Antibodies to the PcrV Antigen of Pseudomonas aeruginosa

• US 20090191186A1
• Filed: 12/01/2008
• Published: 07/30/2009
• Est. Priority Date: 11/30/2007
• Status: Active Grant

• Current Assignee: KaloBios Pharmaceuticals, Inc.
• Original Assignee: KaloBios Pharmaceuticals, Inc.
• Inventors: Yarranton, Geoffrey T., Bebbington, Christopher R., Luehrsen, Kenneth, Baer, Mark
• Granted Patent: US 8,044,181 B2
• Time in Patent Office: Days
• US Class Current: 424/130.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/40 bacterial
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: C07K 16/1214 from Pseudomonadaceae (F)
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The current invention provides high-affinity antibodies to the Pseudomonas aeruginosa PcrV protein that have reduced immunogenicity when administered to treat Pseudomonas aeruginosa infections.

Claims

1. An anti-PcrV comprising a V_L region that has a CDR3 comprising FWGTP (SEQ ID NO:

40), wherein the antibody selectively binds PcrV.

2. The antibody of claim 1, wherein the antibody comprises a V_H region that has a CDR3 comprising the sequence NRGDIYYDFTY (SEQ ID NO:

38).

3. The antibody of claim 1, wherein the V_L region V-segment has at least 80% identity to a human germline V-segment.

4. The antibody of claim 1, wherein the V_L region comprises a FR4 that has at least 90% identity to the FR4 region of a human germline Jkappa1, Jkappa2, Jkappa 3, Jkappa4, or Jkappa5 segment or at least 90% identity to the FR4 region of a human germline Jlambda 1, Jlambda2, Jlambda3, or Jlambda7 segment.

5. The antibody of claim 1, wherein the V_L region comprises a FR4 that has at least 90% identity to the FR4 region of the human JK2 germline gene segment or at least 90% identity to the JL2 germline sequence;

and comprises a V-segment has at least 80% identity to a human germline Vkappa I or Vkappa III sequence, or at least 80% identity to a human germline Vlambda sequence.

6. The antibody of claim 1, wherein the antibody is an antagonist of the Pseudomonas aeruginosa Type III secretion system.

7. The antibody of claim 1, further comprising:

a V_H region that comprises a CDR3 having a sequence NRGDIYYDFTY (SEQ ID NO;

38), a FR4 and a V-segment, wherein the FR4 comprises at least 90% identity to the FR4 region of the human JH3 or human JH6 segment and the V-segment comprises at least 80% identity to the human VH1 18 subclass V-segment or to the human VH30.3 V segment.

8. The antibody of claim 1, wherein the V_H region comprises a CDR3 having a sequence NRGDIYYDFTYA(M/F)DX₁ (SEQ ID NO:

42), wherein X₁ is I, Q, Y, or S.

9. The antibody of claim 8, wherein the V_H region CDR3 has the sequence NRGDIYYDFTYA(M/F)DI (SEQ ID NO:

116).

10. The antibody of claim 1, wherein the FR4 of the V_H region has the sequence WGQGTX₂VTVSS (SEQ ID NO:

45), wherein X₂ is T or M.

21. The antibody of claim 1, wherein the V_H region comprises the V-segment region of an amino acid sequence selected from the group consisting of SEQ ID NOS:

1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 29, and 35.

11. An anti-PcrV antibody that binds to PcrV, comprising:

a V_H region that comprises a CDR3 having a sequence NRGDIYYDFTYAMDX₁ (SEQ ID NO;

43), wherein X₁ is I, Q, Y, or S;

a FR4 and a V-segment, wherein the FR4 comprises at least 90% identity to the FR4 region of the human germline JH3 segment or the FR4 region of the human germine JH6 segment, and the V-segment comprises at least 80% identity to the human germline VH1-18 subclass V-segment or to the human germline VH3-30.3 subclass V segment, with the proviso that when X₁ is Y, the FR4 region is not WGQGTSVTVSS (SEQ ID NO;

44).

12. The antibody of claim 11, wherein the antibody is an antagonist of the Pseudomonas aeruginosa Type III secretion system.

