WO2026027251A1 - Peptides signaux permettant d'augmenter la sécrétion de protéines - Google Patents

Peptides signaux permettant d'augmenter la sécrétion de protéines

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Publication number
WO2026027251A1
WO2026027251A1 PCT/EP2025/070414 EP2025070414W WO2026027251A1 WO 2026027251 A1 WO2026027251 A1 WO 2026027251A1 EP 2025070414 W EP2025070414 W EP 2025070414W WO 2026027251 A1 WO2026027251 A1 WO 2026027251A1
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WIPO (PCT)
Prior art keywords
protein
interest
sequence
host cell
secretion
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Pending
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PCT/EP2025/070414
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English (en)
Inventor
Roland Weis
Victoria KONCAR
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Boehringer Ingelheim RCV GmbH and Co KG
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Boehringer Ingelheim RCV GmbH and Co KG
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Publication of WO2026027251A1 publication Critical patent/WO2026027251A1/fr
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Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the present invention relates to the field of recombinant protein expression.
  • Yeasts in general and Pichia pastoris in particular are popular expression systems for the secretion of recombinant proteins.
  • the initial and crucial step in secretion is the translocation of the recombinant protein into the endoplasmic reticulum (ER). This process is directed by an N-terminal secretion signal fused to the recombinant protein.
  • the signal sequence specifies either a co-translational or post-translational targeting route to the ER on the conventional secretion pathway (Ng et al., 1996). The most commonly used secretion signal in P.
  • yeast pastoris is the Saccharomyces cerevisiae a-mating prepro-leader (MFa) (Lin-Cereghino et al., 2013). This signal mediates post-translational translocation in S. cerevisiae and most likely in P. pastoris too (Fitzgerald and Glick, 2014; Ng et al., 1996). Other secretion signals are continually added to the repertoire and tested with different recombinant proteins.
  • MFa prepro-leader
  • the MFa signal sequence could, in fact, be suboptimal, and it may be preferable to use a co-translational signal sequence (Ng et al., 1996).
  • mammalian antibodies have become the dominant product class within the biopharmaceutical market (Ecker et al., 2015).
  • Antibodies are known to be co- translationally translocated in their native environment (Feige et al., 2010).
  • a trend toward development of smaller antigen-binding fragments e.g. Fab, scFv and VHH is also evident (Nelson and Reichert, 2009; Walsh, 2014).
  • Fab fragments are sometimes inefficiently secreted and therefore only reach low production titers (Looser et al., 2015; Pfeffer et al., 2011). This may be due to the post-translational signal sequence MFa, which has already been reported as causing a bottleneck in translocation (Fitzgerald and Glick, 2014; Zahrl et al., 2018).
  • WO2018165589 and WO2018165594 disclose a recombinant secretion signal comprising an MFa pro-leader originating from Saccharomyces cerevisiae and a signal peptide other than MFa pre-sequence originating from Saccharomyces cerevisiae. Fitzgerald et al.
  • WO2022171827 discloses a secretion signal comprising the KRE1 or SWP1 signal sequence followed by the MFa pro-sequence.
  • a protein of interest which is expressed as part of a fusion protein comprising a signal peptide sequence, signal peptide or presequence (all terms can be used interchangeably) originating from a protein selected from the group consisting of GDT1 , SLP1 , PP7435_Chr4_0694, WSC3, NCR1 , ZRT1 and PP7435_Chr2_0965 fused to a pro-sequence, preferably an a-mating factor (MFa) pro-sequence, is significantly increased.
  • MFa a-mating factor
  • the present invention relates to a secretion signal comprising from N-terminus to C-terminus
  • a pro-sequence preferably an a-mating factor (MFa) pro-sequence.
  • a further aspect of the present invention relates to a nucleic acid molecule encoding a fusion protein comprising from N-terminus to C-terminus
  • a pro-sequence preferably an a-mating factor (MFa) pro-sequence and
  • the secretion signal increases the expression and/or the secretion of a protein of interest fused to the secretion signal of the present invention from a eukaryotic host cell in comparison to a eukaryotic host cell expressing a nucleic acid molecule encoding a protein of interest fused to the wild type Saccharomyces cerevisiae a-mating factor secretion signal, or secretion signal fusions consisting of a signal peptide originating from one of the proteins selected from OST 1 , KRE1 or SWP1 with the pro-sequence of the Saccharomyces cerevisiae a-mating factor.
  • the titers obtained with pre-sequences KRE1 and SWP1 could be increased further by fusing the pre-sequences KRE1 and SWP1 to an MFa pro-sequence (see Tables 7 and 8 of WO2022171827).
  • secretion signals comprising pre-sequences of GDT1 , SLP1 , PP7435_Chr4_0694, WSC3, NCR1 , ZRT1 and PP7435_Chr2_0965 and a pro- sequence showed a significantly higher expression and/or secretion of a protein of interest than the secretion signals disclosed in WO2022171827 comprising pre-sequences KRE1 or SWP1 fused to an MFa pro-sequence.
  • the MFa pro-sequence may comprise an amino acid sequence which is at least 60% identical to SEQ ID NO: 1 , 2 or 3 to 9, preferably SEQ ID NO: 1 , 2 or 3.
  • the MFa pro-sequence may consist of an amino acid sequence which is at least 60% identical to SEQ ID NO: 1 , 2 or 3 to 9, preferably SEQ ID NO: 1 , 2 or 3.
  • the MFa pro-sequence preferably comprises Ser at a position corresponding to position 23 of SEQ ID NO: 1 and/or Glu at a position corresponding to position 64 of SEQ ID NO: 1.
  • the signal peptide sequence originating from GDT1 preferably comprises or consists of an amino acid sequence which is at least 60% identical to SEQ ID No. 10 (MKFGLGSLGLAVALIPIASA), the signal peptide sequence originating from SLP1 preferably comprises or consists of an amino acid sequence which is at least 60% identical to SEQ ID No. 11 (MLVAWFLLLLVSSCIC), the signal peptide sequence originating from PP7435_Chr4_0694 preferably comprises or consists of an amino acid sequence which is at least 60% identical to SEQ ID No. 12 (MQLQYLAVLCALLLNVQS), the signal peptide sequence originating from WSC3 preferably comprises or consists of an amino acid sequence which is at least 60% identical to SEQ ID No.
  • the signal peptide sequence originating from NCR1 preferably comprises or consists of an amino acid sequence which is at least 60% identical to SEQ ID No. 14 (MIILLPLLFLFVAGLVQA), the signal peptide sequence originating from ZRT 1 preferably comprises or consists of an amino acid sequence which is at least 60% identical to SEQ ID No. 15 (MNLKTWITVFIAVAQS) and the signal peptide sequence originating from PP7435_Chr2-0965 preferably comprises or consists of an amino acid sequence which is at least 60% identical to SEQ ID No. 16 (MRLSYECLFSVFLVLAYHLKGTKA).
  • sequence identity refers to the percentage of identical nucleotide or amino acid matches between at least two nucleotide or amino acid sequences aligned using a standardized algorithm. Such an algorithm can, in a standardized and reproducible manner, insert gaps in the compared sequences to optimize the alignment between two sequences, thus achieving a more meaningful comparison of the two sequences.
  • Percent identity between sequences may be determined using one or more computer algorithms or programs known in the prior art or described herein.
  • the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information (NCBI) (Altschul et al., 1990)
  • NCBI National Center for Biotechnology Information
  • BLAST 2 Sequences which is used for direct pairwise compari-son of two nucleotide or amino acid sequences.
  • BLAST 2 Se-quences can also be accessed and used interactively via the NCBI World Wide Web page on the Internet.
  • W word length
  • E expectation
  • B BLOSUM62 scoring matrix
  • the protein of interest may be any protein, polypeptide or peptide or may be selected from the group consisting of an antibody such as a chimeric, humanized or human antibody, or a bispecific antibody, or an antigen-binding antibody fragment such as Fab or F(ab)2, single chain antibodies such as scFv, single domain antibodies such as VHH fragments of camelid or heavy chain antibodiesor domain antibodies (dAbs), an artificial antigen-binding molecule such as a DARPIN, ibody, affibody, humabody, or a mutein based on a polypeptide of the lipocalin family, an enzyme such as a process enzyme, a cytokine, growth factor, hormone, protein antibiotic, fusion protein such as a toxin-fusion protein, a therapeutic peptide, a modified therapeutic peptide, a peptide comprising non-canonical amino acids, interferons, a structural protein, a regulatory protein, and a vaccine antigen, preferably wherein the protein
  • the present invention further relates to an expression cassette comprising the nucleic acid molecules of the invention and a promoter operably linked thereto.
  • the nucleic acid molecule of the invention or said expression cassette may be comprised in a vector, preferably an expression vector, or be integrated within a chromosome or the genome, in particular an artificial chromosome.
  • the present invention further provides for a recombinant eukaryotic host cell comprising a nucleic acid molecule encoding the secretion signal of the present invention and/or a nucleic acid molecule encoding the fusion protein of the present invention, the vector of the invention or the expression cassette of the invention.
  • a recombinant eukaryotic host cell comprising a nucleic acid molecule encoding the secretion signal of the present invention and/or a nucleic acid molecule encoding the fusion protein of the present invention, the vector of the invention or the expression cassette of the invention.
  • the recombinant eukaryotic host cell engineered with such nucleic acid molecule or expression cassette or vector of the invention is genetically engineered to incorporate the respective nucleic acid molecule, vector or expression cassette.
  • the recombinant eukaryotic host cell may be genetically engineered to comprise such nucleic acid molecules, vector or expression cassette within the host cell genome.
  • the recombinant host cell may be a fungal or yeast host cell, preferably a yeast host cell, selected from the group consisting of Komagataella phaffii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp. and Schizosaccharomyces pombe, or a fungal host cell such as Trichoderma reesei, Aspergillus niger.
  • a yeast host cell selected from the group consisting of Komagataella phaffii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp. and
  • the present invention further relates to a method of producing a protein of interest by culturing the host cell of the invention under conditions to express the nucleic acid molecule of the invention wherein the protein of interest is secreted , and isolating the protein of interest from the host cell culture, and optionally purifying and optionally modifying and optionally formulating the protein of interest.
  • the present invention further relates to a method of manufacturing a protein of interest in a eukaryotic host cell, comprising
  • the present invention further relates to a method of increasing the secretion of a protein of interest from a eukaryotic host cell, comprising expressing in said eukaryotic host cell the nucleic acid molecule of the invention and optionally overexpressing one or more component(s) of a signal recognition particle (SRP), thereby increasing the secretion and/or expression of said protein of interest in comparison to said host cell expressing the nucleic acid molecule of the invention but comprising a wild type Saccharomyces cerevisiae a-mating factor secretion signal (such as SEQ ID NO: 1) instead of the secretion signal described herein.
