Anti-OX40 antibodies and their uses

Abstract

This invention relates to novel anti-OX40 antibodies, compositions comprising said anti-OX40 antibodies, nucleic acids encoding said anti-OX40 antibodies, methods for preparing said anti-OX40 antibodies, and uses of said anti-OX40 antibodies.

Description

Technical Field

[0001] This invention relates to novel anti-OX40 antibodies, compositions comprising said anti-OX40 antibodies, nucleic acids encoding said anti-OX40 antibodies, methods for preparing said anti-OX40 antibodies, and uses of said anti-OX40 antibodies. Background Technology

[0002] Antitumor immune responses in patients with solid tumors have been enhanced through treatment with biologics. For example, two anti-PD-1 monoclonal antibodies: nivolumab. And pembrolizumab It has been approved in the US and EU for the treatment of diseases such as unresectable or metastatic melanoma and metastatic non-small cell lung cancer. Patients treated with these drugs have produced antitumor responses as measured by improvements in progression-free survival and / or overall survival. More cancer treatment products and approaches are still needed in this field to complement existing standard of care.

[0003] PD-1 and CTLA-4 exert immunosuppressive effects during T cell activation, thereby inhibiting the T cell's immune killing function against tumor cells. Therefore, blocking monoclonal antibodies targeting these two molecules can relieve this immunosuppression and restore the T cell's anti-tumor immune function. In addition to such inhibitory immune checkpoint molecules, activating immune checkpoint molecules are gradually becoming new targets for drug development.

[0004] Activating immune checkpoint molecules mainly refer to co-stimulatory signaling molecules that activate T cells—T cell co-stimulatory receptors, which belong to the tumor necrosis factor receptor (TNFR) family and are used to regulate the proliferation, activation, and differentiation of T cells. These include OX40, CD40, 4-1BB, and GITR.

[0005] The OX40 receptor, also known as CD134 and TNFRSF4 (tumor necrosis factor receptor superfamily member 4), is a member of the TNFR superfamily of receptors. Unlike CD28, it is not constitutively expressed on resting naive T cells. OX40 is a secondary co-stimulatory immune checkpoint molecule, expressed 24 to 72 hours after activation; its ligand OX40L (also known as CD252 and TNFSF4) is also not expressed on resting antigen-presenting cells, but rather after their activation. OX40 expression depends on complete T cell activation.

[0006] OX40 binds to its ligand OX40L to transmit co-stimulatory signals. The interaction between OX40 and OX40L recruits TNFR-associated (TRAFs) molecules within the intracellular region of OX40, forming a signaling complex containing IKKα and IKKβ, as well as PI3k and PKB (Akt). OX40 also synergizes with TCR signaling to enhance intracellular calcium absorption through an unknown mechanism. 2+ This enhances NFAT nuclear translocation. OX40 can activate the classical NF-κB1 pathway or the non-classical NF-κB2 pathway, PI3k / PKB, and NFAT pathway, thereby regulating genes involved in T cell division and survival, and promoting the transcription of cytokine genes and the expression of cytokine receptors, which are crucial for cell survival. OX40 signaling induces the downregulation of CTLA-4 and Foxp3.

[0007] When OX40 binds to its ligand OX40L, it helps enhance the immune system's response by: 1. increasing the survival and proliferation of effector T cells and memory T cells, and increasing the secretion of cytokines (such as IL-2, IL-4, IL-5, and IFN-γ); 2. reducing the immunosuppressive activity of regulatory T cells, further amplifying the T cell activation effect. In the tumor microenvironment, immune activation can lead to OX40 expression. This can enhance the activation and proliferation of effector T cells and inhibit regulatory T cells, thereby leading to a complex anti-tumor immune response. Several clinical projects using anti-OX40 antibodies to treat cancer can currently be found on the Clinical Trials website.

[0008] There is a need in this field for more novel anti-OX40 antibodies to provide new cancer treatment options. Summary of the Invention

[0009] The present invention satisfies the above-mentioned needs by providing a novel anti-OX40 antibody that specifically binds to and activates OX40.

[0010] In one aspect, the present invention provides an isolated anti-OX40 antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region having a heavy chain CDR1 domain shown in SEQ ID NO:1, a heavy chain CDR2 domain shown in SEQ ID NO:2, and a heavy chain CDR3 domain shown in SEQ ID NO:3; and a light chain variable region having a light chain CDR1 domain shown in SEQ ID NO:9, a light chain CDR2 domain shown in SEQ ID NO:10, and a light chain CDR3 domain shown in SEQ ID NO:11.

[0011] In one aspect, the present invention provides an antibody-drug conjugate comprising the OX40 antibody or its antigen-binding fragment described herein and an additional therapeutic agent; preferably, the anti-OX40 antibody or its antigen-binding fragment is linked to the additional therapeutic agent via a connector.

[0012] In one aspect, the present invention provides a nucleic acid encoding the anti-OX40 antibody described herein or an antigen-binding fragment thereof.

[0013] In one aspect, the present invention provides an expression vector comprising the nucleic acid described herein.

[0014] In one aspect, the present invention provides a host cell comprising the nucleic acid or expression vector described herein.

[0015] In one aspect, the present invention provides a method for generating the anti-OX40 antibody or an antigen-binding fragment thereof as described herein, comprising culturing the host cells described herein under conditions suitable for expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the culture medium.

[0016] In one aspect, the present invention provides a pharmaceutical composition comprising the anti-OX40 antibody described herein or an antigen-binding fragment thereof, or the antibody-drug conjugate described herein, or the nucleic acid described herein, or the expression vector described herein, and a pharmaceutically acceptable vector.

[0017] In one aspect, the present invention provides the anti-OX40 antibody described herein or an antigen-binding fragment thereof, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, for the treatment of cancer.

[0018] In one aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need a therapeutically effective amount of the anti-OX40 antibody described herein or an antigen-binding fragment thereof, or an antibody-drug conjugate described herein, or a pharmaceutical composition described herein, thereby treating the cancer.

[0019] In one aspect, the present invention provides the use of the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, in the preparation of a medicament for treating cancer.

[0020] In one aspect, the present invention provides the anti-OX40 antibody described herein or an antigen-binding fragment thereof, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, for use in one or more of the following: inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), killing cells expressing OX40 (e.g., cells expressing high levels of OX40), enhancing effector T cell function and / or enhancing memory T cell function, reducing tumor immunity, enhancing T cell function, and / or reducing cells expressing OX40.

[0021] In one aspect, the present invention provides the use of the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, in the preparation of a medicament for treating one or more of the following: inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), killing cells expressing OX40 (e.g., cells expressing high levels of OX40), enhancing effector T cell function and / or enhancing memory T cell function, reducing tumor immunity, enhancing T cell function, and / or reducing cells expressing OX40.

[0022] In one aspect, the present invention provides a pharmaceutical combination comprising the anti-OX40 antibody described herein or an antigen-binding fragment thereof, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0023] In one aspect, the present invention provides a kit comprising the anti-OX40 antibody or its antigen-binding fragment as described herein, or the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, preferably further comprising a delivery device.

[0024] In some embodiments, the antibodies of the present invention comprise one or more point mutations in their amino acid sequence, said point mutations being designed to improve the developability of the antibody. In a preferred embodiment, said one or more point mutations make the antibody more stable during expression in host cells, purification during preparation and / or formulation, and / or administration to a subject patient. In a preferred embodiment, said one or more point mutations make the antibody less likely to aggregate during preparation and / or formulation. In some embodiments, the present invention provides therapeutic antibodies with minimized or reduced developability problems, for example by removing or reducing hydrophobicity and / or optimizing charge by replacing one or more amino acids in their sequence (e.g., in one or more of their CDRs). Attached Figure Description

[0025] Figure 1-1 The binding of HFB10-1E1hG1 to the OX40 protein. The EC80 of HFB10-1E1hG1 is 0.71 nM.

[0026] Figure 1-2 HFB10-1E1hG1 binds to human OX40 protein expressed on the surface of 293T cells. EC50 is 2 nM (MFI).

[0027] Figure 1-3 HFB10-1E1hG1 binds to cynomolgus monkey OX40 protein expressed on the surface of 293T cells. The EC50 is 2.9 nM (MFI).

[0028] Figure 1-4 HFB10-1E1hG1 binds to mouse OX40 protein expressed on the surface of 293T cells. HFB10-1E1hG1 does not bind to mouse OX40 protein expressed on the surface of 293T cells (MFI).

[0029] Figure 1-5 HFB10-1E1hG1 binds to human CD40 protein expressed on the surface of 293T cells. HFB10-1E1hG1 does not bind to human CD40 protein expressed on the surface of 293T cells (MFI).

[0030] Figure 1-6 HFB10-1E1hG1, Reference 1, Reference 2, Reference 3, and Reference 4 bound to human OX40 protein expressed on the surface of 293T cells. The EC50 of HFB10-1E1hG1 was 2.02 nM (MFI); the EC50 of Reference 1 was 2.67 nM (MFI); the EC50 of Reference 2 was 4.17 nM (MFI); the EC50 of Reference 3 was 1.92 nM (MFI); and the EC50 of Reference 4 was 2.11 nM (MFI).

[0031] Figure 1-7 HFB10-1E1hG1, Reference 1, Reference 2, Reference 3, and Reference 4 bound to the cynomolgus monkey OX40 protein expressed on the surface of 293T cells. The EC50 of HFB10-1E1hG1 was 2.94 nM (MFI); the EC50 of Reference 1 was 2.91 nM (MFI); the EC50 of Reference 2 was 6.13 nM (MFI); the EC50 of Reference 3 was 2.57 nM (MFI); and the EC50 of Reference 4 was 3.54 nM (MFI).

[0032] Figure 2-1 Activatory activity of HFB10-1E1hG1 in Jurkat reporter cells. The EC50 of HFB10-1E1hG1 is 2.9 nM (MFI of GFP) when cross-linked with anti-human IgG.

[0033] Figure 2-2Activatory activity of HFB10-1E1hG1 in Jurkat reporter cells. The EC50 of HFB10-1E1hG1 without cross-linking with anti-human IgG is significantly greater than that with cross-linking with anti-human IgG. Without cross-linking with anti-human IgG, the trimeric OX40L recombinant protein alone can activate NF-κB signaling with an EC50 of 45 nM (MFI of GFP).

[0034] Figure 2-3 Activatory activity of HFB10-1E1hG1 in Jurkat reporter cells. In the presence of anti-human IgG crosslinking, HFB10-1E1hG1 exhibits a cooperative activating effect with OX40L, and the addition of HFB10-1E1hG1 together with OX40L enhances the MFI of GFP compared to the individual components.

[0035] Figure 2-4 The agonistic activity of HFB10-1E1hG1 in primary CD4+ T cells was measured by IL-2 secretion, and the EC50 was 0.2 nM.

[0036] Figure 2-5 Activatory activity of HFB10-1E1hG1 in primary CD4+ T cells. In primary CD4+ T cells, HFB10-1E1hG1 exhibits a cooperative activating effect with OX40L.

[0037] Figure 3-1 Pharmacokinetic analysis of HFB10-1E1hG1.

[0038] Figure 4-1 In vivo antitumor efficacy of HFB10-1E1hG1. HFB10-1E1hG1 significantly inhibited tumor growth compared to the PBS control.

[0039] Figure 4-2 In vivo antitumor efficacy of HFB10-1E1hG1. HFB10-1E1hG1 did not show any side effect of causing significant weight loss in mice.

[0040] Figure 4-3 In vivo antitumor efficacy (tumor size) of HFB10-1E1hG1 at different doses.

[0041] Figure 4-4 In vivo antitumor efficacy (body weight) of HFB10-1E1hG1 at different doses.

[0042] Figure 5-1Accelerated stability experiments of HFB10-1E1hG1 were conducted. SDS-PAGE results showed that HFB10-1E1hG1 exhibits good stability under different temperature conditions.

[0043] Figure 5-2 Degradation experiments of HFB10-1E1hG1 were conducted. SDS-PAGE results showed that HFB10-1E1hG1 exhibited good stability under different pH conditions.

[0044] Figure 5-3 Oxidative stress experiments of HFB10-1E1hG1. SDS-PAGE results showed that HFB10-1E1hG1 exhibited good stability under oxidative stress conditions.

[0045] Figure 5-4 Freeze-thaw experiment of HFB10-1E1hG1. SDS-PAGE results show that HFB10-1E1hG1 has good stability under freeze-thaw conditions. Detailed Implementation

[0046] In one aspect, the present invention provides an isolated anti-OX40 antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region having a heavy chain CDR1 domain shown in SEQ ID NO:1, a heavy chain CDR2 domain shown in SEQ ID NO:2, and a heavy chain CDR3 domain shown in SEQ ID NO:3; and a light chain variable region having a light chain CDR1 domain shown in SEQ ID NO:9, a light chain CDR2 domain shown in SEQ ID NO:10, and a light chain CDR3 domain shown in SEQ ID NO:11.

[0047] In one embodiment, the anti-OX40 antibody or its antigen-binding fragment described herein comprises the heavy chain variable region shown in SEQ ID NO:4, or a heavy chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:4; and the light chain variable region shown in SEQ ID NO:12, or a light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:12.

[0048] In one embodiment, the anti-OX40 antibody or its antigen-binding fragment described herein further comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is the heavy chain constant region shown in SEQ ID NO:5, or a heavy chain constant region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:5; and / or preferably, the light chain constant region is the light chain constant region shown in SEQ ID NO:13, or a heavy chain constant region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 87%, 88%, 89%, or 100% identity with the sequence shown in SEQ ID NO:5; and / or preferably, the light chain constant region is the light chain constant region shown in SEQ ID NO:13, or a light chain constant region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 85%, 86%, 87%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:5; The sequence shown in NO:13 has a light chain constant region with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0049] In one embodiment, the anti-OX40 antibody or its antigen-binding fragment described herein further comprises a heavy chain signal peptide linked to the heavy chain variable region and / or a light chain signal peptide linked to the light chain variable region; preferably, the heavy chain signal peptide is the heavy chain signal peptide shown in SEQ ID NO:6, or a heavy chain signal peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:6; and / or preferably, the light chain signal peptide is the light chain signal peptide shown in SEQ ID NO:14, or a heavy chain signal peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 87%, 88%, 89%, or 100% identity with the sequence shown in SEQ ID NO:6; and / or preferably, the light chain signal peptide is the light chain signal peptide shown in SEQ ID NO:14, or a heavy chain signal peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 85%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:6; The light chain signal peptide shown in NO:14 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence.

[0050] In one embodiment, the anti-OX40 antibody or its antigen-binding fragment described herein is an IgG antibody or its antigen-binding fragment, preferably an IgG1 antibody or its antigen-binding fragment.

[0051] In one embodiment, the anti-OX40 antibody or its antigen-binding fragment described herein is a monoclonal antibody or its antigen-binding fragment.

[0052] In one embodiment, the anti-OX40 antigen binding fragment described herein is Fab, Fab', F(ab')2, Fv, scFv, or sdAb.

[0053] In one aspect, the present invention provides an antibody-drug conjugate comprising an anti-OX40 antibody or an antigen-binding fragment thereof as described herein and a further therapeutic agent; preferably, the anti-OX40 antibody or the antigen-binding fragment thereof is linked to the further therapeutic agent via a connector.

[0054] In one aspect, the present invention provides a nucleic acid encoding the anti-OX40 antibody described herein or an antigen-binding fragment thereof.

