WO2024256710A2 - Constructions polypeptidiques de lysine modifiées actives contre des bactéries à gram négatif - Google Patents

Constructions polypeptidiques de lysine modifiées actives contre des bactéries à gram négatif Download PDF

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WO2024256710A2
WO2024256710A2 PCT/EP2024/066700 EP2024066700W WO2024256710A2 WO 2024256710 A2 WO2024256710 A2 WO 2024256710A2 EP 2024066700 W EP2024066700 W EP 2024066700W WO 2024256710 A2 WO2024256710 A2 WO 2024256710A2
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seq
lysin
engineered
gram
variant
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Raymond Schuch
Xavier Vila Farres
Steven Swift
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Aurobac Therapeutics SAS
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
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    • C12R2001/38—Pseudomonas
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)

Definitions

  • the present disclosure relates to the field of antimicrobial agents and more specifically to engineered, phage-derived lysins that infect Gram-negative bacteria and the use of these agents in killing Gram-negative bacteria and combatting bacterial infection and contamination.
  • BACKGROUND OF THE DISCLOSURE [004]
  • Gram-negative bacteria are an important cause of serious and potentially life-threatening invasive infections. Pseudomonas infection, for example, presents a major problem in burn wounds, chronic wounds, chronic obstructive pulmonary disorder (COPD), cystic fibrosis, surface growth on implanted biomaterials, and within hospital surface and water supplies where it poses a host of threats to vulnerable patients.
  • COPD chronic obstructive pulmonary disorder
  • Burkholderia species are pervasive throughout the environment and are known to cause lung infections, particularly in cystic fibrosis patients, diabetics, or patients who are otherwise immunocompromised.
  • Melioidosis and glanders are two examples of Gram-negative bacterial infections caused by Burkholderia species (i.e., Burkholderia pseudomallei and Burkholderia mallei, respectively) that are of concern.
  • Burkholderia species i.e., Burkholderia pseudomallei and Burkholderia mallei, respectively
  • Another Gram-negative bacteria responsible for devastating human diseases is Yersinia pestis, a non-motile coccobacillus bacteria that may be found in small mammals and fleas.
  • Yersinia pestis is the causative agent of bubonic, septicemic, and pneumonic plagues.
  • Bacteriophage lysins are molecules produced by bacterial viruses (bacteriophages) that are capable of lysing the bacterial cell wall, resulting in death of the target bacteria. These bacteriophage lysins, or “lysins,” may be highly efficient at killing pathogenic bacteria, including various pathogenic Burkholderia and/or Yersinia species. [007] Accordingly, there is a need for therapies and agents effective in the diagnosis and treatment of bacterial contamination and infection, particularly with respect to Burkholderia and Yersinia species.
  • This application discloses novel lysins, which have been engineered to cross the outer membrane of target Gram-negative pathogens, such as Burkholderia and Yersinia species, as well as other Gram-negative bacteria species as disclosed herein.
  • the peptidoglycan (PG) layer of Gram-negative bacteria is shielded by the outer membrane (OM) and is generally not readily cleaved by native (unmodified) lysins.
  • OM outer membrane
  • these functional domains may include, for example, one or more antimicrobial peptide (AMP) and/or cell penetrating peptide (CPP).
  • the AMP may be selected from BK3 (SEQ ID NO: 19), HIP A (SEQ ID NO: 27), HIP ABC (SEQ ID NO: 28), HIP AB (SEQ ID NO: 181), BK5 (SEQ ID NO: 17), AM1 (SEQ ID NO: 123), AM13 (SEQ ID NO: 124), MMAV- 2 (SEQ ID NO: 125), MFVI-3 (SEQ ID NO: 126), BCM1 (SEQ ID NO: 18), BK3h2 (SEQ ID NO: 178), BK3h3 (SEQ ID NO: 179), ATRA (SEQ ID NO: 21), ATRA-1A ⁇ aa2-9 (SEQ ID NO: 22), ATRA-1A (SEQ ID NO: 23), PMAP-36 (SEQ ID NO: 24), RI18 (SEQ ID NO: 25);
  • the CPP may be selected from Zf5.3 (SEQ ID NO: 38), PRR (SEQ ID NO: 39), PRR 5 (SEQ ID NO: 40), or PRR 3 (SEQ ID NO: 48), or active fragments or variants thereof having at least 80% sequence identity with the CPP.
  • Such AMPs and CPPs may be added to native (unmodified) Gram-negative base lysins or modified Gram-negative base lysins.
  • the native or modified Gram- negative base lysin may be selected from the protein family phage lysozyme pfam00959; peptidase_M15_4 pfam13539; muramidase pfam11860, or peptidase_M15_3 pfam08291, wherein the Gram-negative base lysin is modified, e.g., by making a non-naturally occurring modification to the native amino acid sequence or truncating the native lysin.
  • the AMPs and CPPs may, in addition to those listed above, further be selected from other AMPs and CPPs known in the art, such as RI18, PRR, PRR 3 and PRR 5 .
  • ATRA comprises SEQ ID NO: 21 or is an active fragment thereof or a variant thereof, such as ATRA-1A ⁇ aa2-9, set forth as SEQ ID NO: 22 and/or variant ATRA-1A set forth as SEQ ID NO: 23.
  • BK3 is a full length BK3, e.g., as set forth in SEQ ID NO: 19.
  • BK3 comprises a variant, such as the variant set forth in SEQ ID NO: 20.
  • the present application discloses and claims novel engineered lysins and fragments and variants thereof comprising (a) a Gram-negative base lysin in addition to (b) one or more antimicrobial peptides (AMP) and/or cell-penetrating peptides (CPPs) that can be used, for example, to treat bacterial infections, including infections caused by Gram-negative bacteria, including, but not limited to Burkholderia and Yersinia species.
  • AMP antimicrobial peptides
  • CPPs cell-penetrating peptides
  • the engineered lysins may be included in compositions comprising an excipient or carrier, particularly in pharmaceutical compositions comprising a pharmaceutically acceptable excipient or carrier that can be used, for example, to kill Gram- negative bacteria or treat bacterial infections.
  • compositions comprising an excipient or carrier
  • pharmaceutical compositions comprising a pharmaceutically acceptable excipient or carrier that can be used, for example, to kill Gram- negative bacteria or treat bacterial infections.
  • methods for using the lysins and engineered lysins per se or in a composition or pharmaceutical composition disclosed herein for treating bacterial infections, and, generally, inhibiting the growth, reducing the population, or killing Gram-negative bacteria.
  • polynucleotides encoding the engineered lysins and variants thereof, recombinant vectors comprising the polynucleotide and host cells comprising the recombinant vector disclosed herein are also provided.
  • certain native lysins without addition of any AMP or CPP are active against Gram-positive bacteria, such as Burkholderia. Therefore, in another aspect, pharmaceutical compositions comprising certain native lysins, such as GN569 or GN568, and the use of these certain native lysins for inhibiting the growth, reducing the population, or killing Gram-negative bacteria and treating bacterial infection are also contemplated.
  • the present disclosure is directed to an engineered lysin comprising: I) (a) a Gram-negative base lysin selected from the protein family phage lysozyme pfam00959; peptidase_M15_4 pfam13539; muramidase pfam11860; or peptidase_M15_3 pfam08291; and (b) one or more antimicrobial peptide (AMP) and/or cell penetrating peptide (CPP) selected from BK3 as defined herein, HIP ABC set forth as SEQ ID NO: 28, HIP A set forth as SEQ ID NO: 27, BK5 set forth as SEQ ID NO: 17, BCM1 set forth as SEQ ID NO: 18, Zf5.3 set forth as SEQ ID NO: 37, ATRA as disclosed herein, BK9 set forth as SEQ ID NO: 29, AM13 set forth as SEQ ID NO: 124, MMAV-2
  • the Gram-negative base lysin is from the phage lysozyme pfam00959 protein family, such as GN867 (SEQ ID NO: 13), GN3 (R100Q,R116H) set forth as SEQ ID NO: 14, GN515 (R100D,R116H) set forth as SEQ ID NO: 15, QPI14001 (SEQ ID NO: 9), or GN917 (SEQ ID NO: 10).
  • the Gram-negative base lysin is from the muramidase pfam11860 protein family, such as GN428 (SEQ ID NO: 4) and CAH0532154 (SEQ ID NO: 11). [0018] In some embodiments, the Gram-negative base lysin is from the peptidase_M15_3 pfam08291 protein family, such as GN588 (SEQ ID NO: 5).
  • an engineered lysin comprising: (a) a Gram- negative base lysin selected from GN515(R100D, R116H) (SEQ ID NO: 15), GN3 (SEQ ID NO: 3), GN3(R100Q, R116H) (SEQ ID NO: 14), GN3(R100Q, R116Q) (SEQ ID NO: 177), GN515 (SEQ ID NO: 6), GN568 (SEQ ID NO: 1), GN685 (SEQ ID NO: 2), GN428 (SEQ ID NO: 4), GN588 (SEQ ID NO: 5), GN569 (SEQ ID NO: 7), GN37 (SEQ ID NO: 8), QPI14001 (SEQ ID NO: 9), GN917 (SEQ ID NO: 10), CAH0532154 (SEQ ID NO: 11), GN577 (SEQ ID NO: 12), GN867 (SEQ ID NO: 13),
  • the Gram-negative base lysin is selected from GN515(R100D,R116H) (SEQ ID NO: 15), GN3(R100Q,R116H) (SEQ ID NO: 14), or GN3 (SEQ ID NO: 3).
  • the engineered lysin of the disclosure further comprises one or more outer membrane binding motifs, and in some embodiments, the engineered lysin comprises at least one linker.
  • the outer membrane binding motif is selected from FIRL Bc (SEQ ID NO: 143), FMRL Yp (SEQ ID NO: 144), FMRL Nm (SEQ ID NO: 147), FIRL (SEQ ID NO: 146), or or FIQL BC (SEQ ID NO: 173)
  • the at least one linker is selected from TAGGTAGG (SEQ ID NO: 41), IGEM peptide linker GGSGSGSGSP (SEQ ID NO: 42), GGGGSGGGGSGGGGS (SEQ ID NO: 43), AGAGAGAGAGAGAGAGAS (SEQ ID NO: 44), LSKLGG (SEQ ID NO: 45), LSKLGGLGGGPRRLGG (SEQ ID NO: 46), LGGLGG (SEQ ID NO: 47),
  • the engineered lysin of the disclosure further comprises one or more amino acid extensions selected from MG or LKWI (SEQ ID NO.127). [0023] In some embodiments, the engineered lysin of the disclosure further comprises a cationic peptide, such as KFFKFFKFFK (SEQ ID NO: 36).
  • the engineered lysin comprises: (a) a Gram-negative base lysin selected comprising GN3(R100Q, R116H) (SEQ ID NO: 14), (b) an AMP comprising HIP ABC (SEQ ID NO: 28), and (c) a linker comprising GT; or an active fragment thereof, or a variant thereof having at least 80% sequence identity with said engineered lysin.
  • the engineered lysin comprises GN1119 (SEQ ID NO: 63), or an active fragment thereof, or a variant thereof having at least 80% sequence identity with said engineered lysin.
  • the engineered lysin is selected from GN1074 (SEQ ID NO: 62), GN1119 (SEQ ID NO: 63), GN1125 (SEQ ID NO: 64), or GN1120 (SEQ ID NO: 149).
