Anti-OX40 antibody and its use

Abstract

Anti-OX40 antibodies, compositions comprising anti-OX40 antibodies, nucleic acids encoding anti-OX40 antibodies, methods for preparing anti-OX40 antibodies, and uses of anti-OX40 antibodies are provided.

Description

Technical Field

[0001] The present invention relates to novel anti-OX40 antibodies, compositions containing anti-OX40 antibodies, nucleic acids encoding anti-OX40 antibodies, methods for preparing anti-OX40 antibodies, and uses of anti-OX40 antibodies.

Background Art

[0002] The anti-tumor immune response of solid tumor patients has been enhanced by treatment with certain biological agents. For example, two anti-PD-1 monoclonal antibodies, nivolumab (OPDIVO®) and pembrolizumab (KEYTRUDA®), are approved in the United States and the EU as therapeutic agents for diseases such as unresectable or metastatic melanoma and metastatic non-small cell lung cancer. Treatment of patients with these drugs has resulted in an anti-tumor response, as measured by improvement in progression-free survival and / or overall survival. To complement existing standard treatments, more cancer treatment products and methods are still needed in the art.

[0003] PD-1 and CTLA-4 play an immunosuppressive role in the process of T cell activation, thereby inhibiting the immune killing function of T cells against tumor cells. Therefore, by blocking monoclonal antibodies against these two targets, this immunosuppression can be reduced and the anti-tumor immune function of T cells can be restored. In addition to such inhibitory immune checkpoint molecules, activating immune checkpoint molecules has gradually become a new target for pharmaceutical development.

[0004] Activated immune checkpoint molecules mainly refer to T cell co-stimulatory receptors, which are molecules that transmit co-stimulatory signals in T cell activation, and these belong to the tumor necrosis factor receptor (TNFR) family. In particular, receptors such as OX40, CD40, 4-1BB, and GITR can be used to regulate the proliferation, activation, and differentiation of T cells.

[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 that is expressed 24 to 72 hours after activation. Its ligand, OX40L (also known as CD252 and TNFSF4), is not expressed on resting antigen-presenting cells but is expressed after activation. The expression of OX40 depends on the complete activation of T cells.

[0006] OX40 binds to its ligand OX40L and transmits a co-stimulatory signal. The interaction between OX40 and OX40L can recruit TNFR-associated molecules (TRAF) to the intracellular domain of OX40 to form a signaling complex that includes IKKα and IKKβ, as well as PI3k and PKB (Akt). OX40 can also cooperate with TCR signaling to increase intracellular Ca 2+ and thereby promote the nuclear translocation of NFAT. OX40 activates the classical NF-κB1 pathway or the non-classical NF-κB2 pathway, the PI3k / PKB and NFAT pathways, thereby regulating genes for T cell division and survival while promoting the transcription of cytokine genes and the expression of cytokine receptors. It is essential for cell survival. OX40 signaling can cause the downregulation of CTLA-4 and Foxp3.

[0007] When OX40 binds to its ligand OX40L, the immune response capacity, including the following, is enhanced: 1. increased survival and proliferation of effector T cells and memory T cells, as well as increased secretion of cytokines (such as IL-2, IL-4, IL-5, IFN-γ, etc.); 2. reduced immunosuppressive activity of regulatory T cells, further amplifying the effect of T cell activation. In the tumor microenvironment, immune activation may lead to the expression of OX40. This can promote the activation and proliferation of effector T cells and suppress regulatory T cells, resulting in a complex anti-tumor immune response. Currently, several clinical projects involving anti-OX40 antibodies in cancer treatment can be obtained on the website of Clinical Trials.

[0008] In order to provide new options for cancer treatment, more novel anti-OX40 antibodies are needed in the art.

Summary of the Invention

[0009] The present invention addresses the above need by providing a novel anti-OX40 antibody that can specifically bind to and activate 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 structural domain shown in SEQ ID NO: 1, a heavy chain CDR2 structural domain shown in SEQ ID NO: 2, and a heavy chain CDR3 structural 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 an antigen-binding fragment thereof described herein and an additional therapeutic agent. Preferably, the anti-OX40 antibody or an antigen-binding fragment thereof is linked to the additional therapeutic agent by a linker.

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

[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 described herein or the expression vector described herein.

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

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

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

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

[0019] In one aspect, the present invention provides for 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 drug for treating cancer.

[0020] In one aspect, the present invention 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 performing one or more of the following: inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), killing cells that express OX40 (e.g., cells that express 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 depleting OX40 - expressing cells.

[0021] In one aspect, the present invention 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 the preparation of a drug for performing one or more of the following: inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), killing cells that express OX40 (e.g., cells that express 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 depleting OX40 - expressing cells.

[0022] In one aspect, the present invention provides a pharmaceutical combination comprising the anti - OX40 antibody or antigen - binding fragment thereof described herein, 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 an anti - OX40 antibody or an antigen - binding fragment thereof described herein, or an antibody - drug conjugate described herein, or a pharmaceutical composition described herein, preferably further comprising a drug delivery device.

[0024] In some embodiments, the antibodies of the present invention contain one or more point mutations in their amino acid sequences, and the point mutations are designed to improve the developability of the antibody. In preferred embodiments, the one or more point mutations render the antibody more stable during expression in host cells, during purification during manufacture and / or formulation, and / or during administration to a subject. In preferred embodiments, the one or more point mutations reduce the likelihood of the antibody aggregating during manufacture and / or formulation. In some embodiments, the present invention provides therapeutic antibodies with minimized or reduced developability issues. For example, hydrophobicity and / or optimized charge can be removed or reduced by substituting one or more amino acids in its sequence (e.g., in one or more of its CDRs).

[0025] One embodiment of the present invention is a monoclonal antibody or an antigen - binding fragment thereof, which can specifically bind to the epitope of OX40 corresponding to amino acid residues 56 - 74 of SEQ ID NO: 33 (SEQ ID NO: 34: CSRSQNTVCRPCGPGFYN).

[0026] Another embodiment of the present invention is a monoclonal antibody or an antigen - binding fragment thereof that can specifically bind to OX40, and the binding of the monoclonal antibody or its antigen - binding fragment does not interfere with the binding of OX40 ligand to OX40.

[0027] Another embodiment of the present invention is a monoclonal antibody or an antigen - binding fragment thereof, and the binding of the monoclonal antibody or its antigen - binding fragment does not interfere with the binding of OX40 ligand to OX40 in a trimeric or multimeric aggregated state.

[0028] Another embodiment of the present invention is a monoclonal antibody or an antigen-binding fragment thereof that can specifically bind to OX40, and the binding of the antibody to OX40 results in agonist signaling together with endogenous OX40 ligand signaling.

[0029] Another embodiment of the present invention is a monoclonal antibody or an antigen-binding fragment thereof of any one of the above embodiments, and OX40 is human OX40.

[0030] Another embodiment of the present invention is a monoclonal antibody or an antigen-binding fragment thereof of the above embodiments, and the epitope includes SEQ ID NO: 2.

[0031] Another embodiment of the present invention is a monoclonal antibody or an antigen-binding fragment thereof of the above embodiments, and the antibody binds to OX40 with a K of about 1 nM to about 10 nM D and binds to OX40.

[0032] Another embodiment of the present invention is a monoclonal antibody or an antigen-binding fragment thereof according to the above embodiments, and the binding of the monoclonal antibody or its antigen-binding fragment to OX40 does not down-regulate OX40 expression or reduce the amount of OX40 present on the cell surface.

[0033] Another embodiment of the present invention is a method of treating cancer with a monoclonal antibody, wherein the cancer is a solid tumor or a non-solid tumor, or the cancer is a cancer having OX40 expression on the cell surface of tumor-infiltrating T cells.

[0034] Another embodiment of the present invention is a method of detecting OX40 in a sample, the method comprising contacting the sample with the monoclonal antibody or an antigen-binding fragment thereof of the present invention.

[0035] Another embodiment of the present invention is a method of determining the OX40 level in a subject, the method comprising a) obtaining a sample from a control, and b) contacting the sample with the monoclonal antibody of the present invention or an antigen-binding fragment thereof; c) determining the level of OX40 in the subject.

[0036] The sample is a tissue sample, a blood sample, or a cancer tissue sample.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0038] 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 structural domain shown in SEQ ID NO: 1, a heavy chain CDR2 structural domain shown in SEQ ID NO: 2, and a heavy chain CDR3 structural 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.

[0039] In one embodiment, the anti - OX40 antibody or antigen - binding fragment thereof described herein comprises a 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% homology with SEQ ID NO: 4, and a 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% homology with SEQ ID NO: 12.

[0040] In one embodiment, the anti - OX40 antibody or antigen - binding fragment thereof 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% homology with 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with SEQ ID NO: 13.

[0041] In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof 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, and 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% homology with 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 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% homology with SEQ ID NO: 14.

[0042] In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein is an IgG antibody or antigen-binding fragment thereof, and preferably, an IgG1 antibody or antigen-binding fragment thereof.

[0043] In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein is a monoclonal antibody or antigen-binding fragment thereof.

[0044] In one embodiment, the antigen-binding fragment thereof described herein is Fab, Fab’, F(ab’)2, Fv, scFv or sdAb.

[0045] In one aspect, the present invention provides an antibody-drug conjugate comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein and an additional therapeutic agent. Preferably, the anti-OX40 antibody or antigen-binding fragment thereof is linked to the additional therapeutic agent via a linker.

[0046] In one aspect, the present invention provides a nucleic acid encoding an anti-OX40 antibody or an antigen-binding fragment thereof described herein.

[0047] 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, and 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.

[0048] In one aspect, the present invention provides an expression vector comprising the nucleic acid described herein.

[0049] In one aspect, the present invention provides a host cell comprising the nucleic acid described herein or the expression vector described herein.

[0050] In one aspect, the present invention provides a method for preparing the anti-OX40 antibody or an antigen-binding fragment thereof described herein, comprising culturing the host cell described herein under conditions suitable for expressing the antibody or an antigen-binding fragment thereof, and recovering the expressed antibody or an antigen-binding fragment thereof from the culture medium.

[0051] In one aspect, the present invention provides a pharmaceutical composition comprising the anti-OX40 antibody or an antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the nucleic acid described herein, or the expression vector described herein, and a pharmaceutically acceptable carrier.

[0052] In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein is used for the treatment of 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, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, 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, melanoma, disseminated superficial melanoma, malignant lentigo melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal angiogenesis associated with keloid, edema (e.g., associated with brain tumor), as well as Meigs syndrome, brain tumor and brain cancer, head and neck cancer, and associated metastases.

[0053] In one aspect, the present invention provides a method for the treatment of cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-OX40 antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein, or a pharmaceutical composition described herein, 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, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, 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, melanoma, disseminated superficial melanoma, malignant lentigo melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma and B cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal angiogenesis associated with keloid, edema (e.g., associated with brain tumor), and Meigs syndrome, brain tumor and brain cancer, head and neck cancer, and associated metastases.

[0054] In one aspect, the present invention provides the use of an anti-OX40 antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein, or a pharmaceutical composition described herein, in the preparation of a drug 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, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal tumor), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, 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, melanoma, diffuse superficial melanoma, malignant melanoma of the lentigo type, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal angiogenesis associated with keloid, edema (e.g., associated with brain tumor), and Meigs syndrome, brain tumor and brain cancer, head and neck cancer, and associated metastases.

[0055] In one aspect, the present invention provides the use of an anti-OX40 antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein, or a pharmaceutical composition described herein, 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 depleting OX40-expressing cells.

[0056] In one aspect, the present invention provides the use of an anti - OX40 antibody or an antigen - binding fragment thereof described herein, or an antibody - drug conjugate described herein, or a pharmaceutical composition described herein, in the preparation of a drug for one or more of: 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 depleting OX40 - expressing cells.

[0057] In one aspect, the present invention provides a pharmaceutical combination comprising an anti - OX40 antibody or an antigen - binding fragment thereof described herein, or an antibody - drug conjugate described herein, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0058] In one aspect, the present invention provides a kit comprising an anti - OX40 antibody or an antigen - binding fragment thereof described herein, or an antibody - drug conjugate described herein, or a pharmaceutical composition described herein, and preferably further comprising a drug delivery device.

[0059] Some embodiments of the present invention provide agonist antibodies against OX - 40. It causes no or little down - regulation of the OX - 40 receptor as compared to other agonist anti - OX - 40 antibodies. The lack or reduction of receptor down - regulation may be due to the fact that the agonist antibodies provided by the present invention can recognize their epitopes. The agonist antibodies provided by the present invention may also have optimized binding kinetics, particularly as compared to other agonist anti - OX - 40 antibodies known in the art.

[0060] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form further embodiments of the present invention. These and other aspects of the present invention will be apparent to those of ordinary skill in the art. These and other embodiments of the present invention are described in further detail below.

[0061] Two polynucleotide or polypeptide sequences are said to be "identical" when the sequences of nucleotides or amino acids in the two sequences are the same when described for maximum alignment of enantiomers. Comparison between two sequences is typically performed by comparing the sequences over a comparison window and identifying and comparing local regions of sequence similarity. As used herein, a "comparison window" refers to a segment having at least about 20 (usually about 30 to about 75, or about 40 to about 50) contiguous positions, after optimally aligning the two sequences, one sequence can be compared to a reference sequence having the same number of contiguous positions.

[0062] Optimal alignment of the arrays for comparison can be performed using the suite of programs of the bioinformatics software (Inc., Madison, WI) with default parameters. This program implements several alignment schemes described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Supplement 3, pp. 345 to 358 pages; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626 - 645, Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5:151 - 153; Myers, E.W. and Muller W., 1988, CABIOS 4:11 - 17; Robinson, E.D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406 - 425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Acad. Sci. USA 80:726 - 730.

[0063] In some embodiments, "percent sequence identity / similarity" is determined by comparing two sequences optimally aligned over a comparison window having at least 20 positions, where for optimal alignment of the two sequences, a portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions of 20% or less, typically 5% - 15%, or 10% - 12% as compared to the reference sequence (excluding additions or deletions). The percentage can be calculated as follows: Determine the number of positions in both sequences where the same nucleic acid base or amino acid residue is present to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the reference sequence (i.e., the comparison window size), and then multiply the result by 100 to obtain the percent sequence identity / similarity.

[0064] Alternatively, the variant can also be substantially homologous to the native gene or a part or complement thereof. These polynucleotide variants can hybridize to the naturally occurring DNA sequence encoding the native antibody (or complementary sequence) under moderately stringent conditions.

[0065] Suitable "moderately stringent conditions" include the following: prewashing at 5×SSC, 0.5% SDS, 1.0 mM EDTA, pH 8.0. Hybridize overnight at 50°C - 65°C in 5×SSC. Subsequently, wash twice at 65°C for 20 minutes each, using 2×, 0.5×, and 0.2× SSC containing 0.1% SDS for each wash.

[0066] As used herein, "highly stringent conditions" or "high stringency conditions" refers to one or more of the following conditions: (1) employing low ionic strength and high temperature for washing, e.g., using 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium lauryl sulfate at 50°C; (2) during hybridization, using a denaturing agent such as formamide, e.g., 50% (v / v) formamide containing 0.1% bovine serum albumin / 0.1% polysucrose / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5 and 42°C), 750 mM sodium chloride, 75 mM sodium citrate; or (3) using 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's hybridization solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate at 42°C, and washing at 42°C with 0.2×SSC (sodium chloride / sodium citrate), washing at 55°C with 50% formamide, and then washing at 55°C with a high stringency wash solution of 0.1×SSC containing EDTA. Those skilled in the art know how to optionally adjust temperature, ionic strength, etc. to accommodate factors such as probe length.

[0067] Those skilled in the art understand that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides 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 vary due to differences in codon usage frequency. Further, alleles of genes containing the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that vary due to one or more mutations such as nucleotide deletions, additions, and / or substitutions. The resulting mRNA and proteins may or may not have an altered structure or function. Alleles can be identified using specific standard techniques such as hybridization, amplification, and / or database sequence comparison.

[0068] The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. Those skilled in the art can use the sequences provided herein and commercially available DNA synthesizers to generate the desired DNA sequences.

[0069] To produce a polynucleotide using recombinant methods, as described herein, 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. The polynucleotide can be inserted into the host cell by any means known in the art. To introduce an exogenous polynucleotide, the cell can be transformed by direct uptake, endocytosis, transfection, F - hybridization, or electroporation. Once introduced, the exogenous polynucleotide can be maintained intracellularly as a non - integrating vector (such as a plasmid) or can be integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by specific methods known in the art. See, for example, Sambrook et al., 1989.

[0070] Alternatively, PCR enables the replication of DNA sequences.

[0071] RNA can be obtained by using isolated DNA in a suitable vector and inserting it into a suitable host cell. When the cell replicates and transcribes the DNA into RNA, the RNA can then be isolated using specific methods known to those of skill in the art.

[0072] Suitable cloning and expression vectors can contain various components such as promoters, enhancers, and other transcriptional regulatory sequences. The vector can also be constructed so that antibody variable domains can later be cloned into different vectors.

[0073] Suitable cloning vectors can be constructed according to standard techniques or selected from a number of cloning vectors available in the art. The cloning vector selected may vary depending on the host cell intended to be used. However, useful cloning vectors generally have the ability to replicate themselves. They can have a single target for a specific restriction endonuclease and / or can carry a gene for a marker that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (e.g., pBS SK+), and their derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, as well as shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

[0074] Expression vectors are further provided. An expression vector is typically a specific replicable polynucleotide construct containing a polynucleotide according to the present invention. It is implied that the expression vector must be replicable within the host cell, either as an episome or as an essential part of its chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, adenoviruses, adeno-associated viruses, viral vectors including retroviruses, cosmids, and the expression vectors disclosed in PCT Publication No. 87 / 04462. The carrier component typically may include, but is not limited to, one or more of the following: an origin of replication; one or more marker genes; suitable transcriptional control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more transcriptional control elements such as ribosome binding sites, translation initiation sites, and stop codons are also typically required.

[0075] The polynucleotide of interest and / or the vector containing the polynucleotide can be introduced into the host cell by any of several suitable means. Such means include transfection with electroporation, calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; particle bombardment; lipofection; and infection (e.g., if the vector is an infectious agent such as a poxvirus). The choice of the introduced vector or polynucleotide generally depends on the characteristics of the host cell.

