Branched receptor binding multi-subunit protein complexes for use in bacterial delivery vehicles

Patent metadata

Field	Value
Publication number	US-11208437-B2
Application number	US-201916726033-A
Country	US
Kind code	B2
Filing date	Dec 23, 2019
Priority date	Dec 21, 2018
Publication date	Dec 28, 2021
Grant date	Dec 28, 2021

Abstract

The present disclosure relates generally to bacterial delivery vehicles for use in efficient transfer of a desired payload into a target bacterial cell. More specifically, the present disclosure relates to bacterial delivery vehicles with desired host ranges based on the presence of a chimeric receptor binding protein (RBP) composed of a fusion between the N-terminal region of a RBP derived from a lambda-like bacteriophage and the C-terminal region of a different RBP, and/or the presence of an engineered branched receptor binding multi-subunit polypeptides (“branched-RBP”).

First claim

What is claimed is:

1. An engineered branched receptor binding multi-subunit protein complex (branched-RBP) comprising two or more associated bacteriophage derived receptor binding proteins (RBP):
wherein the two or more associated bacteriophage derived RBPs comprise an interaction domain (ID) that mediates the association of the RBP's with one another to form the branched-RBP, and
wherein at least one of the two or more associated bacteriophage derived RBPs is a chimeric RBP comprising a fusion between the N-terminal domain of a RBP from a lambda or lambda-like bacteriophage and the C-terminal domain of a different RBP and wherein the ID is inserted between the N-terminal domain and the C-terminal domain of the chimeric RBP.

2. The engineered branched-RBP of claim 1 , wherein the association is a non-covalent association.

3. The engineered branched-RBP of claim 1 , wherein at least one of the two or more receptor binding protein (RBP) comprises a chimeric RBP, wherein said chimeric RBP is selected from the group consisting of:
(i) a fusion between the N-terminal domain of a RBP from a lambda or lambda-like bacteriophage and the C-terminal domain of a different RBP, wherein said different RBP is derived from any bacteriophage or bacteriocin,
wherein said N-terminal domain is fused to said C-terminal domain within one of the amino acid regions selected from positions 1-150, 320-460 or 495-560 of the N-terminal RBP with reference to the lambda stf sequence (SEQ ID NO:1) and wherein said chimeric RBP contains an interaction domain inserted between the N-terminal and C-terminal domain: and
(ii) a fusion between the N-terminal domain of a RBP from a lambda or lambda-like bacteriophage and the C-terminal domain of a different RBP, wherein said different RBP is derived from any bacteriophage or bacteriocin,
wherein said RBP from a lambda or lambda-like bacteriophage and the other RBP have homology in one or more of three amino acids regions ranging from positions 1-150, 320-460 and 495-560 of the N-terminal RBP with reference to the lambda stf sequence (SEQ ID NO:1) and
wherein said N-terminal domain is fused to said C-terminal domain within one of the amino acid regions selected from positions 1-150, 320-460 or 495-560 of the N-terminal RBP with reference to the lambda stf sequence (SEQ ID NO:1) and
wherein said chimeric RBP contains an interaction domain inserted between the N-terminal and C-terminal domain.

4. The engineered branched-RBP of claim 3 , wherein the chimeric RBP is selected from the group consisting of;
(i) a chimeric RBP, wherein the N-terminal domain of the chimeric RBP is fused to said C-terminal domain within one of the amino acids regions selected from positions 80-150, 320-460, or 495-560 of the N-terminal RBP with reference to the lambda bacteriophage stf sequence (SEQ ID NO:1) and wherein said chimeric RBP contains an interaction domain, inserted between the N-terminal and C-terminal domain; and
(ii) a chimeric RBP, wherein the N-terminal domain and the C-terminal domain are fused within said region at an insertion site having at least 80% identity with an insertion site selected from the group consisting of amino acids SAGDAS (SEQ ID NO:190), ADAKKS (SEQ ID NO:191), MDETNR (SEQ ID NO:192), SASAAA (SEQ ID NO:193), GAGENS (SEQ ID NO:194), ATLKQI (SEQ ID NO:195), IIQLED (SEQ ID NO:196), GNIIDL (SEQ ID NO:197), IATRV (SEQ ID NO:198), TPGEL (SEQ ID NO:199), GAIIN (SEQ ID NO:200), NQIID (SEQ ID NO:201), GQIVN (SEQ ID NO:202), and VDRAV (SEQ ID NO:203) wherein said chimeric RBP contains an interaction domain inserted between the N-terminal and C-terminal domain.

