Affinity matured anti-OX40 antibodies and uses thereof

Abstract

Provided herein are high affinity antagonistic anti-OX40 antibodies, methods of making such antibodies, and methods of using such antibodies to treat OX40 mediated disorders.

Description

Cross-references to related applications

[0001] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 588,662, filed October 6, 2023, and U.S. Provisional Patent Application No. 63 / 645,656, filed May 10, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure generally relates to affinity-matured anti-OX40 antibodies, their preparation, and their use in treating various OX40-mediated disorders, including inflammatory and autoimmune diseases. Background Technology

[0003] OX40 (TNFRSF4, CD134) is a costimulatory receptor member of the NGFR / TNFR superfamily, primarily expressed on activated T lymphocytes, including CD4 and CD8 T cells, T helper cells (types 1, 2, and 3: Th1, Th2, and Th17), and Forkhead box P3-positive (Foxp3+) CD4+ regulatory T cells (Tregs). Unlike CD28, which is a classic constitutively expressed T cell costimulatory receptor, OX40 is not expressed on naive T lymphocytes. In contrast, transient OX40 expression was transiently induced on CD4 and CD8 T cells 24 hours to 5 days after initial TCR stimulation (Calderhead et al. (1993), J. Immunol. 151(1):5261-71; Gramaglia et al. (1998), J. Immunol., 161(12):6510-6517; Akiba et al. (1999), J. Immunol., 162(12):7058-7066). Furthermore, OX40 appeared to be upregulated on Treg cells, a T cell population crucial for maintaining immune tolerance and fine-tuning T cell activity (Kondelkova et al. (2010), ActaMedica, 53(2):73-77). Typically, the binding of OX40L expressed on antigen-presenting cells (APCs) to OX40 on T cells promotes effector function of T cells.

[0004] OX40 and CD30 are considered crucial for the late or sustained stages of T cell responses. OX40 binding via its ligand OX40L (TNFSF4-CD252) leads to enhanced T cell survival and proliferation, which can contribute to autoimmune diseases. Certain OX40 therapeutic antibodies used for autoimmune diseases often exhibit some level of residual agonistic activity leading to T cell activation and proliferation. Therefore, despite progress in treating OX40-mediated disorders, additional anti-OX40 therapeutics remain needed. Summary of the Invention

[0005] This disclosure is based in part on the discovery of high-affinity, antagonistic anti-OX40 antibodies, their manufacture, and their use in the treatment of OX40-mediated disorders, including inflammatory and autoimmune disorders. Certain high-affinity antibodies disclosed herein do not exhibit detectable agonistic activity (e.g., T-cell activation and proliferation), making them particularly suitable for the treatment of inflammatory and autoimmune disorders.

[0006] Some of the antibodies disclosed herein, including MAB1 and MAB10, exhibit increased affinity for OX40 compared to their parental antibody, telazormab (GBR 830, also known as ISB830). MAB1 and MAB10 also lack agonistic activity compared to baseline antibodies that retain residual agonistic activity, such as nocartelimumab (also known as KHK4083 or AMG451). In vitro, MAB1 and MAB10 demonstrate stronger inhibitory potency against T cell proliferation than telazormab (GBR 830). Furthermore, MAB1 and MAB10 show unique potential for ADCC-mediated reduction of T cell exhaustion and for limiting Treg exhaustion via ADCC compared to nocartelimumab. These and other pharmacological properties support the potential efficacy of MAB1 and MAB10 in the field of OX40 therapy for autoimmune diseases.

[0007] In one aspect, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40). In one example, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) comprising a heavy chain variable domain (VH) containing the amino acid sequence shown in SEQ ID NO: 7, and three light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) comprising a light chain variable domain (VL) containing the amino acid sequence shown in SEQ ID NO: 8. In other examples, the antibody or its antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) of a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7 and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2, and CDR-L3) of a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, or SEQ ID NO: 33. CDR-H1, CDR-H2, and CDR-H3, as well as CDR-L1, CDR-L2, and CDR-L3, can be defined according to the Kabat, Chothia, IMGT, AbM, Contact, or Honnecger (AHo) CDR numbering systems discussed herein, or other numbering systems known in the art. Antibodies or their antigen-binding fragments can be isolated antibodies or antigen-binding fragments.

[0008] In another aspect, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40). The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region comprising CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 38 or 71, wherein X1 is F, T, W, or M; wherein X2 is G, I, V, L, or E; wherein X3 is A, D, E, L, H, T, or F; and wherein X4 is W, P, F, Y, or T. For example, CDR-L3 contains the amino acid sequence shown in SEQ ID NO: 38. For example, CDR-L3 contains the amino acid sequence shown in SEQ ID NO: 71. In some instances, X1 is F or T, X2 is L, G, or E, X3 is A, and X4 is W. The antibody or its antigen-binding fragment can be a separate antibody or antigen-binding fragment.

[0009] In another aspect, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region including CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, or SEQ ID NO: 32. The antibody or antigen-binding fragment thereof may be a separate antibody or antigen-binding fragment.

[0010] In another aspect, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40). The antibody or its antigen-binding fragment includes a heavy chain variable region comprising (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 59, or SEQ ID NO: 65; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, or SEQ ID NO: 66; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 55, SEQ ID NO: 61, or SEQ ID NO: 67; and (b) a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 56, SEQ ID NO: 62, or SEQ ID NO: 68; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 57, amino acid sequence AT, or SEQ ID NO: 69; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 56, SEQ ID NO: 62, or SEQ ID NO: 68; and a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 57, amino acid sequence AT, or SEQ ID NO: 69; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 56, SEQ ID NO: 67, amino acid sequence AT, or SEQ ID NO: 69; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 57, amino acid sequence AT, or SEQ ID NO: 69; and a heavy chain CDR3 comprising the amino acid sequence The light chain CDR3 of the amino acid sequence of NO: 6, SEQ ID NO: 58, or SEQ ID NO: 70. The antibody or its antigen-binding fragment can be an isolated antibody or antigen-binding fragment.

[0011] For example, an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) may include a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0012] For example, an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) may include a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 41, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 42, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0013] For example, an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) may include a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 47, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 48, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0014] For example, an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) may include a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 53, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 54, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 56, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 57, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 58.

[0015] For example, an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) may include a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 59, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 60, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 61, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 62, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0016] For example, an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) may include a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 66, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 67, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 70.

[0017] In some instances, OX40 is human OX40. Therefore, in some instances, the antibody binding OX40 or its antigen-binding fragment is an antibody binding human OX40 or its antigen fragment.

[0018] In some instances, antibodies or antigen-binding fragments thereof that bind to OX40 (e.g., human OX40) are OX40 antagonists. Furthermore, antibodies or antigen-binding fragments thereof that bind to OX40 may be humanized antibodies. CDRs of the antibody or antigen-binding fragment may be located between human or humanized frame sequences. The antibody or antigen-binding fragment may include a heavy chain having an Fc region with amino acid modifications that increase the serum half-life of the antibody or antigen-binding fragment. For example, the antibody or antigen-binding fragment may include an IgG1 heavy chain, for example, having an Fc region with mutations M252Y, S254T, and T256E, numbered according to the EU numbering system.

[0019] In some instances, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7. In other instances, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a light chain variable region having the amino acid sequence of SEQ ID NO: 8. In a further instance, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8. In yet another further instance, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 12, 15, 18, 21, 24, 27, 30, or 33.

[0020] In some other instances, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) comprises a heavy chain having the amino acid sequence of SEQ ID NO: 9, while in other instances, the antibody or antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73. In some instances, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) comprises a light chain having the amino acid sequence of SEQ ID NO: 10. In one particular case, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain having the amino acid sequence of SEQ ID NO: 10. In another particular case, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) comprises a heavy chain having the amino acid sequence of SEQ ID NO: 9 and a light chain having the amino acid sequence of SEQ ID NO: 10.

[0021] In a further example, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) comprises a heavy chain having the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 13, 16, 19, 22, 25, 28, 31, or 34. In a still further example, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) comprises a heavy chain having the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 13, 16, 19, 22, 25, 28, 31, or 34.

[0022] In some instances, antibodies or antigen-binding fragments of OX40 (e.g., human OX40), as disclosed herein, have increased inhibition of T cell proliferation compared to trazolimumab.

[0023] In other instances, antibodies or antigen-binding fragments thereof that bind to OX40 (e.g., human OX40), as disclosed herein, are formulated in pharmaceutical compositions having pharmaceutically acceptable carriers.

[0024] In another aspect, this disclosure provides a nucleic acid encoding a light chain CDR, light chain variable region, or light chain of an antibody or antigen-binding fragment thereof that binds OX40 (e.g., human OX40) as disclosed herein. The nucleic acid may be contained in an expression vector. The expression vector may also include a nucleotide sequence encoding a heavy chain CDR, heavy chain variable region, or heavy chain of an antibody or antigen-binding fragment thereof that binds OX40 (e.g., human OX40) as disclosed herein. The expression vector may further be contained in a host cell. In one example, a nucleic acid encoding the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8 is provided. In another example, a nucleic acid encoding the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10 is provided. In yet another example, a nucleic acid encoding the amino acid sequences of SEQ ID NO: 73 and SEQ ID NO: 10 is provided.

[0025] Furthermore, this disclosure provides a host cell comprising an expression vector having an expression vector having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7 and an expression vector having an expression vector having an amino acid sequence encoding SEQ ID NO: 8. In another example, the host cell comprises an expression vector having an expression vector having an amino acid sequence encoding SEQ ID NO: 9 and an expression vector having an amino acid sequence encoding SEQ ID NO: 10. In yet another example, the host cell comprises an expression vector having an expression vector having an amino acid sequence encoding SEQ ID NO: 73 and an expression vector having an amino acid sequence encoding SEQ ID NO: 10. In yet another example, the host cell comprises an expression vector having an expression vector having an amino acid sequence encoding both SEQ ID NO: 73 and SEQ ID NO: 10.

[0026] In another aspect, this disclosure provides a method for producing an antibody or antigen-binding fragment thereof that binds to human OX40 (e.g., human OX40). In this method, host cells are grown under conditions that cause the host cells to express one or more polypeptides comprising a heavy chain or heavy chain variable region or heavy chain CDR of an antibody or antigen fragment thereof that binds to human OX40 (e.g., human OX40) as described herein, and a light chain or light chain variable region or light chain CDR, and then the antibody or antigen-binding fragment thereof is purified therefrom.

[0027] In another aspect, this disclosure provides a method for treating OX40-mediated disorders in a subject. The method comprises administering to the subject (e.g., a human) a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40).

[0028] In another aspect, this disclosure provides a method for reducing or inhibiting T cell proliferation in a subject (e.g., a human) in need. The method includes administering to the subject (e.g., a human) a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40). The subject may, for example, have an OX40-mediated barrier.

[0029] In this regard, OX40-mediated disorders can include arthritis, rheumatoid arthritis, psoriatic arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peroni disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, nodular prurigo, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, and immune-mediated inflammatory diseases of the central and peripheral nervous systems, such as multiple sclerosis, lupus (such as systemic lupus erythematosus or lupus nephritis), and Guillain-Barré syndrome. Raynaud's syndrome, atopic dermatitis, autoimmune hepatitis, fibrotic alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, chronic spontaneous urticaria (CSU), chronic inducible urticaria (CIU), Wegener's granulomatosis, pancreatitis, trauma (surgery), graft-versus-host disease (GVHD), transplant rejection, cardiovascular diseases (including ischemic diseases such as myocardial infarction and atherosclerosis), intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypoacidity, and neuromyelitis optica. In some cases, OX40-mediated disorders include atopic dermatitis, nodular prurigo, alopecia areata, chronic spontaneous urticaria (CSU) and chronic induced urticaria (CIU), asthma, hidradenitis suppurativa, lupus nephritis, systemic lupus erythematosus, pemphigus vulgaris, psoriatic arthritis, vasculitis, Hashimoto's thyroiditis, systemic sclerosis, scleroderma, scleroderma, chronic pruritus of unknown cause, ankylosing spondylitis, Sjögren's syndrome, psoriasis, or vitiligo.

[0030] In some instances, OX40-mediated disorders are atopic dermatitis. In other instances, OX40-mediated disorders are urticaria, such as chronic spontaneous urticaria or chronic induced urticaria. In other instances, OX40-mediated disorders are autoimmune disorders, such as lupus erythematosus or rheumatoid arthritis. In other instances, OX40-mediated disorders are asthma. In other instances, OX40-mediated disorders are alopecia areata, scleroderma, or hidradenitis suppurativa. Attached Figure Description

[0031] These and other features, aspects, and advantages of this disclosure will be better understood with reference to the following description and accompanying drawings, wherein: Figure 1It is a graph showing the affinity measurements (binding over time) for GBR 830 and nine GBR 830 variants (denoted as MAB1 to MAB9) for human OX40-CRD-avi-his.

[0032] Figure 2A-2D This is a bar chart showing direct binding ELISA data for epitope localization using the indicated human / rat chimeric OX40 construct. Figure 2A The combination with OX40-HRRR-Fc is shown. Figure 2B The combination with OX40-RHRR-Fc is shown. Figure 2C The combination with OX40-HHHR-Fc is shown. Figure 2D Binding to OX40-HHRH-Fc is shown. Low-concentration binding on the OX40 chimeric construct was confirmed using three concentrations of the indicated anti-OX40 antibody (1, 3, and 10 μg / mL). (In the OX40-Fc construct, R = rat module, H = human module). These figures show the OD at 450 nm measured for each condition.

[0033] Figure 3 This is a graph showing the cross-blocking activity of GBR830 and MAB1 on HPB-ALL cells expressing OX40 using geometric mean fluorescence intensity (GeoMFI) of AF647 fluorescence (APC channel). The top curve shows the blocking effect of cold GBR 830 pre-incubation on the binding of labeled MAB1. The bottom curve shows the blocking effect of MAB1 pre-incubation on the binding of GBR 830-AF647. IgG4-AF647 shows no background staining (not shown). Single dots indicate the binding of MAB1-AF647 and GBR 830-AF647 at the highest antibody concentration used.

[0034] Figure 4 This figure shows the FACS binding of anti-OX40 antibody when Jurkat-NFκB cells are incubated with dose-responsive OX40L-Fc or the indicated antibody. Staining was detected using anti-human IgG Fc-AF647 antibody. The figure shows the geometric mean fluorescence intensity (GeoMFI) of AF647 fluorescence (APC channel).

[0035] Figure 5A This is a graph showing the dose-response curves when Jurkat-NFκB cells are incubated with the indicated antibody or OX40L on a CD3 pre-coated plate. The graph shows the luminescent dose response of the indicated antibody. Each point shows the mean of replicates. The curves show a nonlinear regression fit. Figure 5B This is a bar graph showing the luminescence response at the highest tested concentration. The dashed line indicates twice the threshold for the IgG1 control.

[0036] Figure 6A This is a graph showing the dose-response curve of agonistic activity when Jurkat-NFκB cells are incubated with OKT3 and the indicated antibody on a plate. The graph shows the luminescent dose-response of the indicated antibody. Each point shows the mean of replicates. The curves show a nonlinear regression fit. Figure 6B This is a bar graph showing the luminescence response at the highest tested concentration. The dashed line indicates twice the threshold for the IgG1 control.

[0037] Figure 7 This graph shows T-cell agonism as measured by the proliferation index of the indicated antibody at the highest concentration (5 or 10 μg / mL) of soluble antibody tested in all T-cell donors without crosslinking assays. The horizontal line indicated by the arrow shows the threshold (PI>2) considered for agonism. Each point represents the mean PI of triplicate measurements. The green diamond box shows the mean and the 95% confidence interval of the mean. CD28 indicates anti-CD28 used as the soluble reagent. The isotype is the IgG1 isotype control.

[0038] Figure 8 This is a graph illustrating the T-cell agonist effect on T-cell proliferation assessed by radioactivity count per minute (CPM) measurement under indicated conditions in a soluble antibody non-crosslinking assay. Each point represents the mean CPM from three repeated measurements. Boxes indicate the median, 25th, and 75th quartiles, and whisker lines indicate the lowest and highest measures. “OKT3 alone” is the anti-CD3-only (OKT3) condition, “GBR 200” is trastuzumab used as an IgG1 control, “CD28” is an anti-CD28 antibody, and “9B12” is an OX40 agonist antibody, each used at 10 μg / mL. Soluble antibodies GBR 830 and MAB1 were used at 10, 1, and 0.1 μg / mL, respectively. Dashed lines indicate only baseline levels of anti-CD3.

