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Title:
B7-H4 ANTIBODY FORMULATIONS
Document Type and Number:
WIPO Patent Application WO/2019/165077
Kind Code:
A1
Abstract:
The present disclosure provides pharmaceutical compositions comprising antibodies and antigen-binding fragments thereof that specifically bind to human B7-H4 (and optionally cynomolgus monkey, mouse, and/or rat B7-H4). The present disclosure also provides methods for treating disorders, such as cancer, by administering such pharmaceutical compositions.

Inventors:
QUAN, Yong (111 Oyster Point Boulevard, South San Francisco, California, 94080, US)
HUANG, Chin-Yi (111 Oyster Point Boulevard, South San Francisco, California, 94080, US)
GANDA, Harjeet Singh (111 Oyster Point Boulevard, South San Francisco, California, 94080, US)
Application Number:
US2019/018965
Publication Date:
August 29, 2019
Filing Date:
February 21, 2019
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
FIVE PRIME THERAPEUTICS, INC. (111 Oyster Point Boulevard, South San Francisco, California, 94080, US)
International Classes:
C07K16/28; A61P35/00
Domestic Patent References:
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Attorney, Agent or Firm:
STEFFE, Eric K. et al. (Sterne, Kessler Goldstein & Fox P.L.L.C.,1100 New York Avenue, N, Washington District of Columbia, 20005, US)
Download PDF:
Claims:
WHAT IS CLAIMED:

1. A pharmaceutical composition comprising (i) an antibody or antigen-binding

fragment thereof that specifically binds to human B7-H4, (ii) a buffer selected from the group consisting of acetate or citrate, and (iii) a sugar, wherein the pH of the composition is about 4.5 to about 6.

2. A pharmaceutical composition comprising (i) an antibody or antigen-binding

fragment thereof that specifically binds to human B7-H4 and comprises the VH CDR1, VH CDR2, VH CDR3 and VL CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-10, respectively, (ii) a buffer, and (iii) a pH of about 4.5 to about 6.

3. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises no more than 45% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

4. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises about 30% to about 45% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

5. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises no more than 20% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

6. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises about 9% to about 18% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

7. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises no more than 60% of acidic and basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

8. The composition of any one of claims 1-7, wherein composition comprises about 30% to about 40% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

9. The composition of claim 8, wherein the composition comprises about 35% to about 40% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

10. The composition of any one of claims 1-9, wherein the composition comprises about 10% to about 17% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

11. The composition of claim 10, wherein the composition comprises about 11% to about 16% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

12. The composition of any one of claims 1-11, wherein the composition comprises no more than 55% of acidic and basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

13. The composition of any one of claims 1 or 3-12, wherein the antibody or antigen binding fragment thereof comprises the VH CDR1, VH CDR2, VH CDR3 and VL CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-10, respectively.

14. The composition of any one of claims 3-13, wherein the pH of the composition is about 4.5 to about 6.

15. The composition of any one of claims 3-14, wherein the composition comprises a buffer.

16. The composition of claim 2 or 15, wherein the buffer is acetate or citrate.

17. The composition of any one of claims 2-15, wherein the composition further

comprises a sugar.

18. The composition of claim 1 or 17, wherein the sugar is selected from the group

consisting of sucrose, sorbitol, and trehalose.

19. The composition of any one of claims 1, 2, and 15-18, wherein the concentration of the buffer is about 15 to about 25 mM.

20. The composition of claim 19, wherein the concentration of the buffer is about 18 mM to about 22 mM.

21. The composition of claim 20, wherein the concentration of the buffer is about 20 mM.

22. The composition of any one of claims 1 and 17-21, wherein the concentration of the sugar is about 225 mM to about 300 mM.

23. The composition of claim 22, wherein the concentration of the sugar is about 250 mM to about 290 mM.

24. The composition of claim 23, wherein the concentration of the sugar is about 270 mM.

25. The composition of any one of claims 1 and 17-24, wherein the concentration of the sugar is about 10 to about 15 times the concentration of the buffer, optionally wherein the concentration of the sugar is about 13.5 times the concentration of the buffer.

26. The composition of any one of claims 1-25, wherein the composition further

comprises a surfactant.

27. The composition of claim 26, wherein the surfactant is polysorbate, optionally

wherein the polysorbate is polysorbate 20.

28. The composition of claim 26 or 27, wherein the concentration of the surfactant is about 0.025% to about 0.075% weight/volume (w/v).

29. The composition of claim 28, wherein the concentration of the surfactant is about 0.035% to about 0.065% weight/volume (w/v).

30. The composition of claim 29, wherein the concentration of the surfactant is about 0.005% weight/volume (w/v).

31. The composition of any one of claims 1-30, wherein the concentration of the antibody or antigen-binding fragment thereof is about 5 mg/ml to about 30 mg/ml.

32. The composition of claim 31, wherein the concentration of the antibody or antigen binding fragment thereof is about 10 to about 25 mg/ml.

33. The composition of claim 32, wherein the concentration of the antibody or antigen binding fragment thereof is about 20 mg/ml.

34. The composition of any one of claims 1-33, wherein the pH is about 5.0 to about 6.0.

35. The composition of any one of claims 1-33, wherein the pH is about 5.

36. The composition of any one of claims 1-33, wherein the pH is about 5.5.

37. The composition of any one of claims 1-36, wherein the composition is a liquid.

38. The composition of any one of claims 1-37, wherein the composition is for parenteral administration.

39. The composition of any one of claims 1-37, wherein the composition is for

intravenous administration.

40. The composition of any one of claims 1-39, wherein the buffer is acetate and the excipient is sucrose.

41. The composition of any one of claims 1-40 comprising about 20 mM acetate, about 270 mM sucrose, about 20 mg/ml of the antibody or antigen-binding fragment thereof, and about 0.05% polysorbate 20, wherein the pH is about 5.0.

42. The composition of any one of claims 1-40 comprising a concentration of sucrose that is about 13.5 times the concentration of acetate, about 20 mg/ml of the antibody or antigen-binding fragment thereof, and about 0.05% polysorbate 20, wherein the pH is about 5.0.

43. The composition of any one of claims 1-39, wherein the buffer is citrate and the

excipient is sucrose.

44. The composition of any one of claims 1-39 or 43, comprising about 20 mM citrate, about 270 mM sucrose, about 20 mg/ml of the antibody or antigen-binding fragment thereof, and about 0.05% polysorbate 20, wherein the pH is about 5.5.

45. The composition of any one of claims 1-39 or 43 comprising a concentration of sucrose that is about 13.5 times the concentration of citrate, about 20 mg/ml of the antibody or antigen-binding fragment thereof, and about 0.05% polysorbate 20, wherein the pH is about 5.5.

46. The composition of any one of claims 1-45, wherein the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:l 1 and/or a VL comprising the amino acid sequence set forth in SEQ ID NO: 12.

47. The composition of claim 46, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2l and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO:22.

48. The composition of any one of claims 1-47, wherein at least 95% of the antibodies or antigen-binding fragments thereof in the composition are afucosylated.

49. The composition of any one of claims 1-47, wherein fucosylation is undetectable in the composition.

50. The composition of any one of claims 1-49 comprising a full length antibody.

51. The composition of any one of claims 1-49 comprising an antigen-binding fragment.

52. The composition of claim 51, wherein the antigen-binding fragment comprises a Fab, Fab’, F(ab’)2, single chain Fv (scFv), disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab’)3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAh2, (scFv)2, or scFv-Fc.

53. The composition of any one of claims 1-52, wherein the antibody or antigen-binding fragment thereof specifically binds to cynomolgus monkey B7-H4.

54. The composition of any one of claims 1-53, wherein the antibody or antigen-binding fragment thereof specifically binds to rat B7-H4.

55. The composition of any one of claims 1-54, wherein the antibody or antigen-binding fragment thereof specifically binds to mouse B7-H4.

56. The composition of any one of claims 1-55, wherein the antibody or antigen-binding fragment thereof specifically binds to the IgV domain of human B7-H4.

57. The composition of any one of claims 1-56, wherein the pi of the antibody or antigen binding fragment thereof is about 8.2.

58. A pharmaceutical composition consisting of (i) an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2l and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO:22, (ii) about 20 mM acetate, (iii) about 270 mM sucrose, and (iv) about 0.05% weight/volume polysorbate 20, wherein the pH of the composition is about 5.0.

59. A pharmaceutical composition consisting of (i) an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2l and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO:22, (ii) about 20 mM citrate, (iii) about 270 mM sucrose, and (iv) about 0.05% weight/volume polysorbate 20, wherein the pH of the composition is about 5.5.

60. A syringe or vial comprising the pharmaceutical composition of any one of claims 1- 59.

61. A method of treating a B7-H4 expressing cancer in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 1- 59.

62. The method of claim 61, wherein the cancer is a solid tumor.

63. The method of claim 61 or 62, wherein the cancer is selected from the group

consisting of breast cancer, ductal carcinoma, endometrial carcinoma, ovarian cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer, and bladder cancer.

64. The method of claim 63, wherein the breast cancer is triple negative breast cancer or hormone receptor positive breast cancer.

65. The method of claim 63, wherein the non-small cell lung cancer is squamous cell carcinoma.

66. The method of any one of claims 61-65, wherein the subject is human.

67. The method of any one of claims 61-66, wherein the pharmaceutical composition is administered parenterally.

68. The method of any one of claims 61-66, wherein the pharmaceutical composition is administered intravenously.

Description:
B7-H4 ANTIBODY FORMULATIONS

1. FIELD

[0001] Pharmaceutical compositions comprising B7-H4 antibodies and methods of using such formulations are provided.

2. BACKGROUND

[0002] B7-H4 (also known as B7x, B7-S1, and VTCN1) is an immune regulatory molecule that shares homology with other B7 family members, include PD-L1. It is a type I transmembrane protein comprised of both IgV and IgC ectodomains. While B7-H4 expression in healthy tissues is relatively limited at the protein level, B7-H4 is expressed in several solid tumors such as gynecological carcinomas of the breast, ovary, and endometrium. Expression of B7-H4 in tumors tends to correlate with poor prognosis. The receptor for B7-H4 is unknown, but it is believed to be expressed on T cells. B7-H4 is believed to directly inhibit T cell activity.

[0003] Given the expression and function of B7-H4, antibodies that specifically bind to B7-H4 are being developed for therapies involving the modulation of B7-H4, e.g., for the treatment of cancer. Accordingly, there is a need for pharmaceutical compositions comprising B7-H4 antibodies and antigen-binding fragments thereof for administration of such treatments.

3. SUMMARY

[0004] Provided herein are pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4.

[0005] In certain aspects, a pharmaceutical composition comprises (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, (ii) a buffer selected from the group consisting of acetate or citrate, and (iii) a sugar, wherein the pH of the composition is about 4.5 to about 6 or is 4.5 to 6.

[0006] In certain aspects, the antibody or antigen-binding fragment thereof comprises the CDRs of 20502.

[0007] In certain aspects, a pharmaceutical composition comprises (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises the CDRs of 20502, (ii) a buffer, and (iii) a pH of about 4.5 to about 6 or 4.5 to 6.

[0008] In certain aspects, the CDRs of 20502 are the Kabat-defmed CDRs, the Chothia- defined CDRs, or the AbM-defmed CDRs. In certain aspects, the antibody or antigen- binding fragment thereof comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-10, respectively.

[0009] In certain aspects, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises no more than 45% of acidic variants of the antibody or antigen binding fragment thereof after 6 months at 5 °C. In certain aspects, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises no more than 40% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises about 35% to about 45% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises 35% to 45% of acidic variants of the antibody or antigen binding fragment thereof after 6 months at 5 °C. In certain aspects, the composition comprises no more than 20% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

[0010] In certain aspects, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises no more than 20% of basic variants of the antibody or antigen binding fragment thereof after 6 months at 5 °C. In certain aspects, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises about 9% to about 18% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises 9% to 18% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the composition comprises no more than 45% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the composition comprises no more than 40% of acidic variants of the antibody or antigen binding fragment thereof after 6 months at 5 °C.