  • the engineered lysins of the disclosure is selected from GN693 (SEQ ID NO: 55), GN694 (SEQ ID NO: 56), GN751 (SEQ ID NO: 57), GN757 (SEQ ID NO: 58), GN1040 (SEQ ID NO: 59), GN1041 (SEQ ID NO: 60), GN1072 (SEQ ID NO: 61), GN737 (SEQ ID NO: 65), GN808 (SEQ ID NO: 66), GN848 (SEQ ID NO: 67), GN887 (SEQ ID NO: 68), GN907 (SEQ ID NO: 69), GN918 (SEQ ID NO: 70), GN937 (SEQ ID NO: 71), GN585 (SEQ ID NO: 72), GN686 (SEQ ID NO: 73), GN727 (SEQ ID NO: 74), GN758 (SEQ ID NO: 75), GN764 (SEQ ID NO:
  • the engineered lysins of the disclosure is selected from GN1127 (SEQ ID NO: 150), GN1071 (SEQ ID NO: 151), GN1077 (SEQ ID NO: 152), GN1078 (SEQ ID NO: 153), GN1117 (SEQ ID NO: 154), GN1118 (SEQ ID NO: 155), GN1236 (SEQ ID NO: 156), GN1238 (SEQ ID NO: 157), GN1239 (SEQ ID NO: 158), GN1250 (SEQ ID NO: 159), GN1251 (SEQ ID NO: 160), GN1252 (SEQ ID NO: 161), GN1253 (SEQ ID NO: 162), GN1283 (SEQ ID NO: 163), GN1287 (SEQ ID NO: 164), GN1313 (SEQ ID NO: 165), GN1316 (SEQ ID NO: 166), GN1328 (SEQ ID NO: 167),
  • the Gram-negative bacteria demonstrating inhibited growth, population reduction or which is killed by the engineered lysins of the invention is selected from a Burkholderia spp., a Yersinia spp., such as Yersina pestis, a Pseudomonas spp., such as, Pseudomonas aeruginosa, an Acinetobacter spp., such as Acinetobacter baumannii, an Enterobacter spp., such as E. cloacae, an Escherichia spp., such as E. coli, a Serratia spp., such as S.
  • the Burkholderia spp. are selected from Burkholderia cepacia complex species and/or Burkholderia pseudomallei complex species, wherein the Burkholderia cepacia complex species comprise Burkholderia cepacia, Burkholderia cenocepacia, and/or Burkholderia multivorans and wherein the Burkholderia pseudomallei complex species comprise Burkholderia thailandensis, B. pseudomallei and/or Burkholderia humptydooensis.
  • the disclosure is directed to an isolated polynucleotide comprising a nucleic acid molecule encoding the engineered lysin of the disclosure.
  • the isolated polynucleotide comprises cDNA.
  • the disclosure is directed to a recombinant vector comprising the isolated polynucleotide sequence as described herein.
  • the isolated polynucleotide sequence is operatively linked to a heterologous promoter.
  • the recombinant vector is a recombinant expression vector.
  • the present disclosure is also directed to an isolated host cell comprising the recombinant vector as described herein.
  • the present disclosure is directed to a composition
  • a carrier such as a pharmaceutically acceptable carrier, comprising the engineered lysin of the disclosure, or GN568 or GN569 as herein described.
  • the present disclosure is also directed to an AMP comprising AM13 set forth as SEQ ID NO: 124, MMAV-2 set forth as SEQ ID NO: 125, and/or MFVI-3 set forth as SEQ ID NO: 126.
  • the present disclosure is directed to a method of treating a bacterial infection caused by a Gram-negative bacteria, which method comprises administering to a subject in need thereof an effective amount of the engineered lysin as herein described.
  • the Gram-negative bacteria is a Gram-negative ESKAPE bacteria.
  • the present disclosure is directed to a method of killing a Gram-negative bacteria, which method comprises contacting an effective amount of the engineered lysin as herein described.
  • the bacteria is Burkholderia spp., Yersinia spp., Acinetobacter spp., Klebsiella spp., or Pseudomonas spp.
  • the Burkholderia spp. are selected from B. cepacia, B. cenocepacia, B. thailandensis, B. multivorans, or B.
  • the Yersinia spp. is Yersinia pestis.
  • the Acinetobacter spp. is A. baumannii.
  • the Klebsiella spp. is K. pneumoniae.
  • Gram-negative bacteria include Serratia spp., such as Serratia marcescens. BRIEF DESCRIPTION OF THE DRAWING [0042]
  • Figure 1 is a graph showing the log 10 CFU/mL over time for GN751 against B. humptydooensis (BAA-2767), as described in Example 5.
  • Figure 2 is a graph showing the log 10 CFU/mL over time for GN757 against B. humptydooensis (BAA-2767), as described in Example 5.
  • Figure 3 is a graph showing the log 10 CFU/mL over time for GN751 against B. thaillandensis (NR-9909), as described in Example 5.
  • Figure 4 is a graph showing the log 10 CFU/mL over time for GN757 against B. thaillandensis (NR-9909), as described in Example 5.
  • Figure 5 is a graph showing the relative fluorescence units (RFUs) for GN751 against B.
  • REUs relative fluorescence units
  • Figure 6 is a graph showing the relative fluorescence units (RFUs) for GN808 against Y. Pestis (Yokohama D11) over time for varying concentrations of GN808, as described in Example 5.
  • Figure 7 is a bar graph showing the percent of hemolysis for GN751 over a range of concentrations, as described in Example 5.
  • Figure 8 is a bar graph showing the percent of hemolysis for the RR12Wpolar control lysin over a range of concentrations, as described in Example 5.
  • Carrier refers to a solvent, additive, excipient, dispersion medium, solubilizing agent, coating, preservative, isotonic and absorption delaying agent, surfactant, propellant, diluent, vehicle and the like with which an active compound is administered.
  • Such carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • “Pharmaceutically acceptable carrier” refers to any and all solvents, additives, excipients, dispersion media, solubilizing agents, coatings, preservatives, isotonic and absorption delaying agents, surfactants, propellants, diluents, vehicles and the like that are physiologically compatible.
  • the carrier(s) must be “acceptable” in the sense of not being deleterious to the subject to be treated in amounts typically used in medicaments.
  • Pharmaceutically acceptable carriers are compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose.
  • pharmaceutically acceptable carriers are suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response).
  • Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, and emulsions such as oil/water emulsions and microemulsions. Suitable pharmaceutical carriers are described, for example, in Remington’s Pharmaceutical Sciences by E.W. Martin, 18th Edition.
  • the pharmaceutically acceptable carrier may be a carrier that does not exist in nature.
  • Bacteriostatic or “bactericidal activity” refers to the property of causing the death of bacteria or capable of killing bacteria to an extent of at least a 3-log10 (99.9%) or better reduction among an initial population of bacteria over an 18-24 hour period.
  • Bacteriostatic or “bacteriostatic activity” refers to the property of inhibiting bacterial growth, including inhibiting growing bacterial cells, thus causing a 2-log10 (99%) or better and up to just under a 3-log reduction among an initial population of bacteria over an 18-24 hour period.
  • Antibacterial refers to both bacteriostatic and bactericidal agents.
  • Effective amount refers to an amount which, when applied or administered in an appropriate frequency or dosing regimen, is sufficient to prevent, reduce, inhibit, or eliminate bacterial growth or bacterial burden or to prevent, reduce, or ameliorate the onset, severity, duration, or progression of the disorder being treated (for example, Gram-negative bacterial pathogen growth or infection), prevent the advancement of the disorder being treated, cause the regression of the disorder being treated, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy, such as bacteriostatic therapy.
  • Co-administer refers to the administration of two agents, such as an a lysin or an engineered lysin and another antibacterial agent, in a sequential manner, as well as administration of these agents in a substantially simultaneous manner, such as in a single mixture/composition or in doses given separately, but nonetheless administered substantially simultaneously to the subject, for example at different times in the same day or 24-hour period.
  • agents such as an a lysin or an engineered lysin and another antibacterial agent
  • Such co-administration of lysins, or engineered lysins with one or more additional antibacterial agents can be provided as a continuous treatment lasting up to days, weeks, or months. Additionally, depending on the use, the co-administration need not be continuous or coextensive.
  • the lysin, or engineered lysin could be administered only initially within 24 hours of an additional antibacterial agent, and then the additional antibacterial agent use may continue without further administration of the lysin, or engineered lysin.
  • Subject refers to a mammal, a plant, a lower animal, a single cell organism, or a cell culture.
  • the term “subject” is intended to include organisms, e.g., prokaryotes and eukaryotes, which are susceptible to or afflicted with bacterial infections, for example Gram- positive bacterial infections, Gram-negative bacterial infections, or acid-fast bacterial infections.
  • subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human, e.g., a human suffering from, at risk of suffering from, or susceptible to infection by Gram-negative bacteria, whether such infection be systemic, topical or otherwise concentrated or confined to a particular organ or tissue.
  • Polypeptide is used herein interchangeably with the term “peptide” and refers to a polymer made from amino acid residues and generally having at least about 30 amino acid residues. The term includes not only polypeptides in isolated form, but also active fragments and derivatives thereof, including modified variants.
  • polypeptide also encompasses fusion proteins or fusion polypeptides comprising an antimicrobial peptide (AMP), a cell- penetrating peptide (CPP), a lysin, and/or an engineered lysins as described herein and maintaining, for example a lytic function.
  • AMP antimicrobial peptide
  • CPP cell- penetrating peptide
  • lysin a lysin
  • engineered lysins as described herein and maintaining, for example a lytic function.
  • a polypeptide can be a naturally occurring polypeptide or a recombinant, engineered, or synthetically produced polypeptide.
  • a particular AMP, CPP, lysin or engineered lysin polypeptide construct can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (such as those disclosed in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
  • Fusion polypeptide refers to an expression product resulting from the fusion of two or more nucleic acid segments, resulting in a fused expression product typically having two or more domains or segments, which typically have different properties or functionality.
  • fusion polypeptide may also refer to a polypeptide or peptide comprising two or more heterologous polypeptides or peptides covalently linked, either directly or via an amino acid or peptide linker.
  • the polypeptides forming the fusion polypeptide are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C- terminus, N-terminus to N-terminus, or N-terminus to C-terminus.
  • fusion polypeptide can be used interchangeably with the term “fusion protein.”
  • the open-ended expression “a polypeptide comprising” a certain structure includes larger molecules than the recited structure, such as fusion polypeptides.
  • Heterologous refers to nucleotide, peptide, or polypeptide sequences that are not naturally contiguous.
  • the term “heterologous” can be used to describe a combination or fusion of two or more peptides and/or polypeptides wherein the fusion peptide or polypeptide is not normally found in nature, such as for a lysin or engineered lysin polypeptide construct fragment or active fragment thereof and a cationic and/or an AMP, a CPP, a polycationic peptide, an amphipathic peptide, a sushi peptide (Ding et al.
  • a defensin peptide (Ganz, T. Nature Reviews Immunology 3, 710-720 (2003)), and/or a hydrophobic peptide, which may have enhanced lytic activity. Included in this definition are two or more lysin polypeptides or active fragments or variants thereof. These can be used to make a fusion polypeptide with lytic activity.
  • Active fragment refers to a portion of a polypeptide that retains one or more functions or biological activities of the isolated polypeptide from which the fragment was taken, for example bactericidal activity against one or more Gram-negative bacteria, including without limitation Burkholderia and/or Yersinia species.
  • Amphipathic peptide refers to a peptide having both hydrophilic and hydrophobic functional groups.
  • secondary structure may place hydrophobic and hydrophilic amino acid residues at opposite sides (e.g., inner side vs outer side when the peptide is in a solvent, such as water) of an amphipathic peptide.
  • These peptides may in certain embodiments adopt a helical secondary structure, such as an alpha-helical secondary structure.
  • “Cationic peptide” refers to a peptide having a high percentage of positively charged amino acid residues.
  • a cationic peptide has a pKa-value of 8.0 or greater.
