has the ability of being secreted from a eukaryotic cell and/or which comprises a signal peptide is, preferably, provided in form of a nucleic acid (preferably an RNA), i.e., as a nucleic acid encoding the peptidoglycan hydrolase of the invention, as described herein. A mammalian cell, as used herein and in context of the present invention (e.g. in context of the ability of being secreted from a eukaryotic or mammalian cell), may be a cell from any mammalian species including (but not limited to) humans, livestock such as cows, pets such as dogs, sports animals such as horses, endangered animals or zoo animals such as tigers, or laboratory animals such as mice. For example, a mammalian cell may be, inter alia, a human, a cow (e.g. cattle), a horse, a pig, a sheep, a goat, a camel, a yak, a monkey, a dog, a cat, a hamster, a tiger, a polar bear, a mouse, a rat etc. Preferably, said cell is a human cell, as described herein.

Furthermore, the eukaryotic cell, in particular the mammalian cell, is not particularly limited to a certain cell type. Furthermore, the eukaryotic cell, in particular the mammalian cell, may refer to an in vivoceW, an in vitro ce (e.g. a cell line) or an ex vivoceW (e.g. a primary cell). Furthermore, the ability of being secreted from a mammalian cell, is not to be understood in such a way that the peptidoglycan hydrolase needs to have the ability of being secreted from each and every cell type of the corresponding mammalian species. In contrast, it is sufficient in this respect that the peptidoglycan hydrolase has the ability of being secreted from a relevant cell type of the corresponding species, e.g. a model cell type such as HEK293 cells, and/or a cell type which is associated with a bacterial infection to be treated.

Herein and in context of the present invention, the ability of a peptidoglycan hydrolase of being secreted by a human cell is, preferably, measured by determining the amount of the peptidoglycan hydrolase in the supernatant of HEK293 cells (preferably EXPI293 cells) expressing the peptidoglycan hydrolase.

A detailed assay for measuring the ability of a peptidoglycan hydrolase (e.g., a LO482 variant of the present invention) of being secreted by eukaryotic cells, in particular human cells, is provided in Example 6 under the heading "Determination of secretion of LO482 variants from human cells".

Stability, in particular, thermostability Furthermore, the peptidoglycan hydrolase of the invention is, preferably, stable up to a temperature of about 40°C, e.g., 37°C, 38OC, 39OC, 40°C, 41°C or 42OC, preferably about 42°C, more preferably about 44°C or about 47°C. This stability also refers to a sufficient or good thermostability. Preferably, said thermostability is determined by a thermofluor assay, as described herein. In particular, the thermostability of a peptidoglycan hydrolase is measured by determining the melting temperature, i.e.,the inflecting point of the melting curve, in a thermofluor assay. Thus, when a peptidoglycan hydrolase is stable up to a certain temperature, said temperature, refers, in particular, to the melting temperature of said peptidoglycan hydrolase, preferably as determined by a thermofluor assay, as described herein. Preferably, a SYPRO orange dye and a quantitative PCR device are employed in said thermofluor assay.

A detailed assay for measuring the stability, in particular, the thermostability, of a peptidoglycan hydrolase (e.g., a L0482 variant of the present invention) is provided in Example 6: First, a peptidoglycan hydrolase (e.g., a LO4variant of the present invention) is produced in E. coli, as described in Example 6 under the heading "Production of LO482 variants in E. col/'. Then, the peptidoglycan hydrolase (e.g., the LO482 variant of the present invention) is purified from E. coli, as described under the heading "Purification of L0482 variants from E. coll' in Example 6. Finally, the thermostability of the peptidoglycan hydrolase (e.g., the LO482 variant of the present invention) is determined as described in Example 6 under the heading "Determination of protein stability of LO482 variants by the Thermofluor assay".

Solubility Furthermore, the peptidoglycan hydrolase of the invention is, preferably, soluble in an aqueous solution such as PBS.

A peptidoglycan hydrolase can be produced as described, e.g., in Example 6 herein: First, a peptidoglycan hydrolase (e.g., a LO482 variant of the present invention) is produced in E. coli, as described in Example 6 under the heading "Production of LO482 variants in E. coll'. Then, the peptidoglycan hydrolase (e.g., the LO482 variant of the present invention) is purified from E. coli, as described under the heading "Purification of L0482 variants from E. coll' in Example 6.

The solubility of the (purified) peptidoglycan hydrolase can then be measured by methods known in the art. For example, the solubility of a peptidoglycan hydrolase can be measured by determining the opalescence in an aqueous solution such as PBS at increasing concentrations, or by a PEG precipitation method.

Preferably, the solubility of a peptidoglycan hydrolase is measured by the PEG precipitation method described in Li (2013), Protein Sci. 22(8) which is incorporated herein by reference in its entirety.

Low tendency for aggregation The peptidoglycan hydrolase of the invention further has, preferably, a low tendency for aggregation, in particular in an aqueous solution such as PBS.

A peptidoglycan hydrolase can be produced as described herein, e.g., in Example 6. The tendency for aggregation can then be measured by methods known in the art, e.g., by dynamic light scattering or by size exclusion chromatography.

Enhanced pharmaceutical properties Preferably, herein and in context of the present invention, the peptidoglycan hydrolase of the invention has, compared to the peptidoglycan hydrolase of SEQ ID NO: 1, (i) a similar or enhanced bactericidal activity, preferably a similar or enhanced killing activity against Staphylococcus aureus, as described herein, (ii) a similar or enhanced ability of being secreted by a eukaryotic cell, preferably in a yeast cell or a human cell, e.g., a HEK293 cell, as described herein, (iii) a similar or enhanced solubility in an aqueous solution such as PBS, as described herein, (iv) a similar or enhanced stability, preferably an enhanced thermostability, as described herein and/or (v) a similar or reduced tendency to form aggregates in an aqueous solution such as PBS, as described herein.

Preferably, the peptidoglycan hydrolase of the invention has, compared to the peptidoglycan hydrolase of SEQ ID NO: 1: a similar or enhanced killing activity against Staphylococcus aureus, as described herein; a similar or enhanced ability of being secreted by a human cell, as described herein; and/or a similar or enhanced stability (preferably an enhanced thermostability), as described herein. More preferably, the peptidoglycan hydrolase of the invention has, compared to the peptidoglycan hydrolase of SEQ ID NO: 1: an enhanced killing activity against Staphylococcus aureus, as described herein; an enhanced ability of being secreted by a human cell, as described herein; and/or an enhanced thermostability, as described herein.

Herein and in context of the present invention, when the peptidoglycan hydrolase of the invention is compared with the peptidoglycan hydrolase of SEQ ID NO: 1 for a specific feature (e.g., the killing activity against S. aureus), preferably, the exact same assay is employed for the measurements for the peptidoglycan hydrolases that are compared to each other, and only the peptidoglycan hydrolase itself (and/or, where applicable, the corresponding coding sequence) is changed. Moreover, if present, any specific functional domains or peptides within the peptidoglycan hydrolase of the invention which have been purposefully fused to the peptidoglycan hydrolase and which have a negligible sequence identity to LO482 (SEQ ID NO: 1), e.g. below 30%, for example a signal peptide or a PK tag as described herein, should be added the same way to the peptidoglycan hydrolase of SEQ ID NO: for such comparative measurements.

Furthermore, at described herein above, a peptidoglycan hydrolase of the present invention comprising a CHAP domain of the invention may have a reduced propensity of generating resistance in target bacteria, e.g., S. aureus, compared to other peptidoglycan hydrolases not having a CHAP domain such as lysostaphin. 100 Extended pharmacokinetic peptides The peptidoglycan hydrolase of the invention may further comprise an extended pharmacokinetic (PK) peptide. Herein, an extended pharmacokinetic (PK) peptide is also called an "PK tag" or a half-life extension module. As illustrated in Example 9, a PK tag may increase the half-life of a peptidoglycan hydrolase (e.g., in the blood) of a host, in particular a mammal (preferably a human). Thus, addition of a PK tag may further improve the pharmaceutical properties of the peptidoglycan hydrolase of the invention. In particular, addition of a PK peptide may further enhance the anti-bacterial efficiency in vivo.

Suitable PKtags are well known in the art and any of these may be used in context of the present invention.

Preferably, in context of the present invention, the extended pharmacokinetic (PK) peptide is selected from the group consisting of: a human FC domain (e.g. as shown in SEQ ID NO: 294), a C-terminal peptide of human chorionic gonadotropin (e.g. as shown in SEQ ID NO: 295) and a human lysozyme (e.g. as shown in SEQ ID NO: 296).

Thus, in some embodiments, the PK peptide comprises or consists of the sequence of SEQ ID NO: 294 or it has a sequence identity of at least 90% to the sequence of SEQ ID NO: 294.

In some embodiments, the PK peptide comprises or consists of the sequence of SEQ ID NO: 295 or it has a sequence identity of at least 90% to the sequence of SEQ ID NO: 295.

In some embodiments, the PK peptide comprises of consists of the sequence of SEQ ID NO: 296 or it has a sequence identity of at least 90% to the sequence of SEQ ID NO: 296.

Herein and in context of the present invention, the PK peptide may be positioned at the C- or N-terminus of the CHAP domain of the invention, the cell wall binding domain (e.g. a LYSM domain), or the peptidoglycan hydrolase of the invention. The PK peptide may be, preferably, positioned at the C- or N-terminus of the peptidoglycan hydrolase of the invention. However, when a signal peptide is present, the PK peptide is, preferably, positioned C- terminally of the signal peptide (in particular, C-terminally of the signal peptide and N-terminally of all other of the peptidoglycan hydrolase of the invention). Thus, the PK peptide is, more preferably, C-terminally of any signal peptide and N-terminally of all CBDs and EADs contained in the peptidoglycan hydrolase of the invention (e.g. N- terminally of the LYSM domain and the CHAP domain according to the invention), preferably N-terminally of all other parts of the peptidoglycan hydrolase of the invention (i.e. all other parts than the signal peptide).

Furthermore, the peptidoglycan hydrolase of the invention, preferably, comprises between the PK peptide and the CHAP domain or the cell wall binding domain a peptide linker, for example, a glycine-serine linker, as described herein.

A peptidoglycan hydrolase of the invention comprising a PK peptide may be also considered as a fusion protein herein, in particular, wherein one part of the fusion protein refers to the EADs (e.g. the CHAP domain of the invention), and (if present) the CBDs (e.g. a LYSM domain) and any peptide linkers in between, and another part of the fusion protein refers to the PK peptide. 101 Re-glycosylation of peptidoglycan hydrolases of the invention As illustrated in Example 8, re-glycosylation of lysin variants, e.g., a LO482 variant comprising at least one aglycosylation mutation, as described herein, may further enhance stability and/or solubility of the protein.

Thus, the peptidoglycan hydrolase of the invention, e.g. a peptidoglycan hydrolase of the invention having one or more aglycosylation mutations as described herein, may further comprise at least one glycosylation motif which is not present in the peptidoglycan hydrolase of SEQ ID NO: 1. Preferably herein, in particular in context of re- glycosylation, a glycosylation motif consists of the amino acids X!, X2, and X3, wherein X! is asparagine, X2 is any amino acid except proline, and X3 is serine or threonine.

Deimmunization of peptidoglycan hydrolases of the invention As described herein, immunogenicity is a problem of many peptidoglycan hydrolases (e.g. endolysins) of the prior art which may reduce their efficacy and/or safety for medical uses.

As described herein and as illustrated in the appended Examples, the inventors developed approaches to deimmunize peptidoglycan hydrolases of the invention; see, e.g., Example 10.

As used herein and in context of the present invention, in particular in context of peptidoglycan hydrolase proteins, the terms "deimmunization" or "deimmunizing" refer to the removal of T cell epitopes, B-cell epitopes and/or aggregation hot spots in proteins such as the peptidoglycan hydrolases of the invention. Preferably herein and in context of the invention, the deimmunization refers to the removal of T cell epitopes in a peptidoglycan hydrolases of the invention.

Thus, the peptidoglycan hydrolase of the invention is, preferably, deimmunized, as described herein. Furthermore, the peptidoglycan hydrolase of the invention may have less T cell epitopes than the peptidoglycan hydrolase of SEQ ID NO: 1.

As used herein and in context of the present invention, the terms "immunogenic" or "immunogenicity" refer to the ability of a foreign substance, e.g. a peptidoglycan hydrolase of the invention, to induce an immune response in a mammal such as a human.

Herein and in context of the invention the term "immune response" refers to a an adaptive and/or innate immune response, as commonly understood in the art. Furthermore, an immune response may include a cellular and/or a humoral immune response. In context of the peptidoglycan hydrolase of the invention, the immune response comprises, preferably, an adaptive immune response, in particular, involving T cells such as CD8+ and/or CD4+ T cells. More preferably, the immune response, in this context, is mediated, at least partly, by CD4+ T cells. Herein, CD4+ T cells refer, in particular, to T helper cells. Furthermore, CD8+ T cells may be cytotoxic T cells. Furthermore, the immune response may be mediated by antibodies.

Preferably, the peptidoglycan hydrolase of the invention, e.g., a deimmunized peptidoglycan hydrolase of the invention, is less immunogenic than the peptidoglycan hydrolase of SEQ ID NO: 1.

In some embodiments, T cell epitopes in the peptidoglycan hydrolase of the invention are removed which, preferably, reduces the propensity for generating a T cell mediated immune response in a subject, more preferably 102 a CD4+ T cell mediated immune response, as described herein. Furthermore, said T cell mediated immune response may be accompanied by a humoral immune response and, thus, further be mediated by antibodies.

Avoiding or reducing an immune response in a subject to which the peptidoglycan hydrolase of the invention is administered (in form of a protein and/or a nucleic acid such as an RNA) decreases the risk for elimination and/or degradation of the peptidoglycan hydrolase in the subject and thus enhance the efficacy.

Furthermore, avoiding or reducing an immune response in a subject to which the peptidoglycan hydrolase of the invention is administered (in form of a protein and/or a nucleic acid such as an RNA) decreases the risk of adverse side effects associated with an immune response such as fever, pain, fatigue, rash, nausea, auto-immune responses, anaphylaxis etc.

Hence, deimmunization may further enhance the safety and/or efficacy of a peptidoglycan hydrolases of the invention for medical uses, in particular, for use as a pharmaceutical in large and/or diverse patient populations.

The immunogenicity of peptidoglycan hydrolases may be determined by method known in the art. Preferably, in context of the peptidoglycan hydrolases of the invention, in particular in context of removal of T cell epitopes, the immunogenicity is determined by a T cell activation assay with peripheral blood mononuclear cells (PBMCs), more preferably an INFy ELISPOT assay, which is well-known in the art. Furthermore, in particular in context of the removal of B cell epitopes, the immunogenicity may be determined by performing an ELISA on blood samples from healthy donors for preexisting B-cell epitopes.As mentioned above, when the peptidoglycan hydrolase of the invention is compared with the peptidoglycan hydrolase of SEQ ID NO: 1 for a specific feature (e.g., the immunogenicity), preferably, the exact same assay is employed for the measurements for the peptidoglycan hydrolases that are compared to each other, and only the peptidoglycan hydrolase itself (and/or, where applicable, the corresponding coding sequence) is changed.

As illustrated in Example 10, the inventors found deimmunizing amino acid substitutions in LO482 variants which may remove T cell epitopes from the peptidoglycan hydrolase of the invention, e.g. from the H5 variant (SEQ ID NO: 11); see, e.g., Table 5.

Thus, the CHAP domain of the invention may have one or more amino acid substitutions at positions 72, 73, 75 to 77, 79 to 83, 86, 99, 102 to 104, 107,108,114, 115,117, 122 to 124, 130, 131, 133 to 137, 139 to 141,143, 144, 166,169, 170, 173 to 176, 178,185,190 to 192,194, 196,197, 199, 201, 203 to 207, 212 and 213 in SEQ ID NO: 1, or at positions corresponding to these positions, preferably wherein one or more amino acid residues at said positions are substituted with any of the substitute amino acid residues shown for the corresponding positions in the following Table I. The amino acid substitutions shown in Table I are considered as "deimmunizing mutations" or "deimmunizing amino acid substitutions" herein and in context of the present invention).

Table I: Deimmunizina amino acid substitutions Position in SEQ ID NO: 1 Substitute amino acid residueQD, G, Q or SD, E, N or P 103 17 A, I or MD, H, K, N or QA, D, G, H, K, Q, R or WN, Q or WH orTD, G, H, Q or SD, E, Q, S, TDA, D, E or QA, K or MA, D, E, G, K or QAor WD, G or QD, K, L, M, N or QK or 5A, D or GD, G, N, 5 or TDD, H, K, M, N, Q or 5LorVD, E, H, 5 or WA, D, E, K, P, Q or TD, E, N or Q1, M or ND, H, N or TT5A, G, K, Q or 5D, E, H or NDD5 104 69 G, H, N or QsD, H or TD, S or TD orQD or NA, D, G, H, K, N, Q, S or TH orWA, D, M or NA, G or SGM or QYK, M, Q, T, S or V102 A, G, S or T103 A or G104 D, M, QorY107 A or S108 G, K or Y114 QorT115 D, G or S117 D or N122 L123 D, S, T or W124 QorT130 GorY131 N, Q or Y133 QorT134 K, Q or T135 H or S136 Q, E or T137 G 105 139 A, D, Q or S140 D141 D, H or T143 P144 Q166 A, E, V or T169 D170 A, H or S173 H or N174 A, H, K, R, S or T175 N176 A, Q, S or T178 S orT185 D or N190 A, C, D, H, N, S or T191 D, N192 E, G, K, or T194 G, P or T196 QorT197 P or R199 N, SorT201 D or K203 D204 D205 QorV206 Q207 H orW212 D, E, H, K or N213 Y Furthermore, the peptidoglycan hydrolase of the invention, in particular a peptidoglycan hydrolase which has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, may have one or more amino acid substitutions at positions 1, 12, 13, 17, 19, 22 to 25, 28 to 30, 32 to 34, 36 to 45, 48 to 55, 65, 68, 69, 71 to 73, 75 to 77, 79 106 to 83, 86, 99, 102 to 104, 107, 108, 114, 115, 117, 122 to 124, 130, 131, 133 to 137, 139 to 141, 143, 144, 166, 169, 170, 173 to 176, 178, 185, 190 to 192, 194, 196, 197, 199, 201, 203 to 207, 212 and 213 in SEQ ID NO: 1, or at positions corresponding to these positions, preferably wherein one or more amino acid residues at said positions are substituted with any of the substitute amino acid residues shown for the corresponding positions in the above Table 1.

Furthermore, the LYSM domain of the invention may have one or more amino acid substitutions at positions 1,12, 13,17,19, 22 to 25, 28 to 30, 32 to 34, 36 to 45,48 to 51 in SEQ ID NO: 1, or at positions corresponding to these positions, preferably wherein one or more amino acid residues at said positions are substituted with any of the substitute amino acid residues shown for the corresponding positions in the above Table 1.

Furthermore, the LO482-derived peptide linker of the invention may have one or more amino acid substitutions at positions 52 to 55, 65, 68, 69, and 71 in SEQ ID NO: 1, or at positions corresponding to these positions, preferably wherein one or more amino acid residues at said positions are substituted with any of the substitute amino acid residues shown for the corresponding positions in the above Table 1.

Preferably, the CHAP domain of the invention and/or the peptidoglycan hydrolase of the invention comprises any of the deimmunizing mutations (e.g. as just described above) in addition to any particularly beneficial mutations or any most beneficial mutations as described herein as well as any aglycosylation mutations as described herein. This means that, the particularly beneficial mutations, the most beneficial mutations as well as any aglycosylation mutations, as described herein, are, preferably not overwritten by the deimmunizing mutations. Thus, the deimmunizing mutations are, preferably, employed at positions, where no particularly beneficial mutation, most beneficial mutations or aglycosylation mutation, as described herein, is employed. Thus, the CHAP domain of the invention and the peptidoglycan hydrolase of the invention have at positions 82, 85, 86, 130, 136, 155, 169, 1and/or 186 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably, at least one of the most beneficial amino acid substitutions, and at position 73 or at a position corresponding to this position, preferably, an aglycosylation substitution, as described herein. Hence, the CHAP domain of the invention and the peptidoglycan hydrolase of the invention have, preferably, deimmunizing mutations at other positions than at positions 73, 82, 85, 86, 130, 136, 155, 169, 185 and/or 186 in SEQ ID NO: 1 or at positions corresponding to these positions.

Furthermore, the peptidoglycan hydrolase of the invention and the LO482-derived peptide linker of the invention have at position 68 or at a position corresponding to this position, preferably, an aglycosylation substitution, as described herein. Hence, the peptidoglycan hydrolase of the invention has, in certain embodiments, deimmunizing mutations, preferably, at other positions than at positions 68, 73, 82, 85, 86, 130, 136, 155, 169, 185 and/or 1in SEQ ID NO: 1 or at positions corresponding to these positions. Similarly, the L0482-derived peptide linker of the invention has, preferably, deimmunizing mutations, preferably, at another position than at position 68 or at a position corresponding to this position.

Nucleic acids encoding the peptidoglycan hydrolase of the invention Administration of a peptidoglycan hydrolase of the invention in form of a nucleic acid (e.g., an mRNA) encoding said peptidoglycan hydrolase to a subject (e.g., a human) has certain advantages. For example, cells in a subject to which a nucleic acid of the invention has been introduced may continuously produce and secrete the peptidoglycan hydrolase protein of the invention. This may provide a more efficient treatment of the bacterial infection and, for example, provide a higher efficacy in treating difficult to treat bacterial infections such as bacterial 107 biofilms, e.g., Staphylococcus biofilms. Furthermore, the nucleic acid may be introduced into cells at a particular location which may further improve the efficiency and/or safety of the treatment. Furthermore, nucleic acids, in particular RNAs, have further certain practical advantages over proteins with respect to their manufacturing, safety profile and/or adaptability.

Thus, the present invention relates, in some aspects, to a nucleic acid (e.g. an RNA or RNA construct) encoding the peptidoglycan hydrolase of the invention, as described herein. Preferably, said peptidoglycan hydrolase comprises the CHAP domain of the invention, as described herein. Furthermore, said peptidoglycan hydrolase, preferably, comprises a signal peptide, as described herein. Furthermore, said peptidoglycan hydrolase has, preferably, the ability of being secreted from a eukaryotic cell, preferably a human cell, as described herein. More preferably, said peptidoglycan hydrolase has an enhanced ability of being secreted from a eukaryotic cell (preferably a human cell) compared to the peptidoglycan hydrolase of SEQ ID NO: 1, as described herein.

Furthermore, as described herein and as illustrated in the appended Examples, the peptidoglycan hydrolases of the invention may be particularly well adapted for the production in and secretion from eukaryotic cells. Thus, the peptidoglycan hydrolases of the invention may be particularly suitable for administration to a subject (in particular a mammal such as a human) in form of a nucleic acid; see, e.g., Figures 8 and 10.

As used herein, a peptidoglycan hydrolase "in form of a nucleic acid" refers to a nucleic acid encoding the peptidoglycan hydrolase of the invention. Thus, a peptidoglycan hydrolase which is administered in form of a nucleic acid refers to the administration of a nucleic acid encoding the peptidoglycan hydrolase of the invention from which the peptidoglycan hydrolase protein can be expressed.

As used herein, the term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In some embodiments, a nucleic acid is DNA. In some embodiments, a nucleic acid is RNA. In some embodiments, a nucleic acid is a mixture of DNA and RNA. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. The nucleic acid of the invention can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (II) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

The term "nucleoside" (abbreviated herein as "N" in context of nucleic acids) relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups.

The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively. However, uridine in RNA may be also denoted by the letter "T", e.g., in the enclosed sequence listing pursuant to WIPO St. 26. Furthermore, thymidine may be written as "dT" ("d" represents "deoxy") as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in 108 RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they may be represented as dA, dC and dG.

The nucleic acid (e.g. the RNA) of the invention is, preferably, an engineered nucleic acid, preferably an engineered RNA. Thus, the nucleic acid (e.g. the RNA) of the invention is, preferably, a non-natural nucleic acid, preferably a non-natural RNA. In particular, a nucleic acid (e.g. the RNA) may be considered as "engineered", "modified" or "non-natural", when it comprises a sequence that does not occur in nature, e.g. when it encodes a non-natural peptidoglycan hydrolase of the invention.

Furthermore, the RNA of the invention may be codon-optimized for protein expression in cells of a certain mammalian species, preferably a human, as described herein.

Signal peptides Furthermore, in particular in context of the nucleic acid of the invention, the peptidoglycan hydrolase of the invention may further comprise a signal peptide.

A nucleic acid (e.g. an RNA) of the invention encodes, preferably, a peptidoglycan hydrolase of the invention that comprises a signal peptide as described herein. The peptidoglycan hydrolase protein may be initially expressed from the nucleic acid in a eukaryotic cell in an immature form containing the signal peptide. The signal peptide may then be cleaved off during maturation of the protein, in particular, during secretion or export of the peptidoglycan hydrolase from the cell.

Thus, in some embodiments, the peptidoglycan hydrolase of the invention may comprise a signal peptide, in particular, when the peptidoglycan hydrolase is contained in a cell, as described herein.

The terms "signal peptide" and "signal sequence" are used interchangeably herein and in context of the present invention.

In particular, herein and in context of the present invention, a signal peptide targets a polypeptide with which it is associated, i.e., in which it is contained (e.g. the peptidoglycan hydrolase of the invention) to a secretory pathway in a cell. Moreover, a signal peptide may target a polypeptide with which it is associated, i.e., in which it is contained (e.g. the peptidoglycan hydrolase of the invention) to a certain cellular compartment, in particular, a compartment involved in secretion such as the endoplasmic reticulum (ER), the golgi apparatus and/or the plasma membrane. In other words, a signal peptide contained in a polypeptide (e.g. the peptidoglycan hydrolase of the invention) promotes the secretion of said polypeptide from a cell. In context of the present invention, said cell is, preferably, a eukaryotic cell, more preferably a mammalian cell, most preferably a human cell.

Suitable signal peptides are well known in the art and any of these may be used in context of the present invention. Suitable signal peptides, include, inter alia, an N-terminal mouse IgKappa signal peptide (e.g. as shown in SEQ ID NO: 300), a HSV-1 envelope glycoprotein D signal peptide (e.g. as shown in SEQ ID NO: 321, 323,325, 380 or UniProtKB GD_HHV1K), HSV-2 envelope glycoprotein D signal peptide (e.g. as shown in SEQ ID NO: 327 or 329), a Plasmodium falciparum Csp signal peptide (e.g. as shown in SEQ ID NO: 331), an Ebola spike glycoprotein GP signal peptide (e.g. as shown in SEQ ID NO: 333), a SARS-C0V-2- spike signal peptide (e.g. as shown in SEQ ID NO: 335), a human Ig heavy chain signal peptide (e.g. as shown in SEQ ID NO: 337, 344, 346, 348, 350, 352, 354, 356 or 358), a human insulin signal peptide (SEQ ID NO: 378), a human ig kappa chain signal peptide (e.g. as 109 shown in SEQ ID NO: 338, 360 or 362), a Japanese encephalitis PRM signal peptide (e.g. UniProtKB POLG_JAEVM, or as shown in SEQ ID NO: 340), a VSVg protein signal peptide (e.g. as shown in SEQ ID NO: 341), and a TRIO signal peptide (in particular, an Anopheles gambiae TRIO salivary gland protein signal peptide, e.g. UniProtKB Q7PUJ5_ANOGA or as shown in SEQ ID NO: 342).

Exemplary RNA sequences encoding signal peptides are shown in SEQ ID NO: 320, 322, 324, 326, 328, 330, 332, 334, 336, 343, 345, 347, 349, 351, 353, 355, 357, 359 or 361.

Thus, in context of the present invention, the signal peptide may be, for example, from a pathogen such as a bacterium or virus (e.g. a Herpes simplex virus, a Japanese encephalitis virus or an Ebola virus), as described herein. Furthermore, the signal peptide may be from another organism than a human, e.g., from a mouse, as described herein. Thus, in some embodiments, the signaling peptide contained in the peptidoglycan hydrolase of the invention is not human.

Preferably, in context of the present invention, the signal peptide is located at the N-terminus of the peptidoglycan hydrolase, in particular N-terminal of the cell-wall binding domain (e.g. a LYSM domain according to the invention), the enzymatically active domain (e.g. a CHAP domain according to the invention), and any tags or peptides contained in the peptidoglycan hydrolase of the invention such as an extended pharmacokinetic (PK) peptide.

Preferably, in context of the present invention, the signal peptide is cleaved off during secretion or export from the cell.

Preferably, in context of the present invention, the signal peptide is able to direct the peptidoglycan hydrolase of the invention to a secretory pathway in a mammalian cell, preferably a co-translational translocation pathway.

In certain embodiments, the signal peptide contained in the peptidoglycan hydrolase of the invention comprises or consists of the sequence of SEQ ID NO: 378, SEQ ID NO: 300, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 331, SEQ ID NO: 335, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 344, SEQ ID NO: 346, SEQ ID NO: 348, SEQ ID NO: 350, SEQ ID NO: 352, SEQ ID NO: 354, SEQ ID NO: 356, SEQ ID NO: 358, SEQ ID NO: 360 or SEQ ID NO: 362.

In some embodiments, e.g., in context of the RNA of the invention, the signal peptide contained in the peptidoglycan hydrolase of the invention comprises the sequence of SEQ ID NO: 378.

A peptidoglycan hydrolase of the invention comprising a signal peptide may be also considered as a fusion protein herein, in particular, wherein one part of the fusion protein refers to the EADs (e.g. the CHAP domain of the invention), and (if present) the CBDs (e.g. a LYSM domain) and any peptide linkers in between, and another part of the fusion protein refers to the signal peptide.

Regardless of the signal peptide, however, herein and in context of the present invention, the peptidoglycan hydrolase is, preferably, adapted for secretion, as described herein. As described herein, and as illustrated in the appended Examples, the peptidoglycan hydrolase of the invention has, preferably, the ability of being secreted from a eukaryotic cell and may be particularly well adapted to a eukaryotic (preferably human) secretory pathway. More preferably, e.g., in context of the nucleic acid or the RNA of the invention, the peptidoglycan hydrolase of the invention has an enhanced ability of being secreted from a human cell compared to the WT LO482 endolysin (SEQ ID NO: 1). 110 DNA Herein, the term "DNA" relates to a nucleic acid molecule which is entirely or at least substantially composed of deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 21- position of a 3-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non- standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

Thus, in some embodiments, the nucleic acid of the invention is a DNA, as described herein.

In some embodiments, the DNA of the invention is comprised in a plasmid, for example, an expression vector. In particular, the expression vector is suitable for expressing the polypeptide hydrolase of the invention in a cell, or, at least, the vector is suitable for transcribing an mRNA encoding the polypeptide hydrolase of the invention in a cell. Preferably, said cell is a eukaryotic cell, more preferably a human cell, as described herein. Thus, the invention relates, in some embodiments, to a plasmid comprising the nucleic acid of the invention, as described herein.

RNA As used herein, the term "RNA" relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a 3-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered/modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered/modified nucleotides (i.e., altered/modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at ill least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

"RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some preferred embodiments, "RNA" refers to mRNA.

The term "in vitro transcription" or "IVT" as used herein means that the transcription (i.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living/cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).

According to the present disclosure, the term '"RNA" includes "mRNA". According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" which may be generated by using a DNA template. Generally, mRNA encodes a peptide or polypeptide, e.g., a peptidoglycan hydrolase of the invention. mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.

According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

As established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide/polypeptide coding region (e.g. a sequence encoding a peptidoglycan hydrolase of the invention) and a 3' untranslated region (31- UTR).

In some embodiments, RNA is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in 1//&otranscription. The cDNA may be obtained by reverse transcription of RNA.

In some embodiments of the present disclosure, the RNA is "replicon RNA" or simply a "replicon", in particular "self- replicating RNA" or "self-amplifying RNA". In certain embodiments, the replicon or self-replicating RNA is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5'-cap, and a 3' poly(A) tail. The genome of alphaviruses encodes non- structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near 112 the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).

Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication (trans-amplification) systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

In some embodiments of the present disclosure, the RNA (in particular, mRNA) described herein contains one or more modifications, e.g., in order to increase its stability and/or increase translation efficiency and/or decrease immunogenicity and/or decrease cytotoxicity. For example, in order to increase expression of the RNA (in particular, mRNA), it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without altering the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007/036366 and PCT/EP2019/056502, and include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 51- and/or 31- untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the GC content of the RNA). A combination of the above described modifications, i.e., incorporation of a 51-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 51- and/or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and/or pseudouridine (U) or N(l)-methylpseudouridine (m1W) or 5-methyluridine (m5U) for uridine), and codon optimization, has a synergistic influence on the stability of RNA (preferably mRNA) and increase in translation efficiency. Thus, in some embodiments, the RNA (in particular, mRNA) described in the present disclosure contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5'-cap structure, (ii) incorporation of a poly-A sequence, unmasking of a poly-A sequence; (ill) alteration of the 51- and/or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and/or pseudouridine (U) or N(l)- methylpseudouridine (m1W) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.