[0055] In one embodiment, the nucleic acid described herein comprises the heavy chain variable region nucleotide coding sequence shown in SEQ ID NO:20 and / or the light chain variable region nucleotide coding sequence shown in SEQ ID NO:28; preferably, the nucleic acid further comprises the heavy chain constant region nucleotide coding sequence shown in SEQ ID NO:21 and / or the light chain constant region nucleotide coding sequence shown in SEQ ID NO:29.

[0056] In one aspect, the present invention provides an expression vector comprising the nucleic acid described herein.

[0057] In one aspect, the present invention provides a host cell comprising the nucleic acid or expression vector described herein.

[0058] In one aspect, this document provides a method for generating the anti-OX40 antibody or an antigen-binding fragment thereof as described herein, comprising culturing the host cells described herein under conditions suitable for expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the culture medium.

[0059] In one aspect, the present invention provides a pharmaceutical composition comprising the anti-OX40 antibody described herein or an antigen-binding fragment thereof, or the antibody-drug conjugate described herein, or the nucleic acid described herein, or the expression vector described herein, and a pharmaceutically acceptable vector.

[0060] In one embodiment, the anti-OX40 antibody or its antigen-binding fragment described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, is used to treat cancer. In one embodiment, the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal carcinoma), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, etc. Melanoma, superficial diffuse melanoma, malignant lentigines melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with scarring nevi, edema (e.g., associated with brain tumors) and Meigs syndrome, brain tumors and brain cancers, and head and neck cancers, and related metastases.

[0061] In one aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need a therapeutically effective amount of the anti-OX40 antibody or its antigen-binding fragment described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, thereby treating the cancer. In one embodiment, the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal carcinoma), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, etc. Melanoma, superficial diffuse melanoma, malignant lentigines melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with scarring nevi, edema (e.g., associated with brain tumors) and Meigs syndrome, brain tumors and brain cancers, and head and neck cancers, and related metastases.

[0062] In one aspect, the present invention provides the use of the anti-OX40 antibody or its antigen-binding fragment described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, in the preparation of a medicament for treating cancer. In one embodiment, the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal carcinoma), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, etc. Melanoma, superficial diffuse melanoma, malignant lentigines melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with scarring nevi, edema (e.g., associated with brain tumors) and Meigs syndrome, brain tumors and brain cancers, and head and neck cancers, and related metastases.

[0063] In one aspect, this document provides the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein for use in one or more of the following: inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), killing cells expressing OX40 (e.g., cells expressing high levels of OX40), enhancing effector T cell function and / or enhancing memory T cell function, reducing tumor immunity, enhancing T cell function, and / or reducing cells expressing OX40.

[0064] In one aspect, this document provides the use of the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein in the preparation of a medicament for treating one or more of the following: inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), killing cells expressing OX40 (e.g., cells expressing high levels of OX40), enhancing effector T cell function and / or enhancing memory T cell function, reducing tumor immunity, enhancing T cell function, and / or reducing cells expressing OX40.

[0065] In one aspect, the present invention provides a pharmaceutical combination comprising the anti-OX40 antibody described herein or an antigen-binding fragment thereof, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0066] In one aspect, the present invention provides a kit comprising the anti-OX40 antibody or its antigen-binding fragment as described herein, or the antibody-drug conjugate as described herein, or the pharmaceutical composition as described herein, preferably further comprising a delivery device.

[0067] Some embodiments of the present invention provide agonistic antibodies against OX-40 that, compared to other agonistic anti-OX-40 antibodies, do not cause downregulation of the OX-40 receptor or cause less downregulation of the OX-40 receptor. This lack of receptor downregulation or reduced receptor downregulation may be due to epitopes recognized by the agonistic antibodies of the present invention. The agonistic antibodies of the present invention may also have optimized binding kinetics, particularly compared to other agonistic anti-OX-40 antibodies known in the art.

[0068] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the invention are further described in detail below.

[0069] Two polynucleotide or polypeptide sequences are said to be “identical” if the sequences of nucleotides or amino acids are identical when describing a maximum enantiomeric alignment. Comparisons between two sequences are typically performed by comparing the sequences on a comparison window to identify and compare local regions of sequence similarity. As used herein, a “comparison window” refers to a segment with at least about 20 (typically 30 to about 75 or 40 to about 50) consecutive positions, where, after optimized alignment, the two sequences can be compared to a reference sequence having the same number of consecutive positions.

[0070] Optimized alignment of sequences for comparison can be performed using a program from the suite of bioinformatics software (Inc., Madison, WI) with default parameters. This program implements several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, MO (ed.), Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Supplement 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645, Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG & Sharp, PM, 1989, CABIOS 5:151-153; Myers, EW & Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971, Comb. Theor. 11: 105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0071] In some implementations, the "percentage of sequence identity" is determined by comparing two optimized alignment sequences over a comparison window with at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less, typically 5% to 15%, or 10% to 12% of additions or deletions (i.e., gaps) compared to a reference sequence for optimized alignment of the two sequences (which does not contain additions or deletions). This percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to generate a number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0072] Alternatively, variants may be substantially homologous to the natural gene or a portion thereof or complement. These polynucleotide variants can hybridize with naturally occurring DNA sequences encoding natural antibodies (or complementary sequences) under moderately stringent conditions.

[0073] Suitable “moderately stringent conditions” include prewashing in a solution of 5X SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); hybridization overnight at 50°C to 65°C with 5X SSC; followed by two washes at 65°C for 20 minutes each, with each wash using 2X, 0.5X, and 0.2X SSC containing 0.1% SDS.

[0074] As used herein, “highly stringent conditions” or “highly stringent conditions” are those that: (1) employ low ionic strength and high temperature for cleaning, for example, at 50°C with 0.015M sodium chloride / 0.0015M sodium citrate / 0.1% sodium dodecyl sulfate; (2) employ denaturing agents during hybridization, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% sucrose / 0.1% polyvinylpyrrolidone / 50mM sodium phosphate buffer at pH 6.5 and 750mM sodium chloride, 75mM sodium citrate at 42°C; or (3) employ 50% formamide, 5X SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X SSC ... Denhardt hybridization solution, acoustically treated salmon sperm DNA (50 μg / mL), 0.1% SDS and 10% dextran sulfate, and washing with 0.2X SSC (sodium chloride / sodium citrate) at 42°C and 50% formamide at 55°C, followed by washing with a high-toughness washing solution of 0.1X SSC containing EDTA at 55°C. Those skilled in the art will appreciate how temperature, ionic strength, etc., can be optionally adjusted to accommodate factors such as probe length and the like.

[0075] Those skilled in the art will recognize that, due to the degeneracy of the genetic codon, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequences of any natural gene. However, the present invention specifically contemplates polynucleotides that are altered due to differences in codon usage. Furthermore, alleles of genes containing the polynucleotide sequences provided herein are within the scope of the invention. An allele is an endogenous gene that is altered by one or more mutations in nucleotides, such as deletion, addition, and / or substitution. The resulting mRNA and protein may (but need not) have the altered structure or function. Alleles can be identified using standard techniques such as hybridization, amplification, and / or database sequence comparison.

[0076] The polynucleotides of this invention can be obtained using chemical synthesis, recombination methods, or PCR. Methods for the chemical synthesis of polynucleotides are well known in the art and do not require detailed description herein. Those skilled in the art can use the sequences provided herein and commercially available DNA synthesis reagents to generate the desired DNA sequences.

[0077] In the preparation of polynucleotides using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and the vector can be further introduced into a suitable host cell for replication and amplification, as discussed further herein. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed to introduce exogenous polynucleotides through direct uptake, endocytosis, transfection, F-hybridization, or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrative vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide thus amplified can be isolated from the host cell using methods well known in the art. See, for example, Sambrook et al., 1989.

[0078] Alternatively, PCR allows the replication of DNA sequences.

[0079] RNA can be obtained by using isolated DNA in a suitable vector and inserting it into a suitable host cell. As the cell replicates and transcribes the DNA into RNA, the RNA can then be isolated using methods known to those skilled in the art.

[0080] Suitable cloning and expression vectors can include various components, such as promoters, enhancers, and other transcriptional regulatory sequences. The vector can also be constructed to allow for the subsequent cloning of antibody variable domains into different vectors.

[0081] Suitable cloning vectors can be constructed using standard techniques or selected from a large number of cloning vectors available in the art. While the chosen cloning vector can vary depending on the intended host cell, useful cloning vectors will generally be self-replicating, may have a single target against a specific restriction endonuclease, and / or carry a gene targeting a marker that can be used to select clones containing that vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors (such as pSA3 and pAT28). These and many other cloning vectors are available from commercial suppliers such as BioRad, Stragene, and Invitrogen.

[0082] Further, expression vectors are provided. Expression vectors are typically reproducible polynucleotide constructs containing the polynucleotides according to the invention. This implies that the expression vector must be reproducible in the host cell, either as a free gene or as a whole portion of chromosomal DNA. Suitable expression vectors include (but are not limited to) plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, myxosomes, and expression vectors disclosed in PCT Publication WO 87 / 04462. Vector components may typically include (but are not limited to) one or more of the following: a signal sequence; an origin of replication; one or more marker genes; and suitable transcriptional control components (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more transcriptional control components, such as ribosome binding sites, translation initiation sites, and stop codons, are also typically required.

[0083] A vector containing the target polynucleotide and / or the polynucleotide itself may be introduced into a host cell by any of a number of suitable methods, including electroporation, transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-glucan, or other substances; microprojectile bombardment; lipid transfection; and infection (e.g., where the vector is an infectious agent, such as poxvirus). The choice of vector or polynucleotide for introduction will generally depend on the characteristics of the host cell.

[0084] The antibodies of the present invention include human antibodies prepared, expressed, generated, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into host cells (further described in Part II below), antibodies isolated from a recombinant human antibody library (further described in Part III below), antibodies isolated from transgenic animals (e.g., mice) containing human immunoglobulin genes (see, for example, Taylor, LD et al. (1992) Nucl. Acids Res. 20: 6287-6295), or antibodies prepared, expressed, generated, or isolated by any other method involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242).

[0085] The antibodies or antibody moieties of the present invention can be prepared by recombinantly expressing immunoglobulin light chain genes and heavy chain genes in host cells. To recombinantly express the antibody, host cells are transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, such that the light and heavy chains are expressed in the host cells, and preferably secreted into the culture medium from which the antibody can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy chain genes and antibody light chain genes, to introduce these genes into recombinant expression vectors, and then to introduce the vectors into host cells. These standard methods are, for example, those described in the following literature: Sambrook, Fritsch, and Maniatis (eds.), *Molecular Cloning; A Laboratory Manual*, 2nd edition, Cold Spring Harbor, NY, (1989); Ausubel, FM et al. (eds.), *Current Protocols in Molecuar Biology*, Greene Publishing Associates, (1989); and U.S. Patent No. 4,816,397 to Boss et al.

[0086] Antibodies or their antigen-binding fragments can be recombinantly manufactured using suitable host cells. The nucleic acid encoding the antibody or its antigen-binding fragment can be cloned into an expression vector, which can then be introduced into host cells such as *E. coli* cells, yeast cells, insect cells, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, wherein the cells do not additionally produce immunoglobulins to obtain antibody synthesis in the recombinant host cells. Among many cells well known in the art, preferred host cells include CHO cells, human embryonic kidney (HEK) 293 cells, or Sp2.0 cells.

[0087] Antibody fragments can be produced by recombinant methods or by chemical synthesis from the protein hydrolysis or other degradation of full-length antibodies. Peptide fragments of antibodies (especially shorter peptides of up to about 50 amino acids) can be conveniently prepared by chemical synthesis. Methods for the chemical synthesis of proteins and peptides are known in the art and are commercially available.

[0088] The antibodies or antigen-binding fragments of the present invention can be affinity-matured. For example, affinity-matured antibodies can be produced by procedures known in the art (Marks et al., 1992, Bio / Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9; Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and WO2004 / 058184).

[0089] Antibody variants

[0090] In some embodiments, amino acid sequence variants of the antibodies provided herein are included. For example, improved binding affinity and / or other biological properties of the antibody may be desired. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0091] In some embodiments, the antibodies of the present invention contain one or more point mutations in their amino acid sequences, said point mutations being designed to improve the developability of the antibody. For example, Raybould et al., “Five computational developability guidelines for therapeutic antibody profiling,” PNAS, March 5, 2019, 116(10)4025-4030, describe their Therapeutic Antibody Analysis Tool (TAP), a computational tool for building downloadable homology models of variable domain sequences, testing them against five developability guidelines, and reporting potential sequence responsibility and canonical forms. The authors further provide TAP, which is freely available at opig.stats.ox.ac.uk / webapps / sabdab-sabpred / TAP.php. Besides achieving the desired affinity for the antigen, the development of therapeutic monoclonal antibodies faces numerous obstacles. These include inherent immunogenicity, chemical and conformational instability, self-association, high viscosity, multispecificity, and poor expression. For example, high levels of hydrophobicity (particularly in highly variable complementarity-determining regions (CDRs)) have repeatedly been implicated in aggregation, viscosity, and multispecificity. Asymmetry in the net charge of the variable heavy and light chain domains is also associated with self-association and viscosity at high concentrations. Positive and negative charge patches in CDRs are associated with high clearance rates and poor expression levels. Product heterogeneity (e.g., through oxidation, isomerization, or glycosylation) is often caused by specific sequence motifs that are prone to post-translational or co-translational modifications. Computational tools can be used to facilitate the identification of sequence responsibility. Warszawski also describes methods for optimizing antibody affinity and stability through the automated design of variable light-heavy chain interfaces. Warszawski et al. (2019) Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces, PLoS Comput Biol 15(8):e1007207, https: / / doi.org / 10.1371 / journal.pcbi.1007207. Other methods can be used to identify potential developmentability issues of candidate antibodies, and in a preferred embodiment of the invention, such issues can be addressed by introducing one or more point mutations into the candidate antibody using conventional methods, thereby obtaining the optimized therapeutic antibody of the present invention.

[0092] a) Substitution, insertion, and deletion of variants

[0093] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Substitutional mutagenesis sites of interest include HVR and FR. Conserved substitutions are shown in Table A under the heading "Preferred Substitutions". More substantial variations are provided in Table A under the heading "Exemplary Substitutions" and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0094] Table A

[0095] initial residues Exemplary substitution Preferred alternatives Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser;Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Leucine Leu Leu(L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Leucine Leu

[0096] Based on their common side-chain characteristics, amino acids can be grouped as follows:

[0097] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, IIe;

[0098] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0099] (3) Acidic: Asp, Glu;

[0100] (4) Alkaline: His, Lys, Arg;

[0101] (5) Residues that affect chain orientation: Gly, Pro;

[0102] (6) Aromatic: Trp, Tyr, Phe.

[0103] Non-conservative substitution would require replacing one of these categories with a member of another.

[0104] One class of alternative variants involves one or more hypervariable residues of a substitute parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have certain alterations (e.g., improvements) in biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain some biological properties of the parent antibody. Exemplary alternative variants are affinity-matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more HVR residues are mutated, the variant antibody is displayed on a phage, and it is screened for specific biological activities (e.g., binding affinity).

[0105] HVR can be modified (e.g., substituted), for example, to improve antibody affinity. Such modifications can be made to HVR “hotspots,” which are residues encoded by codons that undergo mutations at high frequency during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact the antigen, where binding affinity is tested for the resulting variant VH or VL. Affinity maturation via the construction and reselection of secondary libraries has been documented, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene for maturation selection by a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves an HVR-guided method, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. Alanine scan mutagenesis or modeling can be used, for example, to specifically identify HVR residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are frequently targeted.

