anti-CD137 molecules and uses thereof

文档序号：1301542 发布日期：2020-08-07 浏览：14次中文

阅读说明：本技术 抗cd137分子及其用途 (anti-CD137 molecules and uses thereof ) 是由罗培志杜方勇刘桂中李艳陈俊佘晓红 P·张于 2017-08-21 设计创作，主要内容包括：本公开提供了与人CD137结合的抗体或其抗原结合片段、编码所述抗体或其抗原结合片段的核酸、所述抗体或其抗原结合片段的治疗组合物,以及它们用于增强T细胞功能以上调细胞介导的免疫应答以及用于治疗T细胞功能失调病症,诸如肿瘤免疫,以及用于治疗癌症的用途。(The present disclosure provides antibodies or antigen-binding fragments thereof that bind to human CD137, nucleic acids encoding the antibodies or antigen-binding fragments thereof, therapeutic compositions of the antibodies or antigen-binding fragments thereof, and their use for enhancing T cell function to upregulate cell-mediated immune responses and for treating T cell dysfunctional disorders, such as tumor immunity, and for treating cancer.)

1. An isolated antibody, or an antigen-binding fragment thereof, that binds to the extracellular domain of human CD137, wherein the antibody, or the antigen-binding fragment thereof, binds to SEQ ID NO: 1, one or more amino acid residues within amino acid residues 34-108 of 1.

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment binds to the amino acid sequence of SEQ id no: 1, or one or more amino acid residues within amino acid residues 34-93 of 1.

3. The antibody or antigen-binding fragment of claim 1 or claim 2, wherein the antibody or antigen-binding fragment binds to a polypeptide selected from the group consisting of SEQ ID NO: 1, amino acid residues 34-36, 53-55, and 92-93, or a pharmaceutically acceptable salt thereof.

4. The antibody or antigen-binding fragment of claim 3, wherein the antibody or antigen-binding fragment binds to the amino acid sequence of SEQ ID NO: 1, one or more of amino acid residues 34-36, one or more of amino acid residues 53-55, and one or more of amino acid residues 92-93.

5. The antibody or antigen-binding fragment of any one of claims 1-4, wherein the antibody or antigen-binding fragment does not bind to a polypeptide selected from the group consisting of SEQ ID NOs: 1, amino acid residues 109-112, 125, 126, 135-138, 150 and 151.

6. The antibody or antigen-binding fragment of claim 5, wherein the antibody or antigen-binding fragment does not bind to SEQ ID NO: 1, amino acid residues 109-112, 125, 126, 135-138, 150 and 151.

7. The antibody or antigen-binding fragment of any one of claims 1-6, wherein the antibody or antigen-binding fragment cross-reacts with a CD137 polypeptide from at least one non-human species selected from the group consisting of cynomolgus monkey, mouse, rat, and/or dog.

8. The antibody or antigen-binding fragment of claim 7, wherein the antibody or antigen-binding fragment binds to cynomolgus monkey CD 137.

9. The antibody or antigen-binding fragment of any one of claims 1-8, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region,

wherein the heavy chain variable region comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 711, HVR-H1 comprising the amino acid sequence SEQ ID NO: 735, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 759 HVR-H3; and/or

Wherein the light chain variable region comprises HVR-L1 comprising amino acid sequence SEQ ID NO: 783, HVR-L2 comprising amino acid sequence SEQ ID NO: 807, and HVR-L3 comprising amino acid sequence SEQ ID NO: 831.

10. The antibody or antigen-binding fragment of claim 9, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 41, and/or the light chain variable region comprises the amino acid sequence SEQ ID NO: 42.

11. the antibody or antigen-binding fragment of claim 10, wherein the antibody comprises a heavy chain and a light chain, and wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 617 and/or the light chain comprises the amino acid sequence SEQ id no: 618.

12. the antibody or antigen-binding fragment of any one of claims 1-8, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region,

wherein the heavy chain variable region comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 712, HVR-H1 comprising the amino acid sequence of SEQ ID NO: 736 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 760 HVR-H3; and/or

Wherein the light chain variable region comprises HVR-L1 comprising amino acid sequence SEQ ID NO: 784, HVR-L2 comprising amino acid sequence SEQ ID NO: 808, and HVR-L3 comprising amino acid sequence SEQ ID NO: 832.

13. The antibody or antigen-binding fragment of claim 12, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, and/or the light chain variable region comprises the amino acid sequence SEQ ID NO: 62.

14. the antibody or antigen-binding fragment of claim 13, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 619 and/or the light chain comprises the amino acid sequence SEQ ID NO: 620.

15. the antibody or antigen-binding fragment of any one of claims 1-8, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region,

wherein the heavy chain variable region comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 731, HVR-H1 comprising the amino acid sequence of SEQ ID NO: 755, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 779 HVR-H3; and/or

Wherein the light chain variable region comprises HVR-L1 comprising amino acid sequence SEQ ID NO: 803, HVR-L2 comprising amino acid sequence SEQ ID NO: 827, and HVR-L3 comprising amino acid sequence SEQ ID NO: 851.

16. The antibody or antigen-binding fragment of claim 15, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71, and/or the light chain variable region comprises the amino acid sequence SEQ ID NO: 72.

17. the antibody or antigen-binding fragment of claim 16, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 657 and/or the light chain comprises the amino acid sequence SEQ ID NO: 658.

18. an isolated antibody, or antigen-binding fragment thereof, that binds to the extracellular domain of human CD137, said antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region,

a) wherein the heavy chain variable region comprises HVR-H1, HVR-H2 and HVR-H3,

wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of:

formula (I): x₁TFX₂X₃YX₄IHWV (SEQ ID NO: 2) wherein X1 is F or Y, X2 is S or T, X3 is G, N or S, and X4 is A, G or W;

formula (II): YSIX₁SGX₂X₃WX₄WI (SEQ ID NO: 3) wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S or T; and

FS L STX of formula (III)₁GVX₂VX₃WI (SEQ ID NO: 4) wherein X1Is G or S, X2 is A or G, and X3 is A, G, S or T;

wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of:

l A L IDWX in formula (IV)₁X₂DKX₃YSX₄S L KSR L (SEQ ID NO: 5) wherein X1 is A, D or Y, X2 is D or G, X3 is R, S or Y, and X4 is P or T;

formula (V): IGX₁IYHSGX₂TYYX₃PS L KSRV (SEQ ID NO: 6) wherein X1 is D or E, X2 is N or S, and X3 is N or S, and

formula (VI): VSX₁ISGX₂GX₃X₄TYYADSVKGRF (SEQ ID NO: 7), wherein X1 is A, G, S, V or Y, X2 is A, D, S or Y, X3 is D, G or S, and X4 is S or T; and is

Wherein the HVR-H3 comprises an amino acid sequence according to the formula: formula (VII): ARX₁GX₂X₃X₄VX₅GDWFX₆Y (SEQ ID NO: 8) wherein X1 is E or G, X2 is E or S, X3 is D or T, X4 is A, T or V, X5 is A, I, L, T or V, and X6 is A, D or G, and/or

b) Wherein the light chain variable region comprises HVR-L1, HVR-L2 and HVR-L3,

wherein said HVR-L1 comprises an amino acid sequence according to formula (VIII) X₁ASQX₂X₃X₄X₅X₆X₇X₈(SEQ id no: 9) wherein X1 is Q or R, X2 is D, G or S, X3 is I or V, X4 is G, R, S or T, X5 is P, R, S or T, X6 is A, D, F, S, V or Y, X7 is L or V, and X8 is A, G or N;

wherein said HVR-L2 comprises an amino acid sequence according to formula (IX) X₁ASX₂X₃X₄X₅GX₆(SEQ ID NO: 10) wherein X1 is A or D, X2 is N, S or T, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V, and

wherein the HVR-L3 comprises an amino acid sequence according to a formula selected from the group consisting of:

formula (X): YCQQX₁YX₂X₃X₄T (SEQ ID NO: 11) wherein X1 is A, G, S or Y, X2 is Q, S or Y, X3 is I, L, T or Y, and X4 is I, S, V or W, and

formula (XI): YCX₁QX₂X₃X₄X₅PX₆T (SEQ ID NO: 12), wherein X1 is E or Q, X2 is P, S or Y, X3 is D, L, S, T or Y, X4 is D, E, H, S or T, X5 is D, L T or W, and X6 is L, P, R or V.

19. The antibody or antigen-binding fragment of claim 18, wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 253-312, the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 313-372, the HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 373-432, the HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 433-492, the HVR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 493-552, and/or the HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 553-612.

20. The antibody or antigen-binding fragment of claim 18 or claim 19, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 13. 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, and 131, and/or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14. 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, and 132.

21. The antibody or antigen-binding fragment of claim 18, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of:

formula (XII): x₁TFSX₂YWIHWV (SEQ ID NO: 853), wherein X1 is F or Y, and X2 is N or S;

formula (XIII): YSIX₁SGX₂X₃WX₄WI (SEQ ID NO: 854) wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N or S; and

formula (XIV) FS L STX₁GVX₂VX₃WI (SEQ ID NO: 855) wherein X1 is G or S, X2 is A or G, and X3 is A, G or S;

wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of:

l A L IDWX in formula (IV)₁X₂DKX₃YSX₄S L KSR L (SEQ ID NO: 5) wherein X1 is A, D or Y, X2 is D or G, X3 is R, S or Y, and X4 is P or T, and

formula (XV): VSX₁ISGX₂GX₃X₄TYYADSVKGRF (SEQ ID NO: 856) wherein X1 is G, S, V or Y, X2 is A, D, S or Y, X3 is D, G or S, and X4 is S or T; and is

Wherein the HVR-L1 comprises an amino acid sequence according to formula (XVI) X₁ASQX₂X₃X₄X₅X₆X₇X₈(SEQ ID NO: 857) wherein X1 is Q or R, X2 is D, G or S, X3 is I or V, X4 is G, R, S or T, X5 is P, R, S or T, X6 is A, F, S, V or Y, X7 is L or V, and X8 is A or G;

wherein saidHVR-L2 comprises an amino acid sequence according to the formula (XVII) X₁ASX₂X₃X₄X₅GX₆(SEQ ID NO: 858) wherein X1 is A or D, X2 is N or S, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V, and

wherein said HVR-L3 comprises an amino acid sequence according to the formula (XVIII) YCQQQX₁YX₂X₃WT (SEQ ID NO: 859) wherein X1 is A or G, X2 is S or Y, and X3 is I, L or T.

22. The antibody or antigen-binding fragment of claim 21, wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 709-732, the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 733-756, the HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 757-780, the HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 781-804, the HVR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 805-828, and the HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 829-852.

23. The antibody or antigen-binding fragment of claim 21 or claim 22, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 15. 17, 31, 33, 35, 3/, 39, 41, 43, 45, 4/, 49, 53, 61, 63, 65, 67, 71, 73, 75, 79, 83, 85, and 87, and/or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ id nos: 16. 18, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 62, 64, 66, 68, 72, 74, 76, 80, 84, 86, and 88.

24. The antibody or antigen-binding fragment of any one of claims 21-23, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 613. 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657 and 659, and/or the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 614. 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, and 660.

25. The antibody or antigen-binding fragment of claim 21, wherein said HVR-H1 comprises the amino acid sequence of SEQ ID No. 711 or 731, said HVR-H2 comprises the amino acid sequence of SEQ ID No. 735 or 755, said HVR-H3 comprises the amino acid sequence of SEQ ID No. 759 or 779, said HVR-L1 comprises the amino acid sequence of SEQ ID No. 783 or 803, said HVR-L2 comprises the amino acid sequence of SEQ ID No. 807 or 827, and said HVR-L3 comprises the amino acid sequence of SEQ ID No. 831 or 851.

26. The antibody or antigen-binding fragment of claim 25, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 41 or 71 and the light chain variable region comprises the amino acid sequence SEQ ID NO: 42 or 72.

27. The antibody or antigen-binding fragment of claim 26, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 617 or 657 and the light chain comprises the amino acid sequence SEQ ID NO: 618, or 658.

28. The antibody or antigen-binding fragment of claim 21, wherein said HVR-H1 comprises the amino acid sequence of SEQ ID NO: 712, said HVR-H2 comprises the amino acid sequence of SEQ ID NO: 736, said HVR-H3 comprises the amino acid sequence of SEQ ID NO: 760, said HVR-L1 comprises the amino acid sequence of SEQ ID NO: 784, said HVR-L2 comprises the amino acid sequence of SEQ ID NO: 808, and said HVR-L3 comprises the amino acid sequence of SEQ ID NO: 832.

29. The antibody or antigen-binding fragment of claim 28, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62.

30. the antibody or antigen-binding fragment of claim 29, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 619 and the light chain comprises the amino acid sequence SEQ ID NO: 620.

31. the antibody or antigen-binding fragment of any one of claims 1-30, wherein the antibody or antigen-binding fragment binds human CD137 with a KD of 100nM or less as measured by surface plasmon resonance.

32. The antibody or antigen-binding fragment of claim 31, wherein the antibody or antigen-binding fragment has a K of 50nM or less as measured by surface plasmon resonance_DBinds to human CD 137.