  • cationic peptide in the context of the present disclosure also encompasses polycationic peptides that are synthetically produced peptides composed of mostly positively charged amino acid residues, such as lysine (Lys) and/or arginine (Arg) residues.
  • the amino acid residues that are not positively charged can be neutrally charged amino acid residues, negatively charged amino acid residues, and/or hydrophobic amino acid residues.
  • Hydrophobic group refers to a chemical group such as an amino acid side chain that has low or no affinity for water molecules but higher affinity for oil molecules. Hydrophobic substances tend to have low or no solubility in water or aqueous phases and are typically apolar but tend to have higher solubility in oil phases.
  • hydrophobic amino acids examples include glycine (Gly), alanine (Ala), valine (Val), Leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp).
  • “Augmenting” refers to a degree of activity of an agent, such as antimicrobial activity, that is higher than it would be otherwise. “Augmenting” encompasses additive as well as synergistic (superadditive) effects.
  • Treatment refers to any process, action, application, therapy, or the like, wherein a subject, such as a human being, is subjected to medical aid with the object of curing a disorder, eradicating a pathogen, or improving the subject’s condition, directly or indirectly. Treatment also refers to reducing incidence, alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, reducing the risk of incidence, improving symptoms, improving prognosis, or combinations thereof. “Treatment” may further encompass reducing the population, growth rate, or virulence of a bacteria in the subject and thereby controlling or reducing a bacterial infection in a subject or bacterial contamination of an organ, tissue, or environment.
  • treatment that reduces incidence may, for example, be effective to inhibit growth of at least one Gram-negative bacteria in a particular milieu, whether it be a subject or an environment.
  • treatment of an already established infection refers to inhibiting the growth, reducing the population, killing and/or eradicating, a Gram-negative bacteria responsible for an infection or contamination.
  • Preventing refers to the prevention of the incidence, recurrence, spread, onset or establishment of a disorder such as a bacterial infection. It is not intended that the present disclosure be limited to complete prevention or to prevention of establishment of an infection.
  • the onset is delayed, or the severity of a subsequently contracted disease or the chance of contracting the disease is reduced, and such constitute examples of prevention.
  • Constracted diseases refers to diseases manifesting with clinical or subclinical symptoms, such as the detection of fever, sepsis, or bacteremia, as well as diseases that may be detected by growth of a bacterial pathogen (e.g., in culture) when symptoms associated with such pathology are not yet manifest.
  • the term “derivative” in the context of a peptide or polypeptide or active fragments thereof is intended to encompass, for example, a polypeptide modified to contain one or more chemical moieties other than an amino acid that do not substantially adversely impact or destroy the lytic activity.
  • the chemical moiety can be linked covalently to the peptide, e.g., via an amino terminal amino acid residue, a carboxy terminal amino acid residue, or at an internal amino acid residue.
  • modifications may be natural or non-natural.
  • a non- natural modification may include the addition of a protective or capping group on a reactive moiety, addition of a detectable label, such as antibody and/or fluorescent label, addition or modification of glycosylation, or addition of a bulking group such as PEG (pegylation) and other changes known to those skilled in the art.
  • the non-natural modification may be a capping modification, such as N-terminal acetylations and C-terminal amidations.
  • Exemplary protective groups that may be added to peptides include, but are not limited to, t-Boc and Fmoc.
  • Commonly used fluorescent label proteins such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and mCherry, are compact proteins that can be bound covalently or noncovalently to a peptide or fused to a peptide without interfering with normal functions of cellular proteins.
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • CFP cyan fluorescent protein
  • YFP yellow fluorescent protein
  • mCherry are compact proteins that can be bound covalently or noncovalently to a peptide or fused to a peptide without interfering with normal functions of cellular proteins.
  • a polynucleotide encoding a fluorescent protein may be inserted upstream or downstream of the polynucleotide sequence.
  • a fusion protein e.g., lysin::GFP
  • a fusion protein e.g., lysin::GFP
  • PEG polyethylene glycol conjugation to proteins
  • the term “derivative” encompasses peptides chemically modified by covalent attachment of one or more PEG molecules. It is anticipated that pegylated peptides will exhibit prolonged circulation half-life compared to the unpegylated peptides, while retaining biological and therapeutic activity.
  • Modified variant refers to a lysin or engineered lysin polypeptide construct wherein a non-naturally occurring modification has been made to the amino acid sequence that either enhances the lytic activity or does not substantially adversely impact or destroy the lytic activity of the lysin or engineered lysin polypeptide construct.
  • exemplary modifications that may be made to modified variants include modifying an amino acid of the lysin peptide, such as a positively charged amino acid, from an L-form to a D-form; adding or deleting one or more amino acid residue or residues to the C-terminus and/or the N-terminus, forming fusion polypeptides, and forming charge array variants, wherein amino acid charges have been reordered.
  • an N-terminal methionine residue is deleted from the lysin or engineered lysin polypeptide construct.
  • Cleavage of an initial methionine residue, e.g., by a methionine aminopeptidase enzyme, after protein synthesis is a known mechanism recognized in the art. Wingfield, P., N-Terminal Methionine Processing, Curr. Protoc. Protein Sci. 2017, 88:6.14.1-6.14.3.
  • a modified variant of a lysin or engineered lysin as disclosed herein may include the lysin or engineered lysin with or without an initial methionine residue.
  • the sequence may include the initial methionine residue or the sequence may exclude the initial methionine residue.
  • the HIP ABC AMP identified herein as SEQ ID NO: 28 may contain an initial methionine residue such that the sequence is MKGRGKTGGKARAKWKTRSSRAGLQWPVGRVHRLLRKGNYAHRVGAGAPVWL (SEQ ID NO: 28) or it may exclude the initial methionine residue such that the HIP ABC AMP sequence is KGRGKTGGKARAKWKTRSSRAGLQWPVGRVHRLLRKGNYAHRVGAGAPVWL (amino acid residues 2-52 of SEQ ID NO: 28).
  • HIP ABC (SEQ ID NO: 28) may indicate either a 52 amino acid sequence containing an initial methionine residue or a 51 amino acid sequence containing lysine (K) as the initial residue.
  • Percent amino acid sequence identity refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, such as a specific lysin or engineered construct polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available software such as BLAST or software available commercially, for example from DNASTAR.
  • Two or more polypeptide sequences can be anywhere from 0-100% identical, or any integer value there between.
  • two polypeptides may have at least 80% of the amino acid residues (such as at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92.5%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5%) that are identical.
  • the term “percent (%) amino acid sequence identity” as described herein applies to peptides as well.
  • Two polypeptides having certain percent amino acid sequence identity as disclosed herein will encompass mutated, truncated, fused, or otherwise sequence- modified forms of isolated polypeptides and peptides described herein, and active fragments thereof (e.g., at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92.5%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% identity) as measured for example by one or more methods referenced above) as compared to the reference (wild type or other intact) polypeptide.
  • active fragments thereof e.g., at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • “Inhalable composition” refers to pharmaceutical compositions of the present disclosure that are formulated for direct delivery to the respiratory tract during or in conjunction with routine or assisted respiration (e.g., by intratracheobronchial, pulmonary, and/or nasal administration), including, but not limited to, atomized, nebulized, dry powder, and/or aerosolized formulations.
  • “Outer Membrane” or “OM” refers to a feature of Gram-negative bacteria. The outer membrane is comprised of a lipid bilayer with an internal leaflet of phospholipids and an external amphiphilic leaflet largely consisting of lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • the LPS has three main sections: a hexa-acylated glucosamine-based phospholipid called lipid A, a polysaccharide core and an extended, external polysaccharide chain called O-antigen.
  • the OM presents a non- fluid continuum stabilized by three major interactions, including: i) the avid binding of LPS molecules to each other, especially if cations are present to neutralize phosphate groups; ii) the tight packing of largely saturated acyl chains; and iii) hydrophobic stacking of the lipid A moiety.
  • the resulting structure is a barrier for both hydrophobic and hydrophilic molecules.
  • the peptidoglycan forms a thin layer that is very sensitive to hydrolytic cleavage - unlike the peptidoglycan of Gram-negative bacteria which is 30-100 nanometers (nm) thick and consists of up to 40 layers, the peptidoglycan of Gram-negative bacteria is only 2-3 nm thick and consists of only 1-3 layers.
  • ESKAPE bacteria refers to a group of pathogens that are known for their ability to escape the effects of antimicrobial drugs.
  • ESKAPE stands for the following bacteria: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species.
  • Enterococcus faecium Enterococcus faecium
  • Staphylococcus aureus Klebsiella pneumoniae
  • Klebsiella pneumoniae Acinetobacter baumannii
  • Pseudomonas aeruginosa enterobacter species.
  • Gram-negative ESKAPE bacteria the group of Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species.
  • POLYPEPTIDES Native and Modified Lysins [0077] The present disclosure is directed to isolated polypeptides comprising base lysins and modified base lysins, as well as active fragments and variants thereof as herein described.
  • the N-terminal methionine residue is deleted from the native or modified base lysin or an engineered lysin, e.g., a base lysin, which includes a variant base lysin, as herein described, which is modified to include linker polypeptides, AMPs, CPPs and/or other features as herein described.
  • a base lysin or engineered lysin may include an initial methionine residue or may not include an initial methionine residue.
  • the isolated polypeptides comprise a lysin, variant lysin, active fragments thereof or derivatives thereof, wherein the lysin, variant, fragment, or derivative thereof is combined with at least one antimicrobial peptide (“AMP”) and/or at least one cell- penetrating peptides (“CPP”) to form an engineered lysin, wherein the engineered lysin has lytic activity.
  • AMP antimicrobial peptide
  • CPP cell- penetrating peptides
  • lytic activity encompasses the ability of an engineered lysin polypeptide construct to kill bacteria, such as a Gram-negative bacteria (e.g., P. aeruginosa, B.
  • cepacian, Burkholderia spp., Yersinia spp. reduce the population of bacteria or inhibit bacterial growth (e.g., by penetrating the outer membrane of a Gram-negative bacteria), optionally in the presence of human serum.
  • the present isolated lysins and engineered lysins, fragments, or variants thereof are capable of penetrating the outer membrane of Gram-negative bacteria.
  • the present isolated lysins and engineered lysins, fragments, or variants thereof can degrade peptidoglycan, a major structural component of the bacterial cell wall, resulting in e.g., cell lysis or non-lethal damage that inhibits bacterial growth.
  • the present isolated lysins and engineered lysins, fragments, or variants thereof disclosed herein contain positively charged (and amphipathic) N- and/or C-terminal ⁇ -helical domains that facilitate binding to the anionic outer membrane of a Gram-negative bacteria to effect translocation into the sub-adjacent peptidoglycan.
  • NPN fluorescence can be used as a measurement of the outer membrane permeability.
  • the ability of a lysin to penetrate an outer wall may be assessed by incubating, e.g., NPN with a Gram-negative bacteria, e.g., Burkholderia spp. or Yersinia spp., in the presence of the lysin to be tested for activity.
  • a Gram-negative bacteria e.g., Burkholderia spp. or Yersinia spp.
  • fluorescence induction can be compared to that of established permeabilizing agents, such as EDTA (ethylene diamine tetraacetate) or an antibiotic such as an antibiotic of last resort used in the treatment of Gram-negative bacterial infections, i.e., Polymyxin B (PMB), to assess the level of outer membrane permeability.
  • established permeabilizing agents such as EDTA (ethylene diamine tetraacetate) or an antibiotic such as an antibiotic of last resort used in the treatment of Gram-negative bacterial infections, i.e., Polymyxin B (PMB)
  • PMB Polymyxin B
  • a minimum inhibitory concentration (“MIC”) value (i.e., the minimum concentration of peptide sufficient to suppress at least 80% of the bacterial growth compared to control) may be determined for a lysin and compared to, e.g., a parent lysin or compound inactive in human serum, e.g., T4 phage lysozyme or artilysin GN126.
