Humanized immunoglobulin reactive with b7-2 and methods of treatment therewith Number:6,827,934 from the United States Patent and Trademark Office (PTO) owispatent The invention relates to a humanized anti-B7-2 antibody that comprises a variable region of nonhuman origin and at least a portion of an immunoglobulin of human origin. The invention also pertains to methods of treatment for various autoimmune diseases, transplant rejection, inflammatory disorders and infectious diseases by administering humanized anti-B7-2 and/or anti-B7-1 antibodies.<H immunoglobulin, therewith, Humanized, immun, 6827934.html, Patent, pto, 6827934

Title: Humanized immunoglobulin reactive with b7-2 and methods of treatment therewith

Abstract: The invention relates to a humanized anti-B7-2 antibody that comprises a variable region of nonhuman origin and at least a portion of an immunoglobulin of human origin. The invention also pertains to methods of treatment for various autoimmune diseases, transplant rejection, inflammatory disorders and infectious diseases by administering humanized anti-B7-2 and/or anti-B7-1 antibodies.

Patent Number: 6,827,934 Issued on 12/07/2004 to Co, et al.

Inventors: Co; Man Sung (Cupertino, CA); Vasquez; Maximiliano (Palo Alto, CA); Carreno; Beatriz (Acton, MA); Celniker; Abbie Cheryl (Newton, MA); Collins; Mary (Natick, MA); Goldman; Samuel (Acton, MA); Knight; Andrea (Hampton, NH); O'Hara; Denise (Reading, MA); Rup; Bonita (Reading, MA); Veldman; Geertruida M. (Sudbury, MA); Gray; Gary S. (Brookline, MA)
Assignee: Genetics Institute, LLC (Cambridge, MA)

Appl. No.: 627896

Filed: July 27, 2000

Current U.S. Class: 424/153.1 ; 424/130.1; 424/133.1; 424/141.1; 424/143.1; 424/144.1; 424/173.1; 530/387.1; 530/387.3; 530/388.1; 530/388.2; 530/388.22; 530/388.7; 530/388.73

Field of Search: 424/130.1,133.1,144.1,173.1 530/387.1,388.2,388.73

References Cited [Referenced By]

U.S. Patent Documents


5397703	March 1995	de Boer et al.
5562903	October 1996	Co et al.
5585089	December 1996	Queen et al.
5622701	April 1997	Berg
5624821	April 1997	Winter et al.
5648260	July 1997	Winter et al.
5693762	December 1997	Queen et al.
5747034	May 1998	de Boer et al.
5869050	February 1999	de Boer et al.
6084067	July 2000	Freeman et al.
6130316	October 2000	Freeman et al.
6346248	February 2002	De Boer et al.

Foreign Patent Documents


94/01547	Jan., 1994	WO
95/03408	Feb., 1995	WO
WO 95/34320	Dec., 1995	WO
WO 96/14865	May., 1996	WO
98/19706	May., 1998	WO

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Primary Examiner: Gambel; Phillip
Attorney, Agent or Firm: Finnegan, Henderson, Farabow, Garrett & Dunner, LLP

Parent Case Text

RELATED APPLICATION(S)

This application is a divisional of U.S. application Ser. No. 09/249,011 filed Feb. 12, 1999, the entire teachings of which are incorporated herein by reference.

Claims

What is claimed is:

1. A method of inhibiting the interaction of a first cell bearing a B7-2 receptor with a second cell bearing B7-2, comprising contacting said second cell with an effective amount of a humanized immunoglobulin having binding specificity for B7-2, said immunoglobulin comprising: a) at least one antigen binding region of nonhuman origin and b) a portion of an immunoglobulin heavy chain of human origin derived from the III2R (SEQ ID NOS: 25, 29) variable region and a portion of an immunoglobulin light chain of human origin derived from the H2F (SEQ ID NOS: 26, 30) variable region, wherein humanized immunoglobulin has a binding affinity of at least about 10.sup.7 M.sup.-1.

2. A method of inducing a lack of responsiveness to an antigen in a patient having a transplanted organ, tissue, or cell comprising administering an effective amount of a humanized immunoglobulin having binding specificity for B7-2, said immunoglobulin comprising: a) at least one antigen binding region of nonhuman origin, and b) a portion of an immunoglobulin heavy chain of human origin derived from the III2R (SEQ ID NOS: 25, 29) variable region and a portion of an immunoglobulin light chain of human origin derived from the H2F (SEQ ID NOS: 26, 30) variable region, wherein the immunoglobulin is administered in a carrier and the humanized immunoglobulin has a binding affinity of at least about 10.sup.7 M.sup.-1.

3. A method of reducing transplantation rejection in a patient having a transplanted organ, tissue, or cell, comprising administering a therapeutically effective amount of a humanized immunoglobulin having binding specificity for B7-2, said immunoglobulin comprising: a) at least one antigen binding region of nonhuman origin, and b) a portion of an immunoglobulin heavy chain of human origin derived from the III2R (SEQ ID NOS: 25, 29) variable region and a portion of an immunoglobulin light chain of human origin derived from the H2F (SEQ ID NOS: 26, 30) variable region, wherein the humanized immunoglobulin has a binding affinity of at least about 10.sup.7 M.sup.-1.

4. The method of claim 2, wherein the carrier is pharmaceutical carrier.

5. The method of claim 1, wherein said at least one antigen binding region further comprises one CDR of the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

6. The method of claim 1, wherein said immunoglobulin comprises a light chain encoded by the amino acid sequence of SEQ ID NO: 8 and a heavy chain encoded by the amino acid sequence of SEQ ID NO: 6.

7. The method of claim 6, wherein said immunoglobulin further comprises a constant region comprising a human IgG4 isotype.