Thus, in preferred embodiments, the nucleic acid of the invention is an RNA, as described herein. 113 The RNA or RNA construct of the invention is, preferably, suitable for translating the peptidoglycan hydrolase of the invention encoded by said RNA or RNA construct in a suitable cell, preferably a eukaryotic cell, more preferably a human cell.

In preferred embodiments, the RNA is an mRNA as described herein. In another preferred embodiment, the RNA is a repl icon RNA as described herein.

Preferably, the mRNA according to the present invention is a nucleoside-modified RNA, as described herein.

In some embodiments, the mRNA of the invention comprises (i) a modified nucleoside selected from pseudouridine (w), Nl-methyl-pseudouridine (mly), and 5-methyl- uridine (m5U), preferably Nl-methyl-pseudouridine (mly), in place of uridine, preferably in place of each uridine; (ii) a capO 5' cap, for example, m2720׳G(5')ppSp(5')G; or a capl 5'cap, for example, m270 '־ 3 ׳ Gppp(m12'־°)ApG (ill) a 5' UTR and/or a 3' UTR, and/or (iv) a poly-A sequence comprising preferably at least 100 nucleotides; as described herein.

Furthermore, the present invention relates to an RNA construct comprising in 5' to 3' order: (i) a 5' UTR, preferably, comprising or consisting of a modified human alpha-globin 5'-UTR; (ii) a sequence encoding a peptidoglycan hydrolase of the invention; (iii) a 3' UTR, preferably, comprising or consisting of a first sequence from the amino terminal enhancer ofsplit (AES) messenger RNA and a second sequence from the mitochondrial encoded 125 ribosomal RNA; and (iv) a poly-A sequence, comprising preferably at least about 100 nucleotides.

In preferred embodiments, the 5' UTR of the RNA construct comprises or consists of a sequence according to SEQ ID NO: 371; the 3' UTR of the RNA construct comprises or consists of a sequence according to SEQ ID NO: 374; and/or the polyA tail sequence of the RNA construct is a split polyA tail sequence which, preferably, comprises or consists of a sequence according to SEQ ID NO: 375. In some embodiments, the RNA construct comprises between the 3'UTR and the poly-A sequence a sequence according to SEQ ID NO: 379 which may have a further beneficial effect on the expression level of the encoded peptidoglycan hydrolase.

In some embodiments, the RNA construct further comprises a 5' cap. In some embodiments, the RNA construct is an mRNA, and preferably the mRNA contains a modified nucleoside selected from pseudouridine (w), Nl-methyl- pseudouridine (mly), and 5-methyl-uridine (m5U) in place of uridine, preferably in place of each uridine, as described herein. Preferably, said modified nucleoside is Nl-methyl-pseudouridine (mly).

Herein, the RNA construct of the invention is an embodiment of the RNA of the invention. It is to be understood that all features, aspects and embodiments described in the context of the RNA of the invention are equally applicable to the RNA construct of the invention. 114 In some embodiments, the RNA of the invention is formulated as a particle comprising said RNA, preferably as a lipid nanoparticle (LNP) or lipoplex (LPX).

Further details and embodiments concerning the RNA of the invention, e.g., with respect to the 5' Cap, the poly-A sequence (i.e. poly-A tail), the 5' UTR, the 3' UTR, modified nucleosides and particles such as LNP and LPX are described in the following: '-Cap Preferably, the RNA (in particular, mRNA) of the invention comprises a 5'-cap structure. In some embodiments, the RNA does not have uncapped 5'-triphosphates. In some embodiments, the RNA (in particular, mRNA) may comprise a conventional 5'-cap and/or a 5'-cap analog. The term "conventional 5'-cap" refers to a cap structure found on the 5'-end of an RNA molecule and generally comprises a guanosine 5‘-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'-end of the next nucleotide of the RNA (i.e., the guanosine is connected via a 51 to 5' triphosphate linkage to the rest of the RNA). The guanosine may be methylated at position N7 (resulting in the cap structure m7Gppp). The term "5'-cap analog" includes a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 21- or 3'-position of the m7guanosine structure in order to avoid an integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5'-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphoroth ioate modified 5'-cap analogs at the 3-phosphate (such as m27,2'OG(5')ppSp(5l)G (referred to as beta-S-ARCA or B-S-ARCA)), as described in PCT/EP2019/056502. Providing an RNA (in particular, mRNA) with a 5'-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5'-cap compound, wherein said 5'-cap structure is co- transcriptionally incorporated into the generated RNA (in particular, mRNA) strand, or the RNA (in particular, mRNA) may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

In some embodiments, the RNA (in particular, mRNA) comprises a capO, capl, or cap2, preferably capl. According to the present disclosure, the term "capO" means the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 21. According to the present disclosure, the term "capl" means the structure "m7GpppNm", wherein Nm is any nucleoside bearing an OCH3 moiety at position 21. According to the present disclosure, the term "cap2" means the structure "m7GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCHmoiety at position 21.

In some embodiments, the RNA (in particular, mRNA) comprises a 5'-cap structure selected from the group consisting of m27,2'OG(5')ppSp(5')G (in particular its DI diastereomer), m27,3'OG(5')ppp(5l)G, and m27,3'- OGppp(ml2'-O)ApG. In some embodiments, RNA comprises m27,2'OG(5')ppSp(5')G (in particular its Ddiastereomer) as 5'-cap structure. In some embodiments, the RNA comprises m27,3'-OGppp(ml2'-O)ApG as 5'-cap structure.

The 5'-cap analog beta-S-ARCA (3-S-ARCA) has the following structure: 115 The "Di diastereomer of beta-S-ARCA" or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and thus exhibits a shorter retention time. The HPLC preferably is an analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column, preferably of the format: 5 pm, 4.6 x 250 mm is used for separation, whereby a flow rate of 1.3 ml/min can be applied. In some embodiments, a gradient of methanol in ammonium acetate, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH = 5.9, within 15 min is used. UV-detection (VWD) can be performed at 260 nm and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.The 5'-cap analog m27,3'-OGppp(ml2'-O)ApG (also referred to as m27,3'OG(5')ppp(5l)m2l-OApG) which is a building block of a capl has the following structure: An exemplary capO mRNA comprising 3-S-ARCA and mRNA has the following structure: 116 mRNA An exemplary capl mRNA comprising m27,3'-OGppp(ml2'-O)ApG and mRNA has the following structure: Poly-A tailPreferably, the RNA (in particular, mRNA) of the invention comprises a poly-A sequence, in particular at the 3' end of the RNA.

As used herein, the term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA (in particular, mRNA) molecule. Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs (in particular, mRNAs) described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. RNAs (in particular, mRNAs) disclosed herein can have a poly-A tail attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

It has been demonstrated that a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (S') of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017). 117 The poly-A tail may be of any length. In some embodiments, a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

In some embodiments, a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.

In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly-A tail contained in an RNA (in particular, mRNA) molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50,10 to 30, or 10 to 20 nucleotides in length.

In some embodiments, the poly(A) tail comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence of nucleotides.

In some embodiments, no nucleotides other than A nucleotides flank a poly-A tail at its 3'-end, i.e., the poly-A tail is not masked or followed at its 3'-end by a nucleotide other than A.

In some embodiments, a poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 1and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 1nucleotides. In some embodiments, the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 375. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides. 118 In some embodiments, RNA comprises a poly-A tail comprising the nucleotide sequence of SEQ ID NO: 375, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 375.

Untranslated regions (UTR)In some embodiments, the RNA (in particular, mRNA) of the invention comprises a 5'-UTR and/or a 3'-UTR. The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 51-end, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 51-cap. A 3'-UTR, if present, is located at the 3'-end, downstream of the termination codon of a protein-encoding region, but the term "3'-UTR" does generally not include the poly-A sequence. Thus, the 3'-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. Incorporation of a 3'-UTR into the 3'-non translated region of an RNA (preferably mRNA) molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3'-UTRs (which are preferably arranged in a head-to-tail orientation; cf., e.g., Holtkamp et al., Blood 108,4009-4017 (2006)). The 3'-UTRs may be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced.

In some embodiments, a 5'-UTR is or comprises a modified human alpha-globin 5'-UTR. A particularly preferred 5'- UTR comprises the nucleotide sequence of SEQ ID NO: 371. In some embodiments, a 3'-UTR comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA (SEQ ID NO: 372) and a second sequence from the mitochondrial encoded 12S ribosomal RNA (SEQ ID NO: 373). A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 374. In some embodiments, a 3'-UTR comprises a first sequence comprising, or consisting of, the nucleotide sequence of SEQ ID NO: 372, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 372, and a second sequence comprising, or consisting of, the nucleotide sequence of SEQ ID NO: 373, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 373.

In some embodiments, the RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 371, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 371.

In some embodiments, the RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 374, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 374.

Chemical modificationFurthermore, the RNA (in particular, mRNA) of the invention may have modified ribonucleotides in order to increase its stability and/or decrease immunogenicity and/or decrease cytotoxicity. For example, in some embodiments, uridine in the RNA (in particular, mRNA) of the invention is replaced (partially or completely, preferably completely) by a modified nucleoside. Preferably, the modified nucleoside is a modified uridine. 119 In some preferred embodiments, the modified uridine replacing uridine is selected from the group consisting of pseudouridine (w), Nl-methyl-pseudouridine (mlw), 5-methyl-uridine (m5U), and combinations thereof.

In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (m05U), 5-aza-uridine, 6-aza- uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (h05U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), uridine 5- oxyacetic acid (cm05U), uridine 5-oxyacetic acid methyl ester (mcm05U), 5-carboxymethyl-uridine (cm5U), 1- carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl- pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5- carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, l-propynyl-pseudouridine, 5- taurinomethyl-uridine (Tm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), 1- taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (mls41p), 4- thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m31p), 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza- pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl- pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 w), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a- thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (wm), 2-thio- 2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O- methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara- uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.

An RNA (preferably mRNA) which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine) is referred to herein as "W-modified", whereas the term "ml 1^-modified" means that the RNA (preferably mRNA) contains N(1 )-methylpseudouridine (replacing partially or completely, preferably completely, uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (replacing partially or completely, preferably completely, uridine). Such W- or mlW- or m5U-modified RNAs usually exhibit decreased immunogenicity compared to their unmodified forms and, thus, are preferred in applications where the induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) of the invention contains N(l)-methylpseudouridine replacing completely uridine, in particular in context of RNA used in pharmaceutical compositions and/or for medical uses.

Codon optimization and GC enrichmentThe codons of the RNA (in particular, mRNA) of the invention may further be optimized, e.g., to increase the GC content of the RNA and/or to replace codons which are rare in the cell (or subject) in which the peptide or polypeptide of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject). 120 In some embodiments, the amino acid sequence encoded by the RNA (in particular, mRNA) described in the present disclosure is encoded by a coding sequence which is codon-optimized and/or the G/C content of which is increased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence. In particular, in context of the present invention, the codon- optimization and/or the increase in the G/C content does not change the sequence of the encoded amino acid sequence.

Thus, the term "codon-optimized" refers, in particular, to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, coding regions may be codon- optimized for optimal expression in a subject to be treated using the RNA (in particular, mRNA) described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence oftRNAs in cells. Thus, the sequence of RNA (in particular, mRNA) may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".

In some embodiments, the guanosine/cytosine (G/C) content of the coding region of the RNA (in particular, mRNA) described herein is increased compared to the G/C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and/or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleotides.

In various embodiments, the G/C content of the coding region of the RNA (in particular, mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of the coding region of the wild type RNA.

Non-immunogenic RNAIn some embodiments, the RNA (in particular, mRNA) of the present invention is non-immunogenic.

The term "non-immunogenic RNA" (such as "non-immunogenic mRNA") as used herein refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal such as a human, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., than would have been induced by standard RNA (stdRNA). In certain embodiments, non-immunogenic RNA is rendered non- immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and/or limiting the amount of double-stranded RNA (dsRNA), e.g., by limiting the formation of double-stranded RNA (dsRNA), e.g., during in vitro transcription, and/or by removing double-stranded RNA 121 (dsRNA), e.g., following in vitro transcription. In certain embodiments, non-immunogenic RNA is rendered non- immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and/or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription.

For rendering the non-immunogenic RNA (especially mRNA) non-immunogenic by the incorporation of modified nucleosides, any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleosides comprise a replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl- uridine (m3U), 5-methoxy-uridine (m05U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (h05U), 5-aminoallyl-uridine, 5- halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cm05U), uridine 5-oxyacetic acid methyl ester (mcm05U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5- methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2- thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2- thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (Tm5U), 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), l-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio- uridine (m5s2U), l-methyl-4-thio-pseudouridine (mls41p), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m31p), 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 w), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O- dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (wm), 2-thio-2'-O-methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F- uridine, 2'-OH-a ra-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)uridine. In certain preferred embodiments, the nucleoside comprising a modified nucleobase is pseudouridine (w), Nl-methyl- pseudouridine (mlw) or 5-methyl-uridine (m5U), in particular Nl-methyl-pseudouridine.

In some embodiments, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.

During synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase significant amounts of aberrant products, including double-stranded RNA (dsRNA) are produced due to unconventional activity of the enzyme. 122 dsRNA induces inflammatory cytokines and activates effector enzymes leading to protein synthesis inhibition. Formation of dsRNA can be limited during synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP may be added once or several times during synthesis of mRNA. Also, dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzymatic based method using E. coli RNaselll that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017/182524.

As the term is used herein in context of RNA, "remove" or "removal" refers to the characteristic of a population of first substances, such as non-immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance. However, a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the non-separated mixture of first and second substances.

In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, e.g., dsRNA (especially dsmRNA) is removed from non-immunogenic RNA, such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non- immunogenic RNA (especially mRNA) is free or essentially free of dsRNA. In some embodiments, the non- immunogenic RNA (especially mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises single- stranded nucleoside modified RNA (especially mRNA) and is substantially free of double stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%,, at least 99.993%״ at least 99.994%, , at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

Various methods can be used to determine the amount of dsRNA. For example, a sample may be contacted with dSRNA-specific antibody and the amount of antibody binding to RNA may be taken as a measure for the amount of dsRNA in the sample. A sample containing a known amount of dsRNA may be used as a reference.

For example, RNA may be spotted onto a membrane, e.g., nylon blotting membrane. The membrane may be blocked, e.g., in TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCI, 0.1% (v/v) TWEEN-20) containing 5% (w/v) skim milk powder. For detection of dsRNA, the membrane may be incubated with dSRNA-specific antibody, e.g., dSRNA-specific mouse mAb (English 81 Scientific Consulting, Szirak, Hungary). After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected. 123 In some embodiments, the non-immunogenic RNA (especially mRNA) is translated in a cell more efficiently than standard RNA with the same sequence. In some embodiments, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by a 3-fold factor. In some embodiments, translation is enhanced by a 4-fold factor. In some embodiments, translation is enhanced by a 5- fold factor. In some embodiments, translation is enhanced by a 6-fold factor. In some embodiments, translation is enhanced by a 7-fold factor. In some embodiments, translation is enhanced by an 8-fold factor. In some embodiments, translation is enhanced by a 9-fold factor. In some embodiments, translation is enhanced by a 10- fold factor. In some embodiments, translation is enhanced by a 15-fold factor. In some embodiments, translation is enhanced by a 20-fold factor. In some embodiments, translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In some embodiments, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000- fold. In some embodiments, the factor is 200-1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3-fold factor. In some embodiments, innate immunogenicity is reduced by a 4-fold factor. In some embodiments, innate immunogenicity is reduced by a 5-fold factor. In some embodiments, innate immunogenicity is reduced by a 6-fold factor. In some embodiments, innate immunogenicity is reduced by a 7-fold factor. In some embodiments, innate immunogenicity is reduced by an 8-fold factor. In some embodiments, innate immunogenicity is reduced by a 9-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15-fold factor. In some embodiments, innate immunogenicity is reduced by a 20-fold factor. In some embodiments, innate immunogenicity is reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100- fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor.

The term "exhibits significantly less innate immunogenicity" refers to a detectable decrease in innate immunogenicity. In some embodiments, the term refers to a decrease such that an effective amount of the non- immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a decrease such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the decrease is such that the non- immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA. 124 The term "immunogenicity" refers to the ability of a foreign substance, e.g. an RNA encoding a peptidoglycan hydrolase of the invention, to induce an immune response in a mammal such as a human.

The innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system, as described herein.

RNA deliveryThe RNA of the invention may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNA, or delivery mediated by delivery vehicles.

In some embodiments, after administration of the RNA (in particular, mRNA) compositions/formulations described herein, at least a portion of the RNA is delivered to a target cell, target tissue or target organ. In some embodiments, at least a portion of the RNA is delivered to the cytosol of a target cell. In some embodiments, the RNA is translated by a target cell to produce the encoded peptide or polypeptide. In some embodiments, the target cell is a cell in the liver. In some embodiments, the target cell is a muscle cell.

In some embodiments, after administration of the RNA (in particular, mRNA) compositions/formulations described herein to a subject, at least a portion of the RNA is delivered to cells of the subject for translation of the encoded peptide or polypeptide.

Delivery vehiclesTo overcome the barriers to safe and effective RNA delivery, RNA may be administered with one or more delivery vehicles that protect the RNA from degradation, maximize delivery to on-target cells and minimize exposure to off- target cells. Such RNA delivery vehicles may complex or encapsulate RNA and include a range of materials, including polymers and lipids. In some embodiments, such RNA delivery vehicles may form particles with RNA.

RNA, in particular mRNA, described herein may be present in particles comprising (i) the RNA, and (ii) at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the RNA. Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged RNA are involved in particle formation. This results in complexation and spontaneous formation of RNA particles.

Different types of RNA containing particles have been described previously to be suitable for delivery of RNA in particulate form (cf., e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral RNA delivery vehicles, nanoparticle encapsulation of RNA physically protects RNA from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.

In the context of the present invention, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

RNA particles described herein include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

A lipoplex (LPX) described herein is obtainable from mixing two aqueous phases, namely a phase comprising RNA and a phase comprising a dispersion of lipids. In some embodiments, the lipid phase comprises liposomes. 125 In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.

In some embodiments, liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids employed in formulating liposomes designed for the delivery of RNA are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.

In some embodiments, lipoplexes are multilamellar liposome-based formulations that form upon electrostatic interaction of cationic liposomes with RNAs. In some embodiments, formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact RNA- lipoplexes.

In some embodiments, an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and RNA (especially mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids) and negatively charged RNA (especially mRNA) results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as DOTMA and/or DODMA, and optionally additional lipids, such as DOPE or DSPC.

In general, a lipid nanoparticle (LNP) is typically obtainable from direct mixing of RNA in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.

In some embodiments, LNPs comprise or consist of a cationic/cationically ionizable lipid and helper lipids such as phospholipids, cholesterol, and/or polymer-conjugated lipids (e.g., polyethylene glycol (PEG) lipids). In some embodiments, in the RNA LNPs described herein the RNA (in particular, mRNA) is bound by cationically ionizable lipid that occupies the central core of the LNP. In some embodiments, polymer-conjugated lipid forms the surface of the LNP, along with phospholipids. In some embodiments, cholesterol and cationically ionizable lipid in charged and uncharged forms can be distributed throughout the LNP.

In some embodiments, the particles (e.g., LNPs and LPXs) described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450 nm, from about 100 nm toabout 400 nm, from about 100 nm to about 350 nm, from about 100 nm to about 300 nm, from about 100 nm toabout 250 nm, from about 100 nm to about 200 nm, from about 150 nm to about 1000 nm, from about 150 nm toabout 800 nm, from about 150 nm to about 700 nm, from about 150 nm to about 600 nm, from about 150 nm to 126 about 500 nm, from about 150 nm to about 450 nm, from about 150 nm to about 400 nm, from about 150 nm toabout 350 nm, from about 150 nm to about 300 nm, from about 150 nm to about 250 nm, from about 150 nm toabout 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm toabout 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm toabout 300 nm, from about 200 nm to about 250 nm, or from about 80 to about 150 nm. In some embodiments, the particles (e.g., LNPs and LPXs) described herein have an average diameter that in some embodiments ranges from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 1nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.

RNA particles (especially mRNA particles) described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the RNA particles can exhibit a polydispersity index in a range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

The N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the RNA. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N/P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations may be formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles can be favorable for transfection. In that case, RNA is considered to be completely bound to nanoparticles.

In some embodiments, RNA particles (especially mRNA particles) comprise more than one type of RNA molecules, where the molecular parameters of the RNA molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features, In particulate formulation, it is possible that each RNA species is separately formulated as an individual particulate formulation. In that case, each individual particulate formulation will comprise one RNA species. The individual particulate formulations may be present as separate entities, e.g. in separate containers. Such formulations are obtainable by providing each RNA species separately (typically each in the form of an RNA-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific RNA species that is being provided when the particles are formed (individual particulate formulations). In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual particle formulation. Respective pharmaceutical compositions are referred to as mixed particulate formulations. Mixed particulate formulations according to the present disclosure are obtainable by forming, separately, individual particulate formulations, followed by a step of mixing of the individual particulate formulations. By the step of mixing, a formulation comprising a mixed population of RNA-containing particles is obtainable. Individual particulate populations may be together in one container, comprising a mixed population of individual particulate formulations. Alternatively, it is possible that all RNA species of the pharmaceutical composition are formulated together as a combined particulate formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of all RNA species together with a particle-forming agent, thereby allowing the formation of particles. As opposed to a mixed particulate formulation, a combined 127 particulate formulation will typically comprise particles which comprise more than one RNA species. In a combined particulate composition different RNA species are typically present together in a single particle.

PolymersGiven their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically condense the negatively charged RNA into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. In addition, some investigators have synthesized polymers specifically for nucleic acid delivery. Poly(P־amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

A "polymer," as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. The following disclosure with respect to "polymers" is, in particular, relevant in context of RNA and/or delivery vehicles, as described herein. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties.

If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." It is to be understood that the polymer being employed herein can be a copolymer. The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

In certain embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.

In certain embodiments, polymer may be protamine or polyalkyleneimine.

The term "protamine" refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term "protamine" refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

According to the disclosure, the term "protamine" as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms 128 of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.

In one embodiment, the polyalkyleneimine comprises polyethylenimine and/or polypropylenimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75-102 to 107 Da, preferably 1000 to 105 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.

Preferred according to the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI).

Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid. In one embodiment, cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

Particles described herein may also comprise polymers other than cationic polymers, i.e., non-cationic polymers and/or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

LipidsThe terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. The following disclosure with respect to "lipids" and "lipid-like material" is, in particular, relevant in context of RNA and/or delivery vehicles, as described herein. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

As used herein, the term "hydrophobic" refers to any molecule, moiety or group which is substantially immiscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least 6 carbon atoms. The monovalent radical of a hydrocarbon is referred to as hydrocarbyl herein. The hydrophobic group can have functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.

The term "lipid-like material", "lipid-like compound" or "lipid-like molecule relates to substances, in particular amphiphilic substances, that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term includes molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids. Examples of lipid-like compounds capable of 129 spontaneous integration into cell membranes include functional lipid constructs such as synthetic function-spacer- lipid constructs (FSL), synthetic function-spacer-sterol constructs (FSS) as well as artificial amphipathic molecules. Lipids comprising two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Such lipids have low solubility as monomers and tend to aggregate into planar bilayers that are water insoluble. Traditional surfactant monomers comprising only one linear alkyl chain and a hydrophilic head group are generally cone shaped. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into spherical or elliptoid micelles that are water soluble. While lipids also have the same general structure as surfactants - a polar hydrophilic head group and a nonpolar hydrophobic tail - lipids differ from surfactants in the shape of the monomers, in the type of aggregates formed in solution, and in the concentration range required for aggregation. As used herein, in context of RNA and/or delivery vehicles, the term "lipid" is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.

Cationic/Cationicaiiy ionizable lipidsIn some embodiments, the RNA compositions and formulations and RNA particles described herein comprise at least one cationic or cationically ionizable lipid as particle forming agent. Cationic or cationically ionizable lipids contemplated for use herein include any cationic or cationically ionizable lipids (including lipid-like materials) which are able to electrostatically bind nucleic acid. In some embodiments, cationic or cationically ionizable lipids contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

As used herein, a "cationic lipid" refers to a lipid or lipid-like material having a net positive charge. Cationic lipids bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.

In some embodiments, a cationic lipid has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

As used herein, a "cationically ionizable lipid" refers to a lipid or lipid-like material which has a net positive charge or is neutral, i.e., which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral. For purposes of the present disclosure, cationically ionizable lipids are covered by the term "cationic lipid" unless contradicted by the circumstances.

In some embodiments, the cationic or cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated, e.g., under physiological conditions.

Examples of cationic or cationically ionizable lipids include, but are not limited to N,N-dimethyl-2,3- dioleyloxypropylamine (DODMA), l,2-dioleoyl-3-trimethylammonium propane (DOTAP); l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), 3-(N—(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3- 130 dimethylammonium propanes; l,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2- hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2- dimyristoyl-3-trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy- N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'- oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3- Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[!,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-K- XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9- tetradecenyloxy)-l-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-l-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (PAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-l-aminium (DOBAQ), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3- amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), l,2-dioleoyl-sn-glycero-3- ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-l-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non- 2-en-l-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan-l-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l-amine (DMDMA), Di((Z)- non-2-en-l-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl- ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]- amino}-ethylamino)propionamide (lipidoid 98N12-5), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200).

In some embodiments, the cationic or cationically ionizable lipid is DOTMA. In some embodiments, the cationic or cationically ionizable lipid is DODMA.

DOTMA is a cationic lipid with a quaternary amine headgroup. The structure of DOTMA may be represented as follows: 131 DODMA is an ionizable cationic lipid with a tertiary amine headgroup. The structure of DODMA may be represented as follows: In some embodiments, the cationic or cationically ionizable lipid may comprise from about 10 mol % to about mol %, from about 20 mol % to about 95 mol %, from about 20 mol % to about 90 mol %, from about 30 mol % to about 90 mol %, from about 40 mol % to about 90 mol %, or from about 40 mol % to about 80 mol % of the total lipid present in the particle.

Additional lipidsThe RNA compositions and formulations and RNA particles described herein may also comprise lipids (including lipid-like materials) other than cationic or cationically ionizable lipids (also collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids. Optimizing the formulation of RNA particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to a cationic or cationically ionizable lipid may enhance particle stability and efficacy of RNA delivery.

One or more additional lipids may or may not affect the overall charge of the RNA particles. In some embodiments, the or more additional lipids are a non-cationic lipid or lipid-like material. The non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. As used herein, an "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, a "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.

In some embodiments, the RNA compositions and formulations and RNA particles described herein comprise a cationic or cationically ionizable lipid and one or more additional lipids.

Without wishing to be bound by theory, the amount of the cationic or cationically ionizable lipid compared to the amount of the one or more additional lipids may affect important RNA particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the RNA. Accordingly, in some embodiments, the molar ratio of the cationic or cationically ionizable lipid to the one or more additional lipids is from about 10:0 to about 1:9, about 4:1 to about 1:2, about 4:1 to about 1:1, about 3:1 to about 1:1, or about 3:1 to about 2:1.

In some embodiments, the one or more additional lipids comprised in the RNA compositions and formulations and RNA particles described herein comprise one or more of the following: neutral lipids, steroids, and combinations thereof.

In some embodiments, the one or more additional lipids comprise a neutral lipid which is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines,phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines,phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), 132 dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl- phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl- phosphatidylethanolamine (DPyPE), l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2- dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DPPG), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and further phosphatidylethanolamine lipids with different hydrophobic chains. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.

In some embodiments, the additional lipid comprises one of the following: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2‘- hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

Thus, in some embodiments, the RNA compositions and formulations and RNA particles described herein comprise (1) a cationic or cationically ionizable lipid, and a phospholipid such as DSPC or DOPE or (2) a cationic or cationically ionizable lipid and a phospholipid such as DSPC or DOPE and cholesterol.

In some embodiments, the RNA particles (especially the particles comprising mRNA) described herein comprise (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE or (4) DODMA, DOPE and cholesterol.

DSPC is a neutral phospholipid. The structure of DSPC may be represented as follows: DOPE is a neutral phospholipid. The structure of DOPE may be represented as follows: 133 The structure of cholesterol may be represented as follows: In some embodiments, the additional lipid (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 2 mol % to about 80 mol %, from about 5 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, from about 5 mol % to about mol %, from about 7.5 mol % to about 50 mol %, or from about 10 mol % to about 40 mol % of the total lipid present in the particle. In some embodiments, the additional lipid (e.g., one or more phospholipids and/or cholesterol) comprises about 10 mol %, about 15 mol %, or about 20 mol % of the total lipid present in the particle.

Polymer-conjugated HpidsIn some embodiments, RNA compositions and formulations and RNA particles described herein may comprise at least one polymer-conjugated lipid. A polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto. In some embodiments, a polymer-conjugated lipid is a PEG-conjugated lipid, also referred to herein as pegylated lipid or PEG-lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art. In some embodiments, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine portion.

In some embodiments, a polymer-conjugated lipid is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, a polymer-conjugated lipid can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

Polyethyleneglycol (PEG)-conjugated HpidsIn some embodiments, RNA compositions/formulations and RNA particles described herein comprise a PEG- conjugated lipid.

In some embodiments, the PEG-conjugated lipid (pegylated lipid) is a lipid having the structure of the following general formula: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: 134 each of R12 and R13 is each independently a straight or branched, alkyl or alkenyl chain containing from 10 to carbon atoms, wherein the alkyl/alkenyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

In some embodiments of this formula, each of R12 and R13 is independently a straight alkyl chain containing from to 18 carbon atoms, preferably from 12 to 16 carbon atoms.

In some embodiments of this formula, R12 and R13 are identical. In some embodiments, each of R12 and R13 is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R12 and R13 is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R12 and R13 is a straight alkyl chain containing carbon atoms.

In some embodiments of this formula, R12 and R13 are different. In some embodiments, one of R12 and R13 is a straight alkyl chain containing 12 carbon atoms and the other of R12 and R13 is a straight alkyl chain containing carbon atoms.

In some embodiments of this formula, w has a mean value ranging from 40 to 50, such as a mean value of 45.

In some embodiments of this formula, w is within a range such that the PEG portion of the pegylated lipid has an average molecular weight of from about 400 to about 6000 g/mol, such as from about 1000 to about 5000 g/mol, from about 1500 to about 4000 g/mol, or from about 2000 to about 3000 g/mol. In some embodiments, each of R12 and R13 is a straight alkyl chain containing 14 carbon atoms and w has a mean value of 45.

Various PEG-conjugated lipids are known in the art and include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-0-(2' ,di(tetradecanoyloxy)propyl-l-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as w-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(w methoxy(polyethoxy)ethyl)carbamate, and the like.

In some embodiments, the PEG-conjugated lipid (pegylated lipid) is or comprises 2-[(polyethylene glycol)-2000]- N,N-ditetradecylacetamide. In some embodiments, the pegylated lipid has the following structure:o/X Jx /X /X/X/X. /X^/x/'X pO " " //555555 In some embodiments, the PEG-conjugated lipid (pegylated lipid) is DMG-PEG 2000, e.g., having the following structure: 135 In some embodiments, the PEG-conjugated lipid (pegylated lipid) has the following structure: wherein n has a mean value ranging from 30 to 60, such as about 50. In one embodiment, the PEG-conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N-[(aj-methoxy polyethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol- 2,3-bis(tetradecyloxy)propylcarbamate (2000).

In some embodiments, RNA compositions/formulations described herein may comprise one or more PEG- conjugated lipids or pegylated lipids as described in WO 2017/075531 and WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

In some embodiments, the pegylated lipid comprises from about 1 mol % to about 10 mol %, preferably from about 1 mol % to about 5 mol %, more preferably from about 1 mol % to about 2.5 mol % of the total lipid present in the RNA compositions/formulations and RNA particles described herein.

Lipid nanoparticies (LNPs)In some embodiments, the RNA of the invention is present in the form of lipid nanoparticies (LNPs). LNPs typically comprise four components: cationically ionizable lipid, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer-conjugated lipid such as PEG-lipid. LNPs may be prepared by mixing lipids dissolved in ethanol with RNA in an aqueous buffer.