  • T4 phage lysozyme is commercially available, e.g. from Sigma-Aldrich, Inc.
  • GN126 corresponds to Art-175, which is described in the literature and may be obtained by fusing AMP SMAP-29 to GN lysin KZ144.
  • MIC values may be expressed, e.g., in ⁇ g/mL, and MIC may be determined by any method known in the art.
  • MIC values for a lysin may be determined against a Gram-negative bacterial strain, e.g., a Burkholderia spp in e.g., AST medium containing % CAA (casamino acid media) with 0.02% TWEEN80®, and 0.5micromolar MgSO4, or 25% CAMHB with 0.02% TWEEN80® when the Gram-negative bacterial strain are e.g., Yersinia spp.
  • the present isolated lysins and engineered lysins, fragments, and variants thereof show low toxicity against erythrocytes.
  • lysins of the present disclosure include Gram-negative base lysins.
  • a “Gram-negative base lysin” is an isolated lysin, which may be modified, e.g., by fusing the Gram-negative base lysin with an AMP and/or CPP or other element as herein described, e.g, at the N-terminus or C-terminus.
  • This term includes native lysins, as well as native lysins modified to contain one or more insertions, deletions and/or amino acid substitutions in comparison to a reference lysin polypeptide, e.g., a naturally occurring lysin or a parent lysin, which itself may be a variant lysin.
  • Such variant base lysins may be further modified to include AMPs, CPPs or other features as disclosed, e.g, at the N-terminus or C- terminus.
  • Suitable Gram-negative base lysins include those from the phage lysozyme pfam00959 protein family, such as GN867 set forth as SEQ ID NO: 13, GN3 set forth as SEQ ID NO: 3, QPI14001 set forth as SEQ ID NO: 9 and GN917 set forth as SEQ ID NO: 10.
  • Suitable base lysins without e.g., substitutions include peptidase_M15_4 pfam13539 protein family, such as GN685 set forth as SEQ ID NO: 2, GN37 set forth as SEQ ID NO: 8, GN569 set forth as SEQ ID NO: 7, GN568 set forth as SEQ ID NO: 1 and GN577 set forth as SEQ ID NO: 12.
  • the Gram-negative base lysin is from the phage lysozyme pfam00959 protein family, such as GN867 set forth as SEQ ID NO: 13, GN3 set forth as SEQ ID NO: 3, GN515 set forth as SEQ ID NO: 6, QPI14001 set forth as SEQ ID NO: 9 and GN917 set forth as SEQ ID NO: 10.
  • the Gram-negative base lysin is from the peptidase_M15_4 pfam13539 protein family, such as GN685 set forth as SEQ ID NO: 2, GN568 set forth as SEQ ID NO: 1, GN37 set forth as SEQ ID NO: 8, GN569 set forth as SEQ ID NO: 7, and GN577 set forth as SEQ ID NO: 12.
  • Other suitable Gram-negative base lysins are from the muramidase pfam11860 protein family, such as GN428 set forth as SEQ ID NO: 4, and CAH0532154 set forth as SEQ ID NO: 11.
  • GN588 as set forth in SEQ ID NO: 5 from the peptidase_M15_3 pfam08291 protein family is also suitable for use as a Gram-negative base lysin.
  • the foregoing Gram-negative base lysins are detailed below.
  • Table 1 Exemplary Base Lysins Source of lysin NCBI Amino acid sequence Lysin (protein family) Accession number GN568 Yersinia phage CAJ28446 MEVQPTIEEVSMGFKLGSRSLQRLQG PY100 VHPDLVKVVKRAIEISPVDFTVTEGL (Peptidase_M15_ RTLERQKELFAKGASKTMRSRHLTG 4; pfam13539) HAVDISPLVDGKVSWDWKYYYPMA DAMKQAAKELNIPVEWGGDWKTFK DGPHFQLPYGVYK (SEQ ID NO: 1) GN685 Yersinia phage CAJ28446 MGFKLGSRSLQRLQGVHPDLVKVVK PY100 RAIEISPVDFTVTEGLRTLERQKELFA (Peptidase_M15_ KGASKTMRSRHLTGHAVDISPLVDG 4; pf
  • an engineered lysin includes e.g., substitutions, deletions or insertions relative to a native base lysin, but does not include AMPs, CPPs or additional elements as described herein.
  • the engineered lysin, active fragment thereof or derivative thereof has at least about 80%, such as at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% sequence identity with a reference lysin and/or active fragment thereof described herein.
  • the engineered lysin, active fragment thereof or derivative thereof has at least two amino acid substitution relative to a reference lysin and/or active fragment thereof described herein, and in certain embodiments, the engineered lysin, active fragment thereof or derivative thereof has at least two amino acid substitutions relative to a reference lysin and/or active fragment thereof described herein. In certain embodiments, the engineered lysin, active fragment thereof or derivative thereof has two amino acid substitution relative to a reference lysin and/or active fragment thereof described herein.
  • an engineered lysin includes a base lysin which base lysin includes, e.g., substitutions, deletions or insertions and further includes the AMPs, CPPs and optionally other elements, such as linkers, as described herein or in embodiments, such as those when an engineered lysin includes substitutions, deletions or insertions, but does not include the AMPs, CPPs or other features described herein, function the same as a reference lysin.
  • such engineered lysins retain one or more functional or biological activities of a reference lysin.
  • the modification improves the antibacterial activity in comparison to the reference lysin.
  • the lysin variant has improved in vitro antibacterial activity (e.g., in buffer and/or media) in comparison to the reference lysin. In other embodiments, the lysin variant has improved in vivo antibacterial activity (e.g., in an animal infection model).
  • the modification improves the antibacterial activity of the lysin in the presence of human serum, and in some embodiments, the modification improves the antibacterial activity of the lysin in the absence of human serum.
  • Suitable variant lysins particularly those which may be used as a Gram-negative base lysin, include variants of GN685, GN3, GN4, GN515, and GN569.
  • the variant of GN685 comprises an amino acid substitution of K78H relative to GN685 (in embodiments wherein GN685 includes an initial methionine residue) or amino acid substitutions of K77H relative to GN685 (in embodiments wherein GN685 does not include an initial methionine residue).
  • the variant of GN3 comprises amino acid substitutions of R100Q and R116H relative to GN3 (in embodiments wherein GN3 includes an initial methionine residue) or amino acid substitutions of R99Q and R115H relative to GN3 (in embodiments wherein GN3 does not include an initial methionine residue).
  • the variant of GN3 comprises amino acid substitutions of R100Q and R116Q relative to GN3 (in embodiments wherein GN3 includes an initial methionine residue) or amino acid substitutions of R99Q and R115Q relative to GN3 (in embodiments wherein GN3 does not include an initial methionine residue).
  • the variant of GN4 comprises amino acid substitutions of K100D and R116H relative to GN4 (in embodiments wherein GN4 includes an initial methionine residue) or amino acid substitutions of K99D and R115H relative to GN4 (in embodiments wherein GN4 does not include an initial methionine residue).
  • the GN4 lysin may be obtained from Pseudomonas phage PAJU2 (NCBI Reference Sequence YP 002284361.1, and is described in PCT Publication No. 2019/191633, which reference is incorporated by reference herein in its entirety.
  • the variant of GN515 comprises amino acid substitutions of R100D and R116H relative to GN515 (in embodiments wherein GN515 includes an initial methionine residue) or amino acid substitutions of R99D and R115H relative to GN515 (in embodiments wherein GN515 does not include an initial methionine residue).
  • the variant of GN569 comprises an amino acid substitution of K87H relative to GN569 (in embodiments wherein GN569 includes an initial methionine residue) or amino acid substitutions of K86H relative to GN569 (in embodiments wherein GN569 does not include an initial methionine residue).
  • Exemplary variant lysins are detailed below in Table 2.
  • the variant lysins are obtained by modifying a reference lysin to include a modification resulting in a change in the overall isoelectric point (pI) of the lysin, i.e., the pH at which a molecule has a net neutral charge by, for example, incorporating a single pI- increasing mutation, such as a single point mutation, into a reference lysin.
  • pI isoelectric point
  • the lysin variants of the present disclosure are typically designed to retain an ⁇ -helix domain, the presence or absence of which can be readily determined using various software programs, such as Jpred4 (compio.dundee.ac.uk/jpred), Helical Wheel (hael.net/helical.htm), HeliQuest (zhanglab.ccmb.med.umich.edu/I-TASSER/) and PEP-FOLD 3 (bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3).
  • the ⁇ -helix domain is located at the C-terminus of a lysin.
  • the ⁇ -helix domain is located at the N-terminus of a lysin. More typically, the ⁇ -helix domain is located at the C terminus.
  • the ⁇ -helix domain of the lysins of the present disclosure varies in size between about 20 and 40 amino acids, more typically between about 15 and 33 amino acid residues.
  • Lysin variants may be formed by any method known in the art and as described in WO 2017/049233, which is herein incorporated by reference in its entirety, e.g., by modifying any of the lysins, active fragments thereof and derivatives described herein through site-directed mutagenesis or via mutations in hosts that produce the present lysins which retain one or more of the biological functions as described herein.
  • the present lysin variants may be truncated, chimeric, shuffled or “natural,” and may be in combination as described, for example, in U. S. Patent No.5,604,109, which is incorporated herein in its entirety by reference.
  • Such a mutation is generally made by making the fewest nucleotide changes possible.
  • a substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
  • Such a conservative change generally leads to less change in the structure and function of the resulting protein.
  • a non-conservative change is more likely to alter the structure, activity or function of the resulting protein.
  • the present disclosure should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.
  • amino acid changes or substitutions in the lysin polypeptide sequence can be made to replace or substitute one or more, one or a few, one or several, one to five, one to ten, or such other number of amino acids in the sequence of the lysin(s) provided herein to generate mutants or variants thereof.
  • mutants or variants thereof may be predicted for function or tested for function or capability for anti-bacterial activity as described herein against, e.g., Burkholderia spp. or Yersinia spp., and/or for having comparable activity to the lysin(s) as described and particularly provided herein.
  • changes made to the sequence of lysin, and mutants or variants described herein can be tested using the assays and methods known in the art and described herein.
  • One of skill in the art on the basis of the domain structure of the lysin(s) hereof can predict one or more, one or several amino acids suitable for substitution or replacement and/or one or more amino acids which are not suitable for substitution or replacement, including reasonable conservative or non-conservative substitutions.
  • the present isolated lysins and engineered lysin polypeptide constructs comprise active fragments of lysins or derivatives.
  • active fragment refers to a portion of a full-length lysin, which retains one or more biological activities of the reference lysin.
  • an active fragment of a lysin or variant lysin inhibits the growth, or reduces the population, or kills a Gram-negative bacteria, such as a Burkholderia spp. and/or Yersinia spp. and optionally at least one additional species of Gram-negative bacteria as described herein in the absence or presence of human serum.
  • the isolated lysin or engineered lysin polypeptide construct comprises a lysin selected from GN568, GN685, GN3, GN4, GN428, GN588, GN515, GN37, GN569, QPI14001.1, GN917, CAH0532154, GN577, and GN867 or an active fragment thereof, wherein the lysin or active fragment thereof inhibits the growth, or reduces the population, or kills at least one Gram-negative bacteria, such as Burkholderia spp. or at least one Yersinia spp.
  • the lysin or active fragment thereof contains at least one substitution, deletion, or insertion modification relative to GN568, GN685, GN3, GN4, GN428, GN588, GN515, Peptidase_M15_4, GN37, GN569, QPI14001.1, GN917, CAH0532154, GN577, and GN867.