  • SRP signal recognition particle
  • the method of increasing the expression and/or secretion of a protein of interest from a eukaryotic host cell may additionally comprise
  • the secretion signal may further increase secretion and/or expression of said protein of interest from the eukaryotic host cell in comparison to the protein of interest fused to a wild type Saccharomyces cerevisiae a-mating factor secretion signal or fused to a secretion signal comprising the KRE1 , SWP1 orOSTI signal sequence followed by an MFa pro-sequence (e.g. SEQ ID NO: 1 , SEQ ID No. 2 or SEQ ID No. 3).
  • a pro-sequence e.g. SEQ ID NO: 1 , SEQ ID No. 2 or SEQ ID No. 3
  • the present invention relates to the use of the recombinant host cell of the invention for manufacturing a protein of interest.
  • a protein of interest fused to the inventive secretion signal (the fusion protein) is more efficiently secreted by recombinant host cells when compared to eukaryotic host cells expressing fusion proteins comprising secretion signals OST1-aMFpro, KRE1-aMFpro and SWP1-aMFpro, for instance, or a wild type Saccharomyces cerevisiae a-mating factor secretion signal instead of the secretion signal as defined herein, i.e. the protein of interest comprised in the fusion protein of the invention is secreted while the secretion signal is cleaved off during secretion.
  • a fusion protein comprising from N-terminus to C-terminus (a) a secretion signal, the secretion signal comprising (i) a signal peptide sequence originating from a protein selected from the group consisting of GDT1 , SLP1 , PP7435_Chr4_0694, WSC3, NCR1 , ZRT1 and PP7435_Chr2_0965 and (ii) an a-mating factor (MFa) pro-sequence; and (b) a protein of interest provides superior properties, e.g. an increased secretion of the protein of interest.
  • a secretion signal comprising (i) a signal peptide sequence originating from a protein selected from the group consisting of GDT1 , SLP1 , PP7435_Chr4_0694, WSC3, NCR1 , ZRT1 and PP7435_Chr2_0965 and (ii) an a-mating factor (MFa) pro-sequence
  • the wording “from N-terminus to C-terminus” does not necessarily exclude that the secretion signal, including the signal peptide sequence and the a-mating factor (MFa) pro-sequence, and the protein of interest are separated by one or more amino acids. These one or more amino acids may be a linker or linker sequences.
  • a “linker sequence” (also referred to as a “spacer sequence”) is an amino acid sequence that is introduced between the secretion signal and the fusion protein.
  • the “linker sequence” may also be an amino acid sequence that is introduced between the signal peptide sequence and the a-mating factor (MFa) pro-sequence.
  • linker sequences there is however no linker between the signal peptide sequence and the a-mating factor (MFa) pro-sequence.
  • linker sequences can be composed of flexible residues like glycine and serine or rather rigid residues as alanine-proline repeats. It may be preferred that the linker sequence does not adopt a secondary structure (such as an a-helical structure or a p-sheet) in order to ensure maximal flexibility.
  • a linker sequence can further be a protease cleavage/recognition site, such as recognized by a specific protease e.g., by a member of the subtilisin/kexin-like proprotein convertase (PC) family, a TEV protease or a member of the caspase family such as reversed caspase or circularly permutated caspase variants and variants with improved P1 'tolerance
  • a protease cleavage/recognition site such as recognized by a specific protease e.g., by a member of the subtilisin/kexin-like proprotein convertase (PC) family, a TEV protease or a member of the caspase family such as reversed caspase or circularly permutated caspase variants and variants with improved P1 'tolerance
  • the secretion signal increases expression of the fusion protein and/or the secretion of the protein of interest fused to the secretion signal from a eukaryotic host cell in comparison to a eukaryotic host cell expressing a fusion protein comprising secretion signals OST1-aMFpro, KRE1-aMFpro or SWP1-aMFpro, for instance, or a wild type Saccharomyces cerevisiae a-mating factor secretion signal instead of the secretion signal described herein within the context of the invention.
  • the MFa secretion signal of wild type S. cerevisiae as well as secretion signals OST1-aMFpro, KRE1- aMFpro or SWP1-aMFpro may be used as a control or reference for comparisons.
  • the protein of interest due to (over)expression of the protein of interest as a part of the fusion protein encoded by the nucleic acid of the invention, the protein of interest (POI , after cleavage of the secretion signal and secretion itself) is obtainable in high yields.
  • a high yield also named specific yield
  • the specific yield can also be measured in mg (secreted) POI/g wet biomass, also called wet cell weight, with high yields being considered as 0.25 to 50, or even above this value.
  • “Increased secretion” as used herein relates to a higher amount of detectable protein of interest in the supernatant or culture medium of a host cell and/or higher yield of the protein of interest in comparison to a control; both cultivated under identical conditions (e.g., host cell species, culture medium, cultivation time, cultivation temperature, feeding and expression and induction strategy).
  • a control may be the same host cell, but where in the same host cell the protein of interest is expressed as a fusion protein comprising the MFa secretion signal as well as secretion signals OST1-aMFpro, KRE1-aMFpro and SWP1-aMFpro, for instance, instead of the secretion signal of the present invention.
  • the increase may be expressed in fold change (FC) of secretion and/or yield, e.g. an increase by at least 1.1 -fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 5-fold or at least 10-fold.
  • FC fold change
  • the amount of detectable protein of interest in the supernatant (culture medium) or the culture broth of a host cell can be expressed as volumetric titer in [g of protein of interest / L of supernatant resp.
  • the fold change of secretion is then the ratio of the volumetric titer (also “titer”) or the yield of the cultivation of a host cell to the volumetric titer or the yield of the cultivation of the control.
  • the fusion protein in particular the protein of interest, may further comprise one or more tags, one or more protease recognition/cleavage sites and one or more linkers between the protein of interest, the tag(s) and the cleavage/recognition site(s).
  • Linker(s) are, e.g., defined above.
  • the tags can be tags used for purification and/or to enhance the expression and I or solubility or the detection of the protein of interest. There are many purification, expression enhancing, solubility enhancing tags and tags that allow easy detection and quantification of the protein of interest known to a person skilled in the art.
  • Purification tags are known to a person skilled in the art and may be a protein purification tag, preferably a GST tag, a FLAG tag, a polyarginine tag, a polyhistidine tag, such as a 6His-tag, an MBP tag, an S-tag, an influenza virus HA tag, a thioredoxin tag or a staphylococcal protein A tag.
  • protease cleavage/recognition sites that can be used to cleave off the tags from the protein of interest after expression and/or secretion of the protein of interest from the host cell using the corresponding proteases known to a person skilled in the art, e.g.
  • protease recognition and/or cleavage site such as recognized by a specific protease e.g., by a member of the subtilisin/kexin-like proprotein convertase (PC) family or caspase family such as reversed caspase or circularly permutated caspase variants and variants with improved P1 'tolerance.
  • PC subtilisin/kexin-like proprotein convertase
  • caspase family such as reversed caspase or circularly permutated caspase variants and variants with improved P1 'tolerance.
  • a secretion signal comprises a signal peptide sequence.
  • Signal peptide sequences typically consist of 13 to 36 mostly hydrophobic amino acids flanked by N-terminal basic amino acids and C-terminal polar amino acids.
  • the signal peptide sequence can interact with the signal recognition particle (SRP) or other transport proteins (e.g., SND, GET) that mediates the co- or post-translational translocation of the nascent protein from the cytosol into the lumen of the ER.
  • SRP signal recognition particle
  • GET transport proteins
  • the signal peptide sequence is typically cleaved off and the protein folds and undergoes post-translational modifications. The protein is then delivered from the ER to the Golgi apparatus and then on to secretory vesicles and the cell exterior.
  • a subset of nascent proteins natively destined for secretion carry a secretion signal that also comprises a pro-sequence such as the a-mating factor pro-sequence C- terminal to the signal peptide.
  • Pro-sequences typically consist of hydrophobic amino acids interrupted by charged or polar amino acids.
  • the pro sequence slows down transport and ensures proper folding of the protein, and/or facilitates transport of the protein from the ER to the Golgi apparatus, where the pro sequence is typically cleaved off at a defined recognition site, often a mono- or di-basic sequence (as e.g. K, R or KR, KK, RR, or RK) in a matching N-terminal amino acid sequence environment.
  • a signal peptide sequence originating from” a protein as used herein describes an amino acid sequence, i.e. the signal peptide sequence, which is present in the respective protein as defined herein.
  • “Present in the respective protein” means that the nucleic acid molecule encoding the respective protein comprises a nucleic acid sequence encoding the respective signal peptide and thus the respective protein is expressed as a fusion protein comprising the respective signal peptide.
  • the person skilled in the art can determine the signal peptide of a protein by means of in silico analysis using the website SignalP to predict the presence of a signal peptide within the first 50 amino acids of a given amino acid sequence, including the particular amino acid residue of cleavage (i.e. the C-terminal end of the signal peptide).
  • BLAST Basic Local Alignment Search Tool
  • BLASTp amino acid alignments
  • BLASTn nucleic acid alignments
  • BLASTx translated nucleic acids to amino acids
  • tBLASTn reverse-translated amino acid sequence to nucleic acid alignments
  • signal peptide sequences described herein originate from a protein (prior cleavage of the secretion signal (signal peptide and leader peptide)) selected from the group consisting of GDT1 , SLP1 , PP7435_Chr4_0694, WSC3, NCR1 , ZRT1 and PP7435_Chr2_0965 before secretion and/or before cleavage of the signal peptide sequence. “Originating from” may be used interchangeably with “derived from”.
  • the GDT 1 protein from K phaffii was referenced to a protein of unknown function involved in calcium homeostasis.
  • GDT1 is localized to the cis- and medial-Golgi apparatus and acts as a divalent cation: proton antiporter that exchanges calcium or manganese ions for protons across the Golgi membrane. It mediates the reversible transport of calcium or manganese to the Golgi lumen driven by the proton gradient and possibly the membrane potential generated by V-ATPase.
  • the signal peptide sequence preferably corresponds to amino acids 1-20, as depicted also in SEQ ID NO: 10. Accordingly, the signal peptide sequence originating from a GDT1 protein may comprise or consist of an amino acid sequence being at least 60% identical to SEQ ID NO: 10.
  • the signal peptide sequence originating from a GDT1 protein may preferably comprise or consist of an amino acid sequence of at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, sequence identity to SEQ ID NO: 10.