[0039] Figure 9 This is a graph showing T cell activating activity as measured by proliferation index under indicated conditions in a soluble antibody crosslinking assay. Each point represents the mean PI of triplicate measurements. Boxes indicate the median, 25th, and 75th quartiles, and whisker lines indicate the lowest and highest measures. “OKT3 alone” is the anti-CD3-only (OKT3) condition, “GBR 200” is trastuzumab used as an IgG1 control, “CD28” is an anti-CD28 antibody, and “9B12” is an OX40 activating antibody, each used at 10 μg / mL. Soluble antibodies GBR 830 and MAB1 were used at 10, 1, and 0.1 μg / mL, respectively. Dashed lines indicate what is considered the threshold for activating activity (PI>2).

[0040] Figure 10 This is a graph illustrating the inhibition of T cell proliferation. Isolated T cells were activated via TCR and CD28 triggering, exposed to OX40L, and treated with GBR 830, MAB1, KHK4083, and an allotype control antibody. This graph shows an example of dose-response inhibition of T cell proliferation from a single PBMC donor. Each data point is the mean of the percentage of T cell proliferation inhibition obtained from three replicate experiments from the same PBMC donor, and the standard deviation of the mean is shown. Five independent experiments were performed.

[0041] Figure 11 This figure illustrates ADCC-mediated killing of activated T cells induced by OX40 and an isotype control antibody. Isolated activated T cells were co-cultured with activated NK cells in the presence of GBR 830, MAB1, KHK4083, and an isotype control antibody. This figure shows an example of the dose-response relationship of T cell killing induced by ADCC. Each data point is the mean of the percentage of T cell killing induced by ADCC from three replicate experiments from the same PBMC donor, showing the standard deviation of the mean. Fifteen independent experiments were performed.

[0042] Figure 12 This figure illustrates ADCC-mediated in vitro killing of differentiated Tregs via OX40 and an isotype control antibody. Differentiated Tregs were co-cultured with activated NK cells in vitro in the presence of GBR 830, MAB1, KHK4083, and an isotype control antibody. This figure shows an example of dose-response for ADCC-induced Treg killing. Each data point is the mean of the percentage of T cell killing via ADCC from three replicate experiments using the same PBMC donor, showing the standard deviation of the mean. Five independent experiments were performed.

[0043] Figure 13A The surface plasmon resonance (SPR) sensing image of MAB10 combined with FcRn is shown. Figure 13B The image shows an SPR sensing plot of GBR830 combined with FcRn. Figure 13C The SPR sensing plot of MAB1 combined with FcRn is shown.

[0044] Figure 14A The dose-dependent curves of inhibition of IFNγ release (pg / mL) in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451 (KHK4083 or nocarotelimab) and IgG1 are shown. Figure 14B The dose-dependent curves of inhibition of TNFα release (pg / mL) in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 are shown. Figure 14C The dose-dependent curves of inhibition of IL-5 release (pg / mL) in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 are shown. Figure 14D The dose-dependent curves of inhibition of IL-13 release (pg / mL) in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 are shown. Figure 14E The dose-dependent curves of inhibition of IL-2 release (pg / mL) in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 are shown. Figure 14F The dose-dependent curves of inhibition of IL-4 release (pg / mL) in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 are shown. Figure 14G The dose-dependent curves of inhibition of IL-31 release (pg / mL) in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 are shown. Figure 14H The dose-dependent curves of inhibition of IL-17a release (pg / mL) in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 are shown. Figure 14I The dose-dependent curves of inhibition of IL-21 release (pg / mL) in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 are shown. Figure 14J A dose-dependent curve showing the inhibition of IL-22 release (pg / mL) in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is presented. Figure 14B , 14F The legends to the right of 14J also apply. Figure 14A , 14C 14D, 14E, 14G, 14H and 14I.

[0045] Figure 15A The EC50 (nM) of IFNγ release inhibition in T cell proliferation assays following incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is shown. Figure 15B The EC50 (nM) of TNFα release inhibition in T cell proliferation assays following incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is shown. Figure 15CThe EC50 (nM) of IL-5 release inhibition in T cell proliferation assays following incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is shown. Figure 15D The EC50 (nM) of IL-13 release inhibition in T cell proliferation assays following incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is shown. Figure 15E The EC50 (nM) of IL-2 release inhibition in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is shown. Figure 15F The EC50 (nM) of IL-4 release inhibition in T cell proliferation assays after incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is shown. Figure 15G The EC50 (nM) of IL-31 release inhibition in T cell proliferation assays following incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is shown. Figure 15H The EC50 (nM) of IL-17a release inhibition in T cell proliferation assays following incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is shown. Figure 15I The EC50 (nM) of IL-21 release inhibition in T cell proliferation assays following incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is shown. Figure 15J The EC50 (nM) of IL-22 release inhibition in T cell proliferation assays following incubation with GBR830, MAB1, MAB10, AMG451, and IgG1 is shown. Each symbol represents the EC50 value from one of eight individual T cell donors tested in two independent experiments. Based on goodness-of-fit (R²)... 2 Values ​​>0.7, EC50 values ​​outside the range, or values ​​where the curve has not reached a plateau are excluded. Paired one-way ANOVA is used, followed by Tukey's post-hoc comparisons to compare EC50 values ​​(ns p>0.05, *p<0.05, **0.01). <p<0.05,***p<0.01)。

[0046] Figure 16A The dose-dependent curves of T cell killing induced by anti-OX40 antibody and benchmark antibody AMG451, as assessed in an ADCC assay at a 5:1 effector-target ratio and indicated by LDH release measurement after 4.5 hours, are shown. Figure 16B The dose-dependent curves of regulatory T cell killing induced by anti-OX40 antibody and benchmark antibody AMG451, as assessed in an ADCC assay at a 5:1 effector-target ratio and indicated by LDH release measurement after 4.5 hours, are shown.

[0047] Figure 17A The maximum killing effect on activated T cells by ADCC is shown. Figure 17B The maximum killing effect on regulatory T cells by ADCC is shown. Individual symbols represent values ​​from one of 6 to 8 individual donors tested in 3 to 4 independent experiments. Maximum ADCC values ​​were compared using paired one-way ANOVA followed by Tukey's post-hoc comparisons (ns p>0.05, *p<0.05, **0.01). <p<0.05,***p<0.01)。

[0048] Figure 18A The EC50 (nM) of the indicated antibody against ADCC in activated T cells is shown. Figure 18B The EC50 (nM) of the indicated antibody against ADCC on regulatory T cells is shown. Each symbol represents a value for one of 8 to 6 individual donors tested in 3 to 4 independent experiments. EC50 (nM) of ADCC was compared using paired one-way ANOVA followed by Tukey's post-hoc comparisons (ns p>0.05, *p<0.05, **0.01). <p<0.05,***p<0.01)。

[0049] Figure 19 In vivo pharmacokinetic data of cynomolgus monkeys (n=3) administered 20 mg / kg MAB10 subcutaneously are presented.

[0050] Figure 20A A schematic diagram is shown showing the OX40-OX40L co-stimulatory signaling pathway and its effects on activated T cells and regulatory T cells. Figure 20B This provides a schematic diagram of how MAB10 can target the OX40 receptor, affecting the Th1, Th2, and Th17 / 22 pathways while preserving regulatory T cells.

[0051] Figure 21 The alignment of the VL domains of MAB1-9 is shown. CDR residues according to the Kabat numbering scheme are shown in bold.

[0052] Figure 22 The heavy chains of MAB1, MAB10, and MAB11 are shown in comparison. The YTE and LS variations in MAB10 and MAB11 are shown in bold and underlined, respectively.

[0053] Figure 23AThis is a graph showing the dose-response curves when Jurkat-NFκB cells are incubated with the indicated antibody or OX40L on a CD3 pre-coated plate. The graph shows the luminescent dose response of the indicated antibody. Each point shows the mean of a replicate. The curves show a non-linear regression fit. Figure 23B This is a bar graph showing the luminescent response of each indicated antibody at the highest tested concentration. The dashed line indicates twice the threshold for the IgG1 control.

[0054] Figure 24 Internalization of OX40 on activated T cells is shown. Activated T cells were incubated with 8 μg / mL of Fabfluor red-labeled antibody for 24 h. Internalization was measured using the Incucyte live cell analysis system. Points represent the mean ± SEM of three activated T cell donors from one experiment.

[0055] Figure 25A -C shows the signaling inhibition of anti-OX40 antibody on Jurkat-NF-κB-OX40 engineered cells in the presence of OX40L. The figure shows the mean ± SEM of three independent experiments for the percentage of OX40-OX40L signaling inhibition at different concentrations of the indicated antibody and different doses of OX40L. Figure 25A The concentration of 0.07 μg / mL OX40L was shown. Figure 25B The concentration of 0.3 μg / mL OX40L was shown. Figure 25C The concentration of 5 μg / mL OX40L was shown.

[0056] Figure 26A -D shows the receptor occupancy in Jurkat-NF-κB-OX40 engineered cells in the presence of OX40L and the indicated antibody. The figure shows the mean ± SEM of the percentage of receptor occupancy from three independent experiments (single) at different concentrations of OX40L and in the presence of the indicated antibody. Figure 26A The OX40L is shown without OX. Figure 26B The result is 0.07 μg / mL OX40L. Figure 26C It showed 0.3 μg / mL OX40L. Figure 26D The concentration of 5 μg / mL OX40L was shown. Detailed Implementation

[0057] This disclosure is based in part on the discovery of high-affinity, antagonistic anti-OX40 antibodies, their manufacture, and their use in the treatment of OX40-mediated disorders, including inflammatory and autoimmune disorders. Some high-affinity antibodies do not possess any detectable agonistic properties, making them particularly suitable for the treatment of inflammatory and autoimmune disorders.

[0058] I. Definition Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. For example, the nomenclature and techniques used in relation to topics such as peptide and polynucleotide chemistry and synthesis, molecular and cell biology, protein biology and biochemistry, and immunology described herein are those well-known and commonly used in the art.

[0059] The articles “a,” “one,” and “the,” as used in this disclosure, refer to one or more of the grammatical objects of the article (i.e., at least one), unless the context is inappropriate. For example, “one element” means one or more elements.

[0060] Unless otherwise stated, the term "and / or" is used in this disclosure to mean "and" or "or". Unless otherwise understood from the context, the expression "and / or" relating to three or more of the said objects shall be understood to have the same meaning.

[0061] When the term "about" is used before a quantitative value, the invention also includes the specific quantitative value itself, unless otherwise specifically stated. As used herein, unless otherwise indicated or inferred from the context, the term "about" means a variation of ±10% from the nominal value.

[0062] As used herein, the term “in combination with” means that the described reagents may be mixed, administered simultaneously as single agents, or administered sequentially as single agents to the subject in any order. The term “remission” refers to any therapeutically beneficial outcome of treating an OX40-mediated disease state, such as allergic, asthma, COPD, rheumatoid arthritis, dermatitis, or psoriasis, including prevention, reduction of symptom severity or disease progression, induction of remission, or cure of the disease.

[0063] For example, the term "isolated" in relation to antibody or antigen-binding fragments refers to antibody or antigen-binding fragments that have been removed, for example, from their natural environment, such as in living cells, body fluids, tissues, or inside animals, through purification.

[0064] As used herein, the term "OX40" includes naturally occurring variants, isotypes, and species homologs of OX40, and as used herein, the term "human OX40" includes naturally occurring variants, isotypes, and species homologs of human OX40. In some cases, antibodies of this disclosure may cross-react with OX40 from non-human species. In some embodiments, antibodies may be completely specific to one or more human OX40 proteins and may not exhibit species or other types of non-human cross-reactivity. An exemplary complete amino acid sequence of human OX40 has Swiss-Prot accession number P43489 (TNR4-HUMAN; SEQ ID NO: 72). OX40 is also known as CD134, TNFRSF4, ACT35, or TXGP1 L. Human OX40 is designated as GeneID: 7293 by Entrez Gene and as HGNC: 11918 by HGNC. OX40 is also designated as CD134 (differentiation cluster 134). OX40 can be encoded by a gene designated as TNFRSF4 / OX40. The term "human OX40" includes all known and undiscovered alleles and polymorphic forms of human OX40. The terms "human OX40," "OX40," or "OX40 receptor" are used interchangeably herein.

[0065] As used herein, the terms “OX40 ligand” or “OX40L” are used interchangeably and include OX40 ligand, particularly human OX40 ligand. OX40L is a member of the TNF superfamily, also known as gp34 or CD252. OX40L is also designated as CD252 (differentiation cluster 252) and has sequence database accession numbers P23510 (Swiss-Prot) or Q6FGS4 (Uniprot). OX40L is expressed on the surface of activated B cells, T cells, dendritic cells, and endothelial cells.

[0066] As used herein, the term "OX40-mediated disorder" includes conditions involving OX40 signaling pathways (e.g., activation of OX40 signaling pathways), such as allergies, asthma, COPD, rheumatoid arthritis, psoriasis, atopic dermatitis, urticaria (such as chronic spontaneous urticaria (CSU) or chronic inducible urticaria (CIU)), and diseases related to autoimmunity and inflammation. For OX40-mediated disorders, where activation of OX40 signaling pathways contributes to the pathology of the disorder, OX40 antagonism can be used to treat the OX40-mediated disorder. Other OX40-mediated disorders are disclosed herein.

[0067] As used herein, the phrase “pharmaceutically acceptable” means compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0068] As used herein, the phrase “pharmaceuticalally acceptable carrier” refers to a pharmaceutical agent (e.g., excipient, carrier, buffer, etc.) suitable for contact with tissues in humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption-delaying agents, etc., compatible with drug administration. Standard drug carriers may include, for example, phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. Compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and excipients, see, for example, Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rd ed. 2020).

[0069] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an organism treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., rats, apes, horses, cattle, pigs, dogs, cats, etc.), and more preferably include humans. The antibodies disclosed herein have human therapeutic and veterinary applications. In some instances, the subjects disclosed herein are humans, and the disclosed antibodies are used for human therapy.

[0070] As used herein, the terms “treat,” “treating,” and “treatment” mean treating a disease, disorder, or its symptoms or manifestations in a subject (e.g., a person). This includes: (a) preventing a disease or disorder; (b) suppressing a disease, disorder, etc., i.e., slowing or preventing its progression or development; and (c) alleviating a disease, disorder, etc., for example, causing the remission of a disease state or improving at least one symptom of a disorder. As used herein, “prevent,” “preventing,” and “prevention” mean causing a disease, disorder, or its symptoms or manifestations to not occur in at least some subjects for at least a period of time. Those “in need of treatment” include mammals, such as humans, that already have a disease or disorder, as well as those who are susceptible to a disease or disorder, including those who wish to prevent a disease or disorder.

[0071] As used herein, the phrase “therapeutic effective amount” refers to an amount of active agent (e.g., an anti-OX40 antibody or its antigen-binding fragment disclosed herein) sufficient to achieve a beneficial or desired outcome. An effective amount may be administered in one or more ways, applications, or doses, and is not intended to be limited to a particular formulation or route of administration. A therapeutic effective amount may also be a “prophylactic effective amount,” as prevention can be considered treatment.

[0072] In the context of two or more nucleic acid or polypeptide sequences, the term percentage “identity” refers to two or more sequences or subsequences having a specific percentage of nucleotide or amino acid residues that are identical when compared and aligned using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to the technician) or by means of visual inspection, or by measuring the maximum correspondence. Depending on the application, percentage “identity” may exist in a region of the sequences being compared, such as in a functional domain, or alternatively, across the entire length of the two sequences to be compared.

[0073] For sequence comparisons, a sequence typically serves as a reference sequence to be compared with the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and if necessary, the coordinates of the subsequences are specified, along with the sequence algorithm program parameters. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the specified program parameters.