[0076] The antibodies of the present invention include antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (further described in Section II below), antibodies isolated from a recombinant combinatorial human antibody library (further described in Section III below), antibodies isolated from a human immunoglobulin gene transgenic animal (e.g., a mouse) (see, e.g., Taylor, L.D. et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced or isolated by any other method including splicing a human immunoglobulin gene sequence to another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0077] The antibody or antibody portion of the present invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For recombinant expression of an antibody, one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody are used to transfect a host cell so that the light and heavy chains can be expressed in the host cell. The antibody is also preferably secreted into the medium in which the host cell is cultured, from which the antibody can be recovered. The heavy chain gene of the antibody and the light chain gene of the antibody can be obtained using standard recombinant DNA methods and introduced into a recombinant expression vector, and then the vector can be introduced into a host cell. As used herein, standard methods can be, for example, the methods described in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F.M. et al. (eds.) Current Protocols in Molecuar Biology, Greene Publishing Associates, (1989), and U.S. Patent No. 4,816,397 to Boss et al.

[0078] An antibody or antigen-binding fragment thereof can be produced recombinantly using a suitable host cell. Nucleic acids encoding an antibody or antigen-binding fragment thereof can be cloned into an expression vector and then introduced into a host cell such as an E. coli cell, yeast cell, insect cell, simian COS cell, Chinese hamster ovary (CHO) cell, or myeloma cell that does not additionally produce immunoglobulins in order to achieve antibody synthesis in the recombinant host cell. Among many cells well known in the art, preferred host cells include CHO cells, human embryonic kidney (HEK) 293 cells, and Sp2.0 cells.

[0079] Antibody fragments can be produced by proteolysis or other degradation of full-length antibodies, by recombinant methods, or by chemical synthesis. Polypeptide fragments of antibodies (especially shorter polypeptides 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.

[0080] The antibodies or antigen-binding fragments thereof of the present invention can be affinity matured. For example, affinity matured antibodies can be prepared from 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).

[0081] Antibody variants In some embodiments, the present invention includes amino acid sequence variants of the antibodies provided herein. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions of residues within the amino acid sequence of the antibody and / or insertions and / or substitutions. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct as long as the final construct has the desired characteristics, such as antigen binding.

[0082] In some embodiments, the antibodies of the invention may contain one or more point mutations in their amino acid sequences. The point mutations are designed to enhance the developability of the antibody. For example, Raybould et al., “Five computational developability guidelines for therapeutic antibody profiling,” PNAS, Mar. 5, 2019, 116(10)4025-4030 describes a Therapeutic Antibody Profiling tool (TAP) (a computational tool for constructing homology models of variable domain sequences that are downloadable, testing them against five developability guidelines, and reporting on the potential sequence roles and classical formats). The authors further provide TAP, which is freely available at opig.stats.ox.ac.uk / webapps / sabdab-sabpred / TAP.php. In addition to achieving the desired affinity for the antigen, there are many obstacles in the development of therapeutic monoclonal antibodies. These obstacles include intrinsic immunogenicity, chemical and conformational instability, self-association, high viscosity, multispecificity, insufficient expression, etc. For example, high levels of hydrophobicity, particularly in the hypervariable complementarity-determining regions (CDRs), have repeatedly been involved in aggregation, viscosity, and multispecificity. The asymmetry of the net charge of the heavy and light chain variable domains is also associated with self-association and viscosity at high concentrations. Positively and negatively charged patches in the CDRs are associated with high clearance rates and low expression levels. Product heterogeneity (e.g., due to oxidation, isomerization, or glycosylation) is often caused by specific sequence motifs that are prone to post-translational or co-translational modifications. Computational tools are available to facilitate the identification of sequence roles. Warszawski also describes a method for optimizing the affinity and stability of antibodies by the automated design of the variable light-heavy chain interface.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. Additional methods can be used to identify potential development issues of candidate antibodies. Further, in some preferred embodiments of the present invention, in order to solve such problems, one or more point mutations are introduced into the candidate antibody by conventional methods, thereby obtaining the optimized therapeutic antibody of the present invention.

[0083] a) Substitution, insertion, and deletion mutants In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites for alternative mutagenesis include HVRs and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table A. More substantial changes are provided under the heading "Exemplary Substitutions" in Table A and are further described later with reference to their amino acid side - chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for the desired activity, such as retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. [Table 1]

[0084] According to the general side - chain properties, amino acids can be grouped as follows. (1) Hydrophobic: Nle, Met, Ala, Val, Leu, Ile; (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting the orientation of the lock: Gly, Pro; (6) Aromatics: Trp, Tyr, Phe.

[0085] Non-conservative substitutions involve replacing a member of one of these classes with another class.

[0086] One class of substitution variants involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study have altered (e.g., improved) specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or substantially retain specific biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues can be mutated, the mutant antibodies can be displayed on phage, and screened for a particular biological activity (e.g., binding affinity).

[0087] For example, to improve antibody affinity, changes (e.g., substitutions) can be made to the HVRs. Residues encoded by codons that are mutated frequently during the somatic maturation process, i.e., HVR “hot spots” (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact the antigen can have such changes, in which the resulting mutant VH or VL is tested for binding affinity. Affinity maturation by construction and reselection of a secondary library is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (eds. O’Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants having the desired affinity. Another approach for introducing diversity involves an HVR-directed approach in which multiple HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0088] In some embodiments, substitutions, insertions, or deletions can occur within one or more HVRs so long as such changes do not substantially reduce the ability of the antibody to bind the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made to the HVRs. For example, such changes may be outside of the antigen contact residues of the HVR. In some embodiments of the mutant VH and VL sequences provided above, each HVR is unchanged or contains 1, 2, or 3 or fewer amino acid substitutions.

[0089] One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is what is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and neutralized or negatively charged by amino acid (e.g., alanine or polyalanine) substitution to determine whether the interaction between the antibody and the antigen is affected. Additional substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify the contact points between the antibody and the antigen. As alternative candidates, such contact residues and adjacent residues can be targeted or excluded. Mutants can be screened to determine whether they contain the desired properties.

[0090] Insertions of amino acid sequences include amino acid and / or carboxy-terminal fusions of lengths from 1 residue to polypeptides containing 100 or more residues, as well as insertions within the sequence of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion mutants of the antibody molecule include fusions of the N-terminal or C-terminal of the antibody with an enzyme (e.g., in the case of ADEPT) or a polypeptide that extends the serum half-life of the antibody.

[0091] b) Glycosylation mutants In some embodiments, the antibodies provided herein can be modified to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites to the antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0092] In the case of an antibody comprising an Fc region, the carbohydrate to which it is bound can be altered. Natural antibodies produced by mammalian cells typically contain a branched biantennary oligosaccharide that is typically N-linked to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharide in the antibodies of the invention can be modified to create antibody variants with certain improved properties.

[0093] In one embodiment, an antibody variant is provided that has a carbohydrate structure lacking fucose that binds (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1% - 80%, 1% - 65%, 5% - 65%, or 20% - 40%. The amount of fucose can be determined, for example, as described in WO2008 / 077546, by calculating the average amount of fucose in the sugar chain of Asn297 relative to the total of all sugar structures (complex, hybrid, and high mannose structures, etc.) bound to Asn297 when measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (Eu numbering of the Fe region residues). However, Asn297 can also be located, due to minor sequence variations in the antibody, up to approximately ±3 amino acids upstream or downstream of position 297, for example, between positions 294 - 300. Such fucosylation variants can have improved ADCC function. See, for example, US Patent Publication No. 2003 / 0157108 (Presta, L.), US Patent Publication No. 2004 / 0093621 (Kyowa Hakko Kirin Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, TO2005 / 035778, TO2005 / 053742, TO2002 / 031140, Okazaki et al, J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al, Biotech. Bioeng. 87:614 (2004) can be mentioned. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. 2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al), and knockout cell lines such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (for example, see Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO2003 / 085107).

[0094] For example, there is further provided an antibody variant having a bisected oligosaccharide in which the bisected oligosaccharide bound to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants can have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 (Raju, S.).

[0095] c) Fc region variants In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. The Fc region variants can comprise a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3 or IgG4) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0096] In some embodiments, the invention includes antibody variants that have some, but not all, effector functions. By virtue of the effector functions, it is a desirable candidate for application to the uses described later. The in vivo half-life of an antibody is important. Certain effector functions, such as complement and ADCC, are unwanted or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity), but retains FcRn binding ability. NK cells, which are the major cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat’l Acad. Sci USA 83:7059-7063 (1986) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985)), and U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays (see, for example, ACT I (trademark) non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc., Mountain View, CA) and CytoTox96 non-radioactive cytotoxicity assay (Promega, Madison, WI)) may be used. Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model such as those disclosed in Clynes et al., Proc Nat’l Acad Sci USA 95:652-656 (1998). A Clq binding assay can also be performed to confirm that the antibody cannot bind to Clq and thus lacks CDC activity. See, for example, the Clq and C3c binding ELISAs of WO2006 / 029879 and WO2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), Cragg, M. S. et al., Blood 101:1045-1052 (2003), and Cragg, M. S. and M. J. 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, S. B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0097] Antibodies with reduced effector function include antibodies having one or more substitutions of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc variant in which residues 265 and 297 are each substituted with alanine (U.S. Patent No. 7,332,581).

[0098] Certain antibody variants with improved or reduced binding to FcR have been described (see, for example, U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0099] In some embodiments, the antibody variant may comprise an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) in the Fc region.

[0100] In some embodiments, changes are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0101] Antibodies with an extended half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US2005 / 0014934A1 (Hinton et al.). The neonatal Fc receptor (FcRn) is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al. J. Immunol. 24:249 (1994)). These antibodies may comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 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, those having a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0102] 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 WO94 / 29351, which focus on other examples of Fc region variants.

[0103] d) Cysteine-engineered antibody variants In some embodiments, it may be desirable to generate a cysteine-engineered antibody, such as a “thioMAb,” in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the residues being replaced are present at accessible sites of the antibody. By replacing those residues with cysteine, a reactive thiol group is positioned at an accessible site of the antibody. Further, this can be used to conjugate the antibody to a drug moiety or other moieties such as linker-drug moieties, as further described herein, to generate an immunoconjugate. In some embodiments, cysteine may be replaced with any one or more of the following residues: V205 of the light chain (Kabat numbering), A118 of the heavy chain (EU numbering), and that of the Fc region of the heavy chain S400 (EU numbering). Cysteine-engineered antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541.

[0104] e) Antibody derivatives In some embodiments, the antibodies provided herein can be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homo- or random copolymers), dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof, but are not limited thereto. Polyethylene glycol propionaldehyde may have advantages in manufacture due to its stability in water. The polymer may be of any molecular weight and may 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 molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative is to be used for therapy under defined conditions, etc.

[0105] In another embodiment, a complex of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Nat’l. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength and includes wavelengths that heat the non-proteinaceous moiety to a temperature at which cells near the antibody-non-proteinaceous moiety are killed, although the radiation is not harmful to normal cells.

[0106] Assay The anti - OX40 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art.

[0107] 1. Binding Assays and Other Assays In one aspect, the antibodies of the invention are tested for their antigen - binding activity by known methods such as, for example, 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 shown. The OX40 antibody is iodinated, and a competitive reaction mixture is prepared that contains a fixed concentration of iodinated antibody and a serial dilution of unlabeled OX40 antibody at decreasing concentrations. OX40 - expressing cells (e.g., BT474 cells stably transfected with human OX40) are added to the reaction mixture. After incubation, the cells are washed, and free iodinated OX40 antibody is separated from the OX40 antibody bound to the cells. The level of bound OX40 iodide antibody can be determined, for example, by counting the radioactivity associated with the cells, and the binding affinity is determined using standard methods. In another embodiment, the ability of the OX40 antibody to bind to OX40 expressed on the surface (e.g., on a subset of T cells) is evaluated using flow cytometry. Peripheral blood leukocytes (e.g., from human, cynomolgus monkey, rat, or mouse) are obtained, and the cells are blocked with serum. The labeled OX40 antibody can be added in serial dilutions, and the T cells (using methods known in the art to identify the T - cell subset) are also stained. Following incubation and washing of the sample, the cells are sorted using a flow cytometer, and the data are analyzed using methods well - known in the art. In another embodiment, surface plasmon resonance can be used to analyze OX40 binding. An exemplary surface plasmon resonance method is shown.

[0108] In another aspect, a competitive assay can be used to identify an antibody that competes with any of the anti-OX40 antibodies disclosed herein for binding to OX40. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) to which any of the anti-OX40 antibodies disclosed herein can bind. Detailed exemplary methods for localizing the epitope to which an antibody binds can be found in Morris (1996) “Epitope Mapping Protocols,” Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). A competitive assay is shown.

[0109] In an exemplary competitive assay, a first labeled antibody (that binds to OX, e.g., mab 1A7.gr.1, mab 3C8.gr5) and a second unlabeled antibody (tested to compete with the first antibody for OX40 binding ability) can be incubated with immobilized OX40 in solution. The secondary antibody can be present in the hybridoma supernatant. As a control, the immobilized OX40 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions where the primary antibody can bind to OX40, excess unbound antibody is removed and the amount of label bound to the immobilized OX40 is measured. If the amount of label associated with the immobilized OX40 is substantially decreased in the test sample as compared to the control sample, this indicates that the secondary antibody competes with the primary antibody for binding to OX40. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0110] 2. Activity Assay In one aspect, an assay for identifying a biologically active anti - OX40 antibody is provided. Biologic activity herein can include, for example, binding to OX40 (e.g., binding to human and / or cynomolgus monkey OX40), increasing OX40 - mediated signaling (e.g., increasing NFkB - mediated transcription), depleting cells expressing human OX40 (e.g., T cells), depleting human OX40 - expressing cells by ADCC and / or phagocytosis, enhancing T effector cell function (e.g., CD4+ effector T cells) (e.g., by increasing effector T cell proliferation and / or increasing effector T cell cytokine (e.g., interferon γ) production), enhancing memory T cell function (e.g., CD4+ memory T cells) (e.g., by increasing memory T cell proliferation and / or increasing memory T cell cytokine production (e.g., interferon γ)), 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 having such biologic activity in vivo and / or in vitro are also provided.

[0111] In some embodiments, the antibodies of the invention are tested for such biologic activity.

[0112] T cell co-stimulation can be determined 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 leukocytes (e.g., isolated 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. The isolated T cells are cultured in the presence of antigen-presenting cells (e.g., irradiated L cells expressing CD32 and CD80) and activated by the addition of anti-CD3 antibody in the presence or absence of an OX40 agonist antibody. The effect of the agonist OX40 antibody on T cell proliferation can be measured using methods well known in the art. For example, the Cell Titer Glo kit (Promega) can be used and the results read on a multi-label reader (Perkin Elmer). The effect of the agonist OX40 antibody on T cell function can be determined by analyzing cytokine production by the T cells. In one embodiment, interferon gamma production by CD4+ T cells is determined, for example, by measuring interferon gamma in the cell culture supernatant. Methods for measuring interferon gamma are well known in the art.

[0113] Treg cell function can be assayed using methods known in the art, and exemplary methods are disclosed herein. In one example, the ability of Tregs to suppress the proliferation of effector T cells is assayed. T cells (e.g., isolation of 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 a different reagent. Irradiated antigen-presenting cells (e.g., L cells expressing CD32 and CD80) are co-cultured with the labeled purified naive CD4+ T cells and purified Tregs. The co-culture can be activated with anti-CD3 antibody and tested in the presence or absence of an agonist OMO antibody. After an appropriate time (e.g., 6 days of co-culture), FACS analysis is used to track the level of CD4+ naive T cell proliferation by dye dilution in reduced marker staining (e.g., reduced CFSE marker staining).

[0114] OX40 signaling can be assayed using methods well-known in the art, and exemplary methods are disclosed herein. In one embodiment, transgenic cells are generated that express a reporter gene comprising human OX40 and an NFkB promoter fused to a reporter gene (e.g., β luciferase). Addition of an OX40 agonist antibody to the cells can result in an increase in NFkB transcription, which can be detected using an assay for the reporter gene.

[0115] Phagocytosis can be measured, for example, by using monocytes-derived macrophages or U937 cells, a human histiocytic lymphoma cell line having the form and characteristics of mature macrophages. OX40-expressing cells can be added to monocytes-derived macrophages or U937 cells in the presence or absence of an anti-OX40 agonist antibody. The cells are cultured for an appropriate period, and the percentage of cells double-stained for 1) macrophages or U937 cells and 2) markers of OX40-expressing cells is determined, and then the phagocytosis rate can be determined by dividing the above by the total number of cells showing the marker (e.g., GFP) of cells expressing OX40. The analysis can be performed by flow cytometry. In another embodiment, the analysis can be performed by fluorescence microscopy.

[0116] ADCC can be determined, for example, using methods well known in the art. Exemplary methods are described in the Definitions section. In some embodiments, the OX40 level on OX40-expressing cells is characterized for testing in an ADCC assay. The cells are stained with a detectably labeled anti-OX40 antibody (e.g., PE-labeled), the fluorescence level is determined using flow cytometry, and the results are presented as the median fluorescence intensity (MFI). In another embodiment, ADCC can be analyzed using a CellTiter Glo assay kit, and cell viability / cytotoxicity can be determined by chemiluminescence.

[0117] The reactivity of various antibodies against two allotypes (F158 and V158) of FcγRIA, FcγRIIA, FcγRIIB, and FcγRIIIA can be measured by binding affinity in an ELISA-based ligand binding assay using the corresponding recombinant Fcγ receptors. Purified human Fcγ receptors are expressed as fusion proteins containing the extracellular domain of the receptor γ chain bound to a C-terminal Gly / 6xHis / glutathione S-transferase (GST) polypeptide tag. The binding affinity of antibodies for these human Fcγ receptors can be determined as follows. For low-affinity receptors, namely, F-158 and V-158, which are two allotypes of FcγRIIA (CD32A), FcγRIIB (CD32B), and FcγRIIIA (CD16), antibodies can be obtained as multimers to test by cross-linking with the F(ab’)2 fragment of goat anti-human kappa chain (ICN Biomedical; Irvine, CA) ab’)2 at an approximate molar ratio of antibody pair cross-linking of 1:3. Plates can be coated with anti-GST antibody (Genentech) and blocked with bovine serum albumin (BSA). After washing with phosphate-buffered saline (PBS) containing 0.05% Tween-20 and an ELx405™ plate washer device (Biotek Instruments; Winooski, VT), the Fcγ receptor is added to the plate at 25 ng / well and incubated for 1 hour at room temperature. After washing the plate, serial dilutions of the test antibody can be added as a multimer complex, and then the plate is incubated for 2 hours at room temperature. After washing the plate to remove unbound antibody, the antibody bound to the Fcγ gamma receptor can be detected using the F(ab’)2 fragment of goat anti-human F(ab’)2 conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories; West Grove, PA). Then, the substrate tetramethylbenzidine (TMB) (Kirkegaard and Perry Laboratories; Gaithersburg, MD) is added.Depending on the Fcγ receptor tested, the plate can be incubated at room temperature for 5 - 20 minutes to allow for color development. The reaction is then stopped with 1M H3PO4, and the absorbance is measured at 450 nm using a microplate reader (SpectraMax® 190, Molecular Devices, Sunnyvale, CA). A dose - response binding curve is then generated by plotting the mean absorbance values from duplicate antibody dilutions against the antibody concentration. The value of the effective antibody concentration at which 50% (EC50) of the maximum response from binding to the Fcγ receptor is detected can be determined using SoftMax 190 (Molecular Devices) and fitting the binding curve using a four - parameter equation.