  • the at least one amino acid substitution is a conservative amino acid substitution.
  • the at least one amino acid substitution is chosen from R100D, R100Q, R116H, R116Q, K100D, K78H, or K87H, when the base lysin polypeptide comprises an initial methionine residue.
  • the at least one amino acid substitution is chosen from R99D, R99Q, R115H, K99D, or K77H, K86H, when the base lysin polypeptide does not comprise an initial methionine residue.
  • the base lysin comprises an initial methionine residue and the base lysin is GN3
  • the lysin or active fragment thereof contains two amino acid substitutions of R100Q and R116H or two amino acid substitutions of R100Q and R116Q.
  • the base lysin or active fragment thereof when the base lysin does not comprise an initial methionine residue and the base lysin is GN3, the lysin or active fragment thereof contains two amino acid substitutions of R99Q and R115H or two amino acid substitutions of R99Q and R115Q. In certain embodiments, when the base lysin comprises an initial methionine residue and the base lysin is GN4, the lysin or active fragment thereof contains two amino acid substitutions of K100D and R116H. In certain embodiments, when the base lysin does not comprise an initial methionine residue and the base lysin is GN4, the lysin or active fragment thereof contains two amino acid substitutions of K99D and R115H.
  • the construct comprises an AMP in addition to the lysin polypeptide, e.g., an engineered lysin, which is a lysin-AMP polypeptide construct, optionally further comprising a linker and/or an outer membrane binding motif, as disclosed herein.
  • AMP antimicrobial peptide
  • AMPs include defensins, cathelicidins, sushi peptides, cationic peptides, polycationic peptides, amphipathic peptides, hydrophobic peptides and/or AMP-like peptides, e.g., amurin peptides as described herein. Fragments of AMPs, AMP variants and derivatives of AMPs are also encompassed by this term. [00103]
  • the term “AMP activity” as used herein encompasses the ability of an AMP or fragment or variant thereof to kill bacteria, reduce the population of bacteria or inhibit bacterial growth e.g., by penetrating the outer membrane of a Gram-negative bacteria in the presence and/or absence of human serum.
  • the present AMPs are variant AMPs having at least 80%, such as at least about 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% sequence identity with any of the AMPs described herein, wherein the variant AMP thereof retains an AMP activity.
  • the present AMPs comprise a helical domain, such as an ⁇ - helical domain.
  • the ⁇ -helical domain spans most of the molecule.
  • the ⁇ -helix domain is either interrupted or truncated.
  • the ⁇ -helix domain of the present AMPs may vary in size from between about 3 to 32 amino acids, more typically between about 10 and 25 amino acid residues.
  • the helical domains are required for activity and typically must be retained when fused to a C- or N-terminus of a lysin.
  • helical peptides display amphipathic characteristics and contain a substantial proportion (e.g.
  • hydrophobic residues frequently appearing in repeated patterns.
  • the hydrophilic residues Upon formation of an ⁇ -helical structure, the hydrophilic residues typically end up on the same side of the helix, thereby resulting in a conformation-dependent amphiphilicity.
  • these peptides are unstructured in an aqueous environment, but adopt a helical conformation upon encountering lipid membranes.
  • Peptides belonging to this group typically display an overall positive charge ranging from +2 to +11 and usually kill microbes, such as Gram-negative bacteria, by creating membrane defects, leading to a loss of gradients in electrolytes, signal substances and other factors.
  • Suitable AMPs for use in the present engineered lysin-AMP constructs include, for example BK5, AM1, AM13, MMAV-2, MFV1-3, BCM1, BK3, BK3h2, BK3h3, ATRA, RI18, HIP A , HIP AB , or HIP ABC , HIP Anat , BK9 as detailed in Table 3 below.
  • the AMPs are modified to produce variant AMPs.
  • AMPs typically comprise a helical domain such as an ⁇ -helical domain.
  • the variant AMPs retain the ⁇ -helical domain.
  • any variant AMP is typically accurately identified using various software programs, such as Jpred4 (compio.dundee.ac.uk/jpred), Helical Wheel (hael.net/helical.htm), HeliQuest (zhanglab.ccmb.med.umich.edu/I-TASSER/) and PEP-FOLD 3 (bioserv.rpbs.univ-paris- diderot.fr/services/PEP-FOLD3).
  • AMPs may be prepared according to any method known in the art including as described herein above for the lysins, active fragments thereof and variants thereof.
  • the AMPs for use in the engineered lysins of the present disclosure include a fragment of a larger AMP that retains antibacterial activity.
  • the AMP portion of the lysin-AMP polypeptide construct may include a fragment of porcine myeloid antimicrobial peptide-36 (“PMAP-36”) (SEQ ID NO: 25) that retains antibacterial activity.
  • PMAP-36 is a cathelicidin-related AMP deduced from porcine myeloid cDNA with an amphipathic ⁇ -helical conformation at the N-terminus. Accordingly, suitable PMAP-36 fragments are typically selected from the N-terminus to obtain fragments retaining antibacterial activity.
  • the PMAP-36 fragment of the present disclosure includes the hydrophobic amino acid (Trp) at position 23.
  • the random coil C-terminal is omitted from the PMAP-36 fragment to reduce or eliminate hemolysis that may be caused by PMAP-36.
  • PMAP-36 fragments include RI12 having the amino acid sequence RLKKIGKVLKWK (SEQ ID NO: 30), RI18 having the amino acid sequence RKKTRKRLKKIGKVLKWI (SEQ ID NO: 25), and TI15 having the amino acid sequence TRKRLKKIGKVLKWI (SEQ ID NO: 31).
  • the AMPs of the engineered lysins of the present disclosure include an outer membrane binding motif.
  • a particularly desirable outer membrane binding motif for use with the engineered lysins of the present disclosure includes FIRL (SEQ ID NO:146).
  • FIRL SEQ ID NO: 146
  • the outer membrane binding motif is FIQL Bc set forth as SEQ ID NO: 33, FMRL Yp set forth as SEQ ID NO: 34, or FMRL Nm (FRRL) set forth as SEQ ID NO: 35.
  • outer membrane binding motifs that are disclosed herein include FIRL Bc , (SEQ ID NO: 143), derived from the outer membrane protein assembly factor BamD of Burkholderia cenocepacia (NCBI Accession No.: WP_226210925.1).
  • MIDRFMRL Yp NPTH (SEQ ID NO: 144)
  • MIERFRRLHPQH (FMRL Nm ) (SEQ ID NO: 147) are outer membrane binding motifs derived from the outer membrane protein assembly factor BamD of Yersinia pestis or Neisseria meningitidis (NCBI Accession No: WP_046596486.1 and NCBI Accession No: WP_148082483.1, respectively).
  • peptides which may be incorporated into the engineered lysin polypeptide constructs of the present disclosure includes the cationic peptide KFFKFFKFFK (SEQ ID NO: 36) described in Vaara and Porro, Group of peptides that act synergistically with hydrophobic antibiotics against gram-negative enteric bacteria, Antimicrobial Agents Chemother. 1996, 40(8):1801-1805, which is herein incorporated by reference in its entirety.
  • the foregoing additional peptides are resistant to salts and serum inactivation as described, for example, in Monhanram et al., Salt-resistant short antimicrobial peptides, Peptide Science 2016, 106(3):345-356, which is herein incorporated by reference in its entirety.
  • the construct comprises a cell-penetrating peptide (CPP) in addition to the lysin polypeptide, e.g., an engineered lysin-CPP polypeptide construct, optionally further comprising a linker and/or an outer membrane binding motif, as disclosed herein.
  • CCPP cell-penetrating peptide
  • “Cell-penetrating peptides” or “CPPs” refers to small peptides that have the ability to translocate across biological membranes and may be used, for example, to facilitate intracellular protein delivery.
  • CPPs are also typical short, cationic peptide sequences that have a high affinity for cell membranes and may be either unstructured or alpha- helical.
  • the present CPPs are variant CPPs having at least about 80%, such as at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5% sequence identity with any of the CPPs described herein, wherein the variant CPP thereof retains a CPP activity.
  • the engineered lysin polypeptide constructs, fragments thereof, and variants thereof may comprise both a CPP and an AMP in addition to the lysin polypeptide.
  • Suitable CPPs for use in the present engineered lysin-CPP constructs include, for example ZF5.3 and PRR, PRR 3 , and PRR 5 , as detailed in Table 4 below.
  • the engineered lysin polypeptide construct disclosed herein further comprise at least one linker.
  • the engineered lysin polypeptide constructs of the present disclosure further comprise at least one structure stabilizing component to maintain at least a portion of the structure of the first and/or second component in the construct, e.g., the lysin and/or AMP and/or CPP, substantially the same as in the unconjugated lysin and/or AMP or CPP.
  • the stabilizing structure is also a linker.
  • the at least one structure stabilizing component such as a linker, enables the lysin and AMP or CPP to substantially preserve the three-dimensional structure of the first and/or second protein moieties, such that at least one biological activity of the lysin and/or AMP or CPP is retained.
  • Suitable linkers and structure stabilizing components are disclosed, for example, in PCT Publication Nos. WO 2019/191633 and 2020/046747, both of which are incorporated by referenced herein.
  • Suitable linkers for connecting two polypeptides are known in the art.
  • the linker is a peptide, such as a peptide comprising glycine and serine residues.
  • Specific suitable linkers include, but are not limited to, a TAGGTAGG linker (SEQ ID NO: 41), an IGEM linker GGSGSGSGSGSP (BBa_K1485002) (SEQ ID NO: 42).
  • the linker is a glycine (G) amino acid, and in certain embodiments, the linker is a glycine optionally with a threonine residue having the sequence GT or TG. In certain embodiments, the linker is GT. In certain embodiments, the linker has the amino acid sequence LSKLGG (SEQ ID NO: 45).
  • the linker has the amino acid sequence LSKLGGLGGGPRRLGG (SEQ ID NO: 46). In certain embodiments, the linker has the amino acid sequence LGGLGG (SEQ ID NO: 47). In certain embodiments, the linker has the amino acid sequence SKY. In certain embodiments, the linker has the amino acid sequence LGGKSLG (SEQ ID NO: 174).
  • the structure stabilizing component is a peptide moiety, e.g., an RPP or PP moiety. Such peptide moieties may be included in the present engineered lysin polypeptide constructs to assist in maintaining the structure of the lysin and/or AMP and/or the CPP protein moieties.
  • the structure stabilizing component may be inserted at the C-terminus or N-terminus of a linker.
  • the engineered lysins of the disclosure may further include additional amino acid extensions, such as MG or LKWI (SEQ ID NO: 127).
  • additional amino acid extensions such as MG or LKWI (SEQ ID NO: 127).
  • Engineered Lysin Polypeptide Constructs [00131] In some embodiments, the engineered lysin polypeptide construct comprises a base lysin polypeptide or fragment or variant thereof and at least one AMP or CPP or fragment or variant thereof.
  • the engineered lysin polypeptide construct comprises a lysin polypeptide or fragment or variant thereof and at least one AMP or CMP or fragment or variant thereof and further comprises at least one linker and optionally at least one outer membrane binding motif structure and at least one structure stabilizing component, such as at least two structure stabilizing components.
  • the at least one AMP or CPP or fragment or variant thereof can be at the N-terminus or the C-terminus of the lysin polypeptide or fragment or variant thereof.
  • the optional structure stabilizing component and outer membrane binding motif can be at the N-terminus or the C-terminus of the engineered lysin polypeptide construct.
  • Table 5 depicts specific examples of the engineered lysin polypeptide constructs described herein. For each of the constructs, double underlines correspond to a lysin. Structure stabilizing components, outer membrane binding motifs, Other Amino Acid Extensions, and linkers are italicized. AMPs and CPPs are indicted in bold.