[0074] The optimal alignment of sequences for comparison can be performed, for example, by searching for local homology algorithms in Smith & Waterman, Adv. Appl. Math. 2:482 (1981), homology alignment algorithms in Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), and similarity methods in Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (usually see Ausubel et al., hereinafter). An example of an algorithm suitable for determining the percentage of sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (https: / / ncbi.nlm.nih.gov / blast).

[0075] It should be understood that, unless otherwise understood from the context and use, the expression “at least one of…” individually includes each of the objects described thereafter, as well as various combinations of two or more of the objects described.

[0076] The use of the terms “including,” “having,” or “containing” (including their grammatical equivalents) should generally be understood as open-ended and non-restrictive, for example, not excluding additional unlisted elements or steps unless otherwise specifically stated or understood from the context.

[0077] Where a molecular weight, for example, of a polymer is provided and is not an absolute value, the molecular weight shall be understood as an average molecular weight unless the context otherwise indicates or implies.

[0078] The use of any and all illustrative or exemplary language (e.g., "such as" or "including") in this document is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0079] Throughout this specification, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is further contemplated that the compositions of the present invention are substantially composed of or comprised of said components, and that the processes and methods of the present invention are substantially composed of or comprised of said processing steps.

[0080] In this application, when an element or component is referred to as being included in and / or selected from the list of listed elements or components, it should be understood that the element or component can be any one of the listed elements or components, or the element or component can be selected from a group consisting of two or more of the listed elements or components.

[0081] It should be understood that the order of the steps or the order in which certain actions are performed is irrelevant as long as the invention remains operable. Furthermore, two or more steps or actions can be performed simultaneously.

[0082] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various ways, whether explicitly stated or implicit, without departing from the spirit and scope of the invention. For example, when a particular compound is mentioned, it can be used in various embodiments of the compositions of this disclosure and / or the methods of this disclosure unless the context otherwise requires. In other words, embodiments have been described and depicted in this application in a manner that enables the writing and drawing of a clear and concise application; however, it is intended and will be understood that embodiments can be combined or separated differently without departing from the teachings and invention. For example, it should be understood that all features described and depicted herein are applicable to all aspects of the invention described and depicted herein.

[0083] II. OX40 antibody The co-stimulatory T cell receptor OX40 is primarily expressed on effector T cells and regulatory T cells. Its ligand, OX40L, is expressed on activated antigen-presenting cells, including dendritic cells, endothelial cells, macrophages, and activated B cells. OX40-OX40L engagement is crucial for enhancing effector T cell proliferation and prolonging their survival by inhibiting apoptosis, enhancing T cell effector functions (e.g., cytokine production), and generating T helper memory cells. OX40 co-stimulation inhibits regulatory T cell induction (…) through multiple mechanisms. Figure 20A Not wanting to be bound by theory, the antibodies presented in this article, such as MAB1 and MAB10, target the OX40 receptor, affecting the Th1, Th2, and Th17 / 22 pathways while preserving regulatory T cells. Figure 20B ).

[0084] Typically, antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region contains one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs), which are sequence-hypervariant and / or involved in antigen recognition and / or typically form structurally defined loops) scattered between more conserved regions (called frame regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Heavy chain CDRs 1, 2, and 3 may be referred to herein as, for example, HCDR1, HCDR2, and HCDR3, or, for example, CDR-H1, CDR-H2, and CDR-H3. Light chain CDRs 1, 2, and 3 may be referred herein as, for example, LCDR1, LCDR2, and LCDR3, or, for example, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequences of FR1, FR2, FR3, and FR4 together constitute the “non-CDR region” or “non-extended CDR region” of the VH or VL mentioned herein. The VH and VL regions together define the antigen binding site.

[0085] As mentioned herein, a “heavy chain variable frame region” may contain one or more (e.g., one, two, three, and / or four, preferably four) sequences of heavy chain variable frame regions (e.g., frame 1 (FR1), frame 2 (FR2), frame 3 (FR3), and / or frame 4 (FR4)). As mentioned herein, a “light chain variable frame region” may contain one or more (e.g., one, two, three, and / or four, preferably four) sequences of light chain frame regions (e.g., frame 1 (FR1), frame 2 (FR2), frame 3 (FR3), and / or frame 4 (FR4)).

[0086] The variable regions of the heavy and light chains contain binding sites or binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0087] The phrases “full-length antibody” and “intact antibody” encompass the structure of the naturally occurring biological form that constitutes the antibody, including both variable and constant regions. For example, in most mammals (including humans and mice), full-length IgG antibodies are tetramers and consist of two pairs of identical immunoglobulin chains, each pair having one light chain and one heavy chain. Each light chain contains immunoglobulin domains VL and CL, and each heavy chain contains immunoglobulin domains VH, CH1 (Cγ1), CH2 (Cγ2), and CH3 (Cγ3). In some mammals, such as camels and llamas, IgG antibodies may consist of only two heavy chains, each containing a variable domain linked to the Fc region. Antigen-binding fragments expected for a given antibody include, for example, Fab fragments, Fab' fragments, (Fab')2 fragments, Fv fragments, single-chain Fv molecules (e.g., scFv), minibody, diabody, and triabody.

[0088] Antibodies or antigen-binding fragments that bind to OX40 refer to intact antibodies or antigen-binding fragments that bind to OX40 (e.g., human OX40). The antibodies or antigen-binding fragments preferably have a binding affinity (K0) of OX40 (e.g., human OX40) of 500 nM or stronger, 200 nM or stronger, 150 nM or stronger, 125 nM or stronger, 100 nM, 75 nM, 50 nM, 25 nM, 10 nM, 6 nM or stronger. D ).

[0089] In the context of this specification, the phrase "antagonistic antibody and its antigen-binding fragment" and similar phrases refer to antibodies or antigen-binding fragments capable of inhibiting and / or neutralizing the biological signaling activity of OX40, for example, by blocking or substantially reducing the binding of OX40 to OX40 ligands, thereby inhibiting or reducing OX40-induced signaling pathways and / or inhibiting or reducing OX40-mediated cellular responses, such as lymphocyte proliferation, cytokine expression, or lymphocyte survival.

[0090] "Chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0091] "Humanized antibody" refers to an antibody in which the CDR sequence of a lineage derived from another mammal species (such as a mouse) has been transplanted onto a human frame sequence. Additional frame region modifications can be made within the human frame sequence and within the CDR sequence of a lineage derived from another mammal species.

[0092] The antibodies or antigen-binding fragments of antibodies disclosed herein may be isolated antibodies or antigen-binding fragments, for example, removed from their natural environment. For example, antibodies or their binding fragments may be removed from cells, tissues, body fluids, or animals, for example, by various purification techniques.

[0093] For all human immunoglobulins, the heavy chain constant domain and the light chain constant domain of human κ immunoglobulin are numbered according to the EU numbering system (Edelman et al. (1969) Proc. Natl. Acad. Sci. USA, 63(1): 78-85). For the light chain constant domains of human λ immunoglobulin (IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7), the numbering is according to the Kabat numbering system (Kabat et al. (1991) Sequences of proteins of immunological interest. 5th Edition – US Department of Health and Human Services, NIH publication n° 91-3242), as described by Dariavach et al. (1987) Proc. Natl. Acad. Sci. USA, 84(24): 9074-8 and Frangione et al. (1985) Proc. Natl. Acad. Sci. USA, 82(10): 3415-9.

[0094] In naturally occurring antibodies, the antigen-binding site typically consists of two variable domains that define specificity: one located in the heavy chain (VH) and the other in the light chain (VL). In some cases, specificity may exist only in a single variable domain of a single-domain antibody derived from a heavy chain antibody found in the Camelidae family. The amino acid sequence boundaries of CDRs in the VH and VL domains can be determined by those skilled in the art using any of a variety of known numbering schemes, including Kabat et al., ibid. (“Kabat” numbering scheme); Al-Lazikani et al. (1997) J. Mol. Biol., 273: 927-948 (“Chothia” numbering scheme); Martin (enhanced Chothia or AbM) Abhinandan and Martin (2008) Mol. Immunol., 45(14): 3832-9; MacCalum et al. (1996) J. Mol. Biol., 262: 732-745 (“Contact” numbering scheme); Lefranc et al. (2003) Dev. Comp. Immunol., 27: 55-77 (“IMGT” numbering scheme); and Honegger and Plückthun (2001) J. Mol. Biol., 309: Those described in 657-70 (“AHo” numbering scheme).

[0095] The CDR defined by Kabat is based on sequence variability and is the most commonly used (Kabat et al., ibid.). The CDR defined by Chothia is based on the position of structural loops (Chothia & Lesk (1987) J. Mol. Biol., 196: 901-917). The CDR defined by AbM is a compromise between the Kabat and Chothia numbering schemes and is used by the AbM antibody modeling software of Oxford Molecular (Martin et al. (1989) Proc. Natl. Acad. Sci. USA, 86: 9268-72; Martin et al. (1991) Methods Enzymol., 203: 121-153; Pedersen et al. (1992) Immunomethods, 1: 126-136; Rees et al. (1996) In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172). The CDR defined by Contact is based on the analysis of complex structures available in protein databases (MacCalum et al. (1996) J. Mol. Biol., 262:732-745). IMGT®, the international ImMunoGeneTics information system® (http: / / www.imgt.org), defines CDRs based on the IMGT numbers of all immunoglobulins and T-cell receptor V-regions in all species. (IMGT®, the international ImMunoGeneTics information system®; Lefranc et al. (1991) Nucleic Acids Res., 27(1): 209-12; Ruiz et al. (2000) Nucleic Acids Res., 28(1): 219-21; Lefranc (2001) Nucleic Acids Res., 29(1): 207-9; Lefranc (2003) Nucleic Acids Res., 31(1):307-10; Lefranc et al. (2005) Dev. Comp. Immunol., 29(3): 185-203; Kaas et al. (2007) Briefings in Functional...) Genomics&Proteomics, 6(4): 253-64).Alternatively, CDRs can be defined according to Honegger's numbering scheme, which is based on structural alignment of the antibody's three-dimensional features (AHo; Honegger et al. (2001) J. Mol. Biol., 309: 657-70). Dondelliner et al. (2018) Front. Immunol., 9:2278 describe a comparison of various numbering systems. Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat, Chothia, AbM, Contact, and IMGT schemes. For CDR-H1, residue numbers are provided using both the Kabat and Chothia numbering schemes.

[0096] Antibody sequences, domain numbers, and CDRs can be obtained, for example, using antibody numbering software such as Abnum, available at bioinf.org.uk / abs / abnum / and described in Abhinandan and Martin (2008) Immunology, 45:3832-3839; AbYsis at abysis.org / abysis / index.html; or ANACRI, available at opig.stats.ox.ac.uk / webapps / sabdab-sabpred / sabpred / anarci / and described in Dunbar et al. (2016) Nucleic Acids Res., 44: W474-W478. Descriptions of various antibody numbering schemes are also available at bioinf.org.uk / abs / info.html.

[0097] Table 1. Residues in CDRs according to the indicated numbering scheme

[0098] The C-terminus of *CDR-H1 varies between H32 and H34 when numbered using the Kabat numbering convention, depending on the length of the CDR.

[0099] When referring to residues in the constant region of the antibody heavy chain, the “EU numbering scheme” is typically used (e.g., as reported above by Kabat et al.). Unless otherwise stated, the EU numbering scheme is used to refer to residues in the constant region of the antibody heavy chain as described herein.

[0100] Antibodies are grouped into classes, also known as isotypes, as determined by the genetic identification of constant regions. Human constant light chains are classified as κ (CK) and λ (Cλ) light chains. Heavy chains are classified as μ (mu), δ (delta), γ (gamma), α (alpha), or ε (epsilon), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Therefore, as used herein, “isotype” refers to any class and / or subclass of immunoglobulin defined by the chemical and antigenic characteristics of its constant regions.

[0101] As used herein, unless otherwise stated, the terms "immunoglobulin Fc domain," "Fc," "Fc domain," and "Fc region" refer to a segment of the constant region of the immunoglobulin heavy chain that, alone or in combination with a second immunoglobulin Fc domain, is capable of binding to an Fc receptor. An immunoglobulin Fc domain may include, for example, immunoglobulin CH2 and CH3 domains. An immunoglobulin Fc domain may include, for example, immunoglobulin CH2 and CH3 domains and an immunoglobulin hinge region. The boundaries between the immunoglobulin hinge region, the CH2 domain, and the CH3 domain are well known in the art and can be found, for example, in the PROSITE database (available on the World Wide Web at prosite.expasy.org). Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 and Edelman et al. (1969) Proc Natl Acad Sci USA, 63(1):78-85. Fc can be of the IgA, IgD, IgE, IgG, and IgM classes, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Fc can refer to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, as well as the flexible hinge N-terminus of these domains. For IgA and IgM, Fc may include the J chain. IgG is most commonly used for therapeutic purposes. In humans, this class includes subclasses IgG1, IgG2, IgG3, and IgG4. In mice, this class includes subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3.

[0102] The terms "Fc receptor" and "FcR" are used to describe receptors that bind to the Fc region of an antibody. For example, an FcR can be a naturally occurring human FcR. Typically, an FcR is an FcR that binds to an IgG antibody (γ receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. Other isotypes of immunoglobulins can also be bound by certain FcRs (see, for example, Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). The activating receptor FcγRIIA contains an activating motif (ITAM) based on the tyrosine residue of the immunoreceptor in its cytoplasmic domain. The repressive receptor FcγRIIB contains an repressive motif (ITIM) based on the tyrosine residue of the immunoreceptor in its cytoplasmic domain (reviewed in Daëron (1997) Annu. Rev. Immunol., 15: 203-234). FcRs are reviewed in Ravetch and Kinet (1991) Annu. Rev. Immunol., 9: 457-92; Capel et al. (1994) Immunomethods, 4:25-34; and de Haas et al. (1995) J. Lab. Clin. Med., 126: 330-41.

[0103] The IgG Fc region may also contain one or more mutations that regulate (e.g., increase) its binding to the neonatal Fc receptor (FcRn), thereby prolonging the serum half-life of the OX40 antibody. Such mutations are known in the art and are reviewed in Ramandi et al. (2022) Int. J. Mol. Sci., 23(17): 9604, as well as U.S. Patents 9,803,023 and 8,394,925. In some embodiments, one or more mutations comprise M252Y, S254T, and T256E (“YTE”); M428L and N434S (Xtend TM "LS"); H433K and N434F (NHance ®); M252Y and T256D; T250Q and M428L; T307A, E380A and N434A; T256D and T307Q, or T256D and T307W. These residues are indexed according to EU numbering. Modifications that prolong the serum half-life of OX40 antibodies (e.g., YTE mutations) can allow for less frequent antibody dosing as a therapeutic agent and / or more persistent suppression of unwanted T cell activation and / or proliferation, and promote a reduction in ADCC (antibody-dependent cell-mediated cytotoxicity) (Dall' Acqua et al. (2006) J. Biol. Chem 281(33): 23514-23524). The lower frequency of dosing of antibodies containing serum half-life prolongation modifications compared to parental or control antibodies could be, for example, every 2 to 3 months.

[0104] As used herein, the term "effective function" encompasses biochemical events resulting from the interaction of the antibody's Fc region with an Fc receptor or ligand. Effector functions include FcγR-mediated effector functions, such as ADCC (antibody-dependent cell-mediated cytotoxicity) and ADCP (antibody-dependent cell-mediated phagocytosis), and complement-mediated effector functions, such as CDC (complement-dependent cytotoxicity). Depending on the context, ADCC-mediated killing of immune cells (e.g., T cells) can lead to the release of cytokines and other immunomodulatory molecules. This release can result in fever and chills in the subject. The effector function of an antibody can be altered by changing (i.e., enhancing or reducing, preferably enhancing) the antibody's affinity for effector molecules, such as Fc receptors (FcRs) or complement components. Binding affinity is typically varied by modifying the effector molecule's binding site, and in this case, it is appropriate to target the site of interest in a suitable manner and modify at least a portion of that site. It is further envisioned that alterations to binding sites on antibodies targeting effector molecules do not require a significant change in overall binding affinity, but rather can alter the geometry of the interaction, rendering the effector mechanism ineffective, such as in nonproductive binding. Furthermore, it is envisioned that effector function can be altered by modifying sites that do not directly participate in effector molecule binding but participate in effector function in other ways. By modifying the effector function of antibodies, various aspects of the immune response can be controlled, such as enhancing or suppressing various immune system responses, potentially with beneficial effects in diagnosis and therapy.

