Antibodies and antibody-drug conjugates targeting ox40 and uses thereof

Abstract

Disclosed herein are monoclonal antibodies and antibody-drug conjugates (ADCs) targeting OX40. Polynucleotides encoding the antibodies, pharmaceutical compositions comprising the antibodies or ADCs, and methods ofproducing the antibodies and ADCs are also provided herein. Medical uses of these antibodies and ADCs are also disclosed.

Description

[00376] In subjects having psoriasis, activated T cells, particularly CD4+ and CD8+ T cells, infiltrate the skin and release cytokines that promote inflammation and the overproduction of skin cells. This immune response causes the rapid turnover of skin cells, leading to the characteristic psoriatic plaques and associated symptoms. In some embodiments, the antibodies, ADCs, compositions and methods described herein can also be used to treat psoriasis.

[00377] In subjects having SLE, abnormal T cell activation and dysregulation lead to the production of cytokine and autoantibodies, and immune complex deposition, contributing to inflammation and tissue damage to multiple organs. In some embodiments, the antibodies, ADCs, compositions and methods described herein can also be used to treat SLE.

[00378] In subjects having ankylosing spondylitis, T cells, particularly CD4+ T cells, infiltrate the affected joints and release pro-inflammatory cytokines, promoting inflammation, leading to pain, stiffness, and fusion of the joints. In some embodiments, the antibodies, ADCs, compositions and methods described herein can also be used to treat ankylosing spondylitis.

[00379] Combination therapy using agents with different mechanisms of action can result in additive or synergetic effects. Combination therapy can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and / or increasing the therapeutic index of the agent disclosed herein. In some embodiments, the antibodies, ADCs, compositions and methods described herein can be used in combination with another therapy to treat the autoimmune disease or inflammatory disease. In some embodiments, the additional therapy can be, for example, NonSteroidal Anti-Inflammatory Drugs (NSAIDs), orticosteroids, Disease-Modifying Antirheumatic Drugs (DMARDs), other biologic therapies, immunosuppressants, targeted therapies, or immunomodulatory agents. The additional therapy can be administered prior to, concurrently with, or subsequent to administration of the anti-OX40 antibodies, ADCs, or pharmaceutical compositions described herein. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. A person skilled in the art can readily determine appropriate regimens for administering a pharmaceutical composition described herein and an additional therapy in combination, including the timing and dosing of an additional agent to be used in a combination therapy, based on the needs of the subject being treated.

[00380] In the methods disclosed herein, a therapeutically effective amount of the anti-OX40 antibodies, ADCs, or pharmaceutical compositions disclosed herein is administered to a subject in need of treatment for an autoimmune disease or an inflammatory disease. The subject can be a mammal. In some embodiments, the subject is a human.

[00381] The anti-OX40 antibodies, ADCs, or pharmaceutical compositions provided herein can be administered with medical devices known in the art. For example, in some embodiments, a needleless hypodermic injection device can be used, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use described herein include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art. 6.8 Kits and device

[00382] In some embodiments, the present disclosure provides a variety of kits for conveniently and / or effectively carrying out methods of the present disclosure. Typically, kits will comprise sufficient amounts and / or numbers of components to allow a user to perform multiple treatments of a subject(s) and / or to perform multiple experiments.

[00383] Any of the pharmaceutical compositions or vectors of the present disclosure may be comprised in a kit. In some embodiments, kits can further include reagents and / or instructions for creating and / or synthesizing compounds and / or pharmaceutical compositions of the present disclosure. In some embodiments, kits can also include one or more buffers. In some embodiments, kits of the disclosure can include components for making protein or nucleic acid arrays or libraries and thus, may include, for example, solid supports.

[00384] In some embodiments, kit components can be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component can be placed, and suitably aliquoted. Where there is more than one kit component, (labeling reagent and label may be packaged together), kits can also generally contain second, third or other additional containers into which additional components may be separately placed. In some embodiments, kits can also comprise a second container means for containing sterile, pharmaceutically acceptable buffers and / or other diluents. In some embodiments, various combinations of components can be comprised in one or more vials. Kits of the present disclosure can also typically include means for containing compounds and / or pharmaceutical compositions of the present disclosure, e.g., proteins, nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers can include injection or blow-molded plastic containers into which desired vials are retained.

[00385] In some embodiments, kit components are provided in one and / or more liquid solutions. In some embodiments, liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly used. In some embodiments, kit components can be provided as dried powder(s). When reagents and / or components are provided as dry powders, such powders can be reconstituted by the addition of suitable volumes of solvent. In some embodiments, it is envisioned that solvents can also be provided in another container means.

[00386] In some embodiments, kits can include instructions for employing kit components as well the use of any other reagent not included in the kit. Instructions can include variations that may be implemented. 6.9 Exemplified Embodiments

[00387] Embodiment 1. An antibody or antigen-binding fragment thereof that specifically binds to human 0X40, comprising: (a) a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 15; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO: 16; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs.

[00388] Embodiment 2. The antibody or antigen-binding fragment of Embodiment 1, wherein: (a) the VL CDR1, VL CDR2, VL CDR3 have the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or (b) the VH CDR1, VH CDR2, VH CDR3 have the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs.

[00389] Embodiment 3. The antibody or antigen-binding fragment of Embodiment 1, comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NOs:4, 5, 6, 10, 11 and 12, respectively.

[00390] Embodiment 4. The antibody or antigen-binding fragment of any one of Embodiments 1 to 3 that is humanized.

[00391] Embodiment 5. The humanized antibody or antigen-binding fragment of Embodiment 4, comprising: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15; and / or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[00392] Embodiment 6. The humanized antibody or antigen-binding fragment of Embodiment 5 comprising a VL and a VH having the amino acid sequences of SEQ ID NOs: 15 and 16, respectively.

[00393] Embodiment 7. The antibody or antigen-binding fragment of any one of Embodiments 1 to 6 that is a monoclonal antibody or antigen-binding fragment.

[00394] Embodiment 8. The antibody or antigen-binding fragment of any one of Embodiments 1 to 7 that is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, a (scFv)2, a single domain antibody (sdAb), and a heavy chain antibody (HCAb).

[00395] Embodiment 9. The antibody or antigen-binding fragment of any one of Embodiments 1 to 7 that is an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[00396] Embodiment 10. The antibody or antigen-binding fragment of any one of Embodiments 1 to 9, further comprising: a light chain constant (CL) region and a heavy chain constant (CH) region.

[00397] Embodiment 11. The antibody or antigen-binding fragment of Embodiment 10, wherein the CL region is kappa CL (Ck; SEQ ID NO: 19) or lambda CL (CL; SEQ ID NO:20), or a variant thereof having up to ten amino acids substitutions.

[00398] Embodiment 12. The antibody or antigen-binding fragment of Embodiment 10 or 11, wherein the CH region is IgGl CH (SEQ ID NO:21), IgG2 CH (SEQ ID NO:22), IgG3 CH (SEQ ID NO:23), or IgG4 CH (SEQ ID NO:24), or a variant of any of the above having up to ten amino acids substitutions.

[00399] Embodiment 13. The antibody of Embodiment 9 that is an IgGl antibody.

[00400] Embodiment 14. The antibody of Embodiment 13 comprising a light chain having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:27; and a heavy chain having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29.

[00401] Embodiment 15. The antibody of any one of Embodiments 1 to 14, wherein the antibody (1) does not block the OX40L binding to 0X40; (2) exhibits efficient internalization by 0X40 positive cells; (3) depletes 0X40 positive cells; (4) depletes 0X40 positive regulatory T-cells within TME; (5) exhibits significant ADCC; (6) enhances the anti-tumor immune response; (7) reduces the immunosuppression of TME; or (8) enhances immunological activities in TME; or any combination of (1) to (8).

[00402] Embodiment 16. A polynucleotide encoding a polypeptide of the antibody or antigenbinding fragment of any one of Embodiments 1 to 15.

[00403] Embodiment 17. A vector comprising the polynucleotide of Embodiment 16.

[00404] Embodiment 18. A host cell comprising the polynucleotide of Embodiment 16, or the vector of Embodiment 17.

[00405] Embodiment 19. A method of producing an antibody or antigen-binding fragment thereof that specifically binds human 0X40, comprising culturing the cell of Embodiment 18 under conditions suitable for expression of the antibody or antigen-binding fragment.

[00406] Embodiment 20. The method of Embodiment 19 that comprises isolating the antibody or antigen-binding fragment from the culture.

[00407] Embodiment 21. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of Embodiments 1 to 15, and a pharmaceutically acceptable carrier.

[00408] Embodiment 22. A method of 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 Embodiments 1 to 15.

[00409] Embodiment 23. The method of Embodiment 22, wherein the subject is a human.

[00410] Embodiment 24. Use of the antibody or antigen-binding fragment of any one of Embodiments 1 to 15 in treating cancer.

[00411] Embodiment 25. Use of the antibody or antigen-binding fragment of any one of Embodiments 1 to 15 for the preparation of a medicament for treating cancer.

[00412] Embodiment 26. The method or use of any one of Embodiments 22 to 25, wherein the cancer has 0X40+ tumor infiltrating lymphocytes (TILs).

[00413] Embodiment 27. The method or use of any one of Embodiments 22 to 26, wherein the cancer is an 0X40+ cancer.