[0106] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) may be made to the HVRs that do not materially reduce binding affinity. For example, such changes may occur outside the antigen-contacting residues in the HVRs. In some embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0107] One method for identifying residues or regions in an antibody that can serve as mutagenic targets is called "alanine scan mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, groups of residues or target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions where the initial substitution indicates functional sensitivity. Alternatively / additionally, the contact points between the antibody and antigen can be identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they contain the desired properties.

[0108] Amino acid sequence insertions include fusion of the amino and / or carboxyl ends of peptides ranging in length from 1 residue to 100 or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a peptide that extends the serum half-life of the antibody.

[0109] b) Glycosylation variants

[0110] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to create or eliminate one or more glycosylation sites.

[0111] In cases where the antibody contains an Fc region, the attached carbohydrate can be modified. Naturally occurring antibodies generated from mammalian cells typically contain branched, biantennary oligosaccharides, which are generally attached to the CH2 domain of the Fc region via an N-linked Asn297. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to create antibody variants with certain improved properties.

[0112] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The fucose content is determined by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all glycan structures attached to Asn297 (e.g., complex, heterogeneous, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 (Eu numbering convention for Fe region residues) in the Fc region; however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297 due to minor sequence variations in the antibody, i.e., between positions 294 and 300. Such fucoidylated variants can have improved ADCC function. See, for example, US Patent Publication No. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications involving "defucosylated" or "fucosylated" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; TO 2005 / 035778; TO2005 / 053742; TO2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of generating defucosylation antibodies include LecI3CHO cells with protein fucosylation defects (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108A1, Presta, L; and WO 2004 / 056312A1, Adams et al.), and knockout cell lines, such as α-1,6-fucosylation gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0113] Further antibody variants with bifid oligosaccharides are provided, for example, wherein the bifid oligosaccharide attached to the Fc region of the antibody is bifid via GIcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, WO1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0114] c) Fc region variants

[0115] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby generating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0116] In some embodiments, the present invention covers antibody variants possessing some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, and certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / attenuation of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore potentially lacks ADCC activity), but retains FcRn binding capacity. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Tmmunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest are described in U.S. Patent Nos. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'I Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'I Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, ACT I for flow cytometry). TMNon-radioactive cytotoxicity assays (Cell Technology, Inc., Mountain View, CA; and CytoTox96 non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively / in vivo, ADCC activity of the molecule of interest can be assessed, for example in animal models, such as those disclosed in Clynes et al., Proc Nat'I Acad Sci USA 95:652-656 (1998). Clq binding assays can also be performed to confirm that the antibody cannot bind Clq and therefore lacks CDC activity. See, for example, Clq and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, the CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'I. Immunol. 18(12):1759-1769 (2006)).

[0117] Antibodies with reduced effector function include those with substitutions of one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with residues 265 and 297 substituted with alanine (US Patent No. 7,332,581).

[0118] Certain antibody variants with improved or reduced binding to FcR are described (see, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

[0119] In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as Fc region positions 298, 333, and / or 334 (EU numbering of residues).

[0120] In some implementations, modifications are made to the Fc region that result in altered (i.e., improved or reduced) Clq binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Tmmunol 164:4178-4184 (2000).

[0121] Antibodies with prolonged half-lives and improved binding to the neonatal Fc receptor (FcRn) are described in US2005 / 0014934A1 (Hinton et al.), the neonatal Fc receptor (FcRn) responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol 117:587 (1976) and Kim et al., J. Immunol 24:249 (1994)). These antibodies contain one or more substituted Fc regions that improve Fc region binding to FcRn. Such Fc variants include those that have substitutions at one or more of the following residues in the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitutions at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0122] See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, which focus on other examples of Fc region variants.

[0123] d) Cysteine-engineered antibody variants

[0124] In some embodiments, it may be desirable to create cysteine-engineered antibodies, such as "thioMAb," wherein one or more residues of the antibody are replaced with cysteine ​​residues. In specific embodiments, the substituted residues are located at accessible sites on the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites on the antibody and can be used to conjugate the antibody with other modules, such as drug modules or linker-drug modules, to create immunoconjugates, as further described herein. In some embodiments, any one or more of the following residues may be replaced with cysteine: V205 of the light chain (Kabat numbering); A118 of the heavy chain (EU numbering); and S400 of the Fc region of the heavy chain (EU numbering). Cysteine-engineered antibodies may be generated as described, for example, in U.S. Patent No. 7,521,541.

[0125] e) Antibody derivatives

[0126] In some embodiments, the antibodies provided herein may be further modified to include additional non-protein property modules known in the art and readily available. Suitable modules for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. PEG-propionaldehyde may be advantageous in production due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions that the antibody is to improve, and whether the antibody derivative will be used for treatment under specified conditions.

[0127] In another embodiment, an antibody and a conjugate of a non-protein-natured module that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein-natured module is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm ordinary cells but heat the non-protein-natured module to a temperature at which cells near the antibody-non-protein-natured module are killed.

[0128] Determination method

[0129] The anti-OX40 antibodies provided herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activity using a variety of assays known in the art.

[0130] 1. Combining the assay method with other assay methods

[0131] On one hand, the antigen-binding activity of the antibodies of the present invention is tested, for example by known methods such as ELISA, Western blotting, etc. OX40 binding can be determined using methods known in the art, and exemplary methods are disclosed herein. In one embodiment, binding is measured using a radioimmunoassay. An exemplary radioimmunoassay is illustrated. An OX40 antibody is iodinated, and a competitive reaction mixture is prepared containing a fixed concentration of the iodinated antibody and serially diluted unlabeled OX40 antibody at decreasing concentrations. Cells expressing OX40 (e.g., BT474 cells stably transfected with human OX40) are added to the reaction mixture. After incubation, the cells are washed to separate the free iodinated OX40 antibody from the OX40 antibody bound to the cells. The level of bound iodinated OX40 antibody is determined, for example by a radiometric count of the binding to the cells, and binding affinity is determined using standard methods. In another embodiment, the ability of the OX40 antibody to bind to surface-expressed OX40 (e.g., on a subset of T cells) is assessed using flow cytometry. Peripheral white blood cells (e.g., from humans, cynomolgus monkeys, rats, or mice) were obtained and blocked with serum. Labeled OX40 antibody was added to serial dilutions, and T cells were also stained to identify a subset of T cells (using methods known in the art). After sample incubation and washing, cells were sorted using flow cytometry, and data were analyzed using methods known in the art. In another embodiment, surface plasmon resonance (SPR) can be used to analyze OX40 binding. An exemplary SPR method is illustrated.

[0132] On the other hand, competitive assays can be used to identify antibodies that compete with any of the anti-OX40 antibodies disclosed herein for binding to OX40. In some embodiments, such competitive antibodies bind to the same epitope (e.g., a linear or conformational epitope) as any of the anti-OX40 antibodies disclosed herein. Detailed illustrative methods for locating the epitope bound by the antibody are shown in Morris (1996), “Epitope Mapping Protocols,” Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). One competitive assay is illustrated.

[0133] In an exemplary competitive assay, OX40 is immobilized by incubation in a solution containing a first labeled antibody (which binds to OX, e.g., mab1A7.gr.1, mab3C8.gr5) and a second unlabeled antibody (which is to be tested for its ability to compete with the first antibody for binding to OX40). The second antibody may be present in the hybridoma supernatant. As a control, OX40 is immobilized by incubation in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to OX40, excess unbound antibody is removed, and the amount of label conjugated with the immobilized OX40 is measured. If the amount of label conjugated with the immobilized OX40 in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to OX40. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0134] 2. Activity Assay

[0135] On the one hand, an assay is provided for identifying anti-OX40 antibodies with biological activity. Biological activity may include, for example, binding to OX40 (e.g., binding to human and / or cynomolgus monkey OX40), enhancing OX40-mediated signal transduction (e.g., enhancing NF-κB-mediated transcription), downsampling cells expressing human OX40 (e.g., T cells), downsampling cells expressing human OX40 via ADCC and / or phagocytosis, enhancing T effector cell function (e.g., CD4+ effector T cells) (e.g., by enhancing effector T cell proliferation and / or enhancing effector T cell cytokine production (e.g., interferon-gamma)), enhancing memory T cell function (e.g., CD4+ memory T cells) (e.g., by enhancing memory T cell proliferation and / or enhancing memory T cell cytokine production (e.g., interferon-gamma)), inhibiting Treg suppression that regulates T cell function (e.g., by reducing effector T cell function (e.g., CD4+ effector T cell function)), and binding to human effector cells. Antibodies with such biological activity in vivo and / or in vitro are also provided.

[0136] In some embodiments, the antibodies of the present invention are tested for such biological activity.

[0137] T cell co-stimulation can be measured using methods known in the art, and exemplary methods are disclosed herein. For example, T cells (e.g., memory or effector T cells) can be obtained from peripheral blood cells (e.g., separated from human whole blood using Ficoll gradient centrifugation). Memory T cells (e.g., CD4+ memory T cells) or effector T cells (e.g., CD4+ Teff cells) can be isolated from PBMCs using methods known in the art. For example, the Miltenyi CD4+ memory T cell isolation kit or the Miltenyi naive CD4+ T cell isolation kit can be used. Isolated T cells are cultured in the presence of antigen-presenting cells (e.g., irradiated L cells expressing CD32 and CD80) and activated by adding anti-CD3 antibodies in the presence or absence of OX40 agonist antibodies. The effect of agonist OX40 antibodies on T cell proliferation can be measured using methods known in the art. For example, the Cell Titer Glo kit (Promega) can be used, and the results can be read on a multi-label reader (Perkin Elmer). The effect of agonist OX40 antibodies on T cell function can also be measured by analyzing cytokines produced by T cells. In one embodiment, interferon-γ production in CD4+ T cells is determined, for example, by measuring interferon-γ in the cell culture supernatant. Methods for measuring interferon-γ are well known in the art.

[0138] Treg cell function can be assessed using methods known in the art, and exemplary methods are disclosed herein. In one example, the ability of Tregs to suppress effector T cell proliferation is assessed. T cells (e.g., memory T cells or naive T cells) are isolated from human whole blood using methods known in the art. Purified CD4+ naive T cells are labeled (e.g., with CFSE), and purified Treg cells are labeled with different reagents. Irradiated antigen-presenting cells (e.g., L cells expressing CD32 and CD80) are co-cultured with labeled purified naive CD4+ T cells and purified Tregs. The co-culture is activated using an anti-CD3 antibody and tested in the presence or absence of agonist OMO antibodies. After an appropriate period (e.g., 6 days of co-culture), the level of CD4+ naive T cell proliferation is tracked by dye dilution in reduced marker staining (e.g., reduced CFSE marker staining) using FACS analysis.

[0139] OX40 signaling can be measured using methods known in the art, and exemplary methods are disclosed herein. In one embodiment, transgenic cells expressing human OX40 and a reporter gene (containing an NFkB promoter fused to a reporter gene (e.g., β-luciferase)) are generated. Addition of an OX40 agonist antibody to the cells results in an increase in NFkB transcription, which is detected using a reporter gene assay.

[0140] Phagocytosis can be measured, for example, using monocyte-derived macrophages or U937 cells (a human histiocytic lymphoma cell line with the morphology and characteristics of mature macrophages). Cells expressing OX40 are added to monocyte-derived macrophages or U937 cells in the presence or absence of an anti-OX40 agonist antibody. After culturing the cells for an appropriate period, the percentage of cells double-stained for markers against 1) macrophages or U937 cells and 2) OX40-expressing cells is determined by dividing this percentage by the total number of cells displaying markers for OX40-expressing cells (e.g., GFP). Analysis can be performed by flow cytometry. In another embodiment, analysis can be performed by fluorescence microscopy.

[0141] ADCC can be determined, for example, using methods known in the art. Exemplary methods are described in the definitions section. In some embodiments, the level of OX40 on cells expressing OX40, which are used for testing in the ADCC assay, is characterized. Cells are stained with a detectably labeled anti-OX40 antibody (e.g., PE-labeled), and then the fluorescence level is determined using flow cytometry, with the results presented as median fluorescence intensity (MFI). In another embodiment, ADCC can be analyzed using a CellTiter Glo assay kit, and cell viability / cytotoxicity can be determined using chemiluminescence.

[0142] The binding affinity of various antibodies for two allotypes (F158 and V158) of FcγRIA, FcγRIIA, FcγRIIB, and FcγRIIIA can be measured using an ELISA-based ligand binding assay with the corresponding recombinant Fcγ receptor. The purified human Fcγ receptor was expressed as a fusion protein containing a C-terminal Gly / 6xHis / glutathione S-transferase (GST) polypeptide tag over the extracellular domain of the receptor γ chain. The binding affinity of antibodies for these human Fcγ receptors was determined as follows. For the low-affinity receptors, namely FcγRIIA (CD32A), FcγRIIB (CD32B), and FeγRIIIA (CD16), two allotypes, F-158 and V-158, cross-linking with the F(ab')2 fragment of goat anti-human kappa chain (ICN Biomedical; Irvine, CA) at an approximate molar ratio of 1:3 antibody: cross-linking F(ab')2, was used as the multimeric assay antibody. Plates were coated with anti-GST antibody (Genentech) and blocked with bovine serum albumin (BSA). The assay was performed using phosphate-buffered saline (PBS) containing 0.05% Tween-20 and ELx405. TM After washing with a plate washer (Biotek Instruments; Winooski, VT), 25 ng / well of Fcγ receptor was added to the plate and incubated at room temperature for 1 hour. Following plate washing, serial dilutions of the test antibody were added as a multimeric complex, and the plate was incubated at room temperature for 2 hours. After washing the plate to remove unbound antibody, the antibody bound to the Fcγ receptor was detected using a horseradish peroxidase (HRP)-conjugated goat anti-human F(ab')2 fragment (Jackson ImmunoResearch Laboratories; WestGrove, PA). Then, the substrate, tetramethylbenzidine (TMB) (Kirkegaard and Perry Laboratories; Gaithersburg, MD), was added. Depending on the Fcγ receptor being tested, the plate was incubated at room temperature for 5–20 minutes to allow for color development. The reaction was terminated with IM H3PO4 and read using a microplate reader. The absorbance at 450 nm was measured using a SoftMax 190 (Molecular Devices; Sunnyvale, CA). A dose-response binding curve was generated by plotting the mean absorbance from two antibody dilutions against antibody concentration. The effective antibody concentration (EC50) at which 50% of the maximum response to binding the Fcγ receptor was detected was determined by fitting the binding curve to a four-parameter equation using a SoftMax 190 (Molecular Devices).

[0143] To select antibodies that induce cell death, loss of membrane integrity can be assessed relative to a control by, for example, propidium iodide (PI), trypan blue, or 7AAD uptake. PI uptake assays can be performed in the absence of complement and immune effector cells. Cells expressing OX40 are incubated in a separate culture medium or in a medium containing, for example, a suitable monoclonal antibody at a concentration of about 10 μg / ml. Cells are incubated for a period of time (e.g., 1 or 3 days). After each treatment, cells are washed and aliquoted. In some embodiments, cells are aliquoted into 12x75 tubes (1 ml per tube, 3 tubes per treatment group) with a strainer-capped 35 mm membrane to remove cell clumps. PI (10 μg / ml) is then added to the tubes. FACSCAN can be used. TM Flow cytometer and FACSCONVERT TM CellQuest software (Becton Dickinson) was used to analyze the samples.