[0105] It should be understood that modifications in the CH2 domain can affect the binding of FcR to Fc. Many amino acid modifications in the Fc region are known in the art to be used to selectively alter the affinity of Fc for different Fc-γ (Fcγ) receptors (FcγR or FcR).

[0106] The following are examples of mutations that alter the binding of FcR to Fc: i. S298A / E333A / K334A, S298A / E333A / K334A / K326A (Lu et al. (2011) J. Immunol. Methods, 365(1-2): 132-41).

[0107] ii. F243L / R292P / Y300L / V305I / P396L, F243L / R292P / Y300L / L235V / P396L (Stavenhagen et al. (2007) Cancer Res., 67(18): 8882-90; Nordstrom et al. (2011) BreastCancer Res., 13(6): R123); iii. F243L (Stewart et al. (2011) Protein Eng Des Sel., 24(9): 671-8.), S298A / E333A / K334A (Shields et al. (2001) J. Biol. Chem., 276(9): 6591-604); iv. S239D / I332E / A330L, S239D / I332E (Lazar et al. (2006) Proc. Natl. Acad.Sci. USA, 103(11): 4005-10); v. S239D / S267E, S267E / L328F (Chu et al. (2008) Mol. Immunol., 45(15): 3926-33); vi. S239D / D265S / S298A / I332E, S239E / S298A / K326A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A330L / I332E / S298A, G236A / S239D / D270L / I332E, S239E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D, and other mutations listed in WO2011 / 120134 and WO2011 / 120135. The mutations are listed on page 283 of Therapeutic Antibody Engineering (William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No. 11, ISBN 1 907568 379, Oct 2012). vii. M252Y, S254T and T256E (“YTE”; Dall'Acqua et al. (2002), J. Immunol., 169:5171–5180 and Dall'Acqua et al. (2006) J. Biol. Chem 281(33): 23514-23524); and viii. M428L and N434S (“LS”; Zalevsky et al. (2010) Nat Biotechnol 28: 157–159).

[0108] Fc modification that reduces FcγR and / or complement binding and / or effector function is known in the art. Strategies for engineering antibodies with reduced or silenced effector activity are discussed in Strohl (2009) Curr. Opin. Biotech., 20: 685-691 and Strohl, WR and Strohl LM (2012) “Antibody Fc engineering for optimal antibody performance” In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, pp 225-249. These strategies include reducing effector function through glycosylation modification, using IgG2 / IgG4 scaffolds, or introducing mutations in the hinge or CH2 region of the Fc. For example, U.S. Patent Publication No. 2011 / 0212087 (Strohl), International Patent Publication No. WO 2006 / 105338 (Xencor), U.S. Patent Publication No. 2012 / 0225058 (Xencor), U.S. Patent Publication No. 2012 / 0251531 (Genentech), International Application Publication No. WO1999 / 051642, and Strohl et al. (2012) J. Mol. Biol., 420: 204-219) describe exemplary specific Fc modifications that reduce FcγR or complement binding to Fc. For example, in some embodiments, one or more mutations comprise N297A, N297G, or N297Q mutations. In some implementations, one or more mutations include or further include L234A and L235A (LALA) mutations, L234A, L235A and P329A (LALAPA) mutations, L234A, L235A and P329G (LALAPG) mutations, or L234A, L235E, G237A, A330S and P331S (LALEGAASPS) mutations.

[0109] Specific, non-limiting examples of known amino acid modifications that reduce FcγR or complement binding to Fc include those identified in Table 2 below: Table 2. Modifications that reduce FcγR or complement binding to Fc

[0110] Methods for producing antibodies with little or no fucose at the Fc glycosylation site (Asn 297 EU number, N297) without altering the amino acid sequence are well known in the art. GlymaxX® technology (ProBioGen AG) is based on the introduction of an enzyme into the gene that redirects the cellular pathway of fucose biosynthesis to antibody production. This prevents antibody-producing cells from adding the sugar "fucose" to the N-linked carbohydrate portion of the antibody. (von Horsten et al. (2010) Glycobiology, 20(12): 1607-1618.) Examples of cell lines capable of producing defucosylated antibodies include CHO-DG44 or Lec13 CHO cells with stable overexpression of the bacterial oxidoreductase GDP-6-deoxy-D-lysol-4-hexose reductase (RMD) (see von Horsten et al., ibid.), which lack protein fucosylation (see Rippka et al. (1986) Arch. Biochem. Biophys., 249: 533-545; US Patent Publication No. 2003 / 0157108; WO 2004 / 056312), and knockout cell lines, such as α-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (see Yamane-Ohnuki et al. (2004) Biotech. Bioeng., 87: 614-622; Kanda et al. (2006) Biotechnol. Bioeng., 94:680-688; and WO 2003 / 085107). Another approach to obtaining antibodies with reduced fucosylation levels can be found in U.S. Patent 8,409,572, which teaches the selection of cell lines for antibody production because they are able to produce lower levels of fucosylation on the antibody.

[0111] Antibodies can be completely unfucosylated (meaning they do not contain detectable fucose), or they can be partially unfucosylated, meaning that the antibodies contain less than 95%, less than 85%, less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, less than 25%, less than 15%, or less than 5% of the amount of fucose typically detected against similar antibodies produced by mammalian expression systems.

[0112] The antibodies described herein can be engineered to contain an IgG1 domain with reduced levels or no fucose at position Asn 297 (N297) compared to the naturally occurring IgG1 domain. Such Fc domains are known to have improved ADCC. See Shields et al. (2002) J. Biol. Chem., 277:26733-26740. The amount of fucose can be determined using any suitable method, such as that described in WO 2008 / 077546.

[0113] Antibody variable regions, as well as heavy and light chains In one aspect, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable heavy (VH) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 7, and / or a variable light (VL) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30 or 33.

[0114] In one aspect, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable heavy (VH) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 7, and a variable light (VL) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30 or 33.

[0115] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable heavy (VH) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 7.

[0116] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable light (VL) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 8.

[0117] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable light (VL) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 12.

[0118] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable light (VL) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 15.

[0119] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable light (VL) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 18.

[0120] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable light (VL) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 21.

[0121] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable light (VL) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 24.

[0122] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable light (VL) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 27.

[0123] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable light (VL) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 30.

[0124] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable light (VL) chain sequence containing an amino acid sequence as shown in SEQ ID NO: 33.

[0125] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 7 and a variable light (VL) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 8.

[0126] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 7 and a variable light (VL) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 12.

[0127] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 7 and a variable light (VL) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 15.

[0128] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 7 and a variable light (VL) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 18.

[0129] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 7 and a variable light (VL) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 21.

[0130] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 7 and a variable light (VL) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 24.

[0131] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 7 and a variable light (VL) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 27.

[0132] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 7 and a variable light (VL) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 30.

[0133] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 7 and a variable light (VL) chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 33.

[0134] On the other hand, this disclosure provides variants of the OX40 antibody disclosed herein, wherein amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid changes) are present in the frame region of the variable heavy (VH) chain sequence compared to the disclosed variable heavy (VH) chain sequence (e.g., SEQ ID NO: 7), and / or wherein amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid changes) are present in the frame region of the variable light (VL) chain sequence compared to the disclosed variable light (VL) chain sequence (e.g., SEQ ID NO: 8). The changes may be conserved substitutions of amino acids in the frame region, or non-conserved substitutions of amino acids in the frame region, or deletions of amino acids in the frame region, or insertions of amino acids in the frame region, or combinations thereof. For example, 1-10, e.g., 1-5, e.g., 1 or 2 total amino acid changes, such as substitutions, may be present in the frame region of the variable heavy (VH) chain sequence. For example, there may be 1-10, e.g., 1-5, e.g., 1 or 2 total amino acid changes in the frame region of the variable light (VL) chain. Double sequence alignment using the default settings of the AlignX module in Vector NTI v.9.0.0 (Invitrogen Corp., Carlsbad, Calif.) or other suitable alignment software (such as BLAST, CLUSTAL, or BLOSUM) can be used to identify the number of amino acid changes that have been made in the frame region of the variable heavy (VH) or variable light (VL) chain compared to a reference sequence.

[0135] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable heavy (VH) chain containing an amino acid sequence as shown in SEQ ID NO: 7, except that the variable heavy (VH) chain contains 1, 2, 3, 4 or 5 total amino acid modifications (e.g., 1 or 2 total modifications, such as substitutions) on the variable heavy chain framework region, and a variable light (VL) chain containing an amino acid sequence as shown in SEQ ID NO: 8.

[0136] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable heavy (VH) chain containing an amino acid sequence as shown in SEQ ID NO: 7, and a variable light (VL) chain containing an amino acid sequence as shown in SEQ ID NO: 8, wherein the light chain contains 1, 2, 3, 4 or 5 total amino acid modifications (e.g., 1 or 2 total modifications, such as substitutions) on the light chain framework region.

[0137] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a variable heavy (VH) chain containing the amino acid sequence shown in SEQ ID NO: 7, except that the variable heavy (VH) chain contains 1, 2, 3, 4 or 5 total amino acid modifications (e.g., 1 or 2 total modifications, such as substitutions) in the variable heavy chain framework region, and a variable light (VL) chain containing the amino acid sequence shown in SEQ ID NO: 8, except that the light chain contains 1, 2, 3, 4 or 5 total amino acid modifications (e.g., 1 or 2 total modifications, such as substitutions) in the light chain framework region.

[0138] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable heavy (VH) chain and a variable light (VL) chain, wherein the variable heavy (VH) chain has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 7, for example 99% or greater, wherein the sequence variation is limited to the heavy chain framework region, and the variable light (VL) chain is identical to the amino acid sequence shown in SEQ ID NO: 7. The amino acid sequence shown in NO:8 has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity, for example, 99% or greater, wherein sequence variation is limited to the light chain framework and constant regions.

[0139] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable heavy (VH) chain and a variable light (VL) chain, wherein the variable heavy (VH) chain has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 7, for example 99% or greater, wherein the sequence variation is limited to the heavy chain frame region, and the variable light (VL) chain comprises the amino acid sequence shown in SEQ ID NO: 8.

[0140] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable heavy (VH) chain and a variable light (VL) chain, the variable heavy (VH) chain comprising the amino acid sequence shown in SEQ ID NO: 7, and the variable light (VL) chain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 8, for example 99% or greater, wherein sequence variation is limited to the light chain frame and constant region.

[0141] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a variable heavy (VH) chain sequence comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the amino acid sequence shown in SEQ ID NO: 7, and a variable light (VL) chain sequence comprising light chain CDR1, light chain CDR2 and light chain CDR3 in the amino acid sequence shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30 or 33 (e.g. SEQ ID NO: 8). In some embodiments, the variable heavy chain sequence may further comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, greater than 99% but less than 100%, or 100% sequence identity (e.g., at least 99%) with respect to the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33 (e.g., SEQ ID NO: 8) with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% but less than 100%, or 100% sequence identity (e.g., at least 99%) with respect to the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33 (e.g., SEQ ID NO: 8) with respect to the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33 (e.g., SEQ ID NO: 8) with respect to the amino acid sequence of the heavy ...SEQ ID NO: 8, SEQ ID NO: 8, wherein any sequence variation is present in the frame region. In other embodiments, the heavy chain variable region further comprises the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, in the heavy chain variable frame region, and / or the light chain variable region further comprises the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33, such as the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, in the light chain variable frame region. The CDR can be defined by Kabat. The CDR can be defined by Chothia. The CDR can be defined by AbM. The CDR can be defined by IMGT. The CDR can be defined by Contact. The CDR can be defined by AHo.

[0142] In another aspect, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 9, and / or a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 31 or 34.

[0143] In another aspect, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 9, and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 31 or 34.

[0144] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 9.

[0145] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 10.

[0146] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 13.

[0147] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 16.

[0148] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 19.

[0149] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 22.

[0150] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 25.

[0151] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 28.

[0152] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 31.

[0153] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 34.

[0154] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 9 and a light chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 10.

[0155] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 9 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 13.

[0156] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 9 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 16.

[0157] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 9 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 19.

[0158] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 9 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 22.

[0159] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 9 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 25.

[0160] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 9 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 28.

[0161] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 9 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 31.

[0162] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 9 and a light chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 34.

[0163] In another aspect, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73, and / or a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 31 or 34.

[0164] In another aspect, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73, and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 31 or 34.

[0165] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73.

[0166] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 73 and a light chain sequence comprising an amino acid sequence as shown in SEQ ID NO: 10.

[0167] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 13.

[0168] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 16.

[0169] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 19.

[0170] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 22.

[0171] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 25.

[0172] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 28.

[0173] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 31.

[0174] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain sequence containing an amino acid sequence as shown in SEQ ID NO: 73 and a light chain sequence containing an amino acid sequence as shown in SEQ ID NO: 34.

[0175] On the other hand, this disclosure provides variants of the OX40 antibody disclosed herein, wherein amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid changes) are present in the frame and constant regions of the heavy chain compared to the heavy chain sequence disclosed herein (e.g., SEQ ID NO: 73 or SEQ ID NO: 9); and / or wherein amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid changes) are present in the frame and constant regions of the light chain compared to the light chain sequence disclosed herein (e.g., SEQ ID NO: 10). The changes may be conserved substitutions of amino acids in the frame or constant regions, or non-conserved substitutions of amino acids in the frame or constant regions, or deletions of amino acids in the frame or constant regions, or insertions of amino acids in the frame or constant regions, or combinations thereof. For example, 1-10, e.g., 1-5, total amino acid changes, such as substitutions, may be present in the frame and constant regions of the heavy chain. For example, 1-10, e.g., 1-5, total amino acid changes may be present in the frame and constant regions of the light chain. Double sequence alignment using the default settings of the AlignX module in Vector NTI v.9.0.0 (Invitrogen Corp., Carlsbad, Calif.SUM) or other suitable alignment software (such as BLAST, CLUSTAL, or BLOSUM) can be used to identify the number of amino changes that have been made in the heavy or light chain sequence compared to the heavy or light chain reference sequence.

[0176] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a heavy chain containing an amino acid sequence as shown in SEQ ID NO: 73, except that the heavy chain contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 total amino acid modifications (e.g., 1, 2, 3, 4 or 5 total modifications, such as substitutions) in the heavy chain framework and constant region, and a light chain containing an amino acid sequence as shown in SEQ ID NO: 10.

[0177] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a heavy chain containing an amino acid sequence as shown in SEQ ID NO: 73, and a light chain containing an amino acid sequence as shown in SEQ ID NO: 10, except that the light chain contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 total amino acid modifications (e.g., 1, 2, 3, 4 or 5 total modifications, such as substitutions) in the light chain framework and constant region.

[0178] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof, comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 73, except that the heavy chain contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 total amino acid modifications (e.g., 1, 2, 3, 4 or 5 total modifications, such as substitutions) in the heavy chain frame and constant region, and comprising a light chain containing the amino acid sequence shown in SEQ ID NO: 10, except that the light chain contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 total amino acid modifications (e.g., 1, 2, 3, 4 or 5 total modifications, such as substitutions) in the light chain frame and constant region.

[0179] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, said heavy chain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity, for example 99% or greater, with sequence variation confined to the heavy chain framework and constant regions, said light chain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity, for example 99% or greater, with sequence variation confined to the heavy chain framework and constant regions, said light chain having at least about 80 ....1%, 99 The amino acid sequence shown in NO:10 has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity, for example, 99% or greater, wherein sequence variation is limited to the light chain framework and constant regions.

[0180] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, said heavy chain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity, for example 99% or greater, with sequence variation limited to the heavy chain frame and constant region, said light chain comprising the amino acid sequence shown in SEQ ID NO: 10.

[0181] In some embodiments, this disclosure provides an OX40 antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 73, the light chain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity with an amino acid sequence as shown in SEQ ID NO: 10, for example 99% or greater, wherein sequence variation is limited to the light chain frame and constant regions.

[0182] The variant OX40 antibodies or their antigen-binding fragments disclosed herein retain substantial biological activity, such as specific binding to OX40. For example, such variants can retain similar nanomolar affinity (Kb) for OX40 compared to unmodified reference antibodies. D ).