In some embodiments, the LNP comprises from 35 to 65 mol percent, 40 to 60 mol percent, 40 to 55 mol percent, from 45 to 55 mol percent, or from 45 to 50 mol percent of the cationically ionizable lipid.

In some embodiments, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to mol percent, or from 9 to 11 mol percent.

In some embodiments, the steroid is present in a concentration ranging from 30 to 50 mol percent, from 30 to mol percent, from 35 to 45 mol percent or from 35 to 43 mol percent.

In some embodiments, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer-conjugated lipid.

In some embodiments, the LNP comprises from 45 to 55 mol percent of a cationically ionizable lipid; from 5 to mol percent of a neutral lipid; from 30 to 45 mol percent of a steroid; from 1 to 5 mol percent of a polymer- conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.

In some embodiments, the mol percent is determined based on total mol of lipid present in the lipid nanoparticle. In some embodiments, the mol percent is determined based on total mol of cationically ionizable lipid, neutral lipid, steroid and polymer-conjugated lipid present in the lipid nanoparticle.

In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from 136 the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

In some embodiments, the steroid is cholesterol.

In some embodiments, the polymer conjugated lipid is a pegylated lipid, e.g., a pegylated lipid as described above.

In some embodiments, the cationically ionizable lipid component of the LNPs is selected from the group consisting of 3D-P-DMA, ALC-0366 and ALC-0315.

In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a pegylated lipid.

In some embodiments, RNA described herein is formulated in an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a pegylated lipid.

In some embodiments, RNA described herein is formulated in an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a pegylated lipid.

In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.

In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and PEG2000-C-DMA.

In some embodiments, RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and ALC-0159.

In some embodiments, RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and ALC-0159. 3D-P-DMA: (6Z,16Z)-12-((Z)-dec-4-en-l-yl)docosa-6,16-dien-ll-yl 5-(dimethylamino)pentanoate ALC-0366: ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate) ALC-0315: ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate) / 6-[N-6-(2- hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2-hexyldeca noate 137 DMG-PEG 2000: PEG2000-C-DMA: 3-N-[(a)-Methoxy poly(ethylene glycol)2000) carbamoyl]-l,2-dimyristyloxy-propylamine (MPEG- (2 kDa)-C-DMA or Methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000)) o wherein n has a mean value ranging from 30 to 60, such as about 50.

ALC-0159: 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide / 2-[2-(aj-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide DSPC: l,2-Distearoyl-sn-glycero-3-phosphocholine o Cholesterol: 138 The N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12,to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6.

DosesThe term "dose" as used herein in context of the RNA of the invention refers to a "dose amount" which relates to the amount of RNA administered per administration, i.e., per dosing.

In some embodiments, administration of RNA of the present disclosure may be performed by single administration or boosted by multiple administrations.

In some embodiments, an amount the RNA described herein from 0.1 pg to 300 pg, 0.5 pg to 200 pg, or 1 pg to 100 pg, such as about 1 pg, about 3 pg, about 10 pg, about 30 pg, about 50 pg, or about 100 pg may be administered per dose.

In some embodiments, a regimen described herein includes at least one dose. In some embodiments, a regimen includes a first dose and at least one subsequent dose. In some embodiments, a regimen includes a first dose and two subsequent doses. In some embodiments, the first dose is the same amount as at least one subsequent dose. In some embodiments, the first dose is the same amount as all subsequent doses. In some embodiments, the first dose is a different amount as at least one subsequent dose. In some embodiments, the first dose is a different amount than all subsequent doses. In some embodiments, a regimen comprises two doses. In some embodiments, a regimen consists of two doses. In some embodiments, a regimen comprises three doses. In some embodiments, a regimen consists of three doses.

In one embodiment, the disclosure envisions administration of a single dose. In one embodiment, the disclosure envisions administration of at least two consecutive doses.

Routes of administration of pharmaceutical compositions comprising an RNA of the inventionIn some embodiments, the pharmaceutical compositions comprising an RNA of the invention as described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, intramuscularly, intratumorally, or peritumorally. In some embodiments, the pharmaceutical compositions described herein may be administered intramuscularly. In some embodiments, the pharmaceutical composition comprising an RNA of the invention is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In some embodiments, the pharmaceutical compositions are formulated for systemic administration. In some embodiments, the systemic administration is by intravenous administration. In some embodiments, the pharmaceutical compositions are formulated for intrmuscular administration.

Viral vectors Furthermore, the nucleic acid, plasmid or RNA construct of the invention may be contained in a viral vector. Suitable viral vectors that may be employed in context of the present invention include, inter alia, an adeno-associated virus 139 (AAV) vector, a lentiviral vector, an Adenoviral vector, a Herpes-Simplex Virus vector, and a VSV vector. Thus, the invention relates, in some embodiments, to a viral vector comprising the nucleic acid of the invention, as described herein.

Host cells Furthermore, the nucleic acid of the invention, the RNA construct of the invention, the plasmid of the invention and/or the viral vector of the invention may be contained in a cell, in particular a host cell, e.g. a bacterial cell ora eukaryotic cell. Thus, the invention relates, in some embodiments, to a cell comprising the nucleic acid of the invention, as described herein. Said cell may be a bacterial cell or a eukaryotic cell. Preferably, said cell is a yeast cell such as Pichia pastorisceW, or a mammalian cell, more preferably a human cell.

As used herein, and in context of the present invention, Pichia pastoris also refers to Komagataeiia phaffii.

Pharmaceutical compositions and medical uses of the peptidoglycan hydrolase of the invention Preferably herein and in context of the present invention, the peptidoglycan hydrolase of the invention, the nucleic acid of the invention, the RNA construct of the invention, the plasmid of the invention, the viral vector of the invention and/or the cell (e.g. host cell) of the invention is contained in a pharmaceutical composition.

Thus, the present invention further relates, in some aspects, to a pharmaceutical composition comprising the peptidoglycan hydrolase of the invention.

Furthermore, the present invention relates, in some aspects, to a pharmaceutical composition comprising the nucleic acid of the invention. In particular, said nucleic acid encodes a peptidoglycan hydrolase of the invention, as described herein. In preferred embodiments, said nucleic acid is an RNA of the invention or a RNA construct of the invention, as described herein. As regards the pharmaceutical composition comprising a RNA of the invention or a RNA construct of the invention further details and embodiments are described herein above in context of the RNA.

Preferably, the pharmaceutical composition of the invention comprises, at least, one pharmaceutically acceptable excipient.

As used herein, the term "pharmaceutical composition" relates to a composition comprising a therapeutically effective agent (e.g the peptidoglycan hydrolase of the invention or the nucleic acid of the invention), preferably together with pharmaceutically acceptable excipients such as carriers, diluents or stabilizing agents. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease by administration of said pharmaceutical composition to a subject.

The pharmaceutical compositions of the present disclosure may be in a storable form (e.g., in a frozen or lyophilized/freeze-dried form) or in a "ready-to-use form" (i.e., in a form which can be immediately administered to a subject, e.g., without any processing such as diluting). Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready-to-use or administrable form. Eg, a frozen pharmaceutical composition has to be thawed, or a freeze-dried pharmaceutical composition has to be reconstituted, e.g. by using a suitable solvent (e.g, deionized water, such as water for injection) or liquid (e.g, an aqueous solution). 140 The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

The term "pharmaceutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In some embodiments relating to the treatment of a particular disease, the desired reaction may relate to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in some embodiments, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the pharmaceutical compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the pharmaceutical compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used. As regards the pharmaceutically effective amount of an RNA, suitable doses are further described herein above in context of the RNA.

The pharmaceutical compositions of the present disclosure may contain buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable excipients such as carriers or diluents.

Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.

The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants The term "diluent" relates a diluting and/or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water.

The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carrier include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy- propylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure includes isotonic saline. 141 Pharmaceutically acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

Pharmaceutical excipients, e.g., carriers or diluents, can be selected with regard to the intended route of administration and standard pharmaceutical practice.

In some embodiments, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, or intramuscularly. In some embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In some embodiments, the pharmaceutical compositions are formulated for systemic administration. In some embodiments, the systemic administration is by intravenous administration. In some embodiments, the pharmaceutical compositions are formulated for respiratory/pulmonary administration route and/or administered by respiratory/pulmonary administration route, e.g., by inhalation. In this manner, the peptidoglycan hydrolase or nucleic acid (e.g. RNA) described herein may be locally/regionally or systemically delivered to lungs and/or the respiratory tract. The lungs may also be used as a portal of entry to the body, enabling delivery of the RNA via the airways into the bloodstream. Thus, inhaled formulations may be used for systemic delivery.

Furthermore, the pharmaceutical composition may be formulated, for example, inter alia, as a solution, a suspension, an ointment, a pill such as a tablet or a capsule, a powder, a gel, a foam, a spray, an aerosol, or a suppository.

As described herein, the pharmaceutical composition of the invention is, in particular, used for treating a disease, i.e., a disease in a subject, as described herein.

As used herein, the term "treatment" (and grammatical variations thereof such as "treat" or "treating") further refers to clinical intervention in an attempt to alter the natural course of the individual being treated. Desirable effects of treatment include, but are not limited to, prophylaxis, preventing occurrence or recurrence of disease or symptoms associated with disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, improved prognosis and cure.

Herein and in context of the invention, the subject, e.g. a subject to be treated, a subject having a disease or a subject suspected of having a disease, preferably, a mammal, more preferably a human.

A mammal, as used herein and in context of the present invention or grammatical versions thereof such as "mammalian" (e.g. mammalian cells), may refer, to any mammalian species including (but not limited to) humans, livestock such as cows, pets such as dogs, sports animals such as horses, endangered animals or zoo animals such as tigers, or laboratory animals such as mice. For example, a mammal may be, inter alia, a human, a cow (e.g. cattle), a horse, a pig, a sheep, a goat, a camel, a yak, a monkey, a dog, a cat, a hamster, a tiger, a polar bear, a mouse, a rat etc. Furthermore, the peptidoglycan hydrolase of the invention has, preferably, the ability of being efficiently secreted from a cell of the mammal that is treated. 142 Preferably, the pharmaceutical composition of the invention is used for treating a disease caused by and/or associated with a Staphylococcus infection and/or a subject that has or is suspected of having a Staphylococcus infection. More preferably, the pharmaceutical composition of the invention is used for treating a disease caused by and/or associated with a Staphylococcus aureus infection and/or a subject that has or is suspected of having a Staphylococcus aureus infection.

Accordingly, the peptidoglycan hydrolase of the invention, is, preferably, used for treating a disease caused by and/or associated with a Staphylococcus (preferably, S. aureus) infection and/or a subject that has or is suspected of having a Staphylococcus (preferably, S. aureus) infection.

Similarly, RNA or RNA construct of the invention is, preferably, used for treating a disease caused by and/or associated with a Staphylococcus (preferably, S. aureus) infection and/or a subject that has or is suspected of having a Staphylococcus (preferably, S. aureus) infection.

Herein and in context of the invention, a Staphylococcus aureus infection may be an infection of a skin, soft tissue, bone, lung, sinus and/or urinary tract.

In particular, the pharmaceutical composition of the invention, the peptidoglycan hydrolase of the invention, the nucleic acid of the invention (in particular the RNA or RNA construct of the invention) may be used for treating a bacterial disease, i.e., a disease that is associated with and/or caused by a bacterial infection, preferably an infection with a Staphylococcus species or strain such as Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Staphylococcus warned (S. warned), Staphylococcus capitis (S. capitis), and/or Staphylococcus simuians(S. simulans), more an infection with 5. aureus, as described herein. Furthermore, the bacterial infection may be an infection with a coagulate-negative Staphylococcus species or strain such as S. epidermidis.

Furthermore, the pharmaceutical composition of the invention, the peptidoglycan hydrolase of the invention, the nucleic acid of the invention (in particular the RNA or RNA construct of the invention) may be used for treating a disease selected from the group consisting of: pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, sepsis, a respiratory infection such as sinusitis, pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, abscesses, food poisoning, necrotizing fasciitis, pyomyositis, mediastinitis, infected dermatitis, wound infection, diabetic foot ulcer, septic arthritis, osteoarticular infections, prosthetic infection such as infection of a prosthetic joint or a cardiac device, and urinary tract infections. In particular, said disease is caused by and/or associated with an infection with a Staphylococcus species or strain (preferably S. aureus), as described herein.

Preferably, the pharmaceutical composition of the invention, the peptidoglycan hydrolase of the invention, the nucleic acid of the invention (in particular the RNA or RNA construct of the invention) is used for treating pneumonia, bacteremia, endocarditis, and/or a prosthetic infection.

Furthermore, the pharmaceutical composition of the invention, the peptidoglycan hydrolase of the invention, the nucleic acid of the invention (in particular the RNA or RNA construct of the invention) is particularly useful for treating a bacterial biofilm or free-floating (biofilm-like) aggregate of bacteria; see, e.g., Example 6 and Figure 12.

A biofilm is defined as an aggregate or aggregate of microorganisms (in particular bacteria) attached to a surface. Adherent bacteria are often surrounded and protected by extracellular polymeric substances produced by Gram 143 negative and Gram-positive bacteria. Bacteria are more resistant to antibacterials (in particular antibiotics) through biofilms.

A free-floating aggregate is defined as aggregate of microorganisms (in particular bacteria) that forms (or formed) in suspension, for example, in a synovial fluid during joint infections (e.g., prosthetic joint infections). In the art, such free-floating aggregates are sometimes also denoted as biofilms. The properties of free-floating aggregates are similar to an attached biofilm, as they are more resistant to antibiotics and may secrete different virulence factors. Herein, free-floating aggregates are considered as "biofilm-like" and the same applies to free-floating aggregates as described herein in context of biofilms mutatis mutandis.

As illustrated in Example 6 and Figure 12, peptidoglycan hydrolases of the invention such as H5 (SEQ ID NO: 11) or G1 (SEQ ID NO: 3) may effectively kill biofilms or free-floating aggregates of S. aureus.

Thus, the pharmaceutical composition of the invention, the peptidoglycan hydrolase of the invention, the nucleic acid of the invention (in particular the RNA or RNA construct of the invention) may be used for treating a bacterial disease that is associated with or suspected of forming a biofilm and/or a free-floating aggregate. Preferably, the pharmaceutical composition of the invention, the peptidoglycan hydrolase of the invention, the nucleic acid of the invention (in particular the RNA or RNA construct of the invention) is used for treating a disease caused by and/or associated with a Staphylococcus (preferably, S. aureus) infection and/or a subject that has or is suspected of having a Staphylococcus{ preferably, S. aureus} infection, wherein said Staphylococcus{ preferably Staphylococcus aureus), is present in form of a biofilm and/or a free-floating aggregate or is suspected of forming a biofilm and/or a free-floating aggregate. The biofilm may be present at a site of a Staphylococcus infection, for example, at an endocardium in an endocarditis. The free-floating aggregate may be present in a synovial fluid in a joint infection, e.g., a prosthetic joint infection.

Furthermore, the present invention relates, in some aspects, to a method of treating a disease (e.g. a disease caused by and/or associated with a Staphylococcus infection), as described herein, wherein said method comprises administering an effective amount of the pharmaceutical composition of the invention, the peptidoglycan hydrolase of the invention, or the nucleic acid of the invention (in particular the RNA or RNA construct of the invention) to a subject in need.

Further uses of the peptidoglycan hydrolase of the invention Furthermore, the peptidoglycan hydrolase of the invention may be used for sterilizing a device, preferably a medical device such as a catheter, a pacemaker or a prosthetic joint, in vitro or in vivo. In particular, the peptidoglycan hydrolase may be used to kill or eliminate one or more Staphylococcus species or strains, e.g., S. aureus, on and/or in such a device. Further reference in this respect is made to Choi (2021), Front Microbiol. 12.

Means and methods for screening peptidoglycan hydrolases for bactericidal activity Solidified veast culture media As described herein and as illustrated in the appended Examples, the inventors found solidified yeast culture media dead target bacteria, e.g. dead S. aureus zeWs, which can be advantageously used for screening yeast cells for the secretion of an active peptidoglycan hydrolase, i.e., for identifying peptidoglycan hydrolases with a good killing activity against target bacteria, e.g., S. aureus. 144 Thus, as mentioned herein above, the present invention relates further, in some aspects, to a solidified yeast culture medium comprising substrate particles intermixed with said medium, wherein said substrate particles comprise dead bacterial cells and/or fragments thereof, and/or peptidoglycan particles. Preferably, said substrate particles comprise or essentially consist of dead bacterial cells, as described herein. In particular, said substrate particles have a size which renders the medium turbid, at least when they are used at a suitable concentration as described herein. Thus, the solidified yeast culture medium of the invention is, preferably, turbid.

Preferably, the solidified yeast culture medium of the invention contains dead bacterial cells at an optical density at 620 nm (OD620) of about 1 to 10, preferably about 2 to 4. Such a concentration normally renders the solidified yeast culture medium turbid.

As described herein, and as illustrated in the appended Examples, the solidified yeast culture medium of the invention is particularly useful for screening yeast cells for the secretion of an active peptidoglycan hydrolase, and thus is, preferably, used in such a method.

Preferably, in context of the solidified yeast culture medium of the invention, said culture medium comprises agar, preferably at a concentration of about 0.5% to about 5%, more preferably at a concentration of about 1% to about 2%, e.g., at a concentration of about 1.5%, in particular wherein the % is % weight by volume (w/v).

In particular, in context of the solidified yeast culture medium of the invention, the opacity of the solidified medium is reduced at a location where the substrate particles are lysed or broke down (e.g. because of the secretion of an active peptidoglycan hydrolase from a yeast colony cultured on said solidified yeast culture medium).

Herein, and in context of the present invention, the yeast may be, for example, Pichia pastoris or Saccharomyces (e.g., Saccharomyces cerevisiae). Preferably, herein and in context of the present invention, the yeast is Pichia pastoris.

Furthermore, in context of the solidified yeast culture medium of the invention, the dead bacterial cells are, preferably, dead gram-positive bacterial cells and/or the peptidoglycan in the peptidoglycan particles is, preferably, from gram-positive bacteria. Furthermore, the dead bacterial cells are, preferably, autoclaved bacterial cells. Furthermore, the dead bacterial cells are, preferably, dead Staphylococcus cells and/or the peptidoglycan in the peptidoglycan particles is from a Staphylococcus species or strain. More preferably, the dead bacterial cells are dead Staphylococcus aureus cells and/or the peptidoglycan in the peptidoglycan particles is from Staphylococcus aureus. In particularly preferred embodiments relating to the solidified yeast culture medium of the invention, the substrate particles comprise or essentially consist of autoclaved Staphylococcus aureus cells.

Yeast culture Furthermore, the solidified yeast culture medium of the invention may be used for (or be a part of) a yeast culture, as described herein. In particular, the solidified yeast culture medium of the invention comprises at least one surface for culturing yeast cells on said surface.

Thus, in some embodiments, the invention relates to a yeast culture comprising (i) the solidified yeast culture medium of the invention, and (ii) yeast cells (e.g., at least one yeast colony), on the solidified yeast culture medium, i.e., on a surface of said solidified medium. 145 Furthermore, a yeast cell or yeast colony that is cultured on the solidified yeast culture medium of the invention or that intended for this purpose, preferably, expresses and/or secretes a peptidoglycan hydrolase, as described herein. Preferably, said peptidoglycan hydrolase is an endolysin, as described herein. Preferably said peptidoglycan hydrolase (e.g. said endolysin) has or is suspected to have a killing activity against a gram-positive bacterium, preferably a Staphylococcus species or strain, more preferably Staphylococcus aureus. Furthermore, the peptidoglycan hydrolase may be considered as active when it is able to lyse and/or break down the substrate particles (in particular the peptidoglycan therein) in the solidified yeast culture medium, as described herein. As described herein, and as illustrated in the appended Examples, lysing and/or breaking down the substrate particles renders the solidified yeast culture medium locally translucent or, at least, less opaque.

Method of screening yeast cells for the secretion of an active peptidoglycan hydrolase, i.e., YODA-derived methods.

As described herein, and as illustrated in the appended Examples, yeast on dead aureus (YODA)-derived methods are extremely simple and efficient methods which allows to easily distinguish yeast cells/colonies expressing peptidoglycan hydrolases with a good killing activity against a target bacterium from yeast cells/colonies expressing inactive peptidoglycan hydrolases. Furthermore, YODA-derived methods are very sensitive since the lysins are constantly secreted from the cells.

Hence, as already mentioned above, the present invention relates, in some aspects, to a YODA-derived method, i.e., a method of screening yeast cells for the secretion of an active peptidoglycan hydrolase, as described herein, said method comprising the steps of: a) providing a solidified yeast culture medium of the invention, i.e., a solidified yeast culture medium comprising substrate particles intermixed with said medium, wherein said substrate particles comprise dead bacterial cells and/or fragments thereof, and/or peptidoglycan particles, as described herein; b) culturing yeast cells expressing a peptidoglycan hydrolase on a surface of said solidified medium until at least one yeast colony is detectable, in particular wherein the yeast cells are able to secrete the peptidoglycan hydrolase; c) evaluating whether a halo is apparent around a yeast colony; d) determining that a yeast colony secretes an active peptidoglycan hydrolase when a halo around the colony is apparent, or determining that a yeast colony does not secrete an active peptidoglycan hydrolase when no halo around the colony is apparent.

In particular, a halo, as used herein and in context of the present invention, corresponds to a locally reduced optical density of the solidified medium around a colony, for example, in a radius of about 0.1 to 1 cm from the colony, especially compared to a region of the solidified medium that is free of yeast colonies.

Furthermore, in particular in context of the yeast culture or the method of screening yeast cells for the secretion of an active peptidoglycan hydrolase according to the invention, an active peptidoglycan hydrolase may be considered to have a killing activity against a live bacterium comprising a peptidoglycan in its cell wall which corresponds to the peptidoglycan comprised in the substrate particles in the solidified yeast culture medium of the invention. For example, a peptidoglycan hydrolase secreted from a yeast colony which is able to lyse and/or break down dead 146 Staphylococcus aureus in the solidified yeast culture medium may be considered as an active peptidoglycan hydrolase that has killing activity against live Staphylococcus aureus, as described herein.

Methods for screening peptidoglycan hydrolases having several improved pharmaceutical properties and directed evolution As described herein and as illustrated in the appended Examples, the inventors found a combinatorial screening method which is based on the YODA-derived method of invention and which allows to simultaneously improve, inter alia, the solubility, bactericidal activity and eukaryotic secretion of peptidoglycan hydrolases, e.g., endolysins such as LO482. In particular, said method allows to screen for peptidoglycan hydrolase variants which are adapted to the eukaryotic secretory pathway, and, hence, have an improved expression and secretion profile in eukaryotic cells. Moreover, the combinatorial screening method may be advantageously used for directed evolution of peptidoglycan hydrolases, e.g., endolysins, as described herein.

Thus, in some embodiments, the invention further relates to a combinatorial screening method, i.e., a method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell, said method comprising the steps of: 1) preparing a library of eukaryotic cells expressing peptidoglycan hydrolase variants on the cell surface; II) selecting eukaryotic cells based on a high level of peptidoglycan hydrolase on the cell surface relative to other cells in the library; for example, selecting the 10% of cells in the library with the highest peptidoglycan level on the cell surface; III) performing the method of screening yeast cells for the secretion of an active peptidoglycan hydrolase of the invention, wherein yeast cells that are able to secrete the peptidoglycan hydrolase variants expressed in the eukaryotic cells, selected in step II) are cultured in step b) of said method of screening yeast cells for the secretion of an active peptidoglycan hydrolase; and IV) determining that a yeast colony that has been determined in step d) of said method of screening yeast cells for the secretion of an active peptidoglycan hydrolase to secrete an active peptidoglycan hydrolase produces an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell, as described herein.

In context of said method, the eukaryotic cells are, preferably, yeast cells. However, other eukaryotic cells, e.g. mammalian cells such as human cells, may be also used.

Furthermore, the method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell comprises, preferably, between said steps II) and III) the steps of: IT) isolating DNA encoding at least one peptidoglycan hydrolase variant from the eukaryotic cells, e.g. the yeast cells, selected in step II); and II") introducing the isolated DNA into yeast cells, e.g., by means of transformation. In particular, the isolated DNA is introduced into the yeast cells in a way that the yeast cells are able to secrete the peptidoglycan hydrolase variants, e.g., by employing a suitable expression vector. 147 Furthermore, step I) of the method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell comprises, preferably, comprises (i) fragmenting and reassembling DNA encoding different peptidoglycan hydrolases, preferably endolysins, e.g. endolysin homologues (e.g. by using DNA shuffling), thereby generating a DNA library of peptidoglycan hydrolase variants, and (ii) introducing said DNA library into eukaryotic cells, e.g. by means of transformation, transfection or transduction, thereby generating the library of eukaryotic cellsexpressing peptidoglycan hydrolase variants.

Furthermore, the method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell comprises, preferably, before said step II) a step 1ל of attaching a detectable label to the peptidoglycan hydrolase variants on the cell surface. Preferably, said detectable label is a fluorescent dye or a magnetic particle, preferably a fluorescent dye. Furthermore, the detectable label, is preferably attached by means of immunostaining. In other words, a labeled antibody (preferably a fluorescently labeled antibody) may be used to visualize and determine the level of the peptidoglycan hydrolase variants on the cell surfaces. Furthermore, the peptidoglycan hydrolase variant may comprise a tag to which the antibody binds specifically. Suitable tags, antibodies and labels are well known in the art and any of these may be employed in context of the present invention.

Furthermore, step II) of the method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell, preferably, comprises, sorting (i.e. separating) cells with a high level of peptidoglycan hydrolase on the cell surface relative to other cells in the library from these other cells, e.g., by means of fluorescent activated cell sorting (FACS) or magnetic activated cell sorting (MACS). In particular, FACS is used in case a fluorescent dye is used for labeling the peptidoglycan hydrolase variants, and MACS is used in case a magnetic particle is used for labeling the peptidoglycan hydrolase variants.

Moreover, the combinatorial screening method of the invention, i.e., the inventive method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell, can be used for directed evolution, e.g., when it is repeatedly employed.

Thus, in context of the inventive method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell and/or in context of directed evolution, preferably at least two (e.g. 2, 3, 4 or 5) rounds of steps I) to IV) of said combinatorial screening method are performed. In particular, in step I) of each subsequent round, a further library of eukaryotic cells is prepared, wherein the cells in the library express a different set of peptidoglycan variants compared to the library employed in the preceding round(s). In particular, step I) of a subsequent round comprises, preferably, (i) fragmenting and reassembling DNA encoding different peptidoglycan hydrolases (in particular "DNA shuffling"), wherein at least one, preferably at least 50%, of said peptidoglycan hydrolases has been identified in a preceding round as an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell, thereby generating a further DNA library of peptidoglycan hydrolase variants, and (ii) introducing said further DNA library into eukaryotic cells (e.g. yeast cells), thereby generating a further library of eukaryotic cells (e.g. yeast cells) expressing a different set of peptidoglycan hydrolase variants compared to the library employed in the preceding round(s). Performing at least two rounds of steps I) to IV) of the method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell, as just described, further refers to "directed evolution", as described herein, and as illustrated in the appended Examples. 148 Furthermore, the inventive method of screening yeast cells for the secretion of an active peptidoglycan hydrolase may further comprise a step of obtaining from a yeast colony an active peptidoglycan hydrolase or a nucleic acid encoding an active peptidoglycan hydrolase, as described herein.

Similarly, the inventive method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell may further comprise a step of obtaining from a yeast colony an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell or a nucleic acid encoding an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell, as described herein.

Thus, the present invention further relates, in some aspects, to a peptidoglycan hydrolase or peptidoglycan hydrolase variant that is obtained or obtainable by a method of the invention.

Similarly, the present invention further relates, in some aspects, to a nucleic acid encoding a peptidoglycan hydrolase or peptidoglycan hydrolase variant of the invention, wherein said nucleic acid and/or said peptidoglycan hydrolase or peptidoglycan hydrolase variant is obtained or obtainable by a method of the invention.

Moreover, any peptidoglycan hydrolase of the invention may be obtainable by a method of the invention, as described herein. Similarly, any nucleic acid of the invention may be obtainable by a method of the invention, as described herein.

Items according to the invention The present invention further relates to the following items: 1. A peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain that has (i) a sequence identity of at least 60% to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions as compared to the amino acid sequence from position to position 215 in SEQ ID NO: 1. 2. The peptidoglycan hydrolase of item 1 which has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. 3. The peptidoglycan hydrolase of item 1 or 2, wherein(i) a corresponding segment of said CHAP domain has a sequency identity of at least 80%, preferably at least 90%, to the sequence from position 87 to position 128 in SEQ ID NO: 1, and/or(ii) said CHAP domain has at most six, five, four, three or two, preferably at most one, more preferably no amino acid substitutions or deletions at positions 80, 87, 88,98,99,103,106,110, 114, 122, 126, 128, 137, 182, 202, and 208 of SEQ ID NO: 1 or at positions corresponding to these positions. 4. The peptidoglycan hydrolase of any one of items 1 to 3, wherein said CHAP domain has an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position. 149 . The peptidoglycan hydrolase of item 4, wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than phenylalanine or lysine; and, preferably, wherein said peptidoglycan hydrolase does not have a sequence as shown in any one of SEQ ID NO: 276 to 278. 6. The peptidoglycan hydrolase of item 4 or 5, wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine. 7. The peptidoglycan hydrolase of any one of items 4 to 6, wherein the residue at position 73 in SEQ ID NO: or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, or histidine. 8. The peptidoglycan hydrolase of any one of items 4 to 7, wherein the residue at position 73 in SEQ ID NO: or at a position corresponding to this position is substituted with glycine. 9. The peptidoglycan hydrolase of any one of items 2 to 8, wherein the peptidoglycan hydrolase has (i) a sequence identity of at least 60% to the sequence of SEQ ID NO: 1 and (ii) an amino acid substitution at position 68 in SEQ ID NO: 1 or at a position corresponding to this position.