13. An anti-PcrV antibody that binds to PcrV, comprising:

a V_H region that comprises a CDR3 having a sequence NRGDIYYDFTYAMDX₁ (SEQ ID NO;

43), wherein X₁ is I, Q, Y, or S;

a FR4 and a V-segment, wherein the FR4 comprises at least 90% identity to the FR4 region of the human germline JH3 segment or to the FR4 region of the human germline JH6 segment; and

the V-segment comprises at least 80% identity to the human germline VH1-18 subclass V-segment or to the human germline VH3-30.3 subclass V segment, with the proviso that when X₁ is Y, the FR4 region is not WGQGTSVTVSS (SEQ ID NO;

44);

a V_L region that comprises a CDR3 comprising FW(S/G)TP (SEQ ID NO;

39), a FR4 and a V-segment, wherein the FR4 comprises at least 90% identity to the FR4 region of the human germline JK2 gene segment or to the FR4 region of the human germline JL2 segment; and

the V-segment comprises at least 80% identity to the human germline VKI L12 segment sequence, or at least 80% identity to a Vkappa III L2 segment sequence, or at least 80% identity to a human germline Vlambda2 2c segment sequence or Vlambda3 31 segment sequence.

14. The antibody of claim 13, wherein the antibody is an antagonist of the Type III secretion system.

15. The antibody of claim 13, wherein the V_H region V-segment has at least 80% identity to the human germline VH3-30.3 segment and the heavy chain region CDR1 comprises the sequence X₃X₄X₅X₆H, wherein X₃ is S, T, or N;

X₄ is Y or A;

X₅ is A, G, or P; and

X₆ is M, I, or L; and

the heavy chain region CDR2 comprises the sequence X₇IX₈YX₉GX₁₀X₁₁X₁₂X₁₃Y(A/I)X₁₄SVKG (SEQ ID NO;

49), wherein X₇ is V, F, or N;

X₈ is S or W;

X₉ is D or N;

X₁₀ is S, K, R or Y;

X₁₁ is N, S, D or E;

X₁₂ is K, I, or E;

X₁₃ is Y, S, D or W; and

X₁₄ is D or S.

16. The antibody of claim 15, wherein the V_H region V-segment has at least 90% identity to a VH3-30.3 V segment.

17. The antibody of claim 15, wherein the CDR1 is TAGMH (SEQ ID NO:

50), SYGIH (SEQ ID NO;

51), SYGMH (SEQ ID NO;

52), SYPLH (SEQ ID NO;

53), or NYPMH (SEQ ID NO;

54);

or the CDR2 is VIWYNGKEISYADSVKG (SEQ ID NO;

55), FISYDGSEKYYASSVKG (SEQ ID NO;

56), VISYDGSEKWYADSVKG (SEQ ID NO;

57), VIWYDGRNKYYADSVKG (SEQ ID NO;

58), VIWYDGYNKDYADSVKG (SEQ ID NO;

59), or NIWYDGSSESYIDSVKG (SEQ ID NO;

60).

18. The antibody of claim 15, wherein the CDR1 is TAGMH (SEQ ID NO:

50), SYGIH (SEQ ID NO;

51), SYGMH (SEQ ID NO;

52), SYPLH (SEQ ID NO;

53), or NYPMH (SEQ ID NO;

54);

or the CDR2 is VIWYNGKEISYADSVKG (SEQ ID NO;

55), FISYDGSEKYYASSVKG (SEQ ID NO;

56), VISYDGSEKWYADSVKG (SEQ ID NO;

57), VIWYDGRNKYYADSVKG (SEQ ID NO;

58), VIWYDGYNKDYADSVKG (SEQ ID NO;

59), or NIWYDGSSESYIDSVKG (SEQ ID NO;

60).

20. The antibody of claim 17, wherein the V_H region V-segment comprises at least 80% identity to the human germline VH1-18 subclass V-segment.

23. The antibody of claim 13, wherein the V_L region CDR3 comprises FW(S/G)TPYT (SEQ ID NO:

117).

24. The antibody of claim 23, wherein the V_L region FR4 has the sequence FGQGTKLEIK (SEQ ID NO:

47) or FGGGTKLTVL (SEQ ID NO;

48).

25. The antibody of claim 13, wherein the V_L region V-segment comprises at least 90% identity to a human germline Vkappa 1 L12 or Vkappa III L2 sequence, or at least 90% identity to a human germline Vlambda3 31 or to a Vlambda2 2c sequence.

26. The antibody of claim 13, wherein the V_L region V-segment has at least 80% amino acid sequence identity to the human germline VK1 L12 segment and the CDR1 has the sequence RASX₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁A (SEQ ID NO:

64), where X₁₅ is Q or E;

X₁₆ is S or G;

X₁₇ is I or V;

X₁₈ is S or D;

X₁₉ is S, R, or T;

X₂₀ is W or Y; and

X₂, is L or V; and

the CDR2 has the sequence X₂₁ASX₂₂LX₂₃S (SEQ ID NO;

65), wherein X₂₁ is D or A;

X₂₂ is S, A, or T; and

X₂₃ is E, Q, or K.