  • the engineered lysins of the present disclosure are chemically modified.
  • a chemical modification includes but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Chemical modifications can occur anywhere in a engineered lysin polypeptide construct, including the amino acid side chains, as well as the amino or carboxyl termini.
  • engineered lysin polypeptide constructs comprise an N-terminal acetylation modification.
  • the engineered lysin polypeptide constructs comprise a C-terminal amidation modification. Such modification can be present at more than one site in an engineered lysin polypeptide construct.
  • the engineered lysins are conjugated to a duration enhancing moiety.
  • the duration enhancing moiety is polyethylene glycol.
  • PEG Polyethylene glycol
  • the PEG backbone (CH 2 CH 2 -O-) n , wherein n is a number of repeating monomers, is flexible and amphiphilic.
  • PEG polymer chains can protect such polypeptides from immune response and other clearance mechanisms.
  • pegylation can lead to improved efficacy and safety by optimizing pharmacokinetics, increasing bioavailability, and decreasing immunogenicity and dosing amount and/or frequency.
  • the present disclosure is directed an isolated polynucleotide comprising a nucleic acid molecule encoding an engineered lysin, or a variant or active fragment thereof as described herein.
  • the isolated polynucleotide sequence is a DNA sequence.
  • the isolated polynucleotide is a cDNA sequence.
  • the polynucleotide is selected from SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO:
  • the polynucleotide encodes an engineered polypeptide lysin construct selected from SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 55, S
  • the present disclosure is directed to a vector comprising an isolated polynucleotide comprising a nucleic acid molecule encoding any of the engineered lysin polypeptide constructs or variants or active fragments thereof disclosed herein or a complementary sequence of the present isolated polynucleotides.
  • the vector is a plasmid or cosmid.
  • the vector is a viral vector, wherein additional DNA segments can be ligated into the viral vector.
  • the vector can autonomously replicate in a host cell into which it is introduced.
  • the vector can be integrated into the genome of a host cell upon introduction into the host cell and thereby be replicated along with the host genome.
  • particular vectors referred to herein as “recombinant expression vectors” or “expression vectors”, can direct the expression of genes to which they are operatively linked.
  • a polynucleotide sequence is “operatively linked” when it is placed into a functional relationship with another nucleotide sequence.
  • a promoter or regulatory DNA sequence is said to be “operatively linked” to a DNA sequence that codes for an RNA and/or a protein if the two sequences are operatively linked, or situated such that the promoter or regulatory DNA sequence affects the expression level of the coding or structural DNA sequence.
  • the present disclosure is directed to a vector comprising a nucleic acid molecule that encodes an engineered lysin polypeptide construct chosen from SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO:
  • any system or vector suitable to maintain, propagate or express a polypeptide in a host may be used for expression of the engineered lysin polypeptide constructs disclosed herein or active fragments or variants thereof.
  • the appropriate DNA/polynucleotide sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (2001).
  • tags can also be added to the engineered lysin polypeptide constructs or active fragments or variants thereof to provide convenient methods of isolation, e.g., c-myc, biotin, poly-His, etc. Kits for such expression systems are commercially available.
  • a wide variety of host/expression vector combinations may be employed in expressing the polynucleotide sequences encoding the engineered lysin polypeptide constructs disclosed herein or active fragments or variants thereof. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available.
  • vectors include, among others, chromosomal, episomal and virus derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • chromosomal, episomal and virus derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as
  • the vectors may provide for the constitutive or inducible expression of the engineered lysin polypeptide constructs, or active fragments or variants thereof of the present disclosure.
  • Suitable vectors include but are not limited to derivatives of SV40 and known bacterial plasmids, e.g., E.
  • phage DNAS e.g., the numerous derivatives of phage A, e.g., NM989, and other phage DNA, e.g., M13
  • vectors may comprise various regulatory elements (including promoter, ribosome binding site, terminator, enhancer, various cis-elements for controlling the expression level) wherein the vector is constructed in accordance with the host cell.
  • expression control sequences sequences that control the expression of a polynucleotide sequence operatively linked to it
  • Useful control sequences include, but are not limited to: the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast-mating factors, E.
  • the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the
  • the present disclosure is directed to a host cell comprising any of the vectors disclosed herein including the expression vectors comprising the polynucleotide sequences encoding the engineered lysin polypeptide constructs or active fragments or variants thereof of the present disclosure.
  • a wide variety of host cells are useful in expressing the present polypeptides.
  • Non-limiting examples of host cells suitable for expression of the present polypeptides include well-known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSCl, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.
  • the expression host may be any known expression host cell, in a typical embodiment the expression host is one of the strains of E. coli. These include, but are not limited to, commercially available E.
  • E. coli strains such as Top10 (ThermoFisher Scientific, Inc.), DH5a (Thermo Fisher Scientific, Inc.), XLI-Blue (Agilent Technologies, Inc.), SCSllO (Agilent Technologies, Inc.), JM109 (Promega, Inc.), LMG194 (ATCC), and BL21 (Thermo Fisher Scientific, Inc.).
  • Top10 ThermoFisher Scientific, Inc.
  • DH5a Thermo Fisher Scientific, Inc.
  • XLI-Blue Agilent Technologies, Inc.
  • SCSllO Algilent Technologies, Inc.
  • JM109 Promega, Inc.
  • LMG194 ATCC
  • BL21 Thermo Fisher Scientific, Inc.
  • Efficient expression of the present engineered lysin polypeptide constructs or active fragments or variants thereof depends on a variety of factors such as optimal expression signals (both at the level of transcription and translation), correct protein folding, and cell growth characteristics.
  • optimal expression signals both at the level of transcription and translation
  • correct protein folding and cell growth characteristics.
  • methods for constructing the vector and methods for transducing the constructed recombinant vector into the host cell conventional methods known in the art can be utilized.
  • the engineered lysin polypeptide constructs or active fragments or variants thereof of the present disclosure can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography.
  • High performance liquid chromatography can also be employed for peptide purification, including purification of the engineered lysin polypeptide constructs or active fragments and variants thereof, as well as their component parts, e.g., isolated lysin polypeptides, linkers, stabilizing components, AMPs, and/or CPPs.
  • the vector system used for the production of engineered lysin polypeptide constructs or active fragments or variants of the present disclosure may be a cell-free expression system.
  • Various cell-free expression systems are commercially available, including, but are not limited to those available from Promega, LifeTechnologies, Clonetech, etc.
  • compositions of the present disclosure can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, tampon applications emulsions, aerosols, sprays, suspensions, lozenges, troches, candies, injectants, chewing gums, ointments, smears, time- release patches, liquid absorbed wipes, and combinations thereof.
  • Administration of the compositions of the present disclosure or pharmaceutically acceptable forms thereof may be topical, i.e., the pharmaceutical composition may be applied directly where its action is desired (for example directly to a wound), or systemic.
  • systemic administration can be enteral or oral, i.e., the composition may be given via the digestive tract, parenteral, i.e., the composition may be given by other routes than the digestive tract such as by injection or inhalation.
  • the engineered lysin polypeptide constructs of the present disclosure or active fragments or variants thereof and compositions comprising them can be administered to a subject orally, parenterally, by inhalation, topically, rectally, nasally, buccally, via an implanted reservoir, or by any other known method.
  • the engineered lysin polypeptide constructs of the present disclosure or active fragments or variants thereof can also be administered by means of sustained release dosage forms.
  • the engineered lysin polypeptide constructs of the present disclosure or active fragments or variants thereof can be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions, and dispersions.
  • the composition can be formulated with excipients such as, e.g., lactose, sucrose, corn starch, gelatin, potato starch, alginic acid, and/or magnesium stearate.
  • excipients such as, e.g., lactose, sucrose, corn starch, gelatin, potato starch, alginic acid, and/or magnesium stearate.
  • an engineered lysin polypeptide construct of the present disclosure or active fragments or variants thereof may be mixed with a pharmaceutical excipient to form a solid pre-formulation composition.
  • tablets may be sugar coated or enteric coated by standard techniques.
  • the tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • the topical compositions of the present disclosure may further comprise a pharmaceutically or physiologically acceptable carrier, such as a dermatologically or an otically acceptable carrier.
  • a pharmaceutically or physiologically acceptable carrier such as a dermatologically or an otically acceptable carrier.
  • Such carriers in the case of dermatologically acceptable carriers, may be compatible with skin, nails, mucous membranes, tissues, and/or hair, and can include any conventionally-used dermatological carrier meeting these requirements.
  • the carrier may be compatible with all parts of the ear.
  • Such carriers can be readily selected by one of ordinary skill in the art.
  • Carriers for topical administration of the compositions of the present disclosure include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene and/or polyoxypropylene compounds, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.
  • the active components of the present disclosure may be formulated, for example, in an oleaginous hydrocarbon base, an anhydrous absorption base, a water-in-oil absorption base, an oil-in-water water-removable base, and/or a water-soluble base.
  • the active components of the present disclosure may be formulated, for example, in an aqueous polymeric suspension including such carriers as dextrans, polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels, gellan gums such as Gelrite®, cellulosic polymers such as hydroxypropyl methylcellulose, and carboxy-containing polymers such as polymers or copolymers of acrylic acid, as well as other polymeric demulcents.
  • an aqueous polymeric suspension including such carriers as dextrans, polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels, gellan gums such as Gelrite®, cellulosic polymers such as hydroxypropyl methylcellulose, and carboxy-containing polymers such as polymers or copolymers of acrylic acid, as well as other polymeric demulcents.
  • compositions according to the present disclosure may be in any form suitable for topical application, including aqueous, aqueous- alcoholic or oily solutions; lotion or serum dispersions; aqueous, anhydrous or oily gels; emulsions obtained by dispersion of a fatty phase in an aqueous phase (O/W or oil-in-water) or, conversely, (W/O or water-in-oil); microemulsions or alternatively microcapsules, microparticles or lipid vesicle dispersions of ionic and/or nonionic type; creams; lotions; gels; foams (which may use a pressurized canister, a suitable applicator, an emulsifier, and an inert propellant); essences; milks; suspensions; and patches.
  • aqueous, aqueous- alcoholic or oily solutions including lotion or serum dispersions; aqueous, anhydrous or oily gels; emulsions obtained by dis
  • Topical compositions of the present disclosure may also contain adjuvants such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preserving agents, antioxidants, solvents, fragrances, fillers, sunscreens, odor- absorbers, and dyestuffs.
  • the topical compositions disclosed herein may be administered in conjunction with devices such as transdermal patches, dressings, pads, wraps, matrices, and bandages capable of being adhered to or otherwise associated with the skin or other tissue of a subject, being capable of delivering a therapeutically effective amount of one or more engineered lysin polypeptide construct or active fragment or variant thereof as disclosed herein.
  • the topical compositions of the present disclosure additionally comprise one or more components used to treat topical burns.
  • Such components may include, but are not limited to, a propylene glycol hydrogel; a combination of a glycol, a cellulose derivative, and a water soluble aluminum salt; an antiseptic; and a corticosteroid.
  • Humectants such as solid or liquid wax esters; absorption promoters such as hydrophilic clays or starches; viscosity building agents; and skin-protecting agents may also be added.
  • Topical formulations may be in the form of rinses such as mouthwash. See, e.g., WO 2004/004650.
  • compositions of the present disclosure may also be administered by injection of a therapeutic agent comprising the appropriate amount of an engineered lysin polypeptide construct or active fragment or variant thereof and a carrier.
  • a therapeutic agent comprising the appropriate amount of an engineered lysin polypeptide construct or active fragment or variant thereof and a carrier.