[0144] Cells used in any of the above in vitro assays include cells or cell lines that naturally express OX40 or are engineered to express OX40. Such cells include activated T cells, Treg cells, and activated memory T cells that naturally express OX40. Such cells also include cell lines that express OX40 and cell lines that do not normally express OX40 but have been transfected with nucleic acids encoding OX40. Exemplary cell lines provided herein for use in any of the above in vitro assays include transgenic BT474 cells (a human breast cancer cell line) expressing human OX40.

[0145] It is understood that the immunoconjugates of the present invention can be used to replace or supplement the anti-OX40 antibody for any of the above assays.

[0146] It is understood that anti-OX40 antibodies and other therapeutic agents can be used to perform any of the above assays.

[0147] Formula and Uses

[0148] The antibody or its antigen-binding fragment of the present invention can be formulated into a pharmaceutical composition. This pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient, and / or stabilizer (Remington: The Science and Practice of Pharmacy, 20th Edition, 2000, Lippincott Williams and Wilkins, Ed. KEHoover), in lyophilized formulation or aqueous solution form. The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the specified dose and concentration, and may include buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexahydroxyquaternary ammonium chloride; algaecide; benzaldehyde chloride; phenolic alcohol, butanol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) (Based on) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, aspartic acid, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming opposite ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG). This article further describes pharmaceutically acceptable excipients.

[0149] The antibodies or antigen-binding fragments thereof of the present invention can be used for various therapeutic or diagnostic purposes. For example, the antibodies or antigen-binding fragments thereof of the present invention can be used as affinity purifiers (e.g., for in vitro purification); and as diagnostic agents (e.g., for detecting expression in specific cells, tissues, or serum).

[0150] Exemplary therapeutic uses of the antibodies or antigen-binding fragments thereof of the present invention include the treatment of cancer. The antibodies or antigen-binding fragments thereof of the present invention can also be used for prophylactic treatment.

[0151] For therapeutic applications, the antibodies or antigen-binding fragments of the present invention can be administered to mammals, particularly humans, using conventional techniques such as intravenous (as a bolus or by continuous infusion over time), intramuscular, intraperitoneal, intracerebral, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation. The antibodies or antigen-binding fragments of the present invention can also be appropriately administered via intratumoral, peritumoral, intralesional, or perilesional routes.

[0152] In some embodiments, the antibody or antigen-binding fragment of the present invention is administered subcutaneously. In some embodiments, the antibody or antigen-binding fragment of the present invention is administered intravenously.

[0153] The pharmaceutical composition can be administered to subjects in need at a frequency that varies with the severity of the disease. In the case of preventative treatment, this frequency may vary depending on the subject's susceptibility or predisposition to the disease.

[0154] The composition can be administered to patients in need by bolus injection or by continuous infusion. For example, bolus injection of the antibody presented as a Fab fragment can be administered at doses of 0.0025 to 100 mg / kg body weight, 0.025 to 0.25 mg / kg, 0.010 to 0.10 mg / kg, or 0.10 to 0.50 mg / kg. For continuous infusion, the antibody presented as a Fab fragment can be administered at doses of 0.001 to 100 mg / kg body weight / min, 0.0125 to 1.25 mg / kg / min, 0.010 to 0.75 mg / kg / min, 0.010 to 1.0 mg / kg / min, or 0.10 to 0.50 mg / kg / min for 1 to 24 hours, 1 to 12 hours, 2 to 12 hours, 6 to 12 hours, 2 to 8 hours, or 1 to 2 hours.

[0155] For the administration of antibodies presented as full-length antibodies (with an intact constant region), the dosage may be from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 3 mg / kg to about 10 mg / kg, from about 4 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 20 mg / kg, from about 2 mg / kg to about 20 mg / kg, from about 3 mg / kg to about 20 mg / kg, from about 4 mg / kg to about 20 mg / kg, from about 5 mg / kg to about 20 mg / kg, and about 1 mg / kg. / kg or more, about 2mg / kg or more, about 3mg / kg or more, about 4mg / kg or more, about 5mg / kg or more, about 6mg / kg or more, about 7mg / kg or more, about 8mg / kg or more, about 9mg / kg or more, about 10mg / kg or more, about 11mg / kg or more, about 12mg / kg or more, about 13mg / kg or more, about 14mg / kg or more, about 15mg / kg or more, about 16mg / kg or more, about 17mg / kg or more, about 19mg / kg or more, or about 20mg / kg or more. The frequency of administration will depend on the severity of the condition. The frequency can vary from three times a week to once every two or three weeks.

[0156] Alternatively, the composition can be administered to the patient via subcutaneous injection. For example, a dose of 1 to 100 mg of anti-OX40 antibody can be administered to the patient via subcutaneous or intravenous injection at a frequency of twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months, or once every three months.

[0157] In some embodiments, the half-life of the anti-OX40 antibody in humans is approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days, approximately 15 days, approximately 16 days, approximately 17 days, approximately 18 days, approximately 19 days, approximately 20 days, approximately 21 days, approximately 22 days, approximately 23 days, approximately 24 days, approximately 25 days, approximately 26 days, approximately 27 days, or approximately 28 days. Approximately 29 days, approximately 30 days, from approximately 5 days to approximately 40 days, from approximately 5 days to approximately 35 days, from approximately 5 days to approximately 30 days, from approximately 5 days to approximately 25 days, from approximately 10 days to approximately 40 days, from approximately 10 days to approximately 35 days, from approximately 10 days to approximately 30 days, from approximately 10 days to approximately 25 days, from approximately 15 days to approximately 40 days, from approximately 15 days to approximately 35 days, from approximately 15 days to approximately 30 days, or from approximately 15 days to approximately 25 days.

[0158] In some embodiments, the pharmaceutical composition is administered subcutaneously or intravenously every 2 to 6 weeks at the following doses: from about 0.1 mg / kg to about 10 mg / kg, from about 0.5 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 1.5 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 8 mg / kg, from about 0.5 mg / kg to about 8 mg / kg, from about 1 mg / kg to about 8 mg / kg, from about 1.5 mg / kg to about 8 mg / kg, from about 2 mg / kg to about 8 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 5 mg / kg. g / kg, from about 1 mg / kg to about 5 mg / kg, from about 1.5 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 3.0 mg / kg, about 3.5 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg, about 5.0 mg / kg, about 5.5 mg / kg, about 6.0 mg / kg, about 6.5 mg / kg, about 7.0 mg / kg, about 7.5 mg / kg, about 8.0 mg / kg, about 8.5 mg / kg, about 9.0 mg / kg, about 9.5 mg / kg or about 10.0 mg / kg.

[0159] In some embodiments, the pharmaceutical composition is administered subcutaneously or intravenously every 2 to 6 weeks at a dose of about 2.0 mg / kg. In some embodiments, the pharmaceutical composition is administered subcutaneously or intravenously every 2 to 6 weeks at a dose ranging from about 2.0 mg / kg to about 10.0 mg / kg.

[0160] In one exemplary embodiment, the pharmaceutical composition is administered subcutaneously every 2 weeks.

[0161] The antibodies or antigen-binding fragments of the present invention can be used as monotherapy or in combination with other therapies to treat cancer.

[0162] definition

[0163] Unless otherwise defined herein, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural and plural terms shall include singular. Generally, the terminology and techniques used in connection with cell and tissue cultures, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0164] An "antigen-binding fragment" of an antibody refers to a fragment of a full-length antibody that retains the ability to specifically bind to an antigen (preferably having substantially the same binding affinity). Examples of antigen-binding fragments include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments consisting of two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), which consist of VH domains; and (vi) separated complementarity-determining regions (CDRs), Fvs (dsFv) linked by disulfide bonds, and anti-atopic (anti-Id) antibodies and intracellular antibodies. Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by different genes, they can be linked using a recombinant approach via a synthetic linker, which allows them to be made into a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)); see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Other forms of single-chain antibodies (such as biantibodies) are also included in this invention. Biantibodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but are too short to allow pairing between the two chains on the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and creating two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure 2:1121-1123).

[0165] An antibody “variable domain” refers to a variable region of the antibody light chain (VL) or a variable region of the antibody heavy chain (VH), either alone or in combination. As is known in the art, the variable regions of both the heavy and light chains consist of three complementarity-determining regions (CDRs) connected by four framework regions (FRs), which contribute to the formation of the antibody’s antigen-binding site.

[0166] Residues in the variable domain are numbered according to Kabat, a numbering system used for heavy-chain or light-chain variable domains in antibody compilation. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Using this numbering system, the actual straight-chain amino acid sequence may contain fewer or more amino acids, corresponding to shortening or insertion of the FR or CDR of the variable domain. For a given antibody, the Kabat number of the residues can be determined by comparing the sequence of the antibody with a region homologous to the sequence of a “standard” Kabat number. Various algorithms for assigning Kabat numbers are available. Unless otherwise stated herein, the algorithm used in Abysis (www.abysis.org), published in 2012, is used to assign Kabat numbers to variable regions.

[0167] The specific amino acid residue positions in the antibody (such as complementary residues) are also based on the Kabat number.

[0168] The “complementarity-determining region” (CDR) can be identified according to the definitions of aggregation, AbM, contact, and / or conformational definition of Kabat and Chothia, or any method of CDR determination known in the art. See, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition (Highly Variable Regions); Chothia et al., 1989, Nature 342:877-883 (Structural Ring Structures). The AbM definition of CDR is a compromise between Kabat and Chothia and uses Oxford Molecular’s ​​AbM antibody modeling software. The definition of “contact” in CDR is based on visible antigen contact as described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. The “conformation” definition of a CDR is based on residues that produce enthalpy conducive to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). Other CDR boundary definitions may not strictly adhere to any of the methods described above, but will still overlap with at least a portion of the Kabat CDR; however, they may be shortened or lengthened based on predictions or experimental findings that specific residues or groups of residues, or even all of the CDRs, do not significantly affect antigen binding. As used herein, a CDR may refer to a CDR defined by any method (including combinations of methods) known in the art.

[0169] An antigenic determinant is a range or region in an antigen (Ag) that an antibody specifically binds to; for example, it may contain a range or region of amino acid residues that interact with the antibody (Ab). Antigenic determinants can be linear or non-linear (e.g., conformational).

[0170] When the binding of corresponding antibodies or their antigen-binding fragments is mutually exclusive, the antibody or its antigen-binding fragment binds substantially to the same antigenic determinant as the other antibody or its antigen-binding fragment. That is, the binding of one antibody or its antigen-binding fragment excludes the simultaneous or sequential binding of other antibodies or their antigen-binding fragments. If the antigen can accommodate the simultaneous binding of two corresponding antibodies or their antigen-binding fragments, then the antigenic determinants are considered unique or not substantially identical.

[0171] The term "complementary site" is derived from the above definition of "antigen determinant" by twist angle, and refers to the range or region in an antibody molecule involved in antigen binding, for example, the range or region containing residues that interact with the antigen. Complementary sites can be linear or conformational (such as discontinuous residues in a CDR).

[0172] The antigenic determinants / complementary sites of a given antibody / antigen binding pair can be defined and characterized at different levels of detail using various experimental and computational antigenic determinant localization methods. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen / deuterium exchange mass spectrometry (HX-MS), and various competitive binding methods. Because each method relies on the principle of uniqueness, the description of antigenic determinants is closely related to the method used to determine them. Therefore, the antigenic determinants / complementary sites of a given antibody / antigen pair will be defined differently depending on the localization method employed.

[0173] At its most detailed level, the antigenic determinant / complementary site for the interaction between antibody (Ab) and antigen (Ag) can be defined by the spatial coordinates of the atomic contacts present in the Ag-Ab interaction and information about their relative contribution to binding thermodynamics. To some extent, antigenic determinant / complementary site residues can be characterized by defining the spatial coordinates of the atomic contacts between Ag and Ab. In one aspect, the antigenic determinant / complementary site residue can be defined by specific criteria, such as the distance between atoms in the Ab and Ag (e.g., the distance from the heavy atom of the homologous antibody and the heavy atom of the antigen is equal to or less than approximately). In another aspect, the antigenic determinant / complementary site residue is characterized by participating in hydrogen bonding interactions with the homologous antibody / antigen, or with water molecules that are also hydrogen-bound to the homologous antibody / antigen (water-mediated hydrogen binding). In yet another aspect, the antigenic determinant / complementary site residue is characterized by forming a salt bridge with residues of the homologous antibody / antigen. In yet another aspect, the antigenic determinant / complementary site residue can be characterized by a non-zero change in the masking surface area (BSA) due to interaction with the homologous antibody / antigen. At a less detailed level, the antigenic determinant / complementary site can be characterized by a function, for example, by competitive binding with other Abs. The antigenic determinant / complementary site can also be more generally defined as containing amino acid residues, wherein the characteristic of the interaction between the Ab and Ag is altered by substitution of another amino acid (e.g., alanine scan).

[0174] Since the description and definition of antigenic determinants depend on the antigenic determinant mapping method used and the facts obtained at different levels of detail, it can be inferred that comparisons of antigenic determinants of different antibodies on the same Ag can be performed similarly at different levels of detail. For example, at the amino acid level, antigenic determinants, such as those determined by X-ray structure, are considered identical if they contain the same set of amino acid residues. If the binding of the corresponding antibodies is mutually exclusive, i.e., the binding of one antibody excludes the simultaneous or sequential binding of other antibodies, then antigenic determinants characterized by competitive binding are considered overlapping; and if the antigen can accommodate the simultaneous binding of two corresponding antibodies, then the antigenic determinants are considered distinct (unique).

[0175] The antigenic determinants and complementary sites of a given antibody / antigen pair can be identified using routine methods. For example, the general position of the antigenic determinants can be determined by assessing the ability of the antibody to bind to different fragments or variant peptides, as described more fully earlier herein. Specific residues within the OX40 that can contact specific residues within the antibody can also be determined using routine methods. For example, the antibody / antigen complex can be crystallized. This crystal structure can be determined and used to identify specific sites of interaction between the antibody and antigen.

[0176] The term "specific binding" is well-known in the art, and methods for determining such specific bindings are also well-known in the art. A molecule is considered to exhibit "specific binding" if it reacts or binds to a specific cell or substance more frequently, more rapidly, for a longer duration, and / or with a greater affinity than it reacts or binds to a substitute cell or substance. An antibody or its antigen-binding fragment is considered to "specifically bind" to a target if it binds to the target with greater affinity, binding strength, ease, and / or longer duration compared to binding to other substances.

[0177] For example, an antibody or its antigen-binding fragment that specifically binds to OX40 is an antibody that binds to its homologous antigen with greater affinity, binding strength, ease, and / or longer duration compared to binding to other antigens. For instance, under standard binding assay conditions, an anti-OX40 antibody may specifically bind to human OX40 in a sample but substantially does not recognize or bind to other molecules in that sample. It is also understood that an antibody or its antigen-binding fragment that specifically binds to a first target may or may not specifically bind to a second target. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding. Usually, but not necessarily, the reference to "binding" means specific binding.

[0178] Various analytical modes can be used to select antibodies or antigen-binding fragments that specifically bind to the target molecule. For example, in many assays, solid-phase ELISA, immunoprecipitation, and Biacore... TM (GE Healthcare), KinExA, Fluorescence-Activated Cell Sorting (FACS), Octet TM (FortéBio, Inc.) and Western blot analysis can be used to identify antibodies or antigen-binding fragments that specifically bind to antigens. Typically, specific binding will be at least twice the background signal or noise, more often at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times, or at least 10,000 times the background.