[00414] Embodiment 28. The method or use of any one of Embodiments 22 to 27, wherein the cancer is a liquid cancer.

[00415] Embodiment 29. The method or use of Embodiment 28, wherein the cancer is T cell leukemia or lymphoma.

[00416] Embodiment 30. The method or use of Embodiment 29, wherein the cancer is Peripheral T-cell Lymphoma (PTCL, e.g, adult T-cell leukemia (ATL), angioimmunoblastic T-cell lymphoma (AITL) or T cell acute lymphoblastic leukemia (T-ALL).

[00417] Embodiment 31. The method or use of any one of Embodiments 22 to 27, wherein the cancer is a solid tumor.

[00418] Embodiment 32. The method or use of Embodiment 31, wherein the cancer is sarcoma, breast cancer, non-small cell lung cancer (NSCLC), melanoma, or ovarian cancer.

[00419] Embodiment 33. The method or use of any one of Embodiments 22 to 27, wherein the cancer is an HPV+ cancer or an EBV+ cancer.

[00420] Embodiment 34. The method of Embodiment 33, wherein the cancer is EBV+ B cell lymphoma, Extra-nodal NK / T cell lymphoma (ENKTL; e.g, nasal type), HPV16+ head and neck squamous cell carcinoma (HNSCC), EBV+ Esophageal cancer, or HPV18+ cervical cancer.

[00421] Embodiment 35. A method of treating an autoimmune disease or an inflammatory disease 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 Embodiments 1 to 15.

[00422] Embodiment 36. The method of Embodiment 35, wherein the subject is a human.

[00423] Embodiment 37. Use of the antibody or antigen-binding fragment of any one of Embodiments 1 to 15 in treating an autoimmune disease or an inflammatory disease.

[00424] Embodiment 38. Use of the antibody or antigen-binding fragment of any one of Embodiments 1 to 15 for the preparation of a medicament for treating an inflammatory disease or an inflammatory disease.

[00425] Embodiment 39. The method of use of any one of Embodiments 35 to 38, wherein the inflammatory disease or an inflammatory disease is characterized by excessive T cell activation.

[00426] Embodiment 40. The method of use of Embodiment 39, wherein the autoimmune disease or inflammatory disease is rheumatoid arthritis (RA), inflammation bowl diseases (IBD), Crohn's disease (CD), graft-vs-host diseases (GvHD), multiple sclerosis (MS), type I diabetes, atopic dermatitis, asthma, psoriasis, Sjogren's syndrome (SS), or idiopathic thrombocytopenia (ITP).

[00427] Embodiment 41. The method of any one of Embodiments 22 to 40, wherein the antibody or antigen-binding fragment is administered in combination with an additional therapy.

[00428] Embodiment 42. A kit comprising the antibody or antigen-binding fragment of any one of Embodiments 1 to 15.

[00429] Embodiment 43. An antibody-drug conjugate (“ADC”) comprising an antibody or antigenbinding fragment thereof conjugated to a therapeutic moiety, wherein the antibody or antigen-binding fragment specifically binds human OX40 and comprises: (a) a VL comprising VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50 or SEQ ID NO:52; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or (b) a VH comprising VH CDR1, VH CDR2, and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO:39, SEQ IDN0:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:53, or SEQ ID NO:56; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs.

[00430] Embodiment 44. The ADC of Embodiment 43, wherein the VL and VH have amino acid sequences that are independently at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to (1) SEQ ID NOs:38 and 39, respectively; (2) SEQ ID NOs:40 and 41, respectively; (3) SEQ ID NOs:42 and 43, respectively; (4) SEQ ID NOs:44 and 45, respectively; (5) SEQ ID NOs:46 and 47, respectively; (6) SEQ ID NOs:50 and 51, respectively; (7) SEQ ID NOs:52 and 53, respectively; or (8) SEQ ID NOs:38 and 56, respectively.

[00431] Embodiment 45. The ADC of Embodiment 43 or 44, wherein the antibody comprises a light chain and a heavy chain having amino acid sequences that are independently at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to (1) SEQ ID NOs:59 and 60, respectively; (2) SEQ ID NOs:59 and 61, respectively; (3) SEQ ID NOs:62 and 63, respectively; (4) SEQ ID NOs:62 and 64, respectively; (5) SEQ ID NOs:65 and 66, respectively; or (6) SEQ ID NOs:65 and 67, respectively.

[00432] Embodiment 46. An ADC comprising an antibody or antigen-binding fragment thereof conjugated to a therapeutic moiety, wherein the antibody or antigen-binding fragment specifically binds human OX40 and does not block OX40L binding to OX40.

[00433] Embodiment 47. The ADC of Embodiment 46, comprising the antibody or antigen-binding fragment of any one of Embodiments 1 to 15.

[00434] Embodiment 48. The ADC of any one of Embodiments 43 to 47, wherein the antibody or antigen-binding fragment is conjugated to the therapeutic moiety via a linker.

[00436] Embodiment 50. The ADC of Embodiment 49, wherein the linker is

[00437] Embodiment 51. The ADC of any one of Embodiments 43 to 50, wherein the therapeutic moiety is monomethyl auristatin.

[00438] Embodiment 52. The ADC of Embodiment 51, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE).

[00439] Embodiment 53. The ADC of Embodiment 51, having the structure: antibody or antigen-binding fragment, and p denotes a number ranging from 1 to 16.

[00440] Embodiment 54. The ADC of Embodiment 53, wherein p is number from 1 to 10.

[00441] Embodiment 55. The ADC of Embodiment 53, wherein p is about 4.

[00442] Embodiment 56. The ADC of Embodiment 53, wherein the average value of p in a population of the ADC is about 2.8, about 3.6, about 4, about 4.2, about 4.3, or about 5.

[00443] Embodiment 57. The ADC of any one of Embodiments 53 to 56, having the structure:

[00444] Embodiment 58. The ADC of Embodiment 51, wherein the monomethyl auristatin is monomethyl auristatin F (MMAF).

[00445] Embodiment 59. The ADC of Embodiment 51, having the structure: antibody or antigen-binding fragment, and p denotes a number from 1 to 16.

[00446] Embodiment 60. The ADC of Embodiment 59, wherein p is from 1 to 10.

[00447] Embodiment 61. The ADC of Embodiment 59, wherein p is about 4.

[00448] Embodiment 62. The ADC of Embodiment 59, wherein the average value of p in a population of the ADC is about 2.8, about 3.6, about 4, about 4.2, about 4.3, or about 5.

[00449] Embodiment 63. The ADC of any one of Embodiments 59 to 62, having the structure:

[00450] Embodiment 64. The ADC of any one of Embodiments 43 to 50, wherein the therapeutic moiety is a topoisomerase I inhibitor.

[00451] Embodiment 65. The ADC of Embodiment 64, wherein the topoisomerase I inhibitor is camptothecin, Dxd, SN-38 or exatecan (DX-8951).

[00452] Embodiment 66. The ADC of Embodiment 64, wherein the topoisomerase I inhibitor is Dxd.

[00453] Embodiment 67. The ADC of Embodiment 64, having the structure: antibody or antigen-binding fragment, and p denotes a number from 1 to 16. wherein Ab is the

[00454] Embodiment 68. The ADC of Embodiment 67, wherein p denotes a number from 1 to 10.

[00455] Embodiment 69. The ADC of Embodiment 67, wherein p is about 8.

[00456] Embodiment 70. The ADC of Embodiment 67, wherein the average value of p in a population of the ADC is about 5.3 or about 8.

[00457] Embodiment 71. The ADC of any one of Embodiments 67 to 70, having the structure:

[00458] Embodiment 72. The ADC of Embodiment 64, wherein the topoisomerase I inhibitor is SN-38.

[00459] Embodiment 73. The ADC of Embodiment 64, having the structure: antibody or antigen-binding fragment, and p denotes a number from 1 to 16.

[00460] Embodiment 74. The ADC of Embodiment 73, wherein p denotes a number from 1 to 10.

[00461] Embodiment 75. The ADC of Embodiment 73, wherein p is about 4 or 5.

[00462] Embodiment 76. The ADC of Embodiment 73, wherein the average value of p in a population of the ADC is about 4.5.

[00463] Embodiment 77. The ADC of any one of Embodiments 73 to 76, having the structure:

[00464] Embodiment 78. The ADC of Embodiment 64, wherein the topoisomerase I inhibitor is exatecan.

[00465] Embodiment 79. The ADC of Embodiment 64, having the structure: the antibody or antigen-binding fragment, and p denotes a number from 1 to 16.

[00466] Embodiment 80. The ADC of Embodiment 79, wherein p denotes a number from 1 to 10.

[00467] Embodiment 81. The ADC of Embodiment 79, wherein p is about 4 or 8.

[00468] Embodiment 82. The ADC of Embodiment 79, wherein the average value of p in a population of the ADC is about 4.2 or about 8.

[00469] Embodiment 83. The ADC of any one of Embodiments 79 to 82, having the structure:

[00470] Embodiment 84. The ADC of any one of Embodiments 43 to 50, wherein the therapeutic moiety is a derivative of halichondrin B.

[00471] Embodiment 85. The ADC of Embodiment 84, wherein therapeutic moiety is eribulin.