33. The antibody or antigen-binding fragment of any one of claims 18-32, wherein the antibody or antigen-binding fragment cross-reacts with a CD137 polypeptide from at least one non-human species selected from the group consisting of cynomolgus monkey, mouse, rat, and/or dog.

34. The antibody or antigen-binding fragment of claim 33, wherein the antibody or antigen-binding fragment binds to cynomolgus monkey CD 137.

35. The antibody or antigen-binding fragment of any one of claims 1-34, wherein the activity of human CD137 expressed on human cells is reduced when contacted with the antibody or antigen-binding fragment.

36. The antibody or antigen-binding fragment of any one of claims 1-34, wherein the antibody or antigen-binding fragment has a half-maximal Inhibitory Concentration (IC) of about 100nM or less for blocking in vitro binding of human CD137 to human CD 137L₅₀)。

37. The antibody or antigen-binding fragment of any one of claims 1-34, wherein the antibody or antigen-binding fragment completely blocks in vitro binding of human CD137 to human CD 137L when the antibody or antigen-binding fragment is provided at a concentration of about 1 μ Μ or greater.

38. The antibody or antigen-binding fragment of any one of claims 1-34, wherein the activity of human CD137 expressed on human cells is increased when contacted with the antibody or antigen-binding fragment.

39. The antibody or antigen-binding fragment of claim 38, wherein contacting a human cell expressing CD137 with the antibody or antigen-binding fragment results in increased NF- κ B-dependent transcription.

40. The antibody or antigen-binding fragment of any one of claims 1-39, wherein the antibody comprises a human IgG2Fc region.

41. The antibody or antigen-binding fragment of any one of claims 1-40, wherein the antibody comprises a human IgG4Fc region.

42. The antibody or antigen-binding fragment of claim 41, wherein the human IgG4Fc region comprises the S241P mutation, wherein numbering is according to Kabat.

43. The antibody or antigen-binding fragment of any one of claims 18-20, wherein the heavy chain variable region is encoded by a sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 133. 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, and 251, and/or the light chain variable region is encoded by a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 134. 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, and 252.

44. The antibody or antigen-binding fragment of claim 24, wherein the heavy chain consists of a light chain comprising a sequence selected from the group consisting of SEQ id nos: 661. 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705 and 707, and/or the light chain is encoded by a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 662. 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, and 708.

45. A polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 1-44.

46. A polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 133-252.

47. A vector comprising the polynucleotide of any one of claims 45-46.

48. The vector of claim 47, wherein the vector is an expression vector.

49. A host cell comprising the vector of claim 47 or claim 48.

50. A method of making an antibody or antigen-binding fragment comprising culturing the host cell of claim 49 under conditions suitable for production of the antibody or antigen-binding fragment.

51. The method of claim 50, further comprising recovering the antibody or antigen-binding fragment produced by the cell.

52. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-42, and a pharmaceutically acceptable carrier.

53. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1-42.

54. The method of claim 53, further comprising administering to the subject a therapeutically effective amount of at least one additional therapeutic agent.

55. The method of claim 54, wherein the at least one additional therapeutic agent is selected from the group consisting of: viral gene therapy, immune checkpoint inhibitors, target therapy, radiotherapy and chemotherapy.

56. The method of claim 54 or claim 55, wherein the at least one additional therapeutic agent is selected from the group consisting of pomalidomide, lenalidomide, rildomide, pomalidomide, thalidomide, DNA-alkylated platinum-containing derivative, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CT L A4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD 20 antibody, anti-CD 40 antibody, anti-DR 5 antibody, anti-CD 1d antibody, anti-TIM 3 antibody, anti-S L AMF7 antibody, anti-KIR receptor antibody, anti-OX 40 antibody, anti-HER 2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrotuzumab, radiation therapy, single-dose radiation, split-dose radiation, focal radiation, perioperative radiation, I L-12, IFN α, GM-chimeric CSF, CSF-chimeric antigen receptor, adoptive T-tumor vaccine, and anti-cancer.

Technical Field

The present disclosure relates to antibodies or antigen-binding fragments thereof that bind to human CD137, nucleic acids encoding the antibodies or antigen-binding fragments thereof, therapeutic compositions of the antibodies or antigen-binding fragments thereof, and their anti-tumor uses.

Background

CD137 (also known as CD137 receptor, 4-1BB, TNFRSF9, etc.) is a transmembrane protein of the Tumor Necrosis Factor Receptor Superfamily (TNFRS) current understanding of CD137 suggests that its expression is generally activation dependent and is present in a broad subset of immune cells, including activated NK and NKT cells, regulatory T cells, Dendritic Cells (DCs), stimulated mast cells, differentiating myeloid, monocytes, neutrophils and eosinophils (Wang, 2009, Immunological Reviews 229: 192 @) CD137 expression has also been demonstrated to be on tumor vasculature (Broll, 2001, Amer. J.Clin. Pathol.115 (4): 543- -Seaman, 2007, Cancer 11: 539 554) and endothelial sites of inflammatory or atherosclerotic (Drenkard, EB J.21: 456; Orchison, 137: 35137, CD137, CD 1292, CD137, CD 16, CD137, CD 16, CD137, CD137, CD 3.

Human CD137 is a protein of 255 amino acids (GenBank accession NM-001561; NP-001552; SEQ ID NO.: 1). The protein comprises a signal sequence (amino acid residues 1-17) followed by an extracellular domain (169 amino acids), a transmembrane region (27 amino acids), and an intracellular domain (42 amino acids) (Cheuk ATC et al 2004cancer Therapy 11: 215-. Receptors are expressed on the cell surface in monomeric and dimeric forms and may trimerize with CD137 ligands for signaling.

Many studies of murine and human T cells indicate that CD137 promotes enhanced cell proliferation, survival and cytokine production (Croft, 2009, Nat Rev Immunol 9: 271-.

There is a long-felt unmet need for antibodies that bind to human CD137, increase CD 137-mediated responses and thereby provide potential therapeutic agents for the treatment of various diseases and conditions, including cancer and autoimmune diseases. Furthermore, there is a need for anti-CD137 antibodies that cross-react between different species, such as humans and experimental animals (mice, monkeys, dogs, etc.), to enable animal model studies while providing therapeutic agent candidates.

Disclosure of Invention

It is an object of the present disclosure to provide an isolated binding molecule, such as an antibody or binding fragment thereof, or a derivative thereof, that binds to human CD 137. It is another object of the present disclosure to provide a composition comprising a binding molecule that binds to CD 137. It is yet another object of the present disclosure to provide methods for treating diseases and/or conditions associated with or mediated by CD137 signaling by using one or more binding molecules of the present disclosure. These and other objects of the present disclosure are more fully described herein.

Thus, in one aspect, provided herein are one or more antibodies (e.g., isolated antibodies) or one or more antigen-binding fragments thereof that bind to the extracellular domain of human CD137 and include one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all 10) of the following functional features: (a) binds to SEQ ID NO: 1, one or more amino acid residues within amino acid residues 34-108; (b) does not differ from SEQ ID NO: 1 at one or more amino acid residues within amino acid residues 109-112, 125, 126, 135-138, 150 and 151; (c) at a K of 100nM or less_D(ii) binds to human CD137, (d) has agonist activity on human CD137, (e) does not bind to human OX40, CD40, GITR and/or CD27 receptors at concentrations up to 1000nM, (f) cross-reacts with monkey, mouse, rat and/or dog CD137, (g) does not elicit ADCC effects, (h) is capable of inhibiting tumor cell growth, (i) has therapeutic effect on cancer, and/or (j) blocks binding between CD137 and CD 137L.

Thus, in one aspect, provided herein is an antibody (e.g., an isolated antibody) or antigen-binding fragment thereof that binds to the extracellular domain of human CD 137. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: formula (I): x₁TFX₂X₃YX₄IHWV (SEQ ID NO: 2) wherein X1is F or Y, X2 is S or T, X3 is G, N or S, and X4 is A, G or W; formula (II): YSIX₁SGX₂X₃WX₄WI (SEQ ID NO: 3) wherein X1is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S or T, and formula (III) FS L STX₁GVX₂VX₃WI (SEQ ID NO: 4) wherein X1is G or S, X2 is A or G, and X3 is A, G, S or T; wherein HVR-H2 comprisesAn amino acid sequence according to formula (IV) selected from the group consisting of A IDWX1X 23 KSR (SEQ ID NO: 5) wherein X is or Y, X is D or G, X is or Y, and X is P or T, formula (V) IGX1IYHSGX2 TYX 3KSRV (SEQ ID NO: 6) wherein X is D or E, X is N or S, and formula (VI) VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 7) wherein X is or Y, X is or S, and X is S or T, and wherein HVR-H comprises an amino acid sequence according to formula (VII) ARX1GX2X 4VX5GDWFX 6(SEQ ID NO: 8) wherein X is E or G, X is E or S, X is D or T, X is D or T, and X is P or T, and X is G or T.

In some embodiments, provided herein is an antibody (e.g., an isolated antibody) or antigen-binding fragment thereof that binds to the extracellular domain of human CD137, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H, and HVR-H, wherein HVR-H comprises an amino acid sequence according to a formula selected from the group consisting of formula (XII) X1TFSX2 YHWWIV (SEQ ID NO: 853), wherein X is F or Y, and X is N or S, formula (XIII) YSIX1SGX2X3WX4 (SEQ ID NO: 854), wherein X is S or T, X is H or Y, and X is or S, and formula (XIV) STX1 VX3 (SEQ ID NO: 855), wherein X is G or S, X is A or G, and X is or S, wherein HVR-H comprises an amino acid sequence according to the group consisting of HVX 1 or 5, formula (XV) and wherein X is 2 VXX or VXX 4, and wherein X is 2 or VXX 4, and wherein X is selected from the group consisting of formula (SAX) or VXX 4 is selected from the group consisting of SEQ ID NO, and wherein X is 1 or 5 or VXX is 5 or 5, and wherein X is 5 or VXX, and wherein X is 5 or VXX, and wherein X is shown in the formula (SAX or 5 or VXX) or 5 or V, and wherein SAX is shown in the formula (SAX shown.

In another aspect, provided herein is an antibody (e.g., an isolated antibody) or antigen-binding fragment thereof that binds to the extracellular domain of human CD137, in some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises HVR-1, HVR-2, and HVR-03, wherein HVR-11 comprises an amino acid sequence according to the following formula (VIII) X1ASQX2X3X4X5X6X7X (SEQ ID NO: 9), wherein X is Q or R, X is or S, X is I or V, X is or T, X is or Y, X is or V, and X is or N, wherein HVR-2 comprises an amino acid sequence according to the following formula (IX) X1ASX2X3X4X5GX (SEQ ID NO: 10), wherein X is A or D, X is or T, X is or R, X is or Q, X is S or T, and X is 3X4X5 or Y, Y is X, Y is X, Y is X is.

In some embodiments, provided herein is an antibody (e.g., an isolated antibody) or antigen-binding fragment thereof that binds to the extracellular domain of human CD137, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises HVR-L1, HVR-L2 and HVR-L3, wherein HVR-L1 comprises an amino acid sequence according to the following formula (XVI): X1ASQX2X3X4X5X6X7X8(SEQ ID NO: 857), wherein X1is Q or R, X2 is D, G or S, X3 is I or V, X4 is G, R, S or T, X5 is P, R, S or T, X6 is A, F, S, V or Y, X7 is L or V, and X8 is A or G, wherein R-L2 comprises an amino acid sequence according to the following formula (XVII): formula₁ASX₂X₃X₄X₅GX₆(SEQ ID NO: 858) wherein X1is A or D, X2 is N or S, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V, and wherein HVR-L3 comprises an amino acid sequence according to formula (XVIII): YCQQX₁YX₂X₃WT (SEQ ID NO: 859) wherein X1is A or G, X2 is S or Y, and X3 is I, L orT。

In another aspect, provided herein is an antibody (e.g., an isolated antibody) or antigen-binding fragment thereof that binds to the extracellular domain of human CD137, wherein the heavy chain variable region comprises heavy chain variable regions of VH, VH 348, or VH, and/or light chain variable regions comprising VH1, VH2, V04, VH, V006, V, VH, or VH, or VH, or VH, V, or VH, V150, V, or VH, V, or VH, V, or VH.

In another aspect, provided herein is an antibody (e.g., an isolated antibody) or antigen-binding fragment thereof that binds to the extracellular domain of human CD 137. In some embodiments, the antibody or antigen-binding fragment thereof binds to SEQ ID NO: 1, one or more amino acid residues within amino acid residues 34-108 of 1. In some embodiments, the antibody or antigen-binding fragment binds to SEQ ID NO: 1, or one or more amino acid residues within amino acid residues 34-93 of 1. In some embodiments, the antibody or antigen-binding fragment binds to a polypeptide selected from the group consisting of SEQ ID NOs: 1, amino acid residues 34-36, 53-55, and 92-93, or a pharmaceutically acceptable salt thereof. In some embodiments, the antibody or antigen-binding fragment binds to SEQ ID NO: 1, one or more of amino acid residues 34-36, one or more of amino acid residues 53-55, and one or more of amino acid residues 92-93. In some embodiments, the antibody or antigen binding fragment does not bind to a polypeptide selected from the group consisting of SEQ id nos: 1, amino acid residues 109-112, 125, 126, 135-138, 150 and 151. In some embodiments, the antibody or antigen-binding fragment does not bind to SEQ ID NO: 1, amino acid residues 109-112, 125, 126, 135-138, 150 and 151. In some embodiments, the antibody or antigen-binding fragment cross-reacts with a CD137 polypeptide from at least one non-human species selected from cynomolgus monkey (cynomolgus monkey), mouse, rat, and/or dog. In some embodiments, the antibody or antigen binding fragment binds to cynomolgus monkey CD 137.