[0011] In certain aspects, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises no more than 60% of acidic and basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the composition comprises no more than 55% of acidic and basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the composition comprises no more than 45% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the composition comprises no more than 40% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the composition comprises no more than 20% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

[0012] In certain aspects, the pharmaceutical composition comprises about 30% to about 40% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the pharmaceutical composition comprises 30% to 40% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the pharmaceutical composition comprises about 35% to about 40% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the pharmaceutical composition comprises 35% to 40% of acidic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

[0013] In certain aspects, the pharmaceutical composition comprises about 10% to about 17% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the pharmaceutical composition comprises 10% to 17% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the pharmaceutical composition comprises about 11% to about 16% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In certain aspects, the pharmaceutical composition comprises 11% to 16% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

[0014] In certain aspects, the composition comprises no more than 55% of acidic and basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

[0015] In certain aspects, the antibody or antigen-binding fragment thereof comprises the CDRs of 20502. In certain aspects, the CDRs of 20502 are the Kabat-defmed CDRs, the Chothia-defmed CDRs, or the AbM-defmed CDRs. In certain aspects, the antibody or antigen-binding fragment thereof comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-10, respectively. [0016] In certain aspects, the pH of the composition is about 4.5 to about 6 or is 4.5 to 6.

[0017] In certain aspects, the composition comprises a buffer. In certain aspects, the buffer is acetate or citrate.

[0018] In certain aspects, the composition further comprises a sugar. In certain aspects, the sugar is selected from the group consisting of sucrose, sorbitol, and trehalose.

[0019] In certain aspects, the concentration of the buffer is about 15 to about 25 mM. In certain aspects, the concentration of the buffer is 15 to 25 mM. In certain aspects, the concentration of the buffer is about 18 mM to about 22 mM. In certain aspects, the concentration of the buffer is 18 mM to 22 mM. In certain aspects, the concentration of the buffer is about 20 mM. In certain aspects, the concentration of the buffer is 20 mM.

[0020] In certain aspects, the concentration of the sugar is about 225 mM to about 300 mM. In certain aspects, the concentration of the sugar is 225 mM to 300 mM. In certain aspects, the concentration of the sugar is about 250 mM to about 290 mM. In certain aspects, the concentration of the sugar is 250 mM to 290 mM. In certain aspects, the concentration of the sugar is about 270 mM. In certain aspects, the concentration of the sugar is 270 mM.

[0021] In certain aspects, the concentration of the sugar is about 10 to about 15 times the concentration of the buffer. In certain aspects, the concentration of the sugar is 10 to 15 times the concentration of the buffer. In certain aspects, the concentration of the sugar is about 13.5 times the concentration of the buffer. In certain aspects, the concentration of the sugar is 13.5 times the concentration of the buffer.

[0022] In certain aspects, the composition further comprises a surfactant. In certain aspects, the surfactant is polysorbate. In certain aspects, the polysorbate is polysorbate 20. In certain aspects, the concentration of the polysorbate is about 0.025% to about 0.075% weight/volume (w/v). In certain aspects, the composition further comprises a surfactant. In certain aspects, the surfactant is polysorbate. In certain aspects, the polysorbate is polysorbate 20. In certain aspects, the concentration of the polysorbate is 0.025% to 0.075% weight/volume (w/v). In certain aspects, the concentration of the polysorbate is about 0.035% to about 0.065% weight/volume (w/v). In certain aspects, the concentration of the polysorbate is 0.035% to 0.065% weight/volume (w/v). In certain aspects, the concentration of the polysorbate is about 0.005% weight/volume (w/v). In certain aspects, the concentration of the polysorbate is 0.005% weight/volume (w/v).

[0023] In certain aspects, the concentration of the antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) is about 5 mg/ml to about 30 mg/ml. In certain aspects, the concentration of the antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) is 5 mg/ml to 30 mg/ml. In certain aspects, the concentration of the antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) is about 10 to about 25 mg/ml. In certain aspects, the concentration of the antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) is 10 to 25 mg/ml. In certain aspects, the concentration of the antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) is about 20 mg/ml. In certain aspects, the concentration of the antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) is 20 mg/ml.

[0024] In certain aspects, the pH of the composition is about 5.0 to about 6.0. In certain aspects, the pH of the composition is 5.0 to 6.0. In certain aspects, the pH is about 5. In certain aspects, the pH is 5. In certain aspects, the pH is about 5.5. In certain aspects, the pH is 5.5.

[0025] In certain aspects, the composition is a liquid. In certain aspects, the composition is for parenteral administration. In certain aspects, the composition is for intravenous administration.

[0026] In certain aspects, the buffer is acetate and the excipient is sucrose. In certain aspects, the composition comprises about 20 mM acetate, about 270 mM sucrose, about 20 mg/ml of the antibody or antigen-binding fragment thereof, and about 0.05% polysorbate 20, wherein the pH is about 5.0. In certain aspects, the composition comprises a concentration of sucrose that is about 13.5 times the concentration of acetate, about 20 mg/ml of the antibody or antigen-binding fragment thereof, and about 0.05% polysorbate 20, wherein the pH is about 5.0. In certain aspects, the composition comprises 20 mM acetate, 270 mM sucrose, 20 mg/ml of the antibody or antigen-binding fragment thereof, and 0.05% polysorbate 20, wherein the pH is 5.0. In certain aspects, the composition comprises a concentration of sucrose that is 13.5 times the concentration of acetate, 20 mg/ml of the antibody or antigen binding fragment thereof, and 0.05% polysorbate 20, wherein the pH is 5.0.

[0027] In certain aspects, the buffer is citrate and the excipient is sucrose. In certain aspects, the composition comprises about 20 mM citrate, about 270 mM sucrose, about 20 mg/ml of the antibody or antigen-binding fragment thereof, and about 0.05% polysorbate 20, wherein the pH is about 5.5. In certain aspects, the composition comprises a concentration of sucrose that is about 13.5 times the concentration of citrate, about 20 mg/ml of the antibody or antigen-binding fragment thereof, and about 0.05% polysorbate 20, wherein the pH is about 5.5. In certain aspects, the composition comprises 20 mM citrate, 270 mM sucrose, 20 mg/ml of the antibody or antigen-binding fragment thereof, and 0.05% polysorbate 20, wherein the pH is 5.5. In certain aspects, the composition comprises a concentration of sucrose that is 13.5 times the concentration of citrate, 20 mg/ml of the antibody or antigen binding fragment thereof, and 0.05% polysorbate 20, wherein the pH is 5.5.

[0028] In certain aspects, the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 11 and/or a VL comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2l and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO:22.

[0029] In certain aspects, at least 95% of the antibodies or antigen-binding fragments thereof in the composition are afucosylated. In certain aspects, fucosylation is undetectable in the composition.

[0030] In certain aspects, the composition comprises a full length antibody.

[0031] In certain aspects, the composition comprises an antigen-binding fragment. In certain aspects, the antigen-binding fragment comprises a Fab, Fab’, F(ab’) 2 , single chain Fv (scFv), disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab’) 3 , tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAh 2 , (scFv) 2 , or scFv- Fc.

[0032] In certain aspects, the antibody or antigen-binding fragment thereof specifically binds to cynomolgus monkey B7-H4. In certain aspects, the antibody or antigen-binding fragment thereof specifically binds to rat B7-H4. In certain aspects, the antibody or antigen binding fragment thereof specifically binds to mouse B7-H4.

[0033] In certain aspects, the antibody or antigen-binding fragment thereof specifically binds to the IgV domain of human B7-H4.

[0034] In certain aspects, the pi of the antibody or antigen-binding fragment thereof is about 8.2. In certain aspects, the pi of the antibody or antigen-binding fragment thereof is 8 2

[0035] In certain aspects, a pharmaceutical composition consists of (i) an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2l and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO:22, (ii) about 20 mM acetate, (iii) about 270 mM sucrose, and (iv) about 0.05% weight/volume polysorbate 20, wherein the pH of the composition is about 5.0. In certain aspects, a pharmaceutical composition consists of (i) an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2l and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO:22, (ii) 20 mM acetate, (iii) 270 mM sucrose, and (iv) 0.05% weight/volume polysorbate 20, wherein the pH of the composition is 5.0.

[0036] In certain aspects, a pharmaceutical composition consists of (i) an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2l and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO:22, (ii) about 20 mM citrate, (iii) about 270 mM sucrose, and (iv) about 0.05% weight/volume polysorbate 20, wherein the pH of the composition is about 5.5. In certain aspects, a pharmaceutical composition consists of (i) an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2l and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO:22, (ii) 20 mM citrate, (iii) 270 mM sucrose, and (iv) 0.05% weight/volume polysorbate 20, wherein the pH of the composition is 5.5.

[0037] In certain aspects, a syringe or vial comprises a pharmaceutical composition provided herein.

[0038] In certain aspects, a method of treating a B7-H4 expressing cancer in a subject comprises administering to the subject a pharmaceutical composition provided herein. In certain aspects, the cancer is a solid tumor. In certain aspects, the cancer is selected from the group consisting of breast cancer, ductal carcinoma, endometrial carcinoma, ovarian cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer, and bladder cancer. In certain aspects, the breast cancer is triple negative breast cancer or hormone receptor positive breast cancer. In certain aspects, the non-small cell lung cancer is squamous cell carcinoma.

[0039] In certain aspects, the subject is human.

[0040] In certain aspects, the pharmaceutical composition is administered parenterally. In certain aspects, the pharmaceutical composition is administered intravenously.

4. BRIEF DESCRIPTION OF THE FIGURES

[0041] FIG. 1 shows the unfolding temperature (Tml) of the B7-H4 antibody“20502” (afucosylated) under different pH conditions measured by the UNit system. (See Example 3.) [0042] FIG. 2 shows the impact of buffer pH on aggregate formation of the B7-H4 antibody“20502” (afucosylated) at 40°C as determined by size-exclusion high-performance liquid chromatography (SE-HPLC). The formulation for each pH is listed in Table 13. The percentage of high molecular weight (HMW) was about 0 at TO for all pHs tested. (See Example 3.)

[0043] FIG. 3 shows the impact of buffer pH on fragment formation at 40°C as determined by SE-HPLC. The formulation for each pH is listed in Table 13. The percentage of low molecular weight (LMW) was about 0 at TO for all pHs tested. (See Example 3.) [0044] FIG. 4 shows the impact of buffer pH on aggregate formation (as determined by SE-HPLC) at 40°C in formulations containing 20mM citrate, 270mM sucrose, and 0.05% polysorbate 20 (PS20). (See Example 4.)

[0045] FIG. 5 shows the impact of buffer pH on fragment formation (as determined by SE-HPLC) at 40°C in formulations containing 20mM citrate, 270mM sucrose, and 0.05% PS20. The percentage of LMW was about 0 at TO for all pHs tested. (See Example 4.)

[0046] FIG. 6 shows the impact of buffer pH on acidic variants (as determined by Imaged Capillary Isoelectric Focusing (iCE)) at 40°C. (See Example 4.)

[0047] FIG. 7 shows the impact of buffer types on aggregate formation (as determined by

SE-HPLC) at 40°C. (See Example 5.)

[0048] FIG. 8 shows the impact of buffer types on fragment formation (as determined by SE-HPLC) at 40°C. (See Example 5.)

[0049] FIG. 9 shows the impact of buffer types on acidic variants formation (as determined by iCE) at 40°C. (See Example 5.)

[0050] FIG. 10 shows the impact of buffer types on basic variants formation (as determined by iCE) at 40°C. (See Example 5.)

[0051] FIG. 11 shows the impact of excipients on aggregate formation (as determined by SE-HPLC) at 40°C. (See Example 6.)

[0052] FIG. 12 shows the impact of excipients on fragment formation (as determined by SE-HPLC) at 40°C. (See Example 6.)

[0053] FIG. 13 shows the impact of excipients on acidic variants (as determined by iCE) at 40°C. (See Example 6.)

[0054] FIG. 14 shows the impact of excipients on basic variants (as determined by iCE) at 40°C. (See Example 6.)

[0055] FIG. 15 shows the Differential Scanning Calorimetry (DSC) profile of 20502 (afucosylated) in selected formulation. (See Example 6.)

5. DETAILED DESCRIPTION

[0056] Provided herein are pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4. The pharmaceutical compositions can be stable e.g., under long-term storage conditions, through repeated freeze-thaw cycles (e.g., at least 5 cycles), and/or through agitation. [0057] As provided herein, a pharmaceutical composition comprising a B7-H4 antibody or antigen-binding fragment thereof can have a pH of about 4.5 to about 6, a B7-H4 antibody or antigen-binding fragment thereof (e.g., at a concentration of about 5 to about 25 mg/ml), a buffer (including but not limited to acetate or citrate), an excipient (including but not limited to sucrose, trehalose, and sorbitol), and/or a surfactant (including but not limited to polysorbate, e.g., polysorbate 20 (PS20)).

[0058] In a particular embodiment, a liquid aqueous pharmaceutical composition containing 20 mg/mL of an anti-B7-H4 antibody (e.g., afucosylated antibody 20502) in 20 mM acetate, 270 mM sucrose, and 0.05% PS20 with a pH of 5.0 is provided herein. In another particular embodiment, a liquid aqueous pharmaceutical composition containing 20 mg/mL of an anti-B7-H4 antibody (e.g., afucosylated antibody 20502) in 20 mM citrate, 270 mM sucrose, and 0.05% PS20 with a pH of 5.5 is provided herein.

