Mutant fragments of OspA and methods and uses relating thereto

Patent metadata

Field	Value
Publication number	US-12195501-B2
Application number	US-202117517477-A
Country	US
Kind code	B2
Filing date	Nov 2, 2021
Priority date	Jul 6, 2012
Publication date	Jan 14, 2025
Grant date	Jan 14, 2025

Abstract

The present invention relates to a polypeptide comprising a mutant fragment of an outer surface protein A (OspA), a nucleic acid coding the same, a pharmaceutical composition (particularly for use as a medicament of in a method of treating or preventing a Borrelia infection) comprising the polypeptide and/or the nucleic acid, a method of treating or preventing a Borrelia infection and a method of immunizing a subject.

First claim

What is claimed is:

1. A method for producing a polypeptide comprising the following steps:
a) introducing a vector encoding the polypeptide into a host cell;
b) growing the host cell under conditions allowing for expression of said polypeptide;
c) homogenizing said host cell; and
d) subjecting the host cell homogenate to purification steps,
wherein said polypeptide comprises a mutant fragment of a Borrelia outer surface protein A (OspA),
wherein said mutant OspA fragment comprises a C-terminal domain of a Borrelia OspA, and differs from the corresponding wild-type Borrelia OspA fragment sequence at least by the introduction of at least one disulfide bond that is formed by the introduction of:
i) one cysteine, which forms said disulfide bond with a cysteine residue present within the wild type fragment; or
ii) two cysteines, which together form said disulfide bond.

2. The method of claim 1 , wherein said mutant OspA fragment comprises a C-terminal domain of an OspA of Borrelia afzelii, B. burgdorferi s.s., B. bavariensis , or B. garinii which lacks at least the first 70 N-terminal amino acid residues; and wherein said mutant OspA fragment differs from the corresponding wild-type B. afzelii, B. burgdorferi s.s., B. bavariensis , or B. garinii OspA fragment sequence at least by the introduction of at least one disulfide bond that is formed by the introduction of
i) one cysteine, which forms said disulfide bond with a cysteine residue present within the wild-type fragment; or
ii) two cysteines, which together form said disulfide bond, wherein said two cysteines are substituted for amino acids at
one amino acid at any of positions 182+/−3 and one amino acid at any of positions 269+/−3;
one amino acid at any of positions 182+/−3 and one amino acid at any of positions 272+/−3;
one amino acid at any of positions 244+/−3 and one amino acid at any of positions 259+/−3;
one amino acid at any of positions 141+/−3 and one amino acid at any of positions 241+/−3; and/or
one amino acid at any of positions 165+/−3 and one amino acid at any of positions 265+/−3,

wherein the numbering of the cysteine substitutions corresponds to amino acid positions in the full-length OspA of B. afzelii , K78 as defined by SEQ ID NO: 19 or the homologous amino acids of an OspA from a Borrelia other than B. afzelii.

3. The method of claim 1 , wherein said OspA C-terminal domain is from B. burgdorferi s.s., strain B31, OspA serotype 1; wherein the wild-type sequence of OspA from B. burgdorferi s.s., strain B31 is defined by SEQ ID NO: 20.

4. The method according to claim 3 , wherein said OspA C-terminal domain further differs from the corresponding wild-type sequence by the replacement of the amino acid sequence provided by SEQ ID NO: 17 with the amino acid sequence provided by SEQ ID NO: 18.

5. The method of claim 1 , wherein said mutant OspA fragment is from the OspA C-terminal domain as defined by SEQ ID NO: 179, and comprises at least one introduced disulfide bond between
a) the amino acid at position 182 and the amino acid at position 269;
b) the amino acid at position 182 and the amino acid at position 272;
c) the amino acid at position 244 and the amino acid at position 259;
d) the amino acid at position 141 and the amino acid at position 241; and/or
e) the amino acid at position 165 and the amino acid at position 265;
wherein the numbering corresponds to amino acid positions in the full-length OspA protein as defined by SEQ ID NO: 20.

6. The method of claim 1 , wherein said polypeptide comprises one or more features of the group consisting of
a) an E. coli -derived lipidation signal as defined by MKATKLVLGAVILGSTLLAG (SEQ ID NO: 16);
b) absence of the amino acid sequence GYVLEGTLTAE (SEQ ID NO: 17);
c) a linker peptide led by an N-terminal cysteine residue as a site for lipidation;
d) presence of between 140 and 152 of the most C-terminal contiguous amino acids of the OspA protein; and
e) absence of the N-terminal β-sheet of OspA.