. The peptidoglycan hydrolase of item 9, wherein the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than threonine or serine. 11. The peptidoglycan hydrolase of item 9 or 10, wherein the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine, arginine or alanine. 12. The peptidoglycan hydrolase of any one of items 9 to 11, wherein the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine. 13. The peptidoglycan hydrolase of item 2 or 3, wherein the peptidoglycan hydrolase has (i) a sequence identity of at least 60% to the sequence of SEQ ID NO: 1 and (ii) a pair of amino acid substitutions at positions 68 and 73 in SEQ ID NO: 1 or at positions corresponding to these positions, whereina) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, serine, tyrosine or leucine,b) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with methionine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine,c) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, or 150 d) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or histidine. 14. The peptidoglycan hydrolase of any one of items 2, 3 or 13, wherein the peptidoglycan hydrolase has (i) a sequence identity of at least 60% to the sequence of SEQ ID NO: 1 and (ii) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

. The peptidoglycan hydrolase of any one of items 1 to 14, wherein said CHAP domain has one or more amino acid substitutions at positions 72 to 83, 85, 86, 93, 96, 99,102 to 104,107, 108, 111, 113 to 115, 117, 121 to 125, 129 to 131,133 to 145, 148, 149, 152, 153, 155, 157, 159, 166, 169, 170, 173 to 178, 185, 186, 190 to 194, 196 to 199, 201, 203 to 207, and 212 to 215 in SEQ ID NO: 1, or at positions corresponding to these positions. 16. The peptidoglycan hydrolase of item 15, whereinthe amino acid residue at position 72 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, aspartic acid, histidine or threonine,the amino acid residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine, serine, aspartic acid or threonine,the amino acid residue at position 74 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or proline,the amino acid residue at position 75 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, proline, aspartic acid or glutamine,the amino acid residue at position 76 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, aspartic acid or asparagine,the amino acid residue at position 77 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, aspartic acid, glycine, histidine, lysin, asparagine, glutamine, serine, or threonine, the amino acid residue at position 78 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or tyrosine,the amino acid residue at position 79 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine or tryptophan,the amino acid residue at position 80 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, aspartic acid, methionine or asparagine,the amino acid residue at position 81 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, arginine, serine, alanine or glycine,the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or glycine,the amino acid residue at position 83 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, 151 the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position issubstituted with glycine,the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine or tyrosine, the amino acid residue at position 93 in SEQ ID NO: 1 or at a substituted with alanine or leucine,the amino acid residue at position 96 in SEQ ID NO: 1 or at a substituted with asparagine,the amino acid residue at position 99 in SEQ ID NO: 1 or at a position corresponding to this position is position corresponding to this position is position corresponding to this position issubstituted with lysine, methionine, glutamine, threonine or valine,the amino acid residue at position 102 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, glycine, serine or threonine,the amino acid residue at position 103 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or glycine,the amino acid residue at position 104 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, aspartic acid, methionine, glutamine or tyrosine,the amino acid residue at position 107 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, tyrosine or alanine,the amino acid residue at position 108 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, glycine, lysine or tyrosine,the amino acid residue at position ill in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine,the amino acid residue at position 113 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine,the amino acid residue at position 114 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine,the amino acid residue at position 115 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, aspartic acid, glycine or serine,the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, aspartic acid or asparagine,the amino acid residue at position 121 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine,the amino acid residue at position 122 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine,the amino acid residue at position 123 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, serine, threonine or tryptophan,the amino acid residue at position 124 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, histidine, glutamine or threonine,the amino acid residue at position 125 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 129 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or serine, 152 the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, isoleucine, asparagine or tyrosine,the amino acid residue at position 131 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, glutamine or tyrosine,the amino acid residue at position 133 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, threonine or glutamine,the amino acid residue at position 134 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, valine, lysine or glutamine,the amino acid residue at position 135 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine, histidine or serine,the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, arginine, glutamic acid or threonine,the amino acid residue at position 137 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine,the amino acid residue at position 138 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine,the amino acid residue at position 139 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, aspartic acid, glutamine or serine,the amino acid residue at position 140 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or aspartic acid,the amino acid residue at position 141 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, aspartic acid, histidine or threonine,the amino acid residue at position 142 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine,the amino acid residue at position 143 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with proline,the amino acid residue at position 144 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glutamine,the amino acid residue at position 145 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or leucine,the amino acid residue at position 148 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine or valine,the amino acid residue at position 149 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or threonine,the amino acid residue at position 152 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine,the amino acid residue at position 153 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or serine,the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine,the amino acid residue at position 157 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, 153 the amino acid residue at position 159 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine,the amino acid residue at position 166 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, glutamic acid or threonine,the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or aspartic acid,the amino acid residue at position 170 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, histidine or serine,the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, asparagine or arginine,the amino acid residue at position 174 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, histidine, lysine, arginine, serine or threonine,the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, glutamine or threonine,the amino acid residue at position 176 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, arginine, alanine, glutamine, serine or threonine,the amino acid residue at position 177 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine,the amino acid residue at position 178 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, arginine, serine or threonine,the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, histidine, serine, tyrosine or aspartic acid or asparagine,the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine,the amino acid residue at position 190 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, alanine, cysteine, aspartic acid, histidine, asparagine, serine or threonine, the amino acid residue at position 191 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, arginine, aspartic acid or asparagine,the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, threonine, glutamic acid, glycine or lysine,the amino acid residue at position 193 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid,the amino acid residue at position 194 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, glycine, proline or threonine,the amino acid residue at position 196 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine,the amino acid residue at position 197 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or proline,the amino acid residue at position 198 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine,the amino acid residue at position 199 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, asparagine, serine or threonine, 154 the amino acid residue at position 201 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, serine or lysine,the amino acid residue at position 203 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or valine,the amino acid residue at position 204 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, serine or tyrosine,the amino acid residue at position 205 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or valine,the amino acid residue at position 206 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine,the amino acid residue at position 207 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, valine, histidine or tryptophan,the amino acid residue at position 212 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, aspartic acid, glutamic acid, histidine, lysin or asparagine,the amino acid residue at position 213 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, tyrosine or threonine.the amino acid residue at position 214 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine, and/orthe amino acid residue at position 215 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, valine or serine. 17. The peptidoglycan hydrolase of any one of items 1 to 16, wherein said CHAP domain has one or more amino acid substitutions at positions 72 to 76, 78, 81, 82, 85, 86, 93, 96, 104, 107, 108, 111, 113, 115, 117, 121, 124, 125, 129, 130, 133 to 136, 138, 140 to 142, 144, 145, 148, 149, 152, 153, 155, 157, 159, 169, 173, 175 to 178,185, 186, 190 to 194, 197, 198,199, 201, 203, 204, 207, and 212 to 215 in SEQ ID NO: 1, or at positions corresponding to these positions. 18. The peptidoglycan hydrolase of item 17, whereinthe amino acid residue at position 72 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine,the amino acid residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine or serine,the amino acid residue at position 74 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or proline,the amino acid residue at position 75 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine or proline,the amino acid residue at position 76 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine,the amino acid residue at position 78 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine or tyrosine, 155 the amino acid residue at position 81 in SEQ ID NO: substituted with glutamic acid, arginine or serine,or at a position corresponding to this position is the amino acid residue at position 82 in substituted with serine,the amino acid residue at position 85 in substituted with glycine,the amino acid residue at position 86 in substituted with lysine or methionine, the amino acid residue at position 93 in substituted with alanine or leucine, the amino acid residue at position 96 in SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: or at a or at a or at a or at a or at a position corresponding to this position position corresponding to this position position corresponding to this position position corresponding to this position position corresponding to this position is is is is issubstituted with asparagine, the amino acid residue at position substituted with isoleucine, the amino acid residue at position substituted with serine or tyrosine, the amino acid residue at position substituted with arginine, the amino acid residue at position substituted with serine, the amino acid residue at position substituted with serine, the amino acid residue at position substituted with arginine, the amino acid residue at position substituted with histidine, the amino acid residue at position substituted with valine, the amino acid residue at position 104 in SEQ ID NO: 1 or at a 107 in SEQ ID NO: 1 or at a 108 in SEQ ID NO: 1 or at a ill in SEQ ID NO: 1 or at a 113 in SEQ ID NO: 1 or at a 115 in SEQ ID NO: 1 or at a 117 in SEQ ID NO: 1 or at a 121 in SEQ ID NO: 1 or at a 124 in SEQ ID NO: 1 or at asubstituted with phenylalanine or histidine,the amino acid residue at position 125 in SEQ ID NO: 1 or at a substituted with valine,the amino acid residue at position 129 in SEQ ID NO: 1 or at a substituted with arginine or serine,the amino acid residue at position 130 in SEQ ID NO: 1 or at a substituted with glycine, isoleucine or asparagine,the amino acid residue at position 133 in SEQ ID NO: 1 or at a substituted with arginine or threonine,the amino acid residue at position 134 in SEQ ID NO: 1 or at a substituted with threonine or valine,the amino acid residue at position 135 in SEQ ID NO: 1 or at a substituted with cysteine or histidine, position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is 156 the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine,the amino acid residue at position 138 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine,the amino acid residue at position 140 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or aspartic acid,the amino acid residue at position 141 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine,the amino acid residue at position 142 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine,the amino acid residue at position 144 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid,the amino acid residue at position 145 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or leucine,the amino acid residue at position 148 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine or valine,the amino acid residue at position 149 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or threonine,the amino acid residue at position 152 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine,the amino acid residue at position 153 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine or serine,the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine,the amino acid residue at position 157 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid,the amino acid residue at position 159 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine,the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine,the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, asparagine or arginine,the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, glutamine or threonine,the amino acid residue at position 176 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine,the amino acid residue at position 177 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine,the amino acid residue at position 178 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine or arginine,the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, histidine, serine or tyrosine, 157 the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine,the amino acid residue at position 190 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine,the amino acid residue at position 191 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid or arginine,the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine,the amino acid residue at position 193 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid,the amino acid residue at position 194 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine,the amino acid residue at position 197 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine,the amino acid residue at position 198 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine,the amino acid residue at position 199 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine,the amino acid residue at position 201 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or serine,the amino acid residue at position 203 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or valine,the amino acid residue at position 204 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, serine or tyrosine,the amino acid residue at position 207 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine or valine,the amino acid residue at position 212 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine,the amino acid residue at position 213 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine or threonine.the amino acid residue at position 214 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine or threonine, and/orthe amino acid residue at position 215 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, valine or serine. 19. The peptidoglycan hydrolase of any one of items 1 to 18, wherein said CHAP domain has one or more amino acid substitutions at positions 73, 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191, 192, 194, 198, 204, 212 and 215 in SEQ ID NO: or at positions corresponding to these positions.

. The peptidoglycan hydrolase of item 19, wherein 158 the amino acid residue at position 73 in SEQ ID NO: substituted with glycine or serine,the amino acid residue at position 75 in SEQ ID NO: substituted with phenylalanine,the amino acid residue at position 78 in SEQ ID NO: substituted with tyrosine,the amino acid residue at position 81 in SEQ ID NO: substituted with glutamic acid,the amino acid residue at position 82 in SEQ ID NO: substituted with serine,the amino acid residue at position 85 in SEQ ID NO: substituted with glycine,the amino acid residue at position 86 in SEQ ID NO: substituted with lysine, the amino acid residue at position substituted with isoleucine, the amino acid residue at position substituted with tyrosine, the amino acid residue at position substituted with arginine, the amino acid residue at position substituted with phenylalanine, the amino acid residue at position substituted with valine, the amino acid residue at position substituted with asparagine, the amino acid residue at position substituted with arginine, the amino acid residue at position substituted with histidine, the amino acid residue at position substituted with lysine or arginine, the amino acid residue at position substituted with alanine, the amino acid residue at position substituted with leucine, the amino acid residue at position substituted with tyrosine, the amino acid residue at position substituted with asparagine, the amino acid residue at position substituted with asparagine, 1 or at a position corresponding to this position is 1 or at a position corresponding to this position is 1 or at a position corresponding to this position is 1 or at a position corresponding to this position is 1 or at a position corresponding to this position is 1 or at a position corresponding to this position is 1 or at a position corresponding to this position is 104 in SEQ ID NO: 1 or at a position corresponding to this position is 107 in SEQ ID NO: 1 or at a position corresponding to this position is 115 in SEQ ID NO: 1 or at a position corresponding to this position is 124 in SEQ ID NO: 1 or at a position corresponding to this position is 125 in SEQ ID NO: 1 or at a position corresponding to this position is 130 in SEQ ID NO: 1 or at a position corresponding to this position is 133 in SEQ ID NO: 1 or at a position corresponding to this position is 135 in SEQ ID NO: 1 or at a position corresponding to this position is 136 in SEQ ID NO: 1 or at a position corresponding to this position is 140 in SEQ ID NO: 1 or at a position corresponding to this position is 141 in SEQ ID NO: 1 or at a position corresponding to this position is 155 in SEQ ID NO: 1 or at a position corresponding to this position is 169 in SEQ ID NO: 1 or at a position corresponding to this position is 173 in SEQ ID NO: 1 or at a position corresponding to this position is 159 the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine,the amino acid residue at position 178 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine,the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine,the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine,the amino acid residue at position 191 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine,the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine,the amino acid residue at position 194 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine,the amino acid residue at position 198 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine,the amino acid residue at position 204 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine,the amino acid residue at position 212 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/orthe amino acid residue at position 215 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine. 21. 22.

The peptidoglycan hydrolase of any one of items 1 to 20, wherein said CHAP domain has one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions.

The peptidoglycan hydrolase of item 21, whereinthe amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine,the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine,the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine,the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine,the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine,the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, 160 the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine,the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/orthe amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; and preferably wherein said peptidoglycan hydrolase does not have a sequence as shown in any one of SEQ ID NO: 279 to 293. 23. The peptidoglycan hydrolase of any one of items 1 to 22, wherein said CHAP domain has at least one pair of amino acid substitutionsa) at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions,b) at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, and/orc) at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions. 24. The peptidoglycan hydrolase of item 23, whereinin a) the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to thisposition is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 orat a position corresponding to this position is substituted with glycine;in b) the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 in SEQ ID NO: or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine; and/orin c) the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 in SEQ ID NO: or at a position corresponding to this position is substituted with glycine.

. The peptidoglycan hydrolase of any one of items 1 to 24, wherein said CHAP domain has an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position. 26. The peptidoglycan hydrolase of item 25, wherein the amino acid residue at position 86 in SEQ ID NO: or at a position corresponding to this position is substituted with another amino acid residue than serine. 27. The peptidoglycan hydrolase of item 25 or 26, wherein the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or methionine. 28. The peptidoglycan hydrolase of any one of items 25 to 28, wherein the amino acid residue at position in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine. 29. The peptidoglycan hydrolase of any one of items 1 to 28, wherein said CHAP domain has at least one amino acid substitution or substitution pair selected from the group consisting of the following (i) to (v):(i) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with lysine; 161 (ii) an amino acid substitution at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue at position in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine;(iii) an amino acid substitution at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine;(iv) an amino acid substitution at position 169 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with asparagine; and(v) an amino acid substitution at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; and preferably wherein said peptidoglycan hydrolase does not have a sequence as shown in any one of SEQ ID NO: 279 to 293.

. The peptidoglycan hydrolase of item 29, wherein said CHAP domain has at least 2, preferably at least 3, more preferably at least 4 of the amino acid substitutions or substitution pairs (i) to (v) as defined in item 29. 31. The peptidoglycan hydrolase of item 29 or 30, wherein said CHAP domain has the amino acid substitution (i) as defined in item 29, and at least one, preferably at least two, more preferably at least three of the amino acid substitutions or substitution pairs (ii) to (v) as defined in item 29. 32. The peptidoglycan hydrolase of any one of items 1 to 31, wherein said CHAP domain has an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position. 33. The peptidoglycan hydrolase of item 32, wherein the amino acid residue at position 155 in SEQ ID NO: or at a position corresponding to this position is substituted with tyrosine. 34. The peptidoglycan hydrolase of any one of items 28 to 31, wherein said CHAP domain further has an amino acid substitution at position 155 of SEQ ID NO: 1 or at a position corresponding to this position, and wherein the amino acid residue at said position is substituted with tyrosine.

. The peptidoglycan hydrolase of any one of items 1 to 34, wherein said CHAP domain has a sequence identity of at least 94% to the sequence from position 72 to position 215 in SEQ ID NO: 11. 36. The peptidoglycan hydrolase of any one of items 1 to 35 which has a sequence identity of at least 96% to the sequence of SEQ ID NO: 11. 162 37. The peptidoglycan hydrolase of item 35 or 36, wherein said CHAP domain has one or more amino acid substitutions or substitution pairs as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1, as defined in any one of items 22, 24, 29, 31 and 34. 38. The peptidoglycan hydrolase of any one of items 1 to 37 which is an endolysin. 39. The peptidoglycan hydrolase of any one of items 1 to 38 further comprising a cell wall binding domain,preferably a LYSM domain or a SH3 domain, more preferably a LYSM domain. 40. The peptidoglycan hydrolase of item 39, wherein the cell wall binding domain is derived from an endolysin, preferably an endolysin comprising a LYSM domain or a SH3 domain, more preferably an endolysin comprising a LYSM domain and a CHAP domain, wherein the LYSM domain is, preferably, N-terminally of the CHAP domain. 41. The peptidoglycan hydrolase of item 39 or 40, wherein the cell wall binding domain is derived from an endolysin that has a killing activity against a Staphylococcus species or strain, preferably Staphylococcus aureus. 42. The peptidoglycan hydrolase of any one of items 39 to 41, wherein the cell wall binding domain has the ability to bind to the cell wall of a Staphylococcus species or strain, preferably Staphylococcus aureus. 43. The peptidoglycan hydrolase of any one of items 39 to 42, wherein the cell wall binding domain is a LYSM domain that has a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1. 44. The peptidoglycan hydrolase of item 43, wherein the LYSM domain has one or more amino acid substitutions at positions 1, 8, 10,13, 23 to 25, 30, 33, 37, and 39 to 41 in SEQ ID NO: 1 or at positions corresponding to these positions. 45. The peptidoglycan hydrolase of item 44, whereinthe amino acid residue at position 1 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tryptophan,the amino acid residue at position 8 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine,the amino acid residue at position 10 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine,the amino acid residue at position 13 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid,the amino acid residue at position 23 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine,the amino acid residue at position 24 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine, 163 the amino acid residue at position 25 in SEQ ID NO: 1 or at a substituted with glutamic acid,the amino acid residue at position 30 in SEQ ID NO: 1 or at a substituted with aspartic acid,the amino acid residue at position 33 in SEQ ID NO: 1 or at a substituted with aspartic acid,the amino acid residue at position 37 in SEQ ID NO: 1 or at a substituted with leucine,the amino acid residue at position 39 in SEQ ID NO: 1 or at a substituted with serine,the amino acid residue at position 40 in SEQ ID NO: 1 or at a substituted with threonine, and/orthe amino acid residue at position 41 in SEQ ID NO: 1 or at a position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position is position corresponding to this position issubstituted with serine. 46. The peptidoglycan hydrolase of any one of items 39 to 46, wherein the cell wall binding domain is N- terminally of the CHAP domain. 47. The peptidoglycan hydrolase of any one of items 39 to 46 further comprising a peptide linker between said CHAP domain and said cell wall binding domain. 48. The peptidoglycan hydrolase of item 47, wherein the peptide linker is a glycine-serine linker. 49. The peptidoglycan hydrolase of item 48, wherein the glycine-serine linker comprises one or multiple, e.g.,to 5, copies of the sequence as shown in SEQ ID NO: 297, and wherein said copies are, preferably, directly adjacent to each other. 50. The peptidoglycan hydrolase of item 47, wherein the peptide linker is derived from an endolysin. 51. The peptidoglycan hydrolase of item 50, wherein the peptide linker is derived from an endolysin that hasa killing activity against a Staphylococcus species or strain, preferably Staphylococcus aureus. 52. The peptidoglycan hydrolase of any one of item 50 or 51, wherein the peptide linker has a sequence identity of at least 60% to the sequence from position 52 to position 71 in SEQ ID NO: 1. 53. The peptidoglycan hydrolase of item 52, wherein the peptide linker has one or more amino acid substitutions at positions 53, 55, 56, 58, 63, 65 and 68 in SEQ ID NO: 1 or at positions corresponding to these positions. 54. The peptidoglycan hydrolase of item 53, wherein 164 the amino acid residue at position 53 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine,the amino acid residue at position 55 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine,the amino acid residue at position 56 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine,the amino acid residue at position 58 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine,the amino acid residue at position 63 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine,the amino acid residue at position 65 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and/orthe amino acid residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine, arginine or alanine. 55. The peptidoglycan hydrolase of any one of items 52 to 54, wherein the peptide linker has an amino acid substitution at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, and wherein the amino acid residue at said position is substituted with lysine, methionine, arginine or alanine, preferably lysine. 56. The peptidoglycan hydrolase of any one of items 1 to 55, wherein said bactericidal activity is a killing activity against a Staphylococcus species or strain. 57. The peptidoglycan hydrolase of any one of items 1 to 56, wherein said bactericidal activity is a killing activity against Staphylococcus aureus. 58. The peptidoglycan hydrolase of any one of items 1 to 57, wherein the peptidoglycan hydrolase is able to growth-inhibit a Staphylococcus aureus liquid culture at a concentration of about 40 pg/ml or less, about pg/ml or less or about 10 pg/ml or less, preferably at a concentration of about 4 pg/ml or less, more preferably at a concentration of about 2 pg/ml or less. 59. The peptidoglycan hydrolase of item 57 or 58, wherein said Staphylococcus aureus is resistant to at least one antibiotic, e.g. methicillin, vancomycin, daptomycin and/or linezolid, preferably wherein said Staphylococcus aureus is a methicillin-resistant Staphylococcus aureus (MRSA) strain, a vancomycin- intermediate Staphylococcus aureus (VISA) strain, a vancomycin-resistant Staphylococcus aureus (VRSA) strain, a daptomycin-resistant Staphylococcus aureus (DRSA) strain or a I inezolid-resista nt Staphylococcus aureus (LRSP) strain. 60. The peptidoglycan hydrolase of any one of items 1 to 59 which has the ability to lyse the cell wall of a Staphylococcus species or strain, preferably Staphylococcus aureus, in particular wherein said protein has the ability to break down or cleave peptidoglycan in the cell wall of Staphylococcus aureus. 165 61. The peptidoglycan hydrolase of any one of items 1 to 60 which has the ability of being secreted from a eukaryotic cell when expressed in said cell, for example a yeast cell or a mammalian cell, preferably a human cell. 62. The peptidoglycan hydrolase of any one of items 1 to 61 which is stable up to a temperature of about 40°C, e.g. 37OC, 38OC, 39OC, 40°C, 41°C or 42OC, preferably about 42OC, more preferably about 44OC or about 47OC, as determined by a thermofluor assay. 63. The peptidoglycan hydrolase of any one of items 1 to 62 which has, compared to the peptidoglycan hydrolase of SEQ ID NO: 1,(i) a similar or enhanced bactericidal activity, preferably a similar or enhanced killing activity against Staphylococcus aureus,(ii) a similar or enhanced ability of being secreted by a eukaryotic cell, preferably in a yeast cell or a human cell, e.g., a HEK293 cell,(ill) a similar or enhanced solubility in an aqueous solution such as PBS,(iv) a similar or enhanced stability, preferably an enhanced thermostability, and/or(v) a similar or reduced tendency to form aggregates in an aqueous solution such as PBS. 64. The peptidoglycan hydrolase of any one of items 1 to 63, which has, compared to the peptidoglycan hydrolase of SEQ ID NO: 1 an enhanced killing activity against Staphylococcus aureus, an enhanced ability of being secreted by a human cell, and/or an enhanced thermostability. 65. The peptidoglycan hydrolase of item 63 or 64, wherein the killing activity of a peptidoglycan hydrolase against Staphylococcus aureus is measured by determining the minimum concentration at which the peptidoglycan hydrolase growth-inhibits a Staphylococcus aureus liquid culture (MIC). 66. The peptidoglycan hydrolase of any one of items 63 to 65, wherein the ability of a peptidoglycan hydrolase of being secreted by a human cell is measured by determining the amount of the peptidoglycan hydrolase in the supernatant of HEK293 cells expressing the peptidoglycan hydrolase. 67. The peptidoglycan hydrolase of any one of items 63 to 66, wherein the thermostability of a peptidoglycan hydrolase is measured by determining the melting temperature in a thermofluor assay. 68. The peptidoglycan hydrolase of any one of items 1 to 67, further comprising a signal peptide, preferably a signal peptide that is able to direct the peptidoglycan hydrolase to a secretory pathway in a mammalian cell, preferably a co-translational translocation pathway; preferably, wherein said signal peptide is cleaved off during secretion or export from the cell; and, preferably, wherein said signal peptide is at the N- terminus. 69. The peptidoglycan hydrolase of any one of items 1 to 68, further comprising an extended pharmacokinetic (PK) peptide such as a human FC domain (e.g. as shown in SEQ ID NO: 294), a C-terminal peptide of human chorionic gonadotropin (e.g. as shown in SEQ ID NO: 295) or human lysozyme (e.g. as shown in 166 SEQ ID NO: 296); preferably, wherein said PK peptide is positioned at the C- or N-terminus of said CHAP domain, said cell wall binding domain, or said peptidoglycan hydrolase, and preferably C-terminally of any signal peptide; and, preferably, wherein the peptidoglycan hydrolase comprises between said PK peptide and said CHAP domain or said cell wall binding domain a peptide linker such as a glycine-serine linker, preferably a glycine-serine linker as defined in item 49. 70. The peptidoglycan hydrolase of any one of items 1 to 69, further comprising at least one glycosylation motif which is not present in the peptidoglycan hydrolase of SEQ ID NO: 1; and, preferably, wherein a glycosylation motif consists of the amino acids X!, X2, and X3, wherein X! is asparagine, X2 is any amino acid except proline, and X3 is serine or threonine. 71. The peptidoglycan hydrolase of any one of items 1 to 70, wherein said peptidoglycan hydrolase is deimmunized and/or less immunogenic than the peptidoglycan hydrolase of SEQ ID NO: 1. 72. The peptidoglycan hydrolase of any one of items 1 to 71, wherein said CHAP domain has one or more amino acid substitutions at positions 72, 73, 75 to 77, 79 to 83, 86, 99, 102 to 104, 107, 108, 114, 115, 117, 122 to 124, 130, 131, 133 to 137, 139 to 141, 143, 144, 166, 169, 170, 173 to 176, 178, 185, 1to 192, 194, 196, 197, 199, 201, 203 to 207, 212 and 213 in SEQ ID NO: 1, or at positions corresponding to these positions; and, preferably, wherein said peptidoglycan hydrolase has, in addition, one or more of said amino acid substitutions at positions 1, 12, 13, 17, 19, 22 to 25, 28 to 30, 32 to 34, 36 to 45, 48 to 55, 65, 68, 69 and 71 in SEQ ID NO: 1, or at positions corresponding to these positions;in particular wherein one or more amino acid residues at said positions are substituted with any of the substitute amino acid residues shown for the corresponding positions in the following Table: Position in SEQ ID NO: 1 Substitute amino acid residueQD, G, Q or SD, E, N or PA, I or MD, H, K, N or QA, D, G, H, K, Q, R or WN, Q or WH orTD, G, H, Q or SD, E, Q, S, TDA, D, E or Q 167 32 A, K or MA, D, E, G, K or QAor WD, G or QD, K, L, M, N or QK or 5A, D or GD, G, N, 5 or TDD, H, K, M, N, Q or 5LorVD, E, H, 5 or WA, D, E, K, P, Q or TD, E, N or Q1, M or ND, H, N or TT5A, G, K, Q or 5D, E, H or NDD5G, H, N or Q5D, H or TD, 5 or TD orQD or NA, D, G, H, K, N, Q, S or TH orWA, D, M or N 168 81 A, G or SGM or QYK, M, Q, T, S or V102 A, G, S or T103 A or G104 D, M, QorY107 A or S108 G, K or Y114 QorT115 D, G or S117 D or N122 L123 D, S, T or W124 QorT130 GorY131 N, Q or Y133 QorT134 K, Q or T135 H or S136 Q, E or T137 G139 A, D, Q or S140 D141 D, H or T143 P144 Q166 A, E, V or T169 D170 A, H or S173 H or N 169 174 A, H, K, R, S or T175 N176 A, Q, S or T178 S orT185 D or N190 A, C, D, H, N, S or T191 D, N192 E, G, K, or T194 G, P or T196 QorT197 P or R199 N, SorT201 D or K203 D204 D205 QorV206 Q207 H orW212 D, E, H, K or N213 Y 73. The peptidoglycan hydrolase of any one of items 1 to 72 which is an engineered peptidoglycan hydrolase. 74. The peptidoglycan hydrolase of any one of items 1 to 73 which is an isolated peptidoglycan hydrolase. 75. A nucleic acid encoding the peptidoglycan hydrolase of any one of items 1 to 74. 76. The nucleic acid of item 75, wherein said nucleic acid is a RNA. 77. The nucleic acid of item 76, wherein said RNA is a mRNA. 78. The nucleic acid of item 77, wherein said mRNA is nucleoside-modified. 79. The nucleic acid of item 78, wherein said mRNA comprises 170 (i) a modified nucleoside selected from pseudouridine (w), Nl-methyl-pseudouridine (mly), and 5- methyl-uridine (m5U), preferably Nl-methyl-pseudouridine (mlw), in place of uridine, preferably in place of each uridine;(ii) a capO 5' cap, for example, m27'2'°G(5')ppSp(5l)G; or a capl 5'cap, for example, m270 '־ 3 ׳ Gppp(m12'־°)ApG(iii) a 5' UTR and/or a 3' UTR, and/or(iv) a poly-A sequence comprising, preferably, at least 100 nucleotides. 80. The nucleic acid of any one of items 75 to 79 which is an engineered nucleic acid, preferably an engineered RNA. 81. The nucleic acid of any one of items 75 to 80 which is an isolated nucleic acid, preferably an isolated RNA. 82. The nucleic acid of any one of items 75 to 81 which is codon-optimized for protein expression in cells of acertain mammalian species, preferably a human. 83. An RNA construct comprising in 5' to 3' order:(i) a 5' UTR that, preferably, comprises or consists of a modified human alpha-globin 5'-UTR;(ii) a sequence encoding a peptidoglycan hydrolase of any one of items 1 to 74;(iii) a 3' UTR that, preferably, comprises or consists of a first sequence from the amino terminalenhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 125 ribosomal RNA; and(iv) a poly-A sequence, comprising preferably at least about 100 nucleotides. 84. The RNA construct of item 83, wherein the 5' UTR comprises or consists of a sequence according to SEQ ID NO: 371. 85. The RNA construct of item 83 or 84, wherein the 3' UTR comprises or consists of a sequence according to SEQ ID NO: 374. 86. The RNA construct of any one of items 83 to 85, wherein the polyA tail sequence is a split polyA tail sequence. 87. The RNA construct of item 86, wherein the split polyA tail sequence comprises or consists of a sequence according to SEQ ID NO: 375. 88. The RNA construct of any one of items 83 to 87, further comprising a 5' cap. 89. The RNA construct of any one of items 83 to 88, wherein the RNA is mRNA and wherein, optionally, themRNA comprises modified nucleosides selected from pseudouridine (w), Nl-methyl-pseudouridine (mlw), and 5-methyl-uridine (m5U), preferably Nl-methyl-pseudouridine (mlw), in place of uridine, preferably in place of each uridine. 171 90. The nucleic acid of any one of items 76 to 82 or the RNA construct of any one of items 83 to 89, wherein the RNA is formulated as a particle comprising said RNA, preferably as a lipid nanoparticle (LNP) or lipoplex (LPX). 91. The nucleic acid of any one of items 75 and 80 to 82, wherein said nucleic acid is a DNA. 92. A plasmid comprising the nucleic acid of item 91. 93. A viral vector comprising the nucleic acid of any one of items 75 to 82, 90 and 91, the RNA construct ofany one of items 83 to 90 or the plasmid of item 92. 94. A cell comprising the nucleic acid of any one of items 75 to 82, 90 and 91, the RNA construct of any one of items 83 to 90, the plasmid of item 92 or the viral vector of item 93. 95. The cell of item 94, wherein said cell is a bacterial cell or a eukaryotic cell. 96. The cell of item 94 or 95, wherein said cell is a yeast cell such as Pichia pastoris or a mammalian cell,preferably a human cell. 97. A pharmaceutical composition comprising the peptidoglycan hydrolase of any one of items 1 to 74, the nucleic acid of any one of items 75 to 82, 90 and 91, the RNA construct of any one of items 83 to 90, the plasmid of item 92, the viral vector of item 93 and/or the cell of any one of items 94 to 96; and, preferably, a pharmaceutically acceptable excipient. 98. A pharmaceutical composition comprising the peptidoglycan hydrolase of any one of items 1 to 74. 99. A pharmaceutical composition comprising the RNA of any one of items 76 to 82 and 90 or the RNAconstruct of any one of items 83 to 90. 100. The pharmaceutical composition of items 97 to 99 for use in treating a disease caused by and/or associated with a Staphylococcus infection and/or a subject that has or is suspected of having a Staphylococcus infection, preferably wherein said infection is a Staphylococcus aureus infection. 101. The peptidoglycan hydrolase of any one of items 1 to 74 for use in treating a disease caused by and/or associated with a Staphylococcus aureus infection and/or a subject that has or is suspected of having a Staphylococcus aureus infection. 102. The RNA of any one of items 76 to 82 and 90 or the RNA construct of any one of items 83 to 90 for use in treating a disease caused by and/or associated with a Staphylococcus aureus infection and/or a subject that has or is suspected of having a Staphylococcus aureus infection. 172 103. The pharmaceutical composition for use according to item 100, the peptidoglycan hydrolase for use according to item 101, or the RNA or RNA construct for use according to item 102, wherein said Staphylococcus aureus mfection is an infection of a skin, soft tissue, bone, lung, sinus and/or urinary tract. 104. The pharmaceutical composition, peptidoglycan hydrolase, RNA or RNA construct for use according to item 103, wherein said disease is selected from the group consisting of: pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, sepsis, a respiratory infection such as sinusitis, pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, abscesses, food poisoning, necrotizing fasciitis, pyomyositis, mediastinitis, infected dermatitis, wound infection, diabetic foot ulcer, septic arthritis, osteoarticular infections, prosthetic infection such as infection of a prosthetic joint or a cardiac device, and urinary tract infections; preferably pneumonia, bacteremia, endocarditis, and/or a prosthetic infection. 105. The pharmaceutical composition for use according to any one of items 100,103 and 104, the peptidoglycan hydrolase for use according to any one of items 101, 103 and 104, or the RNA or RNA construct for use according to any one of items 102 to 104, wherein the Staphylococcus, preferably Staphylococcus aureus, is present in form of a biofilm or is suspected of forming a biofilm. 106. A solidified yeast culture medium comprising substrate particles intermixed with said medium, wherein said substrate particles comprise dead bacterial cells and/or fragments thereof, and/or peptidoglycan particles. 107. The solidified yeast culture medium of item 106 containing dead bacterial cells at an optical density at 6nm (OD620) of about 1 to 10, preferably about 2 to 4. 108. A yeast culture comprising (i) the solidified yeast culture medium of item 106 or 107, and (ii) yeast cells, e.g., at least one yeast colony, on the solidified yeast culture medium. 109. The yeast culture of item 108, wherein at least one yeast cell or yeast colony expresses and/or secretes a peptidoglycan hydrolase. 110. A method of screening yeast cells for the secretion of an active peptidoglycan hydrolase, said method comprising the steps of:a) providing a solidified yeast culture medium comprising substrate particles intermixed with said medium, wherein said substrate particles comprise dead bacterial cells and/or fragments thereof, and/or peptidoglycan particles, preferably as defined in item 107;b) culturing yeast cells expressing a peptidoglycan hydrolase on a surface of said solidified medium until at least one yeast colony is detectable, in particular wherein the yeast cells are able to secrete the peptidoglycan hydrolase;c) evaluating whether a halo is apparent around a yeast colony, in particular wherein the halo corresponds to a locally reduced optical density of said solidified medium around said colony, for 173 example, in a radius of about 0.1 to 1 cm from the colony, especially compared to a region of the solidified medium that is free of yeast colonies;d) determining that a yeast colony secretes an active peptidoglycan hydrolase when a halo around the colony is apparent, or determining that a yeast colony does not secrete an active peptidoglycan hydrolase when no halo around the colony is apparent. 111. A method of identifying an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell, said method comprising the steps of:1) preparing a library of eukaryotic cells, preferably yeast cells, expressing peptidoglycan hydrolase variants on the cell surface;II) selecting eukaryotic cells, e.g. yeast cells, based on a high level of peptidoglycan hydrolase on the cell surface relative to other cells in the library; for example, selecting the 10% of cells in the library with the highest peptidoglycan level on the cell surface;III) performing the method of screening yeast cells for the secretion of an active peptidoglycan hydrolase as defined in item 110, wherein yeast cells that are able to secrete the peptidoglycan hydrolase variants expressed in the eukaryotic cells, e.g. the yeast cells, selected in step II) are cultured in step b) of said method; andIV) determining that a yeast colony that has been determined in said step d) to secrete an active peptidoglycan hydrolase produces an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell. 112. The method of item ill further comprising between said steps II) and III) the steps of:IT) isolating DNA encoding at least one peptidoglycan hydrolase variant from the eukaryotic cells, e.g. the yeast cells, selected in step II); andII") introducing the isolated DNA into yeast cells, e.g., by means of transformation, in particular in a way that the yeast cells are able to secrete the peptidoglycan hydrolase variants. 113. The method of item ill or 112, wherein said step I) comprises (i) fragmenting and reassembling DNA encoding different peptidoglycan hydrolases, preferably endolysins, e.g. endolysin homologues, thereby generating a DNA library of peptidoglycan hydrolase variants, and (ii) introducing said DNA library into eukaryotic cells, e.g. by means of transformation, transfection or transduction, thereby generating the library of eukaryotic cells expressing peptidoglycan hydrolase variants. 114. The method of any one of items ill to 113, further comprising before said step II) a step 1ל of attaching a detectable label to the peptidoglycan hydrolase variants on the cell surface, preferably a fluorescent dye, e.g., by means of immunostaining. 115. The method of item 114, wherein in said step II), cells with a high level of peptidoglycan hydrolase on the cell surface relative to other cells in the library are sorted from these other cells, e.g., by means of fluorescent activated cell sorting or magnetic activated cell sorting. 174 116. The method of any one of items ill to 115, wherein at least two rounds of said steps I) to IV) are performed, and wherein in step I) of each subsequent round, a further library of eukaryotic cells is prepared, wherein the cells in the library express a different set of peptidoglycan variants compared to the library employed in the preceding round(s). 117. The method of item 116, wherein step I) of a subsequent round comprises (i) fragmenting and reassembling DNA encoding different peptidoglycan hydrolases, wherein at least one, preferably at least 50%, of said peptidoglycan hydrolases has been identified in a preceding round as an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell, thereby generating a further DNA library of peptidoglycan hydrolase variants, and (ii) introducing said further DNA library into eukaryotic cells or yeast cells, thereby generating a further library of eukaryotic cells or yeast cells expressing a different set of peptidoglycan hydrolase variants compared to the library employed in the preceding round(s). 118. The solidified yeast culture medium of item 106 or 107, the yeast culture of item 108 or 109, or the method of any one of items 110 to 117, wherein said culture medium comprises agar, preferably ata concentration of about 0.5% to about 5%, more preferably at a concentration of about 1% to about 2%, e.g., at a concentration of about 1.5%. 119. The solidified yeast culture medium of any one of items 106,107 and 118, the yeast culture of any one of items 108,109 and 118, or the method of any one of items 110 to 118, wherein the opacity of the solidified medium is reduced at a location where the substrate particles are lysed or broke down. 120. The solidified yeast culture medium of any one of items 106, 107, 118 and 119, the yeast culture of any one of items 108,109,118 and 119, or the method of any one of items 110 to 119, wherein said yeast is Pichia pastoris. 121. The solidified yeast culture medium of any one of items 106,107 and 118 to 120, the yeast culture of any one of items 108, 109 and 118 to 120, or the method of any one of items 110 to 120, wherein the dead bacterial cells are dead gram-positive bacterial cells and/or wherein the peptidoglycan in the peptidoglycan particles is from gram-positive bacteria. 122. The solidified yeast culture medium of any one of items 106,107 and 118 to 121, the yeast culture of any one of items 108, 109 and 118 to 121, or the method of any one of items 110 to 121, wherein the dead bacterial cells are dead Staphylococcus cells and/or wherein the peptidoglycan in the peptidoglycan particles is from a Staphylococcus species or strain. 123. The solidified yeast culture medium of any one of items 106,107 and 118 to 122, the yeast culture of any one of items 108, 109 and 118 to 122, or the method of any one of items 110 to 122, wherein the dead bacterial cells are dead Staphylococcus aureus cells and/or wherein the peptidoglycan in the peptidoglycan particles is from Staphylococcus aureus. 175 124. The solidified yeast culture medium of any one of items 106,107 and 118 to 123, the yeast culture of any one of items 108, 109 and 118 to 123, or the method of any one of items 110 to 123, wherein said substrate particles comprise or essentially consist of autoclaved Staphylococcus aureus cells. 125. The yeast culture of any one of items 108,109 and 118 to 124, or the method of any one of items 110 to 124, wherein the peptidoglycan hydrolase is an endolysin; preferably an endolysin that has or is suspected to have killing activity against a gram-positive bacterium, preferably a Staphylococcus species or strain, more preferably Staphylococcus aureus. 126. The yeast culture of any one of items 108,109 and 118 to 125, or the method of any one of items 110 to 125, wherein the peptidoglycan hydrolase is considered as active when it is able to lyse and/or break down the substrate particles in the solidified yeast culture medium. 127. The yeast culture of item 126 or the method of item 126, wherein an active peptidoglycan hydrolase is considered to have killing activity against a live bacterium comprising a peptidoglycan in its cell wall which corresponds to the peptidoglycan comprised in the substrate particles in said medium; for example, wherein a peptidoglycan hydrolase secreted from a yeast colony which is able to lyse and/or break down dead Staphylococcus aureus in the solidified yeast culture medium may be considered as an active peptidoglycan hydrolase that has killing activity against live Staphylococcus aureus. 128. The method of any one of items 110 to 127, further comprising a step of obtaining from a yeast colony an active peptidoglycan hydrolase or an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell, or a nucleic acid encoding an active peptidoglycan hydrolase or an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell. 129. A peptidoglycan hydrolase or peptidoglycan hydrolase variant obtained or obtainable by the method of item 128. 130. A nucleic acid encoding a peptidoglycan hydrolase or hydrolase variant, wherein said nucleic acid and/or said peptidoglycan hydrolase or hydrolase variant is obtained or obtainable by the method of item 128.