  • the NCR1 protein from K phaffii with UniProt entry A0A1 B2J6W0 is a vacuolar membrane protein that transits through the biosynthetic vacuolar protein sorting pathway, and is involved in sphingolipid metabolism.
  • the signal peptide sequence originating from an NCR1 protein may comprise or consist of an amino acid sequence being at least 60% identical to SEQ ID NO: 14, wherein the signal peptide sequence preferably corresponds to amino acids 1- 18 of said database entry depicted also in SEQ ID NO: 14.
  • the signal peptide sequence originating from an NCR1 protein may preferably comprise or consist of an amino acid sequence of at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, sequence identity to SEQ ID NO: 14.
  • the SLP1 protein or a functional homolog thereof corresponds to an integral membrane protein of unknown function in S. cerevisiae, based on UniProt database entry Q12232.
  • the signal peptide sequence preferably corresponds to amino acids 1-16 of SEQ ID NO: 11.
  • the signal peptide sequence originating from an SLP1 protein may comprise or consist of an amino acid sequence being at least 60% identical to SEQ ID NO: 11.
  • the signal peptide sequence originating from an SLP1 protein may preferably comprise or consist of an amino acid sequence of at least 70%, more preferably at least any one of 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least or 95%, sequence identity to SEQ ID NO: 11.
  • the WSC3 protein or a functional homolog thereof corresponds to a sensor-transducer of the stress-activated PKC1-MPK1 signaling pathway, according to UniProt entry A0A1 B2JEC2, wherein the signal peptide sequence preferably corresponds to amino acids 1-16 of SEQ ID NO: 13.
  • the signal peptide sequence originating from an WSC3 protein may comprise or consist of an amino acid sequence being at least 60% identical to SEQ ID NO: 13.
  • the signal peptide sequence originating from an WSC3 protein may preferably comprise or consist of an amino acid sequence of at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, sequence identity to SEQ ID NO: 13.
  • the PP7435_Chr2-0965 protein or a functional homolog thereof shows low similarity to putative mannoprotein of cell wall with role in response to stress (C. albicans), wherein the signal peptide sequence preferably corresponds to amino acids 1-24 of said database entry depicted also in SEQ ID NO: 16.
  • the signal peptide sequence originating from a PP7435_Chr2-0965 protein may comprise or consist of an amino acid sequence being at least 60% identical to SEQ ID NO: 16.
  • the signal peptide sequence originating from a PP7435_Chr2-0965 protein may preferably comprise or consist of an amino acid sequence of at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, sequence identity to SEQ ID NO: 16.
  • the PP7435_Chr4-0694 or a functional homolog thereof shows similarity to YDR262W (Putative protein of unknown function), wherein the signal peptide sequence preferably corresponds to amino acids 1-18 of said database entry depicted also in SEQ ID NO: 12.
  • the signal peptide sequence originating from a PP7435_Chr4-0694 protein may comprise or consist of an amino acid sequence being at least 60% identical to SEQ ID NO: 12.
  • the signal peptide sequence originating from a PP7435_Chr4-0694 protein may preferably comprise or consist of an amino acid sequence of at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, sequence identity to SEQ ID NO: 12.
  • the ZRT1 protein corresponds to UniProt database entry P32804 for S. cerevisiae or a functional homolog thereof, described as high-affinity zinc transporter of the plasma membrane, wherein the signal peptide sequence preferably corresponds to amino acids 1-16 of said database entry depicted also in SEQ ID NO: 15.
  • the signal peptide sequence originating from a ZRT 1 protein may comprise or consist of an amino acid sequence being at least 60% identical to SEQ ID NO: 15.
  • the signal peptide sequence originating from a ZRT1 protein may preferably comprise or consist of an amino acid sequence of at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, sequence identity to SEQ ID NO: 15.
  • a-Mating factor (MFa) also known as Mating factor alpha-1 , alpha-1 mating pheromone or mating factor alpha, is a hormone, wherein the active factor (MFa without secretion signal) is excreted into the culture medium by haploid cells of the alpha mating type and acts on cells of the opposite mating type (type a).
  • MFa carries a secretion signal comprising a signal peptide sequence (pre-sequence) and a pro-sequence (both cleaved by peptidase and protease).
  • MFa might be from any eukaryotic species, in particular from any yeast, preferably from any yeast of the Saccharomyces genus.
  • Exemplary yeasts include, but are not limited to, Komagataella phaffii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces kudriavzevii, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp. and Schizosaccharomyces pombe.
  • MFa can also be from Trichoderma reesei or Aspergillus niger.
  • the origin is from S. cerevisiae.
  • the pro-sequence may comprise or consist of the amino acids 20-89 of the full-length MFa protein (id est the protein including the signal peptide and the prosequence translated from the mRNA encoding the MFa protein) such as the MFa protein from S. cerevisiae or a functional homolog thereof.
  • the full-length MFa protein corresponds to UniProt database entry P01149, wherein the a-mating factor (MFa) pro-sequence preferably corresponds to amino acids 20-89, more preferably amino acids 20-85 as depicted in SEQ ID NO: 1 , of said database entry.
  • the MFa pro-sequence may comprise or consist of an amino acid sequence which is at least 60% identical to SEQ ID Nos. 1 , 2 or 3 to 9.
  • the MFa pro-sequence preferably comprises or consists of an amino acid sequence of at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, sequence identity to SEQ ID Nos. 1 , 2, or 3 to 9.
  • the MFa pro-sequence preferably has a Ser at a position corresponding to position 23 of SEQ ID NO: 1 and/or Glu at a position corresponding to position 64 of SEQ ID NO: 1. This can further increase secretion.
  • SEQ ID NO: 2 already contains these mutations.
  • a functional homolog is a functional equivalent of a nucleic acid sequence or a peptide, polypeptide or protein described in this document.
  • a functional homolog may be a biologically active sequence that has at least about 60% amino acid sequence identity with a given sequence of a polypeptide as disclosed herein.
  • nucleic acid sequences the degeneracy of the genetic code permits substitution of certain codons by other codons that specify the same amino acid and hence would give rise to the same protein.
  • the nucleic acid sequence can vary substantially since, with the exception of methionine and tryptophan, the known amino acids can be coded for by more than one codon.
  • portions or all of the nucleic acid sequences described herein could be synthesized to give a nucleic acid sequence significantly different from that shown in their indicated sequence. The encoded amino acid sequence thereof would, however, be preserved.
  • the functional homolog may also describe a functional equivalent of an amino acid sequence or a peptide, polypeptide or protein described in this document, which has up to 10 conservative mutations, i.e. the functional homolog can have 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative mutations.
  • the functional homolog in particular for functional homologs of the MFa pro-sequence, can also have 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 14, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, or 27conservative mutations.
  • a “conservative mutation” as used herein is preferably a mutation that results in a point mutation which is e.g., a substitution, insertion or deletion of one amino acid, in particular where the substitution is the replacement of an amino acid residue with a chemically similar amino acid residue, which results in no change of the function of the amino acid sequence or peptide, polypeptide or protein.
  • conservative substitutions are the replacements among the members of the following groups: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan.
  • a conservative mutation can also be any a substitution, insertion or deletion of one amino acid that does not influence the biological activity of the amino acid sequence of a peptide, polypeptide or protein described herein.
  • a functional homolog may have up to 14 conservative mutations.
  • a functional homolog may have up to 13 conservative mutations.
  • a functional homolog may have up to 12 conservative mutations.
  • a functional homolog may have up to 11 conservative mutations.
  • a functional homolog may have 10 conservative mutations.
  • a functional homolog may have up to 9 conservative mutations
  • a functional homolog may have up to 8 conservative mutations.
  • a functional homolog may have up to 7 conservative mutations.
  • a functional homolog may have 6 conservative mutations.
  • a functional homolog may have up to 5 conservative mutations.
  • a functional homolog may have up to 4 conservative mutations.
  • a functional homolog may have up to 3 conservative mutations.
  • a functional homolog may have up to 2 conservative mutations.
  • a functional homolog may have 1 conservative mutation.
  • the present invention further relates to a secretion signal, the secretion signal comprising (i) a signal peptide sequence originating from a GDT1 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • a secretion signal comprising (i) a signal peptide sequence originating from a GDT1 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • the secretion signal comprising a signal peptide sequence originating from a GDT1 protein may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 17.
  • the present invention further relates to a secretion signal, the secretion signal comprising (i) a signal peptide sequence originating from an NCR1 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • a secretion signal comprising (i) a signal peptide sequence originating from an NCR1 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • the secretion signal comprising a signal peptide sequence originating from a NCR1 protein may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 18.
  • the present invention further relates to a secretion signal, the secretion signal comprising (i) a signal peptide sequence originating from an SLP1 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • MFa a-mating factor
  • the signal peptide sequence may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 11 and the a-mating factor (MFa) pro-sequence may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 1 , 2 or 3 to 9.
  • MFa a-mating factor
  • the secretion signal comprising a signal peptide sequence originating from a SLP1 protein may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 19.
  • the present invention further relates to a secretion signal, the secretion signal comprising (i) a signal peptide sequence originating from an WSC3 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • a secretion signal comprising (i) a signal peptide sequence originating from an WSC3 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • the signal peptide sequence may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 13 and the a-mating factor (MFa) pro-sequence may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 1 , 2 or 3 to 9.
  • MFa a-mating factor
  • the secretion signal comprising a signal peptide sequence originating from a WSC3 protein may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 20
  • the present invention further relates to a secretion signal, the secretion signal comprising (i) a signal peptide sequence originating from an PP7435_Chr2-0965 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • a secretion signal comprising (i) a signal peptide sequence originating from an PP7435_Chr2-0965 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • the signal peptide sequence may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 16 and the a-mating factor (MFa) prosequence may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 1 , 2 or 3 to 9.
  • MFa a-mating factor
  • the secretion signal comprising a signal peptide sequence originating from a PP7435_Chr2-0965 protein may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 21.
  • the present invention further relates to a secretion signal, the secretion signal comprising (i) a signal peptide sequence originating from an PP7435_Chr4-0694 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • a secretion signal comprising (i) a signal peptide sequence originating from an PP7435_Chr4-0694 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • the signal peptide sequence may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 12 and the a-mating factor (MFa) prosequence may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 1 , 2 or 3 to 9.
  • MFa a-mating factor
  • the secretion signal comprising a signal peptide sequence originating from a PP7435_Chr4-0694 protein may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 22.
  • the present invention further relates to a secretion signal, the secretion signal comprising (i) a signal peptide sequence originating from an ZRT 1 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • a secretion signal comprising (i) a signal peptide sequence originating from an ZRT 1 protein; and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • the signal peptide sequence may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 15 and the a-mating factor (MFa) pro-sequence may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 1 , 2 or 3 to 9.