7. The method of claim 1 , wherein said mutant OspA fragment is from SEQ ID NO: 179 and comprises an introduced disulfide bond between the amino acid at position 182 and the amino acid at position 269, wherein the numbering corresponds to amino acid positions in the full-length OspA protein as defined by SEQ ID NO: 20.

8. The method of claim 1 , wherein said polypeptide comprises at least two mutant OspA fragments, wherein each mutant OspA fragment comprises a mutant C-terminal domain derived from a different Borrelia OspA sequence.

9. The method of claim 8 , wherein the polypeptide comprises or consists of a heterodimer selected from the group consisting of Lip-S1D4-S2D4 (SEQ ID NO: 185), Lip-S1D1-S2D1 (SEQ ID NO: 186), Lip-S3D4-S4D4 (SEQ ID NO: 187), Lip-S3D1-S4D1 (SEQ ID NO: 188), Lip-S5D4-S6D4 (SEQ ID NO: 189), Lip-S5D1-S6D1 (SEQ ID NO: 190), Lip-S2D4-S1D4 (SEQ ID NO: 191), Lip-S2D1-S1D1 (SEQ ID NO: 192), Lip-S4D4-S3D4 (SEQ ID NO: 193), Lip-S4D1-S3D1 (SEQ ID NO: 194), Lip-S6D4-S5D4 (SEQ ID NO: 195), Lip-S6D1-S5D1 (SEQ ID NO: 196), Lip-S1D4-S2D1 (SEQ ID NO: 197), Lip-S1D1-S2D4 (SEQ ID NO: 198), S3D4-S4D1 (SEQ ID NO: 199), S3D1-S4D4 (SEQ ID NO: 200), S5D4-S6D1 (SEQ ID NO: 201), S5D1-S6D4 (SEQ ID NO: 202), S2D4-S1D1 (SEQ ID NO: 203), S2D1-S1D4 (SEQ ID NO: 204), S4D4-S3D1 (SEQ ID NO: 205), S4D1-S3D4 (SEQ ID NO: 206), S6D4-S5D1 (SEQ ID NO: 207) and Lip-S6D1-S5D4 (SEQ ID NO: 208).

10. The method of claim 1 , wherein said vector comprises a nucleic acid molecule encoding said polypeptide, wherein said nucleic acid molecule is defined by SEQ ID NO: 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136.

11. The method of claim 1 , wherein said vector is pET28b (+).

12. The method of claim 1 , wherein said host cell is E. coli.

13. The method of claim 12 , wherein said E. coli is an E. coli BL21 cell.

14. The method according to claim 1 , wherein said purification steps comprise enriching the polypeptide in a lipid phase separation and purifying over a gel filtration column, and wherein said purification steps optionally further comprise processing over a buffer exchange column.

15. A method for producing a pharmaceutical composition comprising combining a polypeptide with one or more pharmaceutically acceptable carriers or excipients,
wherein said polypeptide comprises a mutant fragment of a Borrelia outer surface protein A (OspA),
wherein said mutant OspA fragment comprises a C-terminal domain of a Borrelia OspA, and differs from the corresponding wild-type Borrelia OspA fragment sequence at least by the introduction of at least one disulfide bond that is formed by the introduction of
i) one cysteine, which forms said disulfide bond with a cysteine residue present within the wild type fragment; or
ii) two cysteines, which together form said disulfide bond.

16. The method of claim 15 , wherein the mutant OspA fragment comprises a C-terminal domain of an OspA of Borrelia afzelii, B. burgdorferi s.s., B. bavariensis , or B. garinii which lacks at least the first 70 N-terminal amino acid residues; and wherein the mutant OspA fragment differs from the corresponding wild-type B. afzelii, B. burgdorferi s.s., B. bavariensis , or B. garinii OspA fragment sequence at least by the introduction of at least one disulfide bond that is formed by the introduction of
i) one cysteine, which forms said disulfide bond with a cysteine residue present within the wild-type fragment; or
ii) two cysteines, which together form said disulfide bond, wherein said two cysteines are sub

Assignees

• Valneva Austria Gmbh

Inventors

• Comstedt Pär
• Hanner Markus
• Lundberg Urban
• Meinke Andreas
• Schüler Wolfgang
• Wizel Benjamin

Classifications

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C07K2317/55

  Fab or Fab' · CPC title

• C07K2317/54

  F(ab')2 · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C07K16/1207

  from Spirochaetales (O), e.g. Treponema, Leptospira · CPC title