[0179] The specificity of antibody binding can be determined by measuring the Kc of the specific binding between the antibody and OX40. D Value and comparison of KD The K value compared to the control antibody known not to bind to OX40 D The value is used for evaluation. Generally speaking, when the K value is... D It is approximately ×10 -5 When M or smaller, it is believed that the antibody "specifically" binds to the antigen.

[0180] When an antibody or its antigen-binding fragment does not bind to an antigen with greater affinity, binding strength, ease, and / or longer duration compared to the binding of other antigens, the antibody or its antigen-binding fragment is considered "substantially non-binding" to that antigen. Typically, this binding will not exceed twice the background signal or noise. Generally, it is 1 × 10⁻⁶. -4 M or larger, 1×10 -3 M or larger, 1×10 -2 M or larger or 1×10 -1 M or larger K D It binds to the antigen.

[0181] As used in this article, the term "competition" for antibodies means that the binding of a first antibody or its antigen-binding moiety to an antigen reduces the binding of a subsequent second antibody or its antigen-binding moiety to the same antigen. Generally, the binding of the first antibody results in steric hindrance, a conformational change, or binding to a common antigenic determinant (or a portion thereof), thus reducing the binding of the second antibody to the same antigen. Standard competitive binding assays can be used to determine whether two antibodies are competing with each other.

[0182] A suitable analysis for antibody competition involves using Biacore technology, which employs surface plasmon resonance (SPR) technology, typically using biosensor systems such as [system name missing], to measure the degree of interaction. For example, SPR can be used in in vitro competitive binding inhibition assays to determine the ability of one antibody to inhibit the binding of a second antibody. Another analysis for measuring antibody competition uses ELISA-based methods. Furthermore, a high-throughput method for “grading” antibodies based on antibody competition is described in WO2003 / 48731. Competition exists if one antibody or its antigen-binding fragment reduces the binding of another antibody or its antigen-binding fragment to OX40. For example, sequential binding competition assays can be used, involving the sequential addition of different antibodies. A first antibody can be added to achieve near-saturation binding. Then, a second antibody is added. If the binding of the second antibody to OX40 is undetectable or is significantly reduced compared to a parallel analysis in the absence of the first antibody (where the value can be set to 100%) (e.g., 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%, or at least about 90%), then the two antibodies are considered to be competing with each other.

[0183] Competitive binding assays can also be performed, where antibody binding to an antigen is relative to binding to another binding partner of the target (such as another antibody or soluble receptor that originally binds to the target). The concentration at which 50% inhibition occurs is called K. i Under ideal conditions, this K i It equals K D Therefore, in general, K i The measurement can be conveniently replaced to provide K. D The upper limit of the binding affinity. Binding affinity associated with different molecular interactions (e.g., a comparison of the binding affinity of different antibodies for a given antigen) can be measured by KA for individual antibody / antigen complexes. D The comparison is made based on the values. K is used against antibodies or other binding partners. D The value can be determined using methods already established in the art.

[0184] An “Fc fusion” protein is a protein in which one or more polypeptides are operatively linked to an Fc polypeptide. An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner. The “Fc region” can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The residues in the Fc region are numbered with an EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin typically contains two constant domains (CH2 and CH3). As is known in the art, the Fc region can exist in dimer or monomeric form.

[0185] The term "therapeutic effective amount" means the amount of an anti-OX40 antibody or its antigen-binding fragment, or a combination containing such an antibody or its antigen-binding fragment, sufficient to achieve the intended purpose. The precise amount will depend on many factors, including (but not limited to) the components and physical characteristics of the therapeutic composition, the intended patient population, individual patient considerations, and can be determined by a person skilled in the art.

[0186] The term "treatment" includes both preventative and therapeutic treatment. Preventative treatment is considered to be administered before the clinical manifestations of a disease, disorder, or condition. Therapeutic treatment includes, for example, reducing or lessening the severity or duration of a disease, disorder, or condition.

[0187] As used in this article, the term “about” refers to + / - 10% of the value.

[0188] Therapeutic methods and compositions

[0189] Any anti-human OX40 antibodies provided in this article can be used in treatments.

[0190] In one aspect, an anti-human OX40 agonist antibody is provided for use as a medicine. In other aspects, an anti-human OX40 agonist antibody is provided for treating cancer. In some embodiments, an anti-human OX40 agonist antibody is provided for use as a treatment method. In some embodiments, a method of treating an individual with cancer using an anti-human OX40 agonist antibody includes administering an effective amount of the anti-human OX40 agonist antibody to the individual. In one such embodiment, the method further includes administering an effective amount of at least one other therapeutic agent to the individual, such as those described below.

[0191] In one aspect, an anti-human OX40 agonist antibody is provided for enhancing immune function (e.g., by upregulating cell-mediated immune responses) in an individual with cancer, comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual. In another aspect, an anti-human OX40 agonist antibody is provided for enhancing T cell function in an individual with cancer, comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual. In another aspect, an anti-human OX40 agonist antibody is provided for reducing cells expressing human OX40 (e.g., OX40-expressing T cells, such as OX40-expressing Tregs), comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual. In some embodiments, reduction is performed via ADCC. In some embodiments, reduction is performed via phagocytosis. An anti-human OX40 agonist antibody is provided for treating an individual with tumor immunity.

[0192] In other aspects, anti-human OX40 agonist antibodies are provided for the treatment of infections (e.g., bacterial, viral, or other pathogen infections). In some embodiments, the invention provides a method of treating an individual with an infection using an anti-human OX40 agonist antibody, comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual. In some embodiments, the infection is a viral and / or bacterial infection. In some embodiments, the infection is a pathogen infection.

[0193] In another aspect, the present invention provides the use of anti-OX40 antibodies in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat cancer. In yet another embodiment, the method of using the medicament to treat cancer includes administering an effective amount of the medicament to an individual having cancer. In one such embodiment, the method further includes administering an effective amount of at least one other therapeutic agent to the individual, such as those described below.

[0194] In one aspect, the drug is used to enhance immune function in an individual with cancer (e.g., by upregulating cell-mediated immune responses), comprising administering an effective amount of the drug to the individual. In another aspect, the drug is used to enhance T cell function in an individual with cancer, comprising administering an effective amount of the drug to the individual. In some embodiments, the T cell dysfunction is cancer. In one aspect, the drug is used to reduce cells expressing human OX40 (e.g., cells expressing high levels of OX40, such as T cells expressing OX40), comprising administering an effective amount of the drug to the individual. In some embodiments, the reduction is performed via ADCC. In some embodiments, the reduction is performed via phagocytosis. In one aspect, the drug is used to treat an individual with tumor immunity.

[0195] In other aspects, a drug is provided for treating an infection (e.g., a bacterial, viral, or other pathogen infection). In some embodiments, the method of using the drug to treat an individual with an infection includes administering an effective amount of the drug to the individual. In some embodiments, the infection is a viral and / or bacterial infection. In some embodiments, the infection is a pathogen infection.

[0196] In another aspect, the present invention provides a method for treating cancer. In one embodiment, the method includes administering an effective amount of an anti-OX40 antibody to an individual having such cancer. In one such embodiment, the method further includes administering an effective amount of at least one other therapeutic agent to the individual, such as those described below. The "individual" according to any of the above embodiments can be a human being.

[0197] In one aspect, a method is provided for enhancing immune function (e.g., by upregulating cell-mediated immune responses) in an individual with cancer, comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual. In another aspect, a method is provided for enhancing T cell function in an individual with cancer, comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual. In another aspect, a method is provided for depleting cells expressing human OX40 (e.g., cells expressing high levels of OX40, such as T cells expressing OX40), comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual. In some embodiments, depletion is performed via ADCC. In some embodiments, depletion is performed via phagocytosis. An anti-human OX40 agonist antibody is provided for treating individuals with tumor immunity.

[0198] In some implementations, examples of cancer further include, but are not limited to, B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small aneuploid NHL, bulkydisease NHL, mantle cell lymphoma, AIDS-associated lymphoma, and Waldenstrom macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with phakomatoses, edema (such as that associated with brain tumors), B-cell proliferative disorders, and Meigs syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, front-line low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B-cell lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytomas and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extra-ganglionic marginal zone (MALT) lymphoma, and nodal marginal zone lymphoma. Zone lymphoma, hairy cell leukemia, multicellular lymphoma and / or plasma cell myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicular central lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive relapsed NHL), relapsed or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary exudative lymphoma, advanced immune cytotoxic NHL, advanced lymphoblastic NHL, advanced small aneuploid cell NHL, bulky lymphoma Diseases include NHL, Burkitt lymphoma, precursor (peripheral) macrogranular lymphocytic leukemia, mycosis fungoides and / or Sezary syndrome, cutaneous lymphoma, anaplastic large cell lymphoma, and angiogenic lymphoma.

[0199] In some embodiments, examples of cancer further include, but are not limited to, B-cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphomas, b) small non-cleaved cell lymphomas / Burkitt's lymphomas (including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma, and non-Burkitt's lymphoma), c) marginal zone lymphomas (including extranodal marginal zone B-cell lymphomas (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B-cell lymphomas, and splenic marginal zone lymphomas), d) mantle cell lymphomas (MCL), e) large cell lymphomas (including diffuse large cell B-cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic lymphoma, etc.). f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell vegetations, plasma cell myeloma, multiple myeloma, plasmacytoma, and / or j) Hodgkin's disease.

[0200] In some embodiments of any method, the cancer is a B-cell proliferative disorder. In some embodiments, the B-cell proliferative disorder is lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed painless NHL, refractory NHL, refractory painless NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), or mantle cell lymphoma. In some embodiments, the B-cell proliferative disorder is NHL, such as painless NHL and / or aggressive NHL. In some embodiments, the B-cell proliferative disorder is painless follicular lymphoma or diffuse large B-cell lymphoma.

[0201] In another aspect, the present invention provides a pharmaceutical formulation comprising any anti-OX40 antibody provided herein, for example, for any of the above-described treatment methods. In one embodiment, the pharmaceutical formulation comprises any anti-OX40 antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any anti-OX40 antibody provided herein and at least one other therapeutic agent, such as those described below.

[0202] In some embodiments of any method of the present invention, the anti-human OX40 agonist antibody suppresses tumor immunity by inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), killing OX40-expressing cells (e.g., cells expressing high levels of OX40), enhancing effector T cell function, and / or enhancing memory T cell function. In some embodiments of any method of the present invention, the anti-human OX40 agonist antibody treats cancer by inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), killing OX40-expressing cells (e.g., cells expressing high levels of OX40), enhancing effector T cell function, and / or enhancing memory T cell function. In some embodiments of any method of the present invention, the anti-human OX40 agonist antibody enhances immune function by inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), killing OX40-expressing cells (e.g., cells expressing high levels of OX40), enhancing effector T cell function, and / or enhancing memory T cell function. In some embodiments of any method of the present invention, the anti-human OX40 agonist antibody enhances T cell function by inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), killing cells expressing OX40 (e.g., cells expressing high levels of OX40), enhancing effector T cell function, and / or enhancing memory T cell function.

[0203] In some embodiments of any method, the anti-human OX40 agonist antibody is a depleting anti-human OX40 agonist antibody. In some embodiments, treatment with the anti-human OX40 agonist antibody results in cell depletion (e.g., depletion of cells expressing OX40, such as depletion of cells expressing high levels of OX40). In some embodiments, depletion is performed via ADCC. In some embodiments, depletion is performed via phagocytosis.

[0204] In some embodiments of any method, the anti-human OX40 agonist antibody inhibits Treg function, for example, by suppressing Treg inhibition of effector and / or memory T cell function (in some embodiments, effector T cell and / or memory T cell proliferation and / or cytokine secretion), relative to Treg function prior to application of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody enhances effector T cell proliferation relative to effector T cell proliferation prior to application of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody enhances memory T cell proliferation relative to effector T cell proliferation prior to application of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody enhances effector T cell cytokine production (e.g., interferon-gamma production) relative to effector T cell cytokine production prior to application of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody enhances memory T cell cytokine production (e.g., interferon-gamma production) relative to memory T cell cytokine production prior to application of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody enhances CD4+ effector T cell proliferation and / or CD8+ effector T cell proliferation relative to prior administration of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody enhances memory T cell proliferation (e.g., CD4+ memory T cell proliferation) relative to prior administration of the OX40 agonist antibody. In some embodiments, CD4+ effector T cells in an individual exhibit enhanced proliferation, cytokine secretion, and / or cytolytic activity relative to prior administration of the anti-human OX40 agonist antibody.

[0205] In some embodiments of any method of the present invention, the number of CD4+ effector T cells is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments, CD4+ effector T cell cytokine secretion is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments of any method, CD8+ effector T cells in an individual exhibit enhanced proliferation, cytokine secretion, and / or cytolytic activity relative to before administration of the anti-human OX40 agonist antibody. In some embodiments, the number of CD8+ effector T cells is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments, CD8+ effector T cell cytokine secretion is increased relative to before administration of the anti-human OX40 agonist antibody.

[0206] In some embodiments of any method of the present invention, the anti-human OX40 agonist antibody binds to human effector cells, for example, to FcγR expressed by human effector cells. In some embodiments, the human effector cell performs ADCC effector function. In some embodiments, the human effector cell performs phagocytic effector function.

[0207] In some embodiments of any method of the present invention, anti-human OX40 agonist antibodies comprising a variant IgG1Fc polypeptide (which contains a mutation that eliminates binding to human effector cells, such as the DANA or N297G mutation) have reduced activity (e.g., CD4+ effector T cell function, such as proliferation) relative to anti-human OX40 agonist antibodies comprising the native IgG1Fc sequence portion. In some embodiments, anti-human OX40 agonist antibodies comprising a variant IgG1Fc polypeptide (which contains a mutation that eliminates binding to human effector cells, such as the DANA or N297G mutation) do not possess substantial activity (e.g., CD4+ effector T cell function, such as proliferation).

[0208] In some embodiments of any method of the present invention, the function of the anti-human OX40 agonist antibody requires antibody cross-linking. In some embodiments, the function is to stimulate the proliferation of CD4+ effector T cells. In some embodiments, antibody cross-linking is determined by providing an anti-human OX40 agonist antibody that adheres to a solid surface (e.g., a cell culture plate). In some embodiments, antibody cross-linking is determined by introducing a mutation (e.g., a DANA or N297S mutation) into the IgG1Fc moiety of the antibody and testing the function of the mutant antibody.

[0209] In some embodiments of any method, memory T cells in the individual exhibit enhanced proliferation and / or cytokine secretion relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, the number of memory T cells is increased relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, the level of cytokine secretion by memory T cells is increased relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments of any method, Tregs in the individual exhibit suppressed effector T cell function (e.g., proliferation and / or cytokine secretion) relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, the number of effector T cells is increased relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, the level of cytokine secretion by effector T cells is increased relative to prior to administration of the anti-human OX40 agonist antibody.

[0210] In some embodiments of any method of the present invention, the number of tumor-infiltrating (invasive) CD4+ effector T cells (e.g., the total number of CD4+ effector T cells, or, for example, the percentage of CD4+ cells in CD45+ cells) is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments of any method of the present invention, the number of tumor-infiltrating (invasive) CD4+ effector T cells expressing interferon-gamma (e.g., the total number of CD4+ cells expressing interferon-gamma, or, for example, the percentage of CD4+ cells expressing interferon-gamma in total CD4+ cells) is increased relative to before administration of the anti-human OX40 agonist antibody.