[0118] To select antibodies that induce cell death, for example, loss of membrane integrity as indicated by uptake of propidium iodide (PI), trypan blue, or 7 - AAD can be evaluated by comparison to a control. The PI uptake assay can be performed in the absence of complement and immune effector cells. OX40 - expressing cells are incubated in medium alone or in medium containing an appropriate monoclonal antibody at a concentration of, for example, about 10 μg / ml. The cells are incubated for a defined period (e.g., 1 day or 3 days). After each treatment, the cells are washed and aliquoted. In some embodiments, the cells are aliquoted into 12×75 test tubes with 35 mm strainer caps (1 ml per test tube, 3 test tubes per treatment group) to remove cell aggregates. Then, PI (10 μg / ml) is added to the test tubes. The samples can be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson).

[0119] Cells for use in any of the above in vitro assays include cells or cell lines that naturally express OX40 or can be 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 a nucleic acid 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) that express human OX40.

[0120] It should be understood that any of the above assays can be performed using the immune complexes of the invention instead of, or in addition to, anti-OX40 antibodies.

[0121] It should be understood that any of the above assays can be performed using anti-OX40 antibodies and other therapeutic agents.

[0122] Formulations and Their Use The antibody or antigen-binding fragment thereof of the present invention can be formulated into a pharmaceutical composition. The pharmaceutical composition can further contain certain pharmaceutically acceptable carriers, excipients and / or stabilizers (Remington: The Science and practice of Pharmacy, 20th Edition, 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover) in the form of a lyoprotectant or an aqueous solution. The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dosage and concentration, and buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexahydroxyquaternary ammonium chloride; aldehyde; bensonin chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other saccharides including glucose, mannose, or dextran; chelating agents such as EDTA; saccharides such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0123] The antibody of the present invention or its antigen-binding fragment can be used for various therapeutic or diagnostic purposes. For example, the antibody of the present invention or its antigen-binding fragment can be used as an affinity purifying agent (e.g., for in vitro purification) and as a diagnostic agent (e.g., for detecting expression in specific cells, tissues, or sera).

[0124] Exemplary therapeutic uses of the antibody of the present invention or its antigen-binding fragment include the treatment of cancer. The antibody of the present invention or its antigen-binding fragment can also be used for prophylactic treatment.

[0125] In the case of therapeutic use, the antibody of the present invention or its antigen-binding fragment can be administered to mammals, particularly humans, by conventional techniques. Such techniques can be intravenous (as a bolus or by continuous infusion over time), intramuscular, intraperitoneal, intracerebral, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or by inhalation. The antibody of the present invention or its antigen-binding fragment can also be administered, if necessary, by intratumoral, peritumoral, intralesional, or perilesional routes.

[0126] In some embodiments, the antibody of the present invention or its antigen-binding fragment is administered subcutaneously. In some embodiments, the antibody of the present invention or its antigen-binding fragment is administered intravenously.

[0127] The pharmaceutical composition can be administered to a subject in need thereof at a frequency that can vary depending on the severity of the disease. In the case of prophylactic treatment, the frequency can vary depending on the subject's susceptibility or predisposition to the disease.

[0128] The composition can be administered to a patient in need thereof as a bolus injection or by continuous infusion. For example, bolus administration of an antibody presented as a Fab fragment can be administered in an amount 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. In the case of continuous infusion, the antibody presented as a Fab fragment can be in an amount 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 and can be administered 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.

[0129] In the case of administration of an antibody presented as a full-length antibody (having an intact constant region), the dosage can be from about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 3 mg / kg to about 10 mg / kg, about 4 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 20 mg / kg, about 2 mg / kg to about 20 mg / kg, about 3 mg / kg to about 20 mg / kg, about 4 mg / kg to about 20 mg / kg, about 5 mg / kg to about 20 mg / kg, about 1 mg / kg or more, about 2 mg / kg or more, about 3 mg / kg or more, about 4 mg / kg or more, about 5 mg / kg or more, about 6 mg / kg or more, about 7 mg / kg or more, about 8 mg / kg or more, about 9 mg / kg or more, about 10 mg / kg or more, about 11 mg / kg or more, about 12 mg / kg or more, about 13 mg / kg or more, about 14 mg / kg or more, about 15 mg / kg or more, about 16 mg / kg or more, about 17 mg / kg or more, about 19 mg / kg or more, or about 20 mg / kg or more. The frequency of administration can depend on the severity of the condition. The frequency can vary from three times a week to once every two or three weeks.

[0130] Alternatively, the composition can be administered to a patient by subcutaneous injection. For example, an anti-OX40 antibody at a dose of 1 to 100 mg can be administered to a patient by 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.

[0131] In some embodiments, the half-life of the anti-OX40 antibody in humans can be about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, from about 5 days to about 40 days, from about 5 days to about 35 days, from about 5 days to about 30 days, from about 5 days to about 25 days, from about 10 days to about 40 days, from about 10 days to about 35 days, from about 10 days to about 30 days, from about 10 days to about 25 days, from about 15 days to about 40 days, from about 15 days to about 35 days, from about 15 days to about 30 days, or from about 15 days to about 25 days.

[0132] In some embodiments, the pharmaceutical composition can be administered subcutaneously or intravenously every 2 to 6 weeks at the following doses: about 0.1 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1.5 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 8 mg / kg, about 0.5 mg / kg to about 8 mg / kg, about 1 mg / kg to about 8 mg / kg, about 1.5 mg / kg to about 8 mg / kg, about 2 mg / kg to about 8 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1.5 mg / kg to about 5 mg / kg, 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.

[0133] In some embodiments, the pharmaceutical composition is administered subcutaneously or intravenously at a dose of about 2.0 mg / kg every 2 to 6 weeks. In some embodiments, the pharmaceutical composition is administered subcutaneously or intravenously at a dose of about 2.0 mg / kg to about 10.0 mg / kg every 2 to 6 weeks.

[0134] In an exemplary embodiment, the pharmaceutical composition is administered subcutaneously every 2 weeks.

[0135] The antibody or antigen-binding fragment thereof of the present invention can be used as a monotherapy or in combination with other therapies for treating cancer.

[0136] Definitions Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. Further, unless the context requires otherwise, the singular forms shall include the plural, and the plural forms shall include the singular. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art.

[0137] 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) the Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) the F(ab’)2 fragment, which is a divalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; (iii) the Fd fragment consisting of the VH and CH1 domains; (iv) the Fv fragment consisting of the VL and VH domains of a single-arm antibody; (v) the dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 341:544-546); and (vi) isolated complementarity-determining regions (CDRs), disulfide-bonded Fv (dsFv), anti-idiotypic (anti-Id) antibodies, and intrabodies. Further, the two domains of the Fv fragment (VL and VH), although encoded by different genes, can be joined by a synthetic linker using recombinant methods. The synthetic linker enables the VL and VH regions to pair to form a single protein chain (referred to as a single-chain Fv (scFv)). See, for example, Bird et al. Science 242:423-426 (1988) and Huston et al., Proc. Nat’l. Acad. Sci. USA 85:5879-5883 (1988). Other forms of single-chain antibodies, such as diabodies, are also included in the present invention. A diabody is a type of bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but in such cases, the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains on the other chain to generate two antigen-binding sites (see, for example, Holliger et al., Proc. Nat’l. Acad. Sci. USA 90:6444-6448 (1993); and Poljak et al., 1994, Structure 2:1121-1123).

[0138] The "variable domain" of an antibody refers to the variable region of an antibody light chain (VL) or the variable region of an antibody heavy chain (VH), either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of three complementarity-determining regions (CDRs) connected by four framework regions (FRs), which contribute to the formation of the antigen-binding site of the antibody.

[0139] The residues of the variable domain are numbered according to the Kabat numbering system. Kabat is a numbering system for the variable domains of heavy or light chains used in the editing of antibodies. See Kabat et al., Sequences of Proteins of Immunological Interest, 5 th th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or CDR of the variable domain. For a given antibody, the Kabat numbering of residues can be determined by aligning the sequence of the antibody with regions homologous to the "standard" Kabat numbering sequence. Various algorithms are available for assigning Kabat numbers. Unless otherwise specified herein, Kabat numbers are assigned to variable regions using the algorithm implemented in Abysis (www.abysis.org) published in 2012.

[0140] The positions of specific amino acid residues of an antibody, such as paratope residues, are also numbered according to the Kabat system.

[0141] "Complementary determining regions" (CDRs) can be identified according to Kabat, Chothia, the integration of both Kabat and Chothia, AbM, contact and / or conformational definitions, or any method of CDR determination well-known in the art. See, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. (hypervariable regions); Chothia et al., 1989, Nature 342:877-883 (structural loop structures). The AbM definition of CDR is a compromise between Kabat and Chothia, and the "contact" definition using the AbM antibody modeling software of Oxford Molecular is based on the obvious antigen contacts described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. The "conformational" definition of CDR is based on the formation of residues contributing to the enthalpy of antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). Still other definitions of CDR boundaries may not strictly follow one of the above methods, but may nevertheless overlap at least in part with the Kabat CDR. However, they may not significantly affect antigen binding, depending on specific residues or groups of residues, or even the entire CDR, that are shortened or extended by prediction or experimental findings. As used herein, CDR can refer to CDRs defined by any method known in the art, including combinations of methods.

[0142] "Epitope" refers to the region or extent of an antigen (Ag) to which an antibody specifically binds, e.g., the region or extent containing amino acid residues that interact with the antibody (Ab). Epitopes can be linear or non-linear (e.g., conformational).

[0143] An antibody or its antigen-binding fragment binds to substantially the same epitope as another antibody or its antigen-binding fragment when the binding of the corresponding antibody or its antigen-binding fragment is mutually exclusive. That is, the binding of one antibody or its antigen-binding fragment prevents the simultaneous or sequential binding of another antibody or its antigen-binding fragment. When an antigen can accommodate the simultaneous binding of two corresponding antibodies or their antigen-binding fragments, the epitopes are considered to be distinct or not substantially identical.

[0144] The term "paratope" is derived from a different perspective from the above definition of "epitope" and refers to the region or extent of the antibody molecule involved in antigen binding, for example, the region or extent containing residues that interact with the antigen. The paratope can be linear or conformational (such as discontinuous residues of CDRs).

[0145] The epitope / paratope of a given antibody / antigen binding pair can be defined and characterized at various levels of detail using a variety of experimental methods and computational epitope mapping 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. Since each method relies on its own principle, the description of the epitope is closely related to the method by which the epitope was determined. Thus, the epitope / paratope of a given antibody / antigen pair can be defined differently depending on the mapping method used.

[0146] At the most detailed level, the epitope / paratope used in the interaction between an antibody (Ab) and an antigen (Ag) can be determined by information regarding the definition of the spatial coordinates of the atomic contacts present in the Ag-Ab interaction and their relative contributions to the thermodynamics of binding. To some extent, the epitope / paratope residues can be characterized by defining the spatial coordinates of the atomic contacts between the Ag and the Ab. In one aspect, the epitope / paratope residues can be defined by specific criteria such as the distance between the atoms of the Ab and the Ag (e.g., the distance from the heavy atoms of the homologous antibody to the heavy atoms of the antigen is equal to or less than its approximate definition). In another aspect, the epitope / paratope residues can be characterized as those involved in hydrogen bond interactions with the homologous antibody / antigen, or similarly, hydrogen bond interactions with water molecules hydrogen-bonded to the antibody / antigen (water-mediated hydrogen bonds). In another aspect, the epitope / paratope residues can be characterized by forming salt bridges with the residues of the homologous antibody / antigen. In yet another aspect, the epitope / paratope residues can be characterized as residues having a non-zero variable in the buried surface area (BSA) due to the interaction with the homologous antibody / antigen. At a less detailed level, the epitope / paratope can be characterized by function, for example, by competitive binding with other Abs. The epitope / paratope can also be more generally defined as including amino acid residues whose substitution by another amino acid changes the characteristics of the interaction between the Ab and the Ag (e.g., alanine scanning).

[0147] Descriptions and definitions of epitopes depend on the epitope mapping method used and the fact that they are obtained at different levels of detail. Therefore, it can be inferred that comparisons of epitopes of different Abs on the same Ag may be similar at different levels of detail. For example, this can be described at the amino acid level, such as epitopes determined from X-ray structures, and if they contain the same set of amino acid residues, they are considered identical. Epitopes characterized by competitive binding can be considered overlapping if the binding of the corresponding antibodies is mutually exclusive, i.e., the binding of one antibody precludes the simultaneous or sequential binding of another antibody. Also, if the antigen can accommodate the simultaneous binding of two corresponding antibodies, the epitopes are considered distinct (unique).

[0148] The epitopes and paratopes of a given antibody / antigen pair can be identified in a routine manner. For example, the general location of an epitope can be determined by assessing the ability of an antibody to bind to various fragments or mutant polypeptides, as already described more fully herein. Specific residues within OX40 that can contact specific residues within an antibody can also be determined using specific routine methods. For example, an antibody / antigen complex can be crystallized. The crystal structure can be determined and used to identify specific sites of interaction between the antibody and the antigen.

[0149] The term "specific binding" is well known in the art, and methods for determining such specific binding are also well known in the art. A molecule is considered to exhibit "specific binding" if it reacts or binds to a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with a higher affinity than when it reacts or binds to alternative cells or substances. An antibody or its antigen-binding fragment "specifically binds" to a target if the antibody or its antigen-binding fragment binds to the target with the characteristics of having a higher affinity, a higher binding capacity, a greater facility, and / or a longer duration compared to other substances.

[0150] For example, an antibody or an antigen-binding fragment thereof that specifically binds to OX40 is an antibody that binds to its cognate antigen with the characteristics of having a higher affinity, a higher binding ability, a higher facility, and / or a longer duration compared to other antigens. For example, under standard binding assay conditions, an anti-OX40 antibody can specifically bind to human OX40 in a sample, but does not substantially recognize or bind to other molecules in the same sample. It should also be understood that an antibody or an antigen-binding fragment thereof that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" as used herein does not necessarily require (although it may include) exclusive binding. Although not necessarily so, "binding" as used herein typically means specifically binding.

[0151] Various assay formats can be used to select an antibody or an antigen-binding fragment thereof that specifically binds to a molecule of interest. For example, among many assays, a solid-phase ELISA immunoassay, immunoprecipitation, Biacore™ (GE Healthcare), KinExA, fluorescence-activated cell sorting (FACS), Octet™ (Forte Bio, Inc.), and Western blot analysis can be used to identify a specific antibody or an antigen-binding fragment thereof that binds to an antigen. Typically, specific binding can be at least 2-fold, more typically at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or at least 10,000-fold of the background signal or noise.

[0152] The specificity of antibody binding is determined by the K D value of the specific binding between the antibody and OX40, and the K D value is compared with the K DIt can be evaluated by comparison with a value. Generally, an antibody binds "specifically" to an antigen when K D is about ×10 -5 M or less.

[0153] If an antibody or its antigen-binding fragment binds to an antigen with the characteristics of having a higher affinity, a higher binding ability, a higher facility, and / or a longer duration compared to an antibody or its antigen-binding fragment that binds to another antigen, the antibody or its antigen-binding fragment "substantially does not bind" to that antigen. Typically, the binding is 2 times or less of the background signal or noise. Generally, it is 1×10 -4 M or more, 1×10 -3 M or more, 1×10 -2 M or more, or 1×10 -1 M or more of K D to bind to the antigen.

[0154] As used herein, the term "compete" with respect to an antibody means that the binding of a first antibody or its antigen-binding portion to an antigen reduces the binding of a subsequent second antibody or its antigen-binding portion to the same antigen. Generally, the binding of the first antibody results in steric hindrance, conformational change, or binding to a common epitope (or a part thereof), and as a result, the binding of the second antibody to the same antigen is reduced. A standard competitive binding assay can be used to determine whether two antibodies compete with each other.

[0155] Suitable assays for antibody competition involve the use of Biacore technology. This technology can use surface plasmon resonance (SPR) technology. The degree of interaction is typically measured using a biosensor system such as the system. For example, SPR can be used in an in vitro competitive binding inhibition assay to determine the ability of one antibody to inhibit the binding of a second antibody. Another assay for measuring antibody competition uses an ELISA-based method. Additionally, a high-throughput method for "fractionating" antibodies based on antibody competition is described in WO2003 / 48731. Competition exists when one antibody or its antigen-binding fragment reduces the binding of another antibody or its antigen-binding fragment to OX40. For example, a sequential binding competition assay can be used where different antibodies are added sequentially. The primary antibody can be added to achieve near-saturating binding, and then the secondary antibody is added. If the binding of the second antibody to OX40 is undetectable or significantly reduced (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%) compared to when the first antibody is absent (the median can be set to 100%), the two antibodies are considered to compete with each other.

[0156] Competitive binding assays can also be performed. The binding of an antibody to an antigen is compared to the binding of the antigen to another binding partner of the target, such as another antibody or a soluble receptor, that binds to the target differently. The concentration at which 50% inhibition occurs is designated as K i and is so-called. Under ideal conditions, this K i is equal to K D Thus, generally, the measurement of K i can conveniently be used to provide an upper limit for K D The binding affinities associated with different molecular interactions (e.g., comparison of the binding affinities of different antibodies for a given antibody) can be compared by comparison of the K D of the individual antibody / antigen complexes. The K DThe value can be determined using methods established in the art.

[0157] An “Fc fusion” protein is a protein in which one or more polypeptides are operably linked to an Fc polypeptide. Fc fusions combine 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. The boundaries of the Fc region of an immunoglobulin heavy chain can vary, but the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxy terminus. Numbering of the residues in the Fc region can be by 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 dimeric or monomeric form.