The invention is also characterized by the following figures, figure legends and the following non-limiting examples.

Brief description of the Figures Figure 1: Lysin domain architectures screened 36 lysins of 13 different domain architectures were screened for killing activity against S. aureus.

Figure 2: Minimal inhibitory concentrations of LO482 against a panel of Staphylococcus strains 5xl05 bacteria were incubated with varying concentrations of lysins. MICs are shown in pg/ml. The MICs were determined as the minimal concentrations where regrowth was inhibited by the lysins. LO466 (i.e. exebacase) was 176 tested for comparison to LO482. MRSA: "methicillin-resistant Staphylococcus aureud' strain; MSSA: "methicillin- sensitive Staphylococcus aureud strain Figure 3. Bactericidal activity of LysM-CHAP expressed and secreted in P.pastoris Supernatants of P.pastoris cultures expressing different LO482 or LO499 constructs were mixed with lysin buffer at the indicated dilutions and incubated with S.aureus cells. Bacterial growth was monitored by measuring the culture OD over the course of 24 hours. Growth curves of S.aureus in the presence of supernatants from LO482 or LO4expressing P.pastoris strains are shown, wt S/N: supernatant from wild type P.pastoris not expressing recombinant lysins.

Figure 4. Yeast on dead aureus (YODA) screening method A) P. pastoris (P p) cells are grown on agar plates containing dead (i.e. autoclaved) S. aureus (S ■a■) cells and 1% methanol for induction of protein expression. Active lysin secreted from a P. pastoris colony degrades the peptidoglycan of the dead S. aureus cells resulting in a halo around the colony. YODA allows to overcome issues of expression, solubility and toxicity of lysins in E. coli Turnaround time: 3-4 days. YODA may be modified to contain other dead bacteria for assaying lysin activity against these bacteria (YODB), in particular, Gram-positives such as C. difficile. B) YODA allowed the detection of enzymatic activity of a broad spectrum of lysin families, including some which are not active when expressed in E. coli.

Figure 5. Active aglycosylated LO482 variants identified by YODA A) Glycosylation can inhibit Ivsin activity: L0482 contains two consensus sites which are N-glycosylated in the secretory pathway. Glycosylation at these sites strongly reduces the enzymatic activity of LO482 when measured in a growth inhibition assay. B) YODA screen for aalvcosvlated LO482 variants: A degenerate codon library was assembled in which the codons for asparagine residues 68 and 73 were mutated to all codons but asparagine and the stop codon (using BNC,VNG and DKS degenerate codons). YODA was used to identify active variants with mutated N-glycosylation motifs. C) YODA selected active aalvcosvlated variants: YODA hits were confirmed to be aglycosylated by SDS-PAGE after purification from P.pastoris supernatants. When produced in E. coli, mutant LO482-18 with mutations N68K, N73G (SEQ ID NO: 2) retained an enzymatic activity in the same range as the wild type LO482 protein (SEQ ID NO: 1) in a growth inhibition assay (8 vs 4pg/ml MIC, respectively).

Figure 6. Poor solubility of certain lysins after expression in E.coii A pilot library of different lysin architectures was expressed in Ecoii. Cells were lysed and the soluble fraction isolated by centrifugation. SDS-PAGE analysis of whole cell extracts and soluble fractions showed that the majority of lysins was poorly soluble when produced in Ecoii. Of note, WT LO482 (SEQ ID NO: 1) showed at least a moderate solubility.

Y?l Figure 7. Directed evolution of lysins A) The lysin of interest (starting point) is mixed with a set of homologues and fragmented by DNAsel treatment. The fragmented DNA pieces are randomly assembled by PCR. DNA shuffling generates a highly diverse library of lysin sequences. B) The lysin library is cloned as a fusion to the SAGI cell wall anchor domain and transformed into P.pastoris. ~1variants are screened for high expression levels by FACS and the top expressing cells are isolated.

Figure 8. Improvement of L0482ag after two rounds of directed evolution A) Frequencies of selected lysin variants in round 1,2 and 3 of directed evolution. 96 (Round 1) or 182 (Round and Round 3) YODA-active P.pastoris clones from the second FACS sort were sequenced after colony PCR. Each lysin sequence was counted and the most frequently observed variants were selected as hits. B) Analysis of the stability of L0482ag and variants thereof by the thermofluor assay. Lysin variants were purified after expression in E.coii and heated in the presence of the SYPRO orange dye. Changes in fluorescence were measured and the inflection point of these melting curves were taken as the Tm (i.e. the melting temperature at which half of the protein remains folded). For L0482ag, no Tm could be determined, likely because the majority of the protein is unfolded at the lowest temperature. Variants from the first two rounds of directed evolution show improved Tms between 37 and 47°C. C) Activity of L0482ag variants against S.aureus. Lysin variants purified after expression in E.coii were incubated at different concentrations with S.aureus cultures. The optical density (OD) at 620 nm of the mixture was measured at 37°C. The minimal inhibitory concentration (MIC) was determined as the concentration at which the OD at 6nm of the culture remained below 0.1 during a 24h time period. L0482ag (SEQ ID NO: 1) showed a MIC of 8pg/ml whereas the hit variants (i.e G1-G4 and H1-H10*; SEQ ID NO: 3-16) displayed 2-16 fold decreased MICs (between 4-0.5pg/ml). *The data point for H3 is missing for technical reasons. It is expected that the MIC of H3 is in the same range as for H1-H10. D) Secretion of L0482ag variants after transfection of lysin coding DNA into EXPI293 cells. Lysin sequences containing an N-terminal mouse IgK signal peptide and optimized for human codon bias were transfected into EXPI293 cells. Secreted protein in the cell supernatants was measured 72h post transfection by Western blotting. Band intensities were quantified by densitometry and plotted as artificial units (A.U.). Secretion of L0482ag could not be detected, while the assayed hit variants (i.e. GI and H1-H10; SEQ ID: 3 and 7-16) showed a strong increase in the amounts of secreted protein. E) Bactericidal activity of certain L0482ag variants, i.e. H3 (SEQ ID NO: 9) and H5 (SEQ ID NO: 11) after secretion from EXPI293 cells. Supernatants from EXPI293 transfections were incubated with different concentrations of S.aureusceWs and cultured at 37°C while monitoring the OD at 620 nm. Delay and complete arrest of growth was observed for variants H3 and H5. Medium: culture medium was mixed with S.aureus instead of supernatants from EXPI293 transfections.

Figure 9. Sequence analysis of directed evolution hits A) Alignment of amino acid sequences of hits from round 1, 2 and 3 of directed evolution. Hits G1-4 (round 1), Hl- (round 2) and 11-30 (round 3) are aligned to L0482ag (SEQ ID NO: 2). Amino acid substitutions relative to L0482ag are indicated. These amino acid substitutions are considered as "particularly beneficial amino acid substitutions", as described herein. Furthermore, the "K"at position 68 and the "G" at position 73 (as also contained 178 in L0482ag) are also considered as "particularly beneficial amino acid substitutions" vis a vis WT LO482 (SEQ ID NO: 1) herein and, furthermore, as preferred "aglycosylation substitutions". Identities to the corresponding amino acid in L0482ag at each position are marked with a dot Deletions are marked with a dash ("-"). The most beneficial amino acid substitutions, as described herein, are highlighted with arrows. Arrows for amino acid substitutions that occur frequently together (i.e. substitution pairs) are connected with a horizontal line. B) Definition of mutation units in hits from round 1, 2 and 3 with highest impact on beneficial lysin properties. Hits Gl-4, H1-H10 and 11-30 were aligned using ClustalOmega. From this alignment, variant H3 emerged as the variant which reflects best the consensus sequence. Mutation units, i.e. single amino acid substitutions and correlated substitution pairs, were identified by visual inspection of the alignment (shown in rows 1-3). The mutation units shown consist of the 5 consensus mutation units as reflected in "H3" and the additional substitution F155Y as in "H5"). The presence of each mutation unit in each hit is indicated with an "X".

Figure 10. Expression of ribolysin variants in human cells mg of mRNA were transfected in HEK293T/17 cells and ribolysin variant expression (i.e., expression of a lysin variant from an mRNA construct) was analyzed by Western Blot. A) Protein levels of different ribolysin variants in supernatants (SN) and cell lysates (L). Cell lysates were 22-fold more concentrated than supernatants. B) Quantification of ribolysin protein levels in supernatants.

Figure 11. The LO482 variant H5 showed enhanced killing kinetics against S. aureus as compared to the WT LO482 lysin Data from an OD reduction assay are shown.

Figure 12. LO482 variants show bactericidal activity against S. aureus biofilms and free-floating aggregates A) Peg biofilm in plasma assay (PBA), data from 3 experiments. B) Free-floating biofilm-like aggregate in synovial fluid assay (FBA), data from 5 experiments.Statistical analysis was performed using One-way ANOVA (Dunnett's multiple comparisons test), p<0.0001; LOD: limit of detection, 500cfu/ml; cfu: colony-forming units.

Examples Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.

Example 1: Selection of the LysM-CHAP architecture as represented by LO482 For the identification of active wildtype endolysins, a set of 36 endolysins with 13 different domain architectures (Figure 1) was compiled from literature and BLAST searches, expressed in E. coli, purified and tested in functional assays, such as growth inhibition assays. Briefly, purified lysins were incubated with 5x105 staphylococcal (i.e. Staphylococcus aureus^ cells in 96 well plates and outgrowth of the culture was monitored for 24 hours by regularly 179 measuring OD620nm. The failure of S. aureusto grow to measurable optical density (i.e. QD620nm > 0.1) at 37OC) indicates the presence of an active endolysin, which is potent enough to eradicate the bacterial cells.Five endolysins, belonging to three different architectures (CHAP-Ami2-SH3, LysM-CHAP and PepM23-SH3, i.e., "lysostaphin") were found to be able to inhibit S. aureus outgrowth in said growth inhibition assays. (Figure 1). Homologues to active lysins belonging to the CHAP-Ami2-SH3, LysM-CHAP and to CHAP-SH3 architectures (a representative of the latter is exebacase, an anti-staphylococcal lysin employed in clinical trials; Fowler (2020). J Clin Invest,130(7)) were identified by BLAST and a total of 65 homologue constructs were ordered as synthetic DNA. The original 36 sequences plus 65 of said homologues as well as internal controls, were expressed in E. coii and the activity of cleared E. co//lysates was interrogated in a growth inhibition assay, wherein each cleared lysate was incubated with 5xl05 S. aureus cells (in cation adjusted Muller-Hinton broth Merck 90922, supplemented with % horse serum, Thermo Scientific 26050070) and S. aureus outgrowth was monitored by measuring OD620nm over a period of 24 hours.LO482 (SEQ ID NO: 1), as well as LO499 (SEQ ID NO: 47), both belonging to the LysM-CHAP domain architecture, were identified to be active against S. aureus m the first screening round. LO482, LO499 and 11 homologues of this domain architecture were included in the 65 sequences that were tested in the second screening round (the first were also included as a control for the screening procedure). Surprisingly, 12 out of the 13 LysM-CHAPs tested (including LO482), were identified to be active against S. aureus which was a much higher fraction compared to other lysin architectures (Figure 1). Furthermore, it has been found that LO482 was highly effective in killing various S. aureus strains including methicillin-resistant Staphylococcus aureus strains (i.e. ATCC43300, A57, B94, and Al), as well as other relevant Staphylococcus species/strains. In addition, it has been found that LO482 had, surprisingly, an overall higher killing activity against the tested Staphylococcus strains (incl. S. aureus) compared to LO466 (SEQ ID NO: 304), i.e. "exebacase" which has been used in clinical trials; Fowler (2020), J Clin Invest,130(7) (see Figure 2).Furthermore, LO482 is relatively small (25kDa) which is advantageous, inter alia, for engineering.The active LysM-CHAP lysins were then expressed in yeast cells, in particular, P.pastoris (see Example 2).

Example 2: Bactericidal activity of LysM-CHAP lysins secreted from P.pastoriscells P.pastoris Xs a well-established eukaryotic expression system for the high level production of recombinant proteins. Proteins in P.pastoris are typically translated, folded and processed by a similar set of factors as in higher eukaryotes, such as human cells. In particular, expression of proteins containing N-terminal signal sequences results in their targeting to the secretory pathway and secretion into the extracellular space. The steps occurring during this process are highly conserved between eukaryotes and involve the translocation of the protein across the membrane of the endoplasmic reticulum (ER), its glycosylation and its trafficking to the plasma membrane. The use of eukaryotic, e.g. human, cells for the delivery of lysins, e.g. by mRNA technology, plasmids or viral vectors, necessitates that lysins are able to pass through the secretory pathway in an active state in those cells. Since the majority of lysins are derived from phages and have evolved to be synthesized in a bacterial cytoplasm, it has been unclear if different lysins can be expressed and secreted in a eukaryotic cell.Therefore, the inventors checked whether LysM-CHAP lysins can be trafficked through the eukaryotic secretory pathway and at the same time maintain their bactericidal activity (e.g. against S.aureus).For this purpose, the inventors employed P.pastorisas an expression system. Moreover, the amino acid sequences (excluding the start methionine) of wild type (WT) LO482 and L0499 were modified by adding the N-terminal pre- and pro sequences of S. cerevisiae alpha mating factor, followed by a Kex2/Stel3 signal peptidase site, a hexa 180 histidine tag and a 3C protease cleavage site (see SEQ ID NO: 48 and 50, respectively). The signal sequences target the lysin constructs to the secretory pathway and are cleaved off at the signal peptidase sites after translocation into the ER. The hexa-histidine tag and 3C sites were further introduced to facilitate lysin purification by affinity capture and removal of the hexa-histidine tag after proteolytic cleavage, respectively. The constructs were codon optimized for expression in P.pastoris (see SEQ ID NO: 49 and 51, respectively) and ordered from Genescript in plasmids conferring antibiotic resistance and control of lysin expression by an AOXI methanol- inducible promoter. Plasmids were linearized by restriction and transformed into a wild type (mut+) P.pastoris strain, followed by selection of transformants on YPD plates containing 100 pg/ml Zeocin. Multiple colonies per construct were transferred each into 3 ml BMGY medium in 24 well plates and grown shaking at 30 °C. After hours, protein expression was induced by exchanging the medium to BMMY (containing 1% methanol). The medium was supplemented with 1% methanol every 24 hours and culture supernatants were harvested after 72 hours of expression, flash frozen in liquid nitrogen and stored at -80 °C.For each transformant, lysin secretion into culture supernatants was estimated by SDS-PAGE and Coomassie staining. Supernatants with highest apparent levels of secretion were serially diluted with lysin buffer (20mM N3POpH 6.0, lOOmM NaCI) and tested for bactericidal activity. For this purpose, 30 pl of supernatants (and dilutions thereof) were mixed with S.aureus cells in 70 pl BHI medium such that the reaction contained 5xl05 cfu/ml. The optical density of these solutions (OD) at 620nm was monitored in a plate reader over the course of 24 hours to quantify bacterial growth. In these experiments, S.aureus cells were able to grow in the presence of supernatants from a parental P.pastoris strain, showing that the culture medium itself does not have lytic activity (negative control). In contrast, S.aureus failed to grow in presence of supernatants from strains expressing the LO482 or LO499 lysin constructs. These experiments show that the level of LO482 or LO499 lysins in undiluted supernatants was sufficiently high to lyse all S.aureus cells in the culture (Figure 3)Taken together, the data demonstrate that LysM-CHAP lysins against S. aureus can be channelled through the secretory pathway of eukaryotic cells and maintain their activity after secretion. Moreover, both tested LysM-CHAP lysins can be expressed in an active form in P. pastoris. In summary, the findings described in Examples 1 and 2 led the inventors to the conclusion, that LysM-CHAP lysins as represented by LO482, are a particularly good starting point for further engineering and directed evolution (see, e.g., Examples 4 and 5).

Example 3: Yeast on dead aureus (YODA) screening method The inventors sought to establish a robust, easy-to-use and high-throughput screening method for determining the secretion and activity of lysins (e.g. against S. aureus) expressed in eukaryotic cells, e.g. in yeast cells such as P.pastoris.To this end, a yeast on dead aureus (YODA) screening method has been developed (Figure 4A):First, a stationary phase suspension culture of S.aureus was sterilized by autoclaving, which destroys most cellular components with exception of the peptidoglycan layer, causing the autoclaved suspensions to retain its turbidity. The resulting dead S. aureus cell (i.e. peptidoglycan) suspension was washed with water by centrifugation and resuspension and then integrated at an optical density at 620nm of 4.0 into yeast plates supplemented with 50mM NaPO4, pH 6.0, yeast nitrogen base, 0.00004% biotin, 1% methanol, and 1.5% agar. P.pastoris cells expressing (and potentially) secreting lysins from derivatives of the pPIC9K or the pPICZ vectors (ThermoFisher Scientific) are plated on the agar (containing the dead S.aureus) and grown into colonies. Secreted lysins cleave the surrounding peptidoglycan in the agar, causing the vicinity of the colony to become translucent. Thus, colonies secreting active lysins can be easily identified by visual inspection for the presence of a halo. The size of the halo depends on the 181 secretion level while its opacity depends on the enzymatic activity of the expressed lysin. If no halo is observed the lysin is either not secreted, not active or both. In case only yeast (e.g. P.pastoris) cells are plated which are capable of secreting a lysin, the presence or absence of a halo around a colony/clone indicates the secretion of an active or inactive variant from that colony/clone, respectively.Indeed, YODA allowed for the detection of enzymatic activity of a diverse set of lysins with different architectures (Figure 4B). For this purpose, P.pastoris strains expressing various lysins with different architectures were plated on YODA plates at a density of 150 colonies per plate and incubated at room temperature for 3-4 days. Enzymatic activity could be detected in the form of halos around lysin-secreting P.pastoris colonies for different lysin constructs.The principle of the YODA method described above is not limited to Ppastoris as a host or S. aureus as a target. For example, lysins may be also expressed in other yeast model organisms, such as S. cerevisae, as well as in other secreting bacteria, such as B. subtiiis. Furthermore, other bacteria, including both gram-positive and gram-negative bacteria, such as Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, Porphyromonas gingivaiis, Helicobacter pylori, Chlamydia trachomatis, Enterobacteriaceae, E. coii, Klebsiella pneumoniae, Salmonella, Shigella, BoreNia, Campylobacter jejuni, Neisseria gonorrhoeae. Chlamydia trachomatis, Vibrio choierae, and Fusobacterium nudeatum, Staphylococci, Streptococci, Staphylococcus aureus, Streptococcus pneumoniae, Ciostridioides difficile, Cutibacterium acnes, Mycobacterium tuberculosis, Gardnerella, Enterococcus faecaiis, Enterococcus faecium, Lactobacillus iners, Bacillus subtiiis and Bacillus anthracis, may be autoclaved and integrated into the agar depending on the target bacterium of the lysins to be screened.Therefore, the above-describe method is not limited to screening or evaluating lysins against S.aureus but it may be easily modified for screening or evaluating lysins against other bacteria (e.g. other Gram-positives such as, inter alia, Ciostridioides difficile). Thus, analogous methods to the "YODA" employing other bacteria for screening of lysins against these other bacteria are contemplated herein as well and encompassed by the term "YODA". Thus, herein and in context of the present invention, the term "YODA" is not only employed in sensu strictu or necessarily limited to S. "aureud' but may also concern other bacteria. Therefore, the term "YODA" may be replaced herein by the term "Yeast on dead bacteria" ("YODB") where suitable. Furthermore, secreting bacteria such as B. subtiiis may be used instead of yeast, further generalizing the method to "Microorganism on dead bacteria", i.e. "MODB". Thus, the terms "YODA" or "YODB" may be replaced herein by the term "Microorganism on dead bacteria" ("MODE") where suitable.However, in context of lysins against S.aureus, e.g., LO482, as described herein, the term "YODA" usually refers to "Yeast on dead aureus" in a strict sense, i.e., wherein yeast cells are used and dead S. aureus anti] or peptidoglycan thereof is comprised in the agar plates.Previously, a method has been described for screening for active lysin variants secreted from P.pastoris colonies using a double agar layer (DAL) assay. In this DAL method, transformed P.pastoris cells are first grown to colonies on agar plates, followed by overlaying the colonies with a molten low percentage top agar solution containing live S.aureus cells (Zhao (2014). Appl Environ Microbiol 80(9)). Notably, in the DAL method, //Ve(and not dead) S. aureus neWs are employed. Furthermore, in the DAL method, the S. aureus cells are only contained in a distinct agar layer (and not in the entire agar plate, or in a homogenous manner). Further differences are apparent when comparing the above-mentioned YODA method to the DAL method described in (Zhao (2014). Appl Environ Microbiol 80(9)). Due to these differences, the YODA (or "YODB") method of the present invention has several advantages over the DAL method, for example: 182 1. Throughput and plate homogeneity are strongly increased because agar plates can be generated in batch and stored at 4°C for up to 1 month. This also provides an easier handling and is more practical.2. Pouring of top agar solutions in the DAL method dissipates yeast colonies, resulting in contamination of potential hits with other clones. In contrast, no pouring is necessary in the YODA method. Therefore, the YODA method is more reliable, robust and efficient.3. Colonies of interest can be picked, grown in culture and characterized in isolation, since the bacterial peptidoglycan substrate is first sterilized before plate pouring. Hence, the risk of contamination with a target bacterium (e.g. S.aureu^ is strongly reduced or eliminated (compared to the use of //Retarget bacteria as in the DAL method) This further enhances efficiency and handling.4. The YODA method is more sensitive than the DAL assay. While the DAL method can detect strong enzymatic activities, such as those observed for lysostaphin, initially weaker lysins either show only faint or no halos at all. In contrast, these weaker lysins show clear halos in the YODA method. Initial bactericidal activity can therefore be detected early in the lysin development process. The reason is likely that in the DAL, live bacteria need to be killed to generate a halo. In other words, in DAL, the yeast growth and lysin secretion rate need to compete with the growth rate of S.aureusto generate well detectable halos.

Accordingly, the YODA/YODB method of the present invention provides a higher throughput and is more practical, more efficient, less error-prone, more sensitive, more robust, and/or more flexible compared to screening methods in the prior art such as the DAL method.

Therefore, the YODA/YODB method of the present invention allows to evaluate the secretion and/or activity (in particular the activity) of lysin variants in a large-scale and/or high-throughput manner. This is highly useful for screening, identifying and/or validating lysin variants with improved properties, e.g., with a greater activity (against a target bacterium such as, inter alia, S.aureus), improved solubility and/or improved secretion from eukaryotic cells, e.g., human cells. Moreover, the YODA/YODB method can be readily combined with yeast display methods (e.g. as described herein) which further improves the screening and identification of lysin variants with desired properties, e.g., a higher activity, better expression in and/or secretion from eukaryotic cells, and/or better solubility in aqueous solutions.

Although the method described herein above is not necessarily limited to the use of dead bacteria, as living bacteria may be incorporated in the agar as well, the use of dead bacteria is preferred.

Example 4. Generation of aglycosylated LO482 variants Native secretory proteins are typically N-and O-glycosylated during their maturation in the secretory pathway. While secretory proteins are evolutionary adapted to these modifications, transgenes may be glycosylated at sites essential for folding and activity, which could potentially lead to the secretion of a less active or even inactive protein. Therefore, the inventors sought to determine whether glycosylation of lysins in eukaryotic cells (e.g. of lysins expressed from a plasmid or mRNA in eukaryotic cells) has any effect on the activity the lysins. To this end, the inventors employed LO482 (SEQ ID NO: 1) which they found to be produced and secreted in an active form in both, P. pastoris and E. col/see Examples 1 and 2).LO482 contains two motifs of N-glycosylation at residues N68 and N73. Secretory expression of LO482 in P. pastoris results in quantitative N-glycosylation, as determined by SDS-PAGE and PNGase F treatment. It was found that these modifications, i.e. glycosylations, do not render the LO482 lysin secreted from P.pastoris inactive but, 183 nevertheless, reduce the activity of the protein as measured by a growth inhibition assay of S.az/rez/scultures (as described in detail in Example 6 below), i.e. MIC=352pg/ml of the WT LO482 produced in P.pastoris ms. 4pg/ml of the WT LO482 produced in Eco//(Figure 5A).While this data further confirms that the WT LO482 can be secreted in an active form from eukaryotic cells (see Example 2), it also indicates that the activity of lysins such as LO482 may be further improved when produced in eukaryotic cells.Therefore, the inventors removed the two N-glycosylation motifs in LO482 in a degenerate codon screen, which is described in the following (Figure 5B):A degenerate codon library of LO482 was generated in which the codons corresponding to N68 and N73 were randomly substituted by codons of all other amino acids but N (asparagine) and the three stop codons (by using BNC, VNG and DKS codons (International Union of Biochemistry (IUB) codon nomenclature)). These LO482 variants were cloned into the pPIC9K vector with the N-terminal pre- and pro sequences of S. cerevisiae alpha mating factor, followed by a Kex2/Stel3 signal peptidase site, a hexa-histidine tag and a 3C protease cleavage site and transformed into GS115 P.pastoris cells, followed by selection of transformants in yeast minimal growth medium (50mM NaPO4, pH6.0, 0.00004% biotin, 1.34% yeast nitrogen base, 2% dextrose). Transformants were then plated on YODA agar, i.e., agar comprising dead S.aureus (see Example 3). Colonies with halos emerged 3 days after plating and were picked for sequence identification by PCR followed by Sanger Sequencing. The most frequent combinations of aglycosylating mutations (N68N73 to KG, KY, and AH) were picked for further characterization. In addition, further combinations were identified (N68N73 to ML, RE, KL and AA). SDS-PAGE of proteins purified by affinity chromatography from P.pastoris supernatants showed that in contrast to wildtype LO482 (as employed in Example 2 above) which is quantitatively modified with N-glycans, all three mutants assayed (i.e. KG, KY and AH) were in fact aglycosylated (Figure 5C). The KG mutant as shown in SEQ ID NO: 2 (i.e. N68K / N73G, also referred to in the present Examples herein as "L0482ag" or "aglycosylated LO482") was further characterized in a growth inhibition assay (as detailed in Example 6), which showed that after Ecoiiexpression this aglycosylated variant was nearly as active as the WT LO482 (MIC=8pg/ml versus 4pg/ml of the WT LO482); see Figure 5C. Therefore, the KG mutant ("L0482ag"; SEQ ID NO: 2), was used as a starting point for the directed evolution procedure described in Example 5 herein below.