[00472] Embodiment 86. The ADC of Embodiment 84, having the structure: Ab is the antibody or antigen-binding fragment, and p denotes a number ranging from 1 to 16.

[00473] Embodiment 87. The ADC of Embodiment 86, wherein p is number from 1 to 10.

[00474] Embodiment 88. The ADC of Embodiment 86, wherein p is about 4 or 5.

[00475] Embodiment 89. The ADC of Embodiment 86, wherein the average value of p in a population of the ADC is about 4.6.

[00476] Embodiment 90. The ADC of any one of Embodiments 86 to 89, having the structure:

[00477] Embodiment 9E An ADC comprising an antibody or antigen-binding fragment thereof conjugated to a therapeutic moiety; wherein the antibody or antigen-binding fragment specifically binds human OX40 and has (1) VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 15; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs, and VH CDR1, VH CDR2 and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO: 16; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs; (2) VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 3 8; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs, and VH CDR1, VH CDR2 and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO:39; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs; (3) VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO:40; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs, and VH CDR1, VH CDR2 and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO:41; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs; (4) VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO:42; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs, and VH CDR1, VH CDR2 and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO: 43; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs; or (5) VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 3 8; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs, and VH CDR1, VH CDR2 and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO:56; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs; and wherein the therapeutic moiety is MMAE, MMAF, Dxd, SN-38, exatecan, or eribulin.

[00478] Embodiment 92. An ADC comprising an antibody or antigen-binding fragment thereof conjugated to a therapeutic moiety; wherein the antibody or antigen-binding fragment specifically binds human 0X40 and has a VL and VH having the amino acid sequences of SEQ ID NOs: 15 and 16, respectively; and wherein the therapeutic moiety is MMAE, MMAF, Dxd, SN-38, exatecan, or eribulin.

[00479] Embodiment 93. The ADC of any one of Embodiments 43 to 92, wherein the ADC (1) does not block the OX40L binding to OX40; (2) exhibits efficient internalization by 0X40 positive cells; (3) depletes 0X40 positive cells; (4) depletes 0X40 positive regulatory T-cells within TME; (5) exhibits significant ADCC; (6) maintains the essential ADCC of the anti-OX40 antibodies or antigen-binding fragments; (7) enhances the anti-tumor immune response; (8) reduces the immunosuppression of TME; or (9) enhances immunological activities in TME; or any combination of (1) to (9).

[00480] Embodiment 94. A method of producing the ADC of any one of Embodiments 43 to 93 comprising conjugating the antibody or antigen-binding fragment to the therapeutic moiety.

[00481] Embodiment 95. A pharmaceutical composition comprising the ADC of any one of Embodiments 43 to 93, and a pharmaceutically acceptable carrier.

[00482] Embodiment 96. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the ADC of any one of Embodiments 43 to 93.

[00483] Embodiment 97. The method of Embodiment 96, wherein the subject is a human.

[00484] Embodiment 98. Use of the ADC of any one of Embodiments 43 to 93 in treating cancer.

[00485] Embodiment 99. Use of the ADC of any one of Embodiments 43 to 93 for the preparation of a medicament for treating cancer.

[00486] Embodiment 100. The method or use of any one of Embodiments 96 to 99, wherein the cancer is an OX40+ cancer.

[00487] Embodiment 101. The method or use of any one of Embodiments 96 to 100, wherein the cancer is a liquid cancer.

[00488] Embodiment 102. The method or use of Embodiment 101, wherein the cancer is T cell leukemia or lymphoma.

[00489] Embodiment 103. The method or use of Embodiment 102, wherein the cancer is Peripheral T-cell Lymphoma (PTCL, e.g, adult T-cell leukemia (ATL), angioimmunoblastic T-cell lymphoma (AITL)), or T cell acute lymphoblastic leukemia (T-ALL).

[00490] Embodiment 104. The method or use of any one of Embodiments 96 to 100, wherein the cancer is a solid tumor.

[00491] Embodiment 105. The method or use of Embodiment 104, wherein the cancer is sarcoma, breast cancer, non-small cell lung cancer (NSCLC), melanoma, or ovarian cancer.

[00492] Embodiment 106. The method or use of any one of Embodiments 96 to 100, wherein the cancer is an HPV+ cancer or an EBV+ cancer.

[00493] Embodiment 107. The method of Embodiment 106, wherein the cancer is EBV+ B cell lymphoma, Extra-nodal NK / T cell lymphoma (ENKTL; e.g, nasal type), HPV16+ head and neck squamous cell carcinoma (HNSCC), EBV+ Esophageal cancer, or HPV18+ cervical cancer.

[00494] Embodiment 108. A method of treating an autoimmune disease or an inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the ADC of any one of Embodiments 43 to 93.

[00495] Embodiment 109. The method of Embodiment 108, wherein the subject is a human.

[00496] Embodiment 110. Use of the ADC of any one of Embodiments 43 to 93 in treating an inflammatory disease or an inflammatory disease.

[00497] Embodiment 111. Use of the ADC of any one of Embodiments 43 to 93 for the preparation of a medicament for treating an inflammatory disease or an inflammatory disease.

[00498] Embodiment 112. The method or use of any one of Embodiments 108 to 111, wherein the autoimmune disease or inflammatory disease is rheumatoid arthritis (RA), inflammation bowl diseases (IBD), Crohn's disease (CD), graft-vs-host diseases (GvHD), multiple sclerosis (MS), type I diabetes, atopic dermatitis, asthma, psoriasis, Sjogren's syndrome (SS), or idiopathic thrombocytopenia (ITP).

[00499] Embodiment 113. The method or use of any one of Embodiments 96 to 112, wherein the ADC is administered in combination with an additional therapy.

[00500] Embodiment 114. A kit comprising the ADC of any one of Embodiments 43 to 93. 6.10 Experimental

[00501] The examples provided below are for purposes of illustration only, which are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. 6.10.1 Example 1: Preparation and characterization of humanized antibody HX011

[00502] Materials and Methods:

[00503] Preparation of chimeric humanized antibodies. A chimeric antibody based on the 9C12 hybridoma clone was constructed by replacing the mouse Fc with human IgGl Fc. Humanized antibodies based on 9C12 were constructed by selecting the most relevant human IgG framework humanization with the fewest modifications of its sequence and putative structure. The CDRs were maintained to ensure maximum affinity to the antigen. All chimeric and humanized antibodies were expressed in engineered recombinant cells for bioactivity validation.

[00504] Cell cultures and reagents'. Jurkat cell line was purchased from ATCC and cultured in RPMI-1640 medium (Hyclone) supplemented with 10% fetal bovine serum. 293T-OX40 was constructed by transfecting a lentiviral vector carrying human OX40 gene into 293T cells. OX40-expressing cell clones were screened with selective antibiotic puromycin treatment. The cells were cultured in High glucose DMEM medium (Gibco) supplemented with 10% fetal bovine serum and puromycin. Jurkat-NFAT(BPS)-CD16A cells were constructed by introducing a lentiviral vector carrying human FCyRIII gene into Jurkat-NFAT (BPS Bioscience) cells. The cells were cultured in RPMI-1640 medium (Hyclone) supplemented with 10% fetal bovine serum and puromycin.

[00505] Antigen binding and ligand blocking by ELISA'. A 96-well polyvinyl microtiter plate was coated with OX40-his in 100 pL carbonate-bicarbonate buffer (200 mM, pH 9.4) overnight at 2-8°C. The plate was washed with PBS-T buffer three times, and the antigen (OX40-his)-coated plate was blocked with 1% BSA-PBS for 60 minutes at 37°C. The plate was washed with PBS-T buffer four times followed by incubation with 100 pL / well of human Fc chimeric antibody (9C12H11) and humanized antibody (HX011) in serial dilutions for 1 hour at 37°C. The plate was then washed five times with PBS-T and incubated with 100 pL of HRP-Goat Anti-Human IgG (H+L) (10000X) in sample dilution buffer (l%o BSA in PBS) for 1 hour at 37°C. The washed plate wells were then incubated with 100 pL / well of substrate solution at 37°C for 10 minutes. The reaction was stopped by adding 50 pL / well of 2M sulfuric acid. The absorbance at 450 nm was recorded using a micro plate reader. ECso values were calculated from best-fit binding curves using GraphPad Prism software.

[00506] For ligand blocking ELISA assay, a 96-well polyvinyl microtiter plate was coated with OX40-his in 100 pL carbonate-bicarbonate buffer (200 mM, pH 9.4) overnight at 2-8°C. The plate was washed with PBS-T buffer three times, and the antigen (OX40-his)-coated plate was blocked with 1% BSA-PBS for 60 minutes at 37°C. The plate was washed with PBS-T buffer for four times and incubated with identical volume of the HX011 or 0X40.21 in five-fold serial dilutions and OX40L-mFc (4 pg / mL) for 1 hour at 37°C. The plate was then washed five times with PBS-T and incubated with 100 pL of HRP-Goat Anti-Mouse (5000X) in sample dilution buffer (l%o BSA in PBS for 1 hour at 37°C. The washed plate wells were then incubated with 100 pL / well of substrate solution at 37°C for 10 minutes. The reaction was stopped by adding 50 pL / well of 2M sulfuric acid. The absorbance at 450 nm was recorded, using a micro plate reader from Molecular Devices. IC50 values were calculated from best-fit binding curves using GraphPad Prism software.