[0158] The term “therapeutically effective amount” means an amount of an anti - OX40 antibody or antigen - binding fragment thereof, or a combination comprising such an antibody or antigen - binding fragment, sufficient to achieve the intended purpose. The exact amount can depend on many factors including, but not limited to, the components and physical characteristics of the therapeutic composition, the intended patient population, considerations of the individual patient, etc., and can be determined by one of ordinary skill in the art.

[0159] The term “treatment” includes prophylactic and / or therapeutic treatment. When administered prior to the clinical symptoms of a disease, disorder or condition, the treatment is considered prophylactic or preventive. Therapeutic treatment includes, for example, reducing or lessening the severity of a disease, disorder or condition, or shortening the length of a disease, disorder or condition.

[0160] As used herein, the term "about" refers to + / - 10% of a value.

[0161] Therapeutic Methods and Compositions Any of the anti-human OX40 antibodies provided herein can be used in a method of treatment.

[0162] In one aspect, the anti-human OX40 agonist antibody is provided for use as a medicament. In yet other aspects, the anti-human OX40 agonist antibody is provided for use in the treatment of cancer. In some embodiments, the anti-human OX40 agonist antibody is provided for use in a method of treatment. In some embodiments, the anti-human OX40 agonist antibody is provided for use in a method of treating an individual having cancer, comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In such an embodiment, the method further comprises, for example, administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

[0163] In one aspect, there is provided an anti-human OX40 agonist antibody for use in enhancing the immune function of an individual having cancer (e.g., by upregulating a cell-mediated immune response) by administering to the individual an effective amount of the anti-human OX40 agonist antibody. In one aspect, there is provided an anti-human OX40 agonist antibody for use in enhancing the T cell function of an individual having cancer by administering to the individual an effective amount of the anti-human OX40 agonist antibody. In one aspect, there is provided an anti-human OX40 agonist antibody for use in depleting human OX40-expressing cells (e.g., OX40-expressing T cells, e.g., OX40-expressing Tregs) by administering to the individual an effective amount of the anti-human OX40 agonist antibody. In some embodiments, depletion is achieved via ADCC. In some embodiments, depletion is achieved by phagocytosis. There is provided an anti-human OX40 agonist antibody for use in the treatment of an individual having tumor immunity.

[0164] In yet other embodiments, the anti-human OX40 agonist antibody is provided for use in the treatment of infectious diseases (e.g., bacterial or viral infections or other pathogenic infections). In some embodiments, the present invention provides an anti-human OX40 agonist antibody for use in a method of treating an individual having an infectious disease, comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In some embodiments, the infectious disease is a viral and / or bacterial infection. In some embodiments, the infectious disease is a pathogen infection.

[0165] In yet another embodiment, the present invention provides the use of an anti-OX40 antibody for the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer. In yet another embodiment, the medicament is for use in a method of treating cancer, comprising administering to an individual having cancer an effective amount of the medicament. In such one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described below.

[0166] In one embodiment, the medicament is for enhancing the immune function of an individual having cancer (e.g., by upregulating a cell-mediated immune response) by administering to the individual an effective amount of the medicament. In one embodiment, the medicament is for enhancing the T cell function of an individual having cancer by administering to the individual an effective amount of the medicament. In some embodiments, the T cell dysfunction disorder is cancer. In one embodiment, the medicament is used to deplete human OX40-expressing cells (e.g., high OX40-expressing cells, e.g., OX40-expressing T cells) by administering to the individual an effective amount of the medicament. In some embodiments, the depletion is achieved via ADCC. In some embodiments, the depletion is achieved by phagocytosis. In one embodiment, the medicament is used to treat an individual having tumor immunity.

[0167] In yet other aspects, drugs for the treatment of infectious diseases (e.g., bacterial or viral infections or other pathogenic infections) are provided. In some embodiments, the drugs are used in a method of treating an individual having an infectious disease, which includes administering an effective amount of the drug to the individual. In some embodiments, the infectious disease is a viral and / or bacterial infection. In some embodiments, the infectious disease is a pathogen infection.

[0168] In yet 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 cancer. In such one embodiment, the method further includes, for example, administering an effective amount of at least one additional therapeutic agent to the individual as described below. The "individual" according to any of the above embodiments can be a human.

[0169] In one aspect, a method for enhancing the immune function (e.g., by upregulating a cell-mediated immune response) of an individual having cancer is provided, which includes administering an effective amount of an anti-human OX40 agonist antibody to the individual. In one aspect, a method for enhancing the T cell function of an individual having cancer is provided, which includes administering an effective amount of an anti-human OX40 agonist antibody to the individual. In one aspect, a method for depleting cells expressing human OX40 (e.g., cells expressing a high level of OX40, e.g., T cells expressing OX40) is provided, which includes administering an effective amount of an anti-human OX40 agonist antibody to the individual. In some embodiments, the depletion is achieved via ADCC. In some embodiments, the depletion is achieved by phagocytosis. An anti-human OX40 agonist antibody for use in the treatment of an individual having tumor immunity is provided.

[0170] In some embodiments, examples of cancers further include B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation, edema (such as those associated with brain tumors), B-cell proliferative disorders, and Meyers syndrome associated with nevus flammeus, but are not limited thereto. More specific examples include relapsed or refractory NHL, frontline low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B 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, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic B-cell lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodular marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or multiple myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicular center lymphoma, 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 B-cell large cell lymphoma, primary effusion lymphoma, progressive immunoblastic NHL, progressive lymphoblastic NHL, progressive small non-cleaved cell NHL, large lesion NHL, Burkitt lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sézary syndrome, cutaneous lymphoma, anaplastic large cell lymphoma, and angiocentric lymphoma, but are not limited thereto.

[0171] In some embodiments, examples of cancers further include, but are not limited to, B cell proliferative disorders, which can include, but are not limited to, lymphoma (e.g., B cell non-Hodgkin lymphoma (NHL)) and lymphocytic leukemia. Such lymphomas and lymphocytic leukemias can include, for example, a) follicular lymphoma, b) small non-cleaved cell lymphoma / Burkitt lymphoma (including endemic Burkitt lymphoma, sporadic Burkitt lymphoma, and non-Burkitt lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B cell lymphoma (including mucosa-associated lymphoid tissue lymphoma, MALT), nodular marginal zone B cell lymphoma, and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B cell large cell lymphoma, angiocentric lymphoma - pulmonary B cell lymphoma), f) hairy cell leukemia, g) lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, h) acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, i) plasma cell neoplasms, plasmacytoma, multiple myeloma, plasmacytosis, and / or j) Hodgkin disease.

[0172] 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 lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent indolent NHL, refractory NHL, refractory indolent 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 indolent NHL and / or aggressive NHL. In some embodiments, the B cell proliferative disorder is indolent follicular lymphoma or diffuse large B cell lymphoma.

[0173] In yet another aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-OX40 antibodies provided herein for use, for example, in any of the above-described methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-OX40 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-OX40 antibodies provided herein and at least one additional therapeutic agent as described, for example, below.

[0174] In some embodiments of any of the methods of the present invention, the anti-human OX40 agonist antibody kills OX40-expressing cells (e.g., cells expressing high levels of OX40) by inhibiting Treg function (e.g., inhibiting the suppressive function of Tregs), increases effector T cell function, and / or increases memory T cell function to suppress tumor immunity. In some embodiments of any of the methods of the present invention, the anti-human OX40 agonist antibody kills OX40-expressing cells (e.g., cells expressing high levels of OX40), increases effector T cell function, and / or enhances memory T cell function to treat cancer. In some embodiments of any of the methods of the present invention, the anti-human OX40 agonist antibody kills OX40-expressing cells (e.g., cells expressing high levels of OX40), increases effector T cell function, and / or enhances memory T cell function to enhance immune function. In some embodiments of any of the methods of the present invention, the anti-human OX40 agonist antibody kills OX40-expressing cells (e.g., cells expressing high levels of OX40), increases effector T cell function, and / or enhances memory T cell function to enhance T cell function.

[0175] In some embodiments of any of the methods, 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 depletion of cells (e.g., depletion of cells expressing OX40, e.g., depletion of cells expressing high levels of OX40). In some embodiments, depletion is achieved via ADCC. In some embodiments, depletion is achieved by phagocytosis.

[0176] In some embodiments of any of the methods, with respect to Treg function prior to administration of an OX40 agonist antibody, the anti-human OX40 agonist antibody inhibits Treg function, for example, by inhibiting Treg suppression of effector and / or memory T cell function (in some embodiments, effector T cell and / or memory T cell proliferation and / or cytokine secretion). In some embodiments of any of the methods, with respect to effector T cell proliferation prior to administration of an OX40 agonist antibody, the anti-human OX40 agonist antibody increases effector T cell proliferation. In some embodiments of any of the methods, with respect to memory T cell proliferation prior to administration of an OX40 agonist antibody, the anti-human OX40 agonist antibody increases memory T cell proliferation. In some embodiments of any method, with respect to effector T cell cytokine production prior to administration of an OX40 agonist antibody, the anti-human OX40 agonist antibody increases effector T cell cytokine production (e.g., gamma interferon production). In some embodiments of any method, with respect to memory T cell cytokine production prior to administration of an OX40 agonist antibody, the anti-human OX40 agonist antibody increases memory T cell cytokine production (e.g., gamma interferon production). In some embodiments of any of the methods, with respect to CD4+ effector T cell proliferation and / or CD8+ effector T cell proliferation prior to administration of an OX40 agonist antibody, the anti-human OX40 agonist antibody increases CD4+ effector T cell proliferation and / or CD8+ effector T cell proliferation. In some embodiments of any of the methods, with respect to memory T cell proliferation prior to administration of an OX40 agonist antibody, the anti-human OX40 agonist antibody increases memory T cell proliferation (e.g., CD4+ memory T cell proliferation). In some embodiments, with respect to proliferation, cytokine secretion, and / or lytic activity prior to administration of an anti-human OX40 agonist antibody, the individual's CD4+ effector T cells have enhanced proliferation, cytokine secretion, and / or lytic activity.

[0177] In some embodiments of any of the methods of the present invention, the number of CD4+ effector T cells is increased compared to that before administration of the anti-human OX40 agonist antibody. In some embodiments, CD4+ effector T cell cytokine secretion is increased compared to that before administration of the anti-human OX40 agonist antibody. In some embodiments of any of the methods of the present invention, the CD8+ effector T cells of an individual have enhanced proliferation, cytokine secretion and / or lytic activity compared to those before administration of the anti-human OX40 agonist antibody. In some embodiments, the number of CD8+ effector T cells is increased compared to that before administration of the anti-human OX40 agonist antibody. In some embodiments, CD8+ effector T cell cytokine secretion is increased compared to that before administration of the anti-human OX40 agonist antibody.

[0178] In some embodiments of any of the methods of the present invention, the anti-human OX40 agonist antibody binds to human effector cells, for example, binds to FcγR expressed by human effector cells. In some embodiments, the human effector cells perform an ADCC effector function. In some embodiments, the human effector cells perform a phagocytic effector function.

[0179] In some embodiments of any of the methods of the present invention, an anti-human OX40 agonist antibody comprising a mutant IgGl Fc polypeptide (comprising a mutation that abrogates binding to human effector cells, such as the DANA or N297G mutation) has reduced activity (e.g., CD4 + effector T cell functions such as proliferation) compared to an anti-human OX40 agonist antibody comprising a native sequence IgGl Fc portion. In some embodiments, an anti-human OX40 agonist antibody comprising a mutant IgGl Fc polypeptide (comprising a mutation that abrogates binding to human effector cells, such as the DANA or N297G mutation) has substantially no activity (e.g., CD4 + effector T cell functions such as proliferation).

[0180] In some embodiments of any of the methods of the invention, antibody cross-linking is required for anti-human OX40 agonist antibody function. In some embodiments, the function is to stimulate CD4+ effector T cell proliferation. In some embodiments, antibody cross-linking is determined by providing an anti-human OX40 agonist antibody that attaches to a solid surface (e.g., a cell culture plate). In some embodiments, antibody cross-linking is determined by introducing a mutation (e.g., DANA or N297S mutation) into the IgG1 Fc portion of the antibody and then testing the function of the mutant antibody.

[0181] In some embodiments of any of the methods, the memory T cells of an individual have enhanced proliferation and / or cytokine secretion compared to those before administration of the anti-human OX40 agonist antibody. In some embodiments, the number of memory T cells is increased compared to that before administration of the anti-human OX40 agonist antibody. In some embodiments, the memory T cell cytokine secretion (level) is increased compared to that before administration of the anti-human OX40 agonist antibody. In some embodiments of any of the methods, the Tregs of an individual have reduced inhibition of effector T cell function (e.g., proliferation and / or cytokine secretion) compared to those before administration of the anti-human OX40 agonist antibody. In some embodiments, the number of effector T cells is increased compared to that before administration of the anti-human OX40 agonist antibody. In some embodiments, the effector T cell cytokine secretion (level) is increased compared to that before administration of the anti-human OX40 agonist antibody.

[0182] In some embodiments of any of the methods of the present invention, the number of intratumoral (infiltrating) CD4+ effector T cells (e.g., the total number of CD4+ effector T cells, or e.g., the percentage of CD4+ cells among CD45+ cells) is increased compared to that before administration of the anti-human OX40 agonist antibody. In some embodiments of any of the methods of the present invention, the number of intratumoral (infiltrating) CD4+ effector T cells expressing interferon-γ (e.g., total interferon gamma expressed by CD4+ cells, or e.g., the percentage of CD4+ cells expressing interferon among all CD4 + cells) is increased compared to that before administration of the anti-human OX40 agonist antibody.

[0183] In some embodiments of any of the methods of the present invention, the number of intratumoral (infiltrating) CD8+ effector T cells (e.g., the total number of CD8+ effector T cells, or e.g., the percentage of CD8+ cells among CD85+ cells) is increased compared to that before administration of the anti-human OX40 agonist antibody. In some embodiments of any of the methods of the present invention, the number of intratumoral (infiltrating) CD8+ effector T cells expressing interferon-γ (e.g., the percentage of CD8+ cells expressing interferon among all CD8+ cells) is increased compared to that before administration of the anti-human OX40 agonist antibody.

[0184] In some embodiments of any of the methods of the present invention, the number of intratumoral (infiltrating) Tregs (e.g., the total number of Tregs or, e.g., the percentage of Fox3p+ cells among CD4+ cells) is decreased compared to that before administration of the anti-human OX40 agonist antibody.

[0185] In some embodiments of any of the methods of the present invention, administration of the anti-human OX40 agonist antibody is combined with 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.

[0186] In some embodiments of any of the methods 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 and include, for example, 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 can be 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.

[0187] In some embodiments of any of the methods of the present invention, the cancer exhibits FcR-expressing cells (e.g., is infiltrated by FcR-expressing cells). Methods for detecting FcR are well known in the art and include, for example, IHC. In some embodiments, the cancer exhibits high levels of FcR-expressing cells. In some embodiments, the FcR is FcyR.

[0188] In some embodiments, the FcR is an activated 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.

[0189] The "individual" according to any of the above embodiments is preferably human.

[0190] The antibodies of the present invention can be used in therapy, alone or in combination with other agents. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent.

[0191] Such combination therapies include co - administration (where two or more therapeutic agents are included in the same formulation or separate formulations) and separate administrations. In this case, administration of the antibody of the present invention can be carried out before, simultaneously with, and / or after the administration of another therapeutic agent and / or drug. In one embodiment, the administration of the anti - OX40 antibody and the administration of the additional therapeutic agent are within about 1 month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days. The antibody of the present invention can also be used in combination with radiation therapy.

[0192] In some embodiments, the anti - human OX40 agonist antibody can be administered in combination with chemotherapy or a chemotherapeutic agent. In some embodiments, the anti - human OX40 agonist antibody can be administered in combination with radiation therapy or a radiation agent. In some embodiments, the anti - human OX40 agonist antibody can be administered in combination with targeted therapy or a targeted therapeutic agent. In some embodiments, the anti - human OX40 agonist antibody can be administered in combination with immunotherapy or an immunotherapeutic agent, such as a monoclonal antibody.

[0193] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a PARP inhibitor (e.g., olaparib, rucaparib, niraparib, cediranib, BMN673, veliparib), trabectedin, nab-paclitaxel (albumin-bound paclitaxel, ABRAXANE), trebananib, pazopanib, cediranib, palbociclib, everolimus, fluorouracil (e.g., FOLFOX, FOLFIRI), IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, Torisel (temsirolimus), Inlyta (axitinib, Pfizer), Afinitor (everolimus, Novartis), Nexavar (sorafenib, Onyx / Bayer), Votrient, pazopanib, axitinib, IMA-901, AGS_003, cabozantinib, vinflunine, an Hsp90 inhibitor (e.g., apatorsin), Ad-GM-CSF (CT-0070), temozolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, axitinib, lenalidomide, bortezomib (VELCADE), amrubicin, carfilzomib, pralatrexate, and / or enzastaurin.

[0194] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a PD-1 axis-binding antagonist. The PD-1 axis-binding antagonists herein 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 the binding of PD-1 to its ligand-binding partner. In certain embodiments, 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 the binding of PD-L1 to its binding partner. In certain embodiments, 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 the binding of PD-L2 to its binding partner. In certain embodiments, 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 can be 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 portion of PD-L1 or PD-L2 or a PD-1-binding portion 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 is selected from the group consisting of YW243.55.S70, MPDL3280A, MEDI4736, and MDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody as described in WO2007 / 005874. The antibody YW243.55.S70 is an anti-PD-L1 antibody as described in WO2010 / 077634A1. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody as described in WO2006 / 121168. Merck 3475, also known as MK-3475, SCH-900475, or pembrolizumab, is an anti-PD-1 antibody as described in WO2009 / 114335. CT-011, also known as hBAT, hBAT-1, or pidilizumab, is an anti-PD-1 antibody as described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor as 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, 0N0-4538, BMS-936558, or nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registration number: 946414-94-4).

[0195] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist to an activating co-stimulatory molecule. In some embodiments, the activating co-stimulatory molecule can include CD40, CD226, CD28, GITR, CD137, CD27, HVEM, and CD127. In some embodiments, the agonist to an activating co-stimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist to an inhibitory co-stimulatory molecule. In some embodiments, the inhibitory co-stimulatory molecule can include CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase. In some embodiments, the antagonist to an inhibitory co-stimulatory molecule is an antagonist antibody that binds to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3 (such as LAG-3-IgG fusion protein (IMP321), etc.), B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase.

