B7-H4 antibody formulations
阅读说明：本技术 B7-h4抗体制剂 (B7-H4 antibody formulations ) 是由 全勇 黄清仪 H·S·甘达 于 2019-02-21 设计创作，主要内容包括：本公开提供了药物组合物,所述药物组合物包含特异性地结合至人B7-H4(和任选的食蟹猴、小鼠和/或大鼠B7-H4)的抗体及其抗原结合片段。本公开还提供了通过施用此类药物组合物来治疗诸如癌症的病症的方法。(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 of treating disorders such as cancer by administering such pharmaceutical compositions.)
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 from 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, VHCDR3 and VL CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 5-10, respectively; (ii) a buffering agent; 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 ℃.
4. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, wherein the composition comprises from about 30% to about 45% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
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 ℃.
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 a basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
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% acidic and basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
8. The composition of any one of claims 1-7, wherein the composition comprises about 30% to about 40% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
9. The composition of claim 8, wherein the composition comprises about 35% to about 40% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
10. The composition of any one of claims 1-9, wherein the composition comprises about 10% to about 17% of the basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
11. The composition of claim 10, wherein the composition comprises about 11% to about 16% of the basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
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 ℃.
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 from about 4.5 to about 6.
15. The composition of any one of claims 3-14, wherein the composition comprises a buffering agent.
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 saccharide 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 buffer is at a concentration of about 18mM to about 22 mM.
21. The composition of claim 20, wherein the buffer is at a concentration of about 20 mM.
22. The composition of any one of claims 1 and 17-21, wherein the sugar is at a concentration of about 225mM to about 300 mM.
23. The composition of claim 22, wherein the sugar is at a concentration of about 250mM to about 290 mM.
24. The composition of claim 23, wherein the sugar is at a concentration of 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 a polysorbate, optionally wherein the polysorbate is polysorbate 20.
28. The composition of claim 26 or 27, wherein the concentration of the surfactant is from 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 5mg/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 20mM acetate, about 270mM sucrose, about 20mg/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 sucrose at a concentration that is about 13.5 times the concentration of acetate, the antibody or antigen-binding fragment thereof at about 20mg/ml, and polysorbate 20 at about 0.05%, 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 20mM citrate, about 270mM sucrose, about 20mg/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 sucrose at a concentration that is about 13.5 times the citrate concentration, the antibody or antigen-binding fragment thereof at about 20mg/ml, and polysorbate 20 at about 0.05%, 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. 11; 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 21; 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 not detectable 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 said antigen binding fragment comprises Fab, Fab ', F (ab')2Single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgG Δ CH2, minibody, F (ab')3Tetravalent antibodies, trivalent antibodies, divalent antibodies, single domain antibodies, DVD-Ig, Fcab, mAb2、(scFv)2Or 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 21, and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO 22; (ii) about 20mM acetate; (iii) about 270mM sucrose; and (iv) about 0.05% w/v 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 21, and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO 22; (ii) about 20mM citrate; (iii) about 270mM sucrose; and (iv) about 0.05% w/v 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, ductal, endometrial, ovarian, non-small cell lung, pancreatic, thyroid, renal, 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 a 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.
1. Field of the invention
Pharmaceutical compositions comprising the B7-H4 antibodies and methods of using such formulations are provided.
2. Background of the invention
B7-H4 (also referred to as B7x, B7-S1 and VTCN1) are immune regulatory molecules with homology to other B7 family members, including PD-L1. It is a type I transmembrane protein composed of IgV and IgC extracellular domains. Although expression of B7-H4 in healthy tissues is relatively limited at the protein level, B7-H4 is expressed in several solid tumors (e.g., gynecological cancers of the breast, ovary, and endometrium). Expression of B7-H4 in tumors is often associated with poor prognosis. The receptor for B7-H4 is unknown, but it is believed that the receptor is expressed on T cells. It is believed that B7-H4 directly inhibits T cell activity.
In view of the expression and function of B7-H4, antibodies that specifically bind to B7-H4 are being developed for therapies involving modulation of B7-H4, e.g., for the treatment of cancer. Accordingly, there is a need for pharmaceutical compositions comprising the B7-H4 antibodies and antigen-binding fragments thereof for use in administering such treatments.
3. Summary of the invention
Provided herein are pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4.
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.
In certain aspects, the antibody or antigen-binding fragment thereof comprises the CDRs of 20502.
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.
In certain aspects, the CDRs of 20502 are Kabat-defined CDRs, Chothia-defined CDRs, or AbM-defined CDRs. In certain aspects, the antibody or antigen-binding fragment thereof comprises 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.
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 ℃. 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 ℃. 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 the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. 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 the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the composition comprises no more than 20% of the basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
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 ℃. 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 the basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. 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 a basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the composition comprises no more than 45% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the composition comprises no more than 40% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
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% acidic and basic variants of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. 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 ℃. In certain aspects, the composition comprises no more than 45% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the composition comprises no more than 40% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the composition comprises no more than 20% of the basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
In certain aspects, the pharmaceutical composition comprises from about 30% to about 40% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the pharmaceutical composition comprises 30% to 40% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the pharmaceutical composition comprises about 35% to about 40% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the pharmaceutical composition comprises 35% to 40% of the acidic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
In certain aspects, the pharmaceutical composition comprises from about 10% to about 17% of the basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the pharmaceutical composition comprises 10% to 17% of the basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the pharmaceutical composition comprises about 11% to about 16% of the basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In certain aspects, the pharmaceutical composition comprises 11% to 16% of the basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
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 ℃.
In certain aspects, the antibody or antigen-binding fragment thereof comprises the CDRs of 20502. In certain aspects, the CDRs of 20502 are Kabat-defined CDRs, Chothia-defined CDRs, or AbM-defined CDRs. In certain aspects, the antibody or antigen-binding fragment thereof comprises heavy chain variable region (VH) Complementarity Determining Region (CDR)1, VH CDR2, VHCDR3, and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs 5-10, respectively.
In certain aspects, the pH of the composition is from about 4.5 to about 6, or from 4.5 to 6.
In certain aspects, the composition comprises a buffering agent. In certain aspects, the buffer is acetate or citrate.
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.
In certain aspects, the buffer is at a concentration of about 15 to about 25 mM. In certain aspects, the buffer is at a concentration of 15 to 25 mM. In certain aspects, the buffer is at a concentration of about 18mM to about 22 mM. In certain aspects, the buffer is at a concentration of 18mM to 22 mM. In certain aspects, the buffer is at a concentration of about 20 mM. In certain aspects, the buffer is at a concentration of about 20 mM.
In certain aspects, the sugar is at a concentration of about 225mM to about 300 mM. In certain aspects, the sugar is at a concentration of 225mM to 300 mM. In certain aspects, the sugar is at a concentration of about 250mM to about 290 mM. In certain aspects, the sugar is at a concentration of 250mM to 290 mM. In certain aspects, the sugar is at a concentration of about 270 mM. In certain aspects, the sugar is at a concentration of 270 mM.
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.
In certain aspects, the composition further comprises a surfactant. In certain aspects, the surfactant is a polysorbate. In certain aspects, the polysorbate is
In certain aspects, the antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) is at a concentration of about 5mg/ml to about 30 mg/ml. In certain aspects, the antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) is at a concentration of 5mg/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 antibody or antigen-binding fragment thereof (including acidic and basic variants thereof) is at a concentration of 20 mg/ml.