Example 5: Directed evolution of lysins Initial screens indicated low solubility of most lysins of various architectures after expression and purification in £09//(Figure 6). Notably, LO482 (SEQ ID NO: 1) showed at least a moderate solubility which was, however, still suboptimal.Low solubility is expected to strongly reduce the potency of a lysin (such as, for example, a lysin expressed from an mRNA or plasmid in patient cells) when used to treat human infections. A tendency to aggregate may further elicit undesired immune responses, resulting in reduced potency upon repeated administration (Ratanji (2014). J Immunotoxicol 11(2)). Furthermore, the bacterial/phage origin of lysins may result in low expression and secretion levels of lysins when expressed as transgenes (e.g. from an mRNA or a plasmid) in mammalian cells (e.g. in a patient).In an attempt to overcome these issues, the inventors developed a directed evolution approach which allows to simultaneously improve the solubility, activity and eukaryotic secretion of lysins.L0482ag (SEQ ID NO: 2) which has been developed in context of the present invention and which has been shown to have a good killing activity against S.aureus (see Example 4) was employed as a starting point for the directed 184 evolution method described herein below. Improved L0482ag variants obtained by the directed evolution approach (i.e. "hits") are further described and characterized in Example 6 below.

Overview: Yeast display method combined with YODA: A yeast display method was developed and combined with YODA (as described in Example 3) to allow for adaption of lysin variants to the eukaryotic secretory pathway during in vitro evolution and to increase the screening throughput (Figure 7). As already indicated above, adaptation of lysins to the eukaryotic secretory pathway is highly desirable, for example, in a situation when a lysin is administered in form of a plasmid or mRNA ("ribolysin") to a patient. In particular, it is desirable that the lysin is expressed in the patient's cells with high efficiency and secreted from the cells in an active and soluble form.Furthermore, the adaptation to the eukaryotic secretory pathway is also important when a lysin is to be produced recombinantly in a mammalian system which may have certain advantages, e.g. over a prokaryotic expression system. These advantages include the higher protein folding capacity of mammalian cells in comparison to prokaryotic cells. Furthermore, lysins targeted against gram-negative bacteria may be toxic to their prokaryotic expression host, e.g. Ecoii. Moreover, post-translational modifications, which will occur when the lysins are expressed in humans will occur similarly in a mammalian cell culture system but not in prokaryotes.In the present Example, P. pastoris was used as an expression platform for directed evolution because the system allows for rapid activity screening of lysin variants by the YODA method (see Examples 2 and 3). Moreover, the yeast and mammalian secretion pathways are highly conserved (Rapoport (2007). Nature 450(7170)). Therefore, any improved variants produced in P.pastoris and selected based on yeast display combined with YODA likely retain their improved and desired properties (at least to a certain extent) when produced in mammalian cells such as human cells (e.g. from mRNA or a plasmid).Screening of lysin secretion levels was achieved using the yeast display method (Boder (2000). Proc Natl Acad Sci USA 97(20)) adapted to P.pastoris, where lysin variants are secreted and anchored to the yeast cell wall via N- terminal fusion to a fragment of the Sagl-protein (De Schutter (2021). Methods Mol Biol 911). In this setup, yeast cells secreting high levels of such fusion proteins can be enriched by flow cytometry-based cell sorting. When further combined with YODA (or YODB), this method allows screening for lysins with high secretion levels and enzymatic activity. Furthermore, yeast display combined with YODA/YODB can be applied in multiple successive cycles, allowing for a strong reduction of false positives in the list of potential hits.

Library generation A variant library of L0482ag (SEQ ID NO: 2) was generated by the DNA shuffling method (Stemmer (1994). Nature 370(6488)), in which DNA sequences are subjected to DNA fragmentation by DNAsel treatment and randomly recombined by the polymerase chain reaction (Figure 7A). As DNA sequence inputs, L0482ag and 11 homologues of L0482 identified bioinformatically and confirmed for activity in a growth inhibition assay (in lysates of Ecoii expressing the respective variants) were chosen (see Example 1). Additionally, the pool of possible mutations was enriched by including six variants of L0482ag, which had been designed for improved stability by the ROSETTA algorithm (Leman (2020). Nat Methods 17(7)). The residue variations at each position present in the parental library are listed in Table 1: 185 Table 1. Sequence variants of L0482ag per position Parental amino acid sequences were aligned to L0482ag and amino acids found at each position in the alignment are listed (columns 2 and 3, respectively). L0482ag was further aligned to its close natural homologues (all sequences with at least 80% sequence coverage and 60% sequence identity). The fraction of amino acids identical to L0482ag at each position was calculated, as detailed in Example 7, and is shown in the third column "conservation". The LYSM domain is from positions 1 to 51; the linker is from positions 52 to 71; the CHAP domain is from positions 72 to 215. parental position L0482ag inputs conservation R N 0.997E Y 0.989A N 0.990P E 0.943K R 0.940T - 0.993Q - 0.9831 N 0.950Y - 0.997T V 0.963V - 0.997K - 1.000K - 0.963G - 0.990D - 0.983T Y 0.967L - 0.997s G 0.977A - 0.9571 M 0.993A - 0.993L KR 0.947K - 0.980Y F 0.941K N 0.977T - 1.000T - 0.997V - 0.993s - 0.967N K 0.9451 - 0.958Q - 0.997N K 0.935T IL 0.939N - 0.993 186 36 N - 0.9901 - 0.997A K 0.955N - 1.000P - 0.990N - 0.990L F 0.9711 - 1.000F L 0.9481 - 0.977G - 0.997Q ־ 0.993K IQV 0.952L - 0.990K - 0.980V - 0.987P L 0.968M F 0.945T 1 0.942P s 0.942L s 0.948V N 0.942E N 0.929P s 0.939K s 0.945P T 0.875K N 0.735T - 0.862V - 0.873s p 0.699s N 0.936N T 0.945 K N 0.939K R 0.939s R 0.942N 0.939s N 0.936 G s 0.980s N 0.500s - 0.958T - 0.971L - 0.984N - 0.984Y - 0.993L - 1.000 187 81 K NS 0.974T s 0.968L - 0.993E A 0.974N GY 0.952R K 0.946G - 1.000W - 1.000D - 0.993F - 0.997D - 0.997G - 0.990s A 0.936Y - 0.997G - 0.997W N 0.974Q- 0.990c - 1.000F - 1.000100 D - 0.993101 L - 0.997102 V - 0.997103 N - 1.000104 V Y 0.990105 Y - 0.997106 W - 1.000107 N Y 0.993108 H - 0.964109 L - 0.964110 Y - 1.000ill G - 0.997112 H - 0.997113 G - 0.997114 L - 1.000115 K R 0.958116 G - 0.997117 Y - 0.971118 G - 0.981119 A - 0.997120 K - 0.993121 D - 1.000122 1 - 1.000123 P AQTV 0.997124 Y F 0.952125 A E 0.945 188 126 N 1.000127 N - 0.997128 F - 1.000129 N R 0.968130 s GN 0.930131 E - 0.997132 A - 0.997133 K T 0.952134 1 V 0.968135 Y - 0.981136 H KRY 0.942137 N־ 1.000138 T 1 0.993139 P - 0.997140 T D 0.984141 F - 1.000142 K־0.997143 A - 0.993144 E - 0.984145 P L 0.971146 G - 0.997147 D - 0.984148 L IV 0.968149 V AT 0.968150 V - 0.987151 F L 0.997152 s - 0.987153 G NS 0.984154 R - 0.984155 F Y 0.933156 G - 0.984157 G - 0.964158 G - 0.987159 Y F 0.964160 G 0.997161 H 0.997162 T - 0.974163 A - 0.993164 1 ־ 0.997165 V - 0.997166 L - 0.974167 N - 0.993168 G c 0.984169 D N 0.946170 Y - 0.997 189 171 D - 0.997172 G - 0.997173 K HN 0.987174 L - 0.997175 M NQ 1.000176 K H 0.993177 F - 1.000178 Q- 0.971179 s - 0.997180 L - 0.997181 D - 0.987182 Q ־ 1.000183 N - 0.997184 W - 0.990185 N Y 0.971186 N G 0.942187 G ־0.987188 G D 0.971189 W s 0.948190 R - 0.967191 K R 0.974192 A QS 0.820193 E D 0.977194 V 1 0.971195 A - 0.997196 H - 0.990197 K R 0.987198 V - 0.997199 V - 0.997200 H - 0.997201 N - 0.967202 Y - 1.000203 E D 0.967204 N DY 0.938205 D 0.990206 M 0.983207 1 Y 0.941208 F - 1.000209 1 ־ 0.997210 R s 0.990211 P - 0.990212 F Y 0.915213 K - 1.000214 K - 0.987215 A - 0.980 190 DNA encoding the variant libraries fused to an N-terminal signal peptide and a C-terminal V5-epitope tag, followed by the sequence of the SAGI membrane anchor domain were subsequently produced in £co//and transformed into P.pastoris GS115 cells.

Screening procedure As a first step in the screening procedure (i.e. the yeast display step), P.pastoris cells (representing the L0482ag variant library) were selected for high surface display levels of lysins. This was achieved by fluorescence activated cell sorting (FACS) after staining the P.pastoris library with a fluorescently labeled antibody directed against the Vepitope tag (Bio-Rad, cat. no. MCA1360A647) (Figure 7B). The top 0.5-10% of cells with fluorescence levels above a control P.pastoris strain expressing an empty vector were sorted. Next, the lysins contained in the sorted P. pastoris ces were screened for activity by the YODA method as described in Example 3. This can be achieved by extracting the lysin-coding DNA from the sorted P. pastoris cells and cloning it into an expression vector which allows the secretion of the lysins from the yeast cells, followed by transformation into P.pastoris GS115 cells. The P. pastoris cells containing the library of sorted L0482ag variants obtained by the yeast display step are then plated on YODA agar (about 10000 colonies per round of directed evolution). Clones with halos were picked (22 clones with the biggest halos in Round 1, 196 clones with the biggest halos in Round 2 and Round 3). Then, the yeast display was repeated with the P. pastoris cells containing the L0482ag variants (enriched for well active and well expressed and secreted lysins) obtained by the initial yeast display and YODA steps as library. This can be achieved by extracting the lysin DNA from P. pastoris cells obtained by the YODA step and cloning it in bulk as N-terminal fusions to SAGI membrane anchor domain as described above, followed by transformation into P.pastoris GS1Acells. P. pastoris cells expressing the enriched L0482ag variants fused to the SAGI membrane anchor domain were then grown and the cells with the highest surface display levels of lysins were sorted by FACS, followed by plating of the sorted cells on YNB agar plates. Colonies were picked (96 for round 1, 196 for rounds 2 and 3) and lysin DNA was sequenced. A hit table was generated in which the frequency of each lysin sequence was counted. The most frequent sequences were selected for validation and used as the input for DNA shuffling in the following round of directed evolution. (Figure 8A).For the three rounds of directed evolution, the molar amount of DNA input for DNA shuffling was specified in the following way:Round 1: 12 parental sequences in equimolar ratio.Round 2: 75% of the sequences composed of the four top rated hits of Round 1 in equimolar ratio;25% of the 12 parental sequences in equimolar ratio.Round 3: 75% of the sequences composed of the 10 top rated hits of Round 2 in amounts correspondingto the frequencies shown in Figure 9A;20% of the four top rated hits of Round 1 in equimolar ratio; and 5% of the 12 parental sequences in equimolar ratio.The sequences of WT LO482, L0482ag and the hits of round 1 (G1-G4), round 2 (H1-H10) and round 3 (11-130) of the directed evolution are as shown in Table 2 and Figure 9A. 191 Table 2: Sequences of LO482, L0482ag and L0482ag hit variants obtained by directed evolution Identifier SEQ ID NO Identifier SEQ ID NO LO482 WT SEQ ID NO: 1 L0482ag SEQ ID NO: 2G1 SEQ ID NO: 3 G2 SEQ ID NO: 4G3 SEQ ID NO: 5 G4 SEQ ID NO: 6Hl SEQ ID NO: 7 H2 SEQ ID NO: 8H3 SEQ ID NO: 9 H4 SEQ ID NO: 10H5 SEQ ID NO: 11 H6 SEQ ID NO: 12H7 SEQ ID NO: 13 H8 SEQ ID NO: 14H9 SEQ ID NO: 15 H10 SEQ ID NO: 16SEQ ID NO: 17 12 SEQ ID NO: 18SEQ ID NO: 19 14 SEQ ID NO: 20SEQ ID NO: 21 16 SEQ ID NO: 22SEQ ID NO: 23 18 SEQ ID NO: 24SEQ ID NO: 25 110 SEQ ID NO: 26Ill SEQ ID NO: 27 112 SEQ ID NO: 28113 SEQ ID NO: 29 114 SEQ ID NO: 30115 SEQ ID NO: 31 116 SEQ ID NO: 32117 SEQ ID NO: 33 118 SEQ ID NO: 34119 SEQ ID NO: 35 120 SEQ ID NO: 36121 SEQ ID NO: 37 122 SEQ ID NO: 38123 SEQ ID NO: 39 124 SEQ ID NO: 40125 SEQ ID NO: 41 126 SEQ ID NO: 42127 SEQ ID NO: 43 128 SEQ ID NO: 44129 SEQ ID NO: 45 130 SEQ ID NO: 46 After hit identification, coding sequences for the respective hits of each round were cloned into E.coii and mammalian expression vectors for further characterization, as detailed in Example 6.

Example 6: Characterization of improved L0482ag variants resulting from directed evolution Production of LO482 variants in E. coii The hits (i.e. G1-G4 and H1-H10 obtained after rounds 1 and 2, respectively, as described in Example 5) were expressed in Ecolifollowed by purification to assess their stability and enzymatic activity. For this purpose, Ecoli BL21(DE3) cells were transformed with the lysin variants cloned with an N-terminal hexahistidine tag and a 30- protease cleavage site into the pET29b vector (SEQ ID NO: 305; Novagen). The pET29b vector shown in SEQ ID NO: 305 contains, as an example, the sequence encoding the lysin variant "GI" at positions 146 to 790. Further lysins (e.g., G2-G4 and H1-H10) can be and were cloned into the pET29b vector instead of G1 as indicated. Furthermore, the pET29b vector contained, inter alia, the following elements in SEQ ID NO: 305: a T7 promoter/ 192 Lac Operator at positions 1 to 42, the start codon (for the polypeptide containing the lysin) at positions 89 to and the corresponding stop codon at positions 791 to 793, an hexahistidine tag at positions 95 to 112, a 30 protease cleavage site at positions 113 to 145, and an 17 terminator at positions 892 to 931.Transformed cultures were grown in 150ml LB medium supplemented with Kanamycin (for antibiotic selection of the pET29b vector) to an optical density at 600nm of 0.6. At this point, protein production was induced by addition of 0.5mM IPTG. Expression was continued at 18°C for 24h, followed by harvest of the cells by centrifugation.

Purification of LO482 variants from E. coli The cells were then resuspended in 100ml lysis buffer (20mM Hepes, pH 7.0, lOmM MgCI2), supplemented with ImM PMSF and Sul benzonase (VWR, 70746-3), followed by sonication (20% duty cycle, 4min, 4oC). Crude lysates were incubated for 5min on ice. The lysates were then supplemented with 300mM NaCI, followed by centrifugation (HOOOxg, 4°C, 30min). Supernatants were incubated for Ihr rotating with 1ml of NiNTA resin (VWR,17-5318-01) which had been equilibrated in lysis buffer. NiNTA resin was washed twice with 50ml wash buffer (20mM Hepes, pH 7.0,300mM NaCI, 20mM Imidazole). Bound protein was eluted with elution buffer (20mM Hepes, pH 7.0,300mM NaCI, 300mM Imidazole; 10ml final volume) and concentrated using lOkDa molecular weight cutoff spin filters (Amicon). The protein concentration was determined using the Bradford reagent and eluates were supplemented with one molar percent of precision protease in order to remove the 6H tag. 3C treated eluates were then dialyzed for 16h at room temperature against 2L of final storage buffer (20mM Hepes, pH 7.0, 150mM NaCI). Precipitates were removed using spin filters with a pore size of 0.22pm. Final protein concentrations were determined using the Bradford assay.

Determination of protein stability of LO482 variants bv the Thermofluor assay Protein stability was determined using the Thermofluor assay. 19pl lysin solutions were mixed with ipl of a 20x solution of SYPRO Orange (Thermo, 56651) in DMSO. The mixtures were transferred to the wells of a 384 well plate. Changes in fluorescence of the SYPRO Orange fluorophore were measured in a qPCR machine (Roche, LightCycler 480) while applying a temperature ramp protocol between 23°C and 95OC. These melting curves were then analyzed in Prism 9.0 (GraphPad) in the following way: The first derivatives were determined with data smoothing across 35 consecutive data points. The derivatives were then fit to Gaussian curves. The mean value of these fits, which corresponds to the inflection point of the melting curve and the temperature, at which 50% of the lysin molecules are unfolded, was defined as the melting temperature Tm.In these experiments, the fluorescence of the SYPRO orange dye did not change drastically in the presence of the parental L0482ag, likely because the majority of the protein was in an unfolded state at the lowest temperature (23°C). For these reasons, no melting temperature could be determined for L0482ag (Figure 8B). In contrast, the fluorescence of the SYPRO Orange dye showed strong changes in response to increases in temperature in the presence of the other purified variants. In these cases, melting temperatures between 37 and 47°C could be determined (Figure 8B). Taken together, these data show that all characterized hit variants (i.e. G1-G4 and Hl- H10; SEQ ID NO: 3 to 46, respectively) have an increased thermal stability over their parental sequence. Assuming that the melting temperature of L0482ag is maximally 23°C, one can conclude that the thermal stability of the variants has been improved by 14-25OC during two rounds of directed evolution. 193 Determination of bactericidal activity of LO482 variants For measurement of bactericidal activity, purified lysin variants (and dilutions thereof) were incubated in 30 pl of final storage buffer mixed with methicillin-resistant S.aureus ATCC43300 cells in 70 pl cation adjusted Muller-Hinton broth ("caMHB"; Merck 90922) medium supplemented with 25% horse serum (Thermo, 26050070) such that the reaction contained 5xl05 cfu/ml. The optical density of these solutions at 620nm was monitored in a plate reader (Tecan Infinite 200 Pro) over the course of 24 hours at 37OC to quantify bacterial growth. The minimal inhibitory concentration (MIC) was determined as the lowest lysin concentration, at which the OD of the reaction at 620 nm stayed below 0.1 over the course of the 24h incubation at 37°C. These experiments showed a MIC for L0482ag of 8pg/ml. All variants showed absence of S.aureus growth at 2 to 16 fold lower lysin concentrations. The lowest MICs were observed at 0.5pg/ml for variants H5 (SEQ ID NO: 11) and H7 (SEQ ID NO: 13) (Figure 8C).

Determination of secretion of LO482 variants from human cells To measure the efficiency at which lysin variants can be secreted after expression in HEK cells (in particular EXPI2cells), variants were optimized for human expression using the GeneArt codon optimization tool and cloned with an N-terminal mouse IgKappa signal peptide (MDMRAPAGIFGFLLVLFPGYRS; SEQ ID NO: 300), followed by the dipeptide (TG) and a 6H-3C tag into the pCDNA3.4 vector (SEQ ID NO: 306; ThermoFisher Scientific). The pCDNA3.4 vector shown in SEQ ID NO: 306 contains, as an example, the sequence encoding the lysin variant "GI" at positions 871 to 1515. Further lysins (e.g., G2-G4 and H1-H10) can be and were cloned into the pCDNA3.4 vector instead of G1 as indicated. Furthermore, the pCDNA3.4 vector contains, inter alia, the following elements in SEQ ID NO: 306: a CMV promoter at positions 47 to 727, a Kozak sequence at positions 742 to 751, a start codon at position 748 to 750, a signal peptide (mouse IgKappa) at positions 748 to 813, the sequence containing the lysin at positions 871 to 1515 and a corresponding stop codon at positions 1516 to 1518, an hexahistidine tag at positions 820 to 837, a 3C protease site at positions 838 to 870, and a WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) at positions 1559 to 2155.Plasmids (i.e. pCDNA3.4 vectors) were transfected at a final concentration of Ipg/ml in 3.2xlOA6 cells per ml of an Expi293 culture in Expi293 medium (Thermo, A14527 and A1435101, respectively) using the Expifectamine transfection kit as described by the manufacturer. Cells were grown shaking at 5% CO2 and at 37°C. Transfected cultures were supplemented with Enhancer solutions 1 and 2 24h after transfection according to the manufacturers instructions. Cells were removed after 72h by centrifugation (300xg, 5min) and the supernatants used for further analysis.Supernatants were analyzed by SDS-PAGE followed by Western blotting. Lysin variants were stained using a primary antibody raised against the 3C tag (Abeam, abl83574), followed by staining with a secondary antibody coupled to HRP (Thermo, G-21234). Stained proteins were detected using the ECL reagent (Thermo, 32106). Band intensities were background corrected and then quantified using ImageJ (NIH).Analysis of the levels of protein secretion from HEK (i.e. EXPI293) cells showed that parental L0482ag (SEQ ID NO: 2) was either not secreted or secreted at levels that could not be detected (Figure 8D). In contrast, variant G1 (SEQ ID NO: 3) could be detected in the supernatants, albeit at low levels. Relative to G1, most of the variants H1-H(SEQ ID NO: showed an improved secretion by up to 10 fold. These data indicate that the parental L0482ag is barely secreted from HEK cells, most likely due to its unfavorable stability. After one round of directed evolution, in which L0482ag variants had been selected for increased secretion in a eukaryotic cell, hit G1 could be secreted 194 from HEK cells in detectable amounts. After the second round of directed evolution, most of the hits H1-H10 (SEQ ID NO: 7 to 16, respectively) showed further enhancements of secretion by up to 10 fold relative to G1.To further test if the improved lysin variants are secreted in an active form, 30 pl of the HEK supernatants were incubated with S.aureus ATCC43300 cells in 70 pl cation adjusted Muller-Hinton broth (caMHB) medium supplemented with 25% horse serum such that the reaction contained 5xl05 cfu/ml, 5xl04 cfu/ml, 5xl03 cfu/ml and 5xl02 cfu/ml. The optical density of these solutions at 620nm was monitored in a plate reader over the course of 24 hours at 37°C to quantify bacterial growth.These experiments showed that supernatants transfected with variants H5 and H3 were able to prevent growth of 5xlOA4 and 5xlOA2 S.aureus cells during the course of 24h, demonstrating that the proteins are secreted in an active state from EXPI293 cells (Figure 8E).

Determination of killing kinetics of LO482 variants by an OD reduction assay The ability of LO482 variants obtained by directed evolution to kill live S. aureus cells was further assessed in an OD reduction assay. An overnight culture of S. aureus ATCC43300 was grown at 37° and diluted 1:100 in Brain- Heart Infusion medium (BHI) the next morning. Bacteria were grown to an optical density at 595 nm of approximately 1, washed once with PBS and readjusted to an OD of 1. WT LO482 (SEQ ID NO: 1) and the variant H5 (SEQ ID NO: 11) were produced and purified as described in this Example 6 and of each lysin a 10 fold stock preparation in PBS was incubated for 3 hours at 37°C. 20 pl per lysin or PBS (negative control) were then added to 180 pl of bacterial suspension to obtain a final concentration of 16 pg/ml per lysin, and the reactions were immediately placed into a Tecan Reader (TECAN infinite plate reader) for the measurement of OD595 every seconds. It was found that both the WT LO482 (SEQ ID NO: 1) and the variant H5 (SEQ ID NO: 11) killed logarithmic phase bacteria effectively. However, "H5" displayed faster killing kinetics than the WT LO482 lysin; see Figure 11.

Determination of anti-biofilm activity of LO482 variants The activity of LO482 variants against staphylococcal biofilms or free-floating biofilm-like aggregates was tested with an assay employing a peg biofilm in plasma or a free-floating aggregate in synovial fluid, respectively. The peg biofilm in plasma assay (PBA) mimics the situation of biofilms as sites of infection such as, for example, in infective endocarditis. The free-floating biofilm-like aggregate in synovial fluid assay (FBA) mimics the situation in joint infections (e.g. prosthetic joint infections).

Peg biofilm in plasma assay (PBA)Disruption of staphylococcal biofilms was assessed using the MBEC Assay® Kit (Innovotech). Overnight cultures of S. aureus strain ATCC 43300 were grown in cation-adjusted Muller-Hinton broth (caMHB, Sigma) at 37°C, 220rpm shaking and adjusted the next morning to an optical density of 0.1 in caMHB supplemented with 10% human Li- Heparin plasma (Innovative Research), previously dialysed in PBS (Thermo Scientific) using a 10 kDa dialyses cassette (Pierce). 100 pl of this bacterial suspension was dispensed into each well of the 96 well plate. The plate lid with 96 pegs attached, was immersed into these wells and biofilms were grown statically for 24 hours at 37°C. After 3 washes with PBS, the pegs were immersed into 100 pl of treatment solutions containing lysins or appropriate controls. Lysins at 50 pg/ml or vancomycin at 256 pg/ml in caMHB supplemented with 50% human Li-Heparin plasma, were incubated for a duration of 2 hours (static at 37OC). The treatment solution was removed and fresh treatment was applied and incubated again for 2 hours at 37°C after which the pegs were washed 3 times with 195 PBS. Biofilm dislodgment was performed by immersing the pegs in 100 pl of TrypLE™ (Gibco) for 20 min and stopping the reaction by the addition of 100 pl of PBS. Quantification of bacterial counts was performed by plating serial dilutions (in saline) on tryptic soy agar plates.

Free-floating biofilm-like aggregate in synovial fluid assay (FBA)The ability to disrupt floating biofilm-like aggregates in bovine synovial fluid was assessed. Overnight cultures of S. aureus stram ATCC 43300 were grown in BHI (Carl Roth) at 37OC, 220 rpm shaking. The next morning, optical density of the bacterial culture was adjusted to 0.1 in caMHB (Sigma) supplemented with 50% bovine synovial fluid (Dunn Labortechnik) and 200 pl of this bacterial suspension was dispensed into single wells in a 96 deep well plate (Nunc™ Non-Treated Multidishes). The plate was incubated for 24 hours @37OC, 500 rpm to allow for the formation of floating biofilm-like aggregates. Treatment was performed by the addition of 22 pl of a 10 fold concentrated stock of lysin or controls, providing a final lysin concentration of 50 pg/ml and a final vancomycin concentration of 256 pg/ml, respectively. After 2 hours of incubation, another 22 pl of treatment was added and incubation was prolonged for 2 hours at 37°C, 500 rpm shaking. The wells were washed as follows: addition of 800 pl PBS per well followed by centrifugation at 2560xg for 20 min. The supernatant was removed and the wash step repeated. Dislodgment of the biofilm-like aggregates was performed by the addition of 100 pl of TrypLE™ (Gibco), incubation for 20 min @ 37°C followed by the addition of 100 pl of PBS. Quantification of bacterial counts was performed by plating serial dilutions (in saline) on tryptic soy agar plates.

ResultsIt was observed that Vancomycin, an antibiotic that is used in the clinics against S. aureus infections and which is active against planktonic bacteria (with a MIC around 1 pg/ml depending on the strain), showed very little or no activity against S. aureus biofilms or free-floating aggregates, respectively, even at a very high concentration of 256 pg/ml. In contrast, both LO482 variants tested, i.e., G1 (SEQ ID NO: 3) and H5 (SEQ ID NO: 11), effectively killed S. aureus biofilms or biofilm-like free-floating aggregates; see Figure 12.

The LO482 variant H5 showed a 2.32-log (i.e. 99.52%) reduction of bacterial cell counts vs. vancomycin and a 3.log (i.e 99.98%) reduction vs untreated biofilms in the PBA assay, which was both statistically highly significant.

In addition, the LO482 variant H5 showed a 3.17-log (i.e. 99.93%) reduction of bacterial cell counts vs. vancomycin and a 3.03 log reduction vs untreated aggregates, (i.e. 99.9%) in the FBA assay which, again, was both statistically highly significant.

The LO482 variant G1 showed a 1.54-log (i.e. 97.11% killing) reduction vs vancomycin and a 2.91 (99.98% killing) log reduction vs untreated reactions which was also both statistically highly significant. The activity of G1 in the FBA assay was not determined.

This demonstrates that both tested LO482 variants obtained by the directed evolution, i.e. G1 and H5, have a high bactericidal activity against S. aureus biofilms while H5 is even more effective than G1. 196 Summary Taken together, the characterizations show that two rounds of directed evolution have yielded strongly improved variants with increased thermal stability and enzymatic activity over L0482ag, while at the same time enabling the secretion from human cells. The two tested LO482 variants H5 and G1 further showed high killing activity against staphylococcal biofilms/free-floating biofilm-like aggregates. H5 was further shown to have enhanced killing kinetics against S. aureus relative to the WT LO482. Moreover, most of the hit variants obtained by the directed evolution had an improved solubility as compared to L0482ag.

Example 7: Identification of conserved positions and segments and beneficial mutations in LO482 variants Homologues of L0482ag were identified in the following manner: A BLAST search against the amino acid sequence of L0482ag (lacking the initiating amino acid methionine) was performed against the NCBI nucleotide database. The 5000 closest homologues of L0482ag were downloaded. Sequences with a coverage below 80% and a sequence identity below 60% were excluded from this selection. The remaining 621 sequences were aligned to L0482ag using Clustal Omega and the fraction of identities to L0482ag per amino acid position was determined using Geneious Prime (Dotmatics, version 2022.2.2).Furthermore, amino acid sequences of input lysins from each round of directed evolution were aligned to L0482ag using the Clustal Omega algorithm. The alignments were then exported into Microsoft Excel to list the amino acids at each position in the lysin sequences. Similarly, amino acid sequences of all variants identified in directed evolution rounds 1 to 3, which had been confirmed to be active by YODA (including but not limited to the G1-G4, H1-Hand 11-130 hit variants described herein), were aligned to L0482ag using Clustal Omega and analyzed by Excel to list the amino acids at each position in the lysin sequence. A combination of the two lists with addition of beneficial aglycosylation mutations identified in Example 4 allowed identification of mutations that can be considered as "beneficial", or at least "permissive", when present in isolation or in combination (Table 3).Moreover, the list of beneficial/permissive mutations was compared with the fraction identities to L0482ag per amino acid position (i.e., the % of the analyzed 621 natural variants having the same amino acid residue at a certain position as WT LO482).It was found that none of the considered variants (either generated upon directed evolution, used as input variant for the directed evolution, or one of the other assessed 621 natural variants) had a variation/mutation at the following positions of L0482 (SEQ ID NO: 1 or 2): 12, 80, 87, 88, 98, 99, 103, 106, 110, 114, 122, 126, 128, 137, 182, 202, and 208 (marked by black fields in Table 3). These positions of SEQ ID NO: 1 are thus considered as conserved positions. Notably, 16/17 of these conserved positions are within the CHAP domain (i.e. positions 72 to 215). Moreover, it has been found that 11/16 of these conserved positions in the CHAP domain are within positions to 128 in SEQ ID NO: 1 and that this CHAP segment contains many further relatively conserved positions (marked by grey fields in Table 3). Said CHAP segment (i.e. positions 87 to 128 in SEQ ID NO: 1) is thus considered as a particularly conserved CHAP segment.