[00507] Binding to target receptor positive cell surface by flow cytometry. OX40 positive 293T cells were incubated with titrated doses of HX011 (approximately 100 nM - 0.00123 nM) at 4°C for 1 hour. Cells were then washed several times with precooled PBS buffer with 2% FBS. Bound antibodies were detected with Alexa Fluor® 647(AF647)-conjugated AffiniPure Goat Anti-Human IgG (H+L) secondary antibody (Jackson Immuno Research) and analyzed by flow cytometry. EC50 values were calculated from best-fit binding curves using GraphPad Prism software.

[00508] Reactivation of SEB stimulated PBMC assay. Human PBMC were isolated from heparinized blood obtained from healthy donors, with local ethical consent. PBMC were obtained by Ficoll density gradient separation followed by stimulation with 50 ng / mL of Staphylococcal enterotoxin B (SEB), then incubated with titrated doses of the HX011 at 37°C and 5% CO2. Culture supernatants harvested at 72 h were assayed by ELISA for the cytokines (e.g.,IL2).

[00509] PBMC-mediated ADCC assay. The Human PBMC were isolated from healthy donors using Ficoll density gradient separation (GE), as the effector cells. We used Jurkat-OX40 cell line (Jurkat cells engineered to express human OX40) as the target cells. Jurkat-OX40 was labeled with carboxyfluorescein succinimidyl ester (CFSE) dye for 20 min, followed by incubation with PBMC at E:T= 25:1 and titrated doses of HX011 for 4 hours in a 96-well culture plate at 37°C. The cells were harvested, and stained with Fixable Viability Stain 780 which stained dead cells. After 15 minutes of incubation, the cells were washed with FACS buffer, and then subjected to FACS analysis using CytoFLEX S.

[00510] Luciferase reporter assay. For ADCC assay, 7.5 / 104 of Jurkat-NFAT(BPS)-CD16A cells (Jurkat cells engineered to express human FcyRIII and NF AT transcription factor) were seeded in 96-well Solid White Flat Bottom Polystyrene TC-treated Microplates together with 1.25 / 104 of 293T-OX40 cells. Then the cells were incubated with titrated doses of the HX011 in serial dilutions (approximately 100 nM - 0.00124 nM) at 37°C at 5% CO2. After 18 hours, Stable-LiteTM Luciferase Assay System reagent (Vazyme-DD 1202-02) was added into each well and the cells were incubated at room temperature for 10 minutes to stabilize luminescent signal, which was then recorded on the 2104 EnVision plate reader. EC50 values were calculated from best-fit binding curves using GraphPad Prism software. For OX40 agnostic assay, Jurkat-NFkB-OX40 (Jurkat cells stably expressing human OX40 on the cell surface and having a luciferase gene driven by NFkB transcription factor) and 293T-CD32B cells (293T cells overexpressing human FcyRIIB) were used following incubation and detecting protocols similar to those described above.

[00511] Immuno-safety-cytokine release storm assay. Responses to therapeutic mAbs were evaluated in both aqueous phase, at titrated doses (69 nM / well-0.69 nM / well) in 96 well microtitre plate, and in solid phase by coating at titrated doses (69 nM / well-0.69 nM / well) in 60 pL of phosphate buffered saline onto 96 well microtitre plates at 4 °C overnight followed by two washes to remove unbound mAb. Human PBMC were isolated by density gradient separation from heparinized blood that was obtained from healthy donors with local ethical consent. Donor PBMC were adjusted to 2 x 106 cells / ml and 100 pL were added to tissue culture plate wells in duplicate sets and incubated for 24 and 48 h in a humidified incubator at 37°C with 5% CO2. Culture supernatants harvested at 24 and 48 h were assayed by Cytometric Bead Array (CBA), according to manufacturer’s instructions, for the cytokines IL-2, IL-4, IL-6, IL-10, TFNa, IL-17a and IFN-y as previously described.

[00512] In vivo Antitumor pharmacology experiments. To prepare the human OX40 knock-in MC-38 colon cancer syngeneic model, the tumor cells were subcutaneously implanted into C57BL / 6 mice (Biocytogen) whose OX40 was replaced with human OX40. When the average tumor volume reached 100 mm3, the mice were randomly grouped and treated. HX011 was intravenously dosed twice weekly with 1,3, 10 mg / kg for 5 weeks. PBS was dosed with the same way as HX011 as control treatment. Tumor volume was measured twice weekly as 0.5 / (long diameter / short diameter)2. Tumor growth inhibition (TGI) was calculated as TGI% = (1 - Vtreatment / VControi)x100%.

[00513] Results:

[00514] Biochemical characterization of the humanized 9C12. Two humanized antibodies of 9C12 clone were assessed, and the one having higher affinity to human 0X40 (“HX011”) was selected for future characterization. Using a luciferase reporter system, the T cell agonistic activity of HX01 Iwas confirmed to be comparable with that of the chimeric antibody (9C12-hu-IgGlFc chimera) with an EC50 of about 9.6 ng / ml (FIG. 1 A). HX011 was also confirmed to bind cell surface 0X40 on 0X40-overexpressing 293T cell with an EC50 of about 1.5 nM (FIG. IB). It provoked IL-2 secretion in PBMC culture after SEB treatment, again confirming its agonistic effect (FIG.IC). A competition ELISA assay was performed, and it was found that different from 0X40.21, a reference agonistic anti-OX40 antibody (Bristol Myers Squibb), HX011 did not interfere with the interaction between human 0X40 and its ligand OX40L (FIG. ID). The fact that HX011 did not competitively block OX40L binding to 0X40 supported that HX011 as an agonist could trigger maximum 0X40-mediated T cell activation.

[00515] HX011 showed ADCC effect in vitro. The strong ADCC effect of HX011 against 0X40 expressing cells was confirmed using different assays: 1) fresh PBMC cultured with 0X40 positive cancer cell line (Jurkat-OX40) demonstrated ADCC against Jurkat-OX40 cells induced by HX011 in a dose dependent manner (FIG.2A); 2) in a luciferase reporter gene assay, HX011 was found to activate FcyRIIIa-expressing Jurkat cell downstream reporter gene (Luciferase) expression when incubated with 0X40 positive target cells with an EC50 of about 0.61 nM (FIG.2B).

[00516] In vivo anti-tumor activity of the HX011. Different doses (1, 3 and 10 mg / kg) of HX011 were administered intravenously into huOX40 HuGEMM syngeneic mouse model. All three doses showed significant antitumor activity; even the lowest dose (1 mg / kg) inhibited tumor growth by more than 90% compared by control (FIG.2C). The results demonstrated the remarkable activity of HX011, a checkpoint agonist, in reducing tumor growth.

[00517] HX011 had low risk to induce cytokine release syndrome (CRS) in vitro. The release of several cytokines, including IL-2 (FIG.3A), IL-6 (FIG.3B), IL-4 (FIG.3C), TFNa (FIG.3D), and IFN-y (FIG.3E) from isolated PBMC was measured in both aqueous and solid phase in the presence of HX011. As shown, HX011 only induced a small amount of IL-6 release at high concentration (69 nM); the amount of IL-2, IL-4, TFNa, and IFN-y release induced by HX011 was not detectable, even at high concentration. These results demonstrated that HX011 was unlikely to provoke CRS if used as a therapeutic in clinic.

[00518] HX011 antibody had robust stability. A cell line that stably expressed HX011 was generated by transfecting vectors encoding HX011 antibody heavy and light chain into CHO cells. After screening, the clone that stably expressed HX011 antibody was isolated. HX011 expressed by this clone was purified using Protein-A affinity chromatography followed by size exclusion chromatography (SEC). The purified antibody showed very high monomer purity (>99%) (FIG. 4, upper panel). The thermal stability and acidic pH stability of HX011 was assessed by placing the antibody at 25°C and 40°C at different acidic pH (3.0 ~ 6.0) for at least 5 days. As shown, the SEC purity of HX011 remained high after five days storage at pH 3.3 ~ 6.0 at 25°C and at pH 3.6 ~ 6.0 at 40°C (see below). Furthermore, HX011 was also found to be stable through multiple freeze-thaw cycle. As shown below, its SEC purity after 5 freeze-thaw cycles at pH 6.0 remained high. Together, all these results demonstrated that HX011 had robust stability under various conditions and external changes. Temperature pH SEC Purity Oh 1 day 5 days 25 °C 3.0 96.7% 67.0% 29.9% 3.3 99.8% 99.9% 99.8% 3.6 99.9% 99.9% 99.9% 6.0 99.8% 99.7% 99.8% 40 °C 3.0 96.7% 34.6% 19.5% 3.3 99.8% 92.3% 56.3% 3.6 99.9% 99.4% 98.1% 6.0 99.8% 99.7% 99.7% pH FT0 FT1 FT2 FT3 FT4 6.0 99.7% 99.8% 99.7% 99.8% 99.8% * FT: freeze-thaw; FTn: freeze-thaw for n cycles.