[0183] Antibody CDR The antibodies or binding fragments thereof that bind to OX40 (e.g., human OX40) disclosed herein may be defined by a CDR sequence in their variable region. The CDR sequence is located between frame sequences, which may be human or humanized, for example.

[0184] For example, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising three heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) and three light chain CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-H1, CDR-H2, and CDR-H3 are contained in a heavy chain variable domain (VH) comprising the amino acid sequence shown in SEQ ID NO: 7, and wherein CDR-L1, CDR-L2, and CDR-L3 are contained in a light chain variable domain (VL) comprising the amino acid sequence shown in SEQ ID NO: 8. CDR-H1, CDR-H2, and CDR-H3, as well as CDR-L1, CDR-L2, and CDR-L3, may be defined according to the Kabat, Chothia, IMGT, AbM, Contact, or Honnecger (AHo) CDR numbering system discussed herein, or other numbering systems known in the art.

[0185] In addition, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising three heavy chain CDR sequences (CDRs) (CDR-H1, CDR-H2 and CDR-H3) containing a heavy chain variable domain (VH) as shown in SEQ ID NO: 7 and / or three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2 and CDR-L3) containing a light chain variable domain (VL) as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30 or 33. In some embodiments, the antibody or antigen-binding fragment thereof binding to OX40 (e.g., human OX40) comprises three heavy chain CDR sequences (CDRs) (CDR-H1, CDR-H2, and CDR-H3) containing a heavy chain variable domain (VH) as shown in SEQ ID NO: 7 and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2, and CDR-L3) containing a light chain variable domain (VL) as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33. In some embodiments, the CDRs are defined according to Kabat. In some embodiments, the CDRs are defined according to Chothia. In some embodiments, the CDRs are defined according to AbM. In some embodiments, the CDRs are defined according to Contact. In some embodiments, the CDRs are defined according to IMGT. In some embodiments, the CDRs are defined according to AHo.

[0186] For example, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising three heavy chain complementarity determination sequences (CDRs) (CDR-H1, CDR-H2 and CDR-H3) comprising a heavy chain variable domain (VH) containing the amino acid sequence shown in SEQ ID NO: 7, and three light chain complementarity determination sequences (CDRs) (CDR-L1, CDR-L2 and CDR-L3) comprising a light chain variable domain (VL) containing the amino acid sequence shown in SEQ ID NO: 8.

[0187] For example, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising three heavy chain complementarity determination sequences (CDRs) (CDR-H1, CDR-H2 and CDR-H3) comprising a heavy chain variable domain (VH) containing the amino acid sequence shown in SEQ ID NO: 7, and three light chain complementarity determination sequences (CDRs) (CDR-L1, CDR-L2 and CDR-L3) comprising a light chain variable domain (VL) containing the amino acid sequence shown in SEQ ID NO: 12.

[0188] For example, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising three heavy chain complementarity determination sequences (CDRs) (CDR-H1, CDR-H2 and CDR-H3) comprising a heavy chain variable domain (VH) containing the amino acid sequence shown in SEQ ID NO: 7, and three light chain complementarity determination sequences (CDRs) (CDR-L1, CDR-L2 and CDR-L3) comprising a light chain variable domain (VL) containing the amino acid sequence shown in SEQ ID NO: 15.

[0189] For example, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising three heavy chain complementarity determination sequences (CDRs) (CDR-H1, CDR-H2 and CDR-H3) comprising a heavy chain variable domain (VH) containing the amino acid sequence shown in SEQ ID NO: 7, and three light chain complementarity determination sequences (CDRs) (CDR-L1, CDR-L2 and CDR-L3) comprising a light chain variable domain (VL) containing the amino acid sequence shown in SEQ ID NO: 18.

[0190] For example, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising three heavy chain complementarity determination sequences (CDRs) (CDR-H1, CDR-H2 and CDR-H3) comprising a heavy chain variable domain (VH) containing an amino acid sequence as shown in SEQ ID NO: 7, and three light chain complementarity determination sequences (CDRs) (CDR-L1, CDR-L2 and CDR-L3) comprising a light chain variable domain (VL) containing an amino acid sequence as shown in SEQ ID NO: 21.

[0191] For example, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) and comprises three heavy chain complementarity determination sequences (CDRs) (CDR-H1, CDR-H2 and CDR-H3) of a heavy chain variable domain (VH) containing an amino acid sequence as shown in SEQ ID NO: 7, and three light chain complementarity determination sequences (CDRs) (CDR-L1, CDR-L2 and CDR-L3) of a light chain variable domain (VL) containing an amino acid sequence as shown in SEQ ID NO: 24.

[0192] For example, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising three heavy chain complementarity determination sequences (CDRs) (CDR-H1, CDR-H2 and CDR-H3) comprising a heavy chain variable domain (VH) containing an amino acid sequence as shown in SEQ ID NO: 7, and three light chain complementarity determination sequences (CDRs) (CDR-L1, CDR-L2 and CDR-L3) comprising a light chain variable domain (VL) containing an amino acid sequence as shown in SEQ ID NO: 27.

[0193] For example, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising three heavy chain complementarity determination sequences (CDRs) (CDR-H1, CDR-H2 and CDR-H3) comprising a heavy chain variable domain (VH) containing the amino acid sequence shown in SEQ ID NO: 7, and three light chain complementarity determination sequences (CDRs) (CDR-L1, CDR-L2 and CDR-L3) comprising a light chain variable domain (VL) containing the amino acid sequence shown in SEQ ID NO: 30.

[0194] For example, this disclosure provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising three heavy chain complementarity determination sequences (CDRs) (CDR-H1, CDR-H2 and CDR-H3) comprising a heavy chain variable domain (VH) containing the amino acid sequence shown in SEQ ID NO: 7, and three light chain complementarity determination sequences (CDRs) (CDR-L1, CDR-L2 and CDR-L3) comprising a light chain variable domain (VL) containing the amino acid sequence shown in SEQ ID NO: 33.

[0195] In some embodiments, the CDR of the aforementioned antibody is defined according to Kabat. In some embodiments, the CDR is defined according to Chothia. In some embodiments, the CDR is defined according to AbM. In some embodiments, the CDR is defined according to Contact. In some embodiments, the CDR is defined according to IMGT. In some embodiments, the CDR is defined according to AHo.

[0196] This disclosure also provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising a heavy chain variable region comprising CDR-H1 of the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 of the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region comprising CDR-L1 of the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 of the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 38, wherein X1 is F, T, W, or M; wherein X2 is G, I, V, L, or E; wherein X3 is A, D, E, L, H, T, or F; and wherein X4 is W, P, F, Y, or T. In some embodiments, X1 is F or T, X2 is L, G, or E, X3 is A, and X4 is W. In some embodiments, X1 is F or T. In some embodiments, X1 is F. In some embodiments, X1 is T. In some embodiments, X2 is L, G, or E. In some embodiments, X2 is G. In some embodiments, X2 is L. In some embodiments, X3 is A. In some embodiments, X4 is W.

[0197] This disclosure also provides an antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40), comprising a heavy chain variable region comprising CDR-H1 of the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 of the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region comprising CDR-L1 of the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 of the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 71, wherein X1 is F, T, W, or M; wherein X2 is G, I, V, L, or E; wherein X3 is A, D, E, L, H, T, or F; and wherein X4 is W, P, F, Y, or T. In some embodiments, X1 is F or T, X2 is L, G, or E, X3 is A, and X4 is W. In some embodiments, X1 is F or T. In some embodiments, X1 is F. In some embodiments, X1 is T. In some embodiments, X2 is L, G, or E. In some embodiments, X2 is G. In some embodiments, X2 is L. In some embodiments, X3 is A. In some embodiments, X4 is W.

[0198] In some instances, the disclosed antibody or antigen-binding fragment that binds to OX40 (e.g., human OX40) may include one or more amino acid sequences provided in Table 3, which are CDRs of the antibody or antigen-binding fragment as defined by Kabat numbers. For example, an antibody or its antigen-binding fragment may include a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, HCDR3 and / or a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, as provided in Table 3.

[0199] Table 3. CDRs defined by Kabat

[0200] For example, in some embodiments, the antibody or antigen-binding fragment of the antibody that binds to OX40 (e.g., human OX40) comprises a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region sequence comprising CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the sequence shown in SEQ ID NO: 6, 11, 14, 17, 20, 23, 26, 29, or 32. In some embodiments, the heavy chain variable region may further comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, greater than 99% but less than 100%, or 100% sequence identity (e.g., at least 99%) with the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7, wherein any sequence variation exists in the frame region, and / or the light chain variable region may further comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% but less than 100%, or 100% sequence identity (e.g., at least 99%) with the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33, wherein any sequence variation exists in the frame region. In some embodiments, the heavy chain variable region further comprises the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 total amino acid modifications (e.g., substitution, e.g., conservative substitution, e.g., deletion) on the heavy chain variable framework region, such as 1 or 2 modifications, and / or the light chain variable region further comprises the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30 or 33, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 total amino acid modifications (e.g., substitution, e.g., conservative substitution, e.g., deletion) on the light chain variable framework region, such as 1 or 2 modifications.

[0201] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the heavy chain variable region may further comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, greater than 99% but less than 100% or 100% sequence identity (e.g., at least 99%) with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7, wherein any sequence variation exists in the frame region, and the light chain variable region may further comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% but less than 100% or 100% sequence identity (e.g., at least 99%) with the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, wherein any sequence variation exists in the frame region. In some embodiments, the heavy chain variable region further comprises the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 total amino acid modifications (e.g., substitution, e.g., conservative substitution, e.g., deletion) on the heavy chain variable framework region, such as 1 or 2 modifications, and / or the light chain variable region further comprises the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 total amino acid modifications (e.g., substitution, e.g., conservative substitution, e.g., deletion) on the light chain variable framework region, such as 1 or 2 modifications.

[0202] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 11.

[0203] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0204] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 17.

[0205] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 20.

[0206] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 23.

[0207] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 26.

[0208] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 29.

[0209] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 32.

[0210] In some instances, the disclosed antibody or antigen-binding fragment that binds to OX40 (e.g., human OX40) may include one or more amino acid sequences provided in Table 4, which are CDRs of the antibody or antigen-binding fragment as defined by Kabat numbers. For example, an antibody or its antigen-binding fragment may include a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, HCDR3 and / or a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, as provided in Table 4.

[0211] Table 4. CDR as defined by Chothia

[0212] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 42, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0213] In some embodiments, the antibody or antigen-binding fragment of the antibody that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising a CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, a CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 42, a CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, a CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, a CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and a CDR-L3 containing the amino acid sequence shown in one of SEQ ID NO: 6, 11, 14, 17, 20, 23, 26, 29, or 32. In some embodiments, the heavy chain variable region may further comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, greater than 99% but less than 100%, or 100% sequence identity (e.g., at least 99%) with the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7, wherein any sequence variation exists in the frame region, and / or the light chain variable region may further comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% but less than 100%, or 100% sequence identity (at least 99%) with the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33 (e.g., SEQ ID NO: 8), wherein any sequence variation exists in the frame region. In some embodiments, the heavy chain variable region further comprises the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, on the heavy chain variable framework region, and / or the light chain variable region further comprises the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33, such as the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, on the light chain variable framework region. CDR-L3 may be SEQ ID NO: 6. Sequence identity may be at least 99%.

[0214] In some instances, the disclosed antibody or antigen-binding fragment that binds to OX40 (e.g., human OX40) may include one or more amino acid sequences provided in Table 5, which are CDRs of the antibody or antigen-binding fragment as defined by Kabat numbers. For example, an antibody or its antigen-binding fragment may include a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, HCDR3 and / or a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, as provided in Table 5.

[0215] Table 5. CDRs defined by AbM

[0216] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 47, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 48, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0217] In some embodiments, the antibody or antigen-binding fragment of the antibody that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising a CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 47, a CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 48, a CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, a CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, a CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and a CDR-L3 containing the amino acid sequence shown in one of SEQ ID NO: 6, 11, 14, 17, 20, 23, 26, 29, or 32. In some embodiments, the heavy chain variable region may further comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, greater than 99% but less than 100%, or 100% sequence identity (e.g., at least 99%) with the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7, wherein any sequence variation exists in the frame region, and / or the light chain variable region may further comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% but less than 100%, or 100% sequence identity (at least 99%) with the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33 (e.g., SEQ ID NO: 8), wherein any sequence variation exists in the frame region. In some embodiments, the heavy chain variable region further comprises the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, on the heavy chain variable framework region, and / or the light chain variable region further comprises the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33, such as the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, on the light chain variable framework region. CDR-L3 may be SEQ ID NO: 6. Sequence identity may be at least 99%.

[0218] In some instances, the disclosed antibody or antigen-binding fragment that binds to OX40 (e.g., human OX40) may include one or more amino acid sequences provided in Table 6, which are CDRs of the antibody or antigen-binding fragment as defined by Kabat numbers. For example, an antibody or its antigen-binding fragment may include a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, HCDR3 and / or a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, as provided in Table 6.

[0219] Table 6. CDRs defined by IMGT

[0220] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 59, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 60, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 61, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 62, CDR-L2 containing the amino acid sequence AT, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0221] In some embodiments, the antibody or antigen-binding fragment of the antibody that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising a CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 59, a CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 60, a CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 61, a CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 62, a CDR-L2 containing the amino acid sequence AT, and a CDR-L3 containing the amino acid sequence shown in one of SEQ ID NO: 6, 11, 14, 17, 20, 23, 26, 29, or 32. In some embodiments, the heavy chain variable region may further comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, greater than 99% but less than 100%, or 100% sequence identity (e.g., at least 99%) with the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7, wherein any sequence variation exists in the frame region, and / or the light chain variable region may further comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% but less than 100%, or 100% sequence identity (at least 99%) with the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33 (e.g., SEQ ID NO: 8), wherein any sequence variation exists in the frame region. In some embodiments, the heavy chain variable region further comprises the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, on the heavy chain variable framework region, and / or the light chain variable region further comprises the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33, such as the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, on the light chain variable framework region. CDR-L3 may be SEQ ID NO: 6. Sequence identity may be at least 99%.

[0222] In some instances, the disclosed antibody or antigen-binding fragment that binds to OX40 (e.g., human OX40) may include one or more amino acid sequences provided in Table 7, which are CDRs of the antibody or antigen-binding fragment as defined by Kabat numbers. For example, an antibody or its antigen-binding fragment may include a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, HCDR3 and / or a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, as provided in Table 7.

[0223] Table 7. CDRs defined by Contact

[0224] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 53, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 54, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 55, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 56, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 57, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 58.

[0225] In some embodiments, the antibody or antigen-binding fragment of the antibody that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising a CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 53, a CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 54, a CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 55, a CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 56, a CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 57, and a CDR-L3 containing the amino acid sequence shown in one of SEQ ID NO: 58, 75, 77, 79, 81, 83, 85, 87, or 89. In some embodiments, the heavy chain variable region may further comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, greater than 99% but less than 100%, or 100% sequence identity (e.g., at least 99%) with the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7, wherein any sequence variation exists in the frame region, and / or the light chain variable region may further comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% but less than 100%, or 100% sequence identity (at least 99%) with the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33 (e.g., SEQ ID NO: 8), wherein any sequence variation exists in the frame region. In some embodiments, the heavy chain variable region further comprises the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, on the heavy chain variable framework region, and / or the light chain variable region further comprises the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33, such as the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, on the light chain variable framework region. CDR-L3 may be SEQ ID NO: 58. Sequence identity may be at least 99%.

[0226] In some instances, the disclosed antibody or antigen-binding fragment that binds to OX40 (e.g., human OX40) may include one or more amino acid sequences provided in Table 8, which are CDRs of the antibody or antigen-binding fragment as defined by Kabat numbers. For example, an antibody or its antigen-binding fragment may include a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, HCDR3 and / or a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, as provided in Table 8.

[0227] Table 8. CDRs defined by AHo

[0228] In some embodiments, the antibody or antigen-binding fragment thereof that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 65, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 66, CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 67, CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 68, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 69, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 70.