8. The method of claim 6, wherein said immunoglobulin further comprises a constant region comprising a human IgG2M3 isotype.

9. The method of claim 1, wherein said immunoglobulin has a portion of the amino acid sequence of its variable region in common with a portion of the amino acid sequence of the variable region of the III2R (SEQ ID NOS: 25, 29) heavy chain.

10. The method of claim 1, wherein said immunoglobulin has a portion of the amino acid sequence of its variable region in common with a portion of the amino acid sequence of the variable region of the H2F (SEQ ID NOS: 26, 30) light chain.

11. The method of claim 2, wherein said at least one antigen binding region further comprises one CDR of the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

12. The method of claim 2, wherein said immunoglobulin comprises a light chain encoded by the amino acid sequence of SEQ ID NO: 8 and a heavy chain encoded by the amino acid sequence of SEQ ID NO: 6.

13. The method of claim 12, wherein said immunoglobulin further comprises a constant region comprising a human IgG4 isotype.

14. The method of claim 12, wherein said immunoglobulin further comprises a constant region comprising a human IgG2M3 isotype.

15. The method of claim 2, wherein said immunoglobulin has a portion of the amino acid sequence of its variable region in common with a portion of the amino acid sequence of the variable region of the III2R (SEQ ID NOS: 25, 29) heavy chain.

16. The method of claim 2, wherein said immunoglobulin has a portion of the amino acid sequence of its variable region in common with a portion of the amino acid sequence of the variable region of the H2F (SEQ ID NOS: 26, 30) light chain.

17. The method of claim 3, wherein said at least one antigen binding region further comprises one CDR of the variable region of 3D1 antibody.

18. The method of claim 3, wherein said immunoglobulin comprises a light chain encoded by the amino acid sequence of SEQ ID NO: 8 and a heavy chain encoded by the amino acid sequence of SEQ ID NO: 6.

19. The method of claim 18, wherein said immunoglobulin further comprises a constant region comprising a human IgG4 isotype.

20. The method of claim 18, wherein said immunoglobulin further comprises a constant region comprising a human IgG2M3 isotype.

21. The method of claim 3, wherein said immunoglobulin has a portion of the amino acid sequence of its variable region in common with a portion of the amino acid sequence of the variable region of the III2R (SEQ ID NOS: 25, 29) heavy chain.

22. The method of claim 3, wherein said immunoglobulin has a portion of the amino acid sequence of its variable region in common with a portion of the amino acid sequence of the variable region of the H2F (SEQ ID NOS: 26, 30) light chain.

23. The method of claim 1, wherein the binding affinity is about 10.sup.9 M.sup.-1.

24. The method of claim 2, wherein the binding affinity is about 10.sup.9 M.sup.-1.

25. The method of claim 3, wherein the binding affinity is about 10.sup.9 M.sup.-1.

26. A method of inhibiting the interaction of a first cell bearing a B7-2 receptor with a second cell bearing B7-2, comprising contacting said second cell with an effective amount of a humanized immunoglobulin having binding specificity for B7-2, said immunoglobulin comprising: a) a light chain which comprises one or more CDR's derived from an antibody of non-human origin which binds to B7-2 and a framework region derived from a human H2F antibody (SEQ ID NOS: 26, 30); and b) a heavy chain which comprises one or more CDR's derived from an antibody of non-human origin which binds to B7-2 and a framework region derived from the human III2R antibody (SEQ ID NOS: 25, 29).

27. The method of claim 26, wherein said one or more CDRs are derived from the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

28. The method of claim 26, wherein said light chain comprises three CDRs derived from an antibody of non-human origin which binds B7-2 and a framework region derived from a human H2F antibody (SEQ ID NOS: 26, 30) and said heavy chain comprises three CDRs derived from an antibody of non-human origin which binds B7-2 and a framework region derived from the human III2R antibody (SEQ ID NOS: 25, 29).

29. The method of claim 26, wherein said immunoglobulin comprises a light chain encoded by the amino acid sequence of SEQ ID NO: 8 and a heavy chain encoded by the amino acid sequence of SEQ ID NO: 6.

30. The method of claim 26, wherein said immunoglobulin further comprises a constant region comprising a human IgG4 isotype.

31. The method of claim 26, wherein said immunoglobulin further comprises a constant region comprising a human IgG2M3 isotype.

32. The method of claim 26, wherein the binding affinity is about 10.sup.9 M.sup.-1.

33. A method of inducing a lack of response to an antigen in a patient having a transplanted organ, tissue, or cell comprising administering an effective amount of a humanized immunoglobulin having binding specificity for B7-2, said immunoglobulin comprising: a) a light chain which comprises one or more CDR's derived from an antibody of non-human origin which binds to B7-2 and a framework region derived from a human H2F antibody (SEQ ID NOS: 26, 30); and b) a heavy chain which comprises one or more CDR's derived from an antibody of non-human origin which binds to B7-2 and a framework region derived from the human III2R antibody (SEQ ID NOS: 25, 29).

34. The method of claim 33, wherein said one or more CDRs are derived from the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

35. The method of claim 33, wherein said light chain comprises three CDRs derived from an antibody of non-human origin which binds B7-2 and a framework region derived from a human H2F antibody (SEQ ID NOS: 26, 30) and said heavy chain comprises three CDRs derived from an antibody of non-human origin which binds B7-2 and a framework region derived from the human III2R antibody (SEQ ID NOS: 25, 29).

36. The method of claim 33, wherein said immunoglobulin comprises a light chain encoded by the amino acid sequence of SEQ ID NO: 8 and a heavy chain encoded by the amino acid sequence of SEQ ID NO: 6.

37. The method of claim 33, wherein said immunoglobulin further comprises a constant region comprising a human IgG4 isotype.

38. The method of claim 33, wherein said immunoglobulin further comprises a constant region comprising a human IgG2M3 isotype.