In certain aspects, the pH of the composition is from 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.
In certain aspects, the composition is a liquid. In certain aspects, the compositions are for parenteral administration. In certain aspects, the compositions are for intravenous administration.
In certain aspects, the buffer is acetate and the excipient is sucrose. In certain aspects, the composition comprises about 20mM acetate, about 270mM sucrose, about 20mg/ml of the antibody or antigen-binding fragment thereof, and about 0.05
In certain aspects, the buffer is citrate and the excipient is sucrose. In certain aspects, the composition comprises about 20mM citrate, about 270mM sucrose, about 20mg/ml of the antibody or antigen-binding fragment thereof, and about 0.05
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 21; and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO. 22.
In certain aspects, at least 95% of the antibodies or antigen-binding fragments thereof in the composition are afucosylated. In certain aspects, fucosylation is not detectable in the composition.
In certain aspects, the composition comprises a full-length antibody.
In certain aspects, the composition comprises an antigen-binding fragment. In certain aspects, the antigen binding fragments comprise Fab, Fab ', F (ab')2Single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgG Δ CH2, minibody, F (ab')3Tetravalent antibodies, trivalent antibodies, divalent antibodies, single domain antibodies, DVD-Ig, Fcab, mAb2、(scFv)2Or scFv-Fc.
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.
In certain aspects, the antibody or antigen-binding fragment thereof specifically binds to the IgV domain of human B7-H4.
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.
In certain aspects, the pharmaceutical composition consists of: (i) an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO 21, and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO 22; (ii) about 20mM acetate; (iii) about 270mM sucrose; and (iv) about 0.05% w/
In certain aspects, the pharmaceutical composition consists of: (i) an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO 21, and/or a light chain comprising the amino acid sequence set forth in SEQ ID NO 22; (ii) about 20mM citrate; (iii) about 270mM sucrose; and (iv) about 0.05% w/
In certain aspects, a syringe or vial comprises a pharmaceutical composition provided herein.
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, ductal, endometrial, ovarian, non-small cell lung, pancreatic, thyroid, renal, 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.
In certain aspects, the subject is a human.
In certain aspects, the pharmaceutical composition is administered parenterally. In certain aspects, the pharmaceutical composition is administered intravenously.
4. Description of the drawings
Figure 1 shows the unfolding temperature (Tm1) of the B7-H4 antibody "20502" (afucosylated) under different pH conditions, as measured by the UNit system. (see example 3.)
Figure 2 shows the effect of buffer pH on aggregate formation of the B7-H4 antibody "20502" (afucosylated) at 40 ℃, as determined by size exclusion high performance liquid chromatography (SE-HPLC). The formulations for each pH are listed in table 13. The percentage of High Molecular Weight (HMW) at T0 was about 0 for all phs tested. (see example 3.)
FIG. 3 shows the effect of buffer pH on fragment formation at 40 ℃ as determined by SE-HPLC. The formulations for each pH are listed in table 13. The percentage of Low Molecular Weight (LMW) at T0 was about 0 for all pH tested. (see example 3.)
Figure 4 shows the effect of buffer pH on aggregate formation (as determined by SE-HPLC) at 40 ℃ in a formulation containing 20mM citrate, 270mM sucrose and 0.05% polysorbate 20(PS 20). (see example 4.)
FIG. 5 shows the effect of buffer pH on fragment formation (as determined by SE-HPLC) at 40 ℃ in a formulation containing 20mM citrate, 270mM sucrose and 0.05
Figure 6 shows the effect of buffer pH on acidic variants at 40 ℃ (as determined by imaging capillary isoelectric focusing (iCE)). (see example 4.)
FIG. 7 shows the effect of buffer type on aggregate formation at 40 deg.C (as determined by SE-HPLC). (see example 5.)
FIG. 8 shows the effect of buffer type on fragment formation at 40 ℃ (as determined by SE-HPLC). (see example 5.)
Figure 9 shows the effect of buffer type on acidic variant formation at 40 ℃ (as determined by iCE). (see example 5.)
Figure 10 shows the effect of buffer type on basic variant formation at 40 ℃ (as determined by iCE). (see example 5.)
FIG. 11 shows the effect of excipient on aggregate formation (as determined by SE-HPLC) at 40 ℃. (see example 6.)
Figure 12 shows the effect of excipient on fragment formation at 40 ℃ (as determined by SE-HPLC). (see example 6.)
Figure 13 shows the effect of excipient on acidic variants at 40 ℃ (as determined by iCE). (see example 6.)
Figure 14 shows the effect of excipient on basic variants at 40 ℃ (as determined by iCE). (see example 6.)
Figure 15 shows the Differential Scanning Calorimetry (DSC) curve of 20502 (afucosylation) in selected formulations. (see example 6.)
5. Detailed description of the preferred embodiments
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, for example, under long term storage conditions, after repeated freeze-thaw cycles (e.g., at least 5 cycles) and/or agitation.
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 25mg/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 a polysorbate, e.g., polysorbate 20(PS 20)).
In a particular embodiment, provided herein is a liquid aqueous pharmaceutical composition having a pH of 5.0 containing 20mg/mL of an anti-B7-H4 antibody (e.g., afucosylated antibody 20502) in 20mM acetate, 270mM sucrose and 0.05
The pharmaceutical compositions provided herein are useful for treating conditions such as cancer.
5.1 terminology
As used herein, the term "B7-H4" refers to mammalian B7-H4 polypeptides, including but not limited to natural B7-H4 polypeptides and isoforms of B7-H4 polypeptides. "B7-H4" encompasses full-length, unprocessed B7-H4 polypeptide as well as forms of B7-H4 polypeptide that result from intracellular processing. "B7-H4 polynucleotide", "B7-H4 nucleotide" or "B7-H4 nucleic acid" refers to a polynucleotide encoding B7-H4.
The term "antibody" means an immunoglobulin molecule that is recognized by at least one antigen recognition site located within the variable region of the immunoglobulin molecule and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof. 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 antibody exhibits the desired biological activity. Antibodies can have any of the following five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) are referred to as α, γ, and μ, respectively, based on the properties of their heavy chain constant structures. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. The antibody may be naked or conjugated to other molecules such as toxins, radioisotopes, and the like.
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 antigen recognition site (e.g., a Complementarity Determining Region (CDR) sufficient to specifically bind an antigen) of an intact antibody. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab ', F (ab')2, and Fv fragments, linear antibodies, and single chain antibodies. Antigen-binding fragments of antibodies can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or can be artificially produced.
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 to 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 binds," "immunospecifically recognizes," and "specifically recognizes" are similar terms in the context of an antibody or antigen-binding fragment thereof. These terms indicate that an antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain, and that binding requires some complementarity between the antigen-binding domain and the epitope. Thus, an antibody that "specifically binds" to human B7-H4(SEQ ID NO:1) can also bind to B7-H4 (e.g., cynomolgus monkey, mouse, and/or rat B7-H4) from other species and/or B7-H4 protein produced by other human alleles, but binds to an unrelated non-B7-H4 protein (e.g., other B7 protein family members, such as PD-L1) to less than about 10% of the binding of the antibody to B7-H4, as measured, for example, by Radioimmunoassay (RIA). In a specific embodiment, the antibodies or antigen-binding fragments thereof used in the formulations provided herein specifically bind to human, cynomolgus monkey, mouse and rat B7-H4.