[0229] In some embodiments, the antibody or antigen-binding fragment of the antibody that binds to OX40 (e.g., human OX40) includes a heavy chain variable region comprising a CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 65, a CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 66, a CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 67, a CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 68, a CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 69, and a CDR-L3 containing the amino acid sequence shown in one of SEQ ID NO: 70, 76, 78, 80, 82, 84, 86, 88, or 90. In some embodiments, the heavy chain variable region may further comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, greater than 99% but less than 100%, or 100% sequence identity (e.g., at least 99%) with the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7, wherein any sequence variation exists in the frame region, and / or the light chain variable region may further comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% but less than 100%, or 100% sequence identity (at least 99%) with the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33 (e.g., SEQ ID NO: 8), wherein any sequence variation exists in the frame region. In some embodiments, the heavy chain variable region further comprises the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, on the heavy chain variable framework region, and / or the light chain variable region further comprises the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33, such as the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8, except that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 total amino acid modifications (e.g., substitution, conservative substitution, deletion), such as 1 or 2 modifications, on the light chain variable framework region. CDR-L3 may be SEQ ID NO: 70. Sequence identity may be at least 99%.

[0230] In other instances, antibodies or antigen-binding fragments thereof that bind to OX40 (e.g., human OX40) include: (a) Heavy chain variable region, which contains: Heavy chain CDR1, comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 59 or SEQ ID NO: 65; Heavy chain CDR2, comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60 or SEQ ID NO: 66; and Heavy chain CDR3, comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 55, SEQ ID NO: 61 or SEQ ID NO: 67; and (b) A light chain variable region, comprising: The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 56, SEQ ID NO: 62 or SEQ ID NO: 68; The light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 57, amino sequence AT, or SEQ ID NO: 69; and The light chain CDR3 contains the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 58 and SEQ ID NO: 70.

[0231] III. Methods for preparing anti-OX40 antibodies The production of anti-OX40 antibodies and their antigen-binding fragments, such as those disclosed herein, is known in the art. For example, DNA molecules encoding light chain variable regions and / or heavy chain variable regions can be chemically synthesized or synthesized via recombinant DNA methods. For example, the sequence of an antibody can be cloned from a hybridoma using suitable synthetic nucleic acid primers via conventional hybridization techniques or polymerase chain reaction (PCR). The resulting DNA molecule encoding the variable region of interest can be ligated to other suitable nucleotide sequences, including, for example, constant region coding sequences and expression control sequences, to produce a conventional gene expression construct (i.e., an expression vector) encoding the desired antibody. The production of the determined gene construct is within the scope of conventional techniques in the art.

[0232] This disclosure provides polynucleotides or polynucleotide sequences encoding the OX40 antibody or antigen-binding fragments disclosed herein.

[0233] In some embodiments, the polynucleotide or polynucleotide group comprises the sequence shown in SEQ ID NO: 39 and / or 40. In some embodiments, the polynucleotide or polynucleotide group encodes an amino acid sequence comprising the amino acid sequences shown in SEQ ID NO: 1, 2, 3, 4, 5, and 6. In some embodiments, the polynucleotide or polynucleotide group encodes the amino acid sequence shown in SEQ ID NO: 7 and / or SEQ ID NO: 8. In some embodiments, the polynucleotide or polynucleotide group encodes the amino acid sequence shown in SEQ ID NO: 9 and / or 10.

[0234] In some embodiments, a polynucleotide set is provided, wherein one polynucleotide comprises the sequence shown in SEQ ID NO: 39, and a separate polynucleotide encodes the sequence shown in SEQ ID NO: 40. In some embodiments, a polynucleotide set is provided, wherein one polynucleotide encodes an amino acid sequence comprising the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, and a separate polynucleotide encodes an amino acid sequence comprising the amino acid sequences shown in SEQ ID NO: 4, 5, and 6. In some embodiments, a polynucleotide set is provided, wherein one polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 7, and a separate polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, a polynucleotide set is provided, wherein one polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 9, and a separate polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 10.

[0235] In some embodiments, the polynucleotide or polynucleotide set encodes an amino acid sequence as shown in SEQ ID NO: 7 and one of SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33. In some embodiments, the polynucleotide or polynucleotide set encodes an amino acid sequence as shown in SEQ ID NO: 9 and one of SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 31, or 34. In one specific example, the polynucleotide or polynucleotide set encodes an amino acid sequence as shown in SEQ ID NO: 9 and one of SEQ ID NO: 10.

[0236] In some embodiments, a polynucleotide set is provided, wherein one polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 7, and a separate polynucleotide encodes the amino acid sequence shown in one of SEQ ID NO: 8, 12, 15, 18, 21, 24, 27, 30, or 33. In some embodiments, a polynucleotide set is provided, wherein one polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 9, and a separate polynucleotide encodes the amino acid sequence shown in one of SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 31, or 34. In one specific example, the polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 9, and a separate polynucleotide encodes the amino acid sequence shown in one of SEQ ID NO: 10.

[0237] In a further embodiment, the polynucleotide or polynucleotide group encodes an amino acid sequence as shown in SEQ ID NO: 73 and one of SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 31 or 34. In one specific example, the polynucleotide or polynucleotide group encodes an amino acid sequence as shown in SEQ ID NO: 73 and one of SEQ ID NO: 10.

[0238] In a further embodiment, a polynucleotide set is provided, wherein one polynucleotide encodes an amino acid sequence as shown in SEQ ID NO: 73, and a separate polynucleotide encodes an amino acid as described in one of SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 31, or 34. In one specific example, a polynucleotide set is provided, wherein one polynucleotide encodes an amino acid sequence as shown in SEQ ID NO: 73, and a separate polynucleotide encodes an amino acid sequence as shown in SEQ ID NO: 10.

[0239] In another aspect, this disclosure provides vectors (e.g., expression vectors) comprising polynucleotides or polynucleotide groups as described herein. In yet another aspect, this disclosure provides host cells (e.g., expression host cells) comprising polynucleotides or polynucleotide groups as described herein, or vectors.

[0240] The polynucleotides or polynucleotide sequences described herein can be incorporated into (linked to) an expression vector, which can be introduced into host cells using conventional transfection or transformation techniques. Exemplary host cells include *E. coli* cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not additionally produce IgG proteins. Transformed host cells can grow under conditions that allow host cells to express genes encoding the variable regions of immunoglobulin light chains and / or heavy chains.

[0241] Specific expression and purification conditions vary depending on the expression system used. For example, if the gene is to be expressed in *E. coli*, it is first cloned into an expression vector by placing the engineered gene downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signaling sequence. The expressed protein can be secreted. The expressed protein can accumulate in refractive bodies or inclusion bodies, which can be harvested after disrupting the cells by Freund's cell crusher or sonication. The refractive bodies are then dissolved, and the protein can be refolded and / or cleaved using methods known in the art.

[0242] If the engineered gene will be expressed in a eukaryotic host cell, such as a CHO cell, it is first inserted into an expression vector containing a suitable eukaryotic promoter, secretion signal, poly-A sequence, and stop codon. Optionally, the vector or gene construct may contain enhancers and introns. In embodiments involving fusion proteins comprising antibodies or portions thereof, the expression vector may optionally contain a sequence encoding all or part of a constant region, enabling expression of all or part of the heavy or light chain. The gene construct can be introduced into eukaryotic host cells using conventional techniques.

[0243] Host cells can express substances containing V L or V H Fragment, V L -V H Heterodimer, V H -V L or V L -V H A single-chain polypeptide, a recombinant antibody or antigen-binding fragment of a complete heavy or light immunoglobulin chain or a portion thereof. In some embodiments, host cells are transfected with a single vector expressing the antibody or its antigen-binding fragment. Alternatively, host cells may be co-transfected with more than one expression vector (e.g., one expression vector expressing a polypeptide containing all or part of the heavy chain or heavy chain variable region, and another expression vector expressing a polypeptide containing all or part of the light chain or light chain variable region).

[0244] Antibodies or their antigen-binding fragments can be produced by culturing host cells transfected with expression vectors encoding such variable regions under conditions that allow for peptide expression. Expression of immunoglobulin heavy and light chains from a single expression vector or from two separate expression vectors is within the scope of ordinary techniques in the art. After expression, the expressed product, such as protein A, protein G, affinity tags such as glutathione S-transferase (GST), or histidine tags, can be harvested and purified or isolated using techniques known in the art.

[0245] IV. Pharmaceutical Compositions Once generated, antibodies or their antigen-binding fragments can be formulated into pharmaceutical compositions.

[0246] For therapeutic use, antibodies or their antigen-binding fragments are combined with pharmaceutically acceptable carriers. Various carriers (e.g., diluents, excipients, etc.) used to formulate and prepare pharmaceutical compositions are known and / or readily available to those skilled in the art. Depending on the application, carriers may be liquid (e.g., sterile liquid) or solid. Carriers may be selected from or contain water, aqueous solvents, non-aqueous solvents, dispersion media, surfactants, antioxidants, buffers, adjuvants, tensioning agents, stabilizers, fillers, lyophilization protectants, metal ions, chelating agents, isotonic and absorption-delaying agents, etc., compatible with drug administration. The use of such media and reagents for pharmaceutically active substances is known in the art. Typically, carriers are approved by the U.S. Food and Drug Administration and conform to the standards of the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or other international pharmacopoeias. Suitable formulations for use in this disclosure can be found, for example, in Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23). rd (ed. 2020). For a brief review of methods used for drug delivery, see, for example, Langer (1990) Science, 249: 1527-1533. The resulting pharmaceutical composition is suitable for administration to subjects (e.g., animals, mammals, such as humans).

[0247] Pharmaceutical compositions may contain formulation materials used to modify, maintain, or preserve, for example, the composition's pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorption, or permeation. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; and hydrophilic polymers (e.g., polyvinylpyrrolidone). Phenolic ketones); low molecular weight peptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic, polyethylene glycol (PEG), dehydrated sorbitol esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, terpineol); stability enhancers (e.g., sucrose or sorbitol); tensile enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery solvents. vehicle); diluents; excipients and / or pharmaceutical adjuvants (see, for example, Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23)). rd ed. 2020).

[0248] In some embodiments, the pharmaceutical composition may contain a sustained-release or controlled-release formulation. Techniques for formulating sustained-release or controlled-release means, such as liposome carriers, biodegradable microparticles or porous beads, and accumulation injection, are also known to those skilled in the art. Sustained-release formulations may include, for example, porous polymer microparticles or semi-permeable polymer matrices in the form of molded articles, such as membranes or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes prepared by any of several methods known in the art.

[0249] Depending on the circumstances, the pharmaceutical composition may contain nanoparticles or lipid droplets, such as polymer nanoparticles, liposomes or micelles (see Anselmo et al. (2016) Bioeng. Transl. Med., 1: 10-29).

[0250] Pharmaceutical compositions containing antibodies or their antigen-binding fragments may be presented in dose units and may be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration include intravenous (IV), subcutaneous, or intramuscular. Other routes may include intraperitoneal, intradermal, inhalation, percutaneous, local, transmucosal, intrathecal, and rectal administration. In some embodiments, the antibody or antigen-binding fragment is administered subcutaneously.

[0251] Useful formulations can be prepared using methods known in the pharmaceutical field. For example, see Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23) rd (ed. 2020). Components of formulations suitable for parenteral administration include sterile diluents, such as water, saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents for injection; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; buffers, such as acetate, citrate, or phosphate; and agents for adjusting osmotic pressure, such as sodium chloride or dextrose.

[0252] For intravenous administration, suitable carriers include physiological saline, antibacterial water, polyethoxylated castor oil, or phosphate-buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be protected against microbial contamination. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof.

[0253] Pharmaceutical formulations are preferably sterile. The formulation can be sterilized, for example, by methods suitable for maintaining the activity and stability of the antibody or its antigen-binding fragment. Sterilization can be accomplished by any suitable method, such as filtration through a sterile filter membrane. In the case of lyophilizing the composition, filter sterilization can be performed before or after lyophilization and reconstitution.

[0254] Depending on the drug substance and formulation, when the dosage form is liquid or solid, the resulting dosage form can remain stable for extended periods, such as 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, or longer. The formulation may be stable at room temperature or higher. The dosage form is expected to be stable in PBS under ambient conditions. Alternatively, the dosage form may be frozen (e.g., liquid or lyophilized) and stable at appropriate temperatures, such as -20°C or -80°C.

[0255] Depending on the circumstances, the dosage form may be formulated as a unit dose, which may include, for example, about 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1 g, 1.5 g, 2.5 g, 5 g, or 10 g of the drug substance. The dose may be in a suitable liquid carrier, for example, for intravenous, intramuscular, or subcutaneous administration.

[0256] The compositions described herein can be administered topically or systemically. It is contemplated that the compositions described herein are typically administered via parenteral administration. Parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. In some embodiments, the pharmaceutical compositions are administered subcutaneously or may be administered intravenously, for example via intravenous infusion. In some embodiments, the synthetic constructs disclosed herein are contemplated that can be administered systemically.

[0257] Typically, the therapeutically effective amount of the active ingredient, such as the antibody or antigen-binding fragment disclosed herein, is in the range of 0.1 mg / kg to 1000 mg / kg, for example, 1 mg / kg to 100 mg / kg, for example, 10 mg / kg to 500 mg / kg, for example, 500 mg / kg to 1000 mg / kg. In some embodiments, the effective amount is in the range of 15 to 50 mg / kg. In some embodiments, the effective amount is 15 mg / kg. In some embodiments, the effective amount is 30 mg / kg. In some embodiments, the effective amount is 50 mg / kg. The amount administered will depend on variables such as the type and extent of the disease or indication to be treated, the patient's overall health, the in vivo potency of the active ingredient, the pharmaceutical formulation, and the route of administration. The initial dose may be increased above the upper limit level to rapidly achieve the desired blood or tissue levels. Alternatively, the initial dose may be less than the optimal value, and the daily dose may be gradually increased during treatment. For example, in a routine Phase I dose escalation study, the human dose may be optimized. Dosing frequency can vary depending on factors such as route of administration, dosage, serum half-life of the synthetic peptide, and the disease, disorder, or condition being treated. Exemplary dosing frequencies are once daily, once weekly, and once every two weeks.

[0258] V. Treatment methods The antagonistic antibodies disclosed herein have numerous in vitro and in vivo diagnostic and therapeutic uses, relating to the diagnosis and treatment of OX40-mediated disorders. For example, these molecules can be administered to cells or human subjects in vitro or in vitro cultures to treat, prevent, and diagnose a variety of OX40-mediated disorders. Preferred subjects are humans, and include subjects suffering from disorders mediated by OX40 activity (OX40-mediated disorders). The antagonistic antibodies of this disclosure, or antibody-binding fragments thereof, can effectively treat subjects regardless of their OX40 co-stimulatory status. In some embodiments, the subject is a human subject expressing low levels of OX40.

[0259] In certain embodiments, antagonistic antibodies are used to treat, prevent, or diagnose various OX40-mediated disorders. Therefore, this disclosure provides a method for treating OX40-mediated disorders in a subject, the method comprising administering to the subject a therapeutically effective amount of an antagonistic antibody or an antigen-binding fragment thereof.

[0260] In specific embodiments, the OX40 antibody disclosed herein binds to T cells. In some embodiments, the T cells are regulatory T (Treg) cells. In some embodiments, the T cells are Th1, Th2, or Th17 / 22 cells. In some embodiments, the T cells are T memory cells. Depending on the situation, the OX40 antibody can inhibit effector T cells. This inhibition can be silencing or reducing T cell activation, independent of ADCC-mediated T cell killing. Inhibition of the OX40 pathway can silence effector T cells such as Th1, Th2, and Th17 / 22 cells, as well as T memory cells, while having a smaller effect on Treg cells. See, for example, Guttman-Yassky E et al. (2019) J. ALLERGY CLIN. IMMUNOL. Aug;144(2):482-493.e7.

[0261] In some embodiments, the OX40 antibody disclosed herein has reduced adverse effects due to ADCC, such as fever and chills, compared to a control OX40 antibody. For example, the antibody is MAB10.