39. The method of claim 33, wherein the binding affinity is about 10.sup.9 M.sup.-1.

40. A method of reducing transplantation rejection in a patient having a transplanted organ, tissue, or cell, comprising administering a therapeutically effective amount of a humanized immunoglobulin having binding specificity for B7-2, said immunoglobulin comprising: a) a light chain which comprises one or more CDR's derived from an antibody of non-human origin which binds to B7-2 and a framework region derived from a human H2F antibody (SEQ ID NOS: 26, 30); and b) a heavy chain which comprises one or more CDR's derived from an antibody of non-human origin which binds to B7-2 and a framework region derived from the human III2R antibody (SEQ ID NOS: 25, 29).

41. The method of claim 40, wherein said one or more CDRs are derived from the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

42. The method of claim 40, wherein said light chain comprises three CDRs derived from an antibody of non-human origin which binds B7-2 and a framework region derived from a human H2F antibody (SEQ ID NOS: 26, 30) and said heavy chain comprises three CDRs derived from an antibody of non-human origin which binds B7-2 and a framework region derived from the human III2R antibody (SEQ ID NOS: 25, 29).

43. The method of claim 40, wherein said immunoglobulin comprises a light chain encoded by the amino acid sequence of SEQ ID NO: 8 and a heavy chain encoded by the amino acid sequence of SEQ ID NO: 6.

44. The method of claim 40, wherein said immunoglobulin further comprises a constant region comprising a human IgG4 isotype.

45. The method of claim 40, wherein said immunoglobulin further comprises a constant region comprising a human IgG2M3 isotype.

46. The method of claim 40, wherein the binding affinity is about 10.sup.9 M.sup.-1.

47. The method of claim 1, wherein the portion of an immunoglobulin light chain of human origin derived from the H2F (SEQ ID NOS: 26, 30) variable region is the framework region.

48. The method of claim 1, wherein the portion of an immunoglobulin heavy chain of human origin derived from the III2R (SEQ ID NOS: 25, 29) variable region is the framework region.

49. The method of claim 2, wherein the portion of an immunoglobulin light chain of human origin derived from the H2F (SEQ ID NOS: 26, 30) variable region is the framework region.

50. The method of claim 2, wherein the portion of an immunoglobulin heavy chain of human origin derived from the III2R (SEQ ID NOS: 25, 29) variable region is the framework region.

51. The method of claim 3, wherein the portion of an immunoglobulin light chain of human origin derived from the H2F (SEQ ID NOS: 26, 30) variable region is the framework region.

52. The method of claim 3, wherein the portion of an immunoglobulin heavy chain of human origin derived from the III2R (SEQ ID NOS: 25, 29) variable region is the framework region.

53. The method of claim 1, wherein said at least one antigen binding region further comprises two CDRs of the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

54. The method of claim 1, wherein said at least one antigen binding region further comprises three CDRs of the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

55. The method of claim 2, wherein said at least one antigen binding region further comprises two CDRs of the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

56. The method of claim 2, wherein said at least one antigen binding region further comprises three CDRs of the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

57. The method of claim 3, wherein said at least one antigen binding region further comprises two CDRs of the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

58. The method of claim 3, wherein said at least one antigen binding region further comprises three CDRs of the variable region of the 3D1 (SEQ ID NOS: 21, 23) antibody.

59. A method of inhibiting the interaction of a first cell bearing a B7-2 receptor with a second cell bearing B7-2, comprising contacting said second cell with an effective amount of a humanized immunoglobulin having binding specificity for B7-2, said immunoglobulin comprising: a) at least one antigen binding region of nonhuman origin and b) a portion of an immunoglobulin heavy chain of human origin derived from the III2R (SEQ ID NOS: 25, 29) or a portion of an immunoglobulin light chain of human origin derived from the H2F (SEQ ID NOS: 26, 30) variable region-and the humanized immunoglobulin has a binding affinity of at least about 10.sup.7 M.sup.-1.

60. A method of inducing a lack of response to an antigen in a patient having a transplanted organ, tissue, or cell comprising administering an effective amount of a humanized immunoglobulin having binding specificity for B7-2, said immunoglobulin comprising; a) at least one antigen binding region of nonhuman origin, and b) a portion of an immunoglobulin heavy chain of human origin derived from the III2R (SEQ ID NOS: 25, 29) or a portion of an immunoglobulin light chain of human origin derived from the H2F (SEQ ID NOS:26, 30) variable region, wherein the immunoglobulin is administered in a carrier, and the humanized antibody has a binding affinity of at least about 10.sup.7 M.sup.-1.

61. A method of reducing transplantation rejection in a patient having a transplanted organ, tissue, or cell, comprising administering a therapeutically effective amount of a humanized antibody having binding specificity for B7-2, said immunoglobulin comprising: a) at least one antigen binding region of nonhuman origin, and b) a portion of an immunoglobulin heavy chain of human origin derived from the III2R (SEQ ID NOS: 25, 29) or a portion of an immunoglobulin light chain of human origin derived from the H2F (SEQ ID NOS: 26, 30) variable region-and the humanized immunoglobulin has a binding affinity of at least about 10.sup.7 M.sup.-1.

62. A method of treating an individual having an inflammatory disorder comprising administering a therapeutically effective amount of the humanized immunoglobulin having binding specificity for B7-2, said immunoglobulin comprising: a) at least one antigen binding region of nonhuman origin, and b) a portion of an immunoglobulin heavy chain of human origin derived from the III2R (SEQ ID NOS: 25, 29) or a portion of an immunoglobulin light chain of human origin derived from the H2F (SEQ ID NOS: 26, 30) variable region, wherein the immunoglobulin is administered in a carrier, and the humanized antibody has a binding affinity of at least 10.sup.7 M.sup.-1.