[0059] The pharmaceutical compositions provided herein can be useful for treating conditions such as cancer.

5.1 Terminology

[0060] As used herein, the term“B7-H4” refers to mammalian B7-H4 polypeptides including, but not limited to, native B7-H4 polypeptides and isoforms of B7-H4 polypeptides. “B7-H4” encompasses full-length, unprocessed B7-H4 polypeptides as well as forms of B7- H4 polypeptides that result from processing within the cell. A“B7-H4 polynucleotide,”“B7- H4 nucleotide,” or“B7-H4 nucleic acid” refer to a polynucleotide encoding B7-H4.

[0061] The term“antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term“antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three- dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc. [0062] The term“antibody fragment” refers to a portion of an intact antibody. An “antigen-binding fragment,”“antigen-binding domain,” or“antigen-binding region,” refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can contain an antigenic recognition site of an intact antibody (e.g., complementarity determining regions (CDRs) sufficient to specifically bind antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, and single chain antibodies. An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.

[0063] The terms“anti-B7-H4 antibody,”“B7-H4 antibody” and“antibody that binds to B7-H4” refer to an antibody that is capable of specifically binding B7-H4 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting B7-H4. As used herein, the terms“specifically binding,”“immunospecifically binding,” “immunospecifically recognizing,” and“specifically recognizing” are analogous terms in the context of antibodies or antigen-binding fragments thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain and that the binding entails some complementarity between the antigen binding domain and the epitope. Accordingly, an antibody that“specifically binds” to human B7-H4 (SEQ ID NO: l) may also bind to B7-H4 from other species (e.g., cynomolgus monkey, mouse, and/or rat B7-H4) and/or B7-H4 proteins produced from other human alleles, but the extent of binding to an un-related, non-B7-H4 protein (e.g., other B7 protein family members such as PD-L1) is less than about 10% of the binding of the antibody to B7-H4 as measured, e.g., by a radioimmunoassay (RIA). In a specific embodiment, an antibody or antigen binding fragment thereof for use in a formulation provided herein specifically binds to human, cynomolgus monkey, mouse, and rat B7-H4.

[0064] A “monoclonal” antibody or antigen-binding fragment thereof refers to a homogeneous antibody or antigen-binding fragment population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal” antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore,“monoclonal” antibody or antigen- binding fragment thereof refers to such antibodies and antigen-binding fragments thereof made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0065] As used herein, the terms “variable region” or“variable domain” are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino- terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0066] The terms“VL” and“VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

[0067] The terms“VH” and“VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.

[0068] The term“Rabat numbering” and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof. In certain aspects, CDRs can be determined according to the Rabat numbering system (see, e.g., Rabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Rabat EA et ak, (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, ET.S. Department of Health and Human Services, NIH Publication No. 91-3242). ETsing the Rabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Rabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). ETsing the Rabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0069] Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35 A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software.

Loop Kabat AbM Chothia

LI L24-L34 L24-L34 L24-L34

L2 L50-L56 L50-L56 L50-L56

L3 L89-L97 L89-L97 L89-L97

Hl H31-H35B H26-H35B H26-H32..34

(Kabat Numbering)

Hl H31-H35 H26-H35 H26-H32

(Chothia Numbering)

H2 H50-H65 H50-H58 H52-H56

H3 H95-H102 H95-H102 H95-H102

[0070] As used herein, the term “constant region” and “constant domain” are interchangeable and have their common meanings in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain. In certain aspects, an antibody or antigen-binding fragment comprises a constant region or portion thereof that is sufficient for antibody- dependent cell-mediated cytotoxicity (ADCC). [0071] As used herein, the term“heavy chain” when used in reference to an antibody can refer to any distinct type, e.g., alpha (a), delta (d), epsilon (e), gamma (g), and mu (m), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGl, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In specific embodiments, the heavy chain is a human heavy chain.

[0072] As used herein, the term“light chain” when used in reference to an antibody can refer to any distinct type, e.g., kappa (K) or lambda (l) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.

[0073] The term“chimeric” antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof wherein the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragments thereof derived from one species of mammals (e.g. mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies or antigen-binding fragments thereof derived from another (usually human) to avoid eliciting an immune response in that species.

[0074] The term“humanized” antibody or antigen-binding fragment thereof refers to forms of non-human (e.g. murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen binding fragments thereof are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (“CDR grafted”) (Jones et ah, Nature 321 :522-525 (1986); Riechmann et ah, Nature 332:323-327 (1988); Verhoeyen et ah, Science 239: 1534-1536 (1988)). In some instances, certain Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody or fragment from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody or antigen-binding fragment thereof can be further modified by the substitution of additional residues either in the Fv framework region and/or within the non-human CDR residues to refine and optimize antibody or antigen-binding fragment thereof specificity, affinity, and/or capability. In general, the humanized antibody or antigen-binding fragment thereof will comprise variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 9l(3):969-973 (1994), and Roguska et al., Protein Eng. 9(l0):895-904 (1996). In some embodiments, a“humanized antibody” is a resurfaced antibody.

[0075] The term “human” antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin gene locus, where such antibody or antigen-binding fragment is made using any technique known in the art. This definition of a human antibody or antigen binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0076] An“afucosylated” antibody or antigen-binding fragment thereof or an antibody or antigen-binding fragment thereof“lacking fucose” refers to an IgGl or IgG3 isotype antibody or antigen-binding fragment thereof that lacks fucose in its constant region glycosylation. Glycosylation of human IgGl or IgG3 occurs at Asn297 as core fucosylated biantennary complex oligosaccharide glycosylation terminated with up to 2 Gal residues. In some embodiments, an afucosylated antibody lacks fucose at Asn297. These structures are designated as GO, Gl (a 1,6 or a 1,3), or G2 glycan residues, depending on the amount of terminal Gal residues. See, e.g., Raju, T. S., BioProcess Int. 1 : 44-53 (2003). CHO type glycosylation of antibody Fc is described, e.g., in Routier, F. FL, Gly coconjugate J. 14: 201- 207 (1997).

[0077] Methods of measuring fucose include any methods known in the art. For purposes herein, fucose is detected by the method described in Example 1 of W02015/017600, which is herein incorporated by reference in its entirety. Briefly, glycan analysis is performed by releasing glycans from the antibody (e.g., by enzymatic release), labeling the gly cans with anthranilic acid (2-AA), and then purifying the labeled glycans. Normal phase HPLC with fluorescent detection is used to separate the glycans and measure the relative amount of each glycan in the antibody. The glycans may be positively identified as lacking or including fucose by mass spectrometry. In some embodiments, fucose is undetectable in a composition comprising a plurality of afucosylated antibodies or antigen-binding fragments thereof. In some embodiments, an afucosylated antibody or antigen-binding fragment thereof has enhanced ADCC activity, which may be measured by the assay provided in Example 12 herein. In some embodiments, an afucosylated antibody or antigen-binding fragment thereof has enhanced affinity for Fc gamma RIIIA. In some embodiments, an afucosylated antibody or antigen-binding fragment thereof has enhanced affinity for Fc gamma RIIIA(Vl58). In some embodiments, an afucosylated antibody or antigen-binding fragment thereof has enhanced affinity for Fc gamma RIIIA(Fl58). Affinity for Fc gamma RIIIA or its alleles may be measure by the assay provided in Example 10 herein.

[0078] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody or antigen-binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of k 0ff /k 0n , whereas KA is calculated from the quotient of k 0n /k 0ff. k on refers to the association rate constant of, e.g., an antibody or antigen-binding fragment thereof to an antigen, and k 0ff refers to the dissociation of, e.g., an antibody or antigen-binding fragment thereof from an antigen. The k on and k 0ff can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA.

[0079] As used herein, an“epitope” is a term in the art and refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody or antigen-binding fragment thereof specifically binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giege R et ak, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251 : 6300-6303). Antibody/antigen-binding fragment thereof: antigen crystals can be studied using well known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale ETniversity, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff EtW et al.,; U.S. 2004/0014194), and BETSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies can be accomplished using any method known to one of skill in the art. See, e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0080] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. As used herein, “substantially pure” refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0081] The terms“polypeptide,”“peptide,” and“protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains.

[0082] As used herein, the term“host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In specific embodiments, the term“host cell” refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0083] The term“pharmaceutical formulation” or“pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The formulation can be sterile.

[0084] The term “drug product” refers to a finished dosage form, e.g., a liquid formulation containing a drug substance, generally, but not necessarily, in association with one or more other ingredients.

[0085] The term“drug substance” refers to an active ingredient, e.g., a B7-H4 antibody or antigen-binding fragment thereof (e.g., afucosylated antibody 20502) that is intended to furnish pharmacological or biological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, but does not include intermediates used in the synthesis of such ingredient.

[0086] As used herein,“buffer” refers to a component in a solution that allows the solution to resist changes in pH b. Buffers include, for example, acetate, citrate, succinate, and histidine.

[0087] A“stable” formulation is one in which the active ingredient (e.g., a B7-H4 antibody or antigen-binding fragment thereof) therein substantially retains its physical stability and/or chemical stability and/or biological activity upon storage. Stability can be measured at a selected condition (e.g., temperature) for a selected time period. Formulations provided herein can be stable at room temperature (about 25° C) for at least 6 months and/or stable at about 2-8° C for at least 1 year. Formulations provided herein can also be stable following freezing (to, e.g., -70° C.) and thawing of the formulation, hereinafter referred to as a“freeze/thaw cycle.” Formulations provided herein can also be stable after agitation.

[0088] The terms“administer”,“administering”,“administration , and the like, as used herein, refer to methods that may be used to enable delivery of a drug, e.g., an anti-B7-H4 antibody or antigen-binding fragment thereof to the desired site of biological action (e.g., intravenous administration). Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington’s, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0089] As used herein, the terms“subject” and“patient” are used interchangeably. The subject can be an animal. In some embodiments, the subject is a mammal such as a non human animal (e.g., cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some embodiments, the subject is a cynomolgus monkey. In some embodiments, the subject is a human.

[0090] The term“therapeutically effective amount” refers to an amount of a drug, e.g., an anti-B7-H4 antibody or antigen-binding fragment thereof effective to treat a disease or disorder in a subject. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumor size or burden; inhibit, to some extent, cancer cell infiltration into peripheral organs; inhibit, to some extent, tumor metastasis; inhibit, to some extent, tumor growth; relieve, to some extent, one or more of the symptoms associated with the cancer; and/or result in a favorable response such as increased progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), or any combination thereof. To the extent the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic.

[0091] Terms such as“treating,”“treatment,”“to treat,”“alleviating,” and“to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a pathologic condition or disorder. Thus, those in need of treatment include those already diagnosed with or suspected of having the disorder. In certain embodiments, a subject is successfully“treated” for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non- tumorigenic state; increased progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), or any combination thereof.

[0092] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, gynecological cancers (e.g., breast cancer (including triple negative breast cancer, ductal carcinoma, ovarian cancer, and endometrial cancer), non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma) and bladder cancer (e.g., urothelial cell carcinoma). The cancer can be a“cancer that expresses B7-H4” or a“B7-H4 expressing cancer.” Such terms refer to a cancer comprising cells that express B7-H4. The cancer may be a primary tumor or may be advanced or metastatic cancer.

[0093] A“refractory” cancer is one that progresses even though an anti-tumor treatment, such as a chemotherapy, is administered to the cancer patient.

[0094] A“recurrent” cancer is one that has regrown, either at the initial site or at a distant site, after a response to initial therapy.

[0095] A“relapsed” patient is one who has signs or symptoms of cancer after remission. Optionally, the patient has relapsed after adjuvant or neoadjuvant therapy.

[0096] As used in the present disclosure and claims, the singular forms“a,”“an,” and “the” include plural forms unless the context clearly dictates otherwise.

[0097] It is understood that wherever embodiments are described herein with the language“comprising,” otherwise analogous embodiments described in terms of“consisting of’ and/or“consisting essentially of’ are also provided. In this disclosure,“comprises,” “comprising,”“containing” and“having” and the like can have the meaning ascribed to them in U.S. patent law and can mean“ includes,”“including,” and the like;“consisting essentially of’ or“consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited are not changed by the presence of more than that which is recited, but excludes prior art embodiments

[0098] Unless specifically stated or obvious from context, as used herein, the term“or” is understood to be inclusive. The term“and/or” as used in a phrase such as“A and/or B” herein is intended to include both“A and B,”“A or B,”“A,” and“B.” Likewise, the term“and/or” as used in a phrase such as“A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0099] As used herein, the terms“about” and“approximately,” when used to modify a numeric value or numeric range, indicate that deviations of 5% to 10% above and 5% to 10% below the value or range remain within the intended meaning of the recited value or range. [00100] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

5.2 Pharmaceutical Compositions Comprising B7-H4 Antibodies

[00101] Provided herein are pharmaceutical compositions (e.g., aqueous pharmaceutical compositions) comprising an anti-B7-H4 antibody or an antigen-binding fragment thereof (e.g., as discussed in Section 5.3 below).