[0262] Exemplary OX40-mediated disorders include infections (viral, bacterial, fungal, and parasitic), infection-associated endotoxic shock, arthritis, rheumatoid arthritis, psoriatic arthritis, asthma, bronchitis, influenza, respiratory syncytial virus, pneumonia, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), Hashimoto's thyroiditis, cryptogenic fibrosis alveolitis (CFA), idiopathic fibrotic interstitial pneumonia, emphysema, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peroni disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, nodular prurigo, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory disorders of the central and peripheral nervous systems (such as multiple sclerosis), lupus (such as systemic erythema) Lupus or lupus nephritis and Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, urticaria such as chronic spontaneous urticaria (CSU) or chronic inducible urticaria (CIU), Wegener's granulomatosis, systemic sclerosis, dermatosclerosis, chronic pruritus (such as chronic pruritus of unknown cause), vitiligo, Sjögren's syndrome, ankylosing spondylitis, pancreatitis, trauma (surgery), graft-versus-host disease (GVHD), transplant rejection, cardiovascular disease (including ischemic diseases such as myocardial infarction and atherosclerosis), intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypoacidity, neuromyelitis optica, alopecia areata, scleroderma, or hidradenitis suppurativa. These exemplary OX40-mediated barriers can be treated with antibodies or binding fragments thereof that bind to OX40 (e.g., human OX40) as disclosed herein.

[0263] In selected examples, OX40-mediated disorders treated with antibodies or fragments thereof that bind to OX40 (e.g., human OX40) disclosed herein are selected from multiple sclerosis, rheumatoid arthritis, colitis, psoriasis, asthma, COPD, idiopathic pulmonary fibrosis (IPF), graft-versus-host disease (GVHD), atherosclerosis, and diabetes. In other selected examples, OX40-mediated disorders are atopic dermatitis, nodular prurigo, alopecia areata, chronic spontaneous urticaria (CSU) and chronic inducible urticaria (CIU), asthma, hidradenitis suppurativa, lupus nephritis, systemic lupus erythematosus, pemphigus vulgaris, psoriatic arthritis, vasculitis, Hashimoto's thyroiditis, systemic sclerosis, scleroderma, scleroderma, chronic pruritus of unknown cause, ankylosing spondylitis, Sjögren's syndrome, psoriasis, or vitiligo.

[0264] In some instances, the OX40-mediated disorder treated with antibodies is graft-versus-host disease (GVHD). In other instances, the OX40-mediated disorder is atopic dermatitis. In other instances, the OX40-mediated disorder is urticaria, such as chronic spontaneous urticaria (CSU), or chronic inducible urticaria (CIU). In other instances, the OX40-mediated disorder is an autoimmune disorder, such as lupus erythematosus or rheumatoid arthritis. In other instances, the OX40-mediated disorder is asthma. In other instances, the OX40-mediated disorder is alopecia areata, scleroderma, or hidradenitis suppurativa.

[0265] This disclosure also provides antibodies for treating pain, particularly pain associated with inflammation.

[0266] Furthermore, antibodies can be used to detect OX40 levels, or the levels of OX40 present on cell surfaces, which may be associated with certain disease symptoms. Alternatively, antibodies can be used to inhibit or block OX40 function, thereby being associated with the prevention or improvement of certain disease symptoms, suggesting that OX40 acts as a disease mediator. This can be achieved by contacting sample and control samples with OX40 antibodies under conditions that allow for the formation of complexes between the antibody and OX40. Any complexes formed between the antibody and OX40 are detected and compared in the sample and control. Given the specific binding of antibodies against OX40, antibodies can be used to specifically detect OX40 expression on cell surfaces, for example, to detect subjects with low levels of OX40 expression.

[0267] This disclosure also provides the use of antagonistic antibodies or fragments thereof as medicaments and their use in the preparation of medicaments for treating OX40-mediated disorders. Antagonistic antibodies or antigen-binding fragments thereof are also provided for methods of treating OX40-mediated disorders, such as those described herein.

[0268] Depending on the circumstances, the antagonistic antibodies disclosed herein can be specifically used to treat OX40-mediated disorders, regardless of the subject's OX40 co-stimulatory status. Furthermore, the antagonistic antibodies or fragments thereof can be used to treat OX40-mediated disorders in subjects expressing low levels of OX40.

[0269] Furthermore, as disclosed herein, antibodies or binding fragments thereof that bind to OX40 (e.g., human OX40) can be administered to subjects who require reduction or inhibition of T cell proliferation. A therapeutically effective amount of the antibody or its binding fragment can be administered to reduce or inhibit T cell proliferation in the subject. The subject may suffer from OX40-mediated disorders as described above.

[0270] Example The subject matter will now be described with reference to the following embodiments. These embodiments are provided for illustrative purposes only, and the claims should not be construed as limiting to these embodiments, but rather as covering any and all variations that become apparent from the teachings provided herein.

[0271] The following are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0272] Example 1: Antibody generation through affinity maturation Materials and methods Generation of Affinity Mature Library The library was designed in scFv form, with diversity limited to CDR-3 of the variable light chain of GBR 830 (terazolizumab, described in U.S. Patent 8,748,585) (SEQ ID NO: 37). CDR diversification was achieved by synthesizing random sequences using a mixture of nucleotides. All 20 amino acids, plus the amber stop codon (TAG), were encoded by the NNK degenerate codon (where N is any of four deoxyribonucleotides and K is G or T). Flexible linkers based on three Gly4Ser peptide repeats were used to assemble the two variable domains. The resulting library contained the GBR 830 variable domain with CDR-L3 bearing the sequence CQXXXXPWTF (Kabat residues 88-98, SEQ ID NO: 71), where X is a randomly occurring naturally occurring amino acid (encoded by the NNK degenerate codon). Diverse scFv fragments were cloned into pGLEN8 (a modified pHEN1 phage vector (Hoogenboom (1991) Nucleic Acids Res., 19(15): 4133-7)), and the resulting ligation reaction was electroporated into *E. coli* XL-1 blue cells. Transformed cells were plated on 2YT-100 μg / mL ampicillin-5% glucose (2YTAG) plates and incubated overnight at 30°C (O / N). Colonies were scraped into 10 mL of 2YT medium, and 15% glycerol (final concentration) was added for storage at -80°C. The diluted transformant cells were plated on 2YTAG plates and incubated overnight at 37°C for titration. The final scFv fragment library reached 6.7 x 10⁻⁶. 6 Total diversity. After replacing the medium with 2YT medium supplemented with ampicillin (100 μg / mL) and kanamycin (25 μg / mL), the samples were tested at 37°C with >10 10Plaque-forming unit (PFU) helper phage (M13K07) was used to infect cells for 30 min, followed by cell culture at 30°C O / N to produce phage. Phage purification was achieved through a two-precipitation step, each time with one-third v / v of 20% PEG-6000 and 2.5 M NaCl. After precipitation, the phage was resuspended in phosphate-buffered saline (PBS).

[0273] Recombinant antigen Recombinant antigens were expressed and purified at Glend Pharmaceuticals SA. Human and cynomolgus monkey OX40 ECDs were expressed either with a polyhistidine sequence fused to their C-terminus or as an Fc fusion (human IgG1 isotype). Library selection was performed on cynomolgus monkey OX40-ECD-Fc and human OX40-ECD-his. Kinetic screening was performed on human OX40-ECD-his and cynomolgus monkey OX40-ECD-his. KD measurements of cynomolgus monkey OX40 were performed using cynomolgus monkey OX40-ECD-his protein. Human OX40 protein, containing only the cysteine-rich (CRD) region of the ECD fused to the avi tag and a polyhistidine sequence at the C-terminus, was found to be more homogeneous and monomeric than human OX40-ECD-his and was used for KD measurements.

[0274] Document Selection Nunc MaxiSorp 5 ml immunoassay tubes were coated with 80 μg of cynomolgus monkey OX40-ECD-Fc dissolved in 4 mL PBS. The purified phage particles (10 μg / mL) were blocked with PBS containing 3% (w / v) skim milk (3% MPBS). 10Plaque-forming units and immunotubes were prepared. The blocked phage was then added to the blocked immunotubes and incubated at room temperature (RT) for 2 hours. The supernatant and unbound phage were discarded, and the immunotubes were washed 20 times with PBS containing 0.1% (v / v) Tween 20 (PBS-Tween 0.1%), followed by 20 washes with PBS. The phage was eluted with 100 mM triethylamine at RT for 10 min and neutralized with 1 M Tris-HCl pH 8 (10% v / v). The eluted phage was used to infect 10 mL of exponentially growing *E. coli* XL-1 blue cells. The infected cells were grown in 2YT medium at 37°C for 30 min, then plated on 2YTAG (2TY medium supplemented with 100 μg / mL ampicillin and 5% glucose) agar plates and incubated overnight at 30°C. Colonies were scraped into 10 mL of 2YT medium and 15% glycerol (v / v) was added for storage at -80°C. XL-1 cells retrieved from the glycerol stock were grown in 2YTAG medium at 37°C and 240 RPM until the OD at 600 nm reached 0.5. Cells were then superinfected with M13K07 helper phage at 37°C for 30 min using a multiplicity of infection (MOI) of 10. The medium was then replaced with 2YTAK medium (2YT medium supplemented with 100 μg / mL ampicillin and 25 μg / mL kanamycin), and the cells were cultured overnight at 30°C and 280 RPM. The next day, phages were precipitated from the cell supernatant using a two-stage precipitation process, each time with one-third v / v of 20% PEG-6000, 2.5 M NaCl, and resuspended in 1.5 mL of PBS. A second round of selection was performed using 1.4 mL of phage recovered from the first round. The same experimental setup was used, except that the coating of the immunosorbent tubes was changed from cynomolgus monkey OX40-ECD-Fc to 80 μg human OX40-ECD.

[0275] ScFv clone screening Binding of scFv clones was assessed by ELISA. Single *E. coli* colonies selected in the second round of selection were picked and cultured in 2-YT ampicillin medium (96-well deep-well plates). ScFv expression was induced by the addition of 1 mM IPTG and incubated at 30°C and 250 RPM for 1 hour. Cells were centrifuged and the culture supernatant was used for screening. RIA / EIA plates were coated with 200 ng / well of human OX40-ECD-his or cynomolgus monkey OX40-ECD-Fc. To distinguish antibodies that might be screened for the Fc fragment, the plates were also coated with unrelated Fc fusion antigens. The plates were then blocked with PBS supplemented with 3% bovine serum albumin (BSA). 80 μL of bacterial culture supernatant was then mixed with 20 μL of 3% BSA and added to the blocking plates at room temperature for 1 hour. The binding of scFv was detected by incubation at RT for 1 h with biotinylated chicken anti-c-myc antibody diluted 1:5000 in PBS-0.3% BSA, followed by staining at RT for 30 min with Pierce high-sensitivity Streptavidin-HRP reagent diluted 1:8000 in PBS-0.3% BSA. The plates were thoroughly washed with PBS supplemented with 0.05% Tween 20 between each incubation step. Finally, the bound scFv was visualized by EIA with 100 μL / well TMB peroxidase at RT for 5 min. Enzyme activity was stopped by adding 50 μL of 2N H2SO4, and OD was measured at 405 nm. Clones that specifically bound both human and cynomolgus monkey OX40 but not unrelated antigen-Fc fusion proteins were sequenced.

[0276] Expression and purification of recombinant antibody fragments cDNAs encoding different antibody constant regions were synthesized by GENEART AG (Regensburg, Germany) and modified using standard molecular biology techniques. PCR products were digested with appropriate DNA restriction enzymes, purified, and ligated into a modified pcDNA3.1 plasmid (Invitrogen) carrying the CMV promoter and bovine growth hormone polyadenylation signal sequence. This expression vector also carried oriP (which is the plasmid origin of replication for EBV) and a mouse VJ2C leader peptide encoding a secretion-encoded polypeptide chain. To reconstruct the cDNA in scFv-Fc form, each scFv cDNA was amplified from its phage library vector by PCR and cloned upstream of the modified pcDNA3.1 vector encoding the small four Gly adapter sequences and the human IgG1 Fc region (human IgG1 hinge, CH2, and CH3 domains).

[0277] To reconstruct the scFv library clones into human IgG1, each scFv clone in the phage library vector was used to amplify its respective VH and VL cDNAs by PCR. The VH PCR product was then cloned upstream of the cDNA encoding the human IgG1 heavy chain (CH1, hinge, CH2, and CH3 domains) in the modified pcDNA 3.1 vector, while the VL PCR product was cloned upstream of the cDNA encoding the human κ constant light chain domain in the modified pcDNA 3.1 vector.

[0278] For transient expression, the recombinant scFv-Fc vector was directly transfected to express the scFv-Fc fusion protein. For antibody expression, equal volumes of the heavy and light chain vectors were co-transfected into suspension-fitted HEK293-EBNA cells using PEI. Cells were typically prepared at 8 million cells per ml in RPMI supplemented with 0.1% Pluronic F-68. Cells were then transfected with a DNA-PEI mixture. Four hours post-transfection, cell cultures were diluted 1:1 in EX-CELL® 293 supplemented with phenol red and 4 mM L-glutamine and incubated for 5 days at 37°C, 5% CO2, and 80% humidity with orbital shaking. Cell-free culture supernatant containing the recombinant protein was prepared by centrifugation and filtration for further purification. CaptivA was used. TM PriMAB resin (rproteinA affinity resin) was used to purify scFv-Fc fusion protein and antibodies. Affinity resin was added to each filtered culture supernatant, and the mixture was gently mixed and incubated overnight at 4°C. The next day, the resin beads were collected into a Poly-Prep column, washed with PBS, and the recombinant protein was eluted with acidic buffer (typically 0.1 M glycine, pH 3). After neutralization with 1 / 10 volume of 1M Tris-HCl (pH 8), the formulation buffer was exchanged for PBS.

[0279] Kinetic screening Binding kinetics were screened using a Biacore T200 instrument at room temperature. A sensor chip (series S CM5) previously coupled with a capture antibody (human antibody capture kit) was used to capture approximately 500 response units (RU) of the test protein (scFv-Fc or antibody), which had been previously diluted to a final concentration of 50 nM in HBS-EP+ buffer (run buffer) at 10 μL / min on flow cell (fc) No. 2. Human OX40-ECD-his or cynomolgus monkey OX40-ECD-his, previously diluted to 1 μM, was injected into fc No. 1 and fc No. 2 (fc No. 1 used as a reference) for 3 min, followed by a 10 min dissociation period in run buffer at a flow rate of 30 μL / min. Regeneration was performed after each cycle using a 1 min injection of 3 M MgCl2. Raw data were analyzed in Biacore evaluation software (v3.0) after the run. The binding curves, after subtracting the blank and reference values, are normalized at the binding event time points, and the decoupling rates are compared.

[0280] Measurement of the affinity of MAB1 IgG1 for human and cynomolgus monkey OX40 Surface plasmon resonance (SPR) analysis was used to measure the binding and dissociation rate constants of the binding kinetics of MAB1 IgG1 and GBR 830. Binding kinetics were measured at RT using a Biacore T200 instrument and analyzed using Biacore T200 evaluation software (v3.0). Analytical data were exported using Biacore T200 kinetic summary software (v3.0). An S-series sensor chip previously conjugated with an anti-human capture antibody (human antibody capture kit) was used to capture 200 RU of MAB1 antibody or GBR 830 on fc No2, both previously diluted to a final concentration of 5 μg / ml in HBS-EP+ buffer (run buffer). Human OX40-CRD-avi-his or cynomolgus monkey OX40-ECD-his, previously diluted in HBS-EP+ buffer, were injected into fc No1 and fc No2 (fc1 was used as a reference); a three-fold dilution series of human OX40-CRD-avi-his and cynomolgus monkey OX40-ECD-his from 1000 nM to 1.37 nM, along with a blank control (0 nM), were flowed through captured GBR 830 or MAB1 IgG. Experimental data were processed using a 1:1 Langmuir model with global Rmax.

[0281] result Antibody 830 (GBR 830) exhibited affinity maturation via phage assay. Nine clones were generated and characterized. The affinity maturation process focused on CDR-L3 diversification. One clone, MAB1, was selected for further characterization. The MAB1 amino acid sequence differs from the GBR 830 antibody by four amino acids in its variable light chain domain, CDR3. The GBR 830 LCDR3 sequence is... (SEQ ID NO: 35), while the MAB1 LCDR3 sequence is (SEQ ID NO: 6).