Description

BACKGROUND OF THE INVENTION

Antigen specific T-cell activation and the initiation of an immune response depend initially on the interaction of the T-cell receptor (TCR) complex with the peptide/major histocompatibility complex (MHC) present on antigen presenting cells (APC). B7 molecules, B7-1 and B7-2, are molecules which are present on APCs. A second "costimulatory" signal, provided by the interaction of B7-1 and B7-2 on the APC with their ligands CD28 and CTLA4 on T-cells, is required to complete T-cell activation and the subsequent regulation of an immune response. A need exists to regulate the B7-1 and B7-2 pathway, referred to as the B7:cD28/CTLA4 pathway. A further need exists to develop treatments for diseases that are affected by this pathway.

SUMMARY OF THE INVENTION

The invention relates to a humanized immunoglobulin having binding specificity for B7-2, wherein the immunoglobulin comprises an antigen binding region of nonhuman origin (e.g. rodent) and at least a portion of human origin (e.g. a human constant region such as an IgG constant region, a human framework region). In one embodiment, the human constant region can also contain a mutation that reduces the effector function of the humanized immunoglobulin. In another embodiment, the humanized immunoglobulin, described herein, can compete with murine 3D1 for binding to B7-2. In a particular embodiment, the antigen binding region of the humanized immunoglobulin is derived from the 3D1 monoclonal antibody.

The humanized immunoglobulin having binding specificity for B7-2 can comprise a constant region of human origin and an antigen binding region, wherein the antigen binding region of nonhuman origin comprises one or more complementarity determining regions (CDRs) of rodent origin (e.g. derived from 3D1 monoclonal antibody) that binds to B7-2, and the portion of an immunoglobulin of human origin is derived from a human framework region (FR). The antigen binding region can further comprise a light chain and a heavy chain, wherein the light and heavy chain each have three CDRs derived from the 3D1 antibody. The FR of the light chain can be derived, for example, from the light chain of the human H2F antibody and the heavy chain can be derived, for example, from the heavy chain of the human III2R antibody. In a particular embodiment, the invention is a humanized immunoglobulin having binding specificity for B7-2 that is derived from the cell line deposited with the American Type Culture Collection (A.T.C.C.), Accession No. CRL-12524.

The invention also embodies a humanized immunoglobulin having a binding specificity for B7-2 comprising a heavy chain and/or a light chain. The light chain comprises a CDR (e.g., CDR1, CDR2 and CDR3) derived from an antibody of nonhuman origin which binds B7-2 and a FR derived from a light chain of human origin (e.g., H2F antibody). The heavy chain comprises a CDR (e.g.,CDR1, CDR2 and CDR3) derived from an antibody of nonhuman origin which binds B7-2 and a FR region derived from a heavy chain of human origin (e.g., the human III2R antibody). The immunoglobulin can further comprise CDR1, CDR2 and CDR3 for the light or heavy chain having the amino acid sequence set forth herein or an amino acid.

One embodiment of the invention is a humanized immunoglobulin light chain having binding specificity for B7-2 comprising CDR1, CDR2 and/or CDR3 of the light chain of murine 3D1 antibody, and a human light chain FR (e.g., H2F antibody). Another embodiment is a humanized immunoglobulin light chain that comprises a variable region shown in FIG. 2B (SEQ ID NO: 8). The invention also relates to an isolated nucleic acid sequence that encodes a humanized variable light chain specific for B7-2 that comprises a nucleic acid, such as the sequence shown in FIG. 2B (SEQ ID NO: 7), a nucleic acid that encodes the amino acid sequence shown in FIG. 2B (SEQ ID NO: 8), a nucleic acid which hybridizes thereto under stringent hybridization conditions, and a nucleic acid which is the complement thereof.

Another embodiment of the invention is a humanized immunoglobulin heavy chain that is specific for B7-2 and comprises CDR1, CDR2 and/or CDR3 of the heavy chain of the 3D1 antibody, and a human heavy chain FR (e.g., III2R antibody). The invention pertains to a humanized immunoglobulin heavy chain that comprises a variable region shown in FIG. 2A (SEQ ID NO: 6). The invention also pertains to an isolated nucleic acid sequence that encodes a humanized variable heavy chain specific for B7-2 that comprises a nucleic acid, such as the sequence shown in FIG. 2A (SEQ ID NO: 5), a nucleic acid that encodes the amino acid sequence shown in FIG. 2A (SEQ ID NO: 6), a nucleic acid which hybridizes thereto under stringent hybridization conditions, and a nucleic acid which is the complement thereof.

In particular, an embodiment of the invention is a humanized immunoglobulin which specifically binds to B7-2 and comprises a humanized light chain comprising three light chain CDRs from the mouse 3D1 antibody and a light chain variable region framework sequence from a human immunoglobulin light chain, and a humanized heavy chain comprising three heavy chain CDRs from the mouse 3D1 antibody and a heavy chain variable region framework sequence from a human immunoglobulin heavy chain. The mouse 3D1 antibody can further have a mature light chain variable domain, such as the mature light chain variable domain shown in FIG. 1B (SEQ ID NO.: 4) and a mature heavy chain variable domain such as the mature heavy chain variable region shown in FIG. 1A (SEQ ID NO.: 2).

The invention includes an expression vector that comprises a fused gene which encodes a humanized immunoglobulin light and/or heavy chain. The gene comprises a nucleotide sequence encoding a CDR derived from a light and/or heavy chain of a nonhuman antibody having binding specificity for B7-2 (e.g., murine 3D1 antibody) and a FR derived from a light and/or heavy chain of human origin.