A "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous population of antibodies or antigen-binding fragments that are involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies which typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" or antigen-binding fragment thereof encompasses 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" antibodies or antigen-binding fragments thereof refer to such antibodies and antigen-binding fragments thereof prepared in any number of ways, including but not limited to, hybridomas, phage selection, recombinant expression, and transgenic animals.
As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region generally refers to a portion of an antibody, generally a portion of a light or heavy chain, typically about the
The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.
The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.
The term "Kabat numbering" and similar terms are art-recognized and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In certain aspects, the CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA and Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. department of health and public service, NIH publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35 (which optionally may comprise one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65(CDR2), and amino acid positions 95 to 102(CDR 3). Using the Kabat 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(CDR 3). In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.
In contrast, Chothia refers to the position of the structural loop (Chothia and Lesk, J.mol.biol.196:901-917 (1987)). The ends of the Chothia CDR-H1 loops, when numbered using the Kabat numbering convention, vary between H32 and H34 depending on the length of the loop (since the Kabat numbering scheme places the insertions at H35A and H35B; the loop ends at 32 if 35A or 35B is not present; the loop ends at 33 if only 35A is present; the loop ends at 34 if both 35A and 35B are present). The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.
As used herein, the terms "constant region" and "constant domain" are interchangeable and have their common meaning in the art. Constant regions are portions of an antibody that are not directly involved in binding of the antibody to an antigen, but may exhibit a variety of effector functions, such as interaction with an Fc receptor, e.g., the carboxy-terminal portion of a light chain and/or heavy chain. The constant regions of immunoglobulin molecules typically have more conserved amino acid sequences relative to immunoglobulin variable domains. In certain aspects, the antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).
As used herein, the term "heavy chain" when used with respect to an antibody can refer to any of the different classes, e.g., α (α), (), γ (γ), and μ (μ), that produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including IgG subclasses, e.g., IgG1, IgG2, IgG3, and IgG4, based on the amino acid sequence of the constant domain. Heavy chain amino acid sequences are well known in the art. In a specific embodiment, the heavy chain is a human heavy chain.
As used herein, the term "light chain" when used with respect to an antibody can refer to any of the different types, e.g., κ (κ) or λ (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.
The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof in which the amino acid sequences are derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to those of an antibody or antigen-binding fragment thereof derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in an antibody or antigen-binding fragment thereof derived from another species (typically human) to avoid eliciting an immune response in that species.
The term "humanized antibody" or antigen-binding fragment thereof refers to a non-human (e.g., murine) antibody or antigen-binding fragment form thereof that is a specific immunoglobulin chain, chimeric immunoglobulin or fragment thereof that contains minimal non-human (e.g., murine) sequences. In general, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins ("CDR grafting") in which residues from the Complementarity Determining Regions (CDRs) are replaced by residues from CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (Jones et al, Nature 321: 522-153525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science 239:1534-1536 (1988)). In some cases, certain Fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding residues in an antibody or fragment from a non-human species having the desired specificity, affinity, and capacity. The humanized antibody or antigen-binding fragment thereof can be further modified by substitution of additional residues in the Fv framework regions and/or within non-human CDR residues to improve and optimize the specificity, affinity, and/or capacity of the antibody or antigen-binding fragment thereof. In general, a humanized antibody or antigen-binding fragment thereof will comprise a variable domain that contains all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody or antigen-binding fragment thereof may further comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically at least a portion of a human immunoglobulin. Examples of methods for producing humanized antibodies are described in U.S. Pat. nos. 5,225,539; roguska et al, Proc. Natl. Acad. Sci., USA,91(3):969-973 (1994); and Roguska et al, Protein Eng.9(10):895-904 (1996). In some embodiments, a "humanized antibody" is a resurfaced antibody.
The term "human" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, wherein such antibody or antigen-binding fragment thereof is prepared using any technique known in the art. This definition of human antibodies or antigen-binding fragments thereof includes whole or full-length antibodies and fragments thereof.
An "afucosylated" antibody or antigen binding fragment thereof or an antibody or antigen binding fragment thereof "lacking fucose" refers to an IgG1 or IgG3 isotype antibody or antigen binding fragment thereof that lacks fucose in its constant region glycosylation. Glycosylation of human IgG1 or IgG3 occurred at Asn297, since the glycosylation of the core fucosylated biantennary complex oligosaccharide ends at up to 2 Gal residues. In some embodiments, the afucosylated antibody lacks fucose at Asn 297. Depending on the amount of terminal Gal residues, these structures were designated G0, Gl (a 1,6 or a1, 3) or G2 glycan residues. See, e.g., Raju, T.S., BioProcess int.1:44-53 (2003). CHO-type glycosylation of antibody Fc is described, for example, in Router, F.FL, Glycoconjugate J.14:201-207 (1997).
Methods of measuring fucose include any method known in the art. For the purposes herein, fucose is detected by the method described in example 1 of WO2015/017600, which is incorporated herein by reference in its entirety. Briefly, glycan analysis is performed by releasing glycans from antibodies (e.g., by enzymatic release), labeling the glycans with anthranilic acid (2-AA), and then purifying the labeled glycans. Normal phase HPLC with fluorescence detection was used to separate glycans and measure the relative content of each glycan in the antibody. Glycans can be unambiguously identified by mass spectrometry as lacking or including fucose. In some embodiments, fucose is undetectable in a composition comprising a plurality of afucosylated antibodies or antigen binding fragments thereof. In some embodiments, the afucosylated antibodies or antigen binding fragments thereof have enhanced ADCC activity, which can be measured by the assay provided in example 12 herein. In some embodiments, the afucosylated antibodies or antigen binding fragments thereof have enhanced affinity for Fc γ RIIIA. In some embodiments, the afucosylated antibody or antigen binding fragment thereof has enhanced affinity for Fc γ RIIIA (V158). In some embodiments, the afucosylated antibody or antigen binding fragment thereof has enhanced affinity for Fc γ RIIIA (F158). The affinity of Fc γ RIIIA or alleles thereof can be measured by the assay provided in example 10 herein.
"binding affinity" generally refers to the strength of the sum 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). As used herein, unless otherwise specified, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an 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 variety of ways known in the art, including but not limited to, equilibrium dissociation constant (KD) and equilibrium association constant (KA). KD is according to koff/konIs calculated based on the quotient of, and KA is based on kon/koffIs calculated by the quotient of (a). k is a radical ofonRefers to, for example, the association rate constant of an antibody or antigen-binding fragment thereof with an antigen, and koffRefers to, for example, dissociation of an antibody or antigen-binding fragment thereof from an antigen. k is a radical ofonAnd koffCan be prepared by techniques known to those of ordinary skill in the art, e.g.Or KinExA.
As used herein, "epitope" is a term in the art and refers to a local 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 be, for example, from two or more non-contiguous regions of one or more polypeptides (conformational, non-linear, non-contiguous, or non-contiguous epitopes). In certain embodiments, the epitope to which the antibody or antigen-binding fragment thereof specifically binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange by binding mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Gieger et al, (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 fragments thereof antigen crystals may be studied using well-known X-ray diffraction techniques and may be improved using computer software such as X-PLOR (Yale University,1992, distributed by Molecular diagnostics, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 and 115, edited by Wyckoff HW et al; U.S.2004/0014194) and BUSTER (Bricogne G (1993) Acta Crystallog Biol Biol Crystallog 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, Carter CW edition; Rovers P et al, (2000) Acta Crystallog D Biol Crystallog 56(Pt10): 1316). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, e.g., Champe M et al, (1995) J biol chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.