[0196] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist, e.g., a blocking antibody against CTLA_4 (also known as CD152). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ipilimumab (also known as MDX-010, MDX-101, or Yervoy®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with tremelimumab (also known as ticilimumab or CP-675,206). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist, e.g., a blocking antibody against B7-H3 (also known as CD276). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with MGA271. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against TGF-β, e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.

[0197] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a therapy that includes adoptive transfer of a chimeric antigen receptor (CAR) that expresses T cells (e.g., cytotoxic T cells or CTLs). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with UCART19. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with WT128z. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with KTE-C19 (Kite). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with CTL019 (Novartis). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a therapy that includes adoptive transfer of T cells that include a dominant negative TGFβ receptor, e.g., a dominant negative TGFβ type I receptor. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a therapy that includes the HER2CREEM regimen (see, e.g., ClinicalTrials.gov Identifier NCT00889954).

[0198] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist to CD19. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with MOR00208. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist to CD38. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with daratumumab.

[0199] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist, for example, an activating antibody against CD137 (also known as TNFRSF9, 4-1BB, or ILA). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with urelumab (also known as BMS-663513). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist, for example, an activating antibody against CD40. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with CP-870893. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist, for example, an agonist of OX40 (also known as CD134). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a different anti-OX40 antibody (e.g., AgonOX). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist of CD27, for example, an activating antibody. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with CDX-1127. In some embodiments, the anti-human OX40 agonist antibody can 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 I-D-MT).

[0200] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist, such as an activating antibody of CD137 (also known as TNFRSF9, 4-1BB, or ILA). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with urelumab (also known as BMS-663513). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist of CD40, such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with CP-870893 or R07009789. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist, such as an activating antibody of OX40 (also known as CD134). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist of CD27, such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with CDX-1127 (also known as varlilumab). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist to 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 an IDO antagonist as described in WO2010 / 005958, the content of which is incorporated herein by reference. In some embodiments, the IDO antagonist is 4-({2-[(aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboxamidine (such as described in Example 23 of WO2010 / 005958). In some embodiments, the IDO antagonist is as follows: [Chemical formula]

[0201] 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 can be administered in combination with an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an anti-NaPi2b antibody-MMAE conjugate (DNIB0600A, RG7599, or also known as refastuzumab vedotin). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with trastuzumab emtansine (T-DM1, ado-trastuzumab emtansine, or also known as KADCYLA®, Genentech). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an anti-MUC16 antibody-MMAE conjugate, DMUC5754A. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an anti-MUC16 antibody-MMAE conjugate, DMUC4064A. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antibody-drug conjugate that targets the endothelin B receptor (EDNBR), for example, an MMAE-conjugated antibody against EDNBR. In some embodiments, the anti-human OX40 agonist antibody can be combined with an antibody-drug conjugate that targets lymphocyte antigen 6 complex, locus E (Ly6E), for example, an MMAE-conjugated antibody against Ly6E (also known as DLYE5953A). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with polatuzumab vedotin. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antibody-drug conjugate that targets CD30.In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ADCETRIS (also known as brentuximab vedotin). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with polatuzumab vedotin.

[0202] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an angiogenesis inhibitor. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antibody against VEGF, such as VEGF-A. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab (also known as AVASTIN®, Genentech). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antibody against angiopoietin 2 (also known as Ang2). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with MEDI3617. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antibody against VEGFR2. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ramucirumab. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a VEGF receptor fusion protein. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with aflibercept. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ziv-aflibercept (also known as VEGF trap or ZALTRAP®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a bispecific antibody against VEGF and Ang2. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with RG7221 (also known as bavencio). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an angiogenesis inhibitor and a PD-1 axis-binding antagonist (e.g., a PD-1-binding antagonist such as an anti-PD-1 antibody, a PD-L1-binding antagonist such as an anti-PD-L1 antibody, and a PD-L2-binding antagonist such as an anti-PD-L2 antibody).In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and a PD-1 axis-binding antagonist (e.g., a PD-1-binding antagonist such as an anti-PD-1 antibody, a PD-L1-binding antagonist such as an anti-PD-L1 antibody, and a PD-L2-binding antagonist such as an anti-PD-L2 antibody). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MDX-1106 (nivolumab, OPDIVO). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and CT-011 (pidilizumab). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and YW243.55.S70. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MPDL3280A. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MEDI4736. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MDX-1105.

[0203] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an anti-tumor agent. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agent that targets CSF-1R (also known as M-CSFR or CD115). In some embodiments, the anti-human OX40 agonist antibody can 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 can 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 can be administered in combination with an interferon, such as interferon-α or interferon-γ. In some embodiments, the anti-human OX40 agonist antibody can 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 administered in combination with GM-CSF (also known as recombinant human granulocyte-macrophage colony-stimulating factor, rhu GM-CSF, sargramostim, or Leukine®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with IL-2 (also known as aldesleukin or Proleukin®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with IL-12. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with IL27. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with IL-15. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ALT-803. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antibody that targets CD20.In some embodiments, the antibody targeting CD20 is obinutuzumab (also known as GAlO1 or Gazyva®) or rituximab. In some embodiments, the anti-human OX40 agonist antibody can 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).

[0204] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of Bruton's tyrosine kinase (BTK). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ibrutinib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with AG-120 (Agios).

[0205] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with onruzumab and a PD-1 axis-binding antagonist (e.g., a PD-1-binding antagonist such as an anti-PD-1 antibody, a PD-L1-binding antagonist such as an anti-PD-1 / PD-L1 antibody, and a PD-L2-binding antagonist such as an anti-PD-L2 antibody).

[0206] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, and in some embodiments, an individualized peptide vaccine. In some embodiments, the peptide cancer vaccine is a multivalent long peptide, a multi-peptide, a peptide mixture, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an adjuvant. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a treatment comprising a TLR agonist such as Poly-ICLC (also known as Hiltonol®), LPS, MPL, or CpG ODN. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with tumor necrosis factor (TNF) α. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with IL-1. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with HMGB1. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an IL-10 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an IL-4 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an IL-13 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an IL-17 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an HVEM antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an ICOS agonist (e.g., administering ICOS-L, or an agonist antibody of ICOS).In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a treatment targeting CX3CL1. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a treatment targeting CXCL9. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a treatment targeting CXCL10. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a treatment targeting CCL5. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an LFA-I or ICAM1 agonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a selectin agonist.

[0207] 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 Zelboraf®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with dabrafenib (also known as Tafinlar®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with encorafenib (LGX818).

[0208] 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 Tarceva®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of EGFR-T790M. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with gefitinib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with afatinib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with cetuximab (also known as Erbitux®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with panitumumab (also known as Vectibix®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with rociletinib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with AZD9291. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of MEK, such as MEK1 (also known as MAP2K1) and / or MEK2 (also known as MAP2K2). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with cobimetinib (also known as CDC-0973 or XL-518). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with trametinib (also known as Mekinist®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with binimetinib.

[0209] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of B-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 can be administered in combination with an inhibitor of ERK (e.g., ERK1 / 2). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with GDC-0994. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of B-Raf, an inhibitor of MEK, and an inhibitor of ERK1 / 2. In some embodiments, the anti-human OX40 agonist antibody can 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 can be administered in combination with one or more MAP kinase pathway inhibitors. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with CK127. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of K-Ras.

[0210] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of c-Met. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ofatumumab (also known as MetMAb). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of anaplastic lymphoma kinase (ALK). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with AF802 (also known as CH5424802 or alectinib). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with crizotinib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ceritinib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of phosphatidylinositol 3-kinase (PI3K). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with buparlisib (BKM-120). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with pictilisib (also known as GDC-0941). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with buparlisib (also known as BKM-120). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with perifosine (also known as KRX-0401). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a delta-selective inhibitor of phosphatidylinositol 3-kinase (PI3K). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with taselisib (also known as GDC-0032).In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with BYL-719. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of Akt. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with MK2206. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with GSK690693. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ipatasertib (also known as CDC-0068). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of mTOR. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with sirolimus (also known as rapamycin). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with temsirolimus (also known as CCI-779 or Torisel®). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with everolimus (also known as RAD001). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with deforolimus (also known as AP-23573, MK_8669, or deforolimus). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with OSI-027. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with AZD8055. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with INK128. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a dual PI3K / mTOR inhibitor. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with XL765.In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with GDC-0980. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with BEZ235 (also referred to as NVP-BEZ235). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with BGT226. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with GSK2126458. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with PF-04691502. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with PF-05212384 (also known as PKI-587).

[0211] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agent that selectively degrades estrogen receptor. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with GDC-0927. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of HER3. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with durvalumab. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of LSD1. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of MDM2. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of BCL2. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with venetoclax. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of CHK1. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with CDC-0575. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of the activated hedgehog signaling pathway. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ERIVEDGE.

[0212] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with radiation therapy. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with gemcitabine. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with nab-paclitaxel (ABRAXANE). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with trastuzumab. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with TVEC. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with IL27. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with cyclophosphamide. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agent that mobilizes T cells to the tumor. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with lirilumab (IPH2102 / BMS-986015). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with idelalisib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with antibodies that target CD3 and CD20. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with REGN1979. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with antibodies that target CD3 and CD19. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with blinatumomab.

[0213] In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a oncolytic virus. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with carboplatin and nab-paclitaxel. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with carboplatin and paclitaxel. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with cisplatin and pemetrexed. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with cisplatin and gemcitabine. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with FOLFOX. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with FOLFIRI.

[0214] The above combination therapies include co-administration (where two or more therapeutic agents are included in the same formulation or separate formulations), and separate administrations. In this case, administration of the antibody of the invention can be carried out before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The antibody of the invention can also be used in combination with radiation therapy.

[0215] The antibodies of the invention (and any other therapeutic agents) can be administered by any suitable means including parenterally, intratracheally, and intranasally, and intralesional administration can be carried out if desired for local therapy. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration. Depending in part on whether the administration is short-term or long-term, the dosage can be by any suitable route (e.g., by injection such as intravenous or subcutaneous injection). A variety of dosage schedules are contemplated herein including, but not limited to, single administration, multiple administrations over multiple time points, bolus administration, and pulse infusion.

[0216] The antibodies of the present invention can be formulated, dosed, and administered in a manner consistent with proper medical practice. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of drug delivery, the method of administration, the dosing schedule, and other factors known to the medical practitioner. The antibodies can, but need not, optionally be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. They are generally used at the same dosage and by the same route of administration as described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and by any route that is determined to be empirically / clinically appropriate.

[0217] For the prevention or treatment of a disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) can depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody is suitable for administration to a patient in a single or series of treatments. Depending on the type and severity of the disease, an antibody in an amount of about 1 μg / kg to about 40 mg / kg can be administered to the patient as an initial candidate dosage, for example, by one or more divided doses or by continuous infusion. A typical daily dosage can be in the range of about 1 μg / kg to about 100 mg / kg or more, depending on the factors described above. In the case of repeated administration over several days or more, the treatment is usually continued until the desired suppression of the disease symptoms occurs, depending on the condition. The dosage can be administered intermittently, for example, weekly or every three weeks (e.g., such that the patient is administered about 2 to about 20 doses, or, for example, about 6 doses of the antibody). Higher initial doses can be administered, followed by one or more lower doses. However, other dosing regimens may also be useful in some cases. The progress of this treatment can be readily monitored by conventional techniques and assays.

[0218] It should be understood that any of the above formulations or treatment methods can be carried out using the immune complex of the present invention instead of, or in addition to, the anti - OX40 antibody.

[0219] In some embodiments, the intravenous (IV) infusion of the anti - OX40 antibody disclosed herein can be administered to patients every 3, 4, 5, 6, 7, or 8 weeks. The dose of the anti - OX40 antibody can be 5 mg, 10 mg, 15 mg, 30 mg, 50 mg, 100 mg, 150 mg, 200 mg, 300 mg, or 500 mg per patient. In some embodiments, the patient can have renal cell carcinoma, hepatocellular carcinoma, or head and neck squamous cell carcinoma.

[0220] In some embodiments, the anti - OX40 antibody can be administered to patients at a dose and dosing regimen that achieve one or more of the following.

[0221] Desired changes in immune cells in the patient's blood and tumor microenvironment (TME) as follows: ● Proliferation (e.g., Ki67) and activation (e.g., CD69 and CD25) markers of CD4+ or CD8+ T cells, measured by flow cytometry of the patient's peripheral blood, ● OX40 receptor levels and anti - OX40 antibody occupancy levels on CD4+CD25+ T cells, measured by flow cytometry of the patient's peripheral blood, and ● Target engagement (e.g., OX40 down - regulation), CD8+ and CD4+ T cell proliferation (e.g., Ki67 up - regulation), measured by multiplex immunohistochemistry (mIHC) or multiplex immunofluorescence (mIF) in pre - and post - treatment tumor biopsies of the patient.

[0222] In some embodiments, the dosage and dosing frequency of the anti-OX40 antibody herein are selected or determined to achieve a patient exposure profile that ensures an appropriate peak-to-trough ratio, minimizes accumulation upon repeated dosing, thereby preventing long-term receptor saturation and subsequent T cell depletion, and as a result, provides sustained immune-mediated antitumor activity in the patient.

[0223] In some embodiments, certain pharmacodynamic (PD) findings such as the desired changes in immune cells and / or the patient's TME as described above are evaluated along with the exposure profile to select or determine the dosage and / or dosing frequency of the anti-OX40 antibody.

Example

[0224] Example 1. Preparation of Anti-OX40 Antibody HFB10-1E1hG1 The nucleotide sequences encoding the heavy and light chains of the synthetic OX40 antibody HFB10-1E1hG1 (SEQ ID NOs: 24 and 32) (Goldwisdom Corporation) were synthesized and cloned into the vector pFUSE, respectively; the plasmid containing both the heavy and light chains was transiently transfected into 293F suspension cells at a 1:1 ratio using PEI to express the full-length antibody. After one week of culture, purification was performed using an AKTA system with a Superdex™ 200 Increase prepacked column.

[0225] Example 2. Binding Properties of HFB10-1E1hG1 Example 2.1 Binding Properties of HFB10-1E1hG1 to OX40 Protein For preliminary testing by ELISA, a 96-well plate containing reference substance 1, reference substance 2, reference substance 3, reference substance 4, HFB10-1E1hG1, and an isotype control was coated overnight with 5 μg / ml of the anti-OX40 antibody. The next day, after blocking the plate with 1% BSA (Sangon Biotech, catalog number A600332-0100) in PBST at 37°C for 2 hours, biotinylated OX40 recombinant protein at a defined concentration was added.

[0226] The EC80 of biotinylated OX40 recombinant protein that binds to the anti-OX40 antibody was measured. As shown in Figure 1-1, the EC80 of reference substance 1 was 0.09 nM, the EC80 of reference substance 2 was 0.22 nM, the EC80 of reference substance 3 was 0.16 nM, the EC80 of reference substance 4 was 0.24 nM, the EC80 of HFB10-1E1hG1 was 0.71 nM, and the EC80 of OX40L was 1.6 nM.

[0227] Example 2.2 Binding characteristics of HFB10-1E1hG1 to OX40 protein expressed on the surface of 293T cells To overexpress human OX40 (Sinobiological, catalog number HG10481-UT), cynomolgus monkey OX40 (Sinobiological, catalog number CG90846-UT), mouse OX40 (Sinobiological, catalog number MG50808-UT), or human CD40 (Sinobiological, catalog number HG10774-UT) was used as a target. DNA plasmids encoding these targets were transiently transfected into 293T cells according to the instructions of Lipusectamine LTX reagent and PLUS reagent (Thermo, catalog number 15338100). 48 hours after transfection, the cells were harvested for later use. To determine the binding affinity, the harvested cells were incubated with the primary antibody HFB10-1E1hG1 at a defined concentration for 1 hour at 4°C. Then, after washing twice with PBS, the cells were incubated with the secondary antibody goat anti-human IgG PE (1:200; Abcam, catalog number ab98596) for 30 minutes at room temperature. Detection was performed using a Beckman CytoFLEX flow cytometer.

[0228] As shown in Figure 1-2, the EC50 of the binding of HFB10-1E1hG1 to human OX40 protein expressed on the surface of 293T cells was 2 nM (MFI).

[0229] As shown in FIGS. 1-3, the EC50 of the binding of HFB10-1E1hG1 to cynomolgus monkey OX40 protein expressed on the surface of 293T cells was 2.9 nM (MFI).

[0230] As shown in FIG. 1-4, HFB10-1E1hG1 did not bind to mouse OX40 protein expressed on the surface of 293T cells (MFI).

[0231] As shown in FIG. 1-5, HFB10-1E1hG1 did not bind to human CD40 protein expressed on the surface of 293T cells (MFI).

[0232] The EC50s of the binding of HFB10-1E1hG1, reference substance 1, reference substance 2, reference substance 3, and reference substance 4 to human OX40 protein expressed on the surface of 293T cells are shown in FIGS. 1-6, respectively. The EC50 of HFB10-1E1hG1 was 2.02 nM (MFI), the EC50 of reference substance 1 was 2.67 nM (MFI), the EC50 of reference substance 2 was 4.17 nM (MFI), the EC50 of reference substance 3 was 1.92 nM (MFI), and the EC50 of reference substance 4 was 2.11 nM (MFI).

[0233] The EC50s of the binding of HFB10-1E1hG1, reference substance 1, reference substance 2, reference substance 3, and reference substance 4 to cynomolgus monkey OX40 protein expressed on the surface of 293T cells are shown in FIGS. 1-7, respectively. The EC50 of HFB10-1E1hG1 was 2.94 nM (MFI), the EC50 of reference substance 1 was 2.91 nM (MFI), the EC50 of reference substance 2 was 6.13 nM (MFI), the EC50 of reference substance 3 was 2.57 nM (MFI), and the EC50 of reference substance 4 was 3.54 nM (MFI).

[0234] Example 3. Agonist Activity of HFB10-1E1hG1 Example 3.1 Agonist Activity of HFB10-1E1hG1 in Jurkat Reporter Protein Anti-OX40 antibodies can be divided into two categories according to their different modes of action: the first class of OX40 agonist activity is independent of Fc receptor cross-linking, while the other class requires Fc receptor cross-linking for OX40 agonist activity. Tumor tissues and surrounding draining regional lymph nodes have more tumor-related inflammatory cells. The Fc receptor FcγR2b is likely to cluster around tumor cells. Therefore, "bridging antibody" agonists have higher tissue selectivity. Only in the intratumoral microenvironment can the antibody have a significant agonist effect, but in the normal tissue parts of the body, the effect remains at a low level. This can improve the safety margin of treatment.