The present invention also relates to a host cell comprising a nucleic acid of the present invention, including one or more constructs comprising nucleic acid of the present invention. In one embodiment, the invention encompasses a host cell comprising a first recombinant nucleic acid that encodes a humanized immunoglobulin light chain and a second recombinant nucleic acid that encodes a humanized immunoglobulin heavy chain. The first nucleic acid comprises a nucleotide sequence encoding a CDR derived from the light chain of murine 3D1 antibody and a FR derived from a light chain of human origin. The second nucleic acid comprises a nucleotide sequence encoding a CDR derived from the heavy chain of murine 3D 1 antibody and a FR derived from a heavy chain of human origin. The invention further relates to a host cell comprising a vector or a nucleic acid that encodes the humanized immunoglobulin, as described herein.

The invention further pertains to methods of preparing a humanized immunoglobulin that comprise maintaining a host cell that encodes a humanized immunoglobulin that is specific for B7-2, as described herein, under conditions appropriate for expression of a humanized immunoglobulin, wherein a humanized immunoglobulin chain (one or more) are expressed and a humanized immunoglobulin is produced. The method further comprises the step of isolating the humanized immunoglobulin.

Additional methods encompassed by the invention include a method of inhibiting the interaction of a first cell bearing a B7-2 receptor with a second cell bearing B7-2, comprising contacting the second cell with an effective amount of a humanized immunoglobulin, as described herein. Accordingly, the invention relates to various methods of treatment. The invention includes a method for modulating an immune response of a patient or treating a patient having a transplanted organ, tissue, cell or the like comprising administering an effective amount of the humanized immunoglobulin, as described herein, in a carrier (e.g., pharmaceutical carrier), wherein the immune response is modulated. The invention pertains to treating acute and/or chronic transplant rejection for a prolonged periods of time (e.g., days, months, years). The invention also pertain to methods of treating a disease associated with modulation of the B7-2 molecule (e.g., autoimmune diseases, infectious diseases, inflammatory disorders, systemic lupus erythematosus, diabetes mellitus, insulitis, arthritis, inflammatory bowel disease, inflammatory dermatitis, and multiple sclerosis), comprising administering to a patient an effective amount (e.g., a therapeutically effective amount) of a humanized immunoglobulin, as described herein, in a carrier. Accordingly, the invention encompasses a pharmaceutical composition comprising the humanized antibody, as described herein.

The invention also embodies a method of making a humanized immunoglobulin specific to B7-2 from a murine antibody specific to B7-2. The method comprises determining the CDRs of an antibody of non-human origin (e.g., murine origin) which has binding specificity for B7-2; obtaining a human antibody having a framework region amino acid sequence suitable for grafting of the CDRs, and grafting the CDRs of an antibody of non-human origin into the FR of the human antibody.

The invention also relates to a method for determining the presence or absence of B7-2 in a sample. The method comprises obtaining the sample to be tested, contacting the sample with a humanized antibody specific to B7-2, or a fragment thereof, sufficiently to allow formation of a complex between B7-2 and the anti-B7-2 antibody, and detecting the presence or absence of the complex formation. The presence of the complex indicates the presence of B7-2 in the sample.

The invention relates to methods for treating a patient having a disease comprising administering a therapeutically effective amount of a humanized immunoglobulin specific to B7-1 and a therapeutically effective amount of a humanized immunoglobulin specific to B7-2. The diseases, as described herein, include, for example, autoimmune diseases, infectious diseases, asthma, inflammatory disorders, systemic lupus erythematosus, diabetes mellitus, insulitis, arthritis, inflammatory bowel disease, inflammatory dermatitis, and multiple sclerosis. This method also pertains to modulating the immune response of a patient having a transplanted organ, tissue, cell or the like comprising administering an effective amount of a humanized immunoglobulin that binds to B7-1 and a humanized immunoglobulin that binds to B7-2. Such diseases are described herein.

The invention also pertains to methods for transplanting cells (e.g., bone marrow, or blood cells or components) to a patient in need thereof comprising obtaining cells (e.g., bone marrow, or blood cells or components) from a donor, contacting the cells with an immunoglobulin specific to B7-1, an immunoglobulin specific to B7-2 and recipient cells, thereby obtaining a mixture. The immunoglobulins and the recipient cells are maintained for a period of time sufficient for tolerance induction. The mixture (e.g., bone marrow or blood cell composition) is then introduced into the patient. The recipient cells comprise a lymphocyte antigen (e.g. lymphocytes that express class 1 antigens (MHCI) or peripheral blood lymphocyte (PBL)). Instead of using recipient cells, the method also comprise utilizing tissue, organs or cells that express MHC Class I antigens, B7-1 and/or B7-2 molecules. The cells can be engineered to express recipient molecules. The cells from the donor can be bone marrow cells or cells/components from blood (e.g., stem cells or immature cells). The B7 immunoglobulins are in contact with the donor bone marrow and the recipient cells for a period of time that is long enough to induce tolerance induction (e.g., about 1 to 48 hours, and, preferably about 36 hours). A patient in need of such a transplant is one who has a disease that is benefitted by or treatable with a bone marrow transplant. Such diseases, for example, are proliferative diseases (e.g. leukemia, lymphoma and cancer), anemia (e.g. sickle-cell anemia, thalassemia, and aplastic anemia) and myeloid dysplasia syndrome (MDS).

The invention includes methods for transplanting bone marrow to a patient having a disease (e.g., proliferative diseases such as leukemia, lymphoma, cancer; anemia (e.g., sickle-cell anemia, thalassemia, and aplastic anemia) and myeloid dysplasia syndrome that is treated with a bone marrow transplant comprising obtaining bone marrow from a donor, and contacting the bone marrow with an immunoglobulin specific to B7-1 and/or an immunoglobulin specific to B7-2 and recipient cells (e.g., lymphocyte). The bone marrow, immunoglobulin(s) and recipient cells are in contact for a period of time sufficient for tolerance induction (e.g., about 1-48 hours, preferably about 36 hours). The method then comprises re-introducing the treated bone marrow to the patient.