In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1 comprising amino acid sequence SEQ ID NO: 731, HVR-H2 comprising amino acid sequence SEQ ID NO: 755, and HVR-H3 comprising amino acid sequence SEQ ID NO: 779, and/or wherein the light chain variable region comprises HVR-L1 comprising amino acid sequence SEQ ID NO: 803, HVR-L2 comprising amino acid sequence SEQ ID NO: 827, and HVR-L3 comprising amino acid sequence SEQ ID NO: 851. in some embodiments, the heavy chain variable region comprises amino acid sequence SEQ ID NO: 71, and/or the light chain variable region comprises amino acid sequence SEQ ID NO: 72. in some embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises amino acid sequence SEQ ID NO: 657, and/or the light chain comprises amino acid sequence SEQ ID NO: 658.

In another aspect, provided herein is an antibody (e.g., an isolated antibody) that binds to the extracellular domain of human CD137, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of formula (I), formula (II), and formula (III), HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of formula (IV), formula (V), and formula (VI), and HVR-H3 comprises an amino acid sequence according to formula (VII), and/or light chain variable region comprises HVR-L, HVR-L2, and HVR-L, wherein HVR-L comprises an amino acid sequence according to formula (VIII), HVR-L comprises an amino acid sequence according to formula (IX), and HVR-L comprises an amino acid sequence according to the formula selected from the group consisting of formula (XVIII), and wherein the isolated antibody comprises an amino acid sequence according to the formula (XVIII), wherein the amino acid sequence selected from the group consisting of HVR-H5926, HVR-H5926, HVR 415, HVR 4153, HVR 4156 comprises an amino acid sequence according to the formula (VII, HVR 24, HVR 4153, HVR 4153, HVR 4148, HVR 4148, HVR 4153, HVR 4148.

In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 253, 312, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 313, 372, HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 373, 432, HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 433, 492, HVR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 493, 552, and/or HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 553, 612 in some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 63, 57, 59, 65, 19, 21, 23, 70.

In some embodiments HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of formula (XII), formula (XIII) and formula (XIV), HVR-H2 comprises an amino acid sequence according to formula (IV) or formula (XV), and HVR-H3 comprises an amino acid sequence according to formula (VII), and/or wherein HVR-L1 comprises an amino acid sequence according to formula (XVI), HVR-L comprises an amino acid sequence according to formula (XVII), and HVR-L3 comprises an amino acid sequence according to formula (XVIII). in some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 709,732, 520,931,264,68, HVR-H4 comprises an amino acid sequence selected from the group consisting of 733,733,756,756,46,46,46,46,46,46,46,46,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,48,70,48,48,48,48,48,70,70,48,70,48,48,48,48,70,70,70,48,70,48,46,46,48,70,48,48,48,48,48,70,70,70,70,48,48,48,48,48,70,48,48,70,70,48,150,150,150,150,150,150,70,150,150,150,150,48,48,150,150,150,150,150,150,48,150,150,150,48,150,150,150,150,150,150,150,150,150,48,150,150,150,150,150,150,150,48,48,150,150,48,150,150,48,48,150,48,48,48,48,48,48,150,48,150,150,48,150,150,150,48,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,48,150,150,48,48,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,48,48,150,150,48,48,48,48,48,48,150,150,150,150,.

In some embodiments, HVR-H1 comprises amino acid sequence SEQ ID NO: 711 or 731, HVR-H2 comprises amino acid sequence SEQ ID NO: 735 or 755, HVR-H3 comprises amino acid sequence SEQ ID NO: 759 or 779, HVR-L1 comprises amino acid sequence SEQ ID NO: 783 or 803, HVR-L2 comprises amino acid sequence SEQ ID NO: 807 or 827, and HVR-L3 comprises amino acid sequence SEQ ID NO: 831 or 851.

In some embodiments, HVR-H1 comprises the amino acid sequence SEQ ID NO: 712, HVR-H2 comprises the amino acid sequence SEQ ID NO: 736, HVR-H3 comprises the amino acid sequence SEQ ID NO: 760, HVR-L1 comprises the amino acid sequence SEQ ID NO: 784, HVR-L2 comprises the amino acid sequence SEQ ID NO: 808, and HVR-L3 comprises the amino acid sequence SEQ ID NO: 832 in some embodiments, the heavy chain variable region comprises the amino acid sequence SEQ ID NO: 61 and the light chain comprises the amino acid sequence SEQ ID NO: 62.

In some embodiments that may be combined with any of the preceding embodiments, the antibody or antigen binding fragment has a K of 100nM or less_DBinds to human CD137 (e.g., as measured by surface plasmon resonance). In some embodiments, the antibody or antigen binding fragment has a K of 50nM or less_DBinds to human CD137 (e.g., as measured by surface plasmon resonance).

In some embodiments that may be combined with any of the preceding embodiments, the antibody or antigen binding fragment cross-reacts with a CD137 polypeptide from at least one non-human species selected from cynomolgus monkey (e.g., GenBank Gene ID 102127961), mouse (e.g., GenBank Gene ID 21942), rat (e.g., GenBank Gene ID 500590), and/or dog (e.g., GenBank Gene ID 608274). In some embodiments, the antibody or antigen binding fragment binds to cynomolgus monkey CD 137.

In some embodiments that may be combined with any of the preceding embodiments, the activity of human CD137 (e.g., when expressed on a cell such as a human cell) is reduced when contacted with the antibody or antigen-binding fragment.

In some embodiments that may be combined with any of the preceding embodiments, the antibody or antigen-binding fragment has a half-maximal Inhibitory Concentration (IC) of about 100nM or less for blocking in vitro binding of human CD137 to human CD 137L₅₀) In some embodiments, the antibody or antigen-binding fragment completely blocks the in vitro binding of human CD137 to human CD 137L when provided at a concentration of about 1 μ Μ or greater in some embodiments that may be combined with any of the preceding embodiments, the activity of human CD137 (e.g., when expressed on a cell such as a human cell) is increased when contacted with the antibody or antigen-binding fragment.

In some embodiments that may be combined with any of the preceding embodiments, the antibody comprises a human IgG2 Fc region. In some embodiments that may be combined with any of the preceding embodiments, the antibody comprises a human IgG4 Fc region. In some embodiments, the human IgG4 Fc region comprises the S241P mutation, wherein numbering is according to Kabat. In some embodiments, the antibody or antigen-binding fragment does not cause ADCC effects.

In another aspect, provided herein is a polypeptide consisting of a sequence comprising an amino acid sequence selected from SEQ ID NOs: 133. 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, and 251, and/or an antibody heavy chain variable region encoded by a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 134. 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, and 252. In some embodiments, provided herein is a polypeptide consisting of a sequence selected from SEQ ID NOs: 661. 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705 and 707, and/or an antibody heavy chain encoded by a polynucleotide comprising a sequence selected from SEQ ID NOs: 662. 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, and 708.

In another aspect, provided herein is a polynucleotide encoding any of the antibodies or antigen-binding fragments described herein. In some embodiments, provided herein is a polynucleotide comprising a sequence selected from SEQ ID NOs: 133-252.

In another aspect, provided herein is a vector comprising any one of the polynucleotides described above. In some embodiments, the vector is an expression vector.

In another aspect, provided herein is a host cell (e.g., a bacterial cell, a yeast cell, an insect cell, a mammalian cell (such as a CHO cell or 293T cell), etc.) comprising any of the polynucleotides or vectors described herein. In some embodiments, provided herein is a method of making an antibody or antigen-binding fragment comprising culturing a host cell under conditions suitable for production of the antibody or antigen-binding fragment. In some embodiments, the method further comprises recovering the antibody or antigen-binding fragment produced by the host cell.

In another aspect, provided herein is a pharmaceutical composition comprising any of the antibodies or antigen-binding fragments (or any derivatives thereof) described herein and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a method of treating abnormal cell growth (e.g., cancer) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the antibodies, antigen-binding fragments, and/or pharmaceutical compositions described herein, in some embodiments, provided herein is a method of reducing tumor cell metastasis in a subject, comprising administering to the subject a therapeutically effective amount of any of the antibodies, antigen-binding fragments, and/or pharmaceutical compositions described herein, in some embodiments, the method further comprises administering to the subject a therapeutically effective amount of at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least 10, etc.) additional therapeutic agent, in some embodiments, at least one additional therapeutic agent is selected from the group consisting of anti-tumor receptor antibodies (e.g., anti-mitomycin), anti-tumor receptor antibodies (anti-trastuximab), anti-tumor receptor antibodies (anti-T-trastuximab) antibodies, anti-tumor cell metastasis antibodies (e), anti-tumor receptor antibodies, anti-T-trastuximab, anti-T-c, anti-T-c, or anti-T.

It will be appreciated that one, some or all of the features of the various embodiments described above and herein may be combined to form further embodiments of the present disclosure. These and other aspects of the disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following detailed description.

Drawings

FIG. 1A shows the hypervariable region (HVR) definitions compared to the Kabat CDR definitions for an exemplary heavy chain variable region (VH) (SEQ ID NO: 13) and an exemplary light chain variable region (V L) (SEQ ID NO: 14).

Figure 1B shows the selection of Fab hits (hits) that cross-react with mouse CD 137.

Figure 2 shows the E L ISA binding assay of exemplary antibodies to human, monkey and mouse CD137 each panel is directed to a different antibody as indicated at the top of the panel.

Figure 3A shows FACS-based binding assays of exemplary antibodies to human, monkey, mouse, and rat CD 137. Each panel is directed to a different antigen as indicated at the top of the panel.

Fig. 3B shows a comparison of species cross-reactivity between exemplary and reference antibodies.

FIG. 4A shows that exemplary antibodies bind to activated human and monkey T cells, but not to naive cellsHuman T cells.

Fig. 4B shows the binding of AG10131 to activated human, monkey, mouse and rat T cells.

Figure 5 shows the binding specificity of an exemplary antibody to CD137, but not other TNFR family members.

Fig. 6A and 6B show that exemplary antibodies block the binding of CD137 to its cognate ligand, CD 137L, obtained by the E L ISA (fig. 6A) and flow cytometry assays (fig. 6B).

Fig. 7A shows epitope mapping results obtained by flow cytometry.

FIG. 7B shows a multiple sequence alignment of a portion of human (SEQ ID NO: 1), cynomolgus monkey (SEQ ID NO: 860) and mouse (SEQ ID NO: 861) CD137 with the target CD137 sequence/region (annotated) identified from the epitope mapping experiment.

Figure 8 shows agonist activity of exemplary antibodies in NF κ B reporter assays.

Figure 9 shows agonist activity of exemplary antibodies in CD8+ T cell proliferation (top panel) and INF- γ secretion (bottom panel).

FIG. 10 shows the anti-tumor efficacy of exemplary antibodies in the H22 mouse liver cancer model, and CD4⁺And CD8⁺Infiltration of T cells in tumors.

Figure 11 shows the anti-tumor efficacy of exemplary antibodies in a CT26 mouse colon cancer model.

Figure 12 shows the anti-tumor efficacy of exemplary antibodies in the EMT6 mouse breast cancer model.

Figure 13 shows that CT26 mice treated with exemplary antibodies remained tumor-free after re-challenge with the same tumor cells.

Figure 14 shows tumor cell killing achieved using splenocytes from tumor rejection re-primed mice.

Figure 15 shows that AG10131 does not show ADCC effect.

Figure 16 shows that the exemplary antibodies show little aggregation at high concentrations.

Figure 17 shows the stability of an exemplary antibody under accelerated stress conditions (acellulated stress conditions).

Fig. 18 shows thermal stability.

FIG. 19 shows that AG10131 has no hematological toxicity in normal mice up to 100mg/kg (BIW) x2 every two weeks.

FIG. 20 shows that AG10131 has no histological liver abnormalities in normal mice up to 100mg/kg (BIW) x2 every two weeks.

FIG. 21 shows that AG10131 has no hematological toxicity in cynomolgus monkeys at 10 mg/kg/week x 4.

FIG. 22 shows that AG10131 has no hepatotoxicity in monkeys at 10 mg/kg/week x 4.

Figure 23 shows the pharmacokinetic profile of AG10131 in monkeys.

Fig. 24 shows the pharmacokinetic profile of AG10131 in rats.

Figure 25 shows the pharmacokinetic profile of various antibodies in mice.