  • the engineered lysin polypeptide construct or active fragment or variant thereof can be administered intramuscularly, intrathecally, subdermally, subcutaneously, or intravenously to treat infections by Gram-negative bacteria, such as those caused by Burkholderia and/or Yersinia spp.
  • the carrier may be comprised of distilled water, a saline solution, albumin, a serum, or any combinations thereof.
  • compositions of parenteral injections can comprise pharmaceutically acceptable aqueous or nonaqueous solutions of engineered lysin polypeptide constructs as disclosed herein or active fragments or variants thereof in addition to one or more of the following: pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • adjuvants e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents
  • liposomal formulations nanoparticles, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • an isotonic formulation may be used.
  • additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose.
  • isotonic solutions such as phosphate buffered saline are preferred.
  • Stabilizers can include gelatin and albumin.
  • a vasoconstriction agent can be added to the formulation.
  • the pharmaceutical preparations according to this type of application may be provided sterile and pyrogen free.
  • the diluent may further comprise one or more other excipient such as ethanol, propylene glycol, an oil, or a pharmaceutically acceptable emulsifier or surfactant.
  • the compositions of the present disclosure are inhalable compositions.
  • the inhalable compositions of the present disclosure can further comprise a pharmaceutically acceptable carrier.
  • the engineered lysin polypeptide constructs of the present disclosure or active fragments or variants thereof may be formulated as a dry, inhalable powder.
  • an inhalation solution comprising engineered lysin polypeptide constructs or active fragments or variants thereof may further be formulated with a propellant for aerosol delivery.
  • solutions may be nebulized.
  • a surfactant can be added to an inhalable pharmaceutical composition of the present disclosure in order to lower the surface and interfacial tension between the medicaments and the propellant.
  • a surfactant may or may not be used.
  • a surfactant may or may not be used, depending, for example, on the solubility of the particular medicament and excipient.
  • the surfactant may be any suitable, non-toxic compound which is non-reactive with the medicament and which reduces the surface tension between the medicament, the excipient and the propellant and/or acts as a valve lubricant.
  • surfactants useful for the invention include, but are not limited to: oleic acid; sorbitan trioleate; cetyl pyridinium chloride; soya lecithin; polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene (10) stearyl ether; polyoxyethylene (2) oleyl ether; polyoxypropylene-polyoxyethylene ethylene diamine block copolymers; polyoxyethylene (20) sorbitan monostearate; polyoxyethylene(20) sorbitan monooleate; polyoxypropylene- polyoxyethylene block copolymers; castor oil ethoxylate; and combinations thereof.
  • propellants useful for the invention include, but are not limited to: dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, and carbon dioxide.
  • excipients for use in inhalable compositions include, but are not limited to: lactose, starch, propylene glycol diesters of medium chain fatty acids; triglyceride esters of medium chain fatty acids, short chains, or long chains, or any combination thereof; perfluorodimethylcyclobutane; perfluorocyclobutane; polyethylene glycol; menthol; lauroglycol; diethylene glycol monoethylether; polyglycolized glycerides of medium chain fatty acids; alcohols; eucalyptus oil; short chain fatty acids; and combinations thereof.
  • the compositions of the present disclosure comprise nasal applications.
  • Nasal applications include applications for direct use, such as nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, as well as applications for indirect use, such as throat lozenges and mouthwashes or gargles, or through the use of ointments applied to the nasal nares or the face, and any combination of these and similar methods of application.
  • the pharmaceutical compositions of the present disclosure comprise a complementary agent, including one or more antimicrobial agents.
  • the therapeutic agent containing an engineered lysin polypeptide construct of the present disclosure or active fragment or variant thereof may further include at least one complementary agent that can also potentiate the bactericidal activity of the construct.
  • the complementary agent is an antimicrobial agent used for the treatment of infections caused by Burkholderia spp.
  • the compositions of the present disclosure may be presented in unit dosage form and may be prepared by any methods well known in the art. The amount of active ingredients that can be combined with a carrier material to produce a single dosage form will vary depending, for example, upon the host being treated, the duration of exposure of the recipient to the infectious bacteria, the size and weight of the subject, and the particular mode of administration.
  • the amount of active ingredients that can be combined with a carrier material to produce a single dosage form may, for example, be that amount of each compound which produces a therapeutic effect. In certain embodiments, out of one hundred percent, the total amount may range from about 1 percent to about ninety-nine percent of active ingredients, such as from about 5 percent to about 70 percent, or from about 10 percent to about 30 percent.
  • Dosages administered may depend on a number of factors such as the activity of infection being treated; the age, health and general physical condition of the subject to be treated; the activity of a particular engineered lysin polypeptide construct or active fragment or variant thereof; in certain embodiments, effective amounts of the engineered lysin polypeptide construct or active fragment or variant thereof to be administered may fall within the range of about 1-50 mg/kg (or 1 to 50 mcg/ml).
  • effective amounts of the engineered lysin polypeptide construct or active fragment or variant thereof to be administered may fall within the range of about 1-50 ⁇ g/mL, such as within the range of about 1-10 ⁇ g/mL, about 1 ⁇ g/mL, or about 10 ⁇ g/mL.
  • the engineered lysin polypeptide construct or active fragment or variant thereof may be administered 1-4 times daily for a period ranging from 1 to 14 days. All such dosages and regimens, however, (whether of the engineered lysin polypeptide construct or active fragment or variant thereof) are subject to optimization.
  • Optimal dosages can be determined by performing in vitro and in vivo pilot efficacy experiments as is within the skill of the art but taking the present disclosure into account.
  • the engineered lysin polypeptide constructs disclosed herein or active fragments or variants thereof may provide a rapid bactericidal and, when used in sub-MIC amounts, may provide a bacteriostatic effect. It is further contemplated that the engineered lysin polypeptide constructs disclosed herein or active fragments or variants thereof may not be associated with evolving resistance. It is believed that existing resistance mechanisms for Gram-negative bacteria do not affect sensitivity to the lytic activity of the present engineered lysin polypeptide constructs or active fragments or variants thereof.
  • time exposure to the lysins and engineered lysin polypeptide constructs disclosed herein or active fragments or variants thereof may influence the desired concentration of active peptide units per ml.
  • Carriers that are classified as “long” or “slow” release carriers such as, for example, certain nasal sprays or lozenges
  • a “short” or “fast” release carrier such as, for example, a gargle
  • mcg high concentration peptide units
  • the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model can also be used to achieve a desirable concentration range and route of administration. Obtained information can then be used to determine the effective doses, as well as routes of administration, in humans. Dosage and administration can be further adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect.
  • a treatment regimen can entail administration daily (e.g., once, twice, thrice, etc. daily), every other day (e.g., once, twice, thrice, etc. every other day), semi-weekly, weekly, once every two weeks, once a month, etc.
  • treatment can be given as a continuous infusion. Unit doses can be administered on multiple occasions. Intervals can also be irregular as indicated by monitoring clinical symptoms.
  • the unit dose can be administered as a sustained release formulation, in which case less frequent administration may be used. Dosage and frequency may vary depending on the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for localized administration, e.g., intranasal, inhalation, rectal, etc., or for systemic administration, e.g., oral, rectal (e.g., via enema), intramuscular (i.m.), intraperitoneal (i.p.), intravenous (i.v.), subcutaneous (s.c.), transurethral, and the like.
  • the engineered lysins and active fragments or variants thereof of the present disclosure can be used in vivo, for example, to treat bacterial infections due to Gram-negative bacteria, such as P. aeruginosa, Burkholderia spp, and/or Yersinia pestis, in a subject, as well as in vitro, for example to reduce the level of bacterial contamination on, for example, a surface, e.g., of a medical device or ex vivo.
  • the engineered lysins and active fragments or variants thereof of the present disclosure can also be used prophylactically, for example, to prevent or ameliorate a bacterial infection due to Gram-negative bacteria, such as P.
  • the present disclosure is directed to a method of treating a bacterial infection caused by Gram-negative bacteria as described herein, comprising administering to a subject in need thereof an effective amount of an engineered lysin of the disclosure.
  • the subject is diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection.
  • the present disclosure is directed to a method of treating a bacterial infection caused by one or more Gram-negative bacteria, e.g., one or more Burkholderia spp.
  • Yersinia spp. comprising administering to a subject in need thereof, e.g., diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection, a lysin or an engineered lysin of the disclosure optionally in a pharmaceutical composition as herein described.
  • the present disclosure is directed to a method of treating a bacterial infection caused by a Gram-negative bacteria that is used or has the potential to be used as a biowarfare agent, as described herein, comprising administering to a subject in need thereof, e.g., diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection, an engineered lysin of the disclosure optionally in a pharmaceutical composition as described herein described.
  • infection and “bacterial infection” are meant to include respiratory tract infections (RTIs), such as respiratory tract infections in patients having cystic fibrosis (CF), including pulmonary exacerbations in CF patients and/or patients with decline in lung function and mortality; lower respiratory tract infections, such as acute exacerbation of chronic bronchitis (ACEB); acute sinusitis; community-acquired pneumonia (CAP); hospital-acquired pneumonia (HAP); nosocomial respiratory tract infections; non-cystic fibrosis lung diseases such as bronchiectasis and/or acute pneumonias; sexually transmitted diseases, such as gonococcal cervicitis and gonococcal urethritis; urinary tract infections; acute otitis media; sepsis including neonatal septicemia and catheter-related sepsis; osteomyelitis; tuberculosis;, and non- tuberculosis mycobacteria infections.
  • RTIs respiratory tract infections
  • CF cystic fibrosis
  • Non-limiting examples of infections caused by Gram-negative bacteria include: A) Nosocomial infections: 1. Respiratory tract infections, especially in cystic fibrosis patients and mechanically-ventilated patients, including pulmonary exacerbations of cystic fibrosis patients and bronchiectasis in non-cystic fibrosis patients; 2. Bacteremia and sepsis; 3. Wound infections, particularly those of burn victims; 4. Urinary tract infections; 5. Post-surgery infections on invasive devises; 6. Endocarditis by intravenous administration of contaminated drug solutions; 7.
  • the one or more species of Gram-negative bacteria of the present methods may include any of the species of Gram-negative bacteria as described herein.
  • the bacteria are Burkholderia spp., Yersinia spp., Acinetobacter spp., Klebsiella spp., or Pseudomonas spp.
  • the bacteria are B. cepacia, B. cenocepacia, B. thailandensis, B. multivorans, or B. humptydooensis.
  • the bacteria are Burkholderia spp., e.g., B. cepacia, B.
  • the Yersinia spp. is Yersinia pestis.
  • the Acinetobacter spp. is A. baumannii.
  • the Klebsiella spp. is K. pneumoniae.
  • Gram-negative bacteria include Serratia spp., such as Serratia marcescens. [00180]
  • infection with Gram-negative bacteria results in a localized infection, such as a topical bacterial infection, e.g., a skin wound.
  • the bacterial infection is a systemic pathogenic bacterial infection.
  • Common Gram-negative pathogens and associated infections are listed in Table A of the present disclosure. These are meant to serve as examples of the bacterial infections that may be treated, mitigated or prevented with the present engineered lysin polypeptide constructs and active fragments and variants thereof and are not intended to be limiting.
  • Table A Medically relevant Gram-negative bacteria and associated diseases Acinetobacter baumanii Wound infections Pseudomonas aeruginosa bloodstream infections and pneumonia Klebsiella pneumoniae UTIs, and bloodstream infections Serratia spp.
  • the engineered lysins and active fragments and variants thereof of the present disclosure are used to treat a subject at risk for acquiring an infection due to a Gram-negative bacterium.
