Lysin-antimicrobial peptide (amp) polypeptide constructs, lysins, isolated polynucleotides encoding same and uses thereof

Abstract

The present disclosure is directed to lysin-AMP polypeptide constructs, isolated lysin polypeptides, and pharmaceutical compositions comprising the isolated polypeptides and/or lysin-AMP polypeptide constructs. Methods of using the lysin-AMP polypeptide constructs, isolated lysin polypeptides and pharmaceutical compositions are also herein provided. In addition, isolated polynucleotides encoding the lysin-AMP polypeptide constructs and isolated lysin polypeptides are disclosed herein.

1 .- 7 . (canceled) 
     
     
         8 . An isolated polypeptide comprising a lysin selected from the group consisting of GN217 lysin (SEQ ID NO: 8), GN394 lysin (SEQ ID NO: 48), GN396 lysin (SEQ ID NO: 50), GN408 lysin (SEQ ID NO: 52), GN418 lysin (SEQ ID NO: 54), and GN486 (SEQ ID NO: 66) or an active fragment thereof or a polypeptide having lytic activity and at least 80% sequence identity with the polypeptide sequence of at least one of SEQ ID NOS: 8, 48, 50, 52, 54, or 66. 
     
     
         9 .- 14 . (canceled) 
     
     
         15 . An isolated polynucleotide sequence comprising a nucleic acid molecule encoding a lysin selected from the group consisting of GN217 lysin (SEQ ID NO: 8), GN394 lysin (SEQ ID NO: 48), GN396 lysin (SEQ ID NO: 50), GN408 lysin (SEQ ID NO: 52), GN418 lysin (SEQ ID NO: 54), and GN486 (SEQ ID NO: 66) or an active fragment thereof or a polypeptide having lytic activity and at least 80% sequence identity with the polypeptide sequence of at least one of SEQ ID NOS: 8, 48, 50, 52, 54, or 66. 
     
     
         16 . The isolated polynucleotide of  claim 15 , wherein the isolated polynucleotide comprises DNA. 
     
     
         17 . The isolated polynucleotide of  claim 15 , wherein the isolated polynucleotide comprises cDNA. 
     
     
         18 . A recombinant vector comprising the isolated polynucleotide sequence according to  claim 15 . 
     
     
         19 . The recombinant vector of  claim 18 , wherein the isolated polynucleotide sequence is operatively linked to a heterologous promoter. 
     
     
         20 . The recombinant vector of  claim 18 , wherein the recombinant vector is a recombinant expression vector. 
     
     
         21 . An isolated host cell comprising the recombinant vector of  claim 18 . 
     
     
         22 . A pharmaceutical composition comprising an isolated lysin and a pharmaceutically acceptable carrier,
 wherein the isolated lysin comprises at least one of:
 (i) GN217 (SEQ ID NO: 8), GN316 variant (SEQ ID NO: 24), GN316 (SEQ ID NO: 22), GN329 (SEQ ID NO: 26), GN333 (SEQ ID NO: 28), GN394 (SEQ ID NO: 48), GN396 (SEQ ID NO: 50), GN408 (SEQ ID NO: 52), GN418 (SEQ ID NO: 54), GN424 (SEQ ID NO: 56), GN425 (SEQ ID NO:58), GN428 (SEQ ID NO: 60), GN431 (SEQ ID NO: 64), GN486 (SEQ ID NO: 66), GN485 (SEQ ID NO: 68), Lysin PaP2_gp17 (SEQ ID NO: 96), 
 (ii) an active fragment thereof, or 
 (iii) a polypeptide having lytic activity and at least 80% sequence identity with the polypeptide sequence of at least one of SEQ ID NOS: 8, 24, 22, 26, 28, 48, 50, 52, 54, 56, 58, 60, 64, 66, 68, or 96. 
   
     
     
         23 - 25 . (canceled) 
     
     
         26 . The pharmaceutical composition of  claim 22 , wherein the isolated lysin is selected from the group consisting of GN316 (SEQ ID NO: 22), GN329 (SEQ ID NO: 26), GN333 (SEQ ID NO: 28), GN424 (SEQ ID NO: 56), GN425 (SEQ ID NO:58), GN428 (SEQ ID NO: 60), GN431 (SEQ ID NO: 64), GN485 (SEQ ID NO: 68), Lysin PaP2_gp117 (SEQ ID NO: 96), an active fragment thereof, or a polypeptide having lytic activity and at least 80% sequence identity with the polypeptide sequence of at least one of SEQ ID NOS: 22, 26, 28, 56, 58, 60, 64, 68, or 96. 
     
     
         27 . The pharmaceutical composition of  claim 26 , wherein the lysin or active fragment thereof or polypeptide comprises at least one amino acid substitution relative to SEQ ID NOS: 22, 26, 28, 56, 58, 60, 64, 68 or 96. 
     
     
         28 . (canceled) 
     
     
         29 . The pharmaceutical composition of  claim 22 , wherein the pharmaceutical composition is formulated as a solution, a suspension, an emulsion, an inhalable powder, an aerosol, or a spray. 
     