An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form that does not occur in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, the isolated antibody, polynucleotide, vector, cell or composition is substantially pure. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified either naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation to 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 will be appreciated that because the polypeptides of the invention are antibody-based, in certain embodiments, the polypeptides may exist as single chains or associated chains.
As used herein, the term "host cell" may be any type of cell, such as a primary cell, a cell in culture, or a cell from a cell line. In particular 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 cells may not be identical to the parent cell transfected with the nucleic acid molecule, for example due to mutations or environmental effects that may occur during passage or integration of the nucleic acid molecule into the genome of the host cell.
The term "pharmaceutical formulation" or "pharmaceutical composition" refers to the following formulation: in a form that allows the biological activity of the active ingredient to be effective, and that is free of additional components that have unacceptable toxicity to the subject to which the formulation is to be administered. The formulation may be sterile.
The term "drug product" refers to a final dosage form, e.g., a liquid formulation comprising a drug substance, typically, but not necessarily, associated with one or more other ingredients.
The term "drug substance" refers to an active ingredient intended to provide a pharmacological or biological activity or other direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, such as the B7-H4 antibody or antigen-binding fragment thereof (e.g., afucosylated antibody 20502), but does not include intermediates used in the synthesis of such ingredient.
As used herein, "buffer" refers to a component of a solution that allows the solution to resist changes in pH b. Buffers include, for example, acetate, citrate, succinate, and histidine.
A "stable" formulation is one in which the active ingredient (e.g., the B7-H4 antibody or antigen-binding fragment thereof) substantially retains its physical and/or chemical stability and/or biological activity upon storage. Stability can be measured under selected conditions (e.g., temperature) over a selected period of time. The formulations provided herein can be stable at room temperature (about 25 ℃) for at least 6 months and/or at about 2 ℃ -8 ℃ for at least 1 year. The formulations provided herein can also be stable after freezing (to, e.g., -70 ℃) and thawing (hereinafter "freeze/thaw cycles"). The formulations provided herein may also be stable upon stirring.
As used herein, the terms "administering", and the like refer to a method (e.g., intravenous administration) that can be used to effect the delivery of a drug, such as an anti-B7-H4 antibody or antigen-binding fragment thereof, to a desired site of biological action. Administration techniques that may be used with The agents and methods described herein are described in, for example, Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.
As used herein, the terms "subject" and "patient" are used interchangeably. The subject may be an animal. In some embodiments, the subject is a mammal, such as a non-human animal (e.g., a 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.
The term "therapeutically effective amount" refers to an amount of a drug, e.g., an anti-B7-H4 antibody or antigen-binding fragment thereof, that is effective to treat a disease or disorder in a subject. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing tumor size or burden; inhibit cancer cell infiltration into peripheral organs to some extent; inhibit tumor metastasis to some extent; inhibit tumor growth to some extent; relieving one or more symptoms associated with the cancer to some extent; and/or produce a favorable response, such as Progression Free Survival (PFS), Disease Free Survival (DFS), Overall Survival (OS), Complete Response (CR), increased Partial Response (PR), or in some cases, Stable Disease (SD), decreased Progressive Disease (PD), decreased 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.
Terms such as "treating," "treatment," "treating," "alleviating," and "reducing" refer to a therapeutic measure capable of curing, slowing, alleviating the symptoms of, and/or arresting the progression of a pathological 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's cancer is successfully "treated" according to the methods of the present invention if the patient exhibits one or more of the following: a reduced number or complete absence of cancer cells; reduction in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs, including, for example, spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; reduction in one or more symptoms associated with a particular cancer; decreased morbidity and mortality; the quality of life is improved; a reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity of the tumor; a decrease in the number or frequency of cancer stem cells in the tumor; differentiation of tumorigenic cells into a non-tumorigenic state; progression Free Survival (PFS), Disease Free Survival (DFS), Overall Survival (OS), Complete Response (CR), increased Partial Response (PR), Stable Disease (SD), decreased Progressive Disease (PD), decreased Time To Progression (TTP), or any combination thereof.
The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancers include, but are not limited to, gynecological cancers (e.g., breast (including triple negative breast, ductal, ovarian, and endometrial), non-small cell lung, pancreatic, thyroid, kidney (e.g., renal cell), and bladder (e.g., urothelial cell) cancers can be "cancers that express B7-H4 (cancer thaxtress B7-H4)" or "cancers that express B7-H4 (B7-H4 expressing cancer)". such terms refer to cancers that contain cells that express B7-H4.
A "refractory" cancer is one that progresses even if an anti-tumor therapy (e.g., chemotherapy) is administered to a cancer patient.
A "recurrent" cancer is one that regrows at the initial site or beyond in response to initial therapy.
By "relapsing" patient is meant a patient with signs or symptoms of cancer after remission. Optionally, the patient relapses after adjuvant or neoadjuvant therapy.
As used in this disclosure and in the claims, the singular forms "a", "an" and "the" include the plural forms unless the context clearly dictates otherwise.
It should be understood that when the language "comprising" is used herein to describe embodiments, other similar embodiments described in terms of "consisting of … …" and/or "consisting essentially of … …" are also provided. In the present disclosure, "comprises", "comprising", "contains", "containing" and "having" and the like may have meanings given to them by us patent law, and may mean "including", and the like; "consisting essentially of … … (consensully of)" or "consisting essentially of … … (consensulinally)" likewise has the meaning attributed to U.S. patent law, and the terms are open-ended, thereby allowing for the presence of more than the recited features, as long as the recited basic or novel features are not altered by the presence of more than the recited features, but preclude prior art embodiments.
As used herein, the term "or" is understood to be inclusive unless explicitly fixed or apparent from the context. The term "and/or" as used herein in phrases such as "a and/or B" is intended to include both "a and B," a or B, "" a, "and" B. Also, the term "and/or" as used in phrases 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).
As used herein, the terms "about" and "approximately" when used to modify a numerical value or numerical range mean that deviations of 5% to 10% above and 5% to 10% below the stated value or range are within the intended meaning of the stated value or range.
Any of the 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
Provided herein are pharmaceutical compositions (e.g., aqueous pharmaceutical compositions) comprising an anti-B7-H4 antibody or antigen-binding fragment thereof (e.g., as discussed in section 5.3 below).
In certain embodiments, the pharmaceutical compositions provided herein are stable to multiple freeze-thaw cycles. A freeze-thaw cycle may include freezing the pharmaceutical composition (e.g., at a temperature of about-70 ℃) and then thawing the pharmaceutical composition (e.g., at room temperature). The pharmaceutical composition may be stable after at least five freeze-thaw cycles. Freeze-thaw cycles (e.g., at least five freeze-thaw cycles) can result in no change in appearance, soluble aggregates, or sub-visible particulate matter.
In certain embodiments, the pharmaceutical compositions provided herein are stable upon stirring. Agitation may include shaking at room temperature (e.g., at about 300 revolutions per minute on an orbital shaker). Agitation can result in no change in appearance, soluble aggregates, charge variant distribution, or sub-visible particulate matter.
In certain embodiments, the pharmaceutical compositions provided herein are stable under long term storage conditions. Long term storage conditions may include storage at about 5 ℃ (e.g., about 2 ℃ to about 8 ℃) for about 6 months or about 1 year. Long term storage conditions may include storage at about 25 ℃ for about 6 months or about 1 year. Long term storage conditions may include storage at about 40 ℃ for about 3 months, about 6 months, or about 1 year.