Advantages of the invention include the ability to regulate or modulate the B7 costimulatory pathway. Manipulation of this costimulatory pathway with a humanized anti-B7-2 and/or anti-B7-1 antibody provides methods of treatments for various diseases. The humanized B7-2 antibody maintains about the same specificity for B7-2 as the murine 3D1 antibody, but with a reduced immunogenicity in humans. Accordingly, the invention can advantageously be used to treat immune-related diseases/disorders or diseases in which the B7-2 molecule plays an important role. Particularly, the invention relates to methods for treating infectious or autoimmune diseases and methods for modulating the immune response for patients with transplanted organs, tissue or cells.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other embodiments, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying figures.

FIG. 1A is a sequence listing illustrating the heavy chain variable region nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of the murine 3D1 antibody, wherein the amino acid sequences of the CDRs (CDR1, CDR2 and CDR3) are underlined and the first amino acids of the mature chains are double underlined.

FIG. 1B is a sequence listing illustrating the light chain variable region nucleic acid and amino acid sequences (SEQ ID NOS: 3 and 4, respectively) of the murine 3D1 antibody wherein, the nucleic and amino acid sequences of the CDRs (CDR1, CDR2 and CDR3) are underlined and the first amino acids of the mature chains are double underlined.

FIG. 2A is a sequence listing illustrating the heavy chain variable region nucleic acid and amino acid sequences (SEQ ID NOs: 5 and 6, respectively) of the humanized 3D1 antibody, wherein the nucleic and amino acid sequences of the CDRs (CDR1, CDR2 and CDR3) are underlined and the first amino acids of the mature chains are double underlined.

FIG. 2B contains the light chain variable region nucleic acid and amino acid sequences (SEQ ID NOs: 7 and 8, respectively) of the humanized 3D1 antibody, wherein the nucleic and amino acid sequences of CDR1, CDR2 and CDR3. The CDRs are underlined and the first amino acids of the mature chains are double underlined.

FIG. 3 is a graph of competitive binding assays. The graph depicts the results of a competitive binding assay of murine or humanized anti-human B7-2 mAbs to CHO expressing rhB7-2 (CHO/hB7-2) on their surface. Increasing concentrations of unlabelled competitor antibodies were incubated with CHO/hB7-2 cells in the presence of radiolabelled tracer murine anti-human B7-2 mAb and the ratio of bound/free antibody was determined.

FIG. 4 is a graph depicting the results of a direct binding assay of murine or humanized anti-human B7-2 mAbs to CHO/hB7-2 cells. Increasing concentrations of radiolabelled antibodies were incubated with CHO or CHO/hB7-2 cells and the amount of specific antibody bound to the CHO/hB7-2 cells was determined.

FIG. 5 is a graph depicting the results of a T cell proliferation assay. Increasing concentrations of murine or humanized anti-human B7-2 mAbs were added to CD28.sup.+ human T cells stimulated with PMA and CHO/hB7-2 cells and the inhibition of T cell proliferation by these mAbs was determined.

FIG. 6 is a graph depicting the results of a one way mixed lymphocyte reaction (MLR) assay. Fixed concentrations of murine or humanized anti-human B7-2 (IgG2.M3 isotype) or hCTLA41 g were added to a mixture of human responder and stimulator PBLs and the proliferation of the responder PBLs was determined on days 3, 4, and 5 by the addition of radiolabelled thymidine.

FIG. 7 is a graph depicting the results of a one way secondary MLR assay using PBLs from a primary MLR as responders and PBLs from the same or a different individual as in the primary MLR as stimulators. The humanized anti-human B7-2 mAb (IgG2.M3 isotype) was added to the primary MLR only. Proliferation of the responder PBLs in the secondary MLR was determined on days 3, 4, and 5 by the addition of radiolabelled thymidine.

FIG. 8 is a graph depicting the results of a one way secondary MLR assay using PBLs from a primary MLR as responders and PBLs from the same or a different individual as in the primary MLR as stimulators. The humanized anti-human B7-1 and B7-2 mAbs (IgG2.M3 isotype) were added to the primary MLR only. Proliferation of the responder PBLs in the secondary MLR was determined on days 3, 4, and 5 by the addition of radiolabelled thymidine.

FIG. 9 is a graph depicting the anti-tetanus response in non-human primates immunized with tetanus toxoid. Cynomolgus monkeys were immunized with purified tetanus toxoid and treated with humanized anti-B7-1 and humanized anti-B7-2 (IgG2.M3 isotype) antibodies. Serum anti-tetanus antibody titers (IgM & IgG) were measured weekly over a 12 week period.

FIG. 10 is a graph showing the serum concentration of anti-B7-1 and anti-B7-2 (IgG2.M3 isotype) mAbs at various times after administration of an I.V. dose of 10 mg/Kg.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a humanized immunoglobulin having binding specificity for B7-2, comprising an antigen binding region of nonhuman origin and at least a portion of an immunoglobulin of human origin. Preferably, the humanized immunoglobulins can bind B7-2 with an affinity of at least about 10.sup.7 M.sup.-1, preferably at least about 10.sup.8 M.sup.-1, and more preferably at least about 10.sup.9 M.sup.-1. In one embodiment, the humanized immunoglobulin includes an antigen binding region of nonhuman origin which binds B7-2 and a constant region derived from a human constant region. The human constant region can have non-human residues in the framework region. In another embodiment, the humanized immunoglobulin which binds B7-2 comprises a complementarity determining region (one or more) of nonhuman origin and a variable framework region (one or more) of human origin, and optionally, a constant region of human origin. Optionally, the FR region of the immunoglobulin can comprise residues of non-human origin. For example, the humanized immunoglobulin can comprise a heavy chain and a light chain, wherein the light chain comprises a complementarity determining region derived from an antibody of nonhuman origin which binds B7-2 and a framework region derived from a light chain of human origin, and the heavy chain comprises a complementarity determining region derived from an antibody of nonhuman origin which binds B7-2 and a framework region derived from a heavy chain of human origin. Also, the invention, individually or in a functional combination, embodies the light chain, the heavy chain, the variable region, the variable light chain and the variable heavy chain.