[0211] In some embodiments of any method of the present invention, the number of tumor-infiltrating (invasive) CD8+ effector T cells (e.g., the total number of CD8+ effector T cells, or, for example, the percentage of CD8+ cells in CD45+ cells) is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments of any method of the present invention, the number of tumor-infiltrating (invasive) CD8+ effector T cells expressing interferon-gamma (e.g., the percentage of CD8+ cells expressing interferon-gamma in total CD8+ cells) is increased relative to before administration of the anti-human OX40 agonist antibody.

[0212] In some embodiments of any method of the present invention, the number of intratumoral (invasive) Tregs (e.g., the total number of Tregs or, for example, the percentage of Fox3p+ cells in CD4+ cells) is reduced relative to before the administration of anti-human OX40 agonist antibody.

[0213] In some embodiments of any method of the present invention, the administration of an anti-human OX40 agonist antibody is combined with the administration of a tumor antigen. In some embodiments, the tumor antigen comprises a protein. In some embodiments, the tumor antigen comprises a nucleic acid. In some embodiments, the tumor antigen is a tumor cell.

[0214] In some embodiments of any method of the present invention, the cancer exhibits human effector cells (e.g., is infiltrated by human effector cells). Methods for detecting human effector cells are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of human effector cells. In some embodiments, the human effector cells are one or more of NK cells, macrophages, and monocytes. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

[0215] In some embodiments of any method of the present invention, the cancer exhibits cells expressing FcR (e.g., infiltrated by cells expressing FcR). Methods for detecting FcR are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits cells expressing high levels of FcR. In some embodiments, the FcR is FcγR. In some embodiments, the FcR is activating FcγR. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

[0216] The "individual" in any of the above embodiments is preferably a person.

[0217] The antibodies of the present invention can be used alone or in combination with other agents in a therapy. For example, the antibodies of the present invention can be co-administered with at least one other therapeutic agent.

[0218] The combination therapies described above encompass combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations) and separate administration, in which case the antibody of the invention may be administered before, simultaneously with, and / or after the administration of other therapeutic agents and / or pharmaceuticals. In one embodiment, the administration of the anti-OX40 antibody and the administration of other therapeutic agents occur within about one month, or about one, two, or three weeks, or about one, two, three, four, five, or six days. The antibody of the invention may also be used in combination with radiotherapy.

[0219] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with chemotherapy or chemotherapeutic agents. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with radiotherapy or radiotherapy agents. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with targeted therapy or targeted therapeutic agents. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with immunotherapy or immunotherapeutic agents, such as monoclonal antibodies.

[0220] In some embodiments, anti-human OX40 agonist antibodies may be combined with PARP inhibitors (e.g., Olaparanib, Rucaparib, Niraparib, Cediranib, BMN673, Veliparib), Trabectedin, nab-paclitaxel (albumin-bound palitaxine, ABRAXANE), Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluorouracil (e.g., FOLFOX, FOLFIRI), IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, Torisel (temsirolimus), Inlyta (axitinib, Pfizer), Afinitor (everolimus, No... Combined administration of vartis, Nexavar (sorafenib, Onyx / Bayer), Votrient, Pazopanib, axitinib, IMA-901, AGS_003, cabozantinib, Vinflunine, Hsp90 inhibitors (e.g., apatorsin), Ad-GM-CSF (CT-0070), Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid (VELCADE), amrubicine, carfilzomib, pralatrexate, and / or enzastaurin.

[0221] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a PD-1 axis binding antagonist. PD-1 axis binding antagonists include, but are not limited to, PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PD-L1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PD-L2” include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand binding partner. In one specific aspect, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding to its binding partner. In one specific aspect, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits PD-L2 from binding to its binding partner. In one specific aspect, the PD-L2 binding partner is PD-1. The antagonist can be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of: MDX-1106 (nivolumab, OPDIVO), Merck... 3475 (MK-3475, pembrolizumab, KEYTRUDAWPCT-011 (Pidilizumab)). In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 binding antagonist... Selected from the following groups: YW243.55.S70, MPDL3280A, MEDI4736, and MDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007 / 005874. Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO2010 / 077634A1. MDX-1106, also known as MDX-1106-04, ON0-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in WO2006 / 121168.Merck3475, also known as MK-3475, SCH-900475, or pembrolizumab, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT, hBAT-1, or pidil izumab, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternative names for “MDX-1106” include MDX-1106-04, ON0-4538, BMS-936558, or nivoilumab. In some implementations, the anti-PD-1 antibody is nivolumab (CAS registration number: 946414-94-4).

[0222] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agonist targeting an activating costimulatory molecule. In some embodiments, the activating costimulatory molecule may include CD40, CD226, CD28, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist targeting the activating costimulatory molecule is an agonist antibody binding to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antagonist targeting an inhibitory costimulatory molecule. In some embodiments, the inhibitory costimulatory molecule may include CTLA-4 (also known as CD152), PD-1, 'I'IM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase. In some embodiments, the antagonist against the inhibitory co-stimulatory molecule is an antagonistic antibody against CTLA-4, PD-1, 'I'IM-3, BTLA, VISTA, LAG-3 (e.g., LAG-3-IgG fusion protein (IMP321)), B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase.

[0223] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antagonist against CTLA-4 (also known as CD152), such as a blocking antibody. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with ipiIimumab (also known as MDX-010, MDX-101, or...). Combined administration. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with tremelimumab (also known as ticilimumab or CP-675, 206). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antagonist against B7-H3 (also known as CD276), such as a blocking antibody. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with MGA271. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antagonist against TGFP, such as metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.

[0224] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a treatment comprising adoptive transfer of T cells expressing a chimeric antigen receptor (CAR), such as cytotoxic T cells or CTLs. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with UCART19. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with WT128z. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with KTE-C19 (Kite). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with CTL019 (Novartis). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a treatment comprising adoptive transfer of T cells containing a dominant-negative TGFI3 receptor, such as a dominant-negative TGFWI receptor. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a treatment comprising the HERCREEM regimen (see, for example, ClinicalTrials.gov Identifier NCT00889954).

[0225] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antagonist targeting CD19. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with MOR00208. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antagonist targeting CD38. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with daratumumab.

[0226] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agonist targeting CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with urelumab (also known as BMS-663513). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agonist targeting CD40, such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with CP-870893. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agonist targeting OX40 (also known as CD134), such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with different anti-OX40 antibodies (e.g., AgonOX). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agonist targeting CD27, such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with CDX-1127. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antagonist against indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as ID-MT).

[0227] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agonist targeting CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with urelumab (also known as BMS-663513). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agonist targeting CD40, such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with CP-870893 or R07009789. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agonist targeting OX40 (also known as CD134), such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agonist targeting CD27, such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with CDX-1127 (also known as varlilumab). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antagonist against indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT). In some embodiments, the IDO antagonist is the IDO antagonist shown in WO2010 / 005958 (the contents of which are explicitly recorded herein). In some embodiments, the IDO antagonist is 4-({2-[(aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazol-3-formamidinium (e.g., as described in Example 23 of WO2010 / 005958). In some embodiments, the IDO antagonist is...

[0228]

[0229] In some embodiments, the IDO antagonist is INCB24360. In some embodiments, the IDO antagonist is Indoximod (the D isomer of 1-methyl-tryptophan). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises mertansine or monomethylorisstatin E (MMAE). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A, RG7599, or Iifastuzumab vedotin). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or KADCYL). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an anti-MUC16 antibody-MMAE conjugate, DMUC5754A. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an anti-MUC16 antibody-MMAE conjugate, DMUC4064A. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antibody-drug conjugate targeting the endothelial angiotensin B receptor (EDNBR), such as an antibody against EDNBR conjugated with MMAE. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antibody-drug conjugate targeting the lymphocyte antigen 6 complex, locus E (Ly6E), such as an antibody against Ly6E conjugated with MMAE (also known as DLYE5953A). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with polatuzumab vedotin. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antibody-drug conjugate targeting CD30. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with ADCETRIS (also known as brentuximab vedotin). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with polatuzumab vedotin.

[0230] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an angiogenesis inhibitor. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antibody targeting VEGF, such as VEGF-A. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with bevacizumab (also known as...). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antibody against angiopoietin 2 (also known as Ang2). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with MEDI3617. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antibody against VEGFR2. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with ramucirumab. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with VEGF receptor fusion protein. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with aflibercept. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with ziv-aflibercept (also known as a VEGF trap or...). Combined administration. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a bispecific antibody against VEGF and Ang2. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with RG7221 (also known as vanucizumab). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an angiogenesis inhibitor and with a PD-1 axis binding antagonist (e.g., PD-1 binding antagonists such as anti-PD-1 antibodies, PD-L1 binding antagonists such as anti-PD-L1 antibodies, and PD-L2 binding antagonists such as anti-PD-L2 antibodies). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with bevacizumab and a PD-1 axis binding antagonist (e.g., PD-1 binding antagonists such as anti-PD-1 antibodies, PD-L1 binding antagonists such as anti-PD-L1 antibodies, and PD-L2 binding antagonists such as anti-PD-L2 antibodies). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with bevacizumab and MDX-1106 (nivolumab, OPDIVO). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with bevacizumab and Merck3475 (MK-3475, pembrolizumab, KEYTRUDA). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with bevacizumab and CT-011 (Pidilizumab). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with bevacizumab and YW243.55.S70. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with bevacizumab and MPDL3280A. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with bevacizumab and MEDI4736. In some implementations, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MDX-1105.

[0231] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antitumor agent. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agent targeting CSF-1R (also known as M-CSFR or CD115). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an anti-CSF-1R antibody (also known as IMC-CS4 or LY3022855). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an anti-CSF-1R antibody, RG7155 (also known as R05509554 or emactuzumab). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with interferon, such as interferon-α or interferon-γ. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with Roferon-A (also known as recombinant interferon α-2a). In some embodiments, the anti-human OX40 agonist antibody can be combined with GM-CSF (also known as recombinant human granulocyte-macrophage colony-stimulating factor, rhu GM-CSF, sargramostim, or... Combined administration. In some embodiments, anti-human OX40 agonist antibodies can be used with IL-2 (also known as aldesleukin or...). Combined administration. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with IL-12. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with IL-27. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with IL-15. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with ALT-803. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antibody targeting CD20. In some embodiments, the antibody targeting CD20 is onuzumab (also known as GAlO1 or...). Or rituximab. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an antibody targeting GITR. In some embodiments, the antibody targeting GITR is TRX518. In some embodiments, the antibody targeting GITR is MK04166 (Merck).

[0232] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of Bruton's tyrosine kinase (BTK). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with ibrutinib. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of vaccine dehydrogenase I (IDH1) and / or vaccine dehydrogenase 2 (IDH2). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with AG-120 (Agios).

[0233] In some implementations, the anti-human OX40 agonist antibody may be administered in combination with onuzumab and PD-1 axis binding antagonists (e.g., PD-1 binding antagonists such as anti-PD-1 antibodies, PD-L1 binding antagonists such as anti-PD-L1 antibodies, and PD-L2 binding antagonists such as anti-PD-L2 antibodies).

[0234] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the peptide cancer vaccine is a multivalent long peptide, multiple peptide, peptide mixture, hybrid peptide, or a dendritic cell vaccine via peptide pulses (see, for example, Yamada et al., Cancer Sci, 104:14-21, 2013). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an adjuvant. In some embodiments, the anti-human OX40 agonist antibody may be administered with a TLR-containing agent, such as Poly-ICLC (also known as...). Treatment with LPS, MPL, or CpG ODN may be administered in combination. In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with tumor necrosis factor (TNF)α. In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with IL-1. In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with HMGBl. In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with an IL-10 antagonist. In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with an IL-4 antagonist. In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with an IL-13 antagonist. In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with an IL-17 antagonist. In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with an HVEM antagonist. In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with an ICOS agonist (e.g., by administration of ICOS-L, or an agonist antibody against ICOS). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a treatment targeting CX3CL1. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a treatment targeting CXCL9. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a treatment targeting CXCL10. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a treatment targeting CCL5. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an LFA-I or ICAM1 agonist. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a selective protein agonist.

[0235] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of B-Raf. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with vemurafenib (also known as...). Combined administration. In some embodiments, anti-human OX40 agonist antibodies can be used in combination with dabrafenib (also known as...). Combined administration. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with encorafenib (LGX818).

[0236] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an EGFR inhibitor. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with erlotinib (also known as...). Combined administration. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an EGFR-T790M inhibitor. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with gefitinib. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with afatinib. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with cetuximab (also known as... Combined administration. In some embodiments, anti-human OX40 agonist antibodies can be used in combination with panitumumab (also known as...). Combined administration. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with rociletinib. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with AZD9291. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with MEK inhibitors, such as MEK1 (also known as MAP2K1) and / or MEK2 (also known as MAP2K2). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with cobimetinib (also known as CDC-0973 or XL-518). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with trametinib (also known as... Combined administration. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with binimetinib.

[0237] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of β-Raf (e.g., vemurafenib or dabrafenib) and an inhibitor of MEK (e.g., MEK1 and / or MEK2) (e.g., cobimetinib or trametinib). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of ERK (e.g., ERK1 / 2). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with GDC-0994. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of β-Raf, an inhibitor of MEK, and an inhibitor of ERK1 / 2. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of EGFR, an inhibitor of MEK, and an inhibitor of ERK1 / 2. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with one or more MAP kinase pathway inhibitors. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with CK127. In some implementations, anti-human OX40 agonist antibodies may be administered in combination with K-Ras inhibitors.

[0238] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of c-Met. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with onartuzumab (also known as MetMAb). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of anaplatinic lymphoma kinase (ALK). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with AF802 (also known as CH5424802 or alectinib). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with crizotinib. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with ceritinib. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of phosphatidylinositol 3-kinase (PI3K). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with buparlisib (BKM-120). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with pictilisib (also known as GDC-0941). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with buparlisib (also known as BKM-120). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with perifosine (also known as KRX-0401). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a delta-selective inhibitor of phosphatidylinositol 3-kinase (PI3K). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with taselisib (also known as GDC-0032). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with BYL-719. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an Akt inhibitor. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with MK2206. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with GSK690693. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with ipatasertib (also known as CDC-0068). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an mTOR inhibitor. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with sirolimus (also known as rapamycin).In some implementations, the anti-human OX40 agonist antibody can be combined with temsirolimus (also known as CCI-779 or...). Combined administration. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with everolimus (also known as RADOO1). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with ridaforoimus (also known as AP-23573, MK_8669, or deforolimus). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with OSI-027. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with AZD8055. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with INK128. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with a dual PI3K / mTOR inhibitor. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with XL765. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with GDC-0980. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with BEZ235 (also known as NVP-BEZ235). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with BGT226. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with GSK2126458. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with PF-04691502. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with PF-05212384 (also known as PKI-587).

[0239] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agent that selectively degrades estrogen receptors. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with GDC-0927. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of HER3. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with duligotuzumab. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of LSD1. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of MDM2. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of BCL2. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with venetoclax. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of CHK1. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with CDC-0575. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an inhibitor of the activated hedgehog signaling pathway. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with ERIVEDGE.

[0240] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with radiotherapy. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with gemcitabine. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with nab-paclitaxel (ABRAXANE). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with trastuzumab. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with TVEC. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with IL27. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with cyclophosphamide. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an agent that recruits T cells to the tumor. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with lirilumab (IPH2102 / BMS-986015). In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with Idelalisib. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with antibodies targeting CD3 and CD20. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with REGN1979. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with antibodies targeting CD3 and CD19. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with blinatumomab.