[00519] Summary:

[00520] Briefly, the study described above included the production and characterization of a novel monoclonal agonistic humanized anti-OX40 IgGl antibody (HX011). Similar to the original mouse mAh clone 9C12, HX011 showed strong binding to human 0X40. In contrast to the reference antibody BMS-986178, HX011 did not compete with OX40L for HX40 binding, which could result in maximized T-cell activation. Indeed, in the humanized syngeneic CRC model, MC38, where syngeneic C57BL / 6 mouse mice were knocked in with huOX40 (“HuGEMM-OX40”), HX011 demonstrated strong in vivo anti-tumor activity (TGI -100% at 1 mg / kg, twice weekly). 6.10.2 Example 2: Preparation and characterization of ADC HX111

[00521] Materials and Methods

[00522] In vivo antitumor activity. All in vivo murine experiments were conducted following the ARRIVE Guideline. Briefly, HuT-102 cells (1 x 107 cells per mouse) or HuT-78 cells (1 x 107 cells per mouse) were subcutaneously inoculated into the right front flanks of female NCG mice (NOD / ShiLtJGpt-Prkdcem26Cd52I12rgem26Cd22 / Gpt, GemPharmatech, China). Once tumors had reached approximately 150 mm3 (small tumor model) or 600 mm3 (large tumor model), mice were randomly assigned to control and treatment groups. On Day 0, mice received an intraperitoneal injection of PBS (10 ml per kg body weight), HX111 (0.2 mg: 10 ml per kg body weight), HX111 (1 mg: 10 ml per kg body weight), HX111 (2 mg: 10 ml per kg body weight), HX111 (5 mg: 10 ml per kg body weight), HX011 (5 mg: 10 ml per kg body weight), or MAL-VC-MMAE (equivalent mole to HX111 at 2 mg / kg or 5 mg / kg) with designated dosing schedules. Tumor volumes and body weight were measured twice per week. Tumor volumes were measured in two dimensions using a caliper, and the volume (mm3) were calculated using the formula: V = (L x W x W) / 2, wherein V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L).

[00523] In vitro cell killing activity. The Hut-102 cells (1,000 cells per well) were incubated with serial dilutions of either a HX011-based ADC or the therapeutic moiety only for 6 days at 37°C in a 5% CO2, humidified incubator. Cell viability was measured with CellCounting-Lite2.0 reagent (Vazyme Biotech Co., Ltd) according to manufacturer’s instructions. IC50 values were determined by using GraphPad software (GraphPad).

[00524] Results

[00525] Preparation of ADC HX11T. OX40-mAb HX011 was conjugated with MC-VC-PAB-MMAE (CAS: 646502-53-6) to form the ADC HX111 at a DAR of about 4. The conjugation ratio was about 97% (FIG.4, lower panel). The purity of HX111 was also confirmed with HPLC (FIG.4, middle panel). HX111 was found to be stable in the presence of plasma.

[00526] Tumor expression of0X40: 0X40 was known as a tumor associated antigen (TAA) of certain leukemia and lymphoma. In this study, the expression of 0X40 in several Tax-expressing tumor models was measured. First, we detected significant surface expression of 0X40 in HuT-102 (Tax+ T cell lymphoma) and HuT-78 cell line (Tax+ T cell leukemia) using flow cytometry (FIG.5A). In contrast, little 0X40 expression was detected in HPB-ALL cells (FIG.5B). Additionally, elevated 0X40 mRNA levels were found in EBV+ DLBCL-PDX (lymphoma) models (FIG.5C). As such, tumor models with elevated 0X40 expression were used to further study the antitumor activity ofHXlll.

[00527] Internalization of HX1H: The anti-OX40 mAh HX011 and the ADC HX111 were incubated with 0X40 positive ATL cells, and the internalization thereof was measured by cell cytometry as described in Example 1. When incubated with either HuT-102 or Jurkat cells engineered to overexpress 0X40, the mAh HX011 was quickly internalized into these cells (FIG.6B). The ADC HX111 was internalized by HuT-102 at the same rate as the mAh HX011 (FIG.6A). This result indicated that the MMAE conjugation had little impact on endocytosis kinetics of the naked monoclonal antibody and supported the therapeutic potential of HX111 as an ADC.

[00528] In vitro cytotoxicity ofHXHT. The cytotoxicity ofHXlll in antigen+ leukemic / lymphoma cell lines was also confirmed. Potent cytotoxicity of HX111 was observed at subnanomolar range in HuT-102 (FIG.7A) and OX40-exprssing Jurkat cells (FIG.7D), supporting that HX111 could effectively treat ATL and other types of OX40-expressing leukemia / lymphoma.

[00529] In addition, in the coculture of both antigen+ and antigen- leukemic / lymphoma cells, HX111 also demonstrated bystander cytotoxicity in the antigen- lines (FIG.8A and 8B). HX111 induced cytotoxicity in 0X40 negative Raji cells in the presence of Hut-102 cells (FIG. 8A), as well as in 0X40 negative Jurkat cells in the presence of Jurkat-OX40-5E9 cells (FIG.8B). Additionally, the greater the ratio of antigen+ cells (i.e., HuT-102 or Jurkat-OX40-5F9): antigen- cells (Raji or Jurkat), the greater the bystander effect.

[00530] In vivo tumor killing activity ofHXHT. HuT-102 and HuT-78 subcutaneous xenograft models were used to study the antitumor activity of HX111 against ATL. Strong anti-tumor activity of HX111 was observed in both small tumor models (FIGs.9A-9B), as well as in large HuT-102 tumor model (FIG. 10). In both the HuT-102 and HuT-78 xenograft model, HX111 induced tumor growth inhibition (TGI)Ztumor regression in a dose-dependent fashion without inducing any abnormalities or body weight loss (FIGs.9A-9B and 10). In addition, in the HuT-102 xenograft model (small tumor), 4 out of 6 mice received HX111 at 1 mg / kg twice per week for 3 weeks and 6 out of 6 mice received HX111 at 5 mg / kg twice per week for 3 weeks showed complete response (CR) (FIG.9A). In the HuT-78 model, HX111 at 5 mg / kg even achieved a TGI of 95.94% on day 14 and induced transient complete response in 1 out of 6 mice (FIG.9B). Furthermore, in mice bearing large HuT-102 tumors, HX111 at 2 mg / kg administrated twice per week, or once a week, or every two weeks all induced similar extent of tumor growth inhibition and complete tumor regression (FIG. 10). Taken together, these results confirmed the in vivo anti-tumor activity of HX111 against the 0X40 positive tumors, such as ATL.

[00531] In vitro cell killing activity ofHXlll, HX011-MMAF andHX011-Dxd. The cytotoxicity of anti-OX40 ADCs against OX40 positive cancer cells HuT-102 were measured. Exemplary anti-0X40 ADCs were prepared by conjugating the anti-OX40 antibody 0X011 with different therapeutic moieties, including MMAE, MMAF, and DXD. As shown, anti-OX40 ADCs, especially HX111 (i.e., HX011-MMAE) and HX011-MMAF showed efficient killing activity against HuT-102 cells (FIG. 11). 6.10.3 Example 3: Treatment of GvHD by ADC HX111

[00532] Materials and Methods:

[00533] Establishment of acute GvHD model: Female NOG (NOD.Cg4Mcdcw,^2rgtelsWJicCrl) mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Receipt NOG mice were sub-lethally irradiated with 3 Gy with an X-Ray source one day prior to human PBMC engraftment. On Day 0, X-ray irradiated mice were injected intravenously with 1 x 107 human PBMCs in 100 pl PBS. Following human PBMC engraftment, mice were treated with either vehicle, HX111 at 0.3 mg / kg, or HX111 at 1 mg / kg twice per week. Body weight and GvHD disease activities were measured twice per week. The GvHD disease activities were scored using the following criteria shown in the table below and shown as a cumulative GvHD score of each category. In the event of a mouse death, the GvHD score was defined as 15. Criteria ScoreO Score 1 (Mild) [Score 2 (Moderate) Score 3(Severe) Weight Loss Normal 5- 10% 10- 20% > 20% Activity Normal Mildly decreased Moderately decreased; delayed passivity / poor grip (delayed or unsuccessful attempt to climb up to the top of the cage when hold the mouse by the tail and place front paws on the edge of the cage top) Stationary until stimulated; poor passivity / no grip (hold the mouse by the tail and place front paw on the edge of the cage top and it falls) Posture Normal Hunching only at rest Moderately hunching, hunching when moving Severe hunching / gait, impaired movement Fur Texture Normal Mild ruffling Moderate ruffling / alopecia Severe ruffling / poor grooming Skin Integrity Normal Scaling of paws, tails or skins Inflamed / thickened skin; scaling / scabbing and thickened skins Obvious areas of denuded skin

[00534] Results

[00535] To evaluate the therapeutic effects of HX111 on GvHD, an acute GvHD mouse model was established by engrafting human PBMCs into X-ray irradiated NOG mice. Following xenograft of human PBMCs, vehicle, HX111 at 0.3 mg / kg, or HX111 at 1 mg / kg was intraperitoneally injected twice per week for 2 weeks. As shown, HX111 treatment significantly alleviated the body weight loss (FIG.12A) and diminished disease activities (FIG.12B) after day 7 in a dose-dependent matter, indicating that HX111 effectively prevented the disease progression of GvHD. Consistently, treatment with HX111 also significantly prolonged the survival of PBMC allograft mice (FIG.12C). Taken together, HX111 demonstrated great therapeutic potential for autoimmune diseases such as acute GvHD. 6.10.4 Example 4: Preparation of additional anti-OX40 ADCs