The invention relates to a humanized B7-2 antibody that possesses substantially the same binding specificity as the murine B7-2 antibody (e.g., 3D1) from which the humanized antibody is made, but with reduced immunogenicity in primates (e.g., humans). The humanized B7-2 antibody has about a lessor, substantially the same, or greater binding affinity as the murine counterpart. See FIGS. 3 and 4.

Naturally occurring immunoglobulins have a common core structure in which two identical light chains (about 24 kD) and two identical heavy chains (about 55 or 70 kD) form a tetramer. The amino-terminal portion of each chain is known as the variable (V) region, also referred to as the "antigen binding" region, and can be distinguished from the more conserved constant (C) regions of the remainder of each chain. Within the variable region of the light chain is a C-terminal portion known as the J region. Within the variable region of the heavy chain, there is a D region in addition to the J region. Most of the amino acid sequence variation in immunoglobulins is confined to three separate locations in the V regions known as hypervariable regions or complementarity determining regions (CDRs) which are directly involved in antigen binding. The variable region is the portion of the antibody that binds to the antigen. The constant region allows for various functions such as the ability to bind to Fc receptors on phagocytic cells, placental cells, mast cells, etc. The light and heavy chains each have a variable region and a constant region. Accordingly, the invention relates to a humanized immunoglobulin having binding specificity to B7-2. The humanized immunoglobulin comprises a light chain and a heavy chain in which two light chains and two heavy chains form the tetramer.

The variable region further constitutes two types of regions, a framework region (FR) and a complementarity determining region (CDR). CDRs are hypervariable regions that contain most of the amino acid sequence variation in between immunoglobulins. Proceeding from the amino-terminus, these regions are designated CDR1, CDR2 and CDR3, respectively. See FIGS. 1A-1B and 2A-2B. The CDRs are connected by more conserved FRs. Proceeding from the amino-terminus, these regions are designated FR1, FR2, FR3, and FR4, respectively. The locations of CDR and FR regions and a numbering system have been defined by Kabat et al. (Kabat, E. A. et al, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991); Kabat, E. A. Structural Concepts in Immunology and Immunochemistry, Second Edition, Holt, Rinehart and Winston, New York (1976); Kabat, E. A. Sequences of Immunoglobulin Chains: Tabulation and Analysis of Amino Acid Sequences of Precurrsors, V-regions, C-regions, J-Chain and .beta.2-Microglobulins, U.S. Department of Health, Education and Welfare, Public Health Service, (1979); Kabat, E. A. Structural Concepts in Immunology and Immunochemistry, Holt, Rinehart and Winston, New York (1968); Kabat, E. A. Experimental Immunochemistry, Second Edition, Springfield, Thomas (1967). During the process of humanizing an immunoglobulin, one or more of the CDRs from an antibody having specificity for B7-2 from a non-human species is grafted into the FRs of a human antibody. In addition, certain non-human framework substitutes can be made according to the methods described herein. The resulting humanized antibody has CDRs from a non-human species such as a mouse and FRs from a human antibody, whereby the humanized antibody maintains its antigenic specificity and affinity to B7-2.

The invention also relates to a humanized immunoglobulin light chain or a humanized immunoglobulin heavy chain. In one embodiment, the invention relates to a humanized light chain comprising one or more light chain CDRs (e.g., CDR1(SEQ ID NO: 16), CDR2 (SEQ ID NO: 18) and/or CDR3 (SEQ ID NO: 20)) of nonhuman origin and a human light chain framework region (See FIG. 2B). In another embodiment, the invention relates to a humanized immunoglobulin heavy chain comprising one or more heavy chain CDRs (e.g., CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 12), and/or CDR3 (SEQ ID NO: 14)) of nonhuman origin and a human heavy chain framework region (See FIG. 2A). The CDRs can be derived from a nonhuman immunoglobulin such as murine heavy (e.g., SEQ ID NO: 1, FIG. 1A) and light (e.g., SEQ ID NO: 3, FIG. 1B) variable region chains which are specific to B7-2.

The invention also embodies the humanized anti-B7-2 antibody expressed by a cell line deposited with the A.T.C.C., 10801 University Boulevard, Manassas, Va. 02110-2209, on May 5, 1998, A.T.C.C. No: CRL-12524. The cell line which expresses the humanized anti-B7-2 antibody, deposited with the A.T.C.C., is designated as a recombinant CHO cell line (PA-CHO-DUKX-1538) expressing the humanized anti-human B7-2 (CD86) monoclonal antibody (#HF2-3D1) of the IgG2.M3 isotype.

Human immunoglobulins can be divided into classes and subclasses, depending on the isotype of the heavy chain. The classes include IgG, IgM, IgA, IgD and IgE, in which the heavy chains are of the gamma (.gamma.), mu (.mu.), alpha (.alpha.), delta (.delta.) or epsilon (.epsilon.) type, respectively. Subclasses include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, in which the heavy chains are of the .gamma.1, .gamma.2, .gamma.3, .gamma.4, .alpha.1 and .alpha.2 type, respectively. Human immunoglobulin molecules of a selected class or subclass may contain either a kappa (.kappa.) or lambda (.lambda.) light chain. See e.g., Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co, Philadelphia, Pa. (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, Mass. (1984).