[0241] In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with an oncolytic virus. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with carboplatin and nab-paclitaxel. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with carboplatin and palitaxel. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with cisplatin and pemetrexed. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with cisplatin and gemcitabine. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with FOLFOX. In some embodiments, the anti-human OX40 agonist antibody may be administered in combination with FOLFIRI.

[0242] The combination therapies described above encompass combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations) and separate administration, in which case the antibody of the invention may be administered before, simultaneously with, and / or after the administration of other therapeutic agents and / or adjuvants. The antibody of the invention may also be used in combination with radiotherapy.

[0243] The antibodies (and any other therapeutic agents) of the present invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if intended for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending in part whether the administration is transient or long-term, dosage can be administered via any suitable route, such as injection, such as intravenous or subcutaneous injection. Various dosage schedules are covered herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0244] The antibodies of this invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of drug delivery, the method of administration, the administration schedule, and other factors known to a medical practitioner. The antibodies are not required but optionally formulated with one or more agents currently used for the prevention or treatment of the disease in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease or treatment, and the other factors described above. These are generally used at the same dosage and route of administration as described herein, or at about 1-99% of the dosage described herein, or at any dosage and route determined empirically / clinically as appropriate.

[0245] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prevention or treatment, previous therapy, the patient's clinical history and response to the antibody, and the attending physician's discretion. The antibody is suitable for administration to the patient in a single or series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 40 mg / kg can be administered to the patient as an initial candidate dose, whether, for example, by single or multiple separate administrations or by continuous infusion. Depending on the factors described above, a typical daily dose can range from about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, treatment will generally continue until the desired suppression of disease symptoms occurs, depending on the situation. Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about 2 to about 20 doses, or for example about 6 doses of antibody). A higher initial loading agent can be administered, followed by one or more lower doses. However, other dosing regimens may be useful. Progress in this therapy is easily monitored using routine techniques and assays.

[0246] It should be understood that the immunoconjugates of the present invention can be used to replace or supplement the anti-OX40 antibody to implement any of the above formulations or therapeutic methods.

[0247] Example

[0248] Example 1. Preparation of anti-OX40 antibody HFB10-1E1hG1

[0249] The heavy and light chain encoding nucleotide sequences (SEQ ID NO: 24 and 32) of the anti-OX40 antibody HFB10-1E1hG1 (Genewiz) were synthesized and cloned into the pFUSE vector. The plasmids containing the heavy and light chains were transiently co-transfected into 293F suspension cells at a 1:1 ratio using PEI to express the full-length antibody. After one week of culture, the cells were analyzed using the Superdex assay on the AKTA system. TM 200 Increase pre-packed column purification.

[0250] Example 2. Binding properties of HFB10-1E1hG1

[0251] Example 2.1 Binding properties of HFB10-1E1hG1 to OX40 protein

[0252] For the pre-test ELISA, 96-well plates were coated overnight with 5 μg / ml anti-OX40 antibody, including references 1, 2, 3, 4, HFB10-1E1hG1, and an isotype control. The next day, the plates were blocked in PBST with 1% BSA (Sangon Biotech, catalog number A600332-0100) at 37°C for 2 hours, and then a defined concentration of biotinylated recombinant OX40 protein was added.

[0253] The EC80 of the binding between biotinylated OX40 recombinant protein and anti-OX40 antibody was measured. The EC80 for reference 1 was 0.09 nM; for reference 2, it was 0.22 nM; for reference 3, it was 0.16 nM; for reference 4, it was 0.24 nM; for HFB10-1E1hG1, it was 0.71 nM; and for OX40L, it was 1.6 nM. Figure 1-1 As shown.

[0254] Example 2.2 Binding properties of HFB10-1E1hG1 with OX40 protein expressed on the surface of 293T cells

[0255] To overexpress targets including human-OX40 (Sinobiological, Cat.HG10481-UT), cynomolgus monkey-OX40 (Sinobiological, Cat.CG90846-UT), mouse-OX40 (Sinobiological, Cat.MG50808-UT), or human CD40 (Sinobiological, Cat.HG10774-UT), DNA plasmids encoding these targets were transiently transfected into 293T cells according to the manufacturer's instructions for Lipusectamine LTX Reagent and PLUS Reagent (Thermo, Cat.15338100). Cells were harvested 48 hours post-transfection for use. To determine binding affinity, the harvested cells were incubated with a defined concentration of the primary antibody HFB10-1E1hG1 at 4°C for 1 hour. Then, after washing twice with PBS, the cells were incubated with the secondary antibody goat anti-human IgG PE (1:200; Abcam, Cat.ab98596) at room temperature for 30 minutes. Detection was performed using a Beckman CytoFLEX flow cytometer.

[0256] The EC50 for the binding of HFB10-1E1hG1 to human OX40 protein expressed on the surface of 293T cells is 2 nM (MFI). For example... Figure 1-2 As shown.

[0257] The EC50 for the binding of HFB10-1E1hG1 to cynomolgus monkey OX40 protein expressed on the surface of 293T cells is 2.9 nM (MFI). Figure 1-3 As shown.

[0258] HFB10-1E1hG1 does not bind to mouse OX40 protein (MFI) expressed on the surface of 293T cells. Figure 1-4 As shown.

[0259] HFB10-1E1hG1 does not bind to human CD40 protein (MFI) expressed on the surface of 293T cells. For example... Figure 1-5 As shown.

[0260] HFB10-1E1hG1, Reference 1, Reference 2, Reference 3, and Reference 4 bind to the EC50 of human OX40 protein expressed on the surface of 293T cells, as shown in Figure 1. Figure 1-6 As shown, the EC50 of HFB10-1E1hG1 is 2.02 nM (MFI); the EC50 of Reference 1 is 2.67 nM (MFI); the EC50 of Reference 2 is 4.17 nM (MFI); the EC50 of Reference 3 is 1.92 nM (MFI); and the EC50 of Reference 4 is 2.11 nM (MFI).

[0261] HFB10-1E1hG1, Reference 1, Reference 2, Reference 3, and Reference 4 bind to the EC50 of cynomolgus monkey OX40 protein expressed on the surface of 293T cells, respectively. Figure 1-7 As shown, the EC50 of HFB10-1E1hG1 is 2.94 nM (MFI); the EC50 of Reference 1 is 2.91 nM (MFI); the EC50 of Reference 2 is 6.13 nM (MFI); the EC50 of Reference 3 is 2.57 nM (MFI); and the EC50 of Reference 4 is 3.54 nM (MFI).

[0262] Example 3. Agonistaltic activity of HFB10-1E1hG1

[0263] Example 3.1 Agonistaltic activity of HFB10-1E1hG1 in Jurkat reporter cells

[0264] Anti-OX40 antibodies can be divided into two categories based on their activation mechanism. The first category exhibits OX40 agonistic activity independent of Fc receptor cross-linking; the second category requires Fc receptor cross-linking to possess OX40 agonistic activity. Tumor tissue and surrounding draining lymph nodes contain more tumor-associated inflammatory cells, and Fc receptor FcγR2b accumulates more around tumor cells. Therefore, "cross-linked antibody" agonists possess higher tissue selectivity; the antibody can only produce significant agonistic effects in the tumor microenvironment, while its activity remains at a low level in normal tissues, thus improving the safety window for treatment.

[0265] Recombinant Jurkat reporter cells constitutively expressing the GFP gene under the control of the NF-κB response element were used. To determine the agonistic activity of HFB10-1E1hG1, 96-well plates were coated overnight with 5 μg / ml anti-human IgG Fc-specific (Sigma, Cat. SAB3701275). Definitive concentrations of HFB10-1E1hG1 were then added to 1×10⁻⁶ cells. 5 Jurkat reporter cells were added together in one well. In another experiment, 50 nM OX40L (Acrobiosystems, Cat.OXL-H52Q8) was added together with HFB10-1E1hG1 to determine the cooperative effect. After 24 hours of incubation, Jurkat reporter cells were harvested, and GFP-positive signals were detected using a Beckman CytoFLEX flow cytometer to indicate the agonistic activity of HFB10-1E1hG1 in Jurkat reporter cells.

[0266] The agonistic activity of HFB10-1E1hG1 is anti-human IgG cross-linked dependent. In the case of anti-human IgG cross-linking, the EC50 of HFB10-1E1hG1 is 2.9 nM (MFI of GFP). Figure 2-1 As shown, the EC50 of HFB10-1E1hG1 is significantly larger than its EC50 when cross-linked with anti-human IgG, without cross-linking. Figure 2-2 As shown. Without cross-linking with anti-human IgG, the trimeric OX40L recombinant protein alone can activate NF-κB signaling at EC50 = 45 nM (the MFI of GFP). Figure 2-2 As shown.

[0267] In the presence of anti-human IgG crosslinking, HFB10-1E1hG1 exhibits a cooperative agonistic effect with OX40L, and the addition of HFB10-1E1hG1 together with OX40L enhances the MFI of GFP compared to the individual components. Figure 2-3 As shown.

[0268] Example 3.2 Actuative activity of HFB10-1E1hG1 in primary CD4+ T cells

[0269] To determine the agonistic activity of HFB10-1E1hG1 in primary CD4+ T cells, 96-well plates were coated overnight with 0.3 μg / ml or 1 μg / ml anti-CD3 antibody (Thermo, Cat. 16-0037-81) and 5 μg / ml anti-human IgG Fc specific antibody (Sigma, Cat. SAB3701275). The following day, primary CD4+ T cells were harvested according to the CD4+ T cell isolation kit (Miltenyi, Cat. 130-045-101). Definitions of HFB10-1E1hG1 and 2 μg / ml anti-CD28 antibody (Thermo, Cat. 16-0289-81) were mixed with 1×10⁻⁶ HFB10-1E1hG1. 5 Primary CD4+ T cells were added together to one well. In another experiment, 50 nM OX40L (Acrobiosystems, Cat.OXL-H52Q8) was added together with HFB10-1E1hG1 to determine the cooperative effect. After 3 days of incubation, the IL-2 secretion level in the supernatant was measured according to the instructions of the Human IL-2 DuoSet ELISA Kit (R&D, Cat.DY202-05) to indicate the agonistic activity of HFB10-1E1hG1 in primary CD4+ T cells.

[0270] To determine the agonistic activity of HFB10-1E1hG1 in primary CD4+ T cells, purified CD4+ T cells were pre-activated with 1 μg / ml anti-CD3 antibody and 2 μg / ml anti-CD28 antibody. Plates were pre-coated with 5 μg / ml anti-human IgG to promote cross-linking with HFB10-1E1hG1. After three days of incubation, the agonistic activity of HFB10-1E1hG1 in primary CD4+ T cells was measured by IL-2 secretion, yielding an EC50 of 0.2 nM. Figure 2-4 As shown.

[0271] In primary CD4+ T cells, HFB10-1E1hG1 exhibited a cooperative agonistic effect with OX40L. Incubation of HFB10-1E1hG1 with 50 nM OX40L enhanced IL-2 secretion compared to the components alone. Figure 2-5 As shown.

[0272] Example 4. Pharmacokinetics of HFB10-1E1hG1

[0273] 384-well plates were coated overnight with 1 μg / ml F(ab')2 goat anti-human IgG Fc specific (Jackson IR, Cat. 109-006-098) in 30 μl / well PBS. After washing three times with PBST buffer, the plates were blocked at 37°C for 1 hour with blocking buffer containing 1 mM EDTA, 0.05% Tween, and 2% BSA in PBS. Then, starting with 1 / 150, mouse serum samples were added serially at 1 / 3 dilutions, at 15 μL / well, and incubated at 37°C for 2 hours. After washing three times with PBST buffer, the secondary antibody peroxidase-goat anti-human IgG (Jackson IR, Cat. 109-035-003) was added at a concentration of 1 / 5000 and incubated at 37°C for 0.5 hours. Then, TMB substrate (Biolegend, Cat. 421101) was added and incubated for another 15 minutes. Finally, ELISA stop solution (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was added. The plate was read at 450 nm using a Multiskan Sky Microplate Spectrophotometer (ThermoFisher).

[0274] hOX40 knock-in mice (131, 132, and 133, purchased from the Shanghai Southern Model Organism Research Center) were administered 10 mg / kg HFB10-1E1hG1 intravenously. Serum was collected at 1 hour, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 196 hours after administration. The half-life of HFB10-1E1hG1 in the serum of hOX40-KI mice was determined to be 24 hours. Figure 3-1As shown. The data points at hour 0 are, from top to bottom, data points 131, 132, and 133.

[0275] Example 5. In vivo antitumor efficacy of HFB10-1E1hG1

[0276] hOX40 knock-in mice were purchased from the Shanghai Southern Model Organism Research Center. After 5 days of quarantine, they were injected with 100 μl of 8×10⁻⁶ PBS. 5 One MC38 tumor cell (provided by Professor Zhang Hongkai of Nankai University) was subcutaneously injected into each mouse. When the tumor size reached 70-100 mm... 3 Anti-OX40 antibody treatment was initiated by intraperitoneal administration of anti-OX40 antibody in 100 μl PBS at a dose of 10 mg / kg every 3 days (q3dx5). Tumor size and mouse weight were measured twice weekly. Tumor size was measured in both directions using calipers and expressed in mm using the following formula. 3 Volume: V = 0.5a × b 2 Where a and b are the long and short diameters of the tumor, respectively.

[0277] Day 7: MC38 tumor cell inoculation in 35 8-week-old mice

[0278] - Obtained MC38 cells from Professor Zhang Hongkai

[0279] - Each mouse was inoculated with 8×10 5 MC38 cells

[0280] Day 0: The average tumor size was 75 mm. 3 (40-120mm 3 )

[0281] - 5 mice per group, 4 groups:

[0282] PBS

[0283] • Reference antibody 1

[0284] ·HFB10-1E1hG1

[0285] • Reference antibody 2

[0286] -Initiated by intraperitoneal administration of Q3D×5 antibody at a dose of 10 mg / kg.

[0287] Day 18: Tumor size in the PBS control group reached 2000 mm. 3 The mice were euthanized.

[0288] -Tissues: Blood, LN, Liver, Spleen, Tumors

[0289] -Phenotype analysis: T cells CD3 / CD4 / CD8; Tregs CD4 / CD25 / Foxp3; NK cells CD3 / CD16 / CD56

[0290] Experiments showed that HFB10-1E1hG1 significantly inhibited tumor growth compared to the PBS control, and did not exhibit any side effect of causing a significant decrease in body weight in mice. Figure 4-1 and Figure 4-2 As shown. In Figure 4-1 In the image, the rightmost data points, from top to bottom, represent PBS, reference antibody 1, HFB10-1E1hG1, and reference antibody 2. Figure 4-2 In the middle, the second data point from left to right, from top to bottom, represents PBS, reference antibody 1, reference antibody 2, and HFB10-1E1hG1.

[0291] A dose-response efficacy study of the antibody was conducted in hOX40 knock-in mice. Specific groupings are as follows:

[0292] Group 1: PBS: Q3D x 5, intraperitoneal;

[0293] Group 2: HFB10-1E1hG1: 1mg / kg, intraperitoneal, Q3 / 4D x 5, intraperitoneal;

[0294] Group 3: HFB10-1E1hG1: 0.1 mg / kg, intraperitoneal, Q3 / 4D x 5, intraperitoneal;

[0295] Group 4: Reference antibody 1: 1 mg / kg, intraperitoneal, Q3D x 5, intraperitoneal.

[0296] A total of 20 hOX40 knock-in mice were used, divided into 4 groups of 5 mice each.

[0297] Mice were inoculated with MC38 tumor cells. The average tumor size was 75 mm. 3 Starting at day 0, the mice were injected with antibodies five times on days 3, 6, 10, and 13. Tumor size and mouse weight were measured twice weekly for up to three weeks or until the tumor size exceeded 2000 mm. 3 .

