Antibodies against protective antigen
The present invention relates to antibodies and related molecules that specifically bind to protective antigen of Bacillus anthracis (PA). Such antibodies have uses, for example, in the prevention and treatment of anthrax and anthrax toxin poisoning. The invention also relates to nucleic acid molecules encoding anti-PA antibodies, vectors and host cells containing these nucleic acids, and methods for producing the same.
1. An isolated antibody or fragment thereof comprising the amino acid sequences of the VHCDR1 domain (residues 26-35 of SEQ ID NO:53), the VHCDR2 domain (residues 50-66 of SEQ ID NO:53), the VHCDR3 domain (residues 99-106 of SEQ ID NO:53), the VLCDR1 domain (residues 156-168 of SEQ ID NO:3), the VLCDR2 domain (residues 184-190 of SEQ ID NO:53), and the VLCDR3 domain (residues 223-233 of SEQ ID NO:53), of the scFv of SEQ ID NO:53, wherein said antibody or fragment thereof specifically binds protective antigen (PA).
2. The antibody or fragment thereof of claim 1 , wherein said antibody or fragment thereof inhibits binding of PA83 to anthrax receptor (ATR).
3. The antibody or fragment thereof of claim 1 , wherein said antibody or fragment thereof inhibits an activity selected from the group consisting of:
(a) binding of PA83 to capillary morphogenesis protein 2 (CMG2);
(b) protease cleavage of PA83 into PA20 and PA63;
(c) heptamerization of PA63;
(d) PA63 binding to edema factor (EF);
(e) PA63 binding to lethal factor (LF);
(f) PA-mediated translocation of EF across a cell membrane; and
(g) PA-mediated translocation of LF across a cell membrane.
4. The antibody or fragment thereof of claim 1 wherein said PA is purified from a bacterial cell culture, and wherein said PA is encoded by a polynucleotide encoding amino acids 1 to 764 of SEQ ID NO:2 operably associated with a regulatory sequence that controls expression of said polynucleotide.
5. The antibody or fragment thereof of claim 1 wherein the antibody or fragment thereof is a monoclonal antibody.
6. The antibody or fragment thereof of claim 1 wherein the antibody or fragment thereof is a human antibody.
7. The antibody or fragment thereof of claim 1 wherein the antibody or fragment thereof is selected from the group consisting of:
(a) a whole immunoglobulin molecule;
(b) an scFv;
(c) a chimeric antibody;
(d) a Fab fragment;
(e) an F(ab′)2; and
(f) a disulfide linked Fv.
8. The antibody or fragment thereof of claim 1 which comprises a heavy chain immunoglobulin constant domain selected from the group consisting of:
(a) a human IgM constant domain;
(b) a human IgG1 constant domain;
(c) a human IgG2 constant domain;
(d) a human IgG3 constant domain;
(e) a human IgG4 constant domain; and
(f) a human IgA constant domain.
9. The antibody or fragment thereof of claim 1 which comprises a light chain immunoglobulin constant domain selected from the group consisting of:
(a) a human Ig kappa constant domain; and
(b) a human Ig lambda constant domain.
10. The antibody or fragment thereof of claim 1 wherein the antibody or fragment thereof has a dissociation constant (K D ) of less than or equal to 10 −9 M.
11. The antibody or fragment thereof of claim 1 wherein the antibody or fragment thereof has a K D less than or equal to 10 −10 M.
12. The antibody or fragment thereof of claim 1 wherein the antibody or fragment thereof is conjugated to a detectable label.
13. The antibody or fragment thereof of any one of claim 1 wherein the antibody or fragment thereof is attached to a solid support.
14. The antibody or fragment thereof of claim 1 wherein the antibody or fragment thereof specifically binds PA in a Western blot.
15. The antibody or fragment thereof of claim 1 wherein the antibody or fragment thereof specifically binds PA in an ELISA.