Table 3. Identification of beneficial/permissive mutations and conserved positions in LO482 variants The column "beneficial/permissive residues" shows amino acid substitutions vis a vis WT LO482 (SEQ ID NO: 1) which are contained in LO482 variants that have been generated in context of the present invention and found to be secreted from yeast cells in active form (in the deglycosylation screen (Example 4) or the directed evolution 197 rounds 1-3 (Example 5)) and/or which were contained in the input variants for the directed evolution. Residues which were merely contained in the input variants are shown in parentheses "()". Mutations shown without parentheses are considered as more beneficial than those shown in parentheses.The column "conservation" shows the % of 621 assessed natural variants of LO482 having the same amino acid residue per position as L0482ag (SEQ ID NO: 2), as also shown in Table 1. The column "conserved position" shows in black fields positions where no mutation/variation was identified at all, and in grey fields positions where no mutation/variation was identified in the input variants or active and secreted variants generated experimentally herein and no variation was observed in at least 99.0% of the 621 natural variants assessed. The LYSM domain isfrom positions 1 to 51; the linker is from positions 52 to 71; the CHAP domain is from positions 72 to 215. A particularly conserved segment has been found within positions 87 to 128. position beneficial/ permissive residues conservation conserved position position beneficial/ permissive residues conservation conserved position NQW 0,997 23 NR 0,980Y 0,989 24 CHN(F) 0,941N 0,990 25 ER(N) 0,977E 0,943 26 A 1,000R 0,940 27 A 0,997IP 0,993 28 A 0,993- 0,983 29 - 0,967N 0,950 30 DS(K) 0,945- 0,997 31 - 0,958AV 0,963 32 R 0,997Al 0,997 33 DS(K) 0,935- 1,000 34 KL) 0,939ER 0,963 35 - 0,993- 0,990 36 DIS 0,990- 0,983 37 LV 0,997M(Y) 0,967 38 G(K) 0,955F 0,997 39 is 1,000LP(G) 0,977 40 LQS 0,990SV 0,957 41 DST 0,990MV 0,993 42 (F) 0,971- 0,993 43 TV 1,000(K)QR 0,947 44 LS 0,948 position beneficial/ permissive residues conservation conserved position TV 0,977- 0,99718111^L 0,993EIQR(V) 0,952-0,990 position beneficial/ permissive residues conservation conserved position (T) 0,945KRMA 0,939(R) 0,939G(R) 0,942DS 0,939 198 50 1 0,980 72 F(N) 0,936- 0,987 73 GSYLEAH 0,980(L) 0,968 74 NP 0,500(T)F 0,945 75 FP 0,958Al 0,942 76 s 0,971HS 0,942 77 - 0,984I(S) 0,948 78 SY 0,984D(N) 0,942 79 - 0,993K(N) 0,929 80 - 1,000(5) 0,939 81 ER(N)S 0,974EI(S) 0,945 82 s 0,968LQ(T) 0,875 83 - 0,993EN 0,735 84 (A) 0,974AM 0,862 85 G(Y) 0,952AL 0,873 86 KM 0,946GNR(P) 0,699 87 - 1,000R(N) 0,936 88-1,000 position beneficial/ permissive residues conservation conserved position position beneficial/ permissive residues conservation conserved position -0,993 ill s 0,997- 0,997 112 - 0,997w- 0,997 113 s 0,997- 0,990 114 - 1,000AL 0,936 115 R 0,958- 0,997 116 - 0,997- 0,997 117 H 0,971N 0,974 118 - 0,981- 0,990 119 - 0,997- 1,000 120 - 0,993•- 1,000 121 V 1,000100-0,993 122-1,000(A)(Q)(T)(V101 - 0,997 123 ) 0,997102 - 0,997 124 FH 0,952103 - 1,000 125 V(E) 0,945104 I(Y) 0,990 126 - 1,000105 - 0,997 127 - 0,997106 - 1,000 128 - 1,000107 SY 0,993 129 RS 0,968108 R 0,964 130 GIN 0,930109 - 0,964 131 - 0,997110-1,000 132-0,997 beneficial/ conserved beneficial/ conserved 199 position permissive residues conservation position position permissive residues conservation position 133 RT 0,952 155 Y 0,933134 TV 0,968 156 - 0,984135 CH 0,981 157 E 0,964136 KR(Y) 0,942 158 - 0,987137 - 1,000 159 F 0,964138 1 0,993 160 - 0,997139 - 0,997 161 - 0,997140 AD 0,984 162 - 0,974141 L 1,000 163 - 0,993142 ER 0,997 164 - 0,997143 - 0,993 165 - 0,997■I■■■144 D 0,984 166 - 0,974145 AL 0,971 167 - 0,993146 - 0,997 168 (C) 0,984147 - 0,984 169 N 0,946148 IV 0,968 170 - 0,997149 AT 0,968 171 - 0,997150 - 0,987 172 - 0,997151 (L) 0,997 173 HNR 0,987152 N 0,987 174 - 0,997153 NS 0,984 175 NQT 1,000154-0,984 176 ER(H) 0,993 position beneficial/ permissive residues conservation conserved position 177 L 1,000178 LR 0,971179 - 0,997180 - 0,997181 - 0,987182 - 1,000183- 0,997184 - 0,990185 FHSY 0,971186 G 0,942187 - 0,987188 (D) 0,971189 (5) 0,948190 Q 0,967191 ER 0,974192 QT(S) 0,820193 D 0,977 position beneficial/ permissive residues conservation conserved position 199 A 0,997200 - 0,997201 DS 0,967202 - 1,000203 DV 0,967204 DSY 0,938205 - 0,990206 - 0,983207 TV(Y) 0,941208 - 1,000209 - 0,997210 (5) 0,990211 - 0,990212 Y 0,915213 RT 1,000214 QT 0,987215 GVS 0,980 200 194 A(I) 0,971195- 0,997196 - 0,990197 R 0,987198 -1 0,997 Next, the inventors identified all mutations occurring in the selected hit variants obtained in rounds 1 to 3 of the directed evolution, i.e., G1 to G4, Hl to H10 and 11 to 130; see Table 2 and SEQ ID NO: 3 to 46. These particularly beneficial mutations are shown in Table 4.

Furthermore, to identify the most beneficial mutations, a consensus sequence was generated using Geneious (Dotmatics, version 2022.2.2) by determining the most frequent residues at each position in the alignment of all selected hit variants (see Figure 9 and black highlights in Table 4). In this analysis, the variant H3 (SEQ ID NO: 9) was found to reflect the consensus sequence. Inspection of the sequence alignment further showed that variant H3 surprisingly contained certain amino acid substitution pairs and individual amino acid substitutions that were present in different combinations in the selected hits. These substitutions pairs or individual substitutions are considered herein as the consensus mutation units. In particular, the following consensus mutation units were found:1. R86K2. T82S and N85G in combination3. S130N and H136K in combination; alternatively, S130N and H136R in combination4. D169N5. N185Y and N186G in combination Furthermore, it has been surprisingly found that all selected hit variants contained the mutation R86K. Moreover, the mutation R86K was further found in 96.08% of all sequenced active and secreted L0482ag variants obtained by the three rounds of directed evolution. This shows that the mutation R86K in reference to SEQ ID NO: 1 or 2 is a particularly important amino acid substitution among the most beneficial mutations in order to improve the pharmaceutical properties of LO482 variants.Since variants H3 (SEQ ID NO: 9) and H5 (SEQ ID NO: 11) were among the variants which showed the best properties in terms of stability, enzymatic activity and secretion in mammalian cells and since all selected and further characterized variants showed strong improvements in these aspects relative to L0482ag (see Figure 8B- E), the individual mutations, and in particular the consensus mutation units, identified above can be deemed to have additive or synergistic beneficial effects on the lysin variants.Furthermore, variant H5 (SEQ ID NO: 11) contained the additional mutation F155Y, which is deemed highly beneficial since F155Y is the only difference between H3 and H5, the latter of which showed better enzymatic activity after secretion from EXPI293 cells (see Figure 10 and the grey highlight in Table 4).

Table 4. Identification of particularly beneficial mutations contained in the selected hit variants obtained in rounds 1 to 3 of the directed evolution. Shown are mutations per position contained in at least one of the variants G1 to G4, Hl to H10 and 11 to 130, i.e. the hits, as described in Table 2 and SEQ ID NO: 3 to 46. Mutations found in G1 to G4 refer to round 1 (R1) mutations, mutations found in Hl to H10 refer to round 2 (R2) mutations and mutations found in 11 to 1 30 refer 201 to round 3 (R3) mutations. Positions which were not modified in G1 to G4, Hl to H10 and 11 to 130 are not shown. Highlighted in black are the most beneficial mutations contained in the consensus sequence, as reflected by H(SEQ ID NO: 9). Highlighted in grey is another one of the most beneficial mutations, namely the only amino acid substitution in the best performing variant characterized, i.e. H5 (SEQ ID NO: 11), vis a vis H3. All variants G1 to G4, Hl to H10 and 11 to 130 contained a "K" at position 68 and a "G" or "S" at position 73. Most variants, including H3 and H5, contained a "K" at position 68 and a "G" at position 73, like the parental L0482ag. The LYSM domain is from positions 1 to 51; the linker is from positions 52 to 71; the CHAP domain is from positions 72 to 215.hit mutations hit mutationsin LYSM domain and linker in CHAP domainposition Ri and R2 R3 R1,R2,R3 position Ri and R2 R3 R1,R2,R3- W W 73-s sN N N 75- F FV V V 78 - Y Y- E E 81 - E ER R R 82 s s sC C C 85 G G G- E E 86 K K K- D D 104 - 1 1- D D 107 Y Y Y- L L 115 R R Rs s s 124 F F F- T T 125 - V V- s s 130 N N N- T T 133 - R R- H H 135 - H H-s s 136 KR KR KR- K K 140- A AT T T 141 - L L-G G 155169 N N N173 N N N175 Q Q Q178 L L185 Y Y Y186 G G G191-R R192 Q Q Q194 A A198 1 1204 s s s212 Y Y Y215-s s 202 Furthermore, the inventors checked whether the particularly beneficial mutations occurred at the active site of LO482, i.e., the active site surface around the catalytic cysteine. To this end, the inventors inspected a published X-ray structure of a CHAP domain in a lysin of the CHAP-AMI-SH3 architecture (LysK) which cleaves S.aureus peptidoglycan; Sanz-Gaitero et al. Virology Journal 2014, 11:133. Based on visual inspection of the residues surrounding the catalytic cysteine in the X-ray structure of said LysK CHAP domain and comparing its amino acid sequence to the sequence of the CHAP domain of LO482, the following positions are thought to form the active site of L0482: positions 94, 97, 99, 117, 118, 119, 156, 159, 160, 182, 183, 184, 186, and 189 of SEQ ID NO: 1.

Interestingly, only one of the particularly beneficial amino acid substitutions (i.e. N186G) contained in the hit variants is located at the active site of the LO482. This individual active site mutation is also contained in the Hvariant (SEQ ID NO: 11) obtained in round 2 of the directed evolution and is therefore among the most beneficial amino acid substitutions found in context of the present invention. Notably, however, also the third round of the directed evolution did not yield any further mutation within the active site of the LO482 variants. Based on the directed evolution data, it appears that the amino acid substitution N186G is the only tolerated mutation within the active site of LO482 lysins which enhances the killing activity against S. aureus, the protein stability and/or the secretion of the lysin from eukaryotic cells. In fact, almost all (31/32) positions which were mutated in the CHAP domain of the hit variants found by three rounds of directed evolution are outside the active site. Hence, the active site is a region of the CHAP domain of LO482 variants that is barely amenable as a target for improving the killing activity and/or other pharmaceutical properties like the secretion from eukaryotic cells.

Example 8: Re-glycosylation of lysin variants Protein glycosylation has been shown to increase protein stability and solubility for eukaryotic proteins (Shental- Bechor (2009). Curr Opin Struct Biol 19(5)). Therefore, one or more identified hit variants from in vitro evolution will be subjected to rational re-glycosylation engineering using a structural model obtained with AlphaFold 2.(Jumper, Evans et al. 2021) as the template. Single amino acid substitutions of surface exposed residues, particularly in loops connecting alpha helices and/or beta sheets (such as those found at residues: 1-5, 11-17, 35- 46, 24-27, 52-72, 84-97, 110-131, 136-146, 153-160, 167-176, 181-195, 202-207 and 210-215), will be mutated to either N or S/T to generate NXS/T glycosylation motifs (where X corresponds to any amino acid but proline). The variants will be interrogated for HEK293 expression/secretion levels, stability and functional activity (e.g., as described in Example 6 above). Variants with engineered single N-glycosylation motifs may be combined and validated in the same manner.

Example 9: Half-life extension of lysin variants Endolysins are small proteins of bacterial or phage origin with relatively short half-lifes in humans (minutes to hours; Cassino (2016), "Results of the First in Human Study of Lysin CF-301 Evaluating the Safety, Tolerability, and Pharmacokinetic (PK) Profile in Healthy Human Volunteers", presented at 26th European Congress of Clinical Microbiology and Infectious Diseases Apr 9-12, 2016). It is contemplated in the context of the present invention that half-life extension modules (i.e. an "extended pharmacokinetic (PK) peptide" / "PKtag"), such as human Fc fragment (e.g. as shown in SEQ ID NO: 294), HSA binding domain, glycosylated peptide tags (e.g. C-terminal peptide of human chorionic gonadotropin (CTP), for example, as shown in SEQ ID NO: 295) or human lysozyme (e.g. as shown in SEQ ID NO: 296) will be fused to the N-terminus or C-terminus (preferably the N-terminus) of selected endolysins obtained by in vitro evolution (see, e.g. Examples 5 and 6 above). Furthermore, the half-life 203 extension module and the lysin are preferably connected by a peptide linker, e.g. a flexible glycin-serine linker, for example as shown in SEQ ID NO: 297 to 299. The fusion proteins will then be interrogated for HEK2expression/secretion level, stability and functional activity (e.g., as described in Example 6 above). For selected constructs the pharmacokinetics in mice will be determined.

Example 10: Deimmunization of lysin variants Phage and bacteria derived endolysins may pose immunogenicity risks, particularly for chronic or repeated systemic applications, due to presence of T-cell epitopes in their amino acid sequences (Zhao (2015). Chern Biol 22(5)). It is therefore contemplated in the context of the present invention that one or more identified lysin hit variants (e.g. as described in Example 6 above) will be subjected to de-immunization in the following way:1. T-cell epitopes will be determined experimentally via MAPPs and/or T-cell activation assays with human PBMCs selected to represent the global HLA distribution. Alternatively or in addition, T-cell epitopes will be predicted using in siiico algorithms such as NetMHCII Pan (Reynisson (2020). J Proteome Res 19(6)).2. A de-immunized endolysin sequence library will be obtained using a Rosetta based algorithm (Rosetta MHC, Yachnin (2021). J Chern Inf Model 61(5)) and as input the experimentally defined T-cell epitopes and structural models of the lead lysins determined by Alphafold 2.0.3. The de-immunized DNA library will be synthesized and expressed in P. pastoris as described in the above Example, e.g., Example 4. Hits with acceptable surface expression levels and YODA activity will be validated experimentally for decreased T-cell epitope presentation in vitro and immunogenicity in vivo.

As a first step, the inventors have generated deimmunized variants of the best performing characterized L0482ag variant, i.e. H5 (SEQ ID NO: 11) in siiico by using, inter alia, the Rosetta based algorithm. The Rosetta based algorithm allows to design deimmunizing mutations that maintain the calculated stability of the protein. It is thus credible that other advantageous properties the lysin (e.g. H5) had before deimmunization, e.g. the bactericidal activity, stability and ability of being secreted from eukaryotic cells, are maintained upon deimmunization according to the invention.In siiico generated deimmunized variants of H5 are shown in SEQ ID NO: 52 to 251. In siiico determined deimmunizing amino acid residues per position are shown in Table 5.

Table 5. Z/7s/7/codetermined deimmunizing amino acid residues. The three dimensional structure of variant H5 was predicted using Alphafold 2.0. The structure and the multiple sequence alignment generated by Alphafold 2.0 were used as the input for ROSETTA-MHC design runs, in which HLA type 1 and HLA type 2 epitopes were mutated while maintaining the overall stability of the protein. Shown are amino acids found at each position which were introduced as deimmunizing mutations in a library of 200 designed variants ("deimmun"). The column "H5" shows the amino acid sequence of H5 (i.e. SEQ ID NO: 11). The LYSM domain is from positions 1 to 51; the linker is from positions 52 to 71; the CHAP domain is from positions 72 to 215. pos H5 deimm pos H5 deimm pos H5 deimm R Q 26 T - 51 V TE - 27 T - 52 P sA -V DEQST 53 M AGKQS 204 4 P - 29 s D 54 T DEHNK - 30 N ADEQ 55 P DT - 31 1 - 56 L -Q- 32 Q AKM 57 V -1 - 33 N ADEGKQ 58 E -Y - 34 T AW 59 P -T -N -K -V - 36 N DGQ 61 P -K DGQS 37 1 DKLMNQ 62 K -K DENP 38 A KS 63 T -G - 39 N ADG 64 V -D -P DGNST 65 s DT - 41 N D 66 s-L AIM 42 L DHKMNQS 67 N -s - 43 1 LV 68 K sA DHKNQ 44 F DEHSW 69 K GHNQ1 - 45 1 ADEKPQT 70 s -A - 46 G - 71 N sL ADGHKQRW 47 Q- 72 s DHTK NQW 48 K DENQ 73 G DNSTY HT 49 L IMN 74 s-K DGHQS 50 K DHNT 75 s DQ pos H5 deimm T DNL ADGHKNQSTN -Y HWL ADMNK AGSs GL MQE -G -K YG-W -D -F -D -G-s-Y -G -W- pos H5 deimm 101 L -102 V AGST103 N AG104 V DMQY105 Y -106 W -107 N AS108 H GKY109 L -110 Y -ill G-112 H -113 G -114 L QT115 K DGS116 G-117 Y DN118 G -119 A-120 K-121 D- pos H5 deimm 126 N -127 N -128 F-129 N -130 N GY131 E NQY132 A -133 K QT134 1 KQT135 Y HS136 K EQT137 N G138 T -139 P ADQS140 T D141 F DHT142 K -143 A p144 E Q145 P -146 G- 205 97 Q- 122 1 LC-123 P DSTWF KMQSTV 124 Y QT100 D-125 A- 147 D -148 L -149 V -150 V- pos H5 deimm 151 F-152 s-153 G-154 R -155 Y -156 G-157 G -158 G -159 Y -160 G -161 H -162 T -163 A -164 1 -165 V -166 L AETV167 N-168 G -169 N D170 Y AHS171 D -172 G-173 K HN174 L AHKRST175 M N pos H5 deimm 176 K AQST177 F -178 Q ST179 s -180 L -181 D -182 Q-183 N -184 W -185 Y DN186 G-187 G-188 G -189 W -190 R ACDHNST191 K DN192 A EGKT193 E -194 V GPT195 A -196 H QT197 K PR198 V -199 V NST200 H- pos H5 deimm 201 N DK202 Y -203 E D204 N D205 D NQV206 M Q207 1 HW208 F-209 1 -210 R -211 P-212 F DEHKN213 K Y214 K -215 A- It is preferred that most or preferably all H5 specific residues at positions characterizing the H5 variant (SEQ ID NO: 11) vis a vis WT L0482 (SEQ ID NO: 1), i.e. at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186, are retained and predominantly other positions are modified with the deimmunizing residues.

Example 11: Ribolysin construct design and mRNA production In !//Zrotranscription of lysin encoding mRNAs was based on the pSTl-T7-AGA-dEarI-hAg-MCS-FI-A30LA70 plasmid- backbone and derivative DNA-constructs. These plasmid constructs contain a 5' UTR (untranslated region, a derivate of the 5'-UTR of homo sapiens hemoglobin subunit alpha l (hAg)), a 3' FI element (where F is a 1nucleotide long 3'-UTR fragment of amino-terminal enhancer of split, mRNA and I is a 142 nucleotide long fragment of mitochondrially encoded 12S RNA both identified in Homo sapiens;'HO 2017/060314) and a poly(A) tail of 1nucleotides, with a linker after 70 nucleotides. 206 Several top-displaying hits from the directed evolution screens (Example 6; Figure 8) were selected for mRNA expression. The insulin signal sequence (SEQ ID NO: 378; derived from human insulin, UniProtKB entry P01308_INS_HUMAN) as well as a DYKDDDDKtag (SEQ ID NO: 307) was added N-terminally, a hexa-histidine tag was added C-terminally; see Tables 6 to 8 and SEQ ID NO: 252 to 275 for the corresponding RNAs and proteins. The signal sequence targets the lysin constructs to the secretory pathway and is cleaved off at the signal peptidase site after translocation into the ER. The DYKDDDDK and the hexa-histidine tags were introduced to facilitate the quantification of translated lysin. The lysin sequences were codon-optimized for expression in Homo sapiens. mRNA was generated by in vitro transcription as described by Kreiter et al. (Kreiter, S. et al. Cancer Immunol. Immunother. 56, 1577-87 (2007)) with substitution of the normal nucleoside uridine by 1-methyl-pseudouridine. Resulting mRNAs are equipped with a Cap 1-structure and double-stranded (dsRNA) molecules were depleted. Purified mRNA was eluted in H20 and stored at -80 °C until further use. In vitro transcription of all described mRNA constructs was carried out at BioNTech SE. A list of all lysin constructs, which were used in subsequent experiments is shown in Table 6.