[00102] In certain embodiments, a pharmaceutical composition provided herein is stable to multiple freeze-thaw cycles. A freeze-thaw cycle can comprise freezing the pharmaceutical composition (e.g., at a temperature of about -70°C) and then thawing the pharmaceutical composition (e.g., at room temperature). The pharmaceutical composition can be stable through at least five freeze-thaw cycles. The freeze-thaw cycles (e.g., the at least five freeze- thaw cycles) can result in no change in appearance, soluble aggregates, or subvisible particulate matter.

[0103] In certain embodiments, a pharmaceutical composition provided herein is stable through agitation. The agitation can comprise shaking (e.g. at about 300 rotations per minute on an orbital shaker) for about three days at room temperature. The agitation can result in no change in appearance, soluble aggregates, charge variant profiles, or subvisible particulate matter.

[0104] In certain embodiments, a pharmaceutical composition provided herein is stable under long-term storage conditions. The long-term storage conditions can comprise storage at about 5°C (e.g., about 2°C to about 8°C) for about 6 months or about 1 year. The long term storage conditions can comprise storage at about 25°C for about 6 months or about 1 year. The long-term storage conditions can comprise storage at about 40°C for about 3 months, about 6 months or about 1 year.

[0105] In certain embodiments, a pharmaceutical composition provided herein is stable to multiple (e.g., at least five) freeze-thaw cycles, stable through agitation, and/or stable under long-term storage conditions.

[0106] In certain embodiments, a pharmaceutical composition provided herein is stable when stored at about -70°C and when stored at about 2°C to about 8°C for about 1 year.

[0107] In certain embodiments, the pharmaceutical composition can contain an B7-H4 antibody or an antigen-binding fragment thereof. In certain embodiments, the concentration of the B7-H4 antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) in the formulation is about 5 mg/ml to about 30 mg/ml. In certain embodiments, the concentration of the B7-H4 antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) in the pharmaceutical composition is about 10 mg/ml to about 25 mg/ml. In certain embodiments, the concentration of the B7-H4 antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) in the pharmaceutical composition is about 20 mg/ml.

[0108] In certain embodiments, the concentration of the B7-H4 antibody or antigen binding fragment thereof (including acidic and basic variants thereof) in the formulation is 5 mg/ml to 30 mg/ml. In certain embodiments, the concentration of the B7-H4 antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) in the pharmaceutical composition is 10 mg/ml to 25 mg/ml. In certain embodiments, the concentration of the B7-H4 antibody or antigen-binding fragment (including acidic and basic variants thereof) thereof in the pharmaceutical composition is 20 mg/ml.

[0109] As provided herein, the pharmaceutical composition can contain a buffer. In certain embodiments, the buffer is acetate. In certain embodiments, the buffer is citrate. In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is about 15 mM to about 25 mM. In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is about 18 mM to about 22 mM. In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is about 20 mM.

[0110] In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is 15 mM to 25 mM. In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is 18 mM to 22 mM. In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is 20 mM.

[0111] As provided herein, the pharmaceutical composition can contain an excipient, for example, a sugar such as sucrose, sorbitol, or trehalose. In some embodiments, the concentration of the excipient (e.g., sucrose) is about 225 mM to about 300 mM. In some embodiments, the concentration of the excipient (e.g., sucrose) is about 250 mM to about 290 mM. In some embodiments, the concentration of the excipient (e.g., sucrose) is about 270.

[0112] In some embodiments, the concentration of the excipient (e.g., sucrose) is 225 mM to mM. In some embodiments, the concentration of the excipient (e.g., sucrose) is 250 mM to 290 mM. In some embodiments, the concentration of the excipient (e.g., sucrose) is 270.

[0113] As provided herein, the pharmaceutical composition can contain a buffer (e.g. acetate or citrate) and an excipient such as a sugar (e.g., sucrose). In some embodiments, the concentration of the excipient such as a sugar (e.g., sucrose) is about 10 to about 15 times the concentration of the buffer (e.g., acetate or citrate). In some embodiments, the concentration of the excipient such as a sugar (e.g., sucrose) is about 13.5 times the concentration of the buffer (e.g., acetate or citrate).

[0114] In some embodiments, the concentration of the excipient such as a sugar (e.g., sucrose) is 10 to 15 times the concentration of the buffer (e.g., acetate or citrate). In some embodiments, the concentration of the excipient such as a sugar (e.g., sucrose) is 13.5 times the concentration of the buffer (e.g., acetate or citrate).

[0115] As provided herein, the pharmaceutical composition can contain a surfactant, for example, a polysorbate. The polysorbate can be, e.g., polysorbate 20 (PS20). In some embodiments, the concentration of the surfactant (e.g., PS20) is about 0.025-0.075% weight by volume (w/v). In some embodiments, the concentration of the surfactant (e.g., PS20) is about 0.035 to about 0.065% w/v. In some embodiments, the concentration of the surfactant (e.g., PS20) is about 0.05% w/v.

[0116] In some embodiments, the concentration of the surfactant (e.g., PS20) is 0.025- 0.075% weight by volume (w/v). In some embodiments, the concentration of the surfactant (e.g., PS20) is 0.035 to 0.065% w/v. In some embodiments, the concentration of the surfactant (e.g., PS20) is 0.05% w/v.

[0117] As provided herein, in some embodiments, the pharmaceutical composition has a pH of about 4.5 to about 6. In some embodiments, the pH of the pharmaceutical composition is about 5 to about 6. In some embodiments, the pH of the pharmaceutical composition is about 5. In some embodiments, the pH of the pharmaceutical composition is about 5.5. In some embodiments, the pH of the pharmaceutical composition is about 6.

[0118] In some embodiments, the pharmaceutical composition has a pH of 4.5 to 6. In some embodiments, the pH of the pharmaceutical composition is 5 to 6. In some embodiments, the pH of the pharmaceutical composition is 5. In some embodiments, the pH of the pharmaceutical composition is 5.5. In some embodiments, the pH of the pharmaceutical composition is 6.

[0119] As provided herein, the pharmaceutical composition can be a liquid. The pharmaceutical composition (e.g., liquid pharmaceutical composition) can be for parenteral administration, e.g., for intravenous administration.

[0120] In one embodiment, the pharmaceutical composition comprises about 5 mg/mL to about 30 mg/mL of a B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in about 15 mM to about 25 mM acetate, about 225 mM to about 300 mM sucrose, and about 0.025% to about 0.075% PS20. In one embodiment, the pharmaceutical composition has a pH of about 4.5 to about 6, e.g., about 5. In one embodiment, the pharmaceutical composition is a liquid.

[0121] In one embodiment, the pharmaceutical composition comprises 5 mg/mL to 30 mg/mL of a B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in 15 mM to 25 mM acetate, 225 mM to 300 mM sucrose, and 0.025% to 0.075% PS20. In one embodiment, the pharmaceutical composition has a pH of 4.5 to 6, e.g., 5. In one embodiment, the pharmaceutical composition is a liquid.

[0122] In one embodiment, the pharmaceutical composition comprises about 10 mg/mL to about 25 mg/mL of a B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in about 18 mM to about 22 mM acetate, about 250 mM to about 290 mM sucrose, and about 0.035% to about 0.065% PS20. In one embodiment, the pharmaceutical composition has a pH of about 4.5 to about 6, e.g., about 5. In one embodiment, the pharmaceutical composition is a liquid.

[0123] In one embodiment, the pharmaceutical composition comprises 10 mg/mL to 25 mg/mL of a B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in 18 mM to 22 mM acetate, 250 mM to 290 mM sucrose, and 0.035% to 0.065% PS20. In one embodiment, the pharmaceutical composition has a pH of 4.5 to 6, e.g., 5. In one embodiment, the pharmaceutical composition is a liquid.

[0124] In one embodiment, the pharmaceutical composition comprises 20 mg/mL of a B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in 20 mM acetate, 270 mM sucrose, and 0.05% PS20. In one embodiment, the pharmaceutical composition has a pH of 5.0. In one embodiment, the pharmaceutical composition is a liquid.

[0125] In one embodiment, the pharmaceutical composition comprises about 5 mg/mL to about 30 mg/mL of a B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in about 15 mM to about 25 mM citrate, about 225 mM to about 300 mM sucrose, and about 0.025% to about 0.075% PS20. In one embodiment, the pharmaceutical composition has a pH of about 4.5 to about 6, e.g. about 5.5. In one embodiment, the pharmaceutical composition is a liquid.

[0126] In one embodiment, the pharmaceutical composition comprises 5 mg/mL to 30 mg/mL of a B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in 15 mM to 25 mM citrate, 225 mM to 300 mM sucrose, and 0.025% to 0.075% PS20. In one embodiment, the pharmaceutical composition has a pH of 4.5 to 6, e.g. 5.5. In one embodiment, the pharmaceutical composition is a liquid.

[0127] In one embodiment, the pharmaceutical composition comprises about 10 mg/mL to about 25 mg/mL of a B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in about 18 mM to about 22 mM citrate, about 250 mM to about 290 mM sucrose, and about 0.035% to about 0.065% PS20. In one embodiment, the pharmaceutical composition has a pH of about 4.5 to about 6, e.g. about 5.5. In one embodiment, the pharmaceutical composition is a liquid.

In one embodiment, the pharmaceutical composition comprises 10 mg/mL to 25 mg/mL of a B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in 18 mM to 22 mM citrate, 250 mM to 290 mM sucrose, and 0.035% to 0.065% PS20. In one embodiment, the pharmaceutical composition has a pH of 4.5 to 6, e.g. 5.5. In one embodiment, the pharmaceutical composition is a liquid.

[0128] In one embodiment, the liquid pharmaceutical composition comprises 20 mg/mL of an antibody or fragment thereof (e.g., afucosylated antibody 20502) in 20 mM citrate, 270 mM sucrose, and 0.05% PS20. In one embodiment, the liquid pharmaceutical composition has a pH of 5.5.

[0129] In some embodiments, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 (e.g., afucosylated antibody 20502), wherein the composition comprises no more than 40% of acidic variants of the antibody or antigen-binding fragment thereof and/or no more than 20% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

[0130] In some embodiments, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 (e.g., afucosylated antibody 20502), wherein the composition comprises about 30% to about 45%, about 30% to about 40%, or about 35% to about 40% acidic variants of the antibody or antigen-binding fragment thereof and/or about 11% to about 16%, about 10% to about 17%, or about 9% to aboutl8% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In some embodiments, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 (e.g., afucosylated antibody 20502), wherein the composition comprises 30% to 45%, 30% to 40%, or 35% to 40% acidic variants of the antibody or antigen-binding fragment thereof and/or 11% to 16%, 10% to 17%, or 9% to 18% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

[0131] In some embodiments, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises no more than 60% or 55% of acidic and basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C. In some embodiments, the composition also comprises no more than 40% of acidic variants and/or no more than 20% of basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 °C.

[0132] In some embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-B7-H4 antibodies or antigen binding fragments thereof and a pharmaceutically acceptable carrier. In specific embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-B7-H4 antibodies or antigen-binding fragments e.g., wherein at least 80% of the antibodies in the composition are afucosylated. In specific embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-B7-H4 antibodies or antigen-binding fragments e.g., wherein at least 85% of the antibodies in the composition are afucosylated. In specific embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-B7-H4 antibodies or antigen-binding fragments e.g., wherein at least 90% of the antibodies in the composition are afucosylated. In specific embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-B7-H4 antibodies or antigen-binding fragments e.g., wherein at least 95% of the antibodies in the composition are afucosylated. In specific embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-B7-H4 antibodies or antigen-binding fragments e.g., wherein at least 96% of the antibodies in the composition are afucosylated. In specific embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-B7-H4 antibodies or antigen-binding fragments e.g., wherein at least 97% of the antibodies in the composition are afucosylated. In specific embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-B7-H4 antibodies or antigen-binding fragments e.g., wherein at least 98% of the antibodies in the composition are afucosylated. In specific embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-B7-H4 antibodies or antigen-binding fragments e.g., wherein at least 99% of the antibodies in the composition are afucosylated. In specific embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises afucosylated anti-B7-H4 antibodies or antigen-binding fragments wherein fucose is undetectable in the composition.