27. The antibody of claim 26, wherein the V_L region V-segment has at least 90% identity to the human germline VKI L12 segment.

28. The antibody of claim 26, wherein the CDR1 has the sequence RASQGISTYLA (SEQ ID NO:

66), RASQGISSWLA (SEQ ID NO;

67), RASQSISRWVA (SEQ ID NO;

     68), or RASEGVDRWLA (SEQ ID NO;

69); and

the CDR2 has the sequence AASSLQS (SEQ ID NO;

70), DASSLKS (SEQ ID NO;

7), AASSLQS, DASALQS (SEQ ID NO;

72), or DASTLQS (SEQ ID NO;

73).

29. The antibody of claim 13, wherein the V_L region V segment has at least 80% amino acid sequence identity to the human germline VKIII L2 sequence and the CDR1 has the sequence RASNSVGAYNLA (SEQ ID NO:

74) or RASQSVSSNLA (SEQ ID NO;

75); and

the CDR2 has the sequence (A/G)AS(T/R)RA(T/P) (SEQ ID NO;

76).

30. The antibody of claim 29, wherein the V_L region V-segment has at least 90% identity to the human germline VKIII L2 segment.

31. The antibody of claim 13, wherein the V_L region V-segment has at least 80% amino acid sequence identity to a human germline Vlambda3 31 segment and the CDR1 has the sequence QGDSLRS(Y/L)YAS (SEQ ID NO:

77), TGTSSDVGAYNYVS (SEQ ID NO;

79), or TGTSSDYVS (SEQ ID NO;

80); and

the CDR2 has the sequence (G/S)KN(N/S)RPS (SEQ ID NO;

78).

32. The antibody of claim 31, wherein the V_L region V-segment has at least 90% identity to the human germline Vlambda3 31 segment.

33. The antibody of claim 13, wherein the V_L region V-segment has at least 80% amino acid sequence identity to a human germline Vlambda2 2c segment and the CDR1 has the sequence TGTSSDVG(G/A)YNYVS (SEQ ID NO:

118) or TGTSSDYVS (SEQ ID NO;

80); and

the CDR2 has the sequence (E/D)V(S/T)(K/N)RPS (SEQ ID NO;

119).

34. The antibody of claim 33, wherein the V_L region V-segment has at least 90% identity to the human germline Vlambda L2 2c segment.

35. The antibody of claim 13, wherein the V_L region comprises the V-segment of an amino acid sequence selected from the group consisting of SEQ ID NO:

2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 37.

36. The antibody of claim 35, wherein the V_L region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 37.

37. The antibody of claim 13, wherein the antibody is a Fab′

fragment.

38. The antibody of claim 13, wherein the antibody is an IgG.

39. The antibody of claim 13, wherein the antibody is PEGylated.

40. The antibody of claim 39, wherein the antibody is di-PEGylated.

41. The antibody of claim 13, wherein the V_H region or the V_L region, or both the V_H and V_L region amino acid sequences comprise a methionine at the N-terminus.

45. A method of treating a patient infected with P. aeruginosa, the method comprising administering an antibody of claim 13.

46. The method of claim 45, wherein the antibody is not immunogenic in humans.

47. The method of claim 45, wherein the antibody is administered intravenously, subcutaneously or by insufflation.

19. An anti-PcrV antibody that binds to PcrV comprising a V_H region that has a CDR3 sequence NRGDIYYDFTYAFDI (SEQ ID NO:

63), a CDR1 sequence DHAIS (SEQ ID NO;

61) and a CDR2 sequence WISPYSGNPNYAQSLQG (SEQ ID NO;

62).

22. The antibody of 21, wherein the V_H region comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:

1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 29, and 35.

42. An anti-PcrV antibody that is an antagonist of the Type III secretion system, comprising:

a V_H region having an amino acid sequence selected from the group consisting of SEQ ID NOS;

1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 29, and 35; and

V_L region having an amino acid sequence selected from the group consisting of SEQ ID NO;

2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 37.

43. The antibody of claim 42, wherein the V_H region or the V_L region, or both the V_H and V_L regions have a methionine present at the N-terminus.

44. The antibody of claim 42, wherein the antibody is a di-PEGylated Fab′

.