16. An isolated cell that produces the antibody or fragment thereof of claim 1 .
17. A method of treatment of anthrax infection or anthrax toxin poisoning comprising administering to an animal the antibody or fragment thereof of claim 1 .
18. The method of claim 17 wherein the animal is a human.
19. A method of passive immunization comprising administering to an animal the antibody or fragment thereof of claim 1 .
20. The method of claim 17 wherein the antibody or fragment thereof is administered in combination with a second antibody or fragment thereof that specifically binds PA.
21. The method of claim 17 wherein the antibody or fragment thereof is administered in combination with an anti-anthrax agent selected from the group consisting of:
(a) a soluble form of the anthrax toxin receptor;
(b) a soluble form of the capillary morphogenesis protein 2 receptor;
(c) an anti-anthrax toxin receptor antibody;
(d) an anti-edema factor antibody; and
(e) an anti-lethal factor antibody.
22. The method of claim 17 wherein the antibody or fragment thereof is administered in combination with an antibiotic.
23. The method of claim 22 wherein the antibiotic is ciprofloxacin hydrochloride.
24. The method of claim 22 wherein the antibiotic is doxycycline.
25. The method of claim 22 wherein the antibiotic is selected from the group consisting of:
(a) penicillin G procaine;
(b) amoxicillan;
(c) ofloxacin; and
(d) levofloxacin.
26. The method of claim 17 wherein the antibody or fragment thereof is administered in combination with a member selected from the group consisting of:
(a) an anti-TNF-alpha antibody; and
(b) an anti-IL-1 beta antibody.
27. A kit comprising the antibody or fragment thereof of claim 1 and a means for administering said antibody to an animal.
28. The kit of claim 27 wherein the animal is a human.
29. An isolated antibody or fragment thereof comprising the amino acid sequence of amino acid residues 1-117 of SEQ ID NO:53 and the amino acid sequence of amino acid residues 134-244 of SEQ ID NO:53 wherein said antibody or fragment thereof specifically binds PA.
30. The antibody or fragment thereof of claim 29 that comprises amino acid residues 1-244 of SEQ ID NO:53.
31. The antibody or fragment thereof of claim 29 that consists of amino acid residues 1-244 of SEQ ID NO:53.
32. The antibody or fragment thereof of claim 29 wherein said PA is purified from a bacterial cell culture, and wherein said PA is encoded by a polynucleotide encoding amino acids 1 to 764 of SEQ ID NO:2 operably associated with a regulatory sequence that controls expression of said polynucleotide.
33. The antibody or fragment thereof of claim 29 wherein the antibody or fragment thereof is a monoclonal antibody.
34. The antibody or fragment thereof of claim 29 wherein the antibody or fragment thereof is a human antibody.
35. The antibody or fragment thereof of claim 29 wherein the antibody or fragment thereof is selected from the group consisting of:
(a) a whole immunoglobulin molecule;
(b) an scFv;
(c) a chimeric antibody;
(d) a Fab fragment;
(e) an F(ab′)2; and
(f) a disulfide linked Fv.
36. The antibody or fragment thereof of claim 29 which comprises a heavy chain immunoglobulin constant domain selected from the group consisting of:
(a) a human IgM constant domain;
(b) a human IgG1 constant domain;
(c) a human IgG2 constant domain;
(d) a human IgG3 constant domain;
(e) a human IgG4 constant domain; and
(f) a human IgA constant domain.
37. The antibody or fragment thereof of claim 29 which comprises a light chain immunoglobulin constant domain selected from the group consisting of:
(a) a human Ig kappa constant domain; and
(b) a human Ig lambda constant domain.
38. The antibody or fragment thereof of claim 29 wherein the antibody or fragment thereof is conjugated to a detectable label.
39. The antibody or fragment thereof of any one of claim 29 wherein the antibody or fragment thereof is attached to a solid support.