[0133] In some embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical composition comprises (i) an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, comprising (a) the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:5-lO, respectively, (b) a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 11 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 12, or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO:2l and a light chain comprising the amino acid sequence of SEQ ID NO:22, and (ii) a pharmaceutically acceptable excipient.

[0134] Also provided herein is a pharmaceutical composition, wherein the pharmaceutical composition comprises (i) antibodies or antigen-binding fragments thereof that specifically bind to human B7-H4 and comprise the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:5-lO, respectively and (ii) a pharmaceutically acceptable excipient, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the antibodies or antigen-binding fragments thereof in the composition are afucosylated. In one embodiment, (i) the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 11 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 12 or (ii) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:2l and a light chain comprising the amino acid sequence of SEQ ID NO:22.

5.3 B7-H4 Antibodies

[0135] Provided herein are pharmaceutical compositions comprising antibodies ( e.g ., monoclonal antibodies, such as chimeric, humanized, or human antibodies) and antigen binding fragments thereof which specifically bind to B7-H4 (e.g., human B7-H4). Exemplary B7-H4 antibodies and antigen-binding fragments thereof that can be used in the pharmaceutical compositions provided herein are known in the art. The amino acid sequences for human, cynomolgus monkey, murine, and rat B7-H4 are known in the art and also provided herein as represented by SEQ ID NOs: l-4, respectively.

Human B7-H4:

MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEP DIKL

SDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNV QLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQP T VVWASQVDQGANF SEV SNTSFELN SENVTMKVV S VLYNVTINNTY SCMIENDIAK AT GDIK VTESEIKRRSHLQLLN SK ASLC V S SFF AIS W ALLPLSP YLMLK (SEQ ID NO: l) Cynomolgus monkey B7-H4:

MASLGQILFWSIISIIFILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEP DIK L SDI VIQ WLKEGVIGL VHEFKEGKDEL SEQDEMFRGRT A VF AD Q VI V GN ASLRLKN V QLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQP T VVWASQVDQGANF SEV SNTSFELN SENVTMKVV S VLYNVTINNTY SCMIENDIAK AT GDIK VTESEIKRRSHLQLLN SK ASLC V S SFL AIS WALLPLAP YLMLK (SEQ ID NO:2)

Murine B7-H4

MA ST GQTTFWSTTNTTTTE AGAT AT IT GF GTSGKFTFTT VTTFT S A GNT GEDGTT , SCTFEPDTKT , NGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNV QLTDAGTYTCYIRTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQP T VAWASQVDQGANF SEV SNTSFELN SENVTMKVV S VLYNVTINNTY SCMIENDIAK AT GDIK VTD SE VKRRS QLQLLN S GP SPC VF S S AF V AGW ALL SL S C CLMLR (SEQ ID NO:3)

Rat B7-H4

MA ST GQTTFWSTTNVTTTE AGATVT ITGFGTSGKTTFTTVTTFTS AGNTGEDGTESCTFEPDTK LN GI VIQ WLKEGIKGL VHEFKEGKDDL S QQHEMFRGRT A VF ADQ V V V GN ASLRLKN VQLTDAGTYTCYIHTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQ PTVAW ASQVDQGANF SEV SNTSFELN SENVTMKVV S VLYNVTINNTYSCMIENDIAK AT GDIK VTD SEVKRRS QLELLN SGPSPCVSSV S A AGW ALL SL S CCLMLR (SEQ ID NO:4)

[0136] In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human and cynomolgus monkey B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human, murine, and rat B7-H4. In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein to human, cynomolgus monkey, murine, and rat B7-H4.

[0137] B7-H4 contains an IgC ectodomain (amino acids 153-241 of SEQ ID NO: l) and an IgV ectodomain (amino acids 35-146 of SEQ ID NO: l). In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to the IgV domain of human B7-H4. Accordingly, provided herein are pharmaceutical compositions comprising antibodies and antigen-binding fragments thereof that specifically bind to a polypeptide consisting of amino acids 35-146 of SEQ ID NO: l .

[0138] In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the six CDRs of the 20502 antibody listed as provided in Tables 1 and 2.

Table 1. VH CDR Amino Acid Sequences 1

Table 2. VL CDR Amino Acid Sequences 2

2 The VL CDRs in Table 2 are determined according to Rabat.

[0139] In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the VH of the 20502 antibody listed in Table 3.

Table 3: Variable Heavy Chain (VH) Amino Acid Sequences

[0140] In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the VL of the 20502 listed in Table 4. Table 4: Variable Light Chain (VL) Amino Acid Sequences

[0141] In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the VH and the VL of the 20502 antibody listed in Tables 3 and 4.

[0142] In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the VH framework regions of the 20502 antibody listed in Table 5.

Table 5. VH FR Amino Acid Sequences 3

3 The VH framework regions described in Table 5 are determined based upon the boundaries of the Kabat numbering system for CDRs. Accordingly, the VH CDRs are determined by Rabat and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0143] In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the VL framework regions of the 20502 antibody listed in Table 6.

Table 6. VL FR Amino Acid Sequences 4

4 The VL fra ework regions described in Table 6 are determined based upon the boundaries of the Kabat numbering system for CDRs. Accordingly, the VL CDRs are determined by Kabat and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. [0144] In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the four VH framework regions and the four VL framework regions of the 20502 antibody listed in Tables 5 and 6.

[0145] In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the heavy chain sequence of the 20502 antibody listed in Table 7.

Table 7: Full-length heavy chain amino acid sequences

[0146] In certain embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the light chain sequence of the 20502 antibody listed in Table 8.

Table 8: Full-length light chain amino acid sequences

[0147] In certain embodiments, an antibody or antigen-binding fragment in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the heavy chain sequence and the light chain sequence of the 20502 antibody listed in Tables 7 and 8.

[0148] In certain aspects, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein is described by its VL domain alone, or its VH domain alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone. See , for example, Rader C et al, (1998) PNAS 95 : 8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of the mouse anti-avP3 antibody by identifying a complementing light chain or heavy chain, respectively, from a human light chain or heavy chain library, resulting in humanized antibody variants having affinities as high or higher than the affinity of the original antibody. See also Clackson T et al. , (1991) Nature 352: 624- 628, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that specifically bind a specific antigen by using a specific VL domain (or VH domain) and screening a library for the complementary VH domain (or VL domain). The screen produced 14 new partners for a specific VH domain and 13 new partners for a specific VL domain, which were strong binders, as determined by ELISA. See also Kim SJ & Hong HJ, (2007) J Microbiol 45: 572-577, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that specifically bind a specific antigen by using a specific VH domain and screening a library ( e.g ., human VL library) for complementary VL domains; the selected VL domains in turn could be used to guide selection of additional complementary (e.g., human) VH domains.

[0149] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g, Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al, (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

[0150] In certain aspects, provided herein are pharmaceutical compositions comprising antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g, human B7-H4) and comprise the Chothia VH and VL CDRs of the 20502 antibody listed in Tables 3 and 4. In certain embodiments, provided herein are pharmaceutical compositions comprising antibodies or antigen-binding fragments thereof that specifically bind to B7-H4 ( e.g ., human B7-H4) and comprise one or more CDRs, in which the Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, provided herein are pharmaceutical compositions comprising antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g., human B7-H4) and comprise combinations of Kabat CDRs and Chothia CDRs.

[0151] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al, (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97. In a particular embodiment, provided herein are pharmaceutical compositions comprising antibodies and antigen-binding fragments thereof that specifically bind to B7-H4 (e.g, human B7-H4) and comprise the IMGT VH and VL CDRs of the 20502 antibody listed in Tables 3 and 4, for example, as described in Lefranc M-P (1999) supra and Lefranc M-P et al, (1999) supra).

[0152] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to MacCallum RM et al, (1996) J Mol Biol 262: 732-745. See also, e.g, Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In a particular embodiment, provided herein are pharmaceutical compositions comprising antibodies or antigen-binding fragments thereof that specifically bind to B7-H4 (e.g, human B7-H4) and comprise VH and VL CDRs of the 20502 antibody listed in Tables 3 and 4 as determined by the method in MacCallum RM et al.

[0153] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers AbM hypervariable regions which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (Oxford Molecular Group, Inc.). In a particular embodiment, provided herein are pharmaceutical compositions comprising antibodies or antigen-binding fragments thereof that specifically bind to B7-H4 (e.g, human B7-H4) and comprise VH and VL CDRs of the 20502 antibody listed in Tables 3 and 4 as determined by the AbM numbering scheme. [0154] In specific aspects, provided herein are pharmaceutical compositions comprising antibodies that comprise a heavy chain and a light chain.

[0155] With respect to the light chain, in a specific embodiment, the light chain of an antibody described herein is a kappa light chain. The constant region of a human kappa light chain can comprise the following amino acid sequence:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQD SKD ST Y SL S S TLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO:23).

[0156] The constant region of a human kappa light chain can be encoded by the following nucleotide sequence:

CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA

AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGC

CAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG

TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC

GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCA

TCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID

NO:24).

[0157] In a particular embodiment, an antibody which immunospecifically binds to a B7- H4 polypeptide ( e.g ., human B7-H4) a pharmaceutical composition described herein comprises a light chain wherein the amino acid sequence of the VL domain comprises the sequence set forth in Table 4, and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region.

[0158] In a particular embodiment, an antibody which immunospecifically binds to B7- H4 (e.g., human B7-H4) in a pharmaceutical composition described herein comprises a heavy chain wherein the amino acid sequence of the VH domain comprises the amino acid sequence set forth in Table 3 and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma (g) heavy chain constant region.

[0159] The constant region of a human IgGi heavy chain can comprise the following amino acid sequence:

AS TKGP S VFPL AP S SK S T S GGT A ALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VL Q S SGL Y SL S SWT VP S S SLGTQT YICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCP AP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPRE PQ VYTLPP SRDELTKNQ V SLT CL VKGF YP SDIAVEWESNGQPENNYKTTPP VLD SDG SFFL Y SKLT VDK SRW Q QGNVF SC S VMHE ALHNH YT QK SL SL SPGK (SEQ ID NO:25).

[0160] The constant region of a human IgGi heavy chain can be encoded by the following nucleotide sequence:

GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCT

CTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG

TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG

CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTC

CAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA

CACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATG

CCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC

CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG

GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC

GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG

CACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC

AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAA

ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC

CCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA

GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG

AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT

ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT

GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT

GTCTCCGGGTAAA. (SEQ ID NO:26)

[0161] In a specific embodiment, an antibody which immunospecifically binds to B7-H4 ( e.g ., human B7-H4) in a pharmaceutical composition described herein comprises a VH domain and a VL domain comprising an amino acid sequence of any VH and VL domain described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of an IgG (e.g., a human IgG) immunoglobulin molecule. In another specific embodiment, an antibody which immunospecifically binds to B7-H4 (e.g., human B7-H4) for use in a pharmaceutical composition described herein comprises a VH domain and a VL domain comprising an amino acid sequence of any VH and VL domain described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of an IgGi (e.g. human IgGi) immunoglobulin molecule.

[0162] Antibodies with reduced fucose content have been reported to have an increased affinity for Fc receptors, such as, e.g, FcyRIIIA Accordingly, in certain embodiments, an antibody or antigen-binding fragment thereof in a pharmaceutical composition described herein has reduced fucose content or lacks fucose (i.e., is“afucosylated”). Such antibodies or antigen-binding fragments thereof can be produced using techniques known to one skilled in the art. For example, they can be expressed in cells deficient or lacking the ability to fucosylate. In a specific example, cell lines with a knockout of both alleles of the al,6- fucosyltransferase gene (FUT8) can be used to produce antibodies or antigen-binding fragments thereof with reduced fucose content. The Potelligent ® system (Lonza) is an example of such a system that can be used to produce antibodies and antigen-binding fragments thereof with reduced fucose content. Alternatively, antibodies or antigen-binding fragments thereof with reduced fucose content or no fucose content can be produced by, e.g. : (i) culturing cells under conditions which prevent or reduce fucosylation; (ii) posttranslational removal of fucose (e.g, with a fucosidase enzyme); (iii) post-translational addition of the desired carbohydrate, e.g, after recombinant expression of a non-glycosylated glycoprotein; or (iv) purification of the glycoprotein so as to select for antibodies or antigen-binding fragments thereof which are not fucosylated. See, e.g, Longmore GD & Schachter H (1982) Carbohydr Res 100: 365-92 and Imai-Nishiya H et ah, (2007) BMC Biotechnol. 7: 84 for methods for producing antibodies thereof with no fucose content or reduced fucose content.