[00536] 3.67 mg of HX011 antibody was replaced into the reduction buffer solution (25 mM of sodium borate, 25 mM of NaCl, 1 mM of DTPA pH 8.0) using 15 mL 30 kD ultrafiltration tube, and the process was repeated 3 times. The final volume was about 1 mL. The replaced sample was transferred to a 1.5ml EP tube, and the protein concentration was measured. A 10-fold molar excess of TCEP was added to the antibody and placed at 37 °C for 1.5h. The sample was then ultrafiltered by a 15mL 30kD tube to replace into coupling buffer (50mM of Tris, 150mM of NaCl, 5mM of EDTA pH7.2) for 4 cycles. The protein concentration was re-measured. Based on the protein concentration, a 12-fold molar excess of MC-GGFG-Exatecan (CAS: 1600418-29-8) were added to the antibody. The sample was mixed and reacted at 25 °C for Ih with shaking. The conjugated protein sample was replaced into a temporary buffer (25 mM phosphate, 150 mM NaCl, pH 6.0), and the activity of each target and purity of the sample were assessed. As shown in FIG. 13 A, HX011-Exd (i. e., HX011 conjugated with MC-GGFG-Exatecan) at a DAR of 8 was obtained; HX011 -Exds at different DARs were also synthesized (e.g, FIG.13B).

[00537] HX011-SN-38 ( / .e., HX011 conjugated with MC-VC-PAB-SN38 (CAS: 1801838-28-7)), HXOll-Eribulin (i.e., HX011 conjugated with Mal-PEG2-VCP-Eribulin (CAS: 2130869-18-8)), HX011-MMAF (i.e., HX011 conjugated with MC-VC-PAB-MMAF (CAS: 863971-17-9)), HX011-Dxd (i.e., HX011 conjugated with deruxtecan (CAS: 1599440-13-7)), HX518-MMAE (i.e., HX518 conjugated with MC-VC-PAB-MMAE (CAS: 646502-53-6)), HX523-1-MMAE (i.e., HX523-1 conjugated with MC-VC-PAB-MMAE (CAS: 646502-53-6)), HX534-MMAE (i.e., HX534 conjugated with MC-VC-PAB-MMAE (CAS: 646502-53-6)), and HX543-MMAE (i.e., HX543 conjugated with MC-VC-PAB-MMAE (CAS: 646502-53-6)) were synthesized similarly to HX011-Exd. The synthesized conjugates were characterized by HPLC analyses (FIG.13C: HX011-SN-38; FIG.13D: HXOll-Eribulin; FIG.13E: HX518-MMAE; FIG.13F: HX523-1-MMAE; FIG.13G: HX534-MMAE; FIG.13H: HX543-MMAE). 6.10.5 Example 5: Characterization of anti-OX40 ADCs

[00538] In vitro antigen binding:

[00539] The ELISA binding assay described in Example 1 was conducted for HX518, HX518-MMAE, HX523-1, HX523-1-MMAE, HX534, HX534-MMAE, HX543, and HX543-MMAE.

[00540] Results: As shown in FIG. 14 and the table below, all tested anti-OX40 antibodies and anti- OX40 ADCs showed comparable antigen binding affinities and EC50 values. Coating 0.5 pg / mL OX40-his Sampling (nM) HX011 HX518 HX518-MMAE HX523-1 HX523-1-MMAE HX534 HX534-MMAE HX543 HX543-MMAE 20 2.937 2.810 2.733 2.802 2.824 2.900 2.826 2.795 2.805 4 2.910 2.753 2.683 2.694 2.760 2.822 2.759 2.758 2.866 0.8 2.911 2.704 2.621 2.672 2.702 2.766 2.706 2.710 2.803 0.16 2.561 2.341 2.095 2.329 2.287 2.475 2.321 2.207 2.228 0.032 1.331 1.135 0.869 1.183 1.039 1.249 1.036 0.951 0.911 0.0064 0.395 0.331 0.246 0.350 0.308 0.360 0.293 0.270 0.263 0.00128 0.125 0.111 0.087 0.113 0.104 0.115 0.100 0.094 0.094 0.000256 0.064 0.059 0.055 0.059 0.058 0.061 0.056 0.056 0.056 Secondary antibody HRP-Goat Anti-Human, 10000X coloring 10 min EC50 nM 0.038 0.045 0.062 0.042 0.051 0.040 0.050 0.057 0.061

[00541] Internalization:

[00542] A sufficient volume of 4X working solution of antibody was prepared in cell culture medium, and a 4X working solution of Zenon™ pHrodo™ iFL IgG Labeling Reagent was also prepared. 25 pL 4X Zenon™ working solution was combined with 25 pL of HX011 and incubated for 5 minutes at room temperature to allow the labeling complexes to form. HuT-102 cells were plated at IxlO5 cells / well in 50 pL culture medium, and 50 pL of the labeling complex was added to each well. The cells were incubated with the labeling complex for 24 hours under standard cell culture conditions. Flow cytometry was used to analyze the cells.

[00543] Results: As shown in FIG.15, HX011, HX111, HX518, HX518-MMAE, HX523-1, HX523-1-MMAE, HX534, HX534-MMAE, HX543, and HX543-MMAE all showed significant internalization by HuT-102 cells, confirming that the conjugation had little impact on endocytosis kinetics of the naked antibodies.

[00544] In vitro cell killing:

[00545] HuT-102 cells were plated at 1x103 cells / well. 100 pL antibody or ADC was added into the cells and incubated at 37°C for 6 days. 50 pl CellCounting-Lite2.0 reagent was in each well. Contents were mixed for 3 minutes on an orbital shaker to induce cell lysis. The plate was incubated at room temperature for 10 minutes to stabilize luminescent signal. Luminescence was recorded on the 2104 EnVision plate reader.

[00546] Results: As shown in FIG.16, ADCs (HX111, HX518-MMAE, HX523-1-MMAE, HX534-MMAE, and HX543-MMAE) showed significant killing activity, whereas the corresponding antibodies did not.

[00547] HX01 l-SN-38, HX011-Exd, and HX011-Eriblin are also expected to exhibit efficient killing activity when subjected to same study. 6.10.6 Example 6: Treatment of GvHD by HX011

[00548] According to the experimental procedures and evaluation methods described in Example 3, mice were treated with either vehicle or HX011 at 3 mg / kg twice per week. Human CD45+ leukocytes and human CD4+ T cells were analyzed by flow cytometry.

[00549] Results: As shown, HX011 treatment significantly alleviated the body weight loss (FIG.17A) and diminished disease activities (FIG. 17B) compared to the vehicle control, indicating that HX011 effectively prevented the disease progression of GvHD. Consistently, treatment with HX011 significantly prolonged the survival of mice (FIG. 17C). In addition, treatment with HX011 significantly decreased the numbers of human CD45+ leukocytes (FIG.18A) and pathogenic human CD4+ T cells (FIG. 18B). Taken together, HX011 demonstrated great therapeutic potential for autoimmune diseases such as acute GvHD. * * *

[00550] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[00551] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. 6.11 References