     
         30 . The pharmaceutical composition of  claim 22 , wherein the pharmaceutical composition further comprises an antibiotic suitable for the treatment of Gram-negative bacteria. 
     
     
         31 . A method of treating a bacterial infection caused by a Gram-negative bacteria, which method comprises:
 administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection, a pharmaceutical composition according to  claim 22 .   
     
     
         32 . The method of  claim 31 , wherein the bacterial infection is a topical or systemic pathogenic bacterial infection. 
     
     
         33 . A method of preventing or treating a bacterial infection comprising:
 co-administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection, a combination of a first effective amount of a pharmaceutical composition according to  claim 22 , and   a second effective amount of an antibiotic suitable for the treatment of a Gram-negative bacterial infection.   
     
     
         34 . A method for augmenting the efficacy of an antibiotic suitable for the treatment of a Gram-negative bacterial infection, comprising:
 co-administering the antibiotic in combination with a composition containing an effective amount of an isolated lysin,   wherein the isolated lysin comprises at least one of:
 (i) GN217 (SEQ ID NO: 8), GN316 variant (SEQ ID NO: 24), GN316 (SEQ ID NO: 22), GN329 (SEQ ID NO: 26), GN333 (SEQ ID NO: 28), GN394 (SEQ ID NO: 48), GN396 (SEQ ID NO: 50), GN408 (SEQ ID NO: 52), GN418 (SEQ ID NO: 54), GN424 (SEQ ID NO: 56), GN425 (SEQ ID NO:58), GN428 (SEQ ID NO: 60), GN431 (SEQ ID NO: 64), GN486 (SEQ ID NO: 66), GN485 (SEQ ID NO: 68), Lysin PaP2_gp17 (SEQ ID NO: 96), or 
 (ii) an active fragment thereof, or 
 (iii) a polypeptide having lytic activity and at least 80% sequence identity with the polypeptide sequence of at least one of SEQ ID NOS: 8, 24, 22, 26, 28, 48, 50, 52, 54, 56, 58, 60, 64, 66, 68, or 96 
   wherein administration of the combination is more effective in inhibiting the growth, or reducing the population, or killing the Gram-negative bacteria in the presence or absence or both in the presence and absence of human serum than administration of either the antibiotic or the lysin individually.   
     
     
         35 . A method of inhibiting the growth, or reducing the population, or killing of at least one species of Gram-negative bacteria which method comprises:
 contacting the bacteria with an effective amount an isolated lysin,   wherein the isolated lysin comprises at least one of:
 (i) GN217 (SEQ ID NO: 8), GN316 variant (SEQ ID NO: 24), GN316 (SEQ ID NO: 22), GN329 (SEQ ID NO: 26), GN333 (SEQ ID NO: 28), GN394 (SEQ ID NO: 48), GN396 (SEQ ID NO: 50), GN408 (SEQ ID NO: 52), GN418 (SEQ ID NO: 54), GN424 (SEQ ID NO: 56), GN425 (SEQ ID NO:58), GN428 (SEQ ID NO: 60), GN431 (SEQ ID NO: 64), GN486 (SEQ ID NO: 66), GN485 (SEQ ID NO: 68), Lysin PaP2_gp17 (SEQ ID NO: 96), or 
 (ii) an active fragment thereof, or 
 (iii) a polypeptide having lytic activity and at least 80% sequence identity with the polypeptide sequence of at least one of SEQ ID NOS: 8, 24, 22, 26, 28, 48, 50, 52, 54, 56, 58, 60, 64, 66, 68, or 96. 
   
     
     
         36 . The method of  claim 31 , wherein the Gram-negative bacteria is selected from the group consisting of  Pseudomonas aeruginosa, Klebsiella  spp.,  Enterobacter  spp.,  Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Yersinia pestis , and  Franciscella tulerensis.    
     
     
         37 . The method of  claim 33 , wherein the antibiotic is selected from one or more of ceftazidime, cefepime, cefoperazone, ceftobiprole, ciprofloxacin, levofloxacin, aminoglycosides, imipenem, meropenem, doripenem, gentamicin, tobramycin, amikacin, piperacillin, ticarcillin, penicillin, rifampicin, polymyxin B, and colistin. 
     
     
         38 . The the pharmaceutical composition  claim 22 , wherein the isolated lysin comprises at least one activity chosen from inhibiting the growth of, reducing a population of, and/or killing in the absence and/or presence of human serum at least one species of Gram-negative bacteria. 
     
     
         39 . The the pharmaceutical composition  claim 38 , wherein the at least one species of Gram-negative bacteria is selected from the group consisting of  Pseudomonas aeruginosa, Klebsiella  spp.,  Enterobacter  spp.,  Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Yersinia pestis , and  Franciscella tulerensis.    
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . The pharmaceutical composition according to  claim 22 , wherein the isolated lysin is selected from the group consisting of GN217 (SEQ ID NO: 8), GN394 (SEQ ID NO: 48), GN396 (SEQ ID NO: 50), GN408 (SEQ ID NO: 52), GN418 (SEQ ID NO: 54), an active fragment thereof, or a polypeptide having lytic activity and at least 80% sequence identity with the polypeptide sequence of at least one of SEQ ID NOs: 8, 48, 50, 52, and 54.