In certain embodiments, the pharmaceutical compositions provided herein are stable for multiple (e.g., at least five) freeze-thaw cycles, stable after agitation, and/or stable under long term storage conditions.
In certain embodiments, the pharmaceutical compositions provided herein are stable when stored at about-70 ℃ and about 2 ℃ to about 8 ℃ for about 1 year.
In certain embodiments, the pharmaceutical composition may comprise a B7-H4 antibody or 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 from about 5mg/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 from about 10mg/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.
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 5mg/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 10mg/ml to 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 20 mg/ml.
As provided herein, the pharmaceutical composition can contain a buffering agent. 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 15mM to about 25 mM. In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is about 18mM to about 22 mM. In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is about 20 mM.
In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is 15mM to 25 mM. In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is 18mM to 22 mM. In certain embodiments, the concentration of the buffer (e.g., acetate or citrate) is 20 mM.
As provided herein, a pharmaceutical composition may contain an excipient, for example, a sugar, such as sucrose, sorbitol, or trehalose. In some embodiments, the concentration of excipient (e.g., sucrose) is about 225mM to about 300 mM. In some embodiments, the concentration of excipient (e.g., sucrose) is about 250mM to about 290 mM. In some embodiments, the concentration of excipient (e.g., sucrose) is about 270.
In some embodiments, the concentration of excipient (e.g., sucrose) is 225mM to mM. In some embodiments, the concentration of excipient (e.g., sucrose) is 250mM to 290 mM. In some embodiments, the concentration of excipient (e.g., sucrose) is 270.
As provided herein, a 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 an excipient, such as a sugar (e.g., sucrose), is about 10 to about 15 times the concentration of a buffer (e.g., acetate or citrate). In some embodiments, the concentration of an excipient, such as a sugar (e.g., sucrose), is about 13.5 times the concentration of a buffer (e.g., acetate or citrate).
In some embodiments, the concentration of an excipient, such as a sugar (e.g., sucrose), is 10 to 15 times the concentration of a buffer (e.g., acetate or citrate). In some embodiments, the concentration of an excipient, such as a sugar (e.g., sucrose), is 13.5 times the concentration of a buffer (e.g., acetate or citrate).
As provided herein, the pharmaceutical composition may contain a surfactant, such as a polysorbate. The polysorbate may be, for example, polysorbate 20(PS 20). In some embodiments, the concentration of surfactant (e.g., PS20) is about 0.025% to 0.075% weight/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 surfactant (e.g., PS20) is about 0.05% w/v.
In some embodiments, the concentration of surfactant (e.g., PS20) is 0.025% to 0.075% weight/volume (w/v). In some embodiments, the concentration of surfactant (e.g., PS20) is 0.035% to 0.065% w/v. In some embodiments, the concentration of surfactant (e.g., PS20) is 0.05% w/v.
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 from 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.
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.
As provided herein, the pharmaceutical composition can be a liquid. The pharmaceutical composition (e.g., liquid pharmaceutical composition) may be for parenteral administration, e.g., for intravenous administration.
In one embodiment, the pharmaceutical composition comprises about 5mg/mL to about 30mg/mL of the B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in about 15mM to about 25mM acetate, about 225mM to about 300mM sucrose, and about 0.025% to about 0.075
In one embodiment, the pharmaceutical composition comprises 5mg/mL to 30mg/mL of B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in 15mM to 25mM acetate, 225mM to 300mM sucrose, and 0.025% to 0.075
In one embodiment, the pharmaceutical composition comprises about 10mg/mL to about 25mg/mL of the B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in about 18mM to about 22mM acetate, about 250mM to about 290mM sucrose, and about 0.035% to about 0.065
In one embodiment, the pharmaceutical composition comprises 10mg/mL to 25mg/mL of B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in 18mM to 22mM acetate, 250mM to 290mM sucrose, and 0.035% to 0.065
In one embodiment, the pharmaceutical composition comprises 20mg/mL of the B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in 20mM acetate, 270mM sucrose and 0.05
In one embodiment, the pharmaceutical composition comprises about 5mg/mL to about 30mg/mL of the B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in about 15mM to about 25mM citrate, about 225mM to about 300mM sucrose, and about 0.025% to about 0.075
In one embodiment, the pharmaceutical composition comprises 5mg/mL to 30mg/mL of B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in 15mM to 25mM citrate, 225mM to 300mM sucrose, and 0.025% to 0.075
In one embodiment, the pharmaceutical composition comprises about 10mg/mL to about 25mg/mL of the B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in about 18mM to about 22mM citrate, about 250mM to about 290mM sucrose, and about 0.035% to about 0.065
In one embodiment, the pharmaceutical composition comprises 10mg/mL to 25mg/mL of B7-H4 antibody or fragment thereof (e.g., afucosylated antibody 20502) in 18mM to 22mM citrate, 250mM to 290mM sucrose, and 0.035% to 0.065
In one embodiment, the liquid pharmaceutical composition comprises 20mg/mL of an antibody or fragment thereof (e.g., afucosylated antibody 20502) in 20mM citrate, 270mM sucrose and 0.05
In some embodiments, the pharmaceutical composition comprises an antibody or antigen-binding fragment thereof (e.g., afucosylated antibody 20502) that specifically binds to human B7-H4, wherein said composition comprises no more than 40% of said antibody or antigen-binding fragment thereof acidic variants and/or no more than 20% of said antibody or antigen-binding fragment thereof basic variants after 6 months at 5 ℃.
In some embodiments, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof (e.g., afucosylated antibody 20502) that specifically binds to human B7-H4, wherein said composition comprises about 30% to about 45%, about 30% to about 40%, or about 35% to about 40% of an acidic variant of said antibody or antigen-binding fragment thereof and/or about 11% to about 16%, about 10% to about 17%, or about 9% to about 18% of a basic variant of said antibody or antigen-binding fragment thereof after 6 months at 5 ℃. In some embodiments, a pharmaceutical composition comprises an antibody or antigen-binding fragment thereof (e.g., afucosylated antibody 20502) that specifically binds to human B7-H4, wherein said composition comprises 30% to 45%, 30% to 40% or 35% to 40% of an acidic variant and/or 11% to 16%, 10% to 17% or 9% to 18% of a basic variant of said antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
In some embodiments, the 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 ℃. In some embodiments, the composition further comprises no more than 40% of the acidic variant and/or no more than 20% of the basic variant of the antibody or antigen-binding fragment thereof after 6 months at 5 ℃.
In some embodiments, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier. In a specific embodiment, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen binding fragment, e.g., wherein at least 80% of the antibodies in the composition are afucosylated. In a specific embodiment, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen binding fragment, e.g., wherein at least 85% of the antibodies in the composition are afucosylated. In a specific embodiment, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen binding fragment, e.g., wherein at least 90% of the antibodies in the composition are afucosylated. In a specific embodiment, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen binding fragment, e.g., wherein at least 95% of the antibodies in the composition are afucosylated. In a specific embodiment, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen binding fragment, e.g., wherein at least 96% of the antibodies in the composition are afucosylated. In a specific embodiment, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen binding fragment, e.g., wherein at least 97% of the antibodies in the composition are afucosylated. In a specific embodiment, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen binding fragment, e.g., wherein at least 98% of the antibodies in the composition are afucosylated. In a specific embodiment, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen binding fragment, e.g., wherein at least 99% of the antibodies in the composition are afucosylated. In a specific embodiment, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises an afucosylated anti-B7-H4 antibody or antigen binding fragment, wherein fucose is not detectable in the composition.