[00552] The following publications are hereby incorporated by reference in their entireties: 1. Ah et al. (2012). PLoS One 7, e44219. 2.      Aranda et al. (2014). Oncoimmunology 3, e27297. 3. Aspeslagh et al. (2016). Eur J Cancer 52, 50-66. 4. Bansal-Pakala et al. (2004). J Immunol 172, 4821-4825. 5. Bordeau et al. (2023). Mol Cancer Ther 22, 459-470. 6. Buchan et al. (2018). Blood 131, 39-48. 7. Chen et al (2019) in Moll and Sebastian (eds.), Target Identification and Validation in Drug Discovery: Methods and Protocols, Methods in Molecular Biology, vol. 1953, Humana Press, New York, NY. 8.      Croft. (2010). Annu Rev Immunol 28, 57-78. 9.      Damayanti et al. (2010). Am JRespir Crit Care Med 181, 688-698. 10. Du et al. (2018). Cell Res. 28, 416-432. 11. Feuer et al. (1996). J. Virol. 70, 4038-4044. 12. Fu et al. (2020). Acta Pharm Sin B 10, 414-433. 13. Griffiths et al. (2020). JImmunother Cancer 8. 14. Gutierrez et al. (2021). Clin Cancer Res. 27, 460-472. 15. Guttman-Yassky et al. (2023). Lancet 401, 204-214. 16. Hassan (2023). LeukRes 131, 107078. 17. Houot and Levy (2009). Blood 113, 3546-3552. 18. Imura et al. (1997). Blood 89, 2951-2958. 19. Ishii et al. (2010). Adv Immunol 105, 63-98. 20. Jin et al. (2022). Sci Rep 12, 3278. 21. Kawano et al. (2005). Leukemia 19, 1384-90. 22. Lahnif et al. (2023). Int J Mol Sci, 24(10), 8543. 23. Lathrop et al. (2004). J Immunol 172, 6735-6743. 24. Lu et al. (2023). Clin Immunol 251, 109637. 25. Lyon et al. (2023). Mol Cancer Ther. OF1-OF10. 26. Maecker et al. (2023). MAbs,15 (1), 2229101. 27. Mao et al. (2023). Eur J Med Chern 257, 115489. 28. Marabelle et al. (2013). J Clin Invest 123, 2447-2463. 29. Matsumura et al. (1997) Arch Dermatol Res 289, 653-656. 30. McKertish and Kayser (2023). Pharmaceutics 15(8). 31. Meric-Bemstam et al. (2023). Mol Cancer Ther. 22(10), 1191-1203.. 32. Montero et al. (2023). J Exp Clin Cancer Res 42(1), 200. 33. Nawaf et al. (2017). J Immunol 199, 974-981. 34. Nuebling et al. (2018). Cancer Immunol Res 6, 209-221. 35. Pakalac / al. (2004). Eur J Immunol 34, 3039-3046. 10.1002 / eji.200425141. 36. Pala et al. (2023). J Allergy Clin Immunol. 37. Parameswaran etal. (2023). Leukemia. 37(10), 2050-2057. 38. Rothfelder et al. (2018). Neoplasia 20, 1150-60. 39. Schroeder and DiPersio (2011). Dis Model Meeh 4, 318-333. 40. Selby et al. (2013). Cancer Immunol Res 1, 32-42. 41. Shimizu et al. (2023). J Clin Oncol 41(29), 4678. 42. Si etal. (2021). Vaccines 9(8), 882. 43. Sonntag et al. (2015). JAutoimmun 62, 55-66. 44. Stewart etal. (1996). Virology 226, 167-175. 45. Sugamura et al. (2004). Nat Rev Immunol 4, 420-431. 10.1038 / nril371. 46. Streeter etal. (2004). Biol Blood Marrow Transplant 10, 298-309. 47. Valzasinaeta / . (2005) Blood 105, 2845-2851. 48.     Weinberg et al. (2004). JLeukoc Biol 75, 962-972. 49.     Yanagida et al. (2020). Hematol Oncol 38, 680-688. 50. Yang et al. (2013). Int J Cancer 132, E74-84. 51. Ye et al. (2020). FASEBJ3A, 12963-12975. 10.1096 / fj.202001203R. 52. Zambraeta / . (2023). Front Pharmacol 14, 1215694. 53. Zhang et al. (2021). Sci TranslMed 13(605), eabg8693.

[00553] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human 0X40, comprising:(a) a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 15; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or(b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO: 16; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs.

2. The antibody or antigen-binding fragment of claim 1, wherein:(a) the VL CDR1, VL CDR2, VL CDR3 have the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or(b) the VH CDR1, VH CDR2, VH CDR3 have the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs.

3. The antibody or antigen-binding fragment of claim 1, comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NOs:4, 5, 6, 10, 11 and 12, respectively.

4. The antibody or antigen-binding fragment of any one of claims 1 to 3 that is humanized.

5. The humanized antibody or antigen-binding fragment of claim 4, comprising:(a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100% sequence identity to the amino acid sequence of SEQ ID NO: 15; and / or(b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16.

6. The humanized antibody or antigen-binding fragment of claim 5 comprising a VL and a VH having the amino acid sequences of SEQ ID NOs: 15 and 16, respectively.

7. The antibody or antigen-binding fragment of any one of claims 1 to 6 that is a monoclonal antibody or antigen-binding fragment.

8. The antibody or antigen-binding fragment of any one of claims 1 to 7 that is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, a (scFv)2, a single domain antibody (sdAb), and a heavy chain antibody (HCAb).

9. The antibody or antigen-binding fragment of any one of claims 1 to 7 that is an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

10. The antibody or antigen-binding fragment of any one of claims 1 to 9, further comprising: a light chain constant (CL) region and a heavy chain constant (CH) region.

11. The antibody or antigen-binding fragment of claim 10, wherein the CL region is kappa CL (Ck; SEQ ID NO: 19) or lambda CL (CL; SEQ ID NO:20), or a variant thereof having up to ten amino acids substitutions.

12. The antibody or antigen-binding fragment of claim 10 or 11, wherein the CH region is IgGl CH (SEQ ID NO:21), IgG2 CH (SEQ ID NO:22), IgG3 CH (SEQ ID NO:23), or IgG4 CH (SEQ ID NO:24), or a variant of any of the above having up to ten amino acids substitutions.

13. The antibody of claim 9 that is an IgGl antibody.

14. The antibody of claim 13 comprising a light chain having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:27; and a heavy chain having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29.

15. The antibody of any one of claims 1 to 14, wherein the antibody (1) does not block the OX40L binding to OX40; (2) exhibits efficient internalization by OX40 positive cells; (3) depletes OX40 positive cells; (4) depletes OX40 positive regulatory T-cells within TME; (5) exhibits significant ADCC; (6) enhances the anti-tumor immune response; (7) reduces the immunosuppression of TME; or (8) enhances immunological activities in TME; or any combination of (1) to (8).

16. A polynucleotide encoding a polypeptide of the antibody or antigen-binding fragment of any one of claims 1 to 15.

17. A vector comprising the polynucleotide of claim 16.

18. A host cell comprising the polynucleotide of claim 16, or the vector of claim 17.

19. A method of producing an antibody or antigen-binding fragment thereof that specifically binds human 0X40, comprising culturing the cell of claim 18 under conditions suitable for expression of the antibody or antigen-binding fragment.

20. The method of claim 19 that comprises isolating the antibody or antigen-binding fragment from the culture.

21. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 15, and a pharmaceutically acceptable carrier.

22. A method of 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 to 15.

23. The method of claim 22, wherein the subject is a human.

24. Use of the antibody or antigen-binding fragment of any one of claims 1 to 15 in treating cancer.

25. Use of the antibody or antigen-binding fragment of any one of claims 1 to 15 for the preparation of a medicament for treating cancer.

26. The method or use of any one of claims 22 to 25, wherein the cancer has 0X40+ tumor infiltrating lymphocytes (TILs).

27. The method or use of any one of claims 22 to 26, wherein the cancer is an 0X40+ cancer.

28. The method or use of any one of claims 22 to 27, wherein the cancer is a liquid cancer.

29. The method or use of claim 28, wherein the cancer is T cell leukemia or lymphoma.

30. The method or use of claim 29, wherein the cancer is Peripheral T-cell Lymphoma (PTCL,e.g, adult T-cell leukemia (ATL), angioimmunoblastic T-cell lymphoma (AITL) or T cell acute lymphoblastic leukemia (T-ALL).

31. The method or use of any one of claims 22 to 27, wherein the cancer is a solid tumor.

32. The method or use of claim 31, wherein the cancer is sarcoma, breast cancer, non-small celllung cancer (NSCLC), melanoma, or ovarian cancer.

33. The method or use of any one of claims 22 to 27, wherein the cancer is an HPV+ cancer or anEBV+ cancer.

34. The method of claim 33, wherein the cancer is EBV+ B cell lymphoma, Extra-nodal NK / T cell lymphoma (ENKTL; e.g., nasal type), HPV16+ head and neck squamous cell carcinoma (HNSCC), EBV+ Esophageal cancer, or HPV18+ cervical cancer.

35. A method of treating an autoimmune disease or an inflammatory disease 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 to 15.

36. The method of claim 35, wherein the subject is a human.

37. Use of the antibody or antigen-binding fragment of any one of claims 1 to 15 in treating an autoimmune disease or an inflammatory disease.

38. Use of the antibody or antigen-binding fragment of any one of claims 1 to 15 for the preparation of a medicament for treating an inflammatory disease or an inflammatory disease.

39. The method of use of any one of claims 35 to 38, wherein the inflammatory disease or an inflammatory disease is characterized by excessive T cell activation.

40. The method of use of claim 39, wherein the autoimmune disease or inflammatory disease is rheumatoid arthritis (RA), inflammation bowl diseases (IBD), Crohn's disease (CD), graft-vs-host diseases (GvHD), multiple sclerosis (MS), type I diabetes, atopic dermatitis, asthma, psoriasis, Sjogren's syndrome (SS), or idiopathic thrombocytopenia (ITP).

41. The method of any one of claims 22 to 40, wherein the antibody or antigen-binding fragment is administered in combination with an additional therapy.

42. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 15.

43. An antibody-drug conjugate (“ADC”) comprising an antibody or antigen-binding fragment thereof conjugated to a therapeutic moiety, wherein the antibody or antigen-binding fragment specifically binds human 0X40 and comprises:(a) a VL comprising VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50 or SEQ ID NO:52; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or(b) a VH comprising VH CDR1, VH CDR2, and VH CDR3 from a VH having the aminoacid sequence of SEQ ID NO:39, SEQ ID N0:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID N0:51, SEQ ID NO:53, or SEQ ID NO:56; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs.

44. The ADC of claim 43, wherein the VL and VH have amino acid sequences that are independently at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to (1) SEQ ID NOs:38 and 39, respectively; (2) SEQ ID NOs:40 and 41, respectively; (3) SEQ ID NOs:42 and 43, respectively; (4) SEQ ID NOs:44 and 45, respectively; (5) SEQ ID NOs:46 and 47, respectively; (6) SEQ ID NOs:50 and 51, respectively; (7) SEQ ID NOs:52 and 53, respectively; or (8) SEQ ID NOs:38 and 56, respectively.