[0282] ScFv clone screening The supernatants of 88 clones were screened by ELISA, and 33 clones showed specific binding to both human and cynomolgus monkey OX40, but not to unrelated antigen-Fc fusion proteins. Of these 33 clones, 21 had unique CDR-L3 amino acid sequences. The unique clones with the highest binding signal were reconstructed as scFv-Fc fusion proteins for further evaluation by SPR. The clones were randomly named MAB1 to MAB9. The variable light chain, light chain, and L3 CDR sequences of MAB1 to MAB9 are provided in SEQ ID NOs: 6, 8, and 10-34.

[0283] Kinetic screening Following the above procedure, MAB1 to MAB9 scFv clones were reconstructed into scFv-Fc fusion proteins. Binding kinetics were assessed using SPR (Synchronization Process). Figure 1 Only one clone, MAB1, showed a significantly slower dissociation rate in human OX40 than its parental clone, GBR 830. Figure 1 ), and showed a slightly better or comparable dissociation rate for cynomolgus monkey OX40 (data not shown).

[0284] Measurement of the affinity of MAB1 IgG1 for human and cynomolgus monkey OX40 The binding affinity of GBR 830 and MAB1 to human and cynomolgus monkey OX40 ECDs was measured using a 1:1 Langmuir binding model. This model assumes a 1:1 interaction between the ligand and the analyte and determines the binding rate (Ka), dissociation rate (Kd), and affinity (KD) for each sample. Using this model, KD is defined as the ratio between the dissociation rate and the binding rate, i.e., KD = Kd / Ka.

[0285] In addition to the KD value, the relative affinity of MAB1 to the parental clone GBR 830 is determined using the following formula: Relative affinity = (KD value) / (KD value) GBR 830 / KD MAB1 ).

[0286] The affinities of MAB1 to human OX40-CRD-avi-his and cynomolgus monkey OX40-ECD-his were measured at 6 nM and 557 nM, respectively; while the affinities of GBR 830 to these two antigens were measured using the same experimental setup at 80 nM and 3 μM (Table 9).

[0287] Table 9. Summary of MAB1 IgG1 characterization

[0288] For both humans and cynomolgus monkeys, the affinity of GBR 830 for the extracellular domain (ECD) of OX40 was measured at 100–80 nM and 5–3 μM, respectively. MAB1 showed an approximately 13-fold improvement in affinity for human OX40, with a mean KD value of 6 nM. MAB1 also showed an approximately 5-fold improvement in affinity for cynomolgus monkey OX40, with a mean KD value of 0.55 μM.

[0289] Example 2: In vitro characterization of MAB1 antagonistic effect Surface plasmon resonance (SPR) studies showed that GBR 830 had a monovalent affinity for OX40 of 80–100 nM, while cell-based epigenetic affinity was measured in the lower nM range: 1.25 ± 0.41 nM on CD4+ T cells and 3.46 ± 0.47 nM on CD8+ T cells. GBR 830 also showed a lack of OX40 agonist activity. One hypothesis for this lack of agonist activity is that its monovalent affinity limits the cross-linking power required for agonist activity. The affinity-matured variant MAB1 showed a 13-fold higher monovalent affinity for OX40 than GBR830. Therefore, affinity-matured antibodies based on GBR 830 could potentially lead to increased OX40 agonist activity, resulting in a trade-off between better antibody binding and increased agonist activity. To assess the potential for increased agonist activity due to increased affinity, the MAB1 antibody was tested in an agonist assay, as described below.

[0290] Materials and methods Epitope localization via ELISA Epitope localization was performed using a binding ELISA. To verify epitope conservation via MAB1, MAB1 binding to the human-rat OX40 CRD chimeric protein was tested by ELISA. Briefly, for example, RHHH indicates OX40 where CRD1 is rat-derived and CRD2, 3, and 4 are human-derived; RHRR corresponds to OX40 where CRD1, 3, and 4 are rat-derived and CRD2 is human-derived; HHHR corresponds to OX40 where CRD1, 2, and 3 are human-derived and CRD4 is rat-derived; HRRR corresponds to OX40 where CRD1 is human-derived and CRD2, 3, and 4 are rat-derived; and HHRH corresponds to OX40 where CRD1, 2, and 4 are human-derived and CRD3 is rat-derived. The human-rat chimeric OX40 extracellular domain protein was coated in DPBS at 2 μg / mL. Serial dilutions (1 / 3) of GBR 830, MAB1, and 7H11 antibodies, starting at 10 μg / mL, were added to OX40 coated plates. HRP-labeled goat anti-human Fab antibody (Jackson Immuno Research) was used as the detection antibody at a 1:2,000 dilution. Plates were blocked and washed with ELISA blocking buffer and ELISA washing buffer, respectively.

[0291] The data (absorbance units) were then plotted against antibody concentration and analyzed using Prism (GraphPad) software.

[0292] Blocking FACS The comparison of cross-blocking between GBR 830 and MAB1 was performed on HPB-ALL. HPB-ALL cells were incubated on ice and in the dark for 30 min with serially diluted cold GBR 830 or MAB1 (starting at 10 μg / mL and diluted 1 / 3). After a washing step (two rounds of centrifugation, with resuspending in flow cytometry buffer in between), cells were resuspended with a single dose of labeled MAB1 (for GBR 830 dose-response, and for MAB1 dose-response, with labeled GBR 830). Plates were incubated on ice and in the dark for 30 min. After the washing step, cells were resuspended in 200 μL of FACS buffer and immediately harvested by flow cytometry.

[0293] The data were then plotted relative to antibody concentration (geometric mean of fluorescence intensity) and analyzed using Prism (GraphPad) software. A nonlinear regression fit was applied after the X=Log(X) transformation, and a sigmoid dose-response fit was applied to all datasets to determine the EC50 values. Data are presented as mean ± SD in all plots.

[0294] Jurkat-OX40-NFκB signal transduction assay The assay is performed using a soluble reagent or a coated test antibody.

[0295] Procedure using antibody as a soluble reagent: A sterile 96-well Chimney-well μCLEAR microplate was coated overnight with OKT3 (5 μg / ml). Control wells without OKT3 coating were included. Jurkat-NFκB cells were loaded at 5 x 10⁻⁶ cells / well. 4 Cells were plated in 50 μL wells on pre-coated OKT3 plates. Serially diluted test antibody or OX40L was added to the cells, and the plates were incubated in a cell culture incubator at 37°C for 5 hours. After incubation, 75 μL of Bio-Glo solution (Promega) was added to the wells, and luminescence was measured using a Synergy Neo microplate reader (Biotek) with the following settings: readout type - endpoint; integration time - 1 min; emission - well; optical position - top; gain 135; readout height - 1.00 mm. The data (luminescence units) were then plotted against antibody concentration and analyzed using Prism (GraphPad) software. Nonlinear regression fitting was applied after X = Log(X) transformation, and sigmoid dose-response fitting was applied to all datasets to determine EC50 values. Each condition was repeated twice.

[0296] Antibody coating procedure: Sterile 96-well Chimney plates were coated overnight with a mixture of OKT3 (5 μg / mL) or OKT3 (5 μg / mL) and anti-OX40 antibody (10 μg / mL serially diluted 1 / 4). Control wells without antibody coating were included. Jurkat-NFκB cells were loaded at 5 x 10⁻⁶ cells / well. 4 Cells were plated in 50 μL wells on antibody-pre-coated plates and incubated at 37°C for 5 hours in a cell culture incubator. The readout procedure was the same as described above.

[0297] T-cell agonist assay Use EasySep according to the manufacturer's instructions. TM The Human T Cell Isolation Kit (StemCell Technologies) purifies human T cells from PBMCs.

[0298] Flow cytometry staining was performed on aliquots of freshly purified T cells to examine T cell purity. Purity was consistently above 95%.

[0299] A mouse anti-human CD3 (OKT3) solution was prepared at 4 μg / mL and distributed into deep-well plates. Other treatments (OX40L, anti-OX40 antibody, IgG control) were diluted at 5 or 10 μg / mL in the corresponding wells of the OKT3 solution to prepare co-coating solutions. 100 μL of each treatment solution was transferred to a 96-well plate and incubated overnight at 4°C.

[0300] Purified T cells (10 5 T cells / well were added to the pre-coated plate. The plate was incubated at 37°C for 3 days, and 0.5 μCu / well was added. 3 H-thymidine was added to the wells. The plates were incubated for another 15–24 hours. The plates were harvested on a Filtermat A filter using a Filtermate 196 collector, and radioactivity was counted using a MicroBeta Trilux counter (Wallac). The proliferation index was calculated using the following formula: Proliferation Index =

[0301] CPM stands for counts of radioactivity per minute.

[0302] Then, data (proliferation index) was plotted for the antibodies (classification) and analyzed using JMP (SAS) software.

[0303] Statistical analysis Statistical analysis of T cell proliferation assay data was performed using JMP software (SAS Institute Inc). Donor-matching analysis was performed to account for donor variability.

[0304] Statistical analysis was performed to compare the effects of various treatments relative to the IgG1 control condition. For each T cell donor, the difference in proliferation index (PI) [treatment X - Hu IgG1 control] was calculated, where treatment X represents the test condition (OX40L, anti-OX40 antibody, anti-CD28 antibody). For each treatment, a one-tailed paired Student's t-test was used to analyze the PI difference against the null hypothesis (H0): the mean of the PI differences across all tested donors is equal to 0. If p < 0.05, H0 was rejected (therefore, the test treatment was considered significantly different from the control condition).

[0305] result GBR 830 and MAB1 bind to OX40 receptors MAB1 is a variant of GBR 830 with a more than 10-fold enhanced affinity for OX40. The affinity of MAB1 (6 nM) is very close to that of humanized 7H11 (5.4 nM), and it exhibits agonistic activity.

[0306] The GBR 830 epitope has previously been characterized as residing within the cysteine-rich domain (CRD)2 of OX40 (see, for example, U.S. Patent 8,748,585). Figure 2 provides direct binding ELISA data using the indicated human / rat chimeric OX40 construct. 7H11 binds to HRRR, HHRH, and HHHR proteins, but not RHRR. Therefore, the 7H11 antibody is a CRD1 domain-specific binder. GBR 830 and MAB1 bind to RHRR, HHHR, and HHRH proteins, but not HRRR. Therefore, GBR 830 and MAB1 are CRD2 domain-specific binders. The isotype control antibody does not bind to any recombinant chimeric protein. Therefore, MAB1 binds to the same CRD2 epitope as GBR 830. Therefore, affinity maturation does not alter the OX40 epitope on CDR2.

[0307] The cross-blocking ability of GBR 830 and MAB1 was also tested using flow cytometry staining on HPB-ALL cell lines expressing OX40. When the concentration of the cold antibody (MAB1) increased, binding of labeled GBR830 (GBR830-AF647) was blocked by MAB1 in a dose-dependent manner, as indicated by decreased geoMFI. Similarly, binding of labeled MAB1 (MAB1-AF647) was partially blocked by GBR 830. Figure 3 This partial blockade may be due to GBR 830 displacing MAB1-AF647 on the cell due to its higher affinity. This result further indicates that GBR 830 and MAB1 share the same epitopes, and that MAB1 exhibits enhanced binding to OX40-expressing cells compared to GBR 830.

[0308] Assay for T-cell activating activity using coated antibodies Purified T cells from different donors were incubated in cell culture plates pre-coated with anti-CD3 (OKT3, to induce OX40 expression on T cells) and anti-OX40 or control antibodies. Agonism was assessed by T cell proliferation as detected by incorporation of 3H-thymidine. A proliferation index (PI) greater than 2 between the OX40 antibody plus OKT3 condition and the OKT3-only condition was considered to reflect agonism, as this threshold effectively distinguished between positive and negative IgG1 controls. PIs greater than 2 have previously been used to characterize the agonism of other TNFR-targeting antibodies (Ralph, Panzenbeck et al., 2016).

[0309] OX40L and 7H11 were used as agonist positive controls. Other OX40 antagonists also included are A26 (UCB Pharma), A10 (Genentech), and 112V8 (corresponding to the sequence of the nocarotelimab antibody (also known as KHK4083 or AMG451) Kirin Pharma). Also included is the OX40 agonist antibody MEDI0562 (humanized 9B12, tavolixizumab, Medimmune).

[0310] Donors were excluded from the analysis if a sample showed agonistic activity with the IgG1 control antibody (PI>2) or no agonistic activity with either the positive controls OX40L and 7H11. Donors were included in the final analysis if a positive control showed agonistic activity. A total of 5 out of 23 donors were excluded. Therefore, 18 donors were included in the final analysis.

[0311] Table 10 summarizes the proportion of responders and provides the mean PI. The data show that the positive controls OX40L and 7H11, as well as the OX40 agonist antibody MEDI0562 (humanized 9B12), exhibited significantly better mean PIs than the IgG1 control antibody (p<0.05). In contrast, the mean PIs of GBR 830 and MAB1 were not different from the controls, indicating that neither antibody induced OX40 activation and therefore did not show detectable agonist activity.

[0312] Table 10. Summary of agonistic effects in coated antibody T cell assays

[0313] Note: Av is the mean stimulation index calculated for all donors included in the final analysis. SD is the standard deviation. Resp indicates the proportion of T cell donors showing SI>2 out of the total included donors for each antibody. P represents the p-value of a paired Student's t-test comparing each treatment to a huIgG1 control. NA: Not applicable; no paired IgG control data available.

[0314] Positive control antibodies OX40L and 7H11 showed agonistic activity as defined by PI>2 in most T cell donors (11 and 17 out of 18, respectively). The OX40 agonist antibody MEDI0562 (humanized 9B12) also showed agonistic activity in 9 out of 11 donors. Interestingly, antibodies A26, described as OX40 antagonists, as well as 112V8 and A10, also showed agonistic activity in a significant proportion of T cell donors. Similarly, two antibodies, Ab 315 and Ab 131 (Imura, Hori et al. 1996), described as blocking OX40L-mediated adhesion of T cells, also showed agonistic activity. In contrast, GBR 830 showed no indication of agonistic activity in any of the 18 T cell donor populations tested. Importantly, MAB1 also showed no agonistic activity in any of the 13 donor cell populations tested.

[0315] Figure 7 The final analysis of all tested antibodies includes proliferation index data for all donors. A horizontal line above zero indicates the threshold for agonistic activity (PI>2). As shown in the figure, MAB1 is below the threshold for agonistic activity.

[0316] Antibodies as T-cell agonists, with or without Fc cross-linking, soluble reagents Assays using OX40 antibody as a soluble reagent were performed in parallel with the coated antibody form. Briefly, purified T cells from six donors were incubated in cell culture plates pre-coated with anti-CD3. OX40 or control antibody was added as a soluble reagent (at 10, 1, and 0.1 μg / mL). Under some conditions, anti-human IgG Fc fragment-specific antibody was added at 5 μg / mL. Activation was assessed by calculating the PI using the coating assay.

[0317] When added as a soluble reagent, no antibodies showed detectable agonistic activity except for the positive control anti-CD28 antibody, which exhibited strong agonistic activity. Figure 8 All six donors showed agonistic activity against the positive control 7H11 in the form of a coated antibody and the OX40 agonist MEDI0562 (humanized 9B12).

[0318] When anti-human IgG-Fc specific cross-linking antibody was added to the soluble antibody, the results were similar. Figure 9 MAB1 did not show agonistic activity in the six donors tested in this experiment.

[0319] Characterization of agonistic activity in Jurkat-NFκB assay The agonistic effect of OX40 antibodies was tested using a Jurkat-OX40-NFκB signaling assay. Jurkat-NFκB cells respond to OX40 signaling by producing luciferase. Cellular responses to OX40L and GBR 830 consistently inhibited OX40L-mediated luciferase production (data not shown). This type of assay is sensitive to the detection of agonistic effects of OX40 antibodies and other TNFR antibodies (see, for example, Voo et al. (2013) J. Immunol., 191(7): 3641-3650; Zhang et al. (2016) J. Biol. Chem., 291(53): 27134-27146; Ralph et al. (2016) J. Immunol., 196 (Suppl1): 70.19-70.19).

[0320] Controlling the binding of the antibody used in this experiment to Jurkat-NFκB cells ( Figure 4 ).like Figure 4 As shown, all antibodies except for the anti-CD28 and