Detailed Description

A. Definition of

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure will have the meanings that are commonly understood by one of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. In general, the nomenclature used in connection with, and the techniques of, antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein, are those well known and commonly used in the art.

As used herein, each of the following terms has its associated meaning in this section.

The articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ -carboxyglutamate, and O-phosphoserine. The term "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, but has a C-terminal carboxyl group, an N-terminal amino group, or a side chain functional group that has been chemically modified to another functional group. The term "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., single chain variable fragments or scfvs) so long as they exhibit the desired biological activity.

The term "antibody" is a art-recognized term and may refer to an antigen binding protein (i.e., an immunoglobulin) having a substantially four polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype_HFollowed by a constant region. The heavy chain constant region comprises three domains: c_H1、C_H2And C_H3. Each light chain has a variable region at the N-terminus (abbreviated herein as V)_I) Followed by a constant region at its other end. The light chain constant region comprises a domain, C_L. Will V_LAnd V_HAlignment and alignment of C_LAligned with the first constant domain of the heavy chain (CH 1). V_HAnd V_LTogether form a single antigen binding site. IgM antibodies consist of 5 basic heterotetrameric units and another polypeptide called the J chain, and thus contain 10 antigen binding sites; while secretory IgA antibodies can polymerize to form multivalent assemblies comprising 2-5 basic 4 chain units and a J chain.

V_HAnd V_LRegions can also be subdivided into regions with high denaturation, based on structural and sequence analysis, called hypervariable regions (HVRs). The HVRs are interspersed with more conserved regions, called framework regions (FW). For comparison, the Kabat CDR definitions of Yvonne Chen et al (Selection and Analysis of an Optimized Anti-VEGF Antibody: crystalline Structure of an Affinity-matched Fab in Complex with Antibody, J.mol.biol. (1999)293, 865-. Each V_HAnd V_LConsisting of three HVRs and four FWs, arranged from amino-terminus to carboxy-terminus, FW1, HVR1, FW2, HVR2, FW3, HVR3, FW4. throughout this disclosure, the three HVRs of the heavy chain are referred to as HVR _ H1, HVR _ H2 and HVR _ H3. similarly, the three HVRs of the light chain are referred to as HVR _ L1, HVR _ L2 and HVR _ L3.

The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 or more amino acids. See generally Fundamental Immunology chapter 7 (Paul, W. eds., 2 nd edition, Raven Press, N.Y. (1989)).

There are five classes of antibodies, IgA, IgD, IgE, IgG and IgM, with heavy chains designated α (alpha), (delta), (epsilon), γ (gamma) and μ (mu), respectively, IgG class antibodies can be further classified into four subclasses IgG1, IgG2, IgG3 and IgG4, respectively, by γ heavy chain Y1-Y4, respectively.

The term "antibody derivative" or "derivative" of an antibody refers to a molecule that is capable of binding to the same antigen (e.g., CD137) to which the antibody binds and comprises the amino acid sequence of the antibody linked to another molecular entity. The amino acid sequence of the antibody comprised in the antibody derivative may be the full length heavy chain, the full length light chain, any one or more portions of the full length heavy chain, any one or more portions of the full length light chain, any other one or more fragments of the antibody, or a complementary antibody of the antibody. The other molecular entity may be a chemical or biological molecule. Examples of another molecular entity include chemical groups, amino acids, peptides, proteins (such as enzymes, antibodies) and chemical compounds. Another molecular entity may have any utility, such as for use as a detection agent, label, marker, pharmaceutical agent, or therapeutic agent. The amino acid sequence of an antibody can be attached or linked to another molecular entity by chemical coupling, genetic fusion, non-covalent association, or other means. The term "antibody derivative" also encompasses chimeric antibodies, humanized antibodies, and molecules derived from modifications of the amino acid sequence of the CD137 antibody, such as conservative amino acid substitutions, additions, and insertions.

The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of an antibody that retain the ability to bind to the antigen (e.g., CD137) to which the antibody binds. Examples of "antigen-binding fragments" of antibodies include (i) Fab fragments, which are composed of V_L、V_H、C_LAnd C_H1Monovalent fragments consisting of domains; (ii) f (ab')₂A fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) from V_HAnd C_H1Domain-forming Fd fragments; (iv) v from one arm of an antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; (v) from V_HdAb fragments consisting of Domain (Ward et al, Nature 34)1: 544-546 (1989)); and (vi) an isolated Complementarity Determining Region (CDR).

The term "binding molecule" encompasses (1) an antibody, (2) an antigen-binding fragment of an antibody, and (3) a derivative of an antibody, each as defined herein.

The term "binding CD137(binding CD 137/bindings CD 137)" or "binding to CD137(binding to CD 137/bindings to CD 137)" refers to a binding molecule as defined herein that has an affinity (K) of 100nM or less in an in vitro assay_D) Binding to human CD137, such as the Biacore assay described in example 4.

The terms "CD 137" and "CD 137 receptor" are used interchangeably herein and include the human CD137 receptor, as well as variants, isoforms, and species homologs thereof. Thus, the binding molecules as defined and disclosed herein may also bind CD137 from species other than human. In other cases, the binding molecule may be completely specific for human CD137 and may not exhibit species or other types of cross-reactivity.

The term "CD 137 antibody" refers to an antibody as defined herein that is capable of binding to the human CD137 receptor.

The term "chimeric antibody" refers to an antibody comprising amino acid sequences derived from different animal species, such as those having a variable region derived from a human antibody and a murine immunoglobulin constant region.

The term "competitive binding" refers to the interaction of two antibodies in their binding to a binding target. A first antibody competes for binding with a second antibody if binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody as compared to binding of the first antibody in the absence of the second antibody. An alternative where the binding of the second antibody to its epitope in the presence of the first antibody is also detectably reduced may be, but need not be. That is, a first antibody may inhibit the binding of a second antibody to its epitope while the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, where each antibody detectably inhibits the binding of another antibody to its cognate epitope, whether to the same, greater or lesser degree, the antibodies are said to "cross-compete" with each other for binding to their respective epitope or epitopes.

The term "epitope" refers to the portion of an antigen to which an antibody (or antigen-binding fragment thereof) binds. Epitopes can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from consecutive amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include varying numbers of amino acids in unique spatial conformations. Methods of determining the spatial conformation of an epitope include, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, deuterium hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or all methods used in combination with antigen and computer modeling of its complex structure with its binding antibody and its variants. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, vol 66, edited by g.e. morris, (1996). Once the desired epitope of an antigen is determined, antibodies to the epitope can be generated, for example, using the techniques described herein. The generation and characterization of antibodies may also clarify information about the desired epitope. From this information, antibodies that bind the same epitope can then be competitively screened. One way to achieve this is to conduct cross-competition studies to find antibodies that compete for binding to each other, i.e., antibodies that compete for binding to the antigen. High-throughput methods based on their cross-competitive "sorting" antibodies are described in PCT publication No. WO 03/48731.

The term "germline" refers to the nucleotide sequence of antibody genes and gene segments as they are passed from parent to offspring via germ cells. Germline sequences differ from nucleotide sequences encoding antibodies in mature B cells that have been altered during B cell maturation by recombination and hypervariable events.

The term "glycosylation site" refers to an amino acid residue that is recognized by a eukaryotic cell as a position for attachment of a sugar residue. The amino acids in which carbohydrates, such as oligosaccharides, are attached are typically asparagine (N-bond), serine (O-bond) and threonine (O-bond) residues. The specific site of attachment is generally marked by an amino acid sequence referred to herein as a "glycosylation site sequence". The glycosylation site sequence for N-linked glycosylation is: -Asn-X-Ser-or-Asn-X-Thr-, wherein X can be any conventional amino acid except proline. The terms "N-linked" and "O-linked" refer to chemical groups that serve as attachment sites between sugar molecules and amino acid residues. The N-linked sugar is attached through an amino group; the O-linked sugar is attached through a hydroxyl group. The term "glycan occupancy" refers to the presence of a carbohydrate moiety attached to a glycosylation site (i.e., the glycan site is occupied). Where there are at least two potential glycosylation sites on the polypeptide, none (occupied by 0-glycan sites), one (occupied by 1-glycan sites), or two (occupied by 2-glycan sites) sites may be occupied by a carbohydrate moiety.

The term "host cell" refers to a cellular system that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2/0, YB 2/0; or human tissue or hybridoma cells, yeast cells and insect cells, as well as cells included within transgenic animals or cultured tissues. The term encompasses not only the particular subject cell, but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".

The term "human antibody" refers to an antibody in which the entire amino acid sequences of both the light and heavy chains are derived from human immunoglobulin genes. The human antibody may comprise a murine carbohydrate chain if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Human antibodies can be made by a variety of means known in the art.

The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues derived from human antibody sequences. A humanized antibody may comprise some or all of the CDRs or HVRs from a non-human animal or synthetic antibody, while the framework and constant regions of the antibody comprise amino acid residues derived from human antibody sequences.

The term "exemplary antibody" refers to any of the antibodies described in this disclosure and designated as those listed in tables 1a and 1 b. These antibodies can belong to any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above are contemplated for V_LAnd V_HAntibodies in all five classes whose regions have identical amino acid sequences. Furthermore, antibodies in the IgG class can belong to any subclass (e.g., IgG1, IgG2, IgG3, and IgG 4). Thus, each of the above identified antibodies in the IgG subclass is contemplated for V_LAnd V_HAntibodies in all four subclasses whose regions have the same amino acid sequence. The amino acid sequences of the heavy chain constant regions of human antibodies in five classes and four IgG subclasses are known in the art. The amino acid sequences of the full-length heavy and light chains of each of the IgG4 subclasses in the exemplary antibodies shown in table 1b are provided in the present disclosure.

The term "isolated antibody" or "isolated binding molecule" refers to an antibody or binding molecule as defined herein which: (1) not associated with a naturally associated component with which it is adjacent in its natural state; (2) free of other proteins from the same species; (3) expressed by cells from different species; or (4) does not occur in nature. Examples of isolated antibodies include CD137 antibodies that have been affinity purified using CD137, CD137 antibodies that have been produced in vitro by hybridomas or other cell lines, and CD137 antibodies derived from transgenic animals.

The term "isolated nucleic acid" refers to a nucleic acid molecule of genomic, cDNA, or synthetic origin, or a combination thereof, that is separate from other nucleic acid molecules that are present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences naturally flanking the nucleic acid (i.e., sequences located at the 5 'and 3' ends of the nucleic acid of interest).

The term "k_a"refers to the association rate of a particular antibody-antigen interactionConstant and the term "k_d"refers to the off-rate constant for a particular antibody-antigen interaction.

The term "K_D"refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. It is composed of k_dAnd k is_aRatio of (i.e., k)_d/k_a) Obtained and expressed as molar concentration (M). K_DUsed as a measure of the binding affinity of an antibody to its binding partner. K_DThe smaller the binding of the antibody, the tighter the binding, or the higher the affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant binds more tightly to a particular antigen than an antibody with a micromolar (μ M) dissociation constant. K of antibody_DValues may be determined using well established methods in the art. Determination of antibody K_DBy using surface plasmon resonance, usually using biosensor systems, such asProvided is a system. Using BIACORE^TMThe assay procedure (BIAcore assay) performed by the system is described in the examples section of the present disclosure.

The term "mammal" refers to any animal species of the mammalian species. Examples of mammals include: a human; laboratory animals such as rats, mice, apes and guinea pigs; domestic animals such as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, and the like.

The term "preventing" with respect to a disease condition in a mammal means preventing or delaying the onset of the disease, or preventing the appearance of clinical or subclinical symptoms thereof.

As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of amino Acids that are identical between the sequences the amino acid sequence identity of a polypeptide can be determined routinely using known computer programs such as Bestfit, FASTA or B L AST (see, e.g., Pearson, Methods enzymol.183: 63-98 (1990); Pearson, Methods mol.biol.132: 185-219 (2000); Altschul et al, J.mol.biol.215: 403-410 (1990); Altschul et al, Nucelic Acids Res.25: 3389-3402 (1997)). when using Bestfit or any other sequence alignment program to determine whether a particular sequence has, e.g., 95% identity to a reference amino acid sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference amino acid sequence and allow the interval of homology of the total number of amino acid residues in the reference sequence to be up to 5% the disclosed Methods for determining the percentage of protein fragments, or variants thereof.

The term "specific binding" or "specific binding to" (antigen to) "when referring to the interaction of a binding molecule (e.g., an antibody) with its binding partner (e.g., an antigen) as defined herein refers to the ability of the binding molecule to distinguish between an antigen of interest from one animal species and an antigen ortholog (antigen orthologue) from a different animal species under a given set of conditions as determined in an in vitro assay, a CD137 binding molecule is said to specifically bind to human CD137 if it binds to less than 50% of EC50 of EC50 of rat or mouse CD 137.

The term "selective binding" or "selective binding to" when referring to the interaction of a binding molecule (e.g. an antibody) with its binding partner (e.g. an antigen) as defined herein, refers to the ability of the binding molecule to distinguish between an antigen of interest from one animal species, such as human CD137, and a different antigen from the same animal species, such as human CD40, under a given set of conditions. A CD137 binding molecule is said to selectively bind to human CD137 if it binds to human CD137 with an EC50 that is less than 10% of its EC50 binding to human CD40 or human CD134, as determined in an in vitro assay.