[0163] In some embodiments, an afucosylated B7-H4 antibody or antigen-binding fragment thereof has enhanced ADCC activity in vitro compared to fucosylated B7-H4 antibodies or antigen-binding fragments thereof having the same amino acid sequence. In some embodiments, the afucosylated B7-H4 antibodies or antigen-binding fragments thereof cause specific lysis that is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points greater than specific lysis with fucosylated B7-H4 antibodies.

[0164] In some embodiments, the B7-H4 antibody or antigen-binding fragment thereof has enhanced affinity for Fc gamma RIIIA compared to fucosylated B7-H4 antibodies or antigen-binding fragments thereof having the same amino acid sequence. In some embodiments, the afucosylated B7-H4 antibodies or antigen-binding fragments thereof bind to Fc gamma RIIIA with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least lO-fold, at least l2-fold, at least l5-fold, at least l7-fold, or at least 20-fold greater affinity than fucosylated B7-H4 antibodies or antigen-binding fragments thereof. In some embodiments, affinity for Fc gamma RIIIA is determined using surface plasmon resonance. In some embodiments, Fc gamma RIIIA is selected from Fc gamma RIIIA(Vl58) and Fc gamma RIIIA(Fl58). In some embodiments, Fc gamma RIIIA is Fc gamma RIIIA(Vl58).

[0165] In some embodiments, the presence of fucose can be determined by a method comprising high performance liquid chromatography (HPLC), capillary electrophoresis, or MALDI-TOF mass spectrometry.

[0166] In specific embodiments, an antibody or antigen-binding fragment thereof (i) comprises the CDR sequences of 20502, the VH and VL sequences of 20502, or the heavy and light chain sequences of 20502 and (ii) is afucosylated.

[0167] In specific embodiments, a composition comprises antibodies or antigen-binding fragments thereof that (i) comprises the CDR sequences of 20502, the VH and VL sequences of 20502, or the heavy and light chain sequences of 20502 and (ii) are afucosylated, e.g., wherein at least 95% of the antibodies in the composition are afucosylated or wherein fucosylation is undetectable in the composition.

[0168] Engineered gly coforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Methods for generating engineered glycoforms in an antibody or antigen-binding fragment thereof described herein include but are not limited to those disclosed, e.g., in Umana P et al, (1999) Nat Biotechnol 17: 176-180; Davies J et al, (2001) Biotechnol Bioeng 74: 288-294; Shields RL et al, (2002) J Biol Chem 277: 26733-26740; Shinkawa T et al, (2003) J Biol Chem 278: 3466-3473; Niwa R et al, (2004) Clin Cancer Res 1 : 6248-6255; Presta LG et al, (2002) Biochem Soc Trans 30: 487- 490; Kanda Y et al, (2007) Glycobiology 17: 104-118; U.S. Patent Nos. 6,602,684; 6,946,292; and 7,214,775; U.S. Patent Publication Nos. US 2007/0248600; 2007/0178551; 2008/0060092; and 2006/0253928; International Publication Nos. WO 00/61739; WO 01/292246; WO 02/311140; and WO 02/30954; Potelligent™ technology (Biowa, Inc. Princeton, N.J.); and GlycoMAb® glycosylation engineering technology (Glycart biotechnology AG, Zurich, Switzerland). See also, e.g., Ferrara C et al, (2006) Biotechnol Bioeng 93: 851-861; International Publication Nos. WO 07/039818; WO 12/130831; WO 99/054342; WO 03/011878; and WO 04/065540.

[0169] In certain embodiments, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody or antigen-binding fragment thereof described herein having two heavy chain constant regions.

[0170] In another particular embodiment, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 (e.g, human B7-H4), comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain comprising the VH CDR1, VL CDR2, and VL CDR3 amino acid sequences of the 20502 antibody listed in Table 1; (ii) the light chain comprises a VL domain comprising the VL CDR1, VH CDR2, and VH CDR3 amino acid sequences of the 20502 antibody listed in Table 2; (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgGi heavy chain; and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa light chain.

[0171] In another particular embodiment, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 ( e.g ., human B7-H4), comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain comprising the amino acid sequence of the VH domain of the 20502 antibody listed in Table 3; (ii) the light chain comprises a VL domain comprising the amino acid sequence of the VL domain of the 20502 antibody listed in Table 4; (iii) and the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgGi heavy chain; and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa light chain.

[0172] In specific embodiments, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 (e.g., human B7-H4) exhibits T cell checkpoint blockade activity. In specific embodiments, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 (e.g, human B7-H4) increases interferon-gamma (IFNy) production in T cells. In specific embodiments, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 (e.g, human B7-H4) increases T cell proliferation. In specific embodiments, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 (e.g, human B7-H4) increases CD4+ T cell proliferation. In specific embodiments, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 (e.g, human B7-H4) increases CD8+ T cell proliferation.

[0173] In specific embodiments, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 (e.g, human B7-H4) exhibits antibody-dependent cellular cytotoxicity (ADCC) activity. In specific embodiments, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 (e.g, human B7-H4) exhibits antibody-dependent cellular cytotoxicity (ADCC) activity on cell lines with at least 300,000 cell surface B7-H4 molecules (e.g., SK- BR-3 cells). In specific embodiments, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 ( e.g ., human B7-H4) exhibits antibody-dependent cellular cytotoxicity (ADCC) activity on cell lines with at least 100,000 cell surface B7-H4 molecules (e.g., HCC1569 cells). In specific embodiments, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7- H4 (e.g., human B7-H4) exhibits antibody-dependent cellular cytotoxicity (ADCC) activity on cell lines with at least 50,000 cell surface B7-H4 molecules (e.g., ZR-75-1 cells). In specific embodiments, an antibody or antigen-binding fragment thereof described herein, which immunospecifically binds to B7-H4 (e.g, human B7-H4) exhibits antibody-dependent cellular cytotoxicity (ADCC) activity on cell lines with at least 30,000 cell surface B7-H4 molecules (e.g., MDA-MB-468 cells). In specific embodiments, an antibody or antigen binding fragment thereof described herein, which immunospecifically binds to B7-H4 (e.g, human B7-H4) exhibits antibody-dependent cellular cytotoxicity (ADCC) activity on cell lines with at least 15,000 cell surface B7-H4 molecules (e.g., HCC1964 cells).

[0174] In a specific aspect, an antigen-binding fragment as described herein, which immunospecifically binds to B7-H4 (e.g, human B7-H4), is selected from the group consisting of a Fab, Fab’, F(ab’) 2 , and scFv, wherein the Fab, Fab’, F(ab’) 2 , or scFv comprises a heavy chain variable region sequence and a light chain variable region sequence of an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein. A Fab, Fab’, F(ab’)2, or scFv can be produced by any technique known to those of skill in the art. In certain embodiments, the Fab, Fab’, F(ab’) 2 , or scFv further comprises a moiety that extends the half-life of the antibody in vivo. The moiety is also termed a“half-life extending moiety.” Any moiety known to those of skill in the art for extending the half-life of a Fab, Fab’, F(ab’) 2 , or scFv in vivo can be used. For example, the half-life extending moiety can include a Fc region, a polymer, an albumin, or an albumin binding protein or compound. The polymer can include a natural or synthetic, optionally substituted straight or branched chain polyalkylene, polyalkenylene, polyoxylalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen, or derivative thereof. Substituents can include one or more hydroxy, methyl, or methoxy groups. In certain embodiments, the Fab, Fab’, F(ab’) 2 , or scFv can be modified by the addition of one or more C-terminal amino acids for attachment of the half- life extending moiety. In certain embodiments, the half-life extending moiety is polyethylene glycol or human serum albumin. In certain embodiments, the Fab, Fab’, F(ab’) 2 , or scFv is fused to an Fc region. 5.4 Antibody Production and Polynucleotides

[0175] Antibodies and antigen-binding fragments thereof that immunospecifically bind to B7-H4 ( e.g ., human B7-H4) can be produced by any method known in the art for the synthesis of antibodies and antigen-binding fragments thereof, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See , e.g., Sambrook J el al. , (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al ^ Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al. , (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0176] In certain aspects, provided herein are pharmaceutical compositions comprising an anti-B7-H4 antibody or antigen-binding fragment, wherein the antibodies or fragments are produced by recombinant expression of a polynucleotide comprising a nucleotide sequence in a host cell.

[0177] In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment in the pharmaceutical compositions provided herein comprises a heavy chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 9 (i.e. SEQ ID NO:27). In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment in the pharmaceutical compositions provided herein comprises a heavy chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 9 (i.e. SEQ ID NO:27) and a nucleotide sequence encoding a human gamma (g) heavy chain constant region. In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment in the pharmaceutical compositions provided herein comprises a heavy chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 9 (i.e. SEQ ID NO:27) and a heavy chain constant domain encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO:26. Table 9: Heavy chain variable region-encoding polynucleotide sequences

[0178] In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment in the pharmaceutical compositions provided herein comprises a light chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 10 (i.e., SEQ ID NO:28). In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment in the pharmaceutical compositions provided herein comprises a light chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 10 (i.e. SEQ ID NO:28) and a nucleotide sequence encoding a human lambda light chain constant region. In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment in the pharmaceutical compositions provided herein comprises a light chain variable region encoded by a polynucleotide comprising the nucleotide sequence shown in Table 10 (i.e., SEQ ID NO:28) and a light chain constant domain encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO:24.

Table 10: Light chain variable region-encoding polynucleotide sequences

[0179] In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment in the pharmaceutical compositions provided herein comprises a variable heavy chain encoded by a polynucleotide comprising the variable heavy chain-encoding nucleotide sequence shown in Table 9 (i.e. SEQ ID NO:27) and a variable light chain encoded by a polynucleotide comprising the variable light chain-encoding nucleotide sequence shown in Table 10 (i.e., SEQ ID NO:28). [0180] In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment in the pharmaceutical compositions provided herein comprises (i) a heavy chain encoded by a polynucleotide comprising the variable heavy chain-encoding nucleotide sequence shown in Table 9 (i.e. SEQ ID NO:27) and a nucleotide sequence encoding a human gamma (g) heavy chain constant region and (ii) a light chain encoded by a polynucleotide comprising the variable light chain-encoding nucleotide sequence shown in Table 10 (i.e. SEQ ID NO:28) and a nucleotide sequence encoding a human lambda light chain constant region.

[0181] In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment in the pharmaceutical compositions provided herein comprises (i) a heavy chain encoded by a polynucleotide comprising the variable heavy chain-encoding nucleotide sequence shown in Table 9 (i.e. SEQ ID NO:27) and the heavy chain constant domain-encoding nucleotide sequence of SEQ ID NO:26 and (ii) a light chain encoded by a polynucleotide comprising the variable light chain-encoding nucleotide sequence shown in Table 10 (i.e., SEQ ID NO:28) and the light chain constant domain-encoding nucleotide sequence of SEQ ID NO:24.

[0182] In certain aspects, the anti-B7-H4 antibodies or antigen-binding fragments in the pharmaceutical compositions provided herein are encoded by polynucleotides encoding anti- B7-H4 antibodies or antigen-binding fragments thereof or a domain thereof that are optimized, e.g ., by codon/RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods to generate optimized nucleic acids encoding an anti-B7-H4 antibody or antigen-binding fragment thereof or a domain thereof (e.g, heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., a codon change that encodes the same amino acid due to the degeneracy of the genetic code) and/or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly.

[0183] Polynucleotides can be, e.g., in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA. DNA can be double-stranded or single- stranded. If single stranded, DNA can be the coding strand or non-coding (anti-sense) strand. In certain embodiments, the polynucleotide is a cDNA or a DNA lacking one or more introns. In certain embodiments, a polynucleotide is a non-naturally occurring polynucleotide. In certain embodiments, a polynucleotide is recombinantly produced. In certain embodiments, the polynucleotides are isolated. In certain embodiments, the polynucleotides are substantially pure. In certain embodiments, a polynucleotide is purified from natural components.

[0184] In certain aspects, vectors ( e.g ., expression vectors) comprise nucleotide sequences encoding anti-B7-H4 antibodies and antigen-binding fragments thereof or a domain thereof for recombinant expression in host cells, preferably in mammalian cells. In certain aspects, cells, e.g. host cells, comprise such vectors for recombinantly expressing anti- B7-H4 antibodies or antigen-binding fragment thereof described herein (e.g., human or humanized antibodies or antigen-binding fragments thereof). Thus, a method for producing an antibody or antigen-binding fragments thereof for use in a pharmaceutical composition described herein can comprise expressing such antibody or antigen-binding fragment thereof in a host cell.