In some embodiments, pharmaceutical compositions are provided, wherein the pharmaceutical compositions comprise (i) an isolated antibody or antigen-binding fragment thereof that specifically binds to human B7-H4, the antibody or antigen-binding fragment thereof comprising (a) the 5-10 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, 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. 21 and a light chain comprising the amino acid sequence of SEQ ID No. 22; and (ii) a pharmaceutically acceptable excipient.
Also provided herein are pharmaceutical compositions, wherein the pharmaceutical compositions comprise (i) an antibody or antigen-binding fragment thereof that specifically binds to human B7-H4 and comprises 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, 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 antibody or antigen-binding fragment thereof in the composition is 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. 21 and a light chain comprising the amino acid sequence of SEQ ID NO. 22.
5.3B 7-H4 antibody
Provided herein are pharmaceutical compositions comprising antibodies (e.g., monoclonal antibodies, such as chimeric, humanized, or human antibodies) and antigen-binding fragments thereof that 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 of human, cynomolgus monkey, murine and rat B7-H4 are known in the art and are also provided herein, represented by SEQ ID NOs: 1-4, respectively.
Human B7-H4:
MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK(SEQ ID NO:1)
cynomolgus monkey B7-H4:
MASLGQILFWSIISIIFILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVIGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFLAISWALLPLAPYLMLK(SEQ ID NO:2)
murine B7-H4
MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIRTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLSCCLMLR(SEQ ID NO:3)
Rat B7-H4
MASLGQIIFWSIINVIIILAGAIVLIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIHTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLELLNSGPSPCVSSVSAAGWALLSLSCCLMLR(SEQ ID NO:4)
In certain embodiments, the antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4. In certain embodiments, the antibodies or antigen-binding fragments thereof in the pharmaceutical compositions provided herein specifically bind to human and cynomolgus monkey B7-H4. In certain embodiments, the antibodies or antigen-binding fragments thereof in the pharmaceutical compositions provided herein specifically bind to human, murine, and rat B7-H4. In certain embodiments, the antibodies or antigen-binding fragments thereof in the pharmaceutical compositions provided herein bind to human, cynomolgus monkey, murine and rat B7-H4.
B7-H4 contains the IgC extracellular domain (amino acids 153-241 of SEQ ID NO:1) and the IgV extracellular domain (amino acids 35-146 of SEQ ID NO: 1). In certain embodiments, the antibodies or antigen-binding fragments thereof in the pharmaceutical compositions provided herein specifically bind to the IgV domain of human B7-H4. Accordingly, provided herein are compositions comprising a pharmaceutical composition comprising an antibody and antigen-binding fragments thereof that specifically binds to a polypeptide consisting of amino acids 35-146 of SEQ ID NO: 1.
In certain embodiments, the 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 in tables 1 and 2.
TABLE 1 VH CDR amino acid sequences1
1The VH CDRs in table 1 were determined according to Kabat.
TABLE 2 VL CDR amino acid sequences2
2The VL CDRs in table 2 were determined according to Kabat.
In certain embodiments, the 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 sequence
In certain embodiments, the antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the VL of 20502 listed in table 4.
Table 4: variable light chain (VL) amino acid sequences
In certain embodiments, the antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the VH and VL of the 20502 antibody listed in tables 3 and 4.
In certain embodiments, the antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the VH framework region of the 20502 antibody listed in table 5.
TABLE 5 VH FR amino acid sequences3
3VH framework regions described in Table 5Is determined based on the boundaries of the Kabat numbering system of the CDRs. Thus, the VH CDRs are determined by Kabat, and the framework regions are the amino acid residues in the variable region surrounding the CDRs, in the form FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4
In certain embodiments, the antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the VL framework region of the 20502 antibody listed in table 6.
TABLE 6 VL FR amino acid sequences4
4The VL framework regions described in table 6 were determined based on the boundaries of the Kabat numbering system of the CDRs. Thus, the VL CDRs are determined by Kabat, and the framework regions are the amino acid residues in the variable region surrounding the CDRs, in the form FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4
In certain embodiments, the 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.
In certain embodiments, the 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 sequence
In certain embodiments, the 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 sequence
In certain embodiments, the antibody or antigen-binding fragment in the pharmaceutical compositions provided herein specifically binds to human B7-H4 and comprises the heavy and light chain sequences of the 20502 antibody listed in tables 7and 8.
In certain aspects, the antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein is described by its VL domain alone, or VH domain alone, or 3 VL CDRs alone, or 3 VH CDRs alone. See, e.g., Rader C et al, (1998) PNAS 95:8910-8915, which is incorporated herein by reference in its entirety, which describes humanization of mouse anti- α v β 3 antibodies by: complementary light or heavy chains from a library of human light or heavy chains, respectively, are identified, thereby generating humanized antibody variants with an affinity as high or higher than that of the original antibody. See also Clackson T et al, (1991) Nature 352: 624-: specific VL domains (or VH domains) are used and the library is screened for complementary VH domains (or VL domains). By ELISA, the screening produced 14 new partners for specific VH domains and 13 new partners for specific VL domains, which were strong binders, as determined by ELISA. See also Kim SJ and Hong HJ, (2007) JMicrobiol 45: 572-: using specific VH domains and screening libraries (e.g., human VL libraries) for complementary VL domains; the selected VL domain may in turn be used to guide the selection of other complementary (e.g. human) VH domains.
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 the immunoglobulin structural loops (see, e.g., Chothia C and Lesk AM, (1987), J Mol Biol 196: 901-. 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
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 antibodies listed in tables 3 and 4. In certain embodiments, provided herein are pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof that specifically binds to B7-H4 (e.g., human B7-H4) and comprises one or more CDRs, wherein 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 a combination of Kabat CDRs and chothia CDRs.
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-. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26 to 35, VH-CDR2 is located at positions 51 to 57, VH-CDR3 is located at positions 93 to 102, VL-CDR1 is located at positions 27 to 32, VL-CDR2 is located at
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-. See also, for example, "Protein Sequence and Structure analysis of Antibody Variable Domains" in Antibody Engineering, Kontermann and Dubel, eds, Chapter 31, p. 422-. In a particular embodiment, provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to B7-H4 (e.g., human B7-H4) and comprises the VH and VL CDRs of the 20502 antibodies listed in tables 3 and 4 as determined by the method of MacCallum RM et al.
In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers to the hypervariable regions of abms that represent a compromise between Kabat CDRs and Chothia structural loops, and used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In a particular embodiment, provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to B7-H4 (e.g., human B7-H4) and comprises the VH and VL CDRs of the 20502 antibodies listed in table 3 and table 4 as determined by the AbM numbering scheme.
In a particular aspect, provided herein are pharmaceutical compositions comprising an antibody comprising a heavy chain and a light chain.
With respect to light chains, in a particular embodiment, the light chain of an antibody described herein is a kappa light chain. The constant region of the human kappa light chain may comprise the amino acid sequence:
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:23)。
the constant region of the human kappa light chain may be encoded by the following nucleotide sequence:
CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT(SEQ ID NO:24)。
in a particular embodiment, the antibody in the pharmaceutical composition described herein that immunospecifically binds to a B7-H4 polypeptide (e.g., human B7-H4) comprises a light chain, wherein the amino acid sequence of the VL domain comprises the sequences listed 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.