[0298] The results of tumor size and body weight responses in mice at different HFB10-1E1hG1 doses are shown in [Figure 1]. Figure 4-3 and Figure 4-4 Experiments showed that 1 mg / kg of HFB10-1E1hG1 exhibited the best in vivo antitumor efficacy.

[0299] Example 6. In vitro characterization of the spreadability of HFB10-1E1hG1

[0300] Example 6.1 Accelerated stability test of HFB10-1E1hG1

[0301] The antibody sample (HFB10-1E1hG1, 3.1 mg / mL, batch number #CP181130004) was concentrated to 10 mg / mL (batch number #JW20181203, batch number #20190624) using a centrifugal filtration device (Millipore, Cat#UFC503096). An appropriate amount of the concentrated antibody sample was transferred to a clean 600 μl tube and incubated at 25°C and 40°C for 7, 14, and 30 days, respectively.

[0302] The incubated samples were analyzed by SEC-HPLC and SDS-PAGE:

[0303] For SEC-HPLC, 50 μg of each treated sample was injected and run at a flow rate of 0.7 mL / min, 40 min / test in 1xPBS buffer, pH 7.4 (diluted with Milli-Q pure water from 10xPBS buffer (Sangon, Cat#E607016-0500)). Absorbance was measured at UV 280 nm (untreated samples stored at 4 °C were also loaded for analysis). The results are shown in Table 1. No significant increase in aggregation or degradation peaks of HFB10-1E1hG1 was observed on the SEC curves after incubation at 25 °C and 40 °C, even up to 30 days, indicating good stability of HFB10-1E1hG1 under these treatment conditions. The slight degradation observed after 30 days of incubation at 40 °C may indicate its instability with prolonged incubation at higher temperatures.

[0304] Table 1 SEC Analysis - Temperature

[0305]

[0306]

[0307] For SDS-PAGE, 4 μg of each treated sample was loaded onto 4–20% gradient gels under both non-reducing and reducing conditions. The gels were run in 150V tris-glycine buffer for 1 hour and stained in staining solution (TaKaRa, Cat#T9320A) for over 1 hour, then destained several times in distilled water, and images were captured on a white light plate (untreated samples stored at 4°C were also loaded for analysis). Figure 5-1 As shown.

[0308] After incubation at 25℃ and 40℃ for 30 days, no obvious aggregation or degradation bands of HFB10-1E1hG1 were observed on the SDS-PAGE images, indicating that HFB10-1E1hG1 has good stability under such treatment conditions. After incubation at 40℃ for 30 days, the observation of non-reduced gel and slight degradation bands on the aggregated bands on the reduced gel may indicate its instability under prolonged incubation at high temperatures.

[0309] Example 6.2 Degradation experiment of HFB10-1E1hG1

[0310] 1. Low pH pressure test:

[0311] Transfer an appropriate amount of antibody (HFB10-1E1hG1, 3.1 mg / mL) to a 600 μL tube, then add 2 M acetic acid at a ratio of 1:20 (v / v, acid relative to antibody sample, final pH adjusted to approximately 3.5). Mix the sample thoroughly and incubate at room temperature for 0, 3, or 6 hours. After incubation, adjust the pH of the antibody solution to 7.4 using neutralization buffer (add 1 M Tris-HCl, pH 9.0 to the incubated sample at a ratio of 13:100, v / v). Analyze the sample by SEC-HPLC and SDS-PAGE as described above (see Accelerated Stability Experiments; note: Untreated samples stored at 4°C were also loaded for analysis).

[0312] 2. High pH pressure test:

[0313] Transfer an appropriate amount of antibody (HFB10-1E1hG1, 3.1 mg / mL) to a 600 μl tube, then add 1 M Tris-HCl (pH 8.5) at a ratio of 1:25 (v / v, final pH adjusted to approximately 8.5). Mix the sample thoroughly and incubate at room temperature for 0 or 6 hours. Analyze the sample as described above by SEC-HPLC and SDS-PAGE (analysis under reduction conditions only).

[0314] SEC analysis results are shown in Table 2. After incubation for up to 6 hours in the corresponding pH 3.5 and pH 8.5 solutions, no increase in aggregation or degradation peaks of HFB10-1E1hG1 was observed on the SEC curves, indicating that HFB10-1E1hG1 has good stability under these treatment conditions.

[0315] Table 2 SEC Analysis - pH

[0316]

[0317] SDS-PAGE analysis, such as Figure 5-2As shown, after incubation in low and high pH buffers for up to 6 hours, no change in HFB10-1E1hG1 was observed on the SDS-PAGE gel, indicating that HFB10-1E1hG1 has good stability under these treatment conditions.

[0318] Example 6.3 Oxidative stress experiment of HFB10-1E1hG1

[0319] Transfer an appropriate amount of antibody (HFB10-1E1hG1, 3.1 mg / mL) to a 600 μl tube, then add H2O2 (0.1% and 1%, respectively) or t-BHP (to a final concentration of 0.1%). Mix the sample thoroughly and incubate at room temperature for 0 or 6 hours. Analyze the sample by SEC-HPLC and SDS-PAGE as previously described.

[0320] Note: 1) The untreated sample stored at 4℃ was used for analysis; 2) The SEC running buffer was prepared in-house and consisted of 100mM NaH2PO4, 150mM NaCl, and pH 6.8.

[0321] SEC analysis is shown in Table 3. After incubation for 6 hours in the corresponding 0.1% H2O2, 1% H2O2 and 0.1% t-BHP (tert-butyl hydroperoxide) solutions, no significant change in HFB10-1E1hG1 was observed on the SEC curve, indicating that HFB10-1E1hG1 has good stability under such treatment conditions.

[0322] Table 3 SEC Analysis - Oxidative Stress

[0323]

[0324]

[0325] SDS-PAGE analysis, such as Figure 5-3 As shown. After incubation for 6 hours in the corresponding 0.1% H2O2, 1% H2O2 and 0.1% t-BHP (tert-butyl hydroperoxide) solutions, no significant changes in HFB10-1E1hG1 were observed on the SDS-PAGE gel, except for slight degradation bands on the non-reducing gel, indicating that HFB10-1E1hG1 has good stability under such treatment conditions.

[0326] Example 6.4 Freeze-thaw experiment of HFB10-1E1hG1

[0327] The antibody sample (HFB10-1E1hG1, 3.1 mg / mL, batch number #CP181130004) was concentrated to 10 mg / mL (batch number #JW20181203) using a centrifugal filter (Millipore, Cat#UFC503096). An appropriate amount of the concentrated antibody sample was transferred to a clean 600 μl tube (3x tubes), and the sample was frozen in liquid nitrogen for 2 minutes, then thawed in a water bath at room temperature. This procedure was repeated two, four, or more times.

[0328] As previously described, the samples were analyzed by SEC-HPLC and SDS-PAGE. Note: 1) The untreated samples stored at 4°C were used for analysis; 2) The SEC run buffer was prepared in-house and consisted of 100 mM NaH2PO4, 150 mM NaCl, and pH 6.8.

[0329] For DSF-based thermal stability analysis: Following the manufacturer's instructions, 3 μg of each treated sample was added to each 25 μl reaction of the ProteoStat assay kit (Enzo Life Sciences, Cat#ENZ-51027-K400) in PCR plates (Bio-Rad plate, Cat#HSP9655; Bio-Rad membrane, Cat#MSB1001). The heating program on the Bio-Rad PCR instrument (C1000touch, CFX96 real-time system) was set as follows: 25°C for 2 minutes, increasing by 0.5°C every 10 seconds to 95°C. Fluorescence absorbance was read using Texas Red mode. The Tm value was correlated with the minimum point of -dF / dT.

[0330] The SEC analysis is shown in Table 4. The DSF analysis is shown in Table 5. The SEC analysis showed that no significant changes in HFB10-1E1hG1 were observed on the SEC curve after up to 5 cycles of freezing / thawing. The DSF analysis showed that the Tm value of HFB10-1E1hG1 did not change significantly after the same treatment, indicating that HFB10-1E1hG1 has good stability under these treatment conditions.

[0331] Table 4 SEC Analysis - Freeze-Thaw

[0332]

[0333] Table 5 DSF Analysis - Freeze-Thaw

[0334]

[0335] SDS-PAGE analysis, such as Figure 5-4As shown, no significant changes in HFB10-1E1hG1 were observed on the SDS-PAGE gel after up to 5 cycles of freeze / thaw treatment, indicating that HFB10-1E1hG1 has good stability under these treatment conditions.

[0336] Example 7

[0337] It has been demonstrated that binding of agonistic antibodies to OX-40 leads to receptor downregulation, but this has been observed in vitro and in clinical trials (Wang et al., Cancer Research 2019). It has been further hypothesized that the loss of target expression following the first injection observed in patients may limit the success of OX-40 agonistic antibodies in clinical trials (Wang et al., Cancer Research 2019). Using ex vivo activated naïve T cells isolated from PBMCs, this invention shows that treatment with HFB10-1E1hG1 results in less reduction of post-treatment OX-40 levels compared to baseline. The unique binding epitope and optimized binding kinetics minimize target degradation, thereby avoiding loss of target expression after the first injection, thus allowing for sustained drug administration in patients.

[0338] References

[0339] Wang,R.,Gao,C.,Raymond,M.,Dito,G.,Kabbabe,D.,Shao,X.,Hilt,E.,Sun,Y.,Pak,I.,Gutierrez,M.,Melero,I.,Spreafico,A.,Carvajal, R.,Og,M.,Olszanski,A.,Milburn,C.,Thudium,K.,Yang,Z.,Feng,Y.,Fracasso,P.,Korman,A.,Aanur,P.,Huang,S.,Quigley,M.(2019).An Integrative Approach to Inform Optimal Administration of OX40 Agonist Antibodies in Patients with Advanced Solid Tumors,ClinicalCancer Research, https: / / dx.doi.org / 10.1158 / 1078-0432.CCR-19-0526.

[0340] Although the principles of the invention have been described above in conjunction with preferred embodiments, it should be clearly understood that this description is by way of example only and is not intended to limit the scope of the invention.

[0341] sequence list

[0342]

[0343]

[0344]

[0345]

[0346]

Claims

1. An isolated anti-OX40 antibody or its antigen-binding fragment, comprising: The heavy chain variable region comprises a heavy chain CDR1 domain consisting of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 domain consisting of the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 domain consisting of the amino acid sequence shown in SEQ ID NO: 3; and The light chain variable region comprises a light chain CDR1 domain consisting of the amino acid sequence shown in SEQ ID NO: 9, a light chain CDR2 domain consisting of the amino acid sequence shown in SEQ ID NO: 10, and a light chain CDR3 domain consisting of the amino acid sequence shown in SEQ ID NO:

11.

2. The anti-OX40 antibody or its antigen-binding fragment according to claim 1, comprising: The heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4, or the heavy chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 4; and The light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 12, or the light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:

12.

3. The anti-OX40 antibody or its antigen-binding fragment according to claim 2, further comprising a heavy chain constant region and a light chain constant region; The heavy chain constant region is a heavy chain constant region composed of the amino acid sequence shown in SEQ ID NO: 5, or a heavy chain constant region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 5; and / or The light chain constant region is a light chain constant region composed of the amino acid sequence shown in SEQ ID NO: 13, or a light chain constant region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:

13.

4. The anti-OX40 antibody or its antigen-binding fragment according to claim 3, further comprising a heavy chain signal peptide linked to the heavy chain variable region and / or a light chain signal peptide linked to the light chain variable region; The heavy chain signal peptide is a heavy chain signal peptide composed of the amino acid sequence shown in SEQ ID NO: 6, or a heavy chain signal peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 6; and / or The light chain signal peptide is a light chain signal peptide composed of the amino acid sequence shown in SEQ ID NO: 14, or a light chain signal peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:

14.

5. The anti-OX40 antibody or its antigen-binding fragment according to claim 1, wherein it is an IgG antibody or its antigen-binding fragment.

6. The anti-OX40 antibody or its antigen-binding fragment according to claim 5, wherein it is an IgG1 antibody or its antigen-binding fragment.

7. The anti-OX40 antibody or its antigen-binding fragment according to claim 1, wherein it is a monoclonal antibody or its antigen-binding fragment.

8. The anti-OX40 antibody or its antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv or scFv.

9. A nucleic acid encoding an anti-OX40 antibody or an antigen-binding fragment thereof according to any one of claims 1-8.

10. The nucleic acid according to claim 9, comprising the heavy chain variable region nucleotide coding sequence shown in SEQ ID NO: 20 and / or the light chain variable region nucleotide coding sequence shown in SEQ ID NO:

28.

11. The nucleic acid according to claim 10, wherein the nucleic acid further comprises the heavy chain constant region nucleotide coding sequence shown in SEQ ID NO: 21 and / or the light chain constant region nucleotide coding sequence shown in SEQ ID NO:

29.

12. An expression vector comprising the nucleic acid according to any one of claims 9-11.

13. A host cell comprising the nucleic acid according to any one of claims 9-11 or the expression vector according to claim 12.

14. A method for producing an anti-OX40 antibody or an antigen-binding fragment thereof according to any one of claims 1-8, comprising culturing a host cell according to claim 13 under conditions suitable for expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the culture medium.

15. A pharmaceutical composition comprising an anti-OX40 antibody or an antigen-binding fragment thereof according to any one of claims 1-8, or a nucleic acid according to any one of claims 9-11, or an expression vector according to claim 12, and a pharmaceutically acceptable vector.

16. The pharmaceutical composition of claim 15, for treating cancer, wherein the cancer is selected from squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, bladder cancer, urethral cancer, breast cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, melanoma, head and neck cancer, and related metastases.

17. The pharmaceutical composition according to claim 16, wherein the squamous cell carcinoma is selected from epithelial squamous cell carcinoma, the lung cancer is selected from small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung, and the gastric cancer is selected from gastrointestinal cancer and gastrointestinal stromal carcinoma.

18. Use of the anti-OX40 antibody or its antigen-binding fragment according to any one of claims 1-8, or the pharmaceutical composition according to claim 15, in the preparation of a medicament for treating cancer, wherein the cancer is selected from gastric cancer, pancreatic cancer, bladder cancer, liver cancer, colorectal cancer, melanoma, and related metastases.

19. The use according to claim 18, wherein the gastric cancer is selected from gastrointestinal cancer and gastrointestinal stromal carcinoma.

20. The anti-OX40 antibody or its antigen-binding fragment according to any one of claims 1-8, which is used for one or more of the following: inhibiting Treg function, killing cells expressing OX40, enhancing effector T cell function and / or enhancing memory T cell function, reducing tumor immunity, enhancing T cell function and / or reducing cells expressing OX40.

21. Use of the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the pharmaceutical composition according to claim 15, in the preparation of a medicament for treating one or more of the following: inhibiting Treg function, killing cells expressing OX40, enhancing effector T cell function and / or enhancing memory T cell function, reducing tumor immunity, enhancing T cell function and / or reducing cells expressing OX40.

22. A pharmaceutical composition comprising an anti-OX40 antibody or an antigen-binding fragment thereof according to any one of claims 1-8, or a pharmaceutical composition according to claim 15, and one or more other therapeutic agents.

23. A kit comprising an anti-OX40 antibody or an antigen-binding fragment thereof according to any one of claims 1-8, or a pharmaceutical composition according to claim 15.

24. The kit according to claim 23, further comprising a drug delivery device.

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