  • MFa a-mating factor
  • the secretion signal comprising a signal peptide sequence originating from a ZRT1 protein may comprise or consist of an amino acid sequence being at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, identical to SEQ ID NO: 23.
  • protein of interest refers to a protein that is produced by means of recombinant technology in a host cell. More specifically, the protein may either be a polypeptide not naturally occurring in the host cell, i.e. a heterologous protein e.g. an artificial protein such as a protein not naturally produced by wild-type cells, or else may be native to the host cell, i.e. a homologous protein to the host cell.
  • Both, heterologous and homologous proteins can be produced, for example, by transformation with a self-replicating vector containing the nucleic acid sequence encoding the POI, or upon integration by recombinant techniques of one or more copies of the nucleic acid sequence encoding the POI into the genome and/or a chromosome of the host cell.
  • Homologous proteins can be produced by recombinant modification of one or more regulatory sequences controlling the expression of the gene encoding the POI, e.g. of the promoter sequence.
  • the proteins of interest referred to herein may be produced by methods of recombinant expression well known to a person skilled in the art.
  • the protein of interest may be a recombinant protein.
  • the POI is usually a eukaryotic or prokaryotic polypeptide, variant or derivative thereof, or an artificial polypeptide, such as a polypeptide not naturally produced by wild-type cells.
  • the POI can be any eukaryotic or prokaryotic protein.
  • the protein can be a naturally secreted protein or an intracellular protein, i.e. a protein which is not naturally secreted.
  • the present invention also includes biologically active fragments of proteins.
  • a POI may be an amino acid chain or present in a complex, such as a dimer, trimer, or higher - mer.
  • the meres may be homo- or hetero-meres or a mixture thereof, the meres can join together to form further di-, tri- or higher meres or higher structures.
  • the POI may also be a multimer or oligomer. Fusion of the POI with the secretion signal of the invention can render any POI to be secreted.
  • the POI may be modified or include one or more non-canonical amino acids.
  • the POI may be a protein that requires co-translational translocation.
  • the POI may also include N- or C-terminal or within the sequence of the POI additional amino acids, tags, protease cleavage I recognition sites, linkers and the like.
  • the protein of interest may be a protein used as nutritional, dietary, digestive, supplements, such as in food products, feed products, or cosmetic products.
  • the food products may be, for example, bouillon, desserts, cereal bars, confectionery, sports drinks, dietary products or other nutrition products.
  • the protein of interest is a food additive.
  • the protein of interest may be used in animal feeds.
  • POI anti-microbial proteins
  • a POI may be an enzyme.
  • Preferred enzymes are those which can be used for industrial application, such as in the manufacturing of a detergent, starch, fuel, textile, pulp and paper, oil, personal care products, or such as for baking, organic synthesis, and the like. Examples of such enzymes include proteases, amylases, lipases, mannanases.
  • a POI may be a therapeutic protein.
  • the POI may be a naturally secreted protein or an intracellular protein, i.e. a protein which is not naturally secreted.
  • the present invention also provides for the recombinant production of functional homologues, functional equivalent variants, derivatives and biologically active fragments of naturally secreted or not naturally secreted proteins.
  • Functional homologues are preferably identical with or correspond to and have the functional characteristics of a sequence.
  • the POI may be structurally similar to the native protein and may be originating from the native protein by addition of one or more amino acids to either or both the C- and N-terminal end or the side-chain of the native protein, substitution of one or more amino acids at one or a number of different sites in the native amino acid sequence, deletion of one or more amino acids at either or both ends of the native protein or at one or several sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the native amino acid sequence.
  • modifications are well known for several of the proteins mentioned above.
  • the protein of interest is a mammalian polypeptide or even more preferably a human polypeptide.
  • the protein of interest is a therapeutic or biopharmaceutical protein.
  • therapeutic proteins which refer to any polypeptide, protein, protein variant, fusion protein and/or fragment thereof which may be administered to a mammal even more preferred to a human.
  • the protein of interest can also be an artificial protein, or a part of a natural protein or of natural proteins or of artificial proteins or a fusion protein. It is envisioned but not required that therapeutic protein according to the present invention is heterologous to the cell, the proteins of interest may be antigens as used for vaccination, vaccines, antigen-binding proteins, immune stimulatory proteins.
  • an antibody fragment may include but not limited to Fv (a molecule comprising the VL and VH), single-chain Fv (scFV) (a molecule comprising the VL and VH connected with by peptide linker), Fab, Fab', F(ab')2, single domain antibody (sdAb) (molecules comprising a single variable domain and 3 CDR), and multivalent presentations thereof.
  • Fv a molecule comprising the VL and VH
  • scFV single-chain Fv
  • Fab' single domain antibody
  • sdAb single domain antibody
  • the antibody or fragments thereof may be murine, human, humanized or chimeric antibody or fragments thereof.
  • therapeutic proteins include an antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragments, such as Fab', F(ab')2, Fv, scFv, di-scFvs, bi-scFvs, tandem scFvs, bispecific tandem scFvs, sdAb, VHH, VH, and VL, or human antibody, humanized antibody, chimeric antibody, IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, intrabody, minibody or synthetic binding proteins constructed using a fibronectin type III domain (FN3) as a molecular scaffold also known as monobody.
  • FN3 fibronectin type III domain
  • the protein of interest may further be selected from the group consisting of an antibody such as a chimeric, humanized or human antibody, or a bispecific antibody, or an antigen-binding antibody fragment such as Fab or F(ab)2, single chain antibodies such as scFv, single domain antibodies such as VHH fragments of camelid or heavy chain antibodies or domain antibodies (dAbs), an artificial antigen-binding molecule such as a DARPIN, ibody, affibody, humabody, or a mutein based on a polypeptide of the lipocalin family, an enzyme such as a process enzyme, a cytokine, growth factor, hormone, protein antibiotic, fusion protein such as a toxin-fusion protein, a structural protein, a regulatory protein, and a vaccine antigen, preferably wherein the protein of interest is a therapeutic protein, a food additive or a feed additive.
  • an antibody such as a chimeric, humanized or human antibody, or a bispecific antibody, or an antigen
  • the protein is an antibody.
  • antibody is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.
  • the archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to be "antibodies.” Numerous antibody coding sequences have been described; and others may be raised by methods well-known in the art.
  • antibodies or antigen binding antibody fragments may be produced by methods known in the art.
  • antibody-producing cells are sensitized to the desired antigen or immunogen.
  • the messenger RNA isolated from antibody producing cells is used as a template to make cDNA using PCR amplification.
  • a library of vectors, each containing one heavy chain gene and one light chain gene retaining the initial antigen specificity, is produced by insertion of appropriate sections of the amplified immunoglobulin cDNA into the expression vectors.
  • a combinatorial library is constructed by combining the heavy chain gene library with the light chain gene library. This results in a library of clones which co-express a heavy and light chain (resembling the Fab fragment or antigen binding fragment of an antibody molecule).
  • the vectors that carry these genes are cotransfected into a host cell. When antibody gene synthesis is induced in the transfected host, the heavy and light chain proteins self-assemble to produce active antibodies that can be detected by screening with the antigen or immunogen.
  • Antibody coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed nucleic acids, and variants thereof.
  • Variant polypeptides can include amino acid (aa) substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function.
  • Variants can be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g., a functional domain, catalytic amino acid residues, etc.).
  • Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and/or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
  • Chimeric antibodies may be made by recombinant means by combining the variable light and heavy chain regions (VK and VH), obtained from antibody producing cells of one species with the constant light and heavy chain regions from another.
  • VK and VH variable light and heavy chain regions
  • chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in order to produce an antibody with predominantly human domains.
  • the production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Patent No. 5,624,659.
  • Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, and incorporate only the complementarity-determining regions of the animal-derived antibody. This is accomplished by carefully examining the sequence of the hyper-variable loops of the variable regions of the monoclonal antibody, and fitting them to the structure of the human antibody chains. Although facially complex, the process is straightforward in practice. See, e.g., U.S. Patent No. 6,187,287.
  • immunoglobulin fragments comprising the epitope binding site (e.g., Fab', F(ab')2, or other fragments) may be synthesized.
  • "Fragment” or minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques.
  • Fv immunoglobulins for use in the present invention may be produced by synthesizing a variable light chain region and a variable heavy chain region. Combinations of antibodies are also of interest, e.g. diabodies, which comprise two distinct Fv specificities.
  • Immunoglobulins may be modified post-translationally, e.g.
  • detectable moieties such as fluorescent dyes, enzymes, substrates, chemiluminescent moieties and the like
  • specific binding moieties such as streptavidin, Avidin, or biotin, and the like
  • therapeutic proteins include blood coagulation factors (VII, VIII, IX), alkaline protease from Fusarium, calcitonin, CD4 receptor darbepoetin, DNase (cystic fibrosis), erythropoetin, eutropin (human growth hormone derivative), follicle stimulating hormone (follitropin), gelatin, glucagon, glucocerebrosidase (Gaucher disease), glucosamylase from A. niger, glucose oxidase from A.
  • the present invention relates to a nucleic acid molecule or sequence encoding a secretion signal of the present invention comprising from N-terminus to C-terminus (i) a signal peptide sequence originating from a protein selected from the group consisting of GDT1 , SLP1 , PP7435_Chr4_0694, WSC3, NCR1 , ZRT1 and PP7435_Chr2_0965 and (ii) a pro-sequence, preferably an a-mating factor (MFa) pro-sequence.
  • MFa a-mating factor
  • Such a fusion protein as described herein may be encoded by a nucleic acid molecule.
  • the nucleic acid molecules of the invention can, e.g., be transformed into a host cell.
  • the present invention relates to a nucleic acid molecule encoding a fusion protein comprising from N-terminus to C-terminus (a) a secretion signal, the secretion signal comprising (i) a signal peptide sequence originating from a protein selected from the group consisting of GDT1 , SLP1 , PP7435_Chr4_0694, WSC3, NCR1 , ZRT1 and PP7435_Chr2_0965; and (ii) an a-mating factor (MFa) pro-sequence; and (b) a protein of interest.
  • a secretion signal comprising (i) a signal peptide sequence originating from a protein selected from the group consisting of GDT1 , SLP1 , PP7435_
  • Encoding means that when the nucleic acid or polynucleotide encoding a protein is expressed, it leads to the production of said protein.