40. An isolated cell that produces the antibody or fragment thereof of claim 29 .
41. A method of treatment of anthrax infection or anthrax toxin poisoning comprising administering to an animal the antibody or fragment thereof of claim 29 .
42. The method of claim 41 wherein the animal is a human.
43. A method of passive immunization comprising administering to an animal the antibody or fragment thereof of claim 29 .
44. The method of claim 41 wherein the antibody or fragment thereof is administered in combination with a second antibody or fragment thereof that specifically binds PA.
45. The method of claim 41 wherein the antibody or fragment thereof is administered in combination with an anti-anthrax agent selected from the group consisting of:
(a) a soluble form of the anthrax toxin receptor;
(b) a soluble form of the capillary morphogenesis protein 2 receptor;
(c) an anti-anthrax toxin receptor antibody;
(d) an anti-edema factor antibody; and
(e) an anti-lethal factor antibody.
46. The method of claim 41 wherein the antibody or fragment thereof is administered in combination with an antibiotic.
47. The method of claim 46 wherein the antibiotic is ciprofloxacin hydrochloride.
48. The method of claim 46 wherein the antibiotic is doxycycline.
49. The method of claim 46 wherein the antibiotic is selected from the group consisting of:
(a) penicillin G procaine;
(b) amoxicillan;
(c) ofloxacin; and
(d) levofloxacin.
50. The method of claim 41 wherein the antibody or fragment thereof is administered in combination with a member selected from the group consisting of:
(a) an anti-TNF-alpha antibody; and
(b) an anti-IL-1 beta antibody.
51. A kit comprising the antibody or fragment thereof of claim 29 .
52. The cell line contained in ATCC Deposit Number PTA-4796.
53. The antibody produced by the cell line of claim 52 .
54. The method of claim 41 wherein the antibody or fragment thereof is administered intravenously (IV).
55. The method of claim 41 wherein the antibody or fragment thereof is administered sub-cutaneously (SC).
56. The method of claim 41 wherein the antibody or fragment thereof is administered intramuscularly (IM).
57. The method of claim 41 for treating anthrax infection or anthrax toxin poisoning wherein the antibody or fragment thereof is administered in a quantity in the range of 1 to 100 milligrams per kilogram of the animal's body weight.
58. The method of claim 57 wherein the antibody or fragment thereof is administered in a quantity in the range of 1 to 10 milligrams per kilogram of the animal's body weight.
59. The method of claim 41 for treating anthrax infection or anthrax toxin poisoning wherein the antibody or fragment thereof is administered in a quantity in the range of 0.1 to 20 milligrams per kilogram of the animal's body weight.
60. The method of claim 59 wherein the antibody or fragment thereof is administered in a quantity in the range of 1 to 10 milligrams per kilogram of the animal's body weight.
61. The method of claim 41 that prevents or reduces bacteremia associated with anthrax infection.
62. The method of claim 17 wherein the antibody or fragment thereof is administered intravenously (IV).
63. The method of claim 17 wherein the antibody or fragment thereof is administered sub-cutaneously (SC).
64. The method of claim 17 wherein the antibody or fragment thereof is administered intramuscularly (IM).
65. The method of claim 17 for treating anthrax infection or anthrax toxin poisoning wherein the antibody or fragment thereof is administered in a quantity in the range of 1 to 100 milligrams per kilogram of the animal's body weight.
66. The method of claim 65 wherein the antibody or fragment thereof is administered in a quantity in the range of 1 to 10 milligrams per kilogram of the animal's body weight.
67. The method of claim 17 for treating anthrax infection or anthrax toxin poisoning wherein the antibody or fragment thereof is administered in a quantity in the range of 0.1 to 20 milligrams per kilogram of the animal's body weight.
68. The method of claim 67 wherein the antibody or fragment thereof is administered in a quantity in the range of 1 to 10 milligrams per kilogram of the animal's body weight.
69. The method of claim 17 that prevents or reduces bacteremia associated with anthrax infection.