[0235] Recombinant Jurkat reporter cells expressing the GFP gene under the control of an NF-kb response element constitutively expressing human OX40 were used in this study. To determine the agonist activity of HFB10-1E1hG1, 96-well plates were coated overnight with 5 μg / ml of anti-human IgG Fc-specific antibody (Sigma, catalog number SAB3701275). A defined concentration of HFB10-1E1hG1 was added to one well together with 1×10 5 of Jurkat reporter cells. In another experiment, 50 nM of OX40L (Acrobiosystems, catalog number OXL-H52Q8) was added together with HFB10-1E1hG1 to determine the synergistic effect. After 24 hours of incubation, the Jurkat reporter cells were harvested. Furthermore, a Beckman CytoFLEX flow cytometer was used to detect the GFP-positive signal, indicating the agonist activity of HFB10-1E1hG1 in Jurkat reporter cells.

[0236] The agonistic activity of HFB10-1E1hG1 depends on anti-human IgG crosslinking. As shown in Figure 2-1, the EC50 of HFB10-1E1hG1 was 2.9 nM (GFP MFI) in the presence of crosslinking with anti-human IgG. As shown in Figure 2-2, when not crosslinked with anti-human IgG, the EC50 of HFB10-1E1hG1 was much higher than when crosslinked with anti-human IgG. As shown in Figure 2-2, the trimeric OX40L recombinant protein alone could activate NF-kb signaling with an EC50 = 45 nM (GFP MFI) without crosslinking with anti-human IgG.

[0237] HFB10-1E1hG1 showed a cooperative agonistic effect with OX40L in the presence of crosslinking with anti-human IgG. As shown in Figure 2-3, by adding HFB10-1E1hG1 together with OX40L, the MFI of GFP was enhanced compared to each component alone.

[0238] Example 3.2 Agonistic Activity of HFB10-1E1hG1 in Primary CD4+ T Cells 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, catalog number 16-0037-81) and 5 μg / ml anti-human IgG Fc-specific antibody (Sigma, catalog number SAB3701275). The next day, primary CD4+ T cells were recovered according to the instructions of the CD4+ T cell isolation kit (Miltenyi, catalog number 130-045-101). A predetermined concentration of HFB10-1E1hG1 and 2 μg / ml anti-CD28 antibody (Thermo, catalog number 16-0289-81) were added at 1×10 5were added to one well together with the primary CD4+ T cells. In another experiment, 50 nM of OX40L (Acrobiosystems, catalog number OXL-H52Q8) was added together with HFB10-1E1hG1 to determine its synergistic effect. After 3 days of incubation, the level of IL-2 secretion in the supernatant was detected according to the instructions of the Human IL-2 DuoSet ELISA Kit (R&D, catalog number DY202-05), indicating the agonist activity of HFB10-1E1hG1 in primary CD4+ T cells.

[0239] To determine the agonist activity of HFB10-1E1hG1 in primary CD4+ T cells, purified CD4+ T cells were pre-activated with 1 μg / ml of anti-CD3 antibody and 2 μg / ml of anti-CD28 antibody. To facilitate cross-linking with HFB10-1E1hG1, the plates were pre-coated with 5 μg / ml of anti-human IgG. After 3 days of incubation, the agonist activity of HFB10-1E1hG1 in primary CD4+ T cells was determined by IL-2, and an EC50 of 0.2 nM was obtained as shown in Figures 2-4.

[0240] In primary CD4+ T cells, HFB10-1E1hG1 showed a synergistic agonist effect with OX40L. As shown in Figure 2-5, incubation of HFB10-1E1hG1 with 50 nM of OX40L enhanced IL-2 secretion compared to the individual components.

[0241] Example 4. Pharmacokinetic study of HFB10-1E1hG1 The 384-well plates were coated overnight with 1 μg / ml of F(ab’)2 goat anti-human IgG Fc-specific antibody (Jackson IR, catalog number 109-006-098) in PBS at 30 μl / well. After washing three times with PBST buffer, the plates were blocked with a blocking buffer containing 1 mM EDTA, 0.05% Tween, and 2% BSA in PBS at 37 °C for 1 hour. Then, mouse serum samples were added at 15 μL / well at serial dilutions starting at 1 / 150 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, catalog number 109-035-003) was added at 1 / 5000 and incubated at 37 °C for 0.5 hour. Then, the TMB substrate (Biolegend, catalog number 421101) was added and incubated for an additional 15 minutes. Next, the ELISA stop solution (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was added. The plates were read at 450 nm using a Multiskan Sky Microplate spectrophotometer (ThermoFisher).

[0242] 10 mg / kg of HFB10-1E1hG1 was administered intravenously to hOX40 knock-in mice (purchased from Shanghai Southern Model Organism Research Center, numbers 131, 132, 133). Mouse sera were collected at 1 hour, 24 hours, 48 hours, 72 hours, 96 hours, and 196 hours after administration. As shown in Figure 3-1, the half-life of HFB10-1E1hG1 in hOX40-KI mouse sera was determined to be 24 hours. The data points at 0 hour were, from top to bottom, data points 131, 132, and 133.

[0243] Example 5. In Vivo Antitumor Effect of HFB10-1E1hG1 hOX40 knock-in mice were purchased from Shanghai Southern Model Organism Research Center. After 5 days of isolation, 8×10 in 100 μl of PBS5 MC38 tumor cells (provided by Professor Zhang Hongkai, Nankai University) were subcutaneously inoculated into each mouse. When the tumor size reached 70 - 100 mm 3 reached, treatment with anti - OX40 antibody was initiated by intraperitoneally administering the anti - OX40 antibody to the mice at 10 mg / kg in 100 μl of PBS 5 times every 3 days. Tumor size and mouse body weight were measured twice a week. Vernier calipers were used to measure the tumor size in both directions. Tumor volume was calculated using the following formula: V = 0.5a×b 2 (where a and b are the major and minor diameters of the tumor, respectively) and expressed in mm 3 . Day 7: MC38 tumor cell inoculation, 35 eight - week - old mice - MC38 cells were obtained from Professor Zhang Hongkai - 8×10 5 MC38 cells per mouse were inoculated Day 0: The average tumor size was 75 mm 3 (40 - 120 mm 3 ) - 5 mice per group, a total of 4 groups: ● PBS ● Reference antibody 1 ● HFB10 - 1E1hG1 ● Reference antibody 2 - Initiated by intraperitoneal administration of 10 mg / kg of the antibody 5 times every 3 days. Day 18: The tumors in the PBS control group reached a size of 2000 mm 3 . The mice were sacrificed. - Tissues: blood, LN, liver, spleen, tumor - Phenotype analysis: T cells CD3 / CD4 / CD8, Treg CD4 / CD25 / Foxp3, NK CD3 / CD16 / CD56

[0244] As shown in FIGS. 4-1 and 4-2, this experiment showed that HFB10-1E1hG1 significantly inhibited tumor growth compared to the PBS control and showed no side effects that could cause significant weight loss in mice. In FIG. 4-1, the data points at the right end were, in order from top to bottom, PBS, reference antibody 1, HFB10-1E1hG1, and reference antibody 2. In FIG. 4-2, the second data point from the left was, in order from top to bottom, PBS, reference antibody 1, reference antibody 2, and HFB10-1E1hG1.

[0245] The dose-response efficacy of the antibody was examined in hOX40 knock-in mice. The specific groups of this test were as follows: Group 1: PBS: 5 times intraperitoneally every 3 days; Group 2: HFB10-1E1hG1: 1 mg / kg, intraperitoneally, 5 times intraperitoneally every 3 days / 4 days; Group 3: HFB10-1E1hG1: 0.1 mg / kg, intraperitoneally, 5 times intraperitoneally every 3 days / 4 days; Group 4: Reference antibody 1: 1 mg / kg, intraperitoneally, 5 times intraperitoneally every 3 days.

[0246] A total of 20 hOX40 knock-in mice were used in this test and divided into 4 groups of 5 mice each.

[0247] Mice were inoculated with MC38 tumor cells. Starting when the average tumor size was 75 mm 3 , antibody injections were performed 5 times on days 0, 3, 6, 10, and 13. Tumor size and mouse body weight were measured twice a week for up to 3 weeks or until the tumor size exceeded 2000 mm 3 .

[0248] The reaction results of tumor size and mouse body weight with different doses of HFB10-1E1hG1 are shown in FIGS. 4-3 and 4-4. This experiment showed that 1 mg / kg of HFB10-1E1hG1 showed the best anti-tumor effect in vivo.

[0249] Example 6. In Vitro Characterization of the Developmental Characteristics of HFB10-1E1hG1 Example 6.1 Accelerated Stability Experiment of HFB10-1E1hG1 The antibody sample (HFB10-1E1hG1, 3.1 mg / mL, lot number CP181130004) was concentrated to 10 mg / mL using a centrifugal filter device (Millipore, catalog number UFC503096) (lot number JW20181203, lot number 20190624). Then, an appropriate amount of the concentrated antibody sample was transferred to a clean 600 μl test tube and incubated at 25 °C and 40 °C for 7 days, 14 days, and 30 days, respectively.

[0250] The incubated samples were analyzed by SEC-HPLC and SDS-PAGE: For SEC-HPLC, 50 μg of each treated sample was injected and tested in 1×PBS buffer at pH 7.4 (diluted from 10×PBS buffer (Sangon, catalog number E607016-0500) with Milli-Q pure water at a flow rate of 0.7 mL / min for 40 minutes per test). Absorbance was detected at UV280 nm (an untreated sample stored at 4 °C was also loaded for analysis). The results are shown in Table 1 below. Since no significant increase in the aggregation or degradation peak of HFB10-1E1hG1 was observed on the SEC curve even after incubation at 25 °C and 40 °C for up to 30 days, it is suggested that HFB10-1E1hG1 has good stability under such treatment conditions. The slight degradation observed after incubation at 40 °C for 30 days may indicate its instability due to long-term incubation at high temperature. [Table 2]

[0251] For SDS-PAGE, 4 μg of each treated sample was loaded onto a 4% - 20% gradient gel under non-reducing and reducing conditions, respectively. The gel was run at 150 V for 1 hour in Tris-glycine buffer, overstained in staining solution (TaKaRa, catalog number T9320A) for over 1 hour, then de-stained several times in distilled water, and imaged on a plate for white light (untreated samples stored at 4 °C were also loaded for analysis). The results are shown in Figure 5-1.

[0252] After incubation at 25 °C and 40 °C for 30 days, no distinct aggregation or degradation bands of HFB10-1E1hG1 were observed in the SDS-PAGE images, suggesting that HFB10-1E1hG1 has good stability under such treatment conditions. After incubation at 40 °C for 30 days, slightly degraded bands along with aggregation bands on non-reducing gels and reducing gels were observed, which may suggest its instability due to long-term incubation at high temperature.

[0253] Example 6.2 Degradation experiment of HFB10-1E1hG1 1. Low pH stress test: An appropriate amount of antibody (HFB10-1E1hG1, 3.1 mg / mL) was transferred to a 600 μl test tube, and then 2 M acetic acid (v / v of acid to antibody sample, adjusted to a final pH of about 3.5) was added at a ratio of 1:20. The sample was mixed well and incubated at room temperature for 0 hours, 3 hours, or 6 hours. After incubation, the pH of the antibody solution was adjusted to 7.4 with neutralization buffer (added to samples incubated with 1 M Tris-HCl at pH 9.0 in a ratio of 13:100 (v / v)). Then, the sample was analyzed by SEC-HPLC and SDS-PAGE as described above (see accelerated stability experiment. Note: Untreated samples stored at 4 °C were also loaded for analysis).

[0254] 2. High pH stress test: An appropriate amount of the antibody (HFB10-1E1hG1, 3.1 mg / mL) was transferred to a 600 μL test tube, and then 1 M Tris-HCl, pH 8.5 (v / v, adjusted to a final pH of approximately 8.5) was added at a ratio of 1:25. The sample was mixed well and incubated at room temperature for 0 hours or 6 hours. The sample was then analyzed by SEC-HPLC and SDS-PAGE (under reducing conditions only) as described above.

[0255] The SEC analysis is 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 the aggregation or degradation peak of HFB10-1E1hG1 was observed on the SEC curve, suggesting that HFB10-1E1hG1 has good stability under such processing conditions. [Table 3]

[0256] The SDS-PAGE analysis is shown in Figure 5-2. After incubation for up to 6 hours in low pH and high pH buffers, no change in HFB10-1E1hG1 was observed on the SDS-PAGE gel, suggesting good stability of HFB10-1E1hG1 under such processing conditions.

[0257] Example 6.3 Oxidative Stress Experiment of HFB10-1E1hG1 An appropriate amount of the antibody (HFB10-1E1hG1, 3.1 mg / mL) was transferred to a 600 μL test tube, and then H2O2 (0.1% and 1% respectively) or t-BHP (final concentration 0.1%) was added. The sample was mixed well and incubated at room temperature for 0 hours or 6 hours. The sample was then analyzed by SEC-HPLC and SDS-PAGE as described above.

[0258] Note: 1) An untreated sample stored at 4°C was loaded for analysis. 2) An SEC running buffer (pH 6.8) containing 100 mM NaH2PO4 and 150 mM NaCl was prepared in-house.

[0259] The SEC analysis was shown in Table 3. After incubation for 6 hours in the corresponding solutions of 0.1% H2O2, 1% H2O2, and 0.1% t-BHP (tert-butyl hydroperoxide), no obvious change in HFB10-1E1hG1 was observed on the SEC curve, suggesting that HFB10-1E1hG1 had good stability under such treatment conditions.

Table 4

[0260] The SDS-PAGE analysis was shown in Figure 5-3. Except for the slightly degraded bands on the non-reducing gel, no obvious change in HFB10-1E1hG1 was observed on the SDS-PAGE gel after incubation for 6 hours in the corresponding solutions of 0.1% H2O2, 1% H2O2, and 0.1% t-BHP (tert-butyl hydroperoxide). This suggests that HFB10-1E1hG1 has good stability under such treatment conditions.

[0261] Example 6.4 Freeze-thaw experiment of HFB10-1E1hG1 The antibody sample (HFB10-1E1hG1, 3.1 mg / mL, lot number CP181130004) was concentrated to 10 mg / mL using a centrifugal filter device (Millipore, catalog number UFC503096) (lot number JW20181203). Next, an appropriate amount of the concentrated antibody sample was transferred to clean 600 μl test tubes (3 × test tubes), and the samples were frozen in liquid nitrogen for 2 minutes and then thawed in a water bath at room temperature. The same procedure was performed 2 more times, 4 times, or more.

[0262] Note: 1) The untreated sample stored at 4°C was loaded for analysis. 2) The SEC running buffer (pH 6.8) containing 100 mM NaH2PO4 and 150 mM NaCl was prepared in-house.

[0263] For DSF-based thermal stability analysis: 3 μg of each treated sample was used in 25 μl reactions each in a PCR plate (Bio-Rad plate, catalog number HSP9655; Bio-Rad membrane, catalog number MSB1001) together with the ProteoStat assay kit (Enzo Life Sciences, catalog number ENZ-51027-K400) according to the manufacturer's instructions. The heating program was set as follows on a Bio-Rad PCR machine (C1000 touch, CFX96 real-time system): raise the temperature to 95°C at 0.5°C every 10 seconds for 2 minutes at 25°C. Fluorescence absorbance was read using the Texas Red mode. The Tm value was associated with the lowest point of -dF / dT.

[0264] SEC analysis is shown in Table 4. DSF analysis is shown in Table 5. SEC analysis showed that no obvious change in HFB10-1E1hG1 was observed on the SEC curve after up to 5 freeze / thaw cycles, indicating that HFB10-1E1hG1 had good stability under such treatment conditions. DSF analysis showed that the Tm value of HFB10-1E1hG1 did not change significantly after the same treatment, suggesting that HFB10-1E1hG1 had good stability under such treatment conditions.

Table 5

Table 6

[0265] SDS-PAGE analysis is shown in Figure 5-4. No obvious change in HFB10-1E1hG1 was observed on the SDS-PAGE gel after up to 5 freeze / thaw cycles, suggesting good stability of HFB10-1E1hG1 under such treatment conditions.

[0266] Example 7 Binding of the agonist antibody to OX-40 has been shown to result in receptor downregulation, as observed in in vitro and clinical trials (Wang et al. Cancer Research 2019). A hypothesis was further proposed that the resulting loss of target expression observed in patients after the first injection could limit the success of OX-40 agonist antibodies in clinical trials (Wang et al. Cancer Research 2019). By using ex vivo activated naive T cells isolated from PBMCs, the present invention showed that treatment with HFB10-1E1hG1 resulted in less reduction of OX-40 levels after treatment compared to baseline. Together with optimized binding kinetics, target degradation is minimized by the unique binding epitope, thereby avoiding loss of target expression after the first injection. Thus, it enables continuous drug administration to patients.

[0267] Example 8 Prediction of the human pharmacokinetics (PK) of HFB10-1E1hG1 The cynomolgus monkey PK data was fitted to a two-compartment model. Figure 6 shows the fitted PK curve and the observed data. The RMSE of the fit was 0.17, and the R 2 was 0.99, indicating good fit to the observed data.

[0268] The human two-compartment PK parameters were predicted from the corresponding monkey parameters based on the rule of exponents (ROE) (Dong et al., 2011).

[0269] Subsequently, the predicted parameters were used to simulate the human PK curve. Figure 7 shows an example of the single-dose human PK profile of 1 mg / kg after a 1-hour intravenous infusion.

[0270] Prediction of the minimum human PAD based on the HFB10-1E1hG1 PK data in hOX40KI mice In hOX40KI mice, the Cmax and AUC(0-72 h) values of HFB10-1E1hG1 at a dose of 10 mg / kg were 160.5 μg / mL and 2591 μg / mL·h, respectively. Assuming linear PK, the Cmax and AUC(0-72 h) values at a dose of 1 mg / kg were 16 μg / mL and 259 μg / mL·h, respectively.

[0271] At a dose of 1 mg / kg, the predicted human Cmax and AUC(0-72 h) values were 23.7 μg / mL and 1213 μg / mL·h, respectively. Assuming human linear PK, the minimum PAD was predicted to be 0.68 mg / kg (based on Cmax) or 0.21 mg / kg (based on AUC(0-72 h)). Conservatively, the minimum human PAD was assumed to be 0.21 mg / kg or a fixed dose of 15 mg (assuming a standard body weight of 70 kg).