  • an “expression cassette” as used herein relates to a distinct component of (vector) DNA comprising a gene such as the nucleic acid molecule of the invention (encoding the fusion protein of the invention) and regulatory sequences such as but not limited to a promoter comprising one or more ribosomal binding sites, a terminator and/or the like operably linked to the nucleic acid molecule of the invention to be expressed by a transfected cell.
  • the expression cassette may direct the host cell's machinery to express protein(s) of interest.
  • an expression cassette is composed of one or more genes and the sequences controlling their expression.
  • the present invention further relates to an expression cassette comprising the nucleic acid molecule of the invention (encoding the fusion protein of the invention) and a promoter operably linked thereto.
  • the expression cassette may be in the form of a vector.
  • the expression cassette may be comprised by a vector.
  • the nucleic acid molecule of the invention and/or the expression cassette and optionally polynucleotide(s) encoding one or more or all component(s) of an SRP can be integrated in a plasmid or vector wherein the nucleic acid molecule of the invention and/or the expression cassette and the polynucleotide(s) encoding one or more or all component(s) of an SRP can be in the same or different plasmids or vectors Accordingly, the present invention also relates to a vector comprising the nucleic acid of the invention or the expression cassette. A vector containing foreign DNA is termed recombinant DNA.
  • plasmid may relate to a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed (e.g., plasmid, cosmid, Lambda phages).
  • plasmid may relate to a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed (e.g., plasmid, cosmid, Lambda phages).
  • a skilled person is able to employ suitable plasmids or vectors depending on the host cell used.
  • the vector is a eukaryotic expression vector, preferably a yeast expression vector.
  • the vector is a eukaryotic expression vector, preferably a yeast expression vector.
  • yeast expression vector examples include Yip type vector, YEp type vector, YRp type vector, YCp type vector, pGPD-2, pAO815, pGAPZ, pGAPZa, pHIL-D2, pHIL-S1 , pPIC3.5K, pPIC9K, pPICZ, pPICZa, pPIC3K, pHWO10, pPUZZLE and 2 pm plasmids.
  • Such vectors are known and are for example described in Gregg et al., Mol Biotechnol. (2000) 16(1):23-52.
  • a “vector” usually comprises selectable markers, a number of restriction enzyme cleavage/recognition sites, a suitable promoter sequence and a transcription terminator, which components are operably linked together.
  • a “vector” may also comprise a bacterial origin of replication and a bacterial selectable marker as remainder from cloning plasmids.
  • the polypeptide coding sequence of interest is operably linked to transcriptional and translational regulatory sequences that provide for expression of the polypeptide in the host cells.
  • a vector or plasmid of the present invention encompass yeast artificial chromosome, which refers to a DNA construct that can be genetically modified to contain a heterologous DNA sequence (e.g., a DNA sequence as large as 3000 kb), that contains telomeric, centromeric, and origin of replication (replication origin) sequences.
  • yeast artificial chromosome refers to a DNA construct that can be genetically modified to contain a heterologous DNA sequence (e.g., a DNA sequence as large as 3000 kb), that contains telomeric, centromeric, and origin of replication (replication origin) sequences.
  • one promoter element can increase the amount of products expressed for multiple sequences attached in tandem. Hence, one promoter element can enhance the expression of one or more recombinant products.
  • the promoter could be an "inducible promoter”, a “de-regulated promoter”, a “de-repressed promoter” or “constitutive promoter” or a mixture thereof.
  • nucleotide sequences encoding the fusion protein of the invention or the secretion signal of the invention are driven by an inducible promoter.
  • inducible promoters are known in the art. Many are described in a review by Gatz, Curr. Op. Biotech., 7: 168 (1996) (see also Gatz, Ann. Rev. Plant. Physiol. Plant Mol. Biol., 48:89 (1997)). Examples include tetracycline repressor system, Lac repressor system, copper-inducible systems, salicylate-inducible systems (such as the PR1 a system), glucocorticoid-inducible (Aoyama et al., 1997), alcohol-inducible systems, e.g., AOX promoters, and ecdysome-inducible systems.
  • Suitable promoter sequences for use with yeast host cells are described in Mattanovich et al., Methods Mol. Biol. (2012) 824:329-58 and include glycolytic enzymes like triosephosphate isomerase (TPI), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH or GAP) and variants thereof, lactase (LAC) and galactosidase (GAL), P.
  • TPI triosephosphate isomerase
  • PGK phosphoglycerate kinase
  • GAP glyceraldehyde-3-phosphate dehydrogenase
  • LAC lactase
  • GAL galactosidase
  • PPGI glucose- 6-phosphate isomerase promoter
  • PPGK 3-phosphoglycerate kinase promoter
  • PPGP glycerol aldehyde phosphate dehydrogenase promoter
  • PTEF translation elongation factor promoter
  • POP1 triose phosphate isomerase
  • PRPS2, PRPS7, PRPS31 , PRPL1 alcohol oxidase promoter
  • PAOX alcohol oxidase promoter
  • PFLD formaldehyde dehydrogenase promoter
  • PICL isocitrate lyase promoter
  • PTHI alpha-ketoisocaproate decarboxylase promoter
  • PSSA1 formaldehyde dehydrogenase promoter
  • PICL isocitrate lyase promoter
  • PTHI alpha-ketoisocaproate decarboxylase promoter
  • PSSA1 formaldehyde dehydrogenase promoter
  • PICL isocitrate lyase promoter
  • PTHI alpha-ketoisocaproate decarboxylase promoter
  • PSSA1 heat shock protein family members
  • PGPM1 6- Phosphogluconate dehydrogenase
  • the GAP promoter, AOX promoter or a promoter originating from GAP or AOX promoter is particularly preferred.
  • AOX promoters can be induced by methanol and/or are repressed by glucose. Promoters originating from the AOX promoter for methanol-induced as well as methanol-free production (regulated by de-repression) are described in WO 2006/089329, EP 1851312 B1 and EP 2199389 B1.
  • Carbon source regulable promoter e.g., de-repressible promoters such as described in W02013050551 (e.g., pG1-pG8, fragments of pG1 , designated pG1a-pG1f), W02017021541 (e.g., pG1-D1240, or pG1-D1427), WO2020144313 (e.g., SEQ ID NO:10-16, or SEQ ID NO:41-45 of WO2020144313), WO2017109082 and WG2024079140 may be used.
  • de-repressible promoters such as described in W02013050551 (e.g., pG1-pG8, fragments of pG1 , designated pG1a-pG1f), W02017021541 (e.g., pG1-D1240, or pG1-D1427), WO2020144313 (e.g., SEQ ID NO:10
  • constitutive promoters such as MDH3, POR1 , PDC1 , FBA1-1 , or GPM1 (Prielhofer et al. 2017, BMC Sys Biol. 11 : 123), or as disclosed in WO2014139608 (e.g., pCS1).
  • suitable promoters include Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1 , ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CLIP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase (PGK), and the maltase gene promoter (MAL).
  • ENO-1 Saccharomyces cerevisiae enolase
  • GAL1 Saccharomyces cerevisiae galactokinase
  • ADH1 Alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
  • TPI
  • yeast host cells Other useful promoters for yeast host cells are described by Romanos et al, 1992, Yeast 8:423-488.
  • the secretion signal of the invention can also be used to improve the secretion and/or expression of endogenous proteins.
  • the nucleic acid sequence of the invention encoding the secretion signal is inserted upstream to the nucleic acid sequence encoding the endogenous proteins and optionally the nucleic acid sequence encoding the endogenous secretion signal is deleted or replaced.
  • Methods for genomic integration are well known to a person skilled in the art.
  • a “host cell” refers to a cell which is capable of protein expression and protein secretion. Such a host cell can be applied in the methods of the present invention. For that purpose, for the host cell to express a polypeptide, a nucleic acid molecule encoding the fusion protein is present or introduced in the cell. Host cells provided by the present invention can be eukaryotes. As will be appreciated by one of skill in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.
  • eukaryotic cells include, but are not limited to, vertebrate cells, mammalian cells, human cells, animal cells, invertebrate cells, plant cells, nematodal cells, insect cells, stem cells, fungal cells or yeast cells.
  • the host cell is a yeast cell.
  • the present invention relates to a host cell comprising the nucleic acid molecule of the invention. Additionally, the present invention relates to a host cell comprising the expression cassette of the invention. The present invention further relates to a host cell comprising the vector or the plasmid of the invention.
  • yeast cells include but are not limited to the Saccharomyces genus (e.g. Saccharomyces cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum), the Komagataella genus (Komagataella pastoris, Komagataella pseudopastoris or Komagataella phaffii), Kluyveromyces genus (e.g. Kluyveromyces lactis, Kluyveromyces marxianus), the Candida genus (e.g. Candida utilis, Candida cacaoi), the Geotrichum genus (e.g. Geotrichum fermentans), as well as Hansenula polymorpha and Yarrowia lipolytica.
  • Saccharomyces genus e.g. Saccharomyces cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum
  • the Komagataella genus Komagataella pastoris, Komagata
  • the eukaryotic host cell of the invention or the eukaryotic host cell used in the methods and uses of the invention can be a fungal or yeast host cell, preferably a yeast host cell, selected from the group consisting of Komagataella phaffii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella spp. and Schizosaccharomyces pombe, or a fungal host cell such as Trichoderma reesei, Aspergillus niger.
  • a yeast host cell selected from the group consisting of Komagataella phaffii (Pichia pastoris), Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia
  • Pichia comprises a number of species, including the species Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta. Most preferred is the species Pichia pastoris.
  • Pichia pastoris has been divided and renamed to Komagataella pastoris and Komagataella phaffii. Therefore Pichia pastoris is synonymous for both Komagataella pastoris and Komagataella phaffii, preferably synonymous for Komagataella phaffii.
  • the host cell is P. pastoris CBS7435 mut s or a subtype thereof, more preferably P.
  • P. pastoris CBS7435 mut s P. pastoris CBS 7435 modifyed to underexpress FLO8 and/or SCJ1 and/o rHCH1 (WO 2020/144313, WO 2015/158800) or P. pastoris CBS7435 mut s SRP#7 (WO 2022/171827) modified to overexpress one or more of all proteins of an/the signal recognition particle (SRP).
  • SRP signal recognition particle
  • the host cell is a Pichia pastoris, Hansenula polymorpha, Trichoderma reesei, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, and Komagataella, and Schizosaccharomyces pombe. It may also be a host cell from Ustilago maydis.
  • recombinant refers to the alteration of genetic material by human intervention. Typically, recombinant refers to the manipulation of DNA or RNA in a virus, cell, plasmid or vector by molecular biology (recombinant DNA technology) methods, including cloning and recombination.
  • a recombinant cell, polypeptide, or nucleic acid can be typically described with reference to how it differs from a naturally occurring counterpart (the "wild-type”).