In a particular embodiment, the antibody in the pharmaceutical composition described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) 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 said heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region.
Human IgG1The constant region of the heavy chain may comprise the amino acid sequence:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:25)。
human IgG1The constant region of the heavy chain may be encoded by the following nucleotide sequence:
GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA。(SEQ ID NO:26)
in a specific embodiment, the antibody in the pharmaceutical composition described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) comprises a VH domain and a VL domain comprising the amino acid sequence of any of the VH and VL domains described herein, and wherein the constant region comprises the amino acid sequence of a constant region of an IgG (e.g., human IgG) immunoglobulin molecule. In another specific embodiment, an antibody that 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 the amino acid sequence of any of the VH and VL domains described herein, and wherein the constant region comprises IgG1(e.g., human IgG)1) The amino acid sequence of the constant region of an immunoglobulin molecule.
Thus, in certain embodiments, the antibodies or antigen binding fragments thereof in the pharmaceutical compositions described herein have reduced fucose content or lack fucose (i.e., "no fucosylation"). such antibodies or antigen binding fragments thereof can be produced using techniques known to those skilled in the artCan be used to produce antibodies or antigen-binding fragments thereof having reduced fucose content.
In some embodiments, the afucosylated B7-H4 antibody or antigen binding fragment thereof has enhanced ADCC activity in vitro compared to a fucosylated B7-H4 antibody or antigen binding fragment thereof having the same amino acid sequence. In some embodiments, the afucosylated B7-H4 antibody or antigen binding fragment thereof causes 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 higher specific lysis compared to specific lysis with the fucosylated B7-H4 antibody.
In some embodiments, the B7-H4 antibody or antigen-binding fragment thereof has increased affinity for Fc γ RIIIA compared to a fucosylated B7-H4 antibody or antigen-binding fragment thereof having the same amino acid sequence. In some embodiments, the afucosylated B7-H4 antibody or antigen binding fragment thereof binds to Fc γ RIIIA with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 17-fold, or at least 20-fold higher affinity than the fucosylated B7-H4 antibody or antigen binding fragment thereof. In some embodiments, surface plasmon resonance is used to determine affinity for Fc γ RIIIA. In some embodiments, Fc γ RIIIA is selected from Fc γ RIIIA (V158) and Fc γ RIIIA (F158). In some embodiments, Fc γ RIIIA is Fc γ RIIIA (V158).
In some embodiments, the presence of fucose can be determined by methods comprising High Performance Liquid Chromatography (HPLC), capillary electrophoresis, or MALDI-TOF mass spectrometry.
In particular embodiments, the 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.
In particular embodiments, a composition comprises an antibody or antigen-binding fragment thereof that (i) comprises a CDR sequence of 20502, a VH and VL sequence of 20502, or a heavy and light chain sequence of 20502, and (ii) is afucosylated, e.g., wherein at least 95% of the antibody in the composition is afucosylated, or wherein fucosylation is not detectable in the composition.
Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Methods for producing engineered glycoforms in the antibodies or antigen binding fragments thereof described herein include, but are not limited to, those disclosed, for example, in:
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.
In another particular embodiment, an antibody or antigen-binding fragment thereof described herein that 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 antibodies listed in table 2; (iii) the heavy chain further comprises a constant heavy chain domain comprising a human IgG1The amino acid sequence of the constant domain of the 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.
In another particular embodiment, an antibody or antigen-binding fragment thereof described herein that 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 20502 antibody listed in table 4; (iii) and the heavy chain further comprises a constant heavy chain domain comprising a human IgG1The amino acid sequence of the constant domain of the heavy chain; and (iv) the light chain further comprises a constant light chain domainAnd a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa light chain.
In particular embodiments, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) exhibits T cell checkpoint blockade activity. In particular embodiments, an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4) described herein increases interferon- γ (IFN γ) production in T cells. In particular embodiments, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to B7-H4 (e.g., human B7-H4) increases T cell proliferation. In particular embodiments, an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4) described herein increases CD4+ T cell proliferation. In particular embodiments, an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4) described herein increases CD8+ T cell proliferation.
In particular embodiments, an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4) described herein exhibits antibody-dependent cellular cytotoxicity (ADCC) activity. In particular embodiments, an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4) described herein exhibits antibody-dependent cellular cytotoxicity (ADCC) activity on a cell line (e.g., SK-BR-3 cells) having at least 300,000 cell surface B7-H4 molecules. In particular embodiments, an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4) described herein exhibits antibody-dependent cellular cytotoxicity (ADCC) activity on a cell line (e.g., HCC1569 cells) having at least 100,000 cell surface B7-H4 molecules. In particular embodiments, an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4) described herein exhibits antibody-dependent cellular cytotoxicity (ADCC) activity on a cell line (e.g., ZR-75-1 cells) having at least 50,000 cell surface B7-H4 molecules. In particular embodiments, an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4) described herein exhibits antibody-dependent cellular cytotoxicity (ADCC) activity on a cell line (e.g., MDA-MB-468 cells) having at least 30,000 cell surface B7-H4 molecules. In particular embodiments, an antibody or antigen-binding fragment thereof that immunospecifically binds to B7-H4 (e.g., human B7-H4) described herein exhibits antibody-dependent cellular cytotoxicity (ADCC) activity on a cell line having at least 15,000 cell surface B7-H4 molecules (e.g., HCC1964 cells).
In a particular aspect, an antigen-binding fragment that immunospecifically binds to B7-H4 (e.g., human B7-H4) as described herein is selected from the group consisting of: fab, Fab ', F (ab')2And scFv, wherein the Fab, Fab ', F (ab')2Or the scFv comprises the heavy chain variable region sequence and the light chain variable region sequence of an anti-B7-H4 antibody or antigen-binding fragment thereof as described herein. Fab, Fab ', F (ab')2 or scFv can be produced by any technique known to those skilled in the art. In certain embodiments, Fab ', F (ab')2Or the scFv further comprises a moiety that extends the half-life of the antibody in vivo. The moiety is also referred to as "half-life extending moiety". The use of known in the art for extending Fab, Fab ', F (ab')2Or any part of the half-life of the scFv. For example, the half-life extending moiety may include an Fc region, a polymer, albumin, or albumin binding protein or compound. The polymer may comprise natural or synthetic, optionally substituted, linear or branched polyalkylene, polyalkenylene, polyoxyalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen or derivatives thereof. The substituents may include one or more hydroxy, methyl or methoxy groups. In certain embodiments, Fab ', F (ab')2Or a scFv. In certain embodiments, the half-life extending moiety is polyethylene glycol or human serum albumin. In certain embodiments, Fab ', F (ab')2Or scFv fused to the Fc region.
5.4 antibody production and polynucleotides
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 synthesizing antibodies and antigen-binding fragments thereof, e.g., by chemical synthesis or by recombinant expression techniques. Unless otherwise indicated, the methods described herein employ molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and techniques conventional in the relevant art within the skill of the art. For example, these techniques are described in, and are fully described in, the references cited herein. See, e.g., Sambrook J et 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 (1987and annual updates); current Protocols in Immunology, John Wiley & Sons (1987and annual updates) Gait (edited) (1984) Oligonucleotide Synthesis: A Practical Approach, IRLPress; eckstein (eds.) (1991) Oligonucleotides and antigens: A practical approach, IRL Press; birren B et al, (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.