[0272] Considerations regarding human dosing frequency To maximize OX40 agonism followed by effector T cell proliferation and regulatory T cell depletion in the tumor microenvironment, it is necessary to consider not saturating the OX40 receptor for an extended period. One strategy to ensure an appropriate time between OX40 agonisms is to avoid significant accumulation of the agonist of interest. Therefore, the PK profiles of HFB10-1E1hG1 were simulated on a schedule of every two weeks (Q2W), every three weeks (Q3W), or every four weeks (Q4W). Figure 8 shows the PK curve simulated at Q4W.

[0273] Next, the Cmax and Cmin accumulation indices for each schedule, as well as the Cmax / Cmin ratio at steady state, were calculated.

[0274] In addition to practical considerations, based on the calculated AI and Cmax / Cmin ratio, a starting dose of 15 mg and a Q4W schedule were selected for the first-in-human trial of HFB10-1E1hG1.

[0275] The human PK of HFB10-1E1hG1 is similar to that of a typical IgG1 monoclonal antibody (mAb), with low clearance (CL, 0.084 mL / hour / kg), a small volume of distribution (Vd ss , 0.084 L / kg), and was predicted to have a typical mAb half-life (T 1 / 2 , 731 hours, approximately 30 days).

[0276] The minimum human PAD of HFB10-1E1hG1 was predicted based on the prediction of human PK from cynomolgus monkey PK, the minimum effective dose from the MC38 tumor efficacy test in hOX40 KI mice, and the exposure data of HFB10-1E1hG1 in hOX40 KI mice. The extrapolation of the exposure / efficacy relationship of KI mice to humans showed a minimum PAD of 0.21 mg / kg in humans. For convenience, an equivalent fixed dose of 15 mg is recommended as the starting dose in humans. Based on the predicted steady-state PK profiles at various dosing frequencies, the recommended dosing frequency is once every 4 weeks (Q4W).

[0277] In this specification, flow cytometry was used to evaluate the binding of HFB10-1E1hG1 to activated primary monkey T cells. The results are shown in Figure 1.

[0278] Bmk1, which can recognize CD4+ T cells, was used as a positive control. HFB10-1E1hG1 was shown to bind to activated CD3 / CD28 primary monkey T cells isolated from three different donors (lot numbers 200107, M20Z013009, and M20Z025008). The EC50 values calculated from the MFI dose-response curves were 0.09 nM, 0.19 nM, and 0.17 nM, respectively.

[0279] The linear detection ranges of the ELISA assays were 125 - 1000 pg / ml in experiment 20200730 and 125 - 2000 pg / ml in experiment 20200731, respectively. The LLOQ for both experiments was 18.75 ng / ml.

[0280] In this specification, using a single administration of 10 mg / kg, the pharmacokinetics of Bmk1 and HFB10-1E1hG1 by intravenous administration at single administrations of 10 mg / kg and 1 mg / kg in WT mice and hOX40-KI mice were evaluated. Plots of the mean concentrations of Bmk1 and HFB10-1E1hG1 against time are shown in Figures 7 and 8 on linear and semi-logarithmic scales.

[0281] Systemic exposure of HFB10-1E1hG1 and Bmk1 was achieved in all treated mice. In WT mice (Figure 7), HFB10-1E1hG1 and Bmk1 showed similar linear clearances with dose proportionality. The dose ratio of HFB10-1E1hG1 to Bmk1 was 1:10, but the ratios of Cmax and AUC(0-72 hours) of HFB10-1E1hG1 were 1:6.8 and 1:7.9, respectively, and the dose ratios of Bmk1 were 1:11.8 and 1:8.6, respectively.

[0282] The serum clearances of HFB10-1E1hG1 at 1 mg / kg and 10 mg / kg were 0.71±0.34 and 1.06±0.77 ml / hour / kg, respectively, in wild-type mice, but 3.09±0.27 ml / hour / kg in hOX40-KI mice. Similar differences in serum clearance were also observed in the PK of Bmk1. The serum clearances of Bmk1 in wild-type mice were 0.45±0.13 and 0.62±0.10 ml / h / kg at 1 mg / kg and 10 mg / kg, respectively, while in hOX40-KI mice, it was 7.24±2.24 ml / h / kg at 10 mg / kg. Since hOX40 is expressed only in hOX40-KI mice, the higher clearance rate in hOX40-KI mice may be due to target-mediated drug disappearance (TMDD).

[0283] The steady-state volume of distribution (Vdss) values of HFB10-1E1hG1 at 1 mg / kg and 10 mg / kg were 0.21 ± 0.03 and 0.26 ± 0.02 L / kg in wild-type mice, respectively, while at 10 mg / kg, it was 0.14 ± 0.01 L / kg in hOX40-KI mice. Similarly, the Vdss values of Bmk1 were 0.19 ± 0.02 and 0.21 ± 0.01 L / kg in wild-type mice, and 0.08 ± 0.03 L / kg in hOX40-KI mice.

[0284] A significant difference in T1 / 2 was also observed between WT mice and hOX40KI mice for these two antibodies. In wild-type mice, the terminal half-life (T 1 / 2 ) of HFB10-1E1hG1 at 10 mg / kg and 1 mg / kg was 239.72 ± 140.48 hours and 274.52 ± 193.12 hours, respectively. The terminal half-lives of Bmk1 at 10 mg / kg and 1 mg / kg were 252.04 ± 51.82 hours and 321.05 ± 75.89 hours, respectively. For HFB10-1E1hG1 and Bmk1 at a dose of 10 mg / kg, their T1 / 2 values were significantly shorter in hOX40KI mice, being 38.65 ± 4.84 hours and 5.45 ± 1.09 hours, respectively.

[0285] Example 9. Pharmacodynamic test Method: For tumor growth, MC-38 tumor cells were subcutaneously inoculated on the right side of 30 female human OX40 knock-in mice (C57BL / 6 background). Nine days after tumor inoculation, 76 - 226 mm 3 (average tumor size 155 mm 3Twenty mice with a tumor size of ) were selected and divided into four groups of five mice each based on their tumor volume by stratified randomization. Treatments from the day of randomization (defined as day 0 (D0)) included: isotype control, 10 mg / kg; HFB10-1E1hG1, 0.1 mg / kg; HFB10-1E1hG1, 1 mg / kg; and HFB10-1E1hG1, 10 mg / kg. All therapeutic agents were administered by intraperitoneal injection on D0, D3, and D7. Tumor size and animal body weight were measured at least three times a week. Six hours after the third administration, tumor samples were collected for flow cytometry (FCM) analysis, blood samples were collected for receptor occupancy (RO) analysis, and plasma samples were sent to HiFiBiO.

[0286] Results: The PD effect of HFB10-1E1hG1 was examined in MC-38 tumor-bearing hOX40KI mice. In both blood and the tumor microenvironment (mainly within the tumor), HFB10-1E1hG1 + resulted in a decrease in OX40 expression, represented as the percentage of positive and MFI of T cells, mainly CD4 + T cells. At the same time, HFB10-1E1hG1 also significantly reduced the Treg population. In the tumor microenvironment, the proportion of CD4 + T cells decreased (mainly caused by the decrease in Treg (CD25 + FOXP3 + cells)), the proliferation of CD4 + T cells (Ki67 + cells) increased, while CD69 + T cells decreased. The expression of PD1 was reduced in CD8

[0287]

[0287] The effect of HFB10-1E1hG1 on OX40 expression in blood samples was analyzed using a mouse anti-hOX40 antibody (clone ACT35) that does not compete with HFB10-1E1hG1. CD4 + 、CD4 + CD25 + and CD11b + total OX40 expression in monocytes was measured (data not shown). HFB10-1E1hG1 treatment resulted in significant downregulation (positive rate and MFI) of OX40 expression in CD4 + and CD4 + CD25 + T cells. No change in OX40 expression was observed in CD11b + cells.

[0288] To evaluate target engagement after HFB10-1E1hG1 treatment, anti-human IgG (hIgG) was used in combination with non-competing Ab ACT35 in FCM analysis. OX40 + CD4 + T cells and OX40 + CD4 + CD25 + The percentage of the hIgG + population on T cells was low. However, the hIgG + CD4 + CD25 + signal expressed as MFI in cells (including Tregs) showed a significant dose-dependent increase in HFB10-1E1hG1. +

[0289] OX40 + CD11b + In cells, high levels of hIgG + signal could be observed in the isotype group and the 10 mg / kg HFB10-1EhG1-treated group. This is correlated with the treatment dose and suggests that hIgG was captured by FcgR on CD11b + cells.

[0290] The infiltrating immune cell population in tumor tissues was analyzed by flow cytometry ("FCM"). All FCM analysis plots in this example were as follows: ● G1: Isotype control group; G2: HFB10-1E1hG1 0.1 mg / kg treatment group; G3: HFB10-1E1hG1 1 mg / kg treatment group; G4: HFB10-1E1hG1 10 mg / kg treatment group. ● Each dot represents the data of an individual animal, the rectangular bar represents the group mean, and the error bar represents the SEM.

[0291] T cells (CD3 + ), CD4 + T, CD8 + T cells and Treg (CD3 + CD4 + CD25 + Foxp3 + ) percentages were measured, and the selected results are shown in Figures 9 and 10. Naive T cells (CD3 + CD44-CD62L + ), memory T cells (CD3 + CD44 + CD62L + ) and effector T cells (CD3 + CD44 + CD62L-) were also measured (data not shown). Under the experimental conditions of the present invention, compared with the isotype control group (G1), the groups treated with HFB10-1E1hG1 (G3 and G4, but not G2) showed a significantly decreased percentage of CD4 + T cells, Treg, naive T cells and naive CD4 + T cells. Furthermore, all HFB10-1E1hG1 treatments resulted in a significant decrease in the percentage of memory T cells and memory CD4 + T cells. All HFB10-1E1hG1 treatment groups showed a decrease in the percentage of memory CD8 + T cells, but only G2 and G3 reached statistical significance. Between treatment groups, T cells (CD3 + ), CD8 +No significant changes were observed in the population of T cells and effector T cells.

[0292] The effect of HFB10-1EhG1 treatment on the OX40 expression, activation and proliferation of different T cell subtypes in tumors was further investigated. The effect of HFB10-1E1hG1 on total OX40 expression was analyzed using a mouse anti-hOX40 antibody (clone ACT35) that does not compete with HFB10-1E1hG1. CD3 + , CD4 + , CD8 + T cells and Tregs (CD4 + CD25 + FoxP3 + ) were measured for OX40 expression (data not shown). CD3 + and CD4 + OX40 expression in T cells was significantly decreased in both the percentage and MFI of the HFB10-1E1hG1-treated group compared to the isotype control group (G1). In the case of Tregs, HFB10-1E1hG1 treatment did not significantly decrease OX40 expression represented by percentage, but significantly decreased OX40 expression represented by MFI (data not shown). In the CD8 + T cells of the HFB10-1E1hG1-treated group, the OX40 expression represented by percentage was significantly decreased, but the decrease in MFI did not reach a significant difference.

[0293] Treatment with HFB10-1E1hG1 (G3 and G4) resulted in a significantly lower percentage of CD69 + CD4 + T cells and PD-1 + CD8 + T cells compared to the isotype control group (G1) (Figure 12). The same treatment also significantly increased the percentage of proliferating (Ki67 + ) CD4 + T cells (Figure 11). All these observations were dose-dependent in the HFB10-1E1hG1-treated group.

[0294] Example 10: Epitope mapping The monoclonal anti-OX40 antibody HFB301001 was developed as an agonist antibody. Table 1 shows the CDR, VH / VL, and HC / LC sequences of HFB301001.

[0295] The binding epitope was characterized by performing epitope binning using a competitive ELISA assay and searching for the OX40 binding epitopes of different anti-OX40 antibodies compared to previously published selected antibodies and the OX40 ligand binding epitope. Briefly, plates were coated with 50 μl of 1 μg / ml anti-OX40 capture antibody, OX40L, or isotype control and incubated overnight at 4°C. The plates were then blocked with 1% BSA in PBST for 2 hours at room temperature. Next, 50 μl / well of biotin-OX40 (lot number 160921111, ChemPartner; Tokyo, JP) was added. Biotin-OX40 was diluted 1- to 3-fold at 8 points starting from 0.5 μg / mL and incubated at 37°C for 1 hour. Next, HRP-conjugated streptavidin (1 / 5000 dilution; 50 μl / well) was added and incubated at 37°C for 1 hour, after which 100 μl / well of TMB was added and incubated at room temperature for 15 minutes. The reaction was stopped by adding 50 μl / well of ELISA stop solution. OD values were determined at a wavelength of 450 nm using a Thermo Multiscan Sky spectrophotometer.

[0296] Hydrogen-deuterium exchange mass spectrometry was used to obtain a more detailed resolution of the bound epitopes. Briefly, hydrogen / deuterium exchange epitope mapping was performed using hydrogen / deuterium exchange mass spectrometry (HDX-MS) to determine the amino acid residues of OX40 (recombinant human OX40) that interact with HFB301001. General descriptions of the H / D exchange method are provided, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259, and Engen and Smith (2001) Anal. Chem. 73:256A-265A. The His-tagged OX40 antigen protein was purchased from Sinobiological (Catalog number 10481-H08H, Sinobiological, Inc., Beijing, CN). The HDX-MS experiments were performed on an integrated HDX / MS platform for peptide mass measurement provided by Novabioassays LLC and a Thermo Q Exactive HF mass spectrometer. 4.5 μL of each sample was incubated with 75 μL of deuterium-labeled buffer (1× PBS, pD 7.4) at 20 °C for 300 seconds, 600 seconds, 1800 seconds, and 3600 seconds. Hydrogen / deuterium exchange was stopped by adding 80 μL of buffer (final pH 2.5) containing 4 M guanidine hydrochloride and 0.85 M TCEP. The reaction-stopped samples were then subjected to on-column pepsin / protease XIII digestion followed by LC-MS analysis. Mass spectra were recorded in the MS-only mode. Then, pepsin / protease XIII digestion, LC-MS, and data analysis were performed. After HDX labeling, 80 μL of buffer (final pH 2.5) containing 4 M guanidine hydrochloride and 0.85 M TCEP was added, and the samples were denatured by incubating at 20 °C for 3 minutes. The mixture was then subjected to on-column pepsin / protease XIII digestion using an internally packed pepsin / protease XIII (w / w, 1:3) column. The resulting peptides were analyzed using a UPLC-MS system consisting of a Waters Acquity UPLC coupled to a Q Exactive™ and Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo, Waltham, MA).The peptides were separated on a 50×1 mm C8 column using a gradient starting from 2% - 33% solvent B (0.2% formic acid in acetonitrile) over 20.5 minutes. Solvent A was water containing 0.1% formic acid. The injection valve, enzyme column, and associated connecting tubes were placed in a cooling box maintained at 15 °C. The second switch valve, C8 column, and associated stainless-steel connecting tubes were placed in a refrigerated circulation box maintained at -6 °C. Peptide identification was performed by searching the MS / MS data of the HFB301001 sequence using Byonics (Protein Metrics, San Carlos, CA) software modified to consider non-specific enzymatic digestion and human glycosylation as common variables. The mass tolerances for precursor ions and product ions were 10 ppm and 0.02 Da, respectively. The raw MS data were processed using HDX WorkBench software (version 3.3) for analyzing H / D exchange MS data (J. Am. Soc. Mass Spectrom. 2012, 23(9), 1512 - 1521). The deuterium incorporation level was calculated using the average mass difference between the deuterated peptide and its unlabeled form (t0). The deuterium incorporation levels of the same peptides were compared in samples of antigen alone and antigen - antibody complex. A relative difference exceeding 5% was considered significant. The epitope region was defined by multiple overlapping peptides showing significant levels of deuterium incorporation. A total of 46 peptides from hOX40 - his were identified from hOX40 - his alone and hOX40 - his forming a complex with the HFB301001 sample, which corresponds to 70% sequence coverage of hOX40 (Figure 13). Peptides showing a percentage difference in deuterium incorporation values exceeding 5% were defined as significantly protected. In the case of hOX40 - his, the peptides corresponding to the region of amino acids 56 - 74 CSRSQNTVCRPCGPGFYND were defined as epitopes because they were significantly protected by HFB301001.As shown in Fig. 13, the epitope region was mapped to the 3D model of the OX40 / OX40L complex adopted from Compaan and Hymowitz. Structure. 2006, 14(8), 1321-30. The epitope of HFB301001 was defined as amino acids 56-74 of SEQ ID NO: 33, CSRSQNTVCRPCGPGFYND (SEQ ID NO: 34).

[0297] Example 11: Agonist Activity of OX40 Ligand To determine the agonist activity of OX40 ligand (OX40L), an OX40 bioassay kit (Promega, catalog number CS197704; Promega; Madison, WI) was used according to the manufacturer's protocol. In this assay, a genetically engineered Jurkat T cell line expressing human OX40 (OX40 effector cells) and a luciferase reporter gene driven by a response element were used. Briefly, OX40 effector cells were cultured in assay buffer the day before use. On the day of the assay, OX40L was serially diluted and added to the plate. The bioluminescence signal was quantified using the Bio-Glo Luciferase Assay System after 5 hours of induction. The results showed that OX40L induced the bioluminescence signal in a dose-dependent manner in OX40 effector cells (Fig. 14A).

[0298] To examine the effect of anti-OX40 antibodies on the agonistic activity of OX40-L, OX40 effector cells were incubated with serial dilutions of anti-OX40 antibodies (HFB301001, Benchmark 1 or Benchmark 2) in the presence of 10 nM OX40L. Benchmark 1 and Benchmark 2 are previously published anti-OX40 antibodies selected because the antibody epitopes on OX40 are the most distal from the cell membrane. Other benchmarks have been described as binding near the ligand-binding pocket of OX40. After 5 hours of induction, the bioluminescence signal was quantified using the Bio-Glo Luciferase Assay System. Both Benchmark 1 and Benchmark 2 blocked OX40L agonism in a dose-dependent manner, while HFB301001 did not (Figure 14B). The results indicated that HFB301001 could recognize a binding site on OX40 different from Benchmark 1 and Benchmark 2. The binding site does not interfere with the agonist function of OX40L.