The term "treating" or "treatment" with respect to a disease condition in a mammal refers to causing a desired or beneficial effect in a mammal having the disease condition. The desired or beneficial effect may include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and/or metastasis, or other effects mediated by the number and/or activity of immune cells, etc.), or a cessation or inhibition of further development of the disease, condition, or disorder. In the context of treating cancer in a mammal, the desired or beneficial effect may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or an increase in survival rate of the mammal. The effect may be subjective or objective. For example, if the mammal is a human, the human may record an improvement in energy or vigor or a reduction in pain as an improved subjective symptom or response to therapy. Alternatively, the clinician may notice a reduction in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers, or imaging findings. Some laboratory signs observable by the clinician for response to treatment include normalization of the test, such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. In addition, the clinician may observe a decrease in detectable tumor markers. Alternatively, other tests may be used to assess objective improvement, such as sonography, nuclear magnetic resonance testing, and positron emission testing.

The term "vector" refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule. The foreign nucleic acid molecule is linked to the vector nucleic acid molecule by recombinant techniques such as ligation or recombination. This allows for the multiplication, selection, further manipulation or expression of the foreign nucleic acid molecule in the host cell or organism. The vector may be a plasmid, phage, transposon, cosmid, chromosome, virus or virion. One type of vector can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicated together with the host genome (e.g., non-episomal mammalian vectors). Another type of vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Another specific type of vector capable of directing the expression of an expressible foreign nucleic acid to which it is operatively linked is often referred to as an "expression vector". Expression vectors typically have control sequences that drive expression of an expressible foreign nucleic acid. Simpler vectors known as "transcription vectors" are only capable of transcription, but not translation: they can replicate in the target cell, but are not expressed. The term "vector" encompasses all types of vectors regardless of their function. Vectors capable of directing the expression of an expressible nucleic acid to which they are operatively linked are generally referred to as "expression vectors".

Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to methods well known in the art and described in various general and more specific references that are cited and discussed throughout the present specification, such references include, for example, Sambrook and Russell, Molecular Cloning, A L absorber apparatus, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002), and Harlow and L ane Antibodies commonly used: A L laboratory, Cold Spring Harbor L antibody Press, Cold Spring Harbor, N.Y. 1990 (1990) enzymatic reaction and purification techniques are according to manufacturer specifications, as well as chemical synthesis and chemical synthesis techniques that are generally used in the art, as described in the art, and chemical synthesis procedures for the purpose of performing the chemical analysis and chemical synthesis of drugs, and chemical delivery procedures that are used in the art, as described in the art, as well as those of chemical synthesis and chemical synthesis procedures described in the art.

As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-ASynthesis (2 nd edition, edited by E.S. Golub and D.R. Gren, Sinauer Associates, Sunderland, Mass. (1991)).

B. Binding molecules that bind to human CD137

The present disclosure provides isolated binding molecules that bind to human CD137, including CD137 antibodies, antigen-binding fragments of CD137 antibodies, and derivatives of CD137 antibodies, in some embodiments, the binding molecules are any of the antibodies described herein, including antibodies described for epitope binding and antibodies described for HVR, variable region (V L, VH), and IgG (e.g., IgG4) light and heavy chain specific amino acid sequences the disclosure relates to binding molecules that bind to human CD137 and have at least one of the following functional properties (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight) (a) bind to human CD137 with a KD of 500nM or less, (b) have agonist activity on human CD137, (c) do not bind to human CD137, CD40, GITR, and/or CD27 receptors, (d) do not bind to human CD137, mouse, or monkey receptors, (c) do not compete with human CD137, mouse, or monkey, bind to human CD137, and (e) block the effects of binding to human CD137 antibodies that do not block the binding to human CD137, CD 19, or rat, and/or rat, further, a method of blocking the binding to blocking the effects of a cancer treatment of a cancer, such as described herein, in some embodiments.

In some embodiments, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues within amino acid residues 34-108 of SEQ ID NO: 1. in some embodiments, the antibody or antigen-binding fragment binds to one or more amino acid residues within amino acid residues 34-93 of SEQ ID NO: 1. in some embodiments, the antibody or antigen-binding fragment binds to one or more amino acid residues selected from the group consisting of amino acid residues 34-36, 53-55, and 92-93 of SEQ ID NO: 1. in some embodiments, the antibody or antigen-binding fragment binds to one or more of amino acid residues 34-36, one or more of amino acid residues 53-55, and one or more of amino acid residues 92-93 of SEQ ID NO: 1. in some embodiments, the antibody or antigen-binding fragment does not bind to one or more of amino acid residues selected from the group consisting of amino acid residues 109, 125, 126, 135, 138, 150, and 151 of SEQ ID NO: 1. in some embodiments, the antibody or antigen-binding fragment binding to a target antigen can be measured using a method such as the resonance assay of binding of antibody binding to amino acid residues 112, 125, 151, antibody binding to a target antigen binding fragment, ISA binding to a target antigen binding fragment, or antigen binding fragment measured by a method comprising measuring binding to the following methods such as the methods of the antibody binding to the antibody or antigen binding to antibody binding to the surface of SEQ ID NO: 112, ISA 112, antibody binding to the following.

In some embodiments, the antibody or antigen-binding fragment binds to human CD137 with a KD of about 500nM or less (e.g., about 500nM or less, about 400nM or less, about 300nM or less, about 200nM or less, about 150nM or less, about 100nM or less, about 90nM or less, about 80nM or less, about 75nM or less, about 70nM or less, about 60nM or less, about 50nM or less, about 40nM or less, about 30nM or less, about 25nM or less, about 20nM or less, about 10nM or less, about 1nM or less, about 0.1nM or less, etc.) in some embodiments, the antibody or antigen-binding fragment binds to human CD137 with a KD of about 100nM or less.

In some embodiments, the antibodies or antigen-binding fragments described herein have agonist activity to human CD137 when cells expressing human CD137 (e.g., human cells) are contacted with the antibodies or antigen-binding fragments, the antibodies or antigen-binding fragments induce one or more (e.g., one or more, two or more, three or more, etc.) activities of human CD137, in some embodiments, when cells expressing human CD137 (e.g., human cells) are contacted with the antibodies or antigen-binding fragments, the respective CD137 activities are known in the art and can include, but are not limited to, induction of NF- κ B-dependent transcription, induction of T cell proliferation, elongation of T cell survival, co-stimulation of activated T cells, induction of cytokine secretion (such as I L-2), and induction of monocyte activation, in some embodiments, one or more CD137 activity is not measured as binding of CD137 to its ligand (e.g., I L-2), in some embodiments, about 10% or more antibody-binding to human CD137, e.g., 10-CD 137-or 10% or more antigen-binding, in some embodiments, the antibody-CD 137-CD-dependent transcription does not increase, e.g., when cells are contacted with the antibodies, 10-or 10-or more-CD-dependent transcription of cells, e.g., when cells are contacted with the antibodies or antigen-CD 137-CD-cell-.

In some embodiments, the antibody or antigen-binding fragment cross-reacts with monkey (e.g., cynomolgus monkey), mouse, rat, and/or dog CD 137. in some embodiments, the antibody or antigen-binding fragment cross-reacts with monkey CD 137. in some embodiments, the antibody or antigen-binding fragment cross-reacts with mouse CD 137. in some embodiments, the antibody or antigen-binding fragment cross-reacts with rat CD 137. in some embodiments, the antibody or antigen-binding fragment cross-reacts with dog CD 137. in some embodiments, the antibody or antigen-binding fragment cross-reacts with monkey and mouse CD137, monkey and rat CD137, monkey and dog CD137, mouse and rat CD137, monkey, mouse and dog CD137, monkey, rat and dog CD137, mouse and dog CD137, or monkey, rat and CD137, dog 137. in some embodiments, the antibody or antigen-binding fragment cross-reacts with monkey, rat, and CD 137. in some embodiments, rat, and dog CD 137. in some embodiments, the antibody or antigen-binding fragment cross-binding reaction is measured at about 100nM under the method of the same in the same as the method of measuring the antibody or antigen-binding activity of about 10nM, e.g., the method of the same under the same.

In some embodiments, the antibody does not cause ADCC effects. Methods of measuring ADCC effects (e.g., in vivo methods) are known in the art, including but not limited to via the methods described in example 11 below. In some embodiments, the antibody does not cause greater than about 10% ADCC effect (does not cause greater than about 10%, greater than about 5%, greater than about 1%, greater than about 0.1%, greater than about 0.01% ADCC effect) relative to a control.

In some embodiments, the antibody or antigen binding fragment is capable of inhibiting tumor cell growth/proliferation. In some embodiments, tumor cell growth/proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) when contacted with an antibody or antigen-binding fragment relative to a corresponding tumor cell not contacted with the antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is capable of reducing tumor volume in a subject upon administration of the antibody or antigen-binding fragment to the subject. In some embodiments, the antibody or antigen-binding fragment is capable of reducing the tumor volume of the subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) relative to the initial tumor volume of the subject (e.g., prior to administration of the antibody or antigen-binding fragment). Methods of monitoring tumor cell growth/proliferation, tumor volume, and/or tumor inhibition are known in the art, including, for example, via the methods described in example 10 below.

In some embodiments, the antibody or antigen binding fragment has a therapeutic effect on cancer. In some embodiments, the antibody or antigen-binding fragment reduces one or more signs or symptoms of cancer. In some embodiments, a subject suffering from cancer enters partial or complete remission when the antibody or antigen-binding fragment is administered.

In another aspect, the disclosure provides an isolated antibody that competes or cross-competes with any of the exemplary antibodies of the disclosure, such as AG10058, AG10059, and/or AG10131, for binding to human CD137 in one particular embodiment, the disclosure provides an isolated antibody that competes or cross-competes with any of the exemplary antibodies of the disclosure for binding to the same epitope on human CD 137.

In some embodiments, an antibody or antigen-binding fragment blocks binding between CD137 and its ligand (e.g., human CD137 and human CD 137L) in vitro in some embodiments, an antibody or antigen-binding fragment has a half maximal inhibitory concentration (IC50) of about 500nM or less (e.g., about 500nM or less, about 400nM or less, about 300nM or less, about 200nM or less, about 100nM or less, about 50nM or less, about 25nM or less, about 10nM or less, about 1nM or less, etc.) for blocking binding of CD137 to its ligand in some embodiments, an antibody or antigen-binding fragment has a half maximal inhibitory concentration (IC50) of about 100nM or less when measured at a binding between about 100nM or more (e.g., about 100nM or more), or about 100nM or more, or about 90nM or more of the antibody or antigen-binding fragment is at least about 95% as measured by a method of blocking binding of a CD137, e.g., a binding of a protein, e.g., CD137, or more, e.g., a cell binding protein that includes at least about 99 μ g., binding between about 99% of a first cell binding between about 99 μ g., a second antibody or antigen binding protein, a cell binding protein, e.g., a cell binding protein, e.g., a cell binding protein, a cell binding protein, e.g., a cell binding protein, a cell binding protein, e.g., a cell binding protein, E.

B-1.CD137 antibodies

In some aspects, the disclosure provides an isolated antibody that binds to SEQ ID No.: 1 to human CD137 at an epitope within amino acid residues 34-108 or 34-93. In some embodiments, the antibody has a K of 50nM or less_DBinding to human CD137, as measured by surface plasmon resonance. In certain embodiments, the antibody can cross-react with at least one non-human species selected from the list consisting of cynomolgus monkey, mouse, rat, and dog.

In one aspect, the disclosure provides an isolated antibody comprising a heavy chain variable region and a light chain variable region, a) wherein the heavy chain variable region comprises HVR-H, HVR-H and HVR-H, wherein HVR-H comprises an amino acid sequence according to formula selected from the group consisting of X1TFX2X3YX4IHWV (SEQ ID NO: 2), wherein X is F or Y, X is S or T, X is or S, and X is or W, formula (II) YSIX1SGX2X3WX4 (SEQ ID NO: 3), wherein X is S or T, X is H or Y, and X is or T, and formula (III) FS STX1 2X3 (SEQ ID NO: 4), wherein X is G or S, X is A or G, and X is or T, wherein HVR-H comprises an amino acid sequence according to formula selected from the group consisting of formula (I) 1, 2X, Y, and 2X is S, Y, or T, or 5 or 2Y, wherein X is H or 5, or 5 or 4 or 5 or 4 or 5 or 2 or 5 or 4 or 5 or 2 or 4 or 5 or V or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence according to formula (or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier or a pharmaceutically acceptable carrier or.

In some embodiments, the antibody may comprise HVR _ H1 having an amino acid sequence selected from the group consisting of SEQ ID NO 253-312, HVR _ H2 having an amino acid sequence selected from the group consisting of SEQ ID NO 313-372, HVR _ H3 having an amino acid sequence selected from the group consisting of SEQ ID NO 373-432, HVR _ L1 having an amino acid sequence selected from the group consisting of SEQ ID NO 433-492, HVR _ L2 having an amino acid sequence selected from the group consisting of SEQ ID NO 493-552, and/or HVR _ L3 having an amino acid sequence selected from the group consisting of SEQ ID NO 553-612.