  • Subjects at risk for acquiring a Gram-negative bacterial infection include, for example, cystic fibrosis patients, neutropenic patients, patients with necrotising enterocolitis, burn victims, patients with wound infections, and, more generally, patients in a hospital setting, in particular surgical patients and patients being treated using an implantable medical device such as a catheter, for example a central venous catheter, a Hickman device, or electrophysiologic cardiac devices, for example pacemakers and implantable defibrillators.
  • Other patient groups at risk for infection with Gram-negative bacteria include without limitation patients with implanted prostheses such a total joint replacement (for example total knee or hip replacement).
  • the engineered lysins or active fragments or variants thereof can be further combined with additional permeabilizing agents of the outer membrane of the Gram-negative bacteria, including, but not limited to metal chelators, such as e.g. EDTA, TRIS, lactic acid, lactoferrin, polymyxins, citric acid (Vaara M. Microbial Rev. 56(3):395-441 (1992), which is herein incorporated by reference in its entirety).
  • metal chelators such as e.g. EDTA, TRIS, lactic acid, lactoferrin, polymyxins, citric acid (Vaara M. Microbial Rev. 56(3):395-441 (1992), which is herein incorporated by reference in its entirety).
  • the present disclosure is directed to a method of inhibiting the growth, or reducing the population, or killing of at least one species of Gram-negative bacteria, the method comprising contacting the bacteria with the lysin or engineered lysin of the invention, or a composition containing an effective amount of the lysins or engineered lysin of the invention or active fragment or variants thereof as described herein, wherein the lysin or engineered lysin polypeptide construct or active fragment or variant thereof inhibits the growth, or reduces the population, or kills at least one species of Gram-negative bacteria.
  • inhibiting the growth, or reducing the population, or killing at least one species of Gram-negative bacteria comprises contacting bacteria with the lysin or engineered lysins or active fragments or variants as described herein, wherein the bacteria are present on a surface of e.g., medical devices, floors, stairs, walls and countertops in hospitals and other health related or public use buildings and surfaces of equipment in operating rooms, emergency rooms, hospital rooms, clinics, and bathrooms and the like.
  • Examples of medical devices that can be protected using the engineered lysin polypeptide constructs or active fragments or variants thereof described herein include but are not limited to tubing and other surface medical devices, such as urinary catheters, mucous extraction catheters, suction catheters, umbilical cannulae, contact lenses, intrauterine devices, intravaginal and intraintestinal devices, endotracheal tubes, bronchoscopes, dental prostheses and orthodontic devices, surgical instruments, dental instruments, tubings, dental water lines, fabrics, paper, indicator strips (e.g., paper indicator strips or plastic indicator strips), adhesives (e.g., hydrogel adhesives, hot-melt adhesives, or solvent-based adhesives), bandages, tissue dressings or healing devices and occlusive patches, and any other surface devices used in the medical field.
  • tubing and other surface medical devices such as urinary catheters, mucous extraction catheters, suction catheters, umbilical cannulae, contact lenses, intrauterine devices, intravaginal and intraintestinal
  • the devices may include electrodes, external prostheses, fixation tapes, compression bandages, and monitors of various types.
  • Medical devices can also include any device which can be placed at the insertion or implantation site such as the skin near the insertion or implantation site, and which can include at least one surface which is susceptible to colonization by Gram-negative.
  • Example 1 Identification of Burkholderia lysins – Screening Native and Engineered Derivatives
  • An initial test panel included isolates of B. cenocepacia, B. cepacia, B. multivorans, and B. thailandensis. The majority of compounds tested were not active (MIC >256 ⁇ g/mL).
  • a total of 220 engineered and native lysins were identified and tested against the following four Burkholderia species: B. cenocepacia, B. multivorans, B. thailandensis, and B. cepacia, as shown below in Table 6.
  • Table 6 Number of native and engineered GN lysins tested against Burkholderia ssp. B. cenocepacia B. multivorans B. thailandensis B. cepacia (NR- (AU24619) (NR-707) (ATCC BAA- 9909) 247) Engineered GN 167 38 58 100 lysins Native peptidases 21 2 21 3 Native lysozymes 13 5 15 5 Native 14 5 15 5 muridamidases Others (including 5 0 6 2 CHAP and amidases) Total: 220 50 115 115 [00190] The native lysins had MICs >256 ⁇ g/mL.
  • GN568 Yersinia phase PY100
  • GN568 15.72 kDa
  • peptidase_M15_4 a 138 amino acid cell wall peptidase from Yersinia phage.
  • GN568 was re-engineered by removing an alternate short start codon to create GN685, which allows for high-level production with >95% purity.
  • GN685 was fused with a series of 8 peptides, including, for example, RI-18, having the amino acid sequence RKKTRKRLKKIGKVLKWI (SEQ ID NO: 25).
  • GN685 was also fused to various linker variants, including FIRL BCC (SEQ ID NO: 32), PRR peptide, and the last four amino acids from RI-18 (to stabilize helix).
  • the activity of GN568 and the engineered constructs was measured against B. cenocepacia, B. thailandensis, and B.
  • cenocepacia GN577+SKY+RI18 SEQ ID NO: 72
  • GN686 Yersinia phage PY100 GN685+RI18
  • SEQ ID NO: 73 GN693 Yersinia phage PY100 GN685+BK5
  • SEQ ID NO: 55 GN694 Yersinia phage PY100 GN685+BCM1
  • aeruginosavorus ATRA-1A+GN37+A+ATRA-1A (SEQ ID NO: 74) GN758 Escherichia phage FIRL BC (SEQ ID NO: 143)+G+BK9+GN428 (SEQ ID NO: 75) GN764 Burkholderia ambifaria GN569(K87H)+RI-18 (SEQ ID NO: 76) GN765 Yersinia phage PY100 GN685(K78H)+RI-18 (SEQ ID NO: 77) GN873 Yersinia enterocolitica FMRL YP (SEQ ID NO: 144)+LSKLGG (SEQ (SEQ ID NO: 78) ID NO: 45)+PRR 5 +GN867 GN916 Yersinia enterocolitica ZF5.3+ LSKLGG (SEQ ID NO: (SEQ ID NO: 79) 45)+PRR+GN867 [00196]
  • B. thailandensis and B. humptydooensis may be used as surrogates for laboratory testing in lieu of the biohazardous B. pseudomallei.
  • PRR 3 RKPRQSPRPQQVRPPRPQVEENQPRPVPV
  • PRR 5 is a PRR homolog from DAS01177.1 TPA: hypothetical protein [Siphoviridae sp.] and indicates (PRR 5 ) PRRPRRGGRRRPHRGRL (SEQ ID NO: 40).
  • Activity of various lysins was evaluated for B. cepacian, B. cenocepacia, B. thailandensis, B. multivorans, and B. humptydooensis, and the results are shown in Table 11 below.
  • Example 3 Direct Lytic Agents’ activity against B. cenocepacia complex
  • Various engineered Gram-negative lysins were evaluated for activity against B. cenocepacia and B. multivorans, and the results are shown in Table 12 below. All of B. cenocepacia, B. cepacia, and B. multivorans, as well as B. contaminans, B. ambifaria, and B. ubonensis are phylogenetically similar as part of the B. cenocepacia complex.
  • Example 4 Direct Lytic Agents’ activity against ESKAPE pathogens, Stenotrophomonas melophilia, Escherichia coli and Serratia marcescens
  • Various engineered Gram-negative lysins were evaluated for activity against P. aeruginosa, A. baumannii, K. pneumoniae, and E. cloacae and the results are shown in Tables 13-15 below.
  • the Gram-negative lysins were evaluated for activity against Serratia marcescens, and the results shown in Table 16 below, and various Gram-negative lysins were evaluated for activity against S. maltophilia and E. coli, and the results shown in Table 17 below.
  • hRBCs human red blood cells
  • humptydooensis demonstrated significant bactericidal activity (reductions of ⁇ 3-log 10 CFU/mL) ⁇ human serum within 24 hours. Bactericidal activity was consistent with rapid outer membrane (OM) permeabilization observed in the NPN assay. No hemolysis of hRBCs was observed with all lead compounds (highest concentration tested was 128 ⁇ g/mL).
  • Table 18 below shows the MICs ( ⁇ g/mL) against Burkholderia cepacia complex organisms, and Table 19 below shows the MICs ( ⁇ g/mL) against Burkholderia pseudomallei complex organisms.
  • MICs typical for native (unmodified) lysins are shown for GN685 (an non-engineered progenitor of GN693). MICs are also indicated for GN370, as disclosed, for example, in in PCT Publication No. WO 2020/046747, incorporated by reference herein. [00212] Table 18 – MICs ( ⁇ g/mL) against Burkholderia cepacia complex organisms B. cepacia B. cenocepacia B.
  • thailandeniss B. humptydooensis Lysin NR-704 NR-9908 NR-9909 ATCC ATCC BAA-2767 700388 GN693 (SEQ ID NO: 55) 1 2 2 1 0.5 GN694 (SEQ ID NO: 56) 1 2 2 1 1 GN751 (SEQ ID NO: 57) 1 1 0.5 1 0.5 GN757 (SEQ ID NO: 58) 2 1 1 2 1 GN1040 (SEQ ID NO: 59) 2 4 2 2 2 1 GN1041 (SEQ ID NO: 60) 2 4 2 1 1 GN1072 (SEQ ID NO: 61) 1 1 1 1 1 0.5 GN1074 (SEQ ID NO: 62) 2 1 ⁇ 0.5 1 1 GN1119 (SEQ ID NO: 63) 1 1 1 1 1 0.5 GN1125 (SEQ ID NO: 64) 0.5 0.5 0.5 1 0.25 GN685 (SEQ ID NO: 2) >256 >
  • MICs were also determined in the absence and presence of human serum (“-” indicates no serum, and “+” indicates human serum) for various Y. pestis strains, as Table 21 below.
  • serum does not inhibit the engineered lysin polypeptide construct activity.
  • a similar effect has been observed for GN370 and exebacase (Schuch et al., 2022, PMID: 35308352; Indiani et al., 2019, PMID: 30670427). The results are shown below in Table 20.
  • Y. pestis was examined using the NPN uptake assay. If the OM is damaged or functionally compromised by pore formation, NPN partitions into the OM, exhibiting strong fluorescence. Rapidly induced fluorescence was observed for each construct examined, consistent with pore formation. Examples for GN751 (SEQ ID NO: 57) and GN808 (SEQ ID NO: 66) are shown for B. thailandensis and Y. pestis in Figure 5 and Figure 6, respectively. [00219] Engineered lysins were tested for hemolytic activity. No significant hemolytic activity was observed for any of the engineered lysins polypeptide constructs tested, with a highest concentration tested of 128 ⁇ g/mL.
  • GN751 SEQ ID NO: 57
  • RR12Wpolar a positive control peptide
  • Table 22 shows the percent hemolysis observed for various engineered lysin constructs tested.

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Abstract

La présente divulgation concerne une lysine modifiée comprenant : (a) une lysine de base à Gram négatif ; et (b) un ou plusieurs peptides antimicrobiens (AMP) et/ou un peptide de pénétration cellulaire (CPP), et éventuellement un motif de liaison à la membrane externe et au moins un lieur, ou un fragment actif de celle-ci, ou un variant de celle-ci ayant au moins 80 % d'identité de séquence. L'invention concerne également des méthodes de traitement d'une infection bactérienne provoquée par au moins une bactérie à Gram négatif et des méthodes d'élimination d'au moins une bactérie à Gram négatif, comprenant l'administration de la lysine modifiée décrite ici.
PCT/EP2024/066700 2023-06-15 2024-06-14 Constructions polypeptidiques de lysine modifiées actives contre des bactéries à gram négatif Ceased WO2024256710A2 (fr)

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