  • a “recombinant cell” or “recombinant host cell” refers to a cell or host cell that has been genetically altered to comprise a nucleic acid sequence which was not native to said cell.
  • a “host cell for manufacturing a protein of interest” refers to a host cell in which nucleic acid sequences encoding a protein of interest may be introduced.
  • the recombinant host cell within the present invention does not necessarily contain the nucleic acid sequences encoding a protein of interest. It is appreciated by a skilled person in the art that the host cells can be provided for inserting desired nucleotide sequences into the host cell, for example, in a kit.
  • polypeptide and “protein” are interchangeably used.
  • polypeptide refers to a protein or peptide that contains two or more amino acids, typically at least 3, preferably at least 20, more preferred at least 30, such as at least 50 amino acids. Accordingly, a polypeptide comprises an amino acid sequence, and, thus, sometimes a polypeptide comprising an amino acid sequence is referred to herein as a “polypeptide comprising a polypeptide sequence”. Thus, herein the term “polypeptide sequence” is interchangeably used with the term “amino acid sequence”.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e.
  • a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • R groups e.g., norleucine
  • modified peptide backbones but retain the same basic chemical structure as a naturally occurring amino acid.
  • Systems to express proteins of interest comprising synthetic amino acids or amino acid analogs are known to a person skilled in the art and include, but are not limited to, the use of an expanded genetic code.
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
  • the present invention further relates to a method of manufacturing a protein of interest in a eukaryotic host cell, comprising (i) optionally genetically engineering a recombinant host cell to comprise the nucleic acid molecule of the invention;
  • Step (i) of the method of manufacturing may also be understood as genetically engineering the recombinant host cell to express a fusion protein of the invention.
  • a host cell is “engineered to express” a given protein, the host cell is manipulated such that the host cell has the capability to express, the nucleic acid molecule of the invention,
  • the present invention further relates to a method of producing a protein of interest by culturing a host cell of the invention under conditions to express the nucleic acid molecule of the invention wherein the protein of interest is secreted and wherein the secretion signal is cleaved from said fusion protein, and isolating the protein of interest from the host cell and optionally purifying and optionally modifying and optionally formulating the protein of interest.
  • a foreign or target polynucleotide such as the nucleic acid molecule of the invention can be inserted into the chromosome and/or genome by various means, e.g., by homologous recombination or by using a hybrid recombinase that specifically targets sequences at the integration sites.
  • the foreign or target polynucleotide described above is typically present in a vector ("inserting vector"). These vectors are typically circular and linearized before used for homologous recombination.
  • the foreign or target polynucleotides may be DNA fragments joined by fusion PCR or synthetically constructed DNA fragments which are then recombined into the host cell.
  • the vectors may also contain markers suitable for selection or screening, an origin of replication, and other elements. It is also possible to use heterologous recombination which results in random or non-targeted integration. Heterologous recombination refers to recombination between DNA molecules with significantly different sequences. Methods of recombinations are known in the art and for example described in Boer et al., Appl Microbiol Biotechnol (2007) 77:513- 523. One may also refer to Principles of Gene Manipulation and Genomics by Primrose and Twyman (7 th edition, Blackwell Publishing 2006) for genetic manipulation of yeast cells.
  • Nucleic acid molecules of the invention may also be present on a vector such as an expression vector.
  • a vector such as an expression vector.
  • Such vectors are known in the art.
  • a promoter is placed upstream of the gene encoding the heterologous protein and regulates the expression of the gene.
  • Multi-cloning vectors are especially useful due to their multi-cloning site.
  • a promoter is generally placed upstream of the multi-cloning site.
  • a vector for integration of the nucleic acid molecule encoding the fusion protein of the invention may be constructed either by first preparing a DNA construct containing the entire DNA sequence coding for the fusion protein of the invention subsequently inserting this construct into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information for the individual elements, such as the DNA binding domain, the activation domain, followed by ligation.
  • recombination methods based on attachment sites (att) and recombination enzymes may be used to insert DNA sequences into a vector. Such methods are described, for example, by Landy (1989) Ann. Rev. Biochem. 58:913-949; and are known to those of skill in the art.
  • Host cells according to the present invention can be obtained by introducing a vector or plasmid comprising the target polynucleotide sequences such as the nucleic acid molecule of the invention into the cells.
  • Techniques for transfecting or transforming eukaryotic cells or transforming prokaryotic cells are well known in the art. These can include lipid vesicle mediated uptake, heat shock mediated uptake, electroporation, calcium phosphate mediated transfection (calcium phosphate/DNA coprecipitation), viral infection, particularly using modified viruses such as, for example, modified adenoviruses, microinjection and electroporation.
  • techniques can include heat shock mediated uptake, bacterial protoplast fusion with intact cells, microinjection and electroporation.
  • Techniques for plant transformation include Agrobacterium mediated transfer, such as by A. tumefaciens, rapidly propelled tungsten or gold microprojectiles, electroporation, microinjection and polyethylyne glycol mediated uptake.
  • the DNA can be single or double stranded, linear or circular, relaxed or supercoiled DNA.
  • the phrase “culturing the (genetically engineered) host cell under conditions to express the nucleic acid molecule of the invention” refers to maintaining and/or growing eukaryotic host cells under conditions (e.g., temperature, pressure, pH, induction (feeding an inductor), growth rate, medium, duration, feeding, depletion of the concentration of a nutrient source, metabolite, etc. or increasing the concentration of a nutrient source, metabolite, etc. or light (LIV-VIS), etc.) appropriate or sufficient to obtain production (expression) of the desired protein of interest..
  • conditions e.g., temperature, pressure, pH, induction (feeding an inductor), growth rate, medium, duration, feeding, depletion of the concentration of a nutrient source, metabolite, etc. or increasing the concentration of a nutrient source, metabolite, etc. or light (LIV-VIS), etc.
  • a host cell according to the invention obtained by engineering a host cell with the nucleic acid molecule of the invention or with the expression cassette of the invention may preferably first be cultivated at conditions to grow efficiently to a large cell number without the burden of expressing a recombinant protein.
  • suitable cultivation conditions are selected and optimized to produce the fusion protein.
  • An inducible promoter may be used that becomes activated as soon as an inductive stimulus is applied, to direct transcription of the gene under its control.
  • An inductive stimulus is preferably the addition of an appropriate agent (e.g. methanol for the AOX-promoter) or the depletion of an appropriate nutrient (e.g., methionine for the MET3-promoter).
  • an appropriate agent e.g. methanol for the AOX-promoter
  • an appropriate nutrient e.g., methionine for the MET3-promoter
  • the addition of ethanol, methylamine, cadmium or copper as well as heat or an osmotic pressure increasing agent can induce the expression depending on the promotors operably linked to the fusion protein.
  • the secretion signal of the invention, the nucleic acid molecules of the invention, and the method of the invention of manufacturing a protein of interest can be applied in any scale such as e.g. microtiter plates, shake flasks, miniaturized fermentation systems, bench scale, pilot scale, large scale and manufacturing scale.
  • the host cell according to the invention may be tested for its expression/secretion capacity or yield by measuring the titer of the protein of interest in the supernatant of the cell culture or the cell homogenate of the cells after cell homogenisation by using standard tests, e.g. ELISA, activity assays, HPLC, Surface Plasmon Resonance (Biacore), Western Blot, capillary electrophoresis (Caliper) or SDS-Page.
  • standard tests e.g. ELISA, activity assays, HPLC, Surface Plasmon Resonance (Biacore), Western Blot, capillary electrophoresis (Caliper) or SDS-Page.
  • the host cells are cultivated in a minimal medium with a suitable carbon source, thereby further simplifying the isolation process significantly.
  • the minimal medium contains a utilizable carbon source (e.g. glucose, glycerol, ethanol or methanol), salts containing the macro elements (potassium, magnesium, calcium, ammonium, chloride, sulfate, phosphate) and trace elements (copper, iodide, manganese, molybdate, cobalt, zinc, and iron salts, and boric acid).
  • a utilizable carbon source e.g. glucose, glycerol, ethanol or methanol
  • salts containing the macro elements potassium, magnesium, calcium, ammonium, chloride, sulfate, phosphate
  • trace elements copper, iodide, manganese, molybdate, cobalt, zinc, and iron salts, and boric acid.
  • the cells may be transformed with one or more of the above-described expression vector(s), and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying the genes encoding the desired sequences.
  • conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying the genes encoding the desired sequences.
  • a number of minimal media suitable for the growth of yeast are known in the art. Any of these media may be supplemented as necessary with salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES, citric acid and phosphate buffer), nucleosides or nucleotides (such as adenosine and thymidine), trace elements, vitamins, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH and the like are those previously used with the host cell selected for expression and are known to the ordinarily skilled artisan. Cell culture conditions for other type of host cells are also known and can be readily determined by the artisan. Descriptions of culture media for various microorganisms are for example contained in the handbook "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C, USA, 1981).
  • Host cells can be cultured (e.g., maintained and/or grown) in liquid media and preferably are cultured, either continuously or intermittently, by conventional culturing methods such as test tube culture, shaking culture (e.g., rotary shaking culture, shake flask culture, etc.), aeration spinner culture, or fermentation.
  • cells are cultured in shake flasks or deep well plates.
  • cells are cultured in a bioreactor (e.g., in a bioreactor cultivation process). Cultivation processes include, but are not limited to, batch, fed-batch and continuous methods of cultivation.
  • batch process and “batch cultivation” refer to a closed system in which the composition of media, nutrients, supplemental additives and the like is set at the beginning of the cultivation and not subject to alteration during the cultivation; however, attempts may be made to control such factors as pH and oxygen concentration to prevent excess media acidification and/or cell death.
  • fed-batch process and “fed-batch cultivation” refer to a batch cultivation with the exception that one or more substrates or supplements are added (e.g., added in increments or continuously) as the cultivation progresses.
  • continuous process and “continuous cultivation” refer to a system in which a defined cultivation media is added continuously to a bioreactor and an equal amount of used or “conditioned” media is simultaneously removed, for example, for recovery of the desired product.
  • conditioned media is simultaneously removed, for example, for recovery of the desired product.
  • host cells are cultured for about 12 to 24 hours, in other embodiments, host cells are cultured for about 24 to 36 hours, about 36 to 48 hours, about 48 to 72 hours, about 72 to 96 hours, about 96 to 120 hours, about 120 to 144 hours, or for a duration greater than 144 hours. In yet other embodiments, culturing is continued for a time sufficient to reach desirable production yields of POI.
  • the methods of the invention may further comprise a step of isolating the expressed POI.
