Chimeric receptor binding proteins resistant to proteolytic degradation

Abstract

The present disclosure provides a chimeric receptor binding protein (RBP) resistant to proteolytic digestion wherein said RBP comprises a portion of a receptor binding protein derived from a bacteriophage fused through a designed linker region consisting of 1 to 70 amino acids, to a portion of a receptor binding protein derived from a different bacteriophage, wherein said linker region is designed to be resistant to proteolytic digestion.

1 . A lambdoid bacterial delivery vehicle comprising a chimeric receptor binding protein (RBP) resistant to proteolytic digestion, 
 wherein said chimeric RBP comprises an N-terminal region of a side tail fiber (STF) protein from a lambdoid bacteriophage, fused through a designed linker region consisting of 1 to 70 amino acids or 1 to 30 amino acids, to a C-terminal region of a RBP protein from a different bacteriophage, wherein said N-terminal region and C-terminal region are fused within an insertion site of the N-terminal STF region, said insertion site having at least 80% identity with a site selected from the group consisting of amino acids SAGDAS (SEQ ID NO: 1), ADAKKS (SEQ ID NO: 2), MDETNR (SEQ ID NO: 3), SASAAA (SEQ ID NO: 4) and GAGENS (SEQ ID NO: 5); and   wherein said designed linker region comprises a helix or helical bundle and wherein said linker region is resistant to proteolytic cleavage.   
     
     
         2 . The bacterial delivery vehicle according to  claim 1 , wherein the designed linker region consists of 1 to 30 amino acids. 
     
     
         3 . The bacterial delivery vehicle according to  claim 1 , wherein said chimeric RBP is resistant to proteolytic digestion by pancreatin, and said linker region is designed to be resistant to proteolytic digestion by pancreatin. 
     
     
         4 . The bacterial delivery vehicle according to  claim 1 , wherein said insertion site has at least 80% identity with sequence GAGENS (SEQ ID NO: 5). 
     
     
         5 . The bacterial delivery vehicle according to  claim 1 , wherein said designed linker region is at the C-terminal end of the insertion site. 
     
     
         6 . The bacterial delivery vehicle according to  claim 1 , wherein said designed linker region is part of the N-terminal region of the C-terminal region of the chimeric RBP. 
     
     
         7 . The bacterial delivery vehicle according to  claim 6 , wherein said N-terminal region or said C-terminal region comprises the sequence of the linker region, said sequence being identical to the corresponding sequence in the N-terminal region or C-terminal region of the RBP from which it is derived, and said sequence restoring resistance to proteolytic digestion to said chimeric RBP compared to a chimeric RBP only differing by the absence of said linker region. 
     
     
         8 . The bacterial delivery vehicle according to  claim 1 , wherein said designed linker region comprises, or consists of, an heterologous amino acid sequence which is not from one of the RBP from which the N-terminal region and the C-terminal region of the chimeric RBP are derived. 
     
     
         9 . The bacterial delivery vehicle according to  claim 1 , wherein said designed linker region comprises, or consists of, an amino acid sequence GSATDVMIQL (SEQ ID NO: 6) or GSATDVMIQLA (SEQ ID NO: 7). 
     
     
         10 . The bacterial delivery vehicle according to  claim 1 , wherein said designed linker region comprises, or consists of, the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 36. 
     
     
         11 . The bacterial delivery vehicle according to  claim 1 , wherein the N-terminal region of said STF protein from said lambdoid bacteriophage corresponds to amino acids 1 to 528 of the lambda STF protein of SEQ ID NO: 8. 
     
     
         12 . The bacterial delivery vehicle according to  claim 1 , wherein the C-terminal region of said STF protein from said different bacteriophage corresponds to amino acids 208 to 875 of the STF protein of SEQ ID NO: 16 or to amino acids 218 to 875 of the STF protein of SEQ ID NO: 16. 
     
     
         13 . The bacterial delivery vehicle according to  claim 12 , wherein said chimeric RBP comprises, or consists of, the sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. 
     
     
         14 . The bacterial delivery vehicle according to  claim 1 , wherein the C-terminal region of said STF protein from said different bacteriophage corresponds to amino acids 28 to 632 of the STF protein of SEQ ID NO: 12 or amino acids 62 to 632 of the STF protein of SEQ ID NO: 12. 
     
     
         15 . The bacterial delivery vehicle according to  claim 14 , wherein said chimeric RBP comprises, or consists of, the sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 38 or SEQ ID NO: 40. 
     