[0185] An expression vector can be transferred to a cell (e.g, host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody or antigen-binding fragment thereof described herein (e.g, an antibody or antigen-binding fragment thereof comprising the six CDRs, the VH, the VL, the VH and the VL, the heavy chain, the light chain, or the heavy and the light chain of 20502) or a domain thereof (e.g, the VH, the VL, the VH and the VL, the heavy chain, or the light chain of 20502).

[0186] In certain embodiments, anti-B7-H4 antibodies or antigen-binding fragment thereof (e.g, an antibody or antigen-binding fragment thereof comprising the CDRs of 20502) in the pharmaceutical compositions provided herein are produced in Potelligent® CHOK1SV cells.

[0187] In some embodiments, anti-B7-H4 antibodies or antigen-binding fragments thereof (e.g, an antibody or antigen-binding fragment thereof comprising the CDRs of 20502) in the pharmaceutical compositions provided herein are produced in a host cell that lacks a functional alpha- l,6-fucosyltransferase gene (FUT8) gene. In some embodiments, the host cell is a CHO cell.

[0188] In specific embodiments, an antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein is isolated or purified. Generally, an isolated antibody or antigen-binding fragment thereof is one that is substantially free of other antibodies or antigen-binding fragments thereof with different antigenic specificities than the isolated antibody or antigen-binding fragment thereof. For example, in a particular embodiment, a preparation of an antibody or antigen-binding fragment thereof described herein is substantially free of cellular material and/or chemical precursors. 5.5 Therapeutic Uses and Methods

[0189] In one aspect, provided herein are methods for modulating one or more immune functions in a subject, comprising administering to a subject in need thereof a pharmaceutical composition provided herein comprising a B7-H4 antibody or antigen-binding fragment thereof.

[0190] In another embodiment, a pharmaceutical composition provided herein comprising an anti-B7-H4 antibody or antigen-binding fragment thereof is administered to a patient (e.g., a human patient) to increase the proliferation of T cells, CD4+ T cells, or CD8+ T cells in the patient. In another embodiment, a pharmaceutical composition provided herein comprising an anti-B7-H4 antibody or antigen-binding fragment thereof is administered to a patient (e.g., a human patient) to increase interferon-gamma (IFNy) production in the patient. In another embodiment, a pharmaceutical composition provided herein comprising an anti- B7-H4 antibody or antigen-binding fragment thereof is administered to a patient (e.g., a human patient) to block the inhibitory activity of B7-H4 against T cells in the patient. In another embodiment, a pharmaceutical composition provided herein comprising an anti-B7- H4 antibody or antigen-binding fragment thereof is administered to a patient (e.g., a human patient) to deplete B7-H4 expressing cancer cells in the patient. In another embodiment, a pharmaceutical composition provided herein comprising an anti-B7-H4 antibody or antigen binding fragment thereof is administered to achieve two or more of the above effects.

[0191] In a certain embodiment, provided herein are methods of treating a cancer, e.g., a B7-H4 expressing cancer comprising administering a pharmaceutical composition comprising an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein to a patient (e.g., a human patient) in need thereof. In a certain embodiment, provided herein are methods of treating a solid tumor, e.g., a B7-H4 expressing solid tumor comprising administering a pharmaceutical composition comprising an anti-B7-H4 antibody or antigen binding fragment thereof provided herein to a patient (e.g., a human patient) in need thereof.

[0192] In a certain embodiment, provided herein are pharmaceutical compositions for treating a cancer selected from the group consisting of: breast cancer (e.g., advanced breast cancer, triple negative breast cancer, hormone receptor (HR) positive breast cancer, or ductal carcinoma), endometrial carcinoma, ovarian cancer, urothelial cancer, non-small cell lung cancer (e.g., squamous cell carcinoma), pancreatic cancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma), and bladder cancer (e.g., urothelial cell carcinoma). In a certain embodiment, provided herein are pharmaceutical compositions for treating advanced breast cancer (including triple-negative breast cancer and hormone receptor (HR) positive breast cancer), ovarian cancer, endometrial cancer, or urothelial cancer. In a certain embodiment, provided herein are pharmaceutical compositions for treating a breast cancer. In a certain embodiment, provided herein are pharmaceutical compositions for treating an ovarian cancer. In a certain embodiment, provided herein are pharmaceutical compositions for treating an endometrial cancer. In a certain embodiment, provided herein are pharmaceutical compositions for treating a urothelial cancer.

[0193] In some embodiments, the cancer is a B7-H4 expressing cancer.

[0194] In another embodiment, a pharmaceutical composition provided herein is administered to a patient (e.g., a human patient) diagnosed with cancer to increase the proliferation of T cells, CD4+ T cells, or CD8+ T cells in the patient. In another embodiment, a pharmaceutical composition provided herein is administered to a patient (e.g., a human patient) diagnosed with cancer to increase interferon-gamma (IFNy) production in the patient. In another embodiment, a pharmaceutical composition provided herein is administered to a patient (e.g., a human patient) diagnosed with cancer to block the inhibitory activity of B7-H4 against T cells in the patient. In another embodiment, a pharmaceutical composition provided herein is administered to a patient (e.g., a human patient) diagnosed with cancer to deplete B7-H4 expressing cancer cells in the patient.

[0195] The pharmaceutical compositions described herein can be delivered to a patient by an intravenous route. Usually, the patient is a human but non-human mammals including transgenic mammals can also be treated.

6. EXAMPLES

[0196] The examples in this Section (i.e., Section 6) are offered by way of illustration, and not by way of limitation.

Example 1: Methods Used in the Formulation Studies

I Antibody Production

[0197] 20502 antibodies were generated in a CHO cell line that lacks the FUT8 gene

(al,6-Fucosyltransferase), so the 20502 antibodies used in all of Examples 2-9 are afucosylated. They lack a terminal fucose at ASN297 in the Fc portion of the antibody.

II. General formulation procedure

[0198] Samples of monoclonal antibody (mAb) were prepared in various formulations by dialysis of the polysorbate-free drug substance (afucosylated 20502) using 20 kD molecular weight cut off (MWCO) dialysis membrane devices. After dialysis, concentration of mAbs was measured by UV spectroscopy using extinction coefficient of 1.47 cm ^g/L] 1 . The protein concentrations of the buffer exchanged samples were adjusted to desired values with dialysis buffer, and 10% PS20 stock solution was spiked into each formulation for a final concentration of 0.05% (w/v) PS20. Formulations were sterile filtered using 0.22 pm filter units and filled into appropriate container/closure systems in a laminar flow hood. Samples were placed at various storage conditions per study design, and their stabilities were analyzed using various methods at specified time points.

III. Analytical methods

[0199] Visual inspection (AD-Gen-002/00): Visual assessment was made against both black and white backgrounds under fluorescence lighting. Samples were examined for color, clarity, and presence of visible particles.

[0200] Protein Concentration (TM- 150-001/00): Protein concentration was determined by UV absorbance at 280 nm using theoretical absorption coefficients of 1.47 cm ^g/L] 1 . Samples were diluted to within the linear range of absorbance with Dulbecco’s Phosphate Buffer Saline (DPBS) and measured against DPBS as blank. Absorbance was measured using Agilent Cary 8454 UV-Vis Spectrophotometer (Agilent Technologies, CA).

[0201] pH (AD-GEN-001/00): Buffer pH was determined using a calibrated Beckman Coulter pHi560 meter (Beckman Coulter, Inc., CA).

[0202] Osmolality (TM-GEN-004/00): Buffer osmolality was measured by vapor pressure using a Wescor VAPRO system (Wescor, Inc., UT).

[0203] Differential Scanning Calorimetry (DSC) Analysis: DSC measurements were performed on a MicroCal VP-Capillary DSC platform (GE Healthcare, UK). The mAb samples were diluted in various formulation buffers to a concentration of 1 mg/mL. Matched formulation buffer was used as a reference. The samples were scanned from l5°C to 1 l0°C at a rate of l°C/min. Data were first normalized for protein concentration, then baseline corrected and buffer subtracted using Origin 7.0 software (OriginLab, MA). Melting transitions were analyzed with cursor initiated DSC peak fit function using non-two-state unfolding model within the origin software.

[0204] Unfolding temperature™ by UNit system: The unfolding temperature (Tm) of a protein provides a measurement of the physical stability of the molecule. Unfolding temperature is defined as the temperature at which equal amounts of native and denatured protein exist in equilibrium. UNit system by Unchained Labs (CA) uses intrinsic fluorescent spectroscopic changes to determine the temperature at which heat-induced unfolding of a protein occurs. Samples at 1 mg/mL were scanned from 20°C to 90°C at a rate of l°C/min. Tm were determined using UNcle software from Unchained Labs (CA).

[0205] Imaged Capillary Isoelectric Focusing (iCE) (TM- 150-003/02): The charge variants were analyzed by Imaged capillary isoelectric focusing (iCE) on a Protein Simple iCE3 instrument with 720 NV auto-sampler (ProteinSimple, CA). Data were analyzed using iCE CFR software, and the relative amounts of main, acidic, and basic peaks were determined by integrating the area of the peaks observed in the profile and used to calculate the percentage of acidic and basic variants.

[0206] Size-Exclusion Chromatography (SEC) (TM- 150-004/00): Samples were analyzed on Agilent 1100 Series HPLC equipped with a diode array detector, and absorbance was monitored at 280 nm. Samples were diluted to 1 mg/ml in mobile phase (100 mM sodium phosphate, 400 mM sodium chloride pH 6.8), and 50 pL was injected onto a pre-equilibrated Sepax Zenix SEC-300 7.8 X 200 mm column (Sepax Technologies, Inc., Delaware). The SEC separation and guard columns were used at 25°C. A flow rate of 1.0 mL/min was used with a l2-min run time. Aggregate, monomer, and fragment peaks were quantified using instrument software for data analysis.

[0207] Capillary Electrophoresis with Sodium Dodecyl Sulfate Gel (CE-SDS) (TM-150- 002/00): CE-SDS was used to determine the purity of the B7-H4 antibody 20502 under reduced and non-reduced conditions. Samples were analyzed on Beckman Coulter PA800 plus system (Beckman Coulter, CA) using an uncoated 50 mM I.D. capillary. Absorbance was monitored at 220 nm. Purity of 20502 under reduced condition was determined by measuring the peak area of heavy and light chain peaks and comparing with the total area of all detected peaks. Purity of 20502 under non-reduced condition was determined by measuring the peak area of the main intact protein peak and comparing it with the total area of all detected peaks.

[0208] Subvisible Particulate Matters by HIAC (AD-GEN-006): An HIAC 9703+ particle counter (Hach, CO), equipped with an HRDL-150 detector and a 1 mL syringe was used. Before use, the system was flushed with particle-free Milli-Q water (Millipore, MA) to generate a clean base line. Four consecutive 0.4 mL aliquots were taken from samples, and particle counts from the last three aliquots were averaged and reported.

Example 2: Biochemical Analysis of Amino Acid Residues of Antibody 20502

[0209] Anti-B7-H4 antibody 20502 is a monoclonal antibody. A knowledge-based formulation development approach was used to identify the appropriate compositions that provide maximal stability for the protein. To do this, both intrinsic properties of the molecule and extrinsic formulation components that could affect the stability of the protein are considered.

[0210] The full length heavy chain and light chain amino acid sequences of antibody 20502 are shown in Table 1 1.

Table 11: Primary Amino Acid Sequences of the Full Length Heavy Chain and Light Chain of Antibody 20502

[021 1] Analysis of 20502 primary sequence revealed that some amino acid residues could potentially undergo biochemical modifications. These include deamidation in asparagine, isomerization in aspartic acid, and oxidation in methionine, cysteine, histidine, tryptophan, phenylalanine, and tyrosine. The asparagines, aspartic acids, and methionines subject to deamidation, isomerization, and oxidation, respectively, are indicated in bold and gray squares in the sequences in Table 1 1. The potential biochemical degradation spots in 20502 are noted in Table 12. Those potential degradation spots are outside the CDR regions.

Table 12: Potential Amino Acid Residues in 20502 for Chemical Degradation Pathways.

[0212] To develop a liquid formulation that could provide good stability for 20502, different conditions such as pH, buffer types, and excipients were evaluated. The stability of the protein was monitored based on biophysical and biochemical properties of 20502 in each example. The details of the studies and their results are described here.

Example 3: Initial pH Screening Study

[0213] Formulation pH affects protein stability. The pH of a formulation can influence biochemical degradation pathways such as deamidation, isomerization, and oxidation, as well as biophysical degradations such as aggregation and fragmentation due to interactions between proteins and with their environment.

[0214] A pH screening study was conducted to determine a pH range that provides maximal stability for 20502 and to understand the degradation mechanisms of the protein under these conditions. The details of formulation composition evaluated in the study are listed in Table 13.