[0299] Example 12: OX40 Receptor Regulation To examine the receptor regulatory effect of anti-OX40 antibodies that bind to the OX40 receptor, human CD4 + T cells were isolated from human peripheral blood mononuclear cells (PBMC) using the Miltenyi Human CD4 + T Isolation Kit (Catalog No. 130-096-533, Miltenyi Biotec, Bergisch Gladbach, Germany). Primary CD4 + T cells were incubated with serial dilutions of a reference antibody or HFB301001, and 0.5 μg / mL of plate-bound anti-CD3 antibody (Clone OKT3, eBioscience, catalog number 16-0037-81) and 2 μg / mL of soluble anti-CD28 antibody (Clone CD28.2, eBioscience, catalog number 16-0289-81, eBioscience, Inc., San Diego, CA) were incubated together to activate them simultaneously. After 3 days of incubation, total OX40 surface expression was detected by staining with anti-OX40 antibody (PerCP-Cy5.5, Clone ACT35, Thermo Fisher, catalog number 17-1347-42). Compared to the isotype control, HFB301001 enhanced OX40 expression, which was stably retained on the cell surface (Figure 15A). In all treatments using the benchmark, OX40 surface expression was U-shaped with increasing antibody concentration. The OX40 level decreased, reached a nadir of approximately 1 nM, and then increased again.

[0300] Example 13: Determination of Binding Affinity To determine the binding affinity of HFB301001 for the recombinant dimeric (mFc-tagged) form of human OX40 protein, biolayer interferometry (BLI) experiments were performed on an Octet QKe instrument. Recombinant human OX40 protein at various concentrations was mixed with the test antibody or isotype control captured by the sensor at pH 7.4 and 25 °C, followed by a dissociation phase. Changes in the binding signal were recorded, and kinetic binding parameters were determined using a 1:1 binding model with mass transfer limitations. The running buffer was PBS, pH 7.4, containing 0.1% BSA and 0.1% Tween 20. Before the kinetic binding experiments, the anti-human Fc capture sensor was rinsed with the running buffer. The instrument was adjusted to a temperature of 25 °C. The test antibody and isotype control antibody were diluted to 200 nM in the running buffer. The OX40-mFc protein was diluted to 250, 125, 62.5, 31.25, 15.125 nM in the running buffer. The binding of the OX40 protein to the antibody was measured in the running buffer at pH 7.4 and 25 °C. The experiment was performed in duplicate. Briefly, the anti-human Fc capture sensor was first baseline in the running buffer for 300 seconds. The sensor was then mixed with the 200 nM test antibody solution for 600 seconds to load the antibody. The sensor was then baseline again in the running buffer for 300 seconds, followed by association with various concentrations of the OX40 antigen for 900 seconds. Dissociation was then performed by immersing the sensor in the running buffer for an additional 900 seconds. In parallel, one sensor was selected for a reference experiment and all loading, association, and dissociation steps were performed in the running buffer. Another sensor was selected for a negative control experiment and the HFB-TT-hG1 isotype control antibody was used in the loading step and 500 nM OX40-mFc protein was used in the association step. Sensorgrams were analyzed by Fortebio data analysis software version 8.2 (ForteBio, Fremont, CA). Signals were first subtracted from the negative control experiment and aligned through the baseline step. Kinetic parameters were obtained by globally fitting these specific sensorgrams to a 1:1 binding model at different antigen concentrations.The equilibrium dissociation constant (KD) was calculated based on the ratio of the dissociation rate constant to the association rate constant (KD = kd / ka). The kinetic binding parameters of the interaction between HFB301001 and human-derived OX40 protein were determined using BLI technology. In this assay, various concentrations of OX40 protein were used to interact with HFB10-1E1 and other antibodies captured at 25 °C and pH 7.4. The following binding affinity table summarizes the calculated kinetic binding parameters. In experiments conducted at 25 °C and pH 7.4, HFB301001 bound to the recombinant human OX40 (hOX40.mFc) protein with high affinity, having a KD value of 4.5 nM. The other two comparative antibodies showed slower dissociation rates, resulting in lower KD values of 0.49 nM for Benchmark 1 and 0.58 nM for Benchmark 2. The kinetic binding parameters of the interaction between HFB301001 and human OX40-mFc were determined by BLI technology at 25 °C and pH 7.4. HFB301001 can specifically bind to the recombinant human OX40-mFc protein with single-digit nM affinity. HFB301001 showed a faster dissociation rate compared to the other comparative antibodies tested. [Table 7]

[0301] Example 14. In Vivo Tumor Model The MC-38 mouse colon cancer model was used in human OX40 (hOX40) knock-in (KI) mice (catalog number NM-HU-00041, Shanghai Model Organisms Center, Inc.). To evaluate the pharmacodynamic (PD) effect of HFB301001 in the MC-38 mouse colorectal cancer model, MC-38 tumor cells (8 × 10 5 cells per mouse) were subcutaneously inoculated on the right side of 45 female hOX40 KI mice to generate tumors. Eight days after tumor inoculation, 104 - 243 mm 3 (average tumor size 181 mm 3Twelve mice with tumors in the range of ) were selected and divided into three groups with four mice in each group based on stratified randomization according to tumor volume. The mice were injected with anti-OX40 antibody HFB301001 at a dose of 10 mg / kg, anti-OX40 antibody benchmark 1 at a dose of 10 mg / kg, and IgG1 isotype control at a dose of 10 mg / kg. All therapeutic agents were administered by intraperitoneal (ip) injection on day D0 (D).

[0302] OX40 expression in CD4+ T cells was measured by flow cytometry 24 hours after the third treatment with 10 mg / kg anti-OX40 antibody. Benchmark 1 induced significant downregulation of OX40 expression in blood CD4 + T cells, while HFB301001 did not (Figure 15B).

[0303] To evaluate the in vivo antitumor activity of HFB301001 in a subcutaneous MC-38 mouse colorectal cancer model, MC-38 tumor cells (8×10 5 cells per mouse) were subcutaneously inoculated on the right side of 26 female hOX40 KI mice to generate tumors. Seven days after tumor inoculation, 101 - 175 mm 3 (mean tumor size 133 mm 3Twenty mice with a tumor size of ) were selected and divided into four treatment groups with five mice in each group based on stratified randomization according to tumor volume. The mice were injected with anti-OX40 antibody HFB301001 at doses of 1 mg / kg and 0.1 mg / kg, anti-OX40 antibody benchmark 1 at 1 mg / kg, and IgG1 isotype control at 10 mg / kg. All therapeutic agents were administered by intraperitoneal injection at D0, D3, D6, D10, and D13. The treatment time points are indicated by arrows, the error bars indicate the standard error of the mean, and significance was calculated by one-way analysis of variance at the last time point (*: p-value < 0.05, **: p-value < 0.01). The tumor size of each treatment group was measured at D0, D3, D6, D10, D12, and D15 after randomization by the diameter (width, length) of the tumor measured using a digital caliper. As shown in Figure 16, HFB301001 resulted in significant tumor growth inhibition compared to the control and was superior to benchmark 1.

[0304] To evaluate the in vivo antitumor activity of HFB301001 in a subcutaneous MC-38 mouse colorectal cancer model, MC-38 tumor cells (8 × 10 5 cells per mouse) were subcutaneously inoculated on the right side of 80 female hOX40 KI mice to generate tumors. Seven days after tumor inoculation, 42 - 147 mm 3 (mean tumor size 82 mm 3Sixty-five mice with tumors in the range of were selected and divided into seven treatment groups, each containing ten mice (excluding the PBS group with only five mice), based on stratified randomization according to tumor volume. The treatment started on the day of randomization (defined as day 0 (D0)). The mice were injected with anti-OX40 antibody HFB301001 at doses of 10 mg / kg, 1 mg / kg, and 0.1 mg / kg, anti-OX40 antibody benchmark 1 at doses of 10 mg / kg and 1 mg / kg, and an IgG1 isotype control at a dose of 10 mg / kg. All therapeutic agents were administered by intraperitoneal injection on D0, D3, D6, D10, and D13. Tumor size and animal body weight were measured at least three times a week from the start of treatment until day 61. Significance was calculated by the log-rank test (*: p-value < 0.05, **: p-value < 0.01). Survival was tracked for 60 days after randomization. The survival rate of mice treated with 10 mg / kg of HFB301001 was significantly higher than that of mice treated with 10 mg / kg of benchmark 1 (Figure 17).

[0305] To further investigate the effect of treatment with anti-OX40 antibody HFB301001, the changes induced in tumor T cells after treatment with anti-OX40 antibody were measured by flow cytometry 24 hours after the third treatment. Eight days after tumor inoculation, 104 - 243 mm 3 (average tumor size 181 mm 3 ) Twelve mice with tumors in the range were selected and divided into three groups, each containing four mice, based on stratified randomization according to tumor volume. A significant increase in KI67 + cells was observed in both CD4 + and CD8 + T cells after treatment with HFB301001 (Figures 18A and 18B). In addition, a significant decrease in PD-1 + cells was observed in both CD4 + and CD8 + T cells after treatment with HFB301001 (Figures 18C and 18D). Furthermore, both benchmark and HFB301001 resulted in a significant decrease in tumor Tregs, and the HFB301001 group showed a more significant decrease (Figure 18E).

[0306] Reference 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., Ong, 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, Clinical Cancer Research, https: / / dx.doi.org / 10.1158 / 1078 - 0432.CCR - 19 - 0526.

[0307] The principles of the present invention have been described above in connection with the preferred embodiments, but it should be understood that this description is not intended to limit the scope of the present invention and is given by way of example only. The present invention includes, without limitation, the following aspects. [Aspect 1] An isolated anti - OX40 antibody or antigen - binding fragment thereof, having a heavy - chain variable region having a heavy - chain CDR1 structural domain shown in SEQ ID NO: 1, a heavy - chain CDR2 structural domain shown in SEQ ID NO: 2, and a heavy - chain CDR3 structural 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, the anti - OX40 antibody or antigen - binding fragment thereof. [Aspect 2] 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% homology with 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% homology with SEQ ID NO: 12, and the anti-OX40 antibody or antigen-binding fragment thereof according to Aspect 1. [Aspect 3] Further comprising 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% homology with 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with SEQ ID NO: 13, the anti-OX40 antibody or antigen-binding fragment thereof according to Aspect 1 or 2. [Aspect 4] 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, 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% homology with 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 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% homology with SEQ ID NO: 14. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 3. [Aspect 5] An IgG antibody or antigen-binding fragment thereof, preferably an IgG1 antibody or antigen-binding fragment thereof. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 4. [Aspect 6] A monoclonal antibody or antigen-binding fragment thereof. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 5 An item. [Aspect 7] The antigen-binding fragment is Fab, Fab’, F(ab’)2, Fv, scFv or sdAb. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 6. [Aspect 8] An antibody-drug conjugate comprising the anti-OX40 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 7 and an additional therapeutic agent, preferably, the anti-OX40 antibody or antigen-binding fragment thereof is linked to the additional therapeutic agent via a linker. Antibody-drug conjugate. [Aspect 9] A nucleic acid encoding the anti-OX40 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 7. [Aspect 10] 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, 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, the nucleic acid according to embodiment 9. [Embodiment 11] An expression vector comprising the nucleic acid according to embodiment 9 or 10. [Embodiment 12] A host cell comprising the nucleic acid according to embodiment 9 or 10 or the expression vector according to embodiment 11. [Embodiment 13] A method for preparing the anti-OX40 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, comprising culturing the host cell according to embodiment 12 under conditions suitable for expressing the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the culture medium. [Embodiment 14] A pharmaceutical composition comprising the anti-OX40 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, or the antibody-drug conjugate according to embodiment 8, or the nucleic acid according to embodiment 9 or 10, or the expression vector according to embodiment 11, and a pharmaceutically acceptable carrier. [Embodiment 15] The anti-OX40 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, or the antibody-drug conjugate according to embodiment 8, or the pharmaceutical composition according to embodiment 14, for use in the treatment of cancer. [Embodiment 16] wherein 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, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal tumor), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, 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, melanoma, disseminated superficial melanoma, malignant melanoma of the lentigo maligna type, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal angiogenesis associated with keloid, edema (e.g., associated with brain tumor), and Meigs syndrome, brain tumor and brain cancer, head and neck cancer, and the anti-OX40 antibody or antigen-binding fragment thereof, or antibody-drug conjugate, or pharmaceutical composition according to any one of aspects 1 to 7 for treating the cancer as described in aspect 15, which is selected from the accompanying metastases X40 antibody or antigen-binding fragment thereof, or antibody-drug conjugate, or pharmaceutical composition. [Aspect 17] A method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-OX40 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 7, or the antibody-drug conjugate according to aspect 8, or the pharmaceutical composition according to aspect 14 for treating the cancer. [Aspect 18] The method according to aspect 17, wherein 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, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, 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, melanoma, disseminated superficial melanoma, malignant melanoma of the skin, acral lentiginous melanoma, nodular melanoma, multiple myeloma and B cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal angiogenesis associated with keloid, edema (e.g., associated with brain tumor), and Meigs syndrome, brain tumor and brain cancer, head and neck cancer, and associated metastases. [Aspect 19] Use of an anti-OX40 antibody or an antigen-binding fragment thereof according to any one of aspects 1 to 7, or an antibody-drug conjugate according to aspect 8, or a pharmaceutical composition according to aspect 14, in the preparation of a drug for treating cancer. [Aspect 20] wherein 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, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, 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, melanoma, disseminated superficial melanoma, malignant lentigo melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal angiogenesis associated with keloid, edema (e.g., associated with brain tumor), and Meigs syndrome, brain tumor and brain cancer, head and neck cancer, and the use according to embodiment 19 selected from the accompanying metastases. [Embodiment 21] Use in one or more of inhibiting Treg function (e.g., inhibiting the inhibitory function of Treg), 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 depleting OX40-expressing cells, of the anti-OX40 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, or the antibody-drug conjugate according to embodiment 8, or the pharmaceutical composition according to embodiment 14. [Embodiment 22] Inhibiting Treg function (e.g., inhibiting the inhibitory function of Treg), 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 depleting OX40-expressing cells Use of an anti - OX40 antibody or an antigen - binding fragment thereof according to any one of aspects 1 - 7, or an antibody - drug conjugate according to aspect 8, or a pharmaceutical composition according to aspect 14, in the preparation of a drug for one or more of thirst - inducing. [Aspect 23] A pharmaceutical combination comprising an anti - OX40 antibody or an antigen - binding fragment thereof according to any one of aspects 1 - 7, or an antibody - drug conjugate according to aspect 8, or a pharmaceutical composition according to aspect 14, and one or more additional therapeutic agents. [Aspect 24] A kit comprising an anti - OX40 antibody or an antigen - binding fragment thereof according to any one of aspects 1 - 7, or an antibody - drug conjugate according to aspect 8, or a pharmaceutical composition according to aspect 14, and preferably further comprising a drug delivery device.

[0308]

Table 8 - 1

Table 8 - 2

Table 8 - 3

Table 8 - 4

Table 8 - 5

Table 8 - 6

Table 8 - 7

Claims

**Claim 1** An isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy-chain variable region having a heavy-chain CDR1 structural domain shown in SEQ ID NO: 1, a heavy-chain CDR2 structural domain shown in SEQ ID NO: 2, and a heavy-chain CDR3 structural 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. **Claim 2** The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy-chain variable region shown in SEQ ID NO: 4, or a heavy-chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 4; and a light-chain variable region shown in SEQ ID NO: 12, or a light-chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:

12. **Claim 3** The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a heavy-chain constant region and a light-chain constant region, wherein the heavy-chain constant region is a heavy-chain constant region shown in SEQ ID NO: 5, or a heavy-chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 5; and / or the light-chain constant region is a light-chain constant region shown in SEQ ID NO: 13, or a light-chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:

13. **Claim 4** The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 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, wherein the heavy-chain signal peptide is a heavy-chain signal peptide shown in SEQ ID NO: 6, or a heavy-chain signal peptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 6; and / or The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the light chain signal peptide is the light chain signal peptide shown in SEQ ID NO: 14, or a light chain signal peptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

14.

5. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is an IgG antibody or antigen-binding fragment thereof.

6. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 5, which is an IgG1 antibody or antigen-binding fragment thereof.

7. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is a monoclonal antibody or antigen-binding fragment thereof .

8. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antigen-binding fragment is Fab, Fab’, F(ab’)2, Fv, scFv or sdAb.

9. An antibody-drug conjugate comprising the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and an additional therapeutic agent.

10. The antibody-drug conjugate according to claim 9, wherein the anti-OX40 antibody or antigen-binding fragment thereof is linked to the additional therapeutic agent via a linker.

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

12. The nucleic acid according to claim 11, 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.

13. The nucleic acid according to claim 12, 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.

14. An expression vector comprising the nucleic acid according to any one of claims 11 to 13.

15. A host cell comprising the nucleic acid according to any one of claims 11 to 13 or the expression vector according to claim 14.

16. A method for preparing an anti-OX40 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, comprising culturing the host cell according to claim 15 under conditions suitable for expressing the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the culture medium.

17. A pharmaceutical composition comprising an anti-OX40 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, or an antibody-drug conjugate according to claim 9 or 10, or a nucleic acid according to any one of claims 11 to 13, or an expression vector according to claim 14, and a pharmaceutically acceptable carrier.

18. An anti-OX40 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, or an antibody-drug conjugate according to claim 9 or 10, or a pharmaceutical composition according to claim 17, for use in the treatment of cancer.

19. The anti-OX40 antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate, or a pharmaceutical composition according to claim 18, wherein the cancer is selected from squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, 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, melanoma, diffuse superficial melanoma, malignant melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal angiogenesis associated with keloid, edema, and Meigs syndrome, brain tumor and brain cancer, head and neck cancer, and associated metastases.

20. Use of an anti-OX40 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, or an antibody-drug conjugate according to claim 9 or 10, or a pharmaceutical composition according to claim 17, in the preparation of a drug for treating cancer.

21. The use according to claim 20, wherein the cancer is selected from squamous cell carcinoma, squamous epithelial cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, 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, melanoma, disseminated superficial melanoma, malignant melanoma of the lentigo maligna type, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal angiogenesis associated with keloid, edema, and Meigs syndrome, brain tumor and brain cancer, head and neck cancer, and associated metastases.

22. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the antibody-drug conjugate according to claim 9 or 10, or the pharmaceutical composition according to claim 17, which is used in one or more of 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 depleting OX40-expressing cells.

23. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the antibody-drug conjugate according to claim 9 or 10, or the pharmaceutical composition according to claim 17, which is used in the preparation of a drug for one or more of 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 depleting OX40-expressing cells.

24. A pharmaceutical combination comprising the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the antibody-drug conjugate according to claim 9 or 10, or the pharmaceutical composition according to claim 17, and one or more additional therapeutic agents.

25. A kit comprising the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the antibody-drug conjugate according to claim 9 or 10, or the pharmaceutical composition according to claim 17.

26. The kit according to claim 25, further comprising a drug delivery device.

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