In certain embodiments, the antibody may comprise V L and/or VH having an amino acid sequence selected from the group consisting of SEQ ID NOS 13-132, which may preferably be encoded by DNA sequences selected from the group consisting of SEQ ID NOS 133-252, respectively.

In some embodiments, the antibody may comprise HVR _ H1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 709-732, HVR _ H2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 733-756, HVR _ H3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 757-780, HVR _ L1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 781-804, HVR _ L2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 805-828, and/or HVR _ L3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 829-852-L.

In certain embodiments, the antibody may comprise a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 613-660 (e.g. IgG such as those of IgG4), which may preferably consist of an amino acid sequence selected from the group consisting of seq id NO: 661-708.

The CD137 antibodies described herein may be of any species, such as IgG, IgM, IgE, IgA, or IgD. Preferably, the CD137 antibody belongs to the IgG class, such as the IgG1, IgG2, IgG3 or IgG4 subclasses. The CD137 antibody can be converted from one class or subclass to another using methods known in the art. Examples for generating antibodies belonging to a desired class or subclassThe method comprises the following steps: isolating nucleic acid encoding the heavy chain of the CD137 antibody and nucleic acid encoding the light chain of the CD137 antibody; separation code V_HThe sequence of the region; will V_HThe sequence is linked to a sequence encoding a heavy chain constant region of a desired class or subclass; expressing the light chain gene and heavy chain construct in a cell; and collecting the CD137 antibody.

Furthermore, the antibodies provided by the present disclosure may be monoclonal or polyclonal, but are preferably monoclonal.

Examples of specific isolated antibodies provided by the present disclosure include those listed in tables 1a and 1 b. The nucleotide and amino acid sequences of the heavy chain variable regions, full-length heavy chain, light chain variable regions of the IgG2 and IgG4 subclasses, and full-length light chains of these antibodies are also provided below.

Antibodies of the present disclosure can be produced by techniques known in the art, including conventional monoclonal antibody methods, e.g., standard somatic hybridization techniques (see, e.g., Kohler and Milstein, Nature 256: 495 (1975)), viral or oncogenic transformation of B lymphocytes, or recombinant antibody techniques, as described in detail below.

Hybridoma production is a very well established procedure. A common animal system for preparing hybridomas is the murine system. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. One well-known method that can be used to prepare the human CD137 antibodies provided by the present disclosure involves XenoMouse^TMUse of animal systems. XenoMouse^TMMice are engineered mouse strains that contain large fragments of human immunoglobulin heavy and light chain loci and are deficient in mouse antibody production. See, e.g., Green et al, Nature Genetics 7: 13-21(1994) and WO 2003/040170. Animals were immunized with the CD137 antigen. The CD137 antigen is an isolated and/or purified CD137, preferably CD 137. It may be a fragment of CD137, such as the extracellular domain of CD137, in particular a polypeptide comprising SEQ ID NO: 1, or 34-93, or a fragment of the CD137 extracellular domain of amino acid residues 34-108 or 34-93. Immunization of animals can be carried out by any method known in the art. See, for exampleHarlow and L ane, Antibodies: A L anaerobic Manual, New York: Cold Spring Harbor Press, 1990. methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. see, e.g., Harlow and L ane, supra and U.S. Pat. No. 5,994,619. CD137 antigen can be administered with an adjuvant to stimulate an immune response. exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immune stimulating complex). following immunization of an animal with CD137 antigen, an antibody-producing immortalized cell line is prepared from cells isolated from the immunized animal.

Such display methods for isolating human antibodies are established in the art, such as Achim Knappik et al, "full Synthetic human combinatorial antibodies L ibraries (HuCA L) Based on modulated sensors frameworks and CDRs random amplified with trinuclear peptides" J.Mol.biol. (2000)296, 57-86; and Michael J.Feldhaus et al, "Flow-cell assay of human antibodies from non-animal Saccharomyces cerevisiae surface biological" Nat Biotechnology (2003) 21: 163-.

B-2 antigen binding fragments

In some other aspects, the disclosure provides antigen binding fragments of any of the CD137 antibodies provided by the disclosure.

An antigen-binding fragment may comprise any sequence of an antibody. In some embodiments, the antigen-binding fragment comprises the amino acid sequence of: (1) a light chain of a CD137 antibody; (2) the heavy chain of the CD137 antibody; (3) a variable region from the light chain of a CD137 antibody; (4) the variable region from the heavy chain of a CD137 antibody; (5) one or more HVRs (two, three, four, five, or six HRVs) of the CD137 antibody; or (6) three HVRs from the light chain of a CD137 antibody and three HVRs from the heavy chain of the CD137 antibody.

In some particular embodiments, the present disclosure provides antigen binding fragments of an antibody selected from those listed in tables 1a and 1 b.

In some other particular embodiments, the antigen-binding fragment of the CD137 antibody comprises: (i) fab fragment consisting of V_L、V_H、C_LAnd C_H1 domain; (ii) f (ab')₂A fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) from V_HAnd C_H1 domain; (iv) v from one arm of an antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; (v) from V_HdAb fragments consisting of domains (Ward et al, (1989) Nature 341: 544-546); (vi) (vii) an isolated CDR, and (vii) a single chain antibody (scFv), which is a V comprising an antibody_HV of region-linked antibodies_LA polypeptide of the region. Bird et al, (1988) Science 242: 423 Asonic 426 and Huston et al, (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883.

In some particular embodiments, the antigen binding fragment is a Fab fragment selected from those listed in table 1 a.

Antibody derivatives

In some further aspects, the disclosure provides derivatives of any of the CD137 antibodies provided by the disclosure.

In one aspect, antibody derivatives are derived from modifications of the amino acid sequences of exemplary antibodies of the present disclosure ("parent antibodies") while retaining the overall molecular structure of the parent antibody amino acid sequences. The amino acid sequence of any region of the parent antibody chain may be modified, such as the framework, HVR, or constant regions. Types of modifications include substitutions, insertions, deletions of one or more amino acids of the parent antibody, or combinations thereof.

In some embodiments, the antibody derivative comprises V_LOr V_HA region which is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NO 13-132 in some embodiments the antibody derivative comprises an HVR _ H1 amino acid sequence region which is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NO 253-312 in some embodiments the antibody derivative comprises an HVR _ H2 amino acid sequence region which is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NO 313-372 in some embodiments the HVR _ H2 amino acid sequence region which is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence as set forth in any one of the HVR 35-H-11 amino acid sequences as set forth in any one of SEQ ID NO 373, at least 95%, 85%, at least 95%, 85%, or at least 95%, or at.

In some embodiments, the antibody derivative comprises the HVR _ L3 amino acid sequence of all Fab hits shown in Table 1a, which can be found in SEQ ID NO 553-612. in some specific embodiments, the derivative comprises 1,2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative or non-conservative substitutions, and/or 1,2, 3, 4, 5,6, 7, 8, 9, 11, 13, 14 or 15 additions and/or deletions to the amino acid sequence shown in any one of SEQ ID NOS 13-132 and 253-612.

In some embodiments, the antibody derivative comprises a heavy chain variable region identical to the amino acid sequence set forth as SEQ ID NO: 613-660 is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in any one of seq id no.

In some embodiments, the antibody derivative comprises a HVR _ H1 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NO 709-732, in some embodiments, the antibody derivative comprises a HVR _ H2 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NO 733-756, in some embodiments, the antibody derivative comprises a HVR _ H3 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NO 757-780, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 90%, at least 75%, at least 90%, at least 75%, at least 90%, at least 75%, or at least 90% as shown in some of the amino acid sequence set forth in some of the amino acid sequence set forth in any one of.

Amino acid substitutions encompass conservative substitutions and non-conservative substitutions. The term "conservative amino acid substitution" refers to the replacement of one amino acid with another amino acid in which the two amino acids share similarities in certain physico-chemical properties, such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, substitution may generally be made within each of the following groups: (a) nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids such as arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids such as aspartic acid and glutamic acid.

Modifications may be made at any position of the amino acid sequence of the antibody, including the HVRs, framework or constant regions. In one embodiment, the disclosure provides an antibody derivative comprising a V of an exemplary antibody of the disclosure_HAnd V_LHVR sequences, also including framework sequences different from those of the exemplified antibodies. Such framework sequences may be derived from genes including germline antibodiesFor example, germline DNA Sequences for Human heavy and light chain variable region genes can be found in the Genbank database or the "VBase" Human germline sequence database (Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, U.S. department of Health and Human Services, NIH publication No. 91-3242 (1991); Tomlinson, I.M. et al, J.mol.biol.227: 776 798 (1992); and Cox, J.P. L. et al, Eur.J.Immunol.24: 827 exemplary 836 (1994)). framework Sequences useful for the construction of antibody derivatives include framework Sequences structurally similar to those used for antibodies of the present disclosure, e.g., similar to those used for V antibodies of the present disclosure_H3-23 framework sequences and/or V_LFor example, the HVR _ H1, HVR _ H2, and HVR _ H3 sequences, as well as HVR _ L1, HVR _ L2, and HVR _ L3 sequences of exemplary antibodies can be grafted onto framework regions having the same sequences found in germline immunoglobulin genes from which the framework sequences are derived, or the HVR sequences can be grafted onto framework regions comprising one or more mutations as compared to the germline sequences.

In a particular embodiment, the antibody derivative is a chimeric antibody comprising the amino acid sequence of an exemplary antibody of the present disclosure. In one example, one or more HVRs from one or more exemplified human antibodies are combined with HVRs from an antibody from a non-human animal (such as a mouse or rat). In another example, all HVRs of a chimeric antibody are derived from one or more exemplified antibodies. In some particular embodiments, a chimeric antibody comprises one, two, or three HVRs from a heavy chain variable region or a light chain variable region of an exemplary antibody. Chimeric antibodies can be generated using conventional methods known in the art.

Another class of modification is that of V_HAnd/or V_LAmino acid residues within the HRV region of the chain are mutated. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce one or more mutations, and the effect on antibody binding or other functional property of interest can be assessed in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. The mutation may be an amino acid addition and/or deletion. In addition, no more than one, two, three, four, or five residues within the HVR region are typically altered. In some embodiments, the antibody derivative comprises 1,2, 3, or 4 amino acid substitutions in the heavy chain HVRs and/or the light chain HVRs. In another embodiment, the amino acid substitution is a change of one or more cysteines in the antibody to another residue, such as, but not limited to, alanine or serine. The cysteine may be a classical or non-classical cysteine. In one embodiment, the antibody derivative has 1,2, 3, or 4 conservative amino acid substitutions in the heavy chain HVR region relative to the amino acid sequence of an exemplary antibody.

Can also be paired with V_HAnd/or V_LFramework residues within the regions are modified. Typically, such framework variants are prepared to reduce the immunogenicity of the antibody. One approach is to "back mutate" one or more framework residues to the corresponding germline sequence. Antibodies that have been somatically mutated may comprise framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to germline sequences from which the antibody was derived. To restore the framework sequences to their germline configuration, somatic mutations can be "back-mutated" to germline sequences by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

In addition, modifications may also be made within the Fc region of exemplary antibodies, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In one example, the hinge region of CH 1is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This process is further described in U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of CH 1is altered, e.g., to facilitate assembly of the light and heavy chains, or to increase or decrease the stability of the antibody. In another instance, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody.

In addition, the antibodies of the present disclosure can be modified to alter their potential glycosylation sites or patterns according to routine experimentation known in the art. In another aspect, the present disclosure provides derivatives of the CD137 antibodies of the present disclosure comprising at least one mutation in the variable region of the light or heavy chain that alters the glycosylation pattern in the variable region. Such antibody derivatives may have increased affinity and/or altered specificity for binding of an antigen. Mutations may add new glycosylation sites in the V-region, alter the position of one or more V-region glycosylation sites, or remove preexisting V-region glycosylation sites. In one embodiment, the present disclosure provides derivatives of CD137 antibodies having potential N-linked glycosylation sites at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation sites in one heavy chain variable region are removed. In another embodiment, the present disclosure provides derivatives of the CD137 antibody having potential N-linked glycosylation sites at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation sites in both heavy chain variable regions are removed. Methods of altering the glycosylation pattern of an antibody are known in the art, such as those described in U.S. patent No. 6,933,368, the disclosure of which is incorporated herein by reference.

In another aspect, the present disclosure provides an antibody derivative comprising a CD137 antibody, or antigen-binding fragment thereof, as described herein, linked to another molecular entity. Examples of another molecular entity include an agent, a peptide or protein, a detection agent or label, and an antibody.