In certain aspects, provided herein are pharmaceutical compositions comprising an anti-B7-H4 antibody or antigen-binding fragment, wherein the antibody or fragment is produced by recombinant expression of a polynucleotide comprising a nucleotide sequence in a host cell.
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 a nucleotide sequence set forth 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 set forth in Table 9 (i.e., SEQ ID NO:27) and a nucleotide sequence encoding a human gamma (γ) 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 set forth 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: polynucleotide sequence encoding heavy chain variable 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 a nucleotide sequence set forth 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 a nucleotide sequence set forth 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 set forth 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: polynucleotide sequence encoding light chain variable region
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 a nucleotide sequence encoding a variable heavy chain set forth in Table 9 (i.e., SEQ ID NO:27) and a variable light chain encoded by a polynucleotide comprising a nucleotide sequence encoding a variable light chain set forth 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 (i) a heavy chain encoded by a polynucleotide comprising a nucleotide sequence encoding a variable heavy chain (i.e., SEQ ID NO:27) set forth in table 9 and a nucleotide sequence encoding a human gamma (γ) heavy chain constant region, and (ii) a light chain encoded by a polynucleotide comprising a nucleotide sequence encoding a variable light chain (i.e., SEQ ID NO:28) set forth in table 10 and a nucleotide sequence encoding a human λ light chain constant region.
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 nucleotide sequence encoding a variable heavy chain shown in Table 9 (i.e., SEQ ID NO:27) and a nucleotide sequence encoding a heavy chain constant domain of SEQ ID NO:26, and (ii) a light chain encoded by a polynucleotide comprising the nucleotide sequence encoding a variable light chain shown in Table 10 (i.e., SEQ ID NO:28) and a nucleotide sequence encoding a light chain constant domain of SEQ ID NO: 24.
In certain aspects, the anti-B7-H4 antibody or antigen-binding fragment in the pharmaceutical compositions provided herein is encoded by an optimized polynucleotide encoding the anti-B7-H4 antibody or antigen-binding fragment thereof or domain thereof, e.g., by codon/RNA optimization, replacement with a heterologous signal sequence, and elimination of mRNA instability elements. Methods of producing optimized nucleic acids encoding anti-B7-H4 antibodies or antigen-binding fragments thereof or domains thereof (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., codon changes encoding the same amino acids due to degeneracy of the genetic code) and/or eliminating suppression regions in the mRNA can thus be optimized by employing the methods described, for example, in U.S. patent nos. 5,965,726; 6,174,666, respectively; 6,291,664, respectively; 6,414,132, respectively; and 6,794,498, respectively.
The polynucleotide may be, for example, in the form of RNA or DNA. DNA includes cDNA, genomic DNA and synthetic DNA. The DNA may be double-stranded or single-stranded. If single-stranded, the DNA may be the coding strand or the non-coding (anti-sense) strand. In certain embodiments, the polynucleotide is a cDNA or DNA lacking one or more introns. In certain embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In certain embodiments, the polynucleotide is recombinantly produced. In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure. In certain embodiments, the polynucleotide is purified from natural components.
In certain aspects, the vectors (e.g., expression vectors) comprise nucleotide sequences encoding anti-B7-H4 antibodies and antigen binding fragments thereof, or domains thereof, for recombinant expression in host cells, preferably in mammalian cells. In certain aspects, the cell (e.g., host cell) comprises such a vector for recombinant expression of an anti-B7-H4 antibody or antigen-binding fragment thereof (e.g., a human or humanized antibody or antigen-binding fragment thereof) described herein. Accordingly, methods for producing an antibody or antigen-binding fragment thereof for use in a pharmaceutical composition described herein can include expressing such an antibody or antigen-binding fragment thereof in a host cell.
The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cell 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 comprising the six CDRs, VH, VL, VH and VL, heavy chain, light chain, or heavy chain and light chain of 20502) or a domain thereof (e.g., VH, VL, VH and VL, heavy chain, or light chain of 20502).
In certain embodiments, in
In some embodiments, the anti-B7-H4 antibody 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 is produced in a host cell that lacks a functional alpha-1, 6-fucosyltransferase gene (FUT8) gene. In some embodiments, the host cell is a CHO cell.
In particular embodiments, the antibody or antigen-binding fragment thereof in the pharmaceutical compositions provided herein is isolated or purified. Typically, an isolated antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that is substantially free of other antibodies or antigen-binding fragments thereof having different antigen specificities than the isolated antibody or antigen-binding fragment thereof. For example, in a particular embodiment, the 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
In one aspect, provided herein is a method for modulating one or more immune functions in a subject, the method comprising administering to a subject in need thereof a pharmaceutical composition provided herein comprising a B7-H4 antibody or antigen binding fragment thereof.
In another embodiment, a pharmaceutical composition comprising an anti-B7-H4 antibody or antigen binding fragment thereof provided herein is administered to a patient (e.g., a human patient) to increase proliferation of T cells, CD4+ T cells, or CD8+ T cells in the patient. In another embodiment, a pharmaceutical composition comprising an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein is administered to a patient (e.g., a human patient) to increase the production of interferon-gamma (IFN γ) in the patient. In another embodiment, a pharmaceutical composition comprising an anti-B7-H4 antibody or antigen binding fragment thereof provided herein 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 comprising an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein 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 comprising an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein is administered to achieve two or more of the above-described effects.
In certain embodiments, provided herein are methods of treating cancer (e.g., a B7-H4-expressing cancer) comprising administering to a patient in need thereof (e.g., a human patient) a pharmaceutical composition comprising an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein. In certain embodiments, provided herein are methods of treating a solid tumor (e.g., a B7-H4-expressing solid tumor) comprising administering to a patient in need thereof (e.g., a human patient) a pharmaceutical composition comprising an anti-B7-H4 antibody or antigen-binding fragment thereof provided herein.
In a certain embodiment, provided herein is a pharmaceutical composition 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 cancer, ovarian cancer, urothelial cancer, non-small cell lung cancer (e.g., squamous cell carcinoma), pancreatic cancer, thyroid cancer, renal cancer (e.g., renal cell carcinoma), and bladder cancer (e.g., urothelial cell carcinoma). In certain embodiments, provided herein are pharmaceutical compositions for the treatment of advanced breast cancer, including triple negative breast cancer and Hormone Receptor (HR) positive breast cancer, ovarian cancer, endometrial cancer, or urothelial cancer. In certain embodiments, provided herein are pharmaceutical compositions for the treatment of breast cancer. In certain embodiments, provided herein are pharmaceutical compositions for the treatment of ovarian cancer. In certain embodiments, provided herein are pharmaceutical compositions for treating endometrial cancer. In certain embodiments, provided herein are pharmaceutical compositions for the treatment of urothelial cancer.
In some embodiments, the cancer is a B7-H4-expressing cancer.
In another embodiment, the pharmaceutical compositions provided herein are administered to a patient (e.g., a human patient) diagnosed with cancer to increase 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 (IFN γ) production in the patient. In another embodiment, the pharmaceutical compositions provided herein are 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, the pharmaceutical compositions provided herein are administered to a patient (e.g., a human patient) diagnosed with cancer to deplete B7-H4-expressing cancer cells in the patient.
The pharmaceutical compositions described herein may be delivered to a patient by intravenous route. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated.
6. Examples of the embodiments
The embodiments in this section (i.e., section 6) are provided by way of illustration and not by way of limitation.
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