  • the POI is secreted from the cells and can be isolated and purified from the culture medium using state of the art techniques. During the process of secretion, the secretion signal is cleaved off. Secretion of the POI from the cells is generally preferred, since the products are recovered from the culture supernatant rather than from the complex mixture of proteins that results when cells are disrupted to release intracellular proteins.
  • a protease inhibitor may be useful to inhibit proteolytic degradation during purification.
  • the composition may be concentrated, filtered, dialyzed, etc., using methods known in the art.
  • the cell culture after fermentation I cultivation can be centrifuged using a separator, a tube centrifuge or filtration (depth filtration, filter press, tangential flow filtration) to separate the cells from the culture supernatant.
  • the supernatant can then be filtered (depth or tangential flow filtration) or concentrated by using a tangential flow filtration (ultra-(dia) filtration).
  • Isolation and purification methods for obtaining the POI may be based on methods utilizing difference in solubility, such as salting out, solvent precipitation, heat precipitation, methods utilizing difference in molecular weight, such as size exclusion chromatography, ultrafiltration and gel electrophoresis, methods utilizing difference in electric charge, such as ion-exchange chromatography, methods utilizing specific affinity, such as affinity chromatography, methods utilizing difference in hydrophobicity, such as hydrophobic interaction chromatography and reverse phase high performance liquid chromatography, methods utilizing difference in isoelectric point, such as isoelectric focusing may be used and methods utilizing certain amino acids, such as IMAC (immobilized metal ion affinity chromatography).
  • solubility such as salting out, solvent precipitation, heat precipitation
  • methods utilizing difference in molecular weight such as size exclusion chromatography, ultrafiltration and gel electrophoresis
  • methods utilizing difference in electric charge such as ion-exchange chromatography
  • methods utilizing specific affinity such as affinity
  • the isolated and purified POI can be identified by conventional methods such as Western Blotting or specific assays for POI activity.
  • the structure of the purified POI can be determined by amino acid analysis, amino-terminal peptide sequencing, primary structure analysis for example by mass spectrometry, RP-HPLC, ion exchange-HPLC, ELISA and the like. It is preferred that the POI is obtainable in large amounts and in a high purity level, thus meeting the necessary requirements for being used as an active ingredient in pharmaceutical compositions or as feed or food additive.
  • isolated means a substance in a form or environment that does not occur in nature.
  • isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature, e.g.
  • cDNA made from mRNA or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).
  • modifying the protein of interest is meant that the POI is chemically or enzymatically modified. There are many methods known in the art to modify proteins. Proteins can be coupled to carbohydrates or lipids.
  • the POI may be PEGylated (the POI chemically coupled to polyethylenglycole) or HESylated (the POI is chemically coupled to hydroxyethyl starch) for half-life extension.
  • the POI may also be coupled with other moieties such as affinity domains for e.g. human serum albumin for half-life extension.
  • the POI also may be treated by a protease or under hydrolytic conditions for cleavage to form the active ingredient from a pre-sequence or to cleave off a tag such as an affinity tag for purification.
  • the POI may also be coupled to other moieties such as toxins, radioactive moieties or any other moiety.
  • the POI may further be treated under conditions to form dimers, trimers and the like.
  • the POI when including non-canonical amino acids, can be modified at these non-canonical amino acids e.g. by coupling to other moieties such as moieties for half-life extension.
  • the term “formulating the protein of interest” refers to bringing the POI to conditions, where the POI can be stored for a longer time.
  • Many different methods known in the art are available to stabilize proteins. By exchanging the buffer in which the POI is existent after purification and I or modification, the POI can be brought under conditions, where it is more stable. Different buffer substances and additives, such as sucrose, mild detergents, stabilizer and the like, known in the art can be used.
  • the POI can also be stabilized by lyophilization, freezing, cooling (e.g. 4-6 °C).
  • formulations can be done by formation of complexes of the POI with lipids or lipoproteins, such as polyplexes, and the like.
  • Some protein may be co-formulated with other proteins.
  • the such formulated POI may additionally be transferred and/or packaged into delivery systems, such as, but not limited to, vials, syringes, inhalers etc.
  • the use of the secretion signal of the invention may increase the secretion of the protein of interest.
  • the present invention also relates to a method of increasing the secretion of a protein of interest from a eukaryotic host cell, comprising expressing in said eukaryotic host cell a nucleic acid molecule of the invention, thereby increasing the secretion of said protein of interest in comparison to a host cell expressing a fusion protein as defined herein but comprising a wild type Saccharomyces cerevisae a-mating factor secretion signal instead of the secretion signal as defined in the nucleic acid molecule of the invention.
  • “Secretion” as used herein relates to the transfer of the protein of interest, which forms part of the fusion protein of the invention, out of the (recombinant) host cell.
  • the signal peptide sequence is cleaved off in the endoplasmic reticulum and the MFa pro-sequence is cleaved off in the Golgi apparatus.
  • secretion is increased, only the secretion of the protein of interest is increased.
  • the titer of the protein of interest in the supernatant of the cell culture can be determined using standard tests, e.g. ELISA, activity assays, HPLC, Surface Plasmon Resonance (Biacore), Western Blot, capillary electrophoresis (Caliper) or SDS-Page.
  • the method of increasing the secretion of a protein of interest from a eukaryotic host cell may further comprise engineering said host cell to incorporate an expression construct to express a nucleic acid molecule of the invention.
  • the method of increasing the secretion of a protein of interest from a eukaryotic host cell may further comprise culturing said host cell under conditions to express the nucleic acid molecule of the invention, wherein the protein of interest is secreted and wherein the secretion signal is cleaved from said fusion protein.
  • the method of increasing the secretion of a protein of interest from a eukaryotic host cell may further comprise isolating the protein of interest from the cell culture.
  • the method of increasing the secretion of a protein of interest from a eukaryotic host cell may further comprise purifying the protein of interest.
  • the method of increasing the secretion of a protein of interest from a eukaryotic host cell may further comprise modifying the protein of interest.
  • the method of increasing the secretion of a protein of interest from a eukaryotic host cell may further comprise formulating the protein of interest.
  • the nucleic acid molecule of the invention may be integrated in a chromosome and/or genome of said host cell or contained in a vector or plasmid, which does not integrate into a chromosome and/or genome of said host cell.
  • the secretion signal described herein can be used to secrete a recombinant protein of interest and/or increase the secretion of a recombinant protein of interest. Accordingly, the present invention also relates to the use of the secretion signal as defined herein for increasing the secretion of a protein of interest from a eukaryotic host cell.
  • the present invention also relates to the use of the secretion signal as defined herein for increasing the secretion of a protein of interest from a eukaryotic host cell, wherein the secretion signal comprises from N- to C-terminus a signal peptide sequence originating from a protein selected from the group consisting of GDT1 , SLP1 , PP7435_Chr4_0694, WSC3, NCR1 , ZRT1 and PP7435_Chr2_0965 protein followed by a pro-sequence, preferably an a-mating factor (MFa) prosequence.
  • MFa a-mating factor
  • the secretion signal may increase secretion of the protein of interest from the eukaryotic host cell in comparison to a eukaryotic host cell expressing a fusion protein of the invention comprising secretion signals OST1-aMFpro, KRE1-aMFpro and SWP1-aMFpro, for instance, or the wild type Saccharomyces cerevisiae a-mating factor secretion signal instead of the secretion signal as defined herein.
  • the recombinant host cell of the invention is useful for the production of various proteins of interest, since they are efficiently secreted to the supernatant, thereby avoiding lysis of the host cells. Accordingly, the present invention further relates to the use of the recombinant host cell of the invention for manufacturing a protein of interest.
  • less than 20 means less than the number indicated.
  • more than or greater than means more than or greater than the indicated number, e.g. more than 80 % means more than or greater than the indicated number of 80 %.
  • the examples provided herein demonstrate that the newly identified signal peptides in combination with the pro-sequence of Saccharomyces cerevisiae alpha-mating factor increase the titer (product per volume in mg/L) and the yield (product per biomass in mg/g biomass measured as wet cell weight) of secreted recombinant proteins compared to known secretion leaders including the commonly used S. cerevisiae alpha-mating factor pre-pro leader.
  • the use of the novel signal peptides led to increased yield of different recombinant proteins and antibody derivatives in the yeast Pichia pastoris. The positive effect was shown in shaking cultures (conducted in 24 deep well plates) and in fed-batch bioreactor cultivations.
  • Example 1 Construction and selection of P. pastoris strains secreting recombinant secretory proteins using the signal peptide sequences of the present invention
  • P. pastoris CBS7435 mut s variant (genome sequenced by Sturmberger et al. 2016) was used as host strain.
  • the genes encoding the POIs e.g. mono-vHH, double-vHH, scR, Phytase
  • the genes encoding the POIs were codon- optimized ATUM and obtained as synthetic DNA.
  • a His6-tag was fused C-terminally to the scR and double-VHH genes for detection. The sequences of these proteins are shown in Table 2 of the description.
  • transformants were selected on YPD (Yeast Extract, Peptone, Dextrose) plates containing the required antibiotics for the integrated selection markers (100 - 300 pg/mL Zeocin for Zeocin resistance marker with Sh ble gene) after incubation for 48h at 28°C and singled under the same conditions.
  • YPD Yeast Extract, Peptone, Dextrose
  • Plasmids were linearized using BglW, prior to electroporation (using a standard transformation protocol as described in Gasser et al. 2013. Future Microbiol. 8(2): 191 -208 - see Example 3) into P. pastoris. Selection of positive transformants was performed on YPhyD plates (per liter: 10 g yeast extract, 20 g phytone-peptone, 20 g glucose, 20 g agar-agar) containing 100 - 300 pg/mL of Zeocin.
  • heterologous proteins were used as reporters: the variable vH region of a monovalent camelid antibody (mono-vHH), the variable VH regions of a bivalent camelid antibody (VHH), a signal chain variable fragment antibody (scFv named scR) and the phytase.
  • the expression of the heterologous POIs was mediated by P. pastoris alcohol oxidase (AOX1) promoter for methanol-induced production. Construction of the plasmids was done by double-restriction digest-mediated assembly of particular novel signal peptides fused to aMF-pro-region and the relevant POI followed by the AOX1 -transcription terminator, all preceded by the AOX1 -promoter.
  • AOX1 P. pastoris alcohol oxidase
  • Example 2 Generation of the P. pastoris strains producing recombinant secretory proteins with the novel signal peptide fusion sequences
  • POI expression plasmids (each 1 pg) were linearized by BglW prior to electroporation into P. pastoris.
  • the P. pastoris strain was made electro competent.