Table 13: Formulations Evaluated for pH Effect on Protein Stability

[0215] To determine the thermal stability of the protein under different pH conditions, unfolding temperatures were measured by intrinsic fluorescent change under different temperature using UNit instrument. The shift in tryptophan fluorescence emission wavelength (Barycentric Mean, BCM) indicates the unfolding event occurs when samples are heated. FIG. 1 shows the measured unfolding temperature (Tml) of 20502 under different pH conditions. The results indicate Tml is pH dependent. At higher pH (e.g., pH > 7), an unfolding temperature of 74°C was observed. At pH 5 and 6, the unfolding temperatures were 66°C and 70°C, respectively. At pH 4, the unfolding temperature was 55°C. In addition, the onset temperature appeared to be ~50°C using pH 4, but the onset temperature was 62°C or above using pH 5-8. These results demonstrate that compositions with pH > 5 will provide better thermal stability than formulations at pH 4.

[0216] The stability of 20502 under different pHs was also evaluated under stressed conditions at 40°C for up to four weeks. All samples remained clear and colorless with no particles observed for the duration of the study. Changes in aggregates and fragments were determined by SE-HPLC. At time zero (TO), no apparent aggregates were observed for 20502 at 1 mg/ml concentration in the formulations studied. After four weeks at 40°C, the soluble aggregates increased noticeably in the pH 4, 7, and 8 formulations; only a small increase of aggregates was observed in pH 5 or 6 formulations (FIG. 2). A similar effect of pH on the formation of fragments was observed (FIG. 3). The results from this study showed that 20502 is most stable around pH 5 - 6 as indicated by SE-HPLC measurement of the indicated properties.

Example 4: Detailed pH Screening Study

[0217] The results of the preliminary pH screening study showed that antibody 20502 was most stable around pH 5- 6. A detailed pH study was carried out to identify the pH that provides maximum stability in the range of 4.5 to 6.5. The details of the formulation compositions are listed in Table 14. This study was conducted using citrate buffer to minimize the possible impact of buffer types on the stability of the mAh. The stability of antibody 20502 at 10 mg/mL was examined based on visual appearance, aggregation, fragmentation (by SE-HPLC), and charge variants (iCE) under stressed (40°C) conditions for up to three weeks.

Table 14: Formulations Evaluated for Detailed pH Effect on Protein Stability

[0218] All samples remained clear and colorless with no particles observed for the duration of the study. SE-HPLC results showed that a low level of aggregate was observed for 20502 at 10 mg/ml concentration at the beginning of the study (TO). Thermal stress of three weeks at 40°C resulted in a pH-dependent increase in aggregation in the order of 4.5 < 5.0 < 5.5 < 6.0 < 6.5, while the fragmentation showed the opposite trend (FIG. 4 and FIG. 5).

[0219] The iCE data showed the acidic variants increased after 3 weeks at 40°C. The increase of acidic variants was pH dependent, with the increasing order of pH at 6.5/6.0/5.5 < 5.0 < 4.5 (FIG. 6).

[0220] The fact that higher pHs were associated with increased aggregation and lower pHs were associated with increased fragmentation increased the difficulty of identifying a desirable pharmaceutical formulation. Collectively, the study results demonstrate that formulations at pH 5-6 provide the best overall stability profile for 20502.

Example 5: Buffer Species Screening Study

[0221] Like pH, buffer types influence protein stability to various extents. The accelerated stability of 20502 at 10 mg/mL was evaluated in acetate, citrate, succinate, and histidine buffers from pH 5.0 to pH 6.5 in 0.5 unit increments based on their pKa, with the aim of determining a buffer that will provide maximal stability at pH 5.0 - 6.5. The detailed formulation compositions are provided in Table 15. These buffers were examined for their effect on protein stability based on appearance, aggregation, fragmentation, and charge variants.

Table 15: Formulation Buffer Species Evaluated for Protein Stability

[0222] All samples remained clear and colorless with no particles observed for the duration of the study. SE-HPLC results shown in FIG. 16 reveal buffer (pH) dependent increase of aggregates in this study. The aggregates were formed at higher rates at higher pH compared to those at lower pH; in the order of histidine (pH 6.5) > succinate (pH 6) > citrate (pH 5.5) > acetate (pH 5). On the other hand, slightly less fragments were observed in acetate (pH 5) and citrate (pH 5.5) buffers compared to those in histidine (pH 6.5) and succinate (pH 6) buffers, though the overall levels were low (0.5% - 0.7%) over 3 weeks at 40°C (FIG. 8).

[0223] The iCE results showed that buffers had some effect on 20502 charge profile as shown in FIG. 9 and FIG. 10. But the overall difference among them was slightly over 10% in acidic variants and about 2% in basic variants upon storage at 40°C for three weeks.

[0224] The 20502 antibody was tested in two other standard formulations containing histidine, but neither resulted in a composition with the desired stability. Based on these results, it was concluded that 20502 was most stable in acetate buffer at pH 5 among all the formulation conditions tested. Citrate buffer at pH 5.5 also provides a relatively stable buffer for 20502.

Example 6: Excipients Selection Study

[0225] Formulation excipients such as bulking agents can affect product stability. To assess the effect of excipients on the stability of antibody 20502, the antibody at 20 mg/mL was formulated into acetate and citrate formulations containing sodium chloride (NaCl), trehalose, sorbitol, or sucrose at isotonic concentrations. Citrate formulations were formulated at pH 5.5 and acetate formulations were formulated at pH 5.0. Two additional acetate formulations with sucrose were also prepared at pHs 4.5 and 5.5. The detailed formulation compositions are provided in Table 16. The excipients were examined for their effect on protein stability based on visual appearance, aggregation, fragmentation and charge variants under the storage conditions of 40°C, 25°C and 5°C.

Table 16: Formulation Excipients Evaluated For Protein Stability

[0226] All samples remained clear and color ess with no particles observed for the 4- week duration of the study. SE-HPLC results shown in FIG. 11 revealed that 20502 aggregates formed at faster rates in the citrate formulations compared to acetate formulations.

Rate of aggregation formation was highest in NaCl formulation, and this was true for both citrate and acetate formulations. Sorbitol, trehalose, and sucrose formulations showed similar changes in aggregation in both citrate and acetate buffers. For fragments, all formulations were similar except for formulation containing acetate and NaCl, which showed slightly more fragments (FIG. 12). Thus, although NaCl is widely used in pharmaceutical compositions, it resulted in increased aggregation and fragmentation of 20502. The amount of fragments observed in this study was slightly higher compared with those in the earlier studies. This could be due to the higher concentration of protein (20 mg/mL) used in this study.

[0227] Formulation excipients had less impact on the 20502 charge profile upon storage. As shown in FIG. 13, less than 10% difference of acidic variants among all the formulations was observed. Similarly, less than 2% difference of basic variants was observed in all the formulations within each buffer type (FIG. 14).

[0228] Based on the results of excipient selection study, sucrose was selected over NaCl due to the aggregates level observed. Sucrose showed similar aggregation and charge profile as sorbitol and trehalose. However, sucrose is more reliable from raw material supply perspective with better quality and regulatory experience.

[0229] Collectively, the results of pH, buffer species, and excipient screening studies indicate that the 20502 antibody was most stable in a formulation containing 20 mM acetate, 270 mM sucrose, and 0.05% PS20 at pH 5.0. The formulation containing 20 mM citrate, 270 mM sucrose, 0.05% PS20 at pH 5.5 also provided good stability.

[0230] A Differential Scanning Calorimetry (DSC) profile of the 20502 antibody in a formulation containing 20 mM acetate, 270 mM sucrose, 0.05% PS20 at pH 5.0 was collected and is shown in FIG. 15. Two peaks were observed, with Tml = 69°C and Tm2 = 84°C.

Example 7: Freeze-thaw Stability of 20502

[0231] A freeze/thaw study was performed by freezing 20502 bulk drug substance formulated as 20 mg/mL protein in 20 mM acetate, 270 mM sucrose, 0.05% PS20 formulation at pH 5.0 at 500 mL scale at -70°C and thawing at ambient temperature through 5 cycles. No apparent changes in appearance, soluble aggregates, or subvisible particulate matter were detected (Table 17).

Table 17: Stability of 20502 Formulation After Freeze-Thaw

Example 8: Agitation Stability of 20502

[0232] Agitation stress was exerted on 20502 by filling 20502 drug substance into 3 cc glass vials, placing the sample vials horizontally on an orbital shaker, and shaking the samples at 300 RPM for 72 hours at room temperature. The 20502 was formulated as 20 mg/mL protein in 20 mM acetate, 270 mM sucrose, 0.05% PS20 formulation at pH 5.0. No apparent changes in appearance, soluble aggregates, charge variants profiles, or subvisible particulate matter were detected in the 20502 formulation samples (Table 18). All studies were conducted at room temperature at 20±5°C. All samples had a clear and colorless appearance with no visible particles.

Table 18: Stability of 20502 Formulation After Agitation For 72 Hours

Example 9: Confirmation Stability Studies

[0233] A stability study was performed to evaluate the stability of 20502 in two formulation (i) a formulation comprising 20 mg/mL protein in 20 mM acetate, 270 mM sucrose, and 0.05% PS20 at pH 5.0and (ii) a formulation containing 20 mg/mL protein in 20 mM citrate, 270 mM sucrose, and 0.05% PS20 at pH 5.5. 1.5 mL of the formulated solutions were filled into 3 cc type 1 glass vials with 13 mm necks, capped with 13 mm West 4023/50 gray bromobutyl serum stoppers, and sealed with aluminum seals. The compatibility of the container closure system with antibody 20502 was evaluated by placing the vials in inverted positions. Storage conditions at 5°C, 25°C, and 40°C were used for this stability study. Six months stability data shown in Table 19 to Table 21 for the acetate formulation and in Table 22 to Table 24 for the citrate formulation.

Table 19: Stability of 20502 Drug Product in Acetate Formulation at 5°C

Abbreviations: NP = Not planned; NT = Not tested; TO = Time zero.

Table 20: Stability of 20502 Drug Product in Acetate Formulation at 25°C

Table 21 : Stability of 20502 Drug Product in Acetate Formulation at 40°C

Table 22: Stability of 20502 Drug Product in Citrate Formulation at 5°C

Table 23: Stability of 20502 Drug Product in Citrate Formulation at 25°C Table 24: Stability of 20502 Drug Product in Citrate Formulation at 40°C

[0234] Stability data of 20502 drug product under the long-term storage condition of 2- 8°C were collected in real-time in acetate and citrate formulations for 6 months. All stability data met the acceptance criteria (Table 19 and Table 22). No clear trend of stability changes was observed in any of the attributes tested. The results demonstrate 20502 drug product is stable in both formulations under the long-term storage condition at 2-8°C for at least 6 months.

[0235] Stability data at the accelerated condition of 25°C were collected in acetate and citrate formulations for 6 months. Overall, storage at the accelerated condition for 6 months resulted in an increase of acidic peaks and decrease of main peak as determined by iCE; slight increase of aggregates and fragments as determined by SE-HPLC; slight decrease of purity as determined by reduced and non-reduced CE-SDS; and slight decrease potency as determined by a cell-based ADCC assay. No change in other product attributes was observed. All stability data were within the acceptance criteria.

[0236] Stability data at the stressed condition of 40°C were collected for 3 months. Some changes were more noticeable in data collected over time. The trends were similar to those shown at the accelerated storage condition (25°C). An increase of acidic peaks and decrease of main and basic peaks by iCE was observed. An increase of aggregate and fragment with a decrease of monomer was observed by SE-HPLC. In addition, the purity decreased under this condition as demonstrated by reduced and non-reduced CE-SDS analysis. Decrease of potency in cell based ADCC assay was also observed. These results are consistent with changes expected for protein therapeutics stored under this condition.

[0237] The above studies were conducted to identify formulation conditions that provide maximal stability for 20502. These include pH screening, buffer species selection, and excipient selection studies. 20502 was most stable in the range of pH 5 - 6 in the acetate or citrate buffer. 20502 was also stable when sucrose was used as excipient. A formulation containing 20 mg/mL protein in 20 mM acetate, 270 mM sucrose, and 0.05% PS20 at pH 5.0 was selected. A backup formulation as 20 mg/mL protein in 20 mM citrate, 270 mM sucrose, 0.05% PS20, pH 5.5 was also selected.

[0238] Antibody 20502 is also stable in the acetate formulation under freeze-thaw and agitation conditions. 20502 in acetate formulation is expected to be stable both as drug substance when stored at -70°C and as drug product when stored at 2-8°C for at least 12 months.

* * *

[0239] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications provided herein in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0240] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0241] Other embodiments are within the following claims.