In some embodiments, the antibody derivative comprises an antibody of the present disclosure linked to an agent. Examples of agents include cytotoxic or other cancer therapeutic agents, and radioisotopes. Specific examples of cytotoxic agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emidine (emetine), mitomycin, etoposide (etoposide), teniposide (teniposide), vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogues or homologues thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil azepamine), alkylating agents (e.g., dichloromethyldiethanamine, thiotepa chlorambucil (thioepa), melphalan (melphalan), carmustine (BSNU) and lomustine (lomustine) (CCNU), cyclophosphamide, busulfan (busulfan), dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and Amtricin (AMC)) and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that may be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine¹³¹Indium, indium¹¹¹Yttrium, yttrium⁹⁰And lutetium¹⁷⁷. Methods for linking antibodies to agents are known in the art, such as using various linker techniques. Examples of linker types include hydrazone, thioether, ester, disulfide and peptide containing linkers. For further discussion of linkers and methods for linking therapeutic agents to antibodies, see also Saito et al, adv. drug deliv. rev.55: 199, 215 (2003); trail et al, Cancer immunol.immunoher.52: 328-337 (2003); payne, Cancer Cell 3: 207-212 (2003); allen, nat.rev.cancer 2: 750- & ltSUB & gt 763 (2002); patan, i, and Kreitman, curr. opin. investig. drugs 3: 1089-; senter, P.D. and Springer, C.J. (2001) adv.drug Deliv.Rev.53: 247-264.

In one particular embodiment, the antibody derivative is a CD137 antibody multimer that is a multimeric form of a CD137 antibody, such as an antibody dimer, trimer, or higher order multimer (highher-order multimer) of a monomeric antibody each monomer within the antibody multimer may be the same or different hi addition, each monomer within the multimer may have the same or different binding specificity the multimerization of the antibody may be achieved by natural aggregation of the antibody, e.g., a percentage of purified antibody preparations (e.g., purified IgG4 molecules) spontaneously form protein aggregates comprising antibody homodimers and other higher order antibody multimers alternatively, antibody homodimers may be formed by Chemical ligation techniques known in the art, such as by using a cross-linker suitable cross-linkers include those that are heterobifunctional, have two different reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester, 4- (maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester and S-acetylthio-acetic acid N-succinimidyl ester) or are homobifunctional (such as bis-succinimidyl acid) and are also available from Chemical ligation, e.g., r Chemical ligation, e.g., rocomponents.

Examples of other antibody derivatives provided by the present disclosure include single chain antibodies, diabodies, domain antibodies, nanobodies, and minibodies (unibodes). "Single chain antibody" (scFv) consisting of a heavy chain variable region comprising a heavy chain variable region_HDomain linked V_LSingle polypeptide chain composition of a domain wherein V_LDomains and V_HThe domains pair to form monovalent molecules. Single chain antibodies can be prepared according to methods known in the art (see, e.g., Bird et al, (1988) Science 242: 423-. A "diabody" consists of two chains, each comprising a heavy chain variable region linked to a light chain variable region on the same polypeptide chain connected by a short peptide linker, wherein the two regions on the same chain do not pair with each other, but with a complementary domain on the other chain, to form a bispecific molecule. Methods for making diabodies are known in the art (see, e.g., Holliger P. et al, (1993) Proc. Natl. Acad. Sci. USA 90: 6444-. Domain antibodies (dabs) are small functional binding units of antibodies that correspond to the variable region of the heavy or light chain of an antibody. Domain antibodies are well expressed in bacterial, yeast and mammalian cell systems. Additional details of domain antibodies and methods of their production are known in the art (see, e.g., U.S. Pat. Nos. 6,291,158; 6,582,915;6,593,081, respectively; 6,172,197, respectively; 6,696,245; european patent 0368684&0616640, respectively; WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 and WO 03/002609). Nanobodies are derived from the heavy chain of an antibody. Nanobodies typically comprise a single variable domain and two constant domains (CH2 and CH3) and retain the antigen binding ability of the original antibody. Nanobodies may be prepared by methods known in the art (see, e.g., U.S. patent No. 6, 765, 087, U.S. patent No. 6,838,254, WO 06/079372). The mini-antibody consists of one light and one heavy chain of the IgG4 antibody. Mini antibodies can be prepared by removing the hinge region of the IgG4 antibody. Additional details of mini-antibodies and methods of making the same can be found in WO 2007/059782.

C. Nucleic acids, vectors, host cells and recombinant methods for producing CD137 antibodies

Another aspect of the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence of a binding molecule provided by the present disclosure. The amino acid sequence encoded by the nucleotide sequence can be any portion of an antibody, such as a HVR, a sequence comprising one, two, or three HVRs, a variable region of a heavy chain, a variable region of a light chain, or can be a full-length heavy chain or a full-length light chain. The nucleic acids of the present disclosure may be, for example, DNA or RNA, and may or may not comprise intron sequences. Typically, the nucleic acid is a cDNA molecule.

In some embodiments, the present disclosure provides an isolated nucleic acid molecule comprising or consisting of a nucleotide sequence encoding an amino acid sequence selected from the group consisting of (1) the amino acid sequence of HVR _ H3 or HVR _ L3 of an exemplary antibody, (2) the variable region of a heavy chain or the variable region of a light chain of an exemplary antibody, or (3) the full-length heavy chain or the full-length light chain of an exemplary antibody.

In other embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO: 13-132, 253-, 612, 613-, 660 and 709-852.

In other embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 133-252 and 661-708 or by the same.

The nucleic acids of the present disclosure can be obtained using any suitable molecular biology technique. For antibodies expressed by hybridomas, the cdnas encoding the light and heavy chains of the antibodies prepared by the hybridomas can be obtained by PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the libraries.

Can be obtained by encoding V_HOperably linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3) to encode V_HIsolated DNA of the region was converted to the full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, e.g., Kabat et al (1991) Sequences of Proteins of immunologicalcatel interest, 5 th edition, U.S. department of health and public service, NIH publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG4 or IgG2 constant region that does not have ADCC effect. The IgG4 constant region sequence may be any of a variety of alleles or allotypes known to be present in different individuals. These allotypes represent the naturally occurring amino acid substitutions in the constant region of IgG 4. For Fab fragment heavy chain genes, the gene encoding V can be_HOperably linked to another DNA molecule encoding only the constant region of heavy chain CH 1.

Can be obtained by encoding V_LOperably linked to another DNA molecule encoding the light chain constant region C L and encoding V_LThe isolated DNA of the region was converted to the full-length light chain gene (as well as the Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (see, e.g., Kabat et al (1991) Sequences of Proteins of immunologicalcatel interest, 5 th edition, U.S. department of health and public service, NIH publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

To generate the scFv gene, V will be encoded_HAnd V_LIs operably linked to a DNA segment encoding a flexible linker, e.g., an amino acid sequence (Gly)₄-Ser)₃Of a fragment of (a) such that V_HAnd V_LThe sequence may be expressed as having V's connected by a flexible linker_LAnd V_HContinuous single-stranded proteins of domains (see, e.g., Bird et al, Science 242: 423-.

The present disclosure also provides a vector comprising a nucleic acid molecule provided by the present disclosure. The nucleic acid molecule may encode an amino acid sequence of a light chain or a portion of a heavy chain (such as a CDR or HVR), a full-length light chain or heavy chain, a polypeptide comprising a portion or the full length of a heavy chain or light chain, or an antibody derivative or antigen-binding fragment. In some embodiments, the vector is an expression vector that can be used to express a binding molecule, such as an antibody or antigen-binding fragment thereof. In some embodiments, provided herein are vectors, wherein a first vector comprises a polynucleotide sequence encoding a heavy chain variable region as described herein and a second vector comprises a polynucleotide sequence encoding a light chain variable region as described herein. In some embodiments, a single vector comprises a polynucleotide encoding a heavy chain variable region as described herein and a light chain variable region as described herein.

To express the binding molecules of the present disclosure, DNA encoding partial or full-length light and heavy chains is inserted into an expression vector such that the DNA molecules are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" refers to the linkage of an antibody gene into a vector such that transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the DNA molecule. The expression vector and expression control sequences chosen should be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, or more typically both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by any suitable method (e.g., linking the antibody gene fragment to complementary restriction sites on the vector, or DN based on homologous recombinationA is connected). The light and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype and subclass by inserting them into expression vectors that already encode the heavy and light chain constant regions of the desired isotype and subclass such that the V_HThe segments are operatively connected to one or more C's within the carrier_HSection and V_LThe segments are operatively connected to C in a carrier_LA sector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain by the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is in-frame with the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

The term "regulatory sequences" is intended to include promoters, enhancers and other Expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain Gene such regulatory sequences are described in, for example, Goeddel (Gene Expression technology. methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)).

In addition to the antibody chain genes and regulatory sequences, the expression vector may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al). For example, typically a selectable marker gene confers resistance to a drug such as G418, hygromycin or methotrexate to a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (used in DHFR-host cells using methotrexate selection/amplification) and the neo gene (used for G418 selection).

To express the light and heavy chains, one or more expression vectors encoding the heavy and light chains are transfected into a host cell by any suitable technique. The term "transfection" in its various forms is intended to encompass a wide variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although the antibodies of the present disclosure can be expressed in prokaryotic or eukaryotic host cells, the antibodies are most typically expressed in eukaryotic cells and typically in mammalian host cells.

The present disclosure also provides a host cell comprising a nucleic acid molecule provided by the present disclosure. The host cell may be virtually any cell for which an expression vector is useful. The cell may be, for example, a higher eukaryotic host cell, such as a mammalian cell; lower eukaryotic host cells, such as yeast cells; and may be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant nucleic acid construct into a host cell can be achieved by calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.

Eukaryotic hosts suitable for transformation include E.coli (E.coli), Bacillus subtilis (Bacillus subtilis), Salmonella typhimurium (Salmonella typhimurium), and various species within the genera Pseudomonas, Streptomyces and Staphylococcus (Staphylococcus).

Mammalian host cells for expression of the binding molecules of the present disclosure include, for example, Chinese Hamster Ovary (CHO) cells (including DHFR-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-. In particular, for use with NS0 myeloma or CHO cells, another expression system is the GS (glutamine synthetase) gene expression system disclosed in WO 87/04462, WO 89/01036 and EP338,841. When an expression vector encoding a gene for an antibody is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the medium in which the host cell is grown. The antibody can be recovered from the culture medium using any suitable protein purification method.

D. Composition comprising a metal oxide and a metal oxide

In other aspects, the present disclosure provides a composition comprising a binding molecule provided by the present disclosure. In one aspect, the composition is a pharmaceutical composition comprising a binding molecule and a pharmaceutically acceptable carrier. The compositions may be prepared by conventional methods known in the art.

In some embodiments, the present disclosure provides a composition comprising an antibody or antigen-binding fragment thereof provided by the present disclosure, and a pharmaceutically acceptable carrier, wherein the antibody comprises a variable domain comprising an HVR amino acid sequence disclosed herein, and wherein the composition comprises no more than about 11%, 10%, 8%, 5%, 3%, or 2% of the antibody or antigen-binding portion thereof glycosylated at an asparagine of the amino acid sequence as compared to the total amount of antibody or antigen-binding portion thereof present in the composition. In another embodiment, the composition comprises at least about 2% of the antibody or antigen-binding portion thereof glycosylated at asparagine of the amino acid sequence as compared to the total amount of antibody or antigen-binding portion thereof present in the composition.

The term "pharmaceutically acceptable carrier" refers to any inactive material suitable for use in a formulation for delivering a binding molecule. The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (such as an antioxidant, antibacterial or antifungal agent), sweetener, absorption retarder, wetting agent, emulsifier, buffer, or the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, dextrose with polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents, such as sugars, polyalcohols, sorbitol, and sodium chloride.

The composition may be in any suitable form, such as liquid, semi-solid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusible solutions), microemulsions, liposomes, dispersions or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A particular form of composition suitable for delivery of the binding molecule is a sterile liquid, such as a solution, suspension or dispersion, for injection or infusion. Sterile solutions can be prepared by incorporating the required amount of antibody into an appropriate vehicle, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the antibody into a sterile vehicle that includes a basic dispersion medium and other carriers. For sterile powders for the preparation of sterile liquids, the methods of preparation include vacuum drying and freeze-drying (lyophilization) to produce a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The various dosage forms of the compositions may be prepared by conventional techniques known in the art.

The relative amounts of binding molecules included in the compositions will vary depending on a variety of factors, such as the particular binding molecule and carrier used, the dosage form, and the release and pharmacodynamic characteristics desired. The amount of binding molecule in a single dosage form will generally be that amount which produces a therapeutic effect, but may be a lesser amount. Generally, this amount will range from about 0.01% to about 99%, from about 0.1% to about 70%, or from about 1% to about 30% relative to the total weight of the dosage form.

In addition to the binding molecule, one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described below. The appropriate amount of additional therapeutic agent to be included in the composition can be readily selected by one of skill in the art and will vary depending on a variety of factors, such as the particular agent and carrier used, the dosage form, and the desired release and pharmacodynamic characteristics. The amount of additional therapeutic agent included in a single dosage form will generally be that amount of agent which produces a therapeutic effect, but may be a lesser amount.