     
         16 . The bacterial delivery vehicle according to  claim 1 , said bacterial delivery vehicle comprising a chimeric RBP comprising or consisting of the sequence of SEQ ID NO: 11 and a chimeric gpJ variant comprising or consisting of the sequence of SEQ ID NO: 27. 
     
     
         17 . The bacterial delivery vehicle according to  claim 1 , wherein said bacterial delivery vehicle comprises a DNA payload of interest. 
     
     
         18 . The bacterial delivery vehicle according to  claim 17 , wherein said DNA payload comprises:
 a nucleic acid of interest selected from the group consisting of a Cas nuclease gene, a Cas 9 nuclease gene, a guide RNA, a CRISPR locus, a toxin gene, a gene expressing an enzyme, a gene encoding a bacterial receptor, a gene encoding a membrane protein, a gene encoding a structural protein, a gene encoding a secreted protein, a gene expressing resistance to an antibiotic, a gene expressing resistance to a drug, a gene expressing a toxic protein, a gene encoding a toxic factor, a gene expressing a virulence protein, a gene expressing a virulence factor, and any of their combinations, or   a nucleic acid of interest encoding a therapeutic protein or an antisense nucleic acid molecule.   
     
     
         19 . The bacterial delivery vehicle according to  claim 18 , wherein said enzyme is selected from the group consisting of a nuclease, a kinase, a TALEN, a ZFN, a meganuclease and a recombinase. 
     
     
         20 . The bacterial delivery vehicle according to  claim 17 , wherein said payload comprises or consists of the nucleic acid sequence of SEQ ID NO: 33 or of the nucleic acid sequence of SEQ ID NO: 42. 
     
     
         21 . The bacterial delivery vehicle according to  claim 1 , for use in in vivo delivery of said DNA payload of interest into a targeted bacterial cell. 
     
     
         22 . A pharmaceutical or veterinary composition comprising:
 a lambdoid bacterial delivery vehicle comprising a chimeric receptor binding protein (RBP) resistant to proteolytic digestion, 
 wherein said chimeric RBP comprises an N-terminal region of a side tail fiber (STF) protein from a lambdoid bacteriophage, fused through a designed linker region consisting of 1 to 70 amino acids or 1 to 30 amino acids, to a C-terminal region of a RBP protein from a different bacteriophage, wherein said N-terminal region and C-terminal region are fused within an insertion site of the N-terminal STF region, said insertion site having at least 80% identity with a site selected from the group consisting of amino acids SAGDAS (SEQ ID NO: 1), ADAKKS (SEQ ID NO: 2), MDETNR (SEQ ID NO: 3), SASAAA (SEQ ID NO: 4) and GAGENS (SEQ ID NO: 5); and 
 wherein said designed linker region comprises a helix or helical bundle and 
   wherein said linker region is resistant to proteolytic cleavage; and   a pharmaceutically acceptable carrier.   
     
     
         23 . The pharmaceutical or veterinary composition according to  claim 22 , wherein said lambdoid bacterial delivery vehicle is for use in in vivo delivery of a DNA payload of interest into a targeted bacterial cell. 
     
     
         24 . A method for in vivo delivery of a DNA payload of interest into a subject comprising administering to said subject a pharmaceutical or veterinary composition comprising:
 a lambdoid bacterial delivery vehicle for use in in vivo delivery of a DNA payload of interest into a targeted bacterial cell, wherein said lambdoid delivery vehicle comprises a chimeric receptor binding protein (RBP) resistant to proteolytic digestion, 
 wherein said chimeric RBP comprises an N-terminal region of a side tail fiber (STF) protein from a lambdoid bacteriophage, fused through a designed linker region consisting of 1 to 70 amino acids or 1 to 30 amino acids, to a C-terminal region of a RBP protein from a different bacteriophage, wherein said N-terminal region and C-terminal region are fused within an insertion site of the N-terminal STF region, said insertion site having at least 80% identity with a site selected from the group consisting of amino acids SAGDAS (SEQ ID NO: 1), ADAKKS (SEQ ID NO: 2), MDETNR (SEQ ID NO: 3), SASAAA (SEQ ID NO: 4) and GAGENS (SEQ ID NO: 5); and 
 wherein said designed linker region comprises a helix or helical bundle and 
   wherein said linker region is resistant to proteolytic cleavage; and   a pharmaceutically acceptable carrier.   
     
     
         25 . The method according to  claim 24 , for treating and/or preventing a disease or disorder caused or mediated by bacteria, wherein a therapeutically effective amount of said pharmaceutical or veterinary composition is administered to a subject having a disease or disorder in need of treatment.