Table 6. Full amino acid and nucleotide sequences of mRNA-encoded hits from directed evolution screens of LO482. The nucleotide sequences shown refer to the mRNAs generated from codon-optimized DNA constructs and encoding various lysins including signal peptide and tags, and further containing a 5' UTR (comprising the deltaEarI-hAg- Kozak sequence shown in SEQ ID NO: 371), a 3' UTR (comprising the 3' FI element shown in SEQ ID NO: 374) and a poly(A) tail (SEQ ID NO: 375), wherein each uridine (shown as "T") in these mRNA sequences was replaced by 1-methyl-pseudouridine.L0482(wt) MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGREAPKTQIYTVKKGDTLSAIALKYKT TVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNNKSNSNSSTLNYLKTLENRGWDFDGSY GWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNSEAKIYHNTPTFKAEPGDLVVFSGRFGGGYG HTAIVLNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIFIRPFKKAAAAGGGGSHH HHHH* (SEQ ID NO: 252)AGCACAAACTAGTAI ILI 1L1GGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAAGAGAGGCTCCCAAAACACAGAT TTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAA CATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCC CATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAACAAGAGTAACAGCAACTC CTCTACACTGAACTATCTCAAGACGTTGGAGAATCGCGGTTGGGACTTTGACGGCAGTTACGGCT GGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGAAAGGCTAT GGGGCCAAAGACATACCTTATGCTAACAATTTCAACTCTGAGGCCAAGATCTACCACAACACTCCT ACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGGCA TACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGA ACTGGAACAATGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAAC GACATGATCTTCATCCGTCCCTTTAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCA CCACCATCATTGAGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCT GGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC CACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCC 207 ACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACT AACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 253)L0482ag MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGREAPKTQIYTVKKGDTLSAIALKYKT TVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKTLENRGWDFDGSY GWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNSEAKIYHNTPTFKAEPGDLWVFSGRFGGGYG HTAIVLNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIFIRPFKKAAAAGGGGSHH HHHH*(SEQ ID NO: 254)AGCACAAACTAGTAI ILI ICIGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAAGAGAGGCTCCCAAAACACAGAT TTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAA CATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCC CATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTC CTCTACACTGAACTATCTCAAGACGTTGGAGAATCGCGGTTGGGACTTTGACGGCAGTTACGGCT GGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGAAAGGCTAT GGGGCCAAAGACATACCTTATGCTAACAATTTCAACTCTGAGGCCAAGATCTACCACAACACTCCT ACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGGCA TACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGA ACTGGAACAATGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAAC GACATGATCTTCATCCGTCCCTTTAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCA CCACCATCATTGAGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCT GGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC CACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCC ACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACT AACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 255)LO482(G1) MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGREAPKTQIYTVKKGDTLSAIALKYKT TVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKGWDFDGSY GWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNSEAKIYHNTPTFKAEPGDLVVFSGRFGGGYG HTAIVLNGNYDGNLQKFQSLDQNWNNGGWRKQEVAHKVVHNYENDMIFIRPFKKAAAAGGGGSHH HHHH* (SEQ ID NO: 256)AGCACAAACTAGTAI ILI 1C1GGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAAGAGAGGCTCCCAAAACACAGAT TTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAA CATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTC CTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGACTTTGACGGCAGTTACGGCT 208 GGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGAAAGGCTAT GGGGCCAAAGACATACCTTATGCTAACAATTTCAACTCTGAGGCCAAGATCTACCACAACACTCCT ACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGGCA TACTGCCATTGTGCTTAATGGGAACTACGATGGCAACCTGCAGAAATTCCAGTCACTGGACCAGAA CTGGAACAATGGTGGTTGGCGAAAACAGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACG ACATGATCTTCATCCGTCCCTTTAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCAC CACCATCATTGAGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTG GGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACC ACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCA CACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTA ACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 257)L0482(G2) MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGREAPKTQIYTVKKGDTLSAIALKYKT TVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKGWDFDGSY GWQCFDLVNVYWNHLYGHGLRGYGAKDIPFANNFNNEAKIYRNTPTFKAEPGDLVVFSGRFGGGYG HTAIVLNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIFIRPFKKAAAAGGGGSHH HHHH* (SEQ ID NO: 258) L0482(G3) AGCACAAACTAGTAI ILI ICIGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAAGAGAGGCTCCCAAAACACAGAT TTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAA CATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCC CATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTC CTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGACTTTGACGGCAGTTACGGCT GGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGCGGGGCTAT GGGGCCAAAGACATACCTTTCGCTAACAATTTCAACAACGAGGCCAAGATCTACCGGAACACTCCT ACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGGCA TACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGA ACTGGAACAATGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAAC GACATGATCTTCATCCGTCCCTTTAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCA CCACCATCATTGAGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCT GGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC CACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCC ACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACT AACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 259)MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGREAPKTQIYTVKKGDTLSAIALKYKT TVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKGWDFDGSY GWQCFDLVNVYWYHLYGHGLKGYGAKDIPYANNFNNEAKIYKNTPTFKAEPGDLVVFSGRYGGGYG 209 HTAIVLNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIFIRPFKKAAAAGGGGSHH HHHH* (SEQ ID NO: 260)AGCACAAACTAGTAI ILI ICIGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAAGAGAGGCTCCCAAAACACAGAT TTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAA CATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCC CATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTC CTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGACTTTGACGGCAGTTACGGCT GGCAATGCTTCGACTTGGTGAATGTGTACTGGTACCACCTGTATGGGCATGGACTGAAAGGCTAT GGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAGATCTACAAGAACACTCCT ACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTACGGCGGAGGCTATGGGC ATACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAG AACTGGAACAATGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAA CGACATGATCTTCATCCGTCCCTTTAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACC ACCACCATCATTGAGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCC TGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCA CCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGC CACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATAC TAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 261)L0482(G4) MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGREAPKTQIYTVKKGDTLSAIALKYKT TVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKTLENKGWDFDGSY GWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNNEAKIYKNTPTFKAEPGDLVVFSGRFGGGYG HTAIVLNGNYDGKLMKFQSLDQNWYGGGWRKAEVAHKVVHNYENDMIFIRPFKKAAAAGGGGSHH HHHH* (SEQ ID NO: 262)AGCACAAACTAGTAI ILI 1C1GGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAGGACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAAGAGAGGCTCCCAAAACACAGAT TTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAA CATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCC CATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTC CTCTACACTGAACTATCTCAAGACGTTGGAGAATAAGGGTTGGGACTTTGACGGCAGTTACGGCT GGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGAAAGGCTAT GGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAGATCTACAAGAACACTCCT ACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGGCA TACTGCCATTGTGCTTAATGGGAACTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGAA CTGGTACGGCGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACG ACATGATCTTCATCCGTCCCTTTAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCAC CACCATCATTGAGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTG 210 GGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACC ACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCA CACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTA ACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 263)LO482(H1) MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGTQNYVVKKGDTLSAIALKYKTTVSNI QNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKGWDFDGSYGWQC FDLVNVYWYHLYGHGLKGYGAKDIPYANNFNSEAKIYHNTPTFKAEPGDLVVFSGRFGGGYGHTAIV LNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIFIRPFKKAAAAGGGGSHHHHHH * (SEQ ID NO: 264)AGCACAAACTAGTAI ILI ICIGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAACACAGAACTACGTGGTCAAGAA GGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAACATCCAGAACACCAA TAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGT CGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTCCTCTACACTGAACTA TCTCAAGAGCTTGGAGGGCAAGGGTTGGGACTTTGACGGCAGTTACGGCTGGCAATGCTTCGACT TGGTGAATGTGTACTGGTACCACCTGTATGGGCATGGACTGAAAGGCTATGGGGCCAAAGACATA CCTTATGCTAACAATTTCAACTCTGAGGCCAAGATCTACCACAACACTCCTACCTTCAAGGCAGAA CCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGGCATACTGCCATTGTGCT TAATGGGGATTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGAACTGGAACAATGGTG GTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACGACATGATCTTCATC CGTCCCTTTAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCACCACCATCATTGAGG ATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCT CCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTC CAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAA ACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGT CAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAT ATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAA (SEQ ID NO: 265)L0482(H2) MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGTQNYVVKKGDTLSAIALKYKTTVSNI QNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKTLENKGWDFDGSYGWQC FDLVNVYWNHLYGHGLKGYGAKDIPYANNFNNEAKIYKNTPTFKAEPGDLVVFSGRFGGGYGHTAIV LNGNYDGKLMKFQSLDQNWYGGGWRKAEVAHKVVHNYENDMIFIRPYKKAAAAGGGGSHHHHHH * (SEQ ID NO: 266)AGCACAAACTAGTAI ILI 1C1GGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAACACAGAACTACGTGGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAACATCCAGAACACCAA TAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGT 211 CGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTCCTCTACACTGAACTA TCTCAAGACGTTGGAGAATAAGGGTTGGGACTTTGACGGCAGTTACGGCTGGCAATGCTTCGACT TGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGAAAGGCTATGGGGCCAAAGACATA CCTTATGCTAACAATTTCAACAACGAGGCCAAGATCTACAAGAACACTCCTACCTTCAAGGCAGAA CCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGGCATACTGCCATTGTGCT TAATGGGAACTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGAACTGGTACGGCGGTG GTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACGACATGATCTTCATC CGTCCCTACAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCACCACCATCATTGAGG ATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCT CCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTC CAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAA ACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGT CAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAT ATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAA (SEQ ID NO: 267)L0482(H3) MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGREAPKTQIYTVKKGDTLSAIALKYKT TVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKGWDFDGSY GWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNNEAKIYKNTPTFKAEPGDLVVFSGRFGGGYG HTAIVLNGNYDGKLMKFQSLDQNWYGGGWRKAEVAHKVVHNYENDMIFIRPFKKAAAAGGGGSHH HHHH* (SEQ ID NO: 268)AGCACAAACTAGTAI ILI ICIGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAAGAGAGGCTCCCAAAACACAGAT TTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAA CATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCC CATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTC CTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGACTTTGACGGCAGTTACGGCT GGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGAAAGGCTAT GGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAGATCTACAAGAACACTCCT ACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGGCA TACTGCCATTGTGCTTAATGGGAACTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGAA CTGGTACGGCGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACG ACATGATCTTCATCCGTCCCTTTAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCAC CACCATCATTGAGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTG GGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACC ACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCA CACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTA ACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 269) 212 L0482(H4) MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGTQNYVVKKGDTLSAIALKYKTTVSNI QNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKGWDFDGSYGWQC FDLVNVYWNHLYGHGLRGYGAKDIPFANNFNNEAKIYRNTPTFKAEPGDLVVFSGRFGGGYGHTAIV LNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIFIRPYKKAAAAGGGGSHHHHHH * (SEQ ID NO: 270)AGCACAAACTAGTAI ILI ICIGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAACACAGAACTACGTGGTCAAGAA GGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAACATCCAGAACACCAA TAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGT CGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTCCTCTACACTGAACTA TCTCAAGAGCTTGGAGGGCAAGGGTTGGGACTTTGACGGCAGTTACGGCTGGCAATGCTTCGACT TGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGCGGGGCTATGGGGCCAAAGACATA CCTTTCGCTAACAATTTCAACAACGAGGCCAAGATCTACCGGAACACTCCTACCTTCAAGGCAGAA CCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGGCATACTGCCATTGTGCT TAATGGGGATTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGAACTGGAACAATGGTG GTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACGACATGATCTTCATC CGTCCCTACAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCACCACCATCATTGAGG ATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCT CCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTC CAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAA ACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGT CAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAT ATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAA (SEQ ID NO: 271)L0482(H5) MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGREAPKTQIYTVKKGDTLSAIALKYKT TVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKGWDFDGSY GWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNNEAKIYKNTPTFKAEPGDLVVFSGRYGGGYG HTAIVLNGNYDGKLMKFQSLDQNWYGGGWRKAEVAHKVVHNYENDMIFIRPFKKAAAAGGGGSHH HHHH* (SEQ ID NO: 272)AGCACAAACTAGTAI ILI 1C1GGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAAGAGAGGCTCCCAAAACACAGAT TTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAA CATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCC CATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTC CTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGACTTTGACGGCAGTTACGGCT GGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGAAAGGCTAT GGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAGATCTACAAGAACACTCCT ACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTACGGCGGAGGCTATGGGC ATACTGCCATTGTGCTTAATGGGAACTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGA 213 Table 7. Lysin-specific amino acid and nucleotide sequences of mRNA-encoded hits from directed evolution screens of LO482 ACTGGTACGGCGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAAC GACATGATCTTCATCCGTCCCTTTAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCA CCACCATCATTGAGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCT GGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC CACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCC ACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACT AACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 273)L0482(H6) MALWMRLLPLLALLALWGPDPAAAMKDYKDDDDKGGGGSGREAPKTQIYTVKKGDTLSAIALKYKT TVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKTLENKGWDFDGSY GWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNNEAKIYKNTPTFKAEPGDLVVFSGRFGGGYG HTAIVLNGDYDGKLMKFQSLDQNWYGGGWRKAEVAHKVVHNYENDMIFIRPFKKAAAAGGGGSHH HHHH* (SEQ ID NO: 274)AGCACAAACTAGTAI ILI 1L1GGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCCCTGTGGA TGAGACTGCTGCCTCTGCTTGCTCTGCTGGCACTGTGGGGACCTGATCCTGCTGCTGCTATGAAG GACTACAAAGACGATGACGACAAGGGAGGAGGCGGCTCCGGAAGAGAGGCTCCCAAAACACAGAT TTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACCGTATCAAA CATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCAAGGTTCC CATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAGCGGCTC CTCTACACTGAACTATCTCAAGACGTTGGAGAATAAGGGTTGGGACTTTGACGGCAGTTACGGCT GGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGAAAGGCTAT GGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAGATCTACAAGAACACTCCT ACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGGCA TACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGA ACTGGTACGGCGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAAC GACATGATCTTCATCCGTCCCTTTAAGAAAGCGGCGGCCGCCGGAGGAGGCGGCTCCCACCACCA CCACCATCATTGAGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCT GGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC CACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCC ACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACT AACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGCAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 275) The amino acid and RNA sequences shown correspond to the amino acid and codon optimized RNA sequences of the lysin part contained in the full amino acid and RNA sequences shown in Table 6, respectively. Again, each uridine (shown as "T") in these sequences was replaced by 1-methyl-pseudouridine. 214 L0482(wt) REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNNKS NSNSSTLNYLKTLENRGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNSEAKIYH NTPTFKAEPGDLVVFSGRFGGGYGHTAIVLNGDYDGKLM KFQSLDQNWNNGGWRKAEVAH KWH NY ENDMIFIRPFKKA (SEQ ID NO: 1)AGAGAGGCTCCCAAAACACAGATTTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTG AAATACAAGACCACCGTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCAT TGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAAT AACAAGAGTAACAGCAACTCCTCTACACTGAACTATCTCAAGACGTTGGAGAATCGCGGTTGGGAC TTTGACGGCAGTTACGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGG CATGGACTGAAAGGCTATGGGGCCAAAGACATACCTTATGCTAACAATTTCAACTCTGAGGCCAAG ATCTACCACAACACTCCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTT GGCGGAGGCTATGGGCATACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTC CAGTCACTGGACCAGAACTGGAACAATGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACGACATGATCTTCATCCGTCCCTTTAAGAAAGCG (SEQ ID NO: 308)L0482ag REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKS NSGSSTLNYLKTLENRGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNSEAKIYH NTPTFKAEPGDLVVFSGRFGGGYGHTAIVLNGDYDGKLM KFQSLDQNWNNGGWRKAEVAH KWH NY ENDMIFIRPFKKA (SEQ ID NO: 2)AGAGAGGCTCCCAAAACACAGATTTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTG AAATACAAGACCACCGTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCAT TGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAAT AAGAAGAGTAACAGCGGCTCCTCTACACTGAACTATCTCAAGACGTTGGAGAATCGCGGTTGGGAC TTTGACGGCAGTTACGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGG CATGGACTGAAAGGCTATGGGGCCAAAGACATACCTTATGCTAACAATTTCAACTCTGAGGCCAAG ATCTACCACAACACTCCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTT GGCGGAGGCTATGGGCATACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTC CAGTCACTGGACCAGAACTGGAACAATGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTC CACAATTACGAGAACGACATGATCTTCATCCGTCCCTTTAAGAAAGCG (SEQ ID NO: 309)LO482(G1) REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKS NSGSSTLNYLKSLEGKGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNSEAKIYHN TPTFKAEPGDLWVFSGRFGGGYGHTAIVLNGNYDGNLQKFQSLDQNWNNGGWRKQEVAHKVVHNYE NDMIFIRPFKKA (SEQ ID NO: 3)AGAGAGGCTCCCAAAACACAGATTTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTG AAATACAAGACCACCGTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCAT TGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAAT AAGAAGAGTAACAGCGGCTCCTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGAC TTTGACGGCAGTTACGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGG CATGGACTGAAAGGCTATGGGGCCAAAGACATACCTTATGCTAACAATTTCAACTCTGAGGCCAAG ATCTACCACAACACTCCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTT GGCGGAGGCTATGGGCATACTGCCATTGTGCTTAATGGGAACTACGATGGCAACCTGCAGAAATTC 215 CAGTCACTGGACCAGAACTGGAACAATGGTGGTTGGCGAAAACAGGAAGTAGCCCACAAGGTCGTC CACAATTACGAGAACGACATGATCTTCATCCGTCCCTTTAAGAAAGCG (SEQ ID NO: 310)L0482(G2) REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKS NSGSSTLNYLKSLEGKGWDFDGSYGWQCFDLVNVYWNHLYGHGLRGYGAKDIPFANNFNNEAKIYR NTPTFKAEPGDLVVFSGRFGGGYGHTAIVLNGDYDGKLM KFQSLDQNWNNGGWRKAEVAH KWH NY ENDMIFIRPFKKA (SEQ ID NO: 4)AGAGAGGCTCCCAAAACACAGATTTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTG AAATACAAGACCACCGTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCAT TGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAAT AAGAAGAGTAACAGCGGCTCCTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGAC TTTGACGGCAGTTACGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGG CATGGACTGCGGGGCTATGGGGCCAAAGACATACCTTTCGCTAACAATTTCAACAACGAGGCCAAG ATCTACCGGAACACTCCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTT GGCGGAGGCTATGGGCATACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTC CAGTCACTGGACCAGAACTGGAACAATGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACGACATGATCTTCATCCGTCCCTTTAAGAAAGCG (SEQ ID NO: 311)L0482(G3) REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKS NSGSSTLNYLKSLEGKGWDFDGSYGWQCFDLVNVYWYHLYGHGLKGYGAKDIPYANNFNNEAKIYKN TPTFKAEPGDLWVFSGRYGGGYGHTAIVLNGDYDGKLM KFQSLDQNWNNGGWRKAEVAH KWH NYE NDMIFIRPFKKA (SEQ ID NO: 5)AGAGAGGCTCCCAAAACACAGATTTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTG AAATACAAGACCACCGTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCAT TGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAAT AAGAAGAGTAACAGCGGCTCCTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGAC TTTGACGGCAGTTACGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGTACCACCTGTATGGG CATGGACTGAAAGGCTATGGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAG ATCTACAAGAACACTCCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTAC GGCGGAGGCTATGGGCATACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTC CAGTCACTGGACCAGAACTGGAACAATGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACGACATGATCTTCATCCGTCCCTTTAAGAAAGCG (SEQ ID NO: 312)L0482(G4) REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKS NSGSSTLNYLKTLENKGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNNEAKIYK NTPTFKAEPGDLVVFSGRFGGGYGHTAIVLNGNYDGKLMKFQSLDQNWYGGGWRKAEVAHKVVHNY ENDMIFIRPFKKA (SEQ ID NO: 6)AGAGAGGCTCCCAAAACACAGATTTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTG AAATACAAGACCACCGTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCAT TGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAAT AAGAAGAGTAACAGCGGCTCCTCTACACTGAACTATCTCAAGACGTTGGAGAATAAGGGTTGGGAC TTTGACGGCAGTTACGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGG CATGGACTGAAAGGCTATGGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAG ATCTACAAGAACACTCCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTT 216 GGCGGAGGCTATGGGCATACTGCCATTGTGCTTAATGGGAACTACGATGGCAAACTGATGAAATTC CAGTCACTGGACCAGAACTGGTACGGCGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACGACATGATCTTCATCCGTCCCTTTAAGAAAGCG (SEQ ID NO: 313)LO482(H1) TQNYVVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSS TLNYLKSLEGKGWDFDGSYGWQCFDLVNVYWYHLYGHGLKGYGAKDIPYANNFNSEAKIYHNTPTFK AEPGDLVVFSGRFGGGYGHTAIVLNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIF IRPFKKA (SEQ ID NO: 7)ACACAGAACTACGTGGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACC GTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCA AGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAG CGGCTCCTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGACTTTGACGGCAGTTA CGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGTACCACCTGTATGGGCATGGACTGAAAGG CTATGGGGCCAAAGACATACCTTATGCTAACAATTTCAACTCTGAGGCCAAGATCTACCACAACACT CCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGG CATACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAG AACTGGAACAATGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAAC GACATGATCTTCATCCGTCCCTTTAAGAAAGCG (SEQ ID NO: 314)L0482(H2) TQNYVVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSS TLNYLKTLENKGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNNEAKIYKNTPTFK AEPGDLVVFSGRFGGGYGHTAIVLNGNYDGKLMKFQSLDQNWYGGGWRKAEVAHKVVHNYENDMIF IRPYKKA (SEQ ID NO: 8)ACACAGAACTACGTGGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACC GTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCA AGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAG CGGCTCCTCTACACTGAACTATCTCAAGACGTTGGAGAATAAGGGTTGGGACTTTGACGGCAGTTA CGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGAAAGG CTATGGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAGATCTACAAGAACACT CCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGG CATACTGCCATTGTGCTTAATGGGAACTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAGA ACTGGTACGGCGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACG ACATGATCTTCATCCGTCCCTACAAGAAAGCG (SEQ ID NO: 315)L0482(H3) REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKS NSGSSTLNYLKSLEGKGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNNEAKIYK NTPTFKAEPGDLVVFSGRFGGGYGHTAIVLNGNYDGKLMKFQSLDQNWYGGGWRKAEVAHKVVHNY ENDMIFIRPFKKA (SEQ ID NO: 9)AGAGAGGCTCCCAAAACACAGATTTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTG AAATACAAGACCACCGTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCAT TGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAAT AAGAAGAGTAACAGCGGCTCCTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGAC TTTGACGGCAGTTACGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGG CATGGACTGAAAGGCTATGGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAG 217 ATCTACAAGAACACTCCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTT GGCGGAGGCTATGGGCATACTGCCATTGTGCTTAATGGGAACTACGATGGCAAACTGATGAAATTC CAGTCACTGGACCAGAACTGGTACGGCGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGT CCACAATTACGAGAACGACATGATCTTCATCCGTCCCTTTAAGAAAGCG (SEQ ID NO: 316)L0482(H4) TQNYVVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSS TLNYLKSLEGKGWDFDGSYGWQCFDLVNVYWNHLYGHGLRGYGAKDIPFANNFNNEAKIYRNTPTFK AEPGDLVVFSGRFGGGYGHTAIVLNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIF IRPYKKA (SEQ ID NO: 10)ACACAGAACTACGTGGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTGAAATACAAGACCACC GTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCATTGGCCAGAAGCTCA AGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAATAAGAAGAGTAACAG CGGCTCCTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGAC 1 1 1GACGGCAGTTA CGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGGCATGGACTGCGGGG CTATGGGGCCAAAGACATACCTTTCGCTAACAATTTCAACAACGAGGCCAAGATCTACCGGAACACT CCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTTGGCGGAGGCTATGGG CATACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTCCAGTCACTGGACCAG AACTGGAACAATGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAAC GACATGATCTTCATCCGTCCCTACAAGAAAGCG (SEQ ID NO: 317)L0482(H5) REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKS NSGSSTLNYLKSLEGKGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNNEAKIYK NTPTFKAEPGDLVVFSGRYGGGYGHTAIVLNGNYDGKLM KFQSLDQNWYGGGWRKAEVAH KWH NY ENDMIFIRPFKKA (SEQ ID NO: 11)AGAGAGGCTCCCAAAACACAGATTTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTG AAATACAAGACCACCGTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCAT TGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAAT AAGAAGAGTAACAGCGGCTCCTCTACACTGAACTATCTCAAGAGCTTGGAGGGCAAGGGTTGGGAC TTTGACGGCAGTTACGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGG CATGGACTGAAAGGCTATGGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAG ATCTACAAGAACACTCCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTAC GGCGGAGGCTATGGGCATACTGCCATTGTGCTTAATGGGAACTACGATGGCAAACTGATGAAATTC CAGTCACTGGACCAGAACTGGTACGGCGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACGACATGATCTTCATCCGTCCCTTTAAGAAAGCG (SEQ ID NO: 318)L0482(H6) REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKS NSGSSTLNYLKTLENKGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNNEAKIYK NTPTFKAEPGDLVVFSGRFGGGYGHTAIVLNGDYDGKLMKFQSLDQNWYGGGWRKAEVAHKVVHNY ENDMIFIRPFKKA (SEQ ID NO: 12)AGAGAGGCTCCCAAAACACAGATTTACACCGTCAAGAAGGGAGATACGCTTAGCGCCATTGCACTG AAATACAAGACCACCGTATCAAACATCCAGAACACCAATAACATAGCCAATCCCAATCTCATCTTCAT TGGCCAGAAGCTCAAGGTTCCCATGACTCCACTGGTCGAACCGAAACCTAAGACAGTGTCCTCCAAT AAGAAGAGTAACAGCGGCTCCTCTACACTGAACTATCTCAAGACGTTGGAGAATAAGGGTTGGGAC TTTGACGGCAGTTACGGCTGGCAATGCTTCGACTTGGTGAATGTGTACTGGAATCACCTGTATGGG 218 CATGGACTGAAAGGCTATGGGGCCAAAGACATACCTTATGCTAACAATTTCAACAACGAGGCCAAG ATCTACAAGAACACTCCTACCTTCAAGGCAGAACCAGGAGATCTGGTTGTGTTTAGCGGACGGTTT GGCGGAGGCTATGGGCATACTGCCATTGTGCTTAATGGGGATTACGATGGCAAACTGATGAAATTC CAGTCACTGGACCAGAACTGGTACGGCGGTGGTTGGCGAAAAGCGGAAGTAGCCCACAAGGTCGTCCACAATTACGAGAACGACATGATCTTCATCCGTCCCTTTAAGAAAGCG (SEQ ID NO: 319) Example 12: In vitro expression of RNA-encoded lysin variants In vitro expression and secretion of several RNA-encoded lysin (i.e. ribolysin) variants of LO482 (see Tables 6 and 7) was analyzed by lipofection of mRNA into HEK293T/17 cells and subsequent quantification of secreted His- tagged protein by Western Blot (see Table 8). One day prior to lipofection, 0.9xl06 HEK293T/17 cells were seeded in 3 ml DMEM (Life Technologies GmbH, cat. no. 31966-021) + 10 % fetal bovine serum (FBS, Biochrom GmbH, cat. No. SO115) in 6-well plates. For lipofection, 5 pg of mRNA were transfected. mRNA was formulated using the RiboJuice™ mRNA Transfection Kit (Sigma Aldrich, cat. no. TR-1013) according to the manufacturer's instructions. hours post lipofection, supernatants were collected and cells were lysed in RIPA buffer (Santa Cruz Biotechnology Inc., cat. No. sc-24948) and stored at -20°C until further use. The levels of secreted lysins were quantified by Western Blot through detection by an anti-His antibody (Abeam, cat. no. abll87 for C-terminal His-tag). Expression levels were quantified by quantification of chemiluminescent signals using Image Lab 6.

Table 8. Details on RNA-encoded LO482 variants Construct The SEQ IDs refer to the generated RNAs (wherein all uridines are replaced by 1-methyl-pseudouridines) pST4-AG-CAC-hAg-Kozak-SS(hINS)-FLAG-hL0482-His-F-I-No_Lig3-A30LA70 (provides SEQ ID NO: 253) Length of RNA[nt](T7 start to end of Poly-A- tail)1271 Predicted MWof the lysinprotein [kDa](without signal peptide)27.21 pST4-AG-CAC-hAg-Kozak-SS(hINS)-FLAG-hL0482ag-His-F-I-No_Lig3-A30LA70 (provides SEQ ID NO: 255)1271 27.17 pST4-AG-CAC-hAg-K0zak-SS(hINS)-FLAG-hL0482(Gl)-His-F-I-No_Lig3-A30LA70 (provides SEQ1271 27.11 ID NO: 257)pST4-AG-CAC-hAg-K0zak-SS(hINS)-FLAG- 1271 27.13hL0482(G2)-His-F-I-No_Lig3-A30LA70 (provides SEQID NO: 259)pST4-AG-CAC-hAg-K0zak-SS(hINS)-FLAG- 1271 27.15hL0482(G3)-His-F-I-No_Lig3-A30LA70 (provides SEQID NO: 261)pST4-AG-CAC-hAg-K0zak-SS(hINS)-FLAG- 1271 27.15hL0482(G4)-His-F-I-No_Lig3-A30LA70 (provides SEQID NO: 263) 219 pST4-AG-CAC-hAg-K0zak-SS(hINS)-FLAG- hL0482(Hl)-His-F-I-No_Lig3-A30LA70 (provides SEQID NO: 265) 1256 26.54 pST4-AG-CAC-hAg-K0zak-SS(hINS)-FLAG- hL0482(H2)-His-F-I-No_Lig3-A30LA70 (provides SEQID NO: 267) 1256 26.58 pST4-AG-CAC-hAg-K0zak-SS(hINS)-FLAG- hL0482(H3)-His-F-I-No_Lig3-A30LA70 (provides SEQID NO: 269) 1271 27.08 pST4-AG-CAC-hAg-K0zak-SS(hINS)-FLAG- hL0482(H4)-His-F-I-No_Lig3-A30LA70 (provides SEQID NO: 271) 1256 26.56 pST4-AG-CAC-hAg-K0zak-SS(hINS)-FLAG- hL0482(H5)-His-F-I-No_Lig3-A30LA70 (provides SEQID NO: 273) 1271 27.1 pST4-AG-CAC-hAg-K0zak-SS(hINS)-FLAG-hL0482(H6)-His-F-I-No_Lig3-A30LA70 (SEQ ID NO:275) 1271 27.15 Protein expression from mRNA was visible at the expected size of approximately 27 kDa and was demonstrated for all LO482 variants (Figure 10); however, the distribution between cell lysate and supernatant differed significantly between variants. While wt and aglycosylated (ag) LO482 displayed only faint bands in the supernatants, all variants obtained by the directed evolution tested could be found at considerable levels in the supernatant and thus were well secreted by the HEK293T/17 cells. In particular, variants G1, G3, G4, H3 and H5 showed more than 20x higher protein levels in the supernatant compared to wt.In summary, secretion of mRNA-encoded LO482 by human cells was successfully improved by using the directed evolution approach.

Claims

220 Claims

1. A peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain that has (i) a sequence identity of at least 83% to the sequence from position 72 to position 215 in SEQ ID NO: 1; (ii) an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and (iii) one or more amino acid substitutions at positions 86, 82, 85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

2. The peptidoglycan hydrolase of claim 1 which has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1.

3. The peptidoglycan hydrolase of claim 2 which further comprises an amino acid substitution or a deletion at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, preferably wherein the amino acid residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine, arginine or alanine, more preferably with lysine

4. The peptidoglycan hydrolase of any one of claims 1 to 3, wherein the residue at position 73 in SEQ ID NO: or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably with glycine. 221

5. The peptidoglycan hydrolase of any one of claims 1 to 4, wherein said CHAP domain has at least one, preferably at least two or more, of the following consensus mutation units 1) to V): an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; II) an amino acid substitution pair at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; III) an amino acid substitution pair at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 130 in SEQ ID NO: or at a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine; IV) an amino acid substitution at position 169 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine; and/or V) an amino acid substitution pair at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 185 in SEQ ID NO: or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

6. The peptidoglycan hydrolase of claim 5, wherein the CHAP domain further has an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the phenylalanine at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

7. The peptidoglycan hydrolase of any one of claims 1 to 6, wherein said CHAP domain has an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine.

8. The peptidoglycan hydrolase of any one of claims 1 to 7, wherein said CHAP domain has a sequence identity of at least 94% to the sequence from position 72 to position 215 in SEQ ID NO: 11.

9. The peptidoglycan hydrolase of any one of claims 1 to 7, wherein said CHAP domain has (a) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; 222 (b) at least one, preferably two, amino acid substitution(s) at positions 82 and 85 in SEQ ID NO: or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; and/or (c) an amino acid substitution at position 169 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine.

10. The peptidoglycan hydrolase of claim 9, wherein the CHAP domain further comprises one or more amino acid substitutions at positions 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and/or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

11. The peptidoglycan hydrolase of any one of claims 1 to 10 further comprising at least one cell wall binding domain, preferably at least one cell wall binding domain derived from an endolysin, more preferably a LYSM domain and/or a SH3 domain.

12. The peptidoglycan hydrolase of any one of claims 1 to 11 comprising a LYSM domain that has a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1.

13. The peptidoglycan hydrolase of claim 12, wherein the LYSM domain has one or more amino acid substitutions at positions 1, 8, 10,13, 23 to 25, 30, 33, 37, and 39 to 41 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 1 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tryptophan, the amino acid residue at position 8 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with valine, the amino acid residue at position 13 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 23 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, 223 the amino acid residue at position 24 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with cysteine, the amino acid residue at position 25 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 30 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 33 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, the amino acid residue at position 37 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 39 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 40 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with threonine, and/or the amino acid residue at position 41 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine.

14. The peptidoglycan hydrolase of any one of claims 1 to 13 which has a killing activity against at least one Staphylococcus species or strain, preferably against Staphylococcus aureus, more preferably against a Staphylococcus aureus strain that is resistant to at least one antibiotic such as methicillin.

15. The peptidoglycan hydrolase of claim 14, wherein said Staphylococcus species or strain, preferably said Staphylococcus aureus, is present in form of a biofilm or is suspected of forming a biofilm.

16. The peptidoglycan hydrolase of any one of claims 1 to 15 which is stable up to a temperature of about 40°C.

17. The peptidoglycan hydrolase of any one of claims 1 to 16, which has, compared to the peptidoglycan hydrolase of SEQ ID NO: 1, an enhanced killing activity against Staphylococcus aureus, an enhanced ability of being secreted by a human cell, and/or an enhanced thermostability.

18. The peptidoglycan hydrolase of any one of claims 1 to 17 further comprising an extended pharmacokinetic (PK) peptide such as a human FC domain, a C-terminal peptide of human chorionic gonadotropin or human lysozyme.

19. The peptidoglycan hydrolase of any one of claims 1 to 18 further comprising a signal peptide, preferably at the N-terminus.

20. A nucleic acid encoding the peptidoglycan hydrolase of any one of claims 1 to 19. 224

21. The nucleic acid of claim 20, wherein said peptidoglycan hydrolase comprises a signal peptide, preferably at the N-terminus.

22. The nucleic acid of claim 20 or 21 which is an RNA.

23. The nucleic acid of any one of claims 20 to 22 which is an RNA construct comprising in 5' to 3' order: (i) a 5' UTR that comprises or consists of a modified human alpha-globin 5‘-UTR; (ii) a sequence encoding a peptidoglycan hydrolase of any one of claims 1 to 19; (iii) a 3' UTR that comprises or consists of a first sequence from the amino terminal enhancer of split(AES) messenger RNA and a second sequence from the mitochondrial encoded 125 ribosomal RNA;and (iv) a poly-A sequence; and, preferably, wherein said RNA construct further comprises (v) a 5' cap and/or (vi) a modified nucleoside selected from pseudouridine (w), Nl-methyl-pseudouridine (mly), and 5- methyl-uridine (m5U), preferably Nl-methyl-pseudouridine (mly), in place of uridine, preferably in place of each uridine.

24. A pharmaceutical composition comprising the peptidoglycan hydrolase of any one of claims 1 to 19 and/or the nucleic acid of any one of claims 20 to 23; and, preferably, a pharmaceutically acceptable excipient.

25. The peptidoglycan hydrolase of any one of claims 1 to 19, the nucleic acid of any one of claims 20 to or the pharmaceutical composition of claim 24 for use in treating a disease caused by and/or associated with a Staphylococcus infection and/or a subject that has or is suspected of having a Staphylococcus infection.

26. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to claim 25, wherein said infection is a Staphylococcus aureus infection.

27. 2ר. A pharmaceutical composition comprising a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain that has (i) a sequence identity of at least 60% to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions as compared to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1; and, preferably, wherein said pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

28. The pharmaceutical composition of claim 27, wherein said CHAP domain has an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, preferably, wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, more preferably with glycine. 225

29.

30.

31. The pharmaceutical composition of claim TI or 28, wherein said peptidoglycan hydrolase has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. The pharmaceutical composition of claim 29, wherein said peptidoglycan hydrolase further comprises an amino acid substitution or a deletion at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, preferably, wherein the amino acid residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine, arginine or alanine, more preferably with lysine. The pharmaceutical composition of any one of claims T1 to 30, wherein said CHAP domain has one or more amino acid substitutions at positions 73, 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169,173, 175, 178, 185, 186, 191, 192, 194,198, 204, 212 and 215 in SEQ ID NO: 1 or at positions corresponding to these positions, and wherein the amino acid residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine or serine, the amino acid residue at position 75 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 78 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 81 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 104 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 107 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 115 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 124 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 125 in SEQ ID NO: 1 or at a position corresponding to this position issubstituted with valine, 226 the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 133 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 135 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with histidine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 140 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 141 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 178 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 191 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 194 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, the amino acid residue at position 198 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with isoleucine, 227 the amino acid residue at position 204 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 212 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue at position 215 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine.

32. The pharmaceutical composition of any one of claims 27 to 31, wherein said CHAP domain has one or more amino acid substitutions at positions 86, 82, 85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: or at positions corresponding to these positions, and wherein the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably with lysine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and/or the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

33. The pharmaceutical composition of any one of claims 27 to 32, wherein said CHAP domain has an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, preferably wherein the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine.

34. The pharmaceutical composition of any one of claims 27 to 33, wherein said CHAP domain has at least one, preferably at least two or more, of the following consensus mutation units I) to V): 228 an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine;II) an amino acid substitution pair at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine;III) an amino acid substitution pair at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 130 in SEQ ID NO: or at a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine;IV) an amino acid substitution at position 169 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine; and/orV) an amino acid substitution pair at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 185 in SEQ ID NO: or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

35. The pharmaceutical composition of any one of claims 27 to 34, wherein said CHAP domain has an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position, preferably wherein the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

36. The pharmaceutical composition of any one of claims 27 to 35, wherein (i) a corresponding segment of said CHAP domain has a sequency identity of at least 80%, preferably at least 90%, to the sequence from position 87 to position 128 in SEQ ID NO: 1, and/or (ii) said CHAP domain has at most six, five, four, three or two, preferably at most one, more preferably no amino acid substitutions or deletions at positions 80, 87, 88,98,99,103,106,110, 114, 122, 126, 128, 137, 182, 202, and 208 of SEQ ID NO: 1 or at positions corresponding to these positions.

37. The pharmaceutical composition of any one of claims 27 to 36, wherein said peptidoglycan hydrolase further comprises at least one cell wall binding domain as defined in claim 11, preferably a LYSM domain as defined in claim 12 or 13.

38. The pharmaceutical composition of any one of claims 27 to 37, wherein said peptidoglycan hydrolase has a killing activity against at least one Staphylococcus species or strain, preferably against Staphylococcus aureus, more preferably against a Staphylococcus aureus *cam that is resistant to at least one antibiotic such as methicillin. 229

39. The pharmaceutical composition of claim 38, wherein said Staphylococcus species or strain, preferably said Staphylococcus aureus, is present in form of a biofilm or is suspected of forming a biofilm.

40. The pharmaceutical composition of any one of claims 27 to 39, wherein the peptidoglycan hydrolase is stable up to a temperature of about 40°C.

41. The pharmaceutical composition of any one of claims 27 to 40, wherein the peptidoglycan hydrolase has, compared to the peptidoglycan hydrolase of SEQ ID NO: 1, an enhanced killing activity against Staphylococcus aureus, an enhanced ability of being secreted by a human cell, and/or an enhanced thermostability.

42. The pharmaceutical composition of any one of claims 27 to 41, wherein said CHAP domain has a sequence identity of at least 80% to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1.

43. The pharmaceutical composition of any one of claims 27 to 42, wherein said CHAP domain has a sequence identity of at least 90% to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1.

44. The pharmaceutical composition of any one of claims 27 to 43, wherein said CHAP domain has a sequence identity of at least 94% to the sequence from position 72 to position 215 in SEQ ID NO: 11.

45. The pharmaceutical composition of any one of claims 27 to 44, wherein the peptidoglycan hydrolase further comprises an extended pharmacokinetic (PK) peptide such as a human FC domain, a C-terminal peptide of human chorionic gonadotropin or human lysozyme.

46. The pharmaceutical composition of any one of claims 27 to 45, wherein the peptidoglycan hydrolase further comprises a signal peptide, preferably at the N-terminus.

47. A pharmaceutical composition comprising a nucleic acid encoding a peptidoglycan hydrolase as defined in any one of claims 27 to 46.

48. The pharmaceutical composition of claim 47, wherein the peptidoglycan hydrolase comprises a signal peptide, preferably at the N-terminus.

49. The pharmaceutical composition of claim 47 or 48, wherein said nucleic acid is an RNA.

50. The pharmaceutical composition of any one of claims 47 to 49, wherein said nucleic acid is an RNAconstruct comprising in 5' to 3' order: (i) a 5' UTR that comprises or consists of a modified human alpha-globin 5‘-UTR; (ii) a sequence encoding a peptidoglycan hydrolase as defined in any one of claims 25 to 45; (iii) a 3' UTR that comprises or consists of a first sequence from the amino terminal enhancer of split(AES) messenger RNA and a second sequence from the mitochondrial encoded 125 ribosomal RNA;and (iv) a poly-A sequence; and, preferably, wherein said RNA construct further comprises 230 (v) a 5' cap and/or (vi) a modified nucleoside selected from pseudouridine (w), Nl-methyl-pseudouridine (mly), and 5- methyl-uridine (m5U), preferably Nl-methyl-pseudouridine (mly), in place of uridine, preferably in place of each uridine.

51. The pharmaceutical composition of any one of claims 27 to 50 for use in treating a disease caused by and/or associated with a Staphylococcus infection and/or a subject that has or is suspected of having a Staphylococcus infection.

52. The pharmaceutical composition for use according to claim 51, wherein said infection is a Staphylococcus aureus infection.

53. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to claim 25 or or the pharmaceutical composition for use according to claim 51 or 52, wherein the Staphylococcus is present in form of a biofilm and/or a free-floating aggregate, or is suspected of forming a biofilm and/or a free-floating aggregate.

54. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to any one of claims 25, 26 and 53 or the pharmaceutical composition for use according to any one of claims 51 to 53, wherein said Staphylococcus infection is a Staphylococcus aureus infection of a skin, soft tissue, bone, lung, sinus and/or urinary tract.

55. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to any one of claims 25, 26, 53 and 54 or the pharmaceutical composition for use according to any one of claims 51 to 54, wherein said disease is selected from the group consisting of: pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, sepsis, a respiratory infection such as sinusitis, pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, abscesses, food poisoning, necrotizing fasciitis, pyomyositis, mediastinitis, infected dermatitis, wound infection, diabetic foot ulcer, septic arthritis, osteoarticular infections, prosthetic infection such as infection of a prosthetic joint or a cardiac device, and urinary tract infections.

56. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to claim 55, wherein said disease is pneumonia, bacteremia, endocarditis, or a prosthetic infection.