E. Use of binding molecules and pharmaceutical compositions

The binding molecules and pharmaceutical compositions provided by the present disclosure may be used for therapeutic, diagnostic, or other purposes, such as modulating immune responses, treating cancer, enhancing the efficacy of other cancer therapies, enhancing vaccine efficacy, or treating autoimmune diseases. Thus, in other aspects, the present disclosure provides methods of using the binding molecules or pharmaceutical compositions. In one aspect, the present disclosure provides a method of treating a disorder in a mammal comprising administering to a mammal in need of treatment a therapeutically effective amount of a binding molecule provided by the present disclosure. The binding molecule may be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist. In some embodiments, the mammal is a human.

In some embodiments, the disorder is cancer. CD137 is implicated in a variety of cancers, whether malignant or benign and whether primary or secondary, that can be treated or prevented using the methods provided by the present disclosure. Examples of such cancers include lung cancers such as bronchial carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (noncancerous), and sarcoma (cancerous); heart cancers such as myxoma, fibroma, and rhabdomyoma; bone cancers such as osteochondrosis, chondroma (condomas), chondroblastoma, chondroromyxoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, ewing's sarcoma, and reticulocytoma; brain cancers such as glioma (e.g., glioblastoma multiforme), anaplastic astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pinealoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germ cell tumor, teratoma, dermato-cysts, and hemangioma; cancers in the digestive system, such as leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, polyps in the large intestine, and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; renal cancer, transitional cell carcinoma such as renal adenocarcinoma, renal cell carcinoma, suprarenal adenoid tumor, and renal pelvis; bladder cancer; blood cancers such as acute lymphocytic (lymphoblastic) leukemia, acute myelogenous (myelocytic, myelogenous, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myelogenous (myeloid, myelogenous, granulocytic) leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, and chronic granulocytic leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, kaposi's sarcoma, and paget's disease; head and neck cancer; eye-related cancers such as retinoblastoma and intraocular melanoma; cancers of the male reproductive system, such as benign prostatic hypertrophy, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonic carcinoma, and choriocarcinoma); breast cancer; cancers of the female reproductive system such as uterine cancer (endometrial cancer), cervical cancer (cervical cancer), ovarian cancer (ovarian cancer), vulvar cancer, vaginal cancer, fallopian tube cancer, and vesicular fetal mass; thyroid cancer (including papillary, follicular, anaplastic or medullary); pheochromocytoma (adrenal gland); non-cancerous growth of parathyroid gland; pancreatic cancer; and blood cancers such as leukemia, myeloma, non-hodgkin's lymphoma, and hodgkin's lymphoma.

In some other embodiments, the disorder is an autoimmune disease. Examples of autoimmune diseases that can be treated with binding molecules include autoimmune encephalomyelitis, lupus erythematosus and rheumatoid arthritis. The binding molecules may also be used to treat inflammation (such as allergic asthma) and chronic graft-versus-host disease,

in another aspect, the present disclosure provides a method of enhancing an immune response in a mammal, comprising administering to the mammal a therapeutically effective amount of a binding molecule provided by the present disclosure, in some embodiments, the binding molecule is a CD137 antibody or antigen binding fragment thereof and the mammal is a human in another embodiment, the binding molecule is a CD137 agonist antibody or antigen binding fragment thereof the term "enhancing an immune response" or grammatical variations thereof refers to stimulating, increasing, enhancing or boosting any response of the immune system of a mammal the immune response may be a cellular response (i.e., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (i.e., antibody-mediated response) and may be a primary immune response or a secondary immune response examples of enhanced immune response include an increase in CD4+ helper T cell activity and production of cytolytic T cells the enhancement of immune response may be assessed using a variety of in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, release of cytokines (e.g., I L-2), production of tumor cell agonists, antibodies, production of immune cells in embodiments, in which the immune responses may be enhanced by a cell binding to a cell-mediated immune response, such as a CD137, in an immune cell binding molecule, an immune response to a tumor cell-enhancing vaccine, in an embodiment, or a vaccine, an immune response may be enhanced in an immune response to a human, or a vaccine, in an immune response to a microorganism, such as an immune cell binding molecule, in an immune system, or a vaccine, in an immune system, and a method of enhancing vaccine, such as an immune system, and a method of enhancing immune system for enhancing a method for enhancing immune system for enhancing a vaccine, for enhancing a method for enhancing a vaccine, for enhancing immune system for enhancing a method for enhancing immune system for the production of a cell, for the production of a human, for the production of a vaccine, for the production of a human, for the production of a microorganism, for the.

In practicing the methods of treatment, the binding molecule can be administered alone as a monotherapy or in combination with one or more additional therapeutic agents or therapies. Thus, in another aspect, the present disclosure provides a combination therapy comprising a binding molecule in combination with one or more additional therapies or therapeutic agents for separate, sequential or simultaneous administration. The term "additional therapy" refers to a therapy that does not employ the binding molecules provided by the present disclosure as therapeutic agents. The term "additional therapeutic agent" refers to any therapeutic agent other than the binding molecules provided by the present disclosure. In a particular aspect, the present disclosure provides a combination therapy for treating cancer in a mammal comprising administering to the mammal a therapeutically effective amount of a binding molecule provided by the present disclosure in combination with one or more additional therapeutic agents. In another embodiment, the mammal is a human.

A wide variety of cancer therapeutics can be used in combination with the binding molecules provided by the present disclosure. One of ordinary skill in the art will recognize the existence and development of other cancer therapies that can be used in combination with the methods and binding molecules of the present disclosure, and will not be limited to those forms of therapy described herein. Examples of classes of additional therapeutic agents that may be used in combination therapy for the treatment of cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonal therapeutic agents.

The term "chemotherapeutic agent" refers to a chemical or biological substance that may cause cancer cell death or interfere with the growth, division, repair and/or function of cancer cells examples of chemotherapeutic agents include those disclosed in WO 2006/129163 and US 20060153808. specific chemotherapeutic agents include (1) alkylating agents such as oncoclonidine (chlorembucil) (L EUKERAN), cyclophosphamide (mcyclophosphamide) (CYTOXAN), Ifosfamide (IFEPE), mechlorethamine hydrochloride (MUSTARGEGEGEN), thiotepa (THEP L), cetrimide (TIFO), antineoplastic (CETAXE), antineoplastic (CERTAIN), antineoplastic (CENOVELOCARN), antineoplastic (CER), antineoplastic (VEGENOVELOCR), antineoplastic (CANTRIFO), antineoplastic (CANTRIFON), antineoplastic (CANTRIFON-L, antineoplastic), antineoplastic (CANTE-CANTRIFON), antineoplastic (CANTE), antineoplastic (CANTE-VECETACETACETACERTASE (CANTE), CETACETACETACETACETACETACETACER), CETACETACETACETACETAE (CANTE (CANTRIFO (CANTE), CETACETACETACETACETACETACER), CETACETACETAE (CANTE), CETACETACETAE), CETACETACETACETACETACETACETACETACETACETACETACETAE (CACHEN), CETACETACETAE (CANTE), CETACETAE (CANTE), CETACETACETACETACETACETACETACETACETAE (CACHEN), CETACETACETAE (CANTE (CACHEN), CETAE (CACHEN), CETACETACETACETACETACETAE (CACHEN), CETAE (CACHEN), CETACETACETAE (CACHEN-CETACETACETACETAE (CACHE-CETAE (CACHEN), CETAE-CETACETAE (CACHE-CETAE), CETACETAE (CACHEN-CETAE (CACHEN), CETACETACETACETACETAE (CACHE-CETAE (CACHEN), CETAE (CACHEN-CETAE (CACHEN), CACHE-CETAE (CACHEN-CETAE-CETACETACETAE (CACHEN), CETAE-CETAE (CACHEN), CETACETACETACETACETACETACETAE (CACHEN-CETAE (CACHEN), CETACETACETAE-CETAE (CACHEN), CETAE-CETAE (CACHE-CETAE (CACHE-CETAE (CACHEN), CACHEN-CETAE (CACHEN), CETACETACETAE (CACHEN-CETAE-CETACETAE-CETAE (CACHEN-CETAE-CETACETAE-CETAE-CETACETACETAX (CACHEN), CACHEN-CETAE (CACHEN), CER (CACHEN), CETACER (CACHEN), CETACE.

Examples of immunotherapeutic agents include BCG (BCG), cytokines such as interferons, vaccines such as MyVax personalized immunotherapy, Onyvax-P, Oncophage, GRNVAC1, Favld, Provenge, GVAX, L ovaxin C, BiovaxID, GMXX, and NeuVax, and antibodies such as alemtuzumab (CAMPATH), Bevacizumab (AVASTIN), Cetuximab (ERBITUX), gemtuzumab ozogamicin (gemtuzumab ozogamicin) (MY L OTARG), ibritumomab tiuxetan (ZE L IN), Pactibizumab (VECTIX), rituximab (RITUN, XAHERA), HERYTHROCAT (HE TERC), IBITUN (IBITUDIUXET), antibodies such as those disclosed IN WO 2003/4939), agonists disclosed against receptors such as WO 2003/4939, EP 369/EP 2009, EP 4939, EP-EP 2009, EP-11 agonists disclosed IN WO 364939, EP-11, EP-.

The term "hormone therapy agent" refers to a chemical or biological substance that inhibits or eliminates the production of hormones, or inhibits or counteracts the effect of hormones on the growth and/or survival of cancer cells examples of such agents suitable for use in the methods herein include those disclosed in US20070117809 examples of specific hormone therapy agents include tamoxifen (NO L VADEX), toremifene (Fareston), fulvestrant (FAS L ODEX), Anastrozole (ARIMIDEX), exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate (MEGACE), goserelin (ZO L ADEX), and leuprolide (L UPRON).

Combination therapy for the treatment of cancer also encompasses the combination of binding molecules with surgery to remove the tumor. The binding molecule can be administered to the mammal before, during, or after surgery.

Combination therapy for the treatment of cancer also encompasses the combination of binding molecules with radiotherapy such as ionizing (electromagnetic) radiotherapy (e.g., X-rays or gamma rays) and particle beam radiotherapy (e.g., high linear energy radiation). The radiation source may be external or internal to the mammal. The binding molecule can be administered to the mammal before, during, or after radiation therapy.

The binding molecules and compositions provided by the present disclosure may be administered by any suitable enteral or parenteral route of administration. The term "enteral route of administration" refers to administration via any part of the gastrointestinal tract. Examples of intestinal routes include the oral, mucosal, buccal and rectal routes, or the intragastric route. By "parenteral route" of administration is meant a route of administration other than the enteral route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal, subcutaneous, or topical administration. The antibodies and compositions of the present disclosure can be administered using any suitable method, such as by oral ingestion, nasogastric tube, gastrostomy tube (gastrostomy tube), injection, infusion, implantable infusion pump, and osmotic pump. Suitable routes of administration and methods may vary depending on a variety of factors, such as the particular antibody used, the desired rate of absorption, the particular formulation or dosage form used, the type or severity of the condition being treated, the particular site of action, and the condition of the patient, and may be readily selected by one of skill in the art.

The term "therapeutically effective amount" of a binding molecule refers to an amount effective for the intended therapeutic purpose. For example, in the context of enhancing an immune response, a "therapeutically effective amount" is any amount that is effective in stimulating, augmenting, raising or boosting any response of the mammalian immune system. In the context of treating a disease, a "therapeutically effective amount" is any amount sufficient to cause any desired or beneficial effect in the mammal being treated. In particular, in the treatment of cancer, examples of desired or beneficial effects include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or increased survival of a mammal. A therapeutically effective amount of a CD137 antibody typically ranges from about 0.001 to about 500mg/kg, and more typically from about 0.01 to about 100mg/kg of mammalian body weight. For example, the amount can be about 0.3mg/kg, 1mg/kg, 3mg/kg, 5mg/kg, 10mg/kg, 50mg/kg, or 100mg/kg of the mammalian body weight. In some embodiments, a therapeutically effective amount of a CD137 antibody is in the range of about 0.01-30mg/kg of mammalian body weight. In some other embodiments, a therapeutically effective amount of a CD137 antibody is in the range of about 0.05-15mg/kg of mammalian body weight. The precise dosage level to be administered can be readily determined by one of skill in the art and will depend upon a variety of factors such as the type and severity of the condition to be treated, the particular binding molecule used, the route of administration, the time of administration, the duration of the treatment, the particular additional therapy used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and like factors well known in the medical arts.

The binding molecules or compositions are typically administered in multiple instances. The interval between single doses may be, for example, 1 time per week, 1 time per month, 1 time per 3 months or 1 time per year. Exemplary treatment regimens are such that administration is 1 time per week, 1 time every 2 weeks, 1 time every 3 weeks, 1 time every 4 weeks, 1 time per month, 1 time every 3 months, or 1 time every 3 to 6 months. A typical dosage regimen for CD137 antibodies includes intravenous administration of 1mg/kg body weight or 3mg/kg body weight using one of the following dosing regimens: (i) 1 time every 4 weeks, 6 doses, then 1 time every 3 months; (ii) every 3 weeks for 1 time; (iii)3mg/kg body weight 1 time, then 1mg/kg body weight 1 time every 3 weeks.

The disclosure will be more fully understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. The contents of all figures and all references, patents, and published patent applications cited throughout this disclosure are expressly incorporated herein by reference in their entirety.

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