45. The ADC of claim 43 or 44, wherein the antibody comprises a light chain and a heavy chain having amino acid sequences that are independently at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to (1) SEQ ID NOs:59 and 60, respectively; (2) SEQ ID NOs:59 and 61, respectively; (3) SEQ ID NOs:62 and 63, respectively; (4) SEQ ID NOs:62 and 64, respectively; (5) SEQ ID NOs:65 and 66, respectively; or (6) SEQ ID NOs:65 and 67, respectively.

46. An ADC comprising an antibody or antigen-binding fragment thereof conjugated to a therapeutic moiety, wherein the antibody or antigen-binding fragment specifically binds human OX40 and does not block OX40L binding to OX40.

47. The ADC of claim 46, comprising the antibody or antigen-binding fragment of any one of claims 1 to 15.

48. The ADC of any one of claims 43 to 47, wherein the antibody or antigen-binding fragment isconjugated to the therapeutic moiety via a linker.49.The ADC of claim 48, wherein the linker isOH51. The ADC of any one of claims 43 to 50, wherein the therapeutic moiety is monomethyl auristatin.

52. The ADC of claim 51, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE).

53. The ADC of claim 51, having the structure:Ab is the antibody or antigen-binding fragment, and p denotes a number ranging from 1to 16.

54. The ADC of claim 53, wherein p is number from 1 to 10.

55. The ADC of claim 53, wherein p is about 4.

56. The ADC of claim 53, wherein the average value of p in a population of the ADC is about2.8, about 3.6, about 4, about 4.2, about 4.3, or about 5.

57. The ADC of any one of claims 53 to 56, having the structure:

58. The ADC of claim 51, wherein the monomethyl auristatin is monomethyl auristatin F (MMAF).

59. The ADC of claim 51, having the structure:Ab is the antibody or antigen-binding fragment, and p denotes a number from 1 to 16.

60. The ADC of claim 59, wherein p is from 1 to 10.

61. The ADC of claim 59, wherein p is about 4.

62. The ADC of claim 59, wherein the average value of p in a population of the ADC is about 2.8, about 3.6, about 4, about 4.2, about 4.3, or about 5.

63. The ADC of any one of claims 59 to 62, having the structure:

64. The ADC of any one of claims 43 to 50, wherein the therapeutic moiety is a topoisomerase I inhibitor.

65. The ADC of claim 64, wherein the topoisomerase I inhibitor is camptothecin, Dxd, SN-38 or exatecan (DX-8951).

66. The ADC of claim 64, wherein the topoisomerase I inhibitor is Dxd.

67. The ADC of claim 64, having the structure:is the antibody or antigen-binding fragment, and p denotes a number from 1 to 16.

68. The ADC of claim 67, wherein p denotes a number from 1 to 10.

69. The ADC of claim 67, wherein p is about 8.

70. The ADC of claim 67, wherein the average value of p in a population of the ADC is about5.3 or about 8.

71. The ADC of any one of claims 67 to 70, having the structure:

72. The ADC of claim 64, wherein the topoisomerase I inhibitor is SN-38.

73. The ADC of claim 64, having the structure:Ab is the antibody or antigen-binding fragment, and p denotes a number from 1 to 16.

74. The ADC of claim 73, wherein p denotes a number from 1 to 10.

75. The ADC of claim 73, wherein p is about 4 or 5.

76. The ADC of claim 73, wherein the average value of p in a population of the ADC is about4.5.

77. The ADC of any one of claims 73 to 76, having the structure:

78. The ADC of claim 64, wherein the topoisomerase I inhibitor is exatecan.

79. The ADC of claim 64, having the structure:wherein Ab is the antibody or antigen-binding fragment, and p denotes a number from 1 to 16.

80. The ADC of claim 79, wherein p denotes a number from 1 to 10.

81. The ADC of claim 79, wherein p is about 4 or 8.

82. The ADC of claim 79, wherein the average value of p in a population of the ADC is about4.2 or about 8.

83. The ADC of any one of claims 79 to 82, having the structure:

84. The ADC of any one of claims 43 to 50, wherein the therapeutic moiety is a derivative of halichondrin B.

85. The ADC of claim 84, wherein therapeutic moiety is eribulin.

86. The ADC of claim 84, having the structure:wherein Ab is the antibody or antigen-binding fragment, and p denotes a number ranging from 1 to 16.

87. The ADC of claim 86, wherein p is number from 1 to 10.

88. The ADC of claim 86, wherein p is about 4 or 5.

89. The ADC of claim 86, wherein the average value of p in a population of the ADC is about4.6.

90. The ADC of any one of claims 86 to 89, having the structure:

91. An ADC comprising an antibody or antigen-binding fragment thereof conjugated to a therapeutic moiety; wherein the antibody or antigen-binding fragment specifically binds human 0X40 and has(1) VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 15; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs, and VH CDR1, VH CDR2 and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO: 16; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs;(2) VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO:38; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs, and VH CDR1, VH CDR2 and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO :3 9; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs;(3) VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO:40; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs, and VH CDR1, VH CDR2 and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO:41; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs;(4) VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence ofSEQ ID NO:42; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs, and VH CDR1, VH CDR2 and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO:43; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs; or(5) VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO:38; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs, and VH CDR1, VH CDR2 and VH CDR3 from a VH having the amino acid sequence of SEQ ID NO:56; or a variant thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs;and wherein the therapeutic moiety is MMAE, MMAF, Dxd, SN-38, exatecan, or eribulin.

92. An ADC comprising an antibody or antigen-binding fragment thereof conjugated to a therapeutic moiety; wherein the antibody or antigen-binding fragment specifically binds human 0X40 and has a VL and VH having the amino acid sequences of SEQ ID NOs: 15 and 16, respectively; and wherein the therapeutic moiety is MMAE, MMAF, Dxd, SN-38, exatecan, or eribulin.

93. The ADC of any one of claims 43 to 92, wherein the ADC (1) does not block the OX40L binding to 0X40; (2) exhibits efficient internalization by 0X40 positive cells; (3) depletes 0X40 positive cells; (4) depletes 0X40 positive regulatory T-cells within TME; (5) exhibits significant ADCC; (6) maintains the essential ADCC of the anti-OX40 antibodies or antigenbinding fragments; (7) enhances the anti-tumor immune response; (8) reduces the immunosuppression of TME; or (9) enhances immunological activities in TME; or any combination of (1) to (9).

94. A method of producing the ADC of any one of claims 43 to 93 comprising conjugating the antibody or antigen-binding fragment to the therapeutic moiety.

95. A pharmaceutical composition comprising the ADC of any one of claims 43 to 93, and a pharmaceutically acceptable carrier.

96. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the ADC of any one of claims 43 to 93.

97. The method of claim 96, wherein the subject is a human.

98. Use of the ADC of any one of claims 43 to 93 in treating cancer.

99. Use of the ADC of any one of claims 43 to 93 for the preparation of a medicament fortreating cancer.

100. The method or use of any one of claims 96 to 99, wherein the cancer is an 0X40+ cancer.

101. The method or use of any one of claims 96 to 100, wherein the cancer is a liquid cancer.

102. The method or use of claim 101, wherein the cancer is T cell leukemia or lymphoma.

103. The method or use of claim 102, wherein the cancer is Peripheral T-cell Lymphoma (PTCL,e.g, adult T-cell leukemia (ATL), angioimmunoblastic T-cell lymphoma (AITL)), or T cell acute lymphoblastic leukemia (T-ALL).

104. The method or use of any one of claims 96 to 100, wherein the cancer is a solid tumor.

105. The method or use of claim 104, wherein the cancer is sarcoma, breast cancer, non-small celllung cancer (NSCLC), melanoma, or ovarian cancer.

106. The method or use of any one of claims 96 to 100, wherein the cancer is an HPV+ cancer or an EBV+ cancer.

107. The method of claim 106, wherein the cancer is EBV+ B cell lymphoma, Extra-nodal NK / Tcell lymphoma (ENKTL; e.g., nasal type), HPV16+ head and neck squamous cell carcinoma (HNSCC), EBV+ Esophageal cancer, or HPV18+ cervical cancer.

108. A method of treating an autoimmune disease or an inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the ADC of any one of claims 43 to 93.

109. The method of claim 108, wherein the subj ect is a human.

110. Use of the ADC of any one of claims 43 to 93 in treating an inflammatory disease or aninflammatory disease.

111. Use of the ADC of any one of claims 43 to 93 for the preparation of a medicament fortreating an inflammatory disease or an inflammatory disease.

112. The method or use of any one of claims 108 to 111, wherein the autoimmune disease or inflammatory disease is rheumatoid arthritis (RA), inflammation bowl diseases (IBD), Crohn's disease (CD), graft-vs-host diseases (GvHD), multiple sclerosis (MS), type I diabetes, atopic dermatitis, asthma, psoriasis, Sjogren's syndrome (SS), or idiopathicthrombocytopenia (ITP).

113. The method or use of any one of claims 96 to 112, wherein the ADC is administered in combination with an additional therapy.

114. A kit comprising the ADC of any one of claims 43 to 93.

Images