The terms "HF2.3D1" and "3D1" refer to a murine immunoglobulin specific to B7-2. The terms "humanized HF2.3D1," "humanized 3D1" and "hu3D1" refer to a humanized immunoglobulin specific to B7-2.

The terms "immunoglobulin" or "antibody" include whole antibodies and biologically functional fragments thereof. Such biologically functional fragments retain at least one antigen binding function of a corresponding full-length antibody (e.g., for B7-2) and preferably, retain the ability to inhibit the interaction of B7-2 with one or more of its receptors (e.g., CD28, CTLA-4). In a preferred embodiment, biologically functional fragments can inhibit binding of B7-2 for manipulation of the co-stimulatory pathway. Examples of biologically functional antibody fragments which can be used include fragments capable of binding to an B7-2, such as single chain antibodies, Fv, Fab, Fab' and F(ab').sub.2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can be used to generate Fab or F(ab').sub.2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding the heavy chain of an F(ab').sub.2 fragment can be designed to include DNA sequences encoding the CH.sub.1 domain and hinge region of the heavy chain. The invention includes single chain antibodies (e.g., a single chain FV) that contain both portions of the heavy and light chains.

The term "humanized immunoglobulin" as used herein refers to an immunoglobulin comprising portions of immunoglobulins of different origin, wherein at least one portion is of human origin. For example, the humanized antibody can comprise portions derived from an immunoglobulin of nonhuman origin with the requisite specificity, such as a mouse, and from immunoglobulin sequences of human origin (e.g., chimeric immunoglobulin). These portions can be joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain). Another example of a humanized immunoglobulin of the invention is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR derived from an antibody of nonhuman origin and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes). Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized immunoglobulin. See, e.g., Cabilly et al, U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al, European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, E. A. et al., European Patent Application No. 0,519,596 A1. See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988)), regarding single chain antibodies.

As embodied in the exemplified antibody of the present invention, the term "humanized immunoglobulin" also refers to an immunoglobulin comprising a human framework, at least one CDR from a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, e.g., at least about 60-90%, preferably at least 95% identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR's, are substantially identical to corresponding parts of one or more native human immunoglobulin sequences. In some instances, the humanized immunoglobulin, in addition to CDRs from a non-human antibody, would include additional non-human residues in the human framework region.

The design of humanized immunoglobulins can be carried out as follows. When an amino acid falls under the following categories, the framework amino acid of a human immunoglobulin to be used (acceptor immunoglobulin) is replaced by a framework amino acid from a CDR-providing non-human immunoglobulin (donor immunoglobulin):

(a) the amino acid in the human framework region of the acceptor immunoglobulin is unusual for human immunoglobulin at that position, whereas the corresponding amino acid in the donor immunoglobulin is typical for human immunoglobulin in that position:

(b) the position of the amino acid is immediately adjacent to one of the CDR's; or

(c) the amino acid is capable of interacting with the CDR's in a tertiary structure immunoglobulin model (see, Queen et al., op. cit., and Co et al., Proc. Natl. Acad. Sci. USA 88, 2869 (1991)).

For a detailed description of the production of humanized immunoglobulins, See Queen et al., op. cit. and Co et al, op. cit. and U.S. Pat. Nos. 5,585,089; 5,693,762, 5,693,761, and 5,530,101.

Usually, the CDR regions in humanized antibodies are substantially identical, and more usually, identical to the corresponding CDR regions in the mouse antibody from which they were derived. Although not usually desirable, it is sometimes possible to make one or more conservative amino acid substitutions of CDR residues without appreciably affecting the binding affinity of the resulting humanized immunoglobulin. Occasionally, substitutions of CDR regions can enhance binding affinity.

Other than for the specific amino acid substitutions discussed above, the framework regions of humanized immunoglobulins are usually substantially identical, and more usually, identical to the framework regions of the human antibodies from which they were derived. Of course, many of the amino acids in the framework region make little or no direct contribution to the specificity or affinity of an antibody. Thus, many individual conservative substitutions of framework residues can be tolerated without appreciable change of the specificity or affinity of the resulting humanized immunoglobulin.

The antigen binding region of the humanized immunoglobulin (the non-human portion) can be derived from an immunoglobulin of nonhuman origin, referred to as a donor immunoglobulin, having specificity for B7-2. For example, a suitable antigen binding region can be derived from the murine HF2.3D1 monoclonal antibody. U.S. Ser. No. 08/101,624, filed on Jul. 26, 1993, Ser. No. 08/109,393, filed Aug. 19, 1993 and Ser. No. 08/147,773, filed Nov. 3, 1993, entitled, "B7-2:CTLA4/CD28 Counter Receptor". See also, Freeman, et al., WO 95/03408, "B7-2: CTLA4/CD 28 Counter Receptor, published on Feb. 2, 1995. Other sources include B7-2-specific antibodies obtained from nonhuman sources, such as rodent (e.g., mouse and rat), rabbit, pig, goat or non-human primate (e.g., monkey) or camelid animals (e.g., camels and llamas).

Additionally, other polyclonal or monoclonal antibodies, such as antibodies which bind to the same or similar epitope as the murine HF2.3D1 antibody, can be made (e.g., Kohler et al., Nature, 256:495-497 (1975); Harlow et al., 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor, N.Y.); and Current Protocols in Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel et al., Eds. (John Wiley & Sons: New York, N.Y.), Chapter 11 (1991)). For example, antibodies can be raised against an appropriate immunogen in a suitable mammal such as a mouse, rat, rabbit, sheep, or camelid. Cells bearing B7-2, membrane fractions containing B7-2, immunogenic fragments of B7-2, and a B7-2 peptide conjugated to a suitable carrier are examples of suita