  • the strain was inoculated into 50 mL YPD media (main culture) for 16-20 hours (28°C; 120 rpm) and harvested at an optical density (ODeoo) from 0.7 - 0.9 by centrifugation (Eppendorf AG, Germany) for 5 minutes at 3,300g and 6°C in a 50 mL falcon tube.
  • the cell pellet was resuspended in 40 mL ice cold distilled sterile water, and centrifuged again (5min; 3,300g; 6°C).
  • the electroporation was performed at 2 kV for 4 milliseconds (Gene Pulser, Bio-Rad Laboratories, Inc, USA). After transformation the electroporated cells were suspended in 800 pL YPhyD media and regenerated for 2 hours on 28°C shaking at 450 rpm on a Thermoshaker (Eppendorf AG, Germany).
  • YPhyD plates per liter: 10 g yeast extract, 20 g phytone-peptone, 20 g glucose, 20 g agar-agar
  • Zeocin CBS7435 mut s background
  • Example 3 Small scale cultivation (screening) of the P. pastoris strains producing recombinant secretory proteins with the novel signal peptide sequences
  • BMM2 induction medium 200 mM sodium phosphate buffer, 1.34% YNB, 4x 10' 5 % biotin and 1% methanol
  • BMM10 200 mM sodium phosphate buffer, 1.34% YNB, 4x 10' 5 % biotin and 5% methanol
  • the volumetric titer (also referred to as “titer”) is the content of the protein of interest (POI) in the supernatant of the cultivations as described in above and in example 5 in g/L or mg/L and the like, calculated against the non-authentic calibrator BSA (bovine serum albumin) applied at known concentration. Titer fold changes are given respective to the reference clones using pre-pro-secretion signal of the S. cerevisiae MFa, or fusion secretion signals of OST1 (Barrero et al. 2018), KRE1 or SWP1 (WO2022171827) fused to the pro-region of the S. cerevisiae MFa secretion signal (SEQ IDs NO: 1).
  • the ‘LabChip GX/GXII Touch System’ (PerkinElmer) was used for quantitative analysis of secreted protein titer in culture supernatants.
  • the consumables ‘Protein Express Lab Chip’ (760499, Revvity) and ‘Protein Express Reagent Kit’ (CLS960008, PerkinElmer) were used. Briefly, 5 pL of culture supernatant were mixed with 8 pL of reducing sample buffer. This mixture was denatured at 95 °C for 5 minutes, 32 pL water (Milli-Q® or equivalent) were added and centrifuged at 3,300 g for 2 min at 22°C, and applied to the instrument.
  • the fluorescently labelled samples were analyzed according to protein size in the instrument, using an electrophoretic system based on microfluidics. Internal standards enabled approximate allocations to size in kDa and external standards (BSA at known concentrations) generate approximate concentrations of detected signals.
  • Example 4 Bioreactor cultivations of the P. pastoris strains producing recombinant secretory proteins with the novel signal peptide sequences
  • Clones expressing the protein of interest (POI) by using the signal peptide fusion sequences of the present invention as well as control strains using the pre-pro-region secretion signal of S. cerevisiae MFa or the fusion sequences of signal peptides OST1 , KRE1 or SWP1 with the pro-region of the MFa secretion signal (Example 2) were selected after small scale screening cultivations (Example 3). The selected clones were further evaluated in larger cultivation volumes by fed batch bioreactor cultivations.
  • Clones were inoculated into wide-necked, baffled, covered 300 mL shake flasks filled with 50 mL of YPhyG (per liter: 20.0 g Phytone-Peptone, 10.0 g Bacto-Yeast Extract, 20.0 g glycerol) and shaken at 110 rpm at 28°C overnight (pre-culture 1).
  • Pre-culture 2 200 mL YPhyG in a 2000 mL widenecked, baffled, covered shake flask was inoculated from pre-culture 1 in a way that the ODeoo (optical density measured at 600 nm) reached approximately 20 (measured against YPhyG media) in late afternoon. Incubation of pre-culture 2 was performed at 110 rpm at 28°C, as well.
  • P. pastoris was grown on glycerol to produce biomass and the culture was subsequently subjected to glycerol feeding (60% w/w + 12 ml/L PTM1) followed by extended glycerol feeding to accumulate more biomass prior to subsequent methanol feeding.
  • Ammonia solution (25%) was used for pH control and antifoaming agent PPG 2000 was added if required (probe-controlled).
  • the temperature was set to 28°C. Over the period of the last hour before initiating the production phase it was decreased to 24°C and kept at this level throughout the remaining process, while the pH dropped to 5.0 and was kept at this level. Oxygen saturation was set to 30% throughout the whole process (cascade control: stirrer, flow, oxygen supplementation). Stirring was applied between 700 and 1200 rpm and a flow range (air) of 1.0 - 2.0 L min -1 was chosen.
  • biomass was generated (p ⁇ 0.30 h -1 ) up to a wet cell weight (WCW) of approximately 110-120 g L’ 1 .
  • WCW wet cell weight
  • the classical batch phase (biomass generation) would last about 13- 14 hours.
  • Example 5 Results of the P. pastoris strains producing recombinant secretory proteins with the novel signal peptide fusion sequences from screening (microscale, deep-well plate) and fed-batch bioreactor cultivations
  • Example 4 The secretion improvement obtained with the novel signal peptide fusion sequences (Example 4) is measured by titer fold-change values (also named FC titer) that refer to the respective control clones secreting the POI using the MFa secretion signal (Example 1) in the same strain background (CBS7435 mutS WT). Titer fold change is understood as the quotient of the titer of the respective fermentation or small scale cultivation divided by the titer of the control.
  • FC titer also named FC titer
  • Table 3 Screening (microscale) and bioreactor results obtained by using the candidate signal peptides fused to an MFa pro-sequence for the secretion of mono-vHH as reporter POI compared to benchmark secretion signals MFa-pre-pro or the signal peptides OST1 , KRE1 and SWP1 fused to MFa-pro in CBS7435 mutS WT upon methanol-induced production with the wildtype AOX1 -promoter. Titer FCs were calculated compared to the MFa-pre-pro secretion signal control by using the final sampling point).
  • results of bioreactor cultivations supported findings from screening, in that the fusion of the secretion signals of the present invention (GDT1, SLP1, PP7435_Chr4_0694, WSC3, NCR1, ZRT1 and PP7435_Chr2_0965) with the pro-region of MFa increased the secretion of POI mono-vHH relative to the basic benchmark MFa-pre-pro as well as calibrator secretion signals OST1, SWP1 and KRE1 all fused to the pro-region of MFa.
  • GDT1, SLP1, PP7435_Chr4_0694, WSC3, NCR1, ZRT1 and PP7435_Chr2_0965 increased the secretion of POI mono-vHH relative to the basic benchmark MFa-pre-pro as well as calibrator secretion signals OST1, SWP1 and KRE1 all fused to the pro-region of MFa.
  • Table 4 Screening (microscale) and bioreactor results obtained by using the candidate signal peptides fused to an MFa pro-sequence for the secretion of phytase as reporter POI compared to benchmark secretion signals MFa-pre-pro or the signal peptides, OST1 , KRE1 and SWP1 fused to MFa-pro in CBS7435 mutS WT upon methanol-induced production with the wildtype AOX1 -promoter. Titer FCs were calculated compared to the MFa-pre-pro secretion signal control by using the final sampling point).
  • results of bioreactor cultivations supported findings from screening, in that the fusion of the secretion signals of the present invention with the pro-region of MFa increased the secretion of POI phytase relative to the basic benchmark MFa-pre-pro as well as calibrator secretion signals OST1 , SWP1 and KRE1 all fused to the pro-region of MFa. Also, the inability of the fusion secretion sequences of the signal peptides FET3, WBP1 , MSB2, PEP1 and ERV25 to improve secretion of POI phytase above levels achieved with MFa-pre-pro identified in screening was supported by results of bioreactor cultivations.
  • Table 5 Screening (microscale) and bioreactor results obtained by using the candidate signal peptides fused to an MFa pro-sequence for the secretion of scR as reporter POI compared to benchmark secretion signals MFa-pre-pro or the signal peptides, OST1 , KRE1 and SWP1 fused to MFa-pro in CBS7435 mutS WT upon methanol-induced production with the wildtype AOX1 -promoter. Titer FCs were calculated compared to the MFa-pre-pro secretion signal control by using the final sampling point;
  • results of bioreactor cultivations supported findings from screening, in that the fusion of the secretion signals of the present invention with the pro-region of MFa increased the secretion of POI scR relative to the basic benchmark MFa-pre-pro as well as calibrator secretion signals OST1 , SWP1 and KRE1 all fused to the pro-region of MFa. Also, the inability of the fusion secretion sequences of the signal peptides FET3, WBP1 , MSB2, PEP1 and ERV25 to improve secretion of POI scR above levels achieved with MFa-pre-pro identified in screening was supported by results of bioreactor cultivations.
  • Table 6 Screening (microscale) and bioreactor results obtained by using the candidate signal peptides fused to an MFa pro-sequence for the secretion of dual-vHH as reporter POI compared to benchmark secretion signals MFa-pre-pro or the signal peptides, OST1 , KRE1 and SWP1 fused to MFa-pro in CBS7435 mutS WT upon methanol-induced production with the wildtype AOX1 -promoter. Titer FCs were calculated compared to the MFa-pre-pro secretion signal control by using the final sampling point;
  • results of bioreactor cultivations supported findings from screening, in that the fusion of the signal peptides of the present invention with the pro-region of MFa increased the secretion of POI dual- vHH relative to the basic benchmark MFa-pre-pro as well as calibrator secretion signals OST1 , SWP1 and KRE1 all fused to the pro-region of MFa. Also, the inability of the fusion secretion sequences of the signal peptides FET3, WBP1 , MSB2, PEP1 and ERV25 to improve secretion of POI dual-vHH above levels achieved with MFa-pre-pro identified in screening was supported by results of bioreactor cultivations.

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Abstract

La présente invention concerne des signaux de sécrétion augmentant la sécrétion et/ou l'expression de protéines d'intérêt, le signal de sécrétion comprenant de l'extrémité N-terminale à l'extrémité C-terminale une séquence peptidique signal provenant d'une protéine choisie dans le groupe constitué par GDT1, SLP1, PP7435_Chr4_0694, WSC3, NCR1, ZRT1 et PP7435_Chr2_0965, et une proséquence, de préférence une proséquence de facteur de conjugaison α (MFα).
PCT/EP2025/070414 2024-08-01 2025-07-17 Peptides signaux permettant d'augmenter la sécrétion de protéines Pending WO2026027251A1 (fr)

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