5. The engineered branched-RBP of claim 3 , wherein the C-terminal domain of the different RBP has a depolymerase activity against an encapsulated bacterial strain.

6. The engineered branched-RBP of claim 1 , wherein one or more of the IDs is selected from the group consisting of SEQ ID NOS.: 131-134 and 280-281.

7. The engineered branched-RBP complex of claim 1 , wherein the at least one or more receptor binding protein (RBP) comprises a chimeric RBP selected from the group consisting of:
(i) a chimeric RBP comprising a fusion between the N-terminal domain of a RBP from a lambda bacteriophage and the C-terminal domain of a different RBP, wherein said different RBP is derived from any bacteriophage or bacteriocin; and
wherein said N-terminal domain is fused to said C-terminal domain within one of the amino acids regions selected from positions 1-150, 320-460 or 495-560 of the N-terminal RBP with reference to the lambda stf sequence (SEQ ID NO:1) and wherein said chimeric RBP contains an interaction domain inserted between the N-terminal and C-terminal domain; and
(ii) a chimeric RBP comprising a fusion between the N-terminal domain of a RBP from a lambda bacteriophage and the C-terminal domain of a different RBP, wherein said different RBP is derived from any bacteriophage or bacteriocin,
wherein said RBP from a lambda bacteriophage and the other RBP have homology in one or more of three amino acids regions ranging from positions 1-150, 320-460, and 495-560 of the N-terminal RBP with reference to the lambda stf sequence (SEQ ID NO:1); and
wherein said N-terminal domain is fused to said C-terminal domain within one of the amino acid regions selected from positions 1-150, 320-460, or 495-560 of the N-terminal RBP with reference to the lambda stf sequence (SEQ ID NO:1); and wherein said chimeric RBP contains an interaction domain inserted between the N-terminal and C-terminal domain.

8. The engineered branched-RBP of claim 7 , wherein said N-terminal domain of the chimeric RBP is fused to said C-terminal domain within one of the amino acids regions selected from positions 80-150, 320-460, or 495-560 of the N-terminal RBP with reference to the lambda stf sequence (SEQ ID NO:1) and wherein said chimeric RBP contains an interaction domain inserted between the N-terminal and C-terminal domain.

9. The engineered branched-RBP of claim 7 , wherein the N-terminal domain and the C-terminal domain are fused within said region at an insertion site having at least 80% identity with insertion site selected from the group consisting of amino acids SAGDAS (SEQ ID NO:190), ADAKKS (SEQ ID NO:190), MDETNR (SEQ ID NO:191), SASAAA (SEQ ID NO:192), GAGENS (SEQ ID NO:193), ATLKQI (SEQ ID NO:195), IIQLED (SEQ ID NO:196), GNIIDL (SEQ ID NO:197), IATRV (SEQ ID NO:198), TPGEL (SEQ ID NO:199), GAIIN (SEQ ID NO:200), NQIID (SEQ ID NO:201), GQIVN (SEQ ID NO:202), and VDRAV (SEQ ID NO:203) wherein said chimeric RBP contains an interaction domain inserted between the N-terminal and C-terminal domain.

10. The engineered branched-RBP of claim 1 , wherein at least one of the two or more associated receptor binding proteins (RBP) comprises a chimeric RBP comprising an amino acid sequence selected from the group consisting SEQ ID NO: 2, 4, 7, 9, 12, 15, 17, 20, 23, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 56, 59, 135 to 144, 147, 150, 151, 154, 157, 160, 163, 215, 216, 219, 221, 223, 225, 227, 229, 232, 325, 237, 239, 241, 282 and 283.

11. A bacterial delivery vehicle comprising an engineered branched-RBP of claim 1 .

12. The bacterial delivery vehicle of claim 11 wherein said bacterial delivery vehicle is a bacteriophage or is a packaged phagemid.

13. The bacterial delivery vehicle of claim 11 , wherein the chimeric RBP is selected from the group consisting of:
(i) a chimeric RBP that comprises a fusion between the N-terminal domain of a RBP from a lambda or lambda-like bacteriophage and the C-terminal domain of a different RBP and
wherein said N-terminal domain is fused to said C

Assignees

• Eligo Bioscience

Inventors

• Fernandez Rodriguez Jesus

Classifications

• A61K35/76

  Viruses; Subviral particles; Bacteriophages · CPC title

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N2795/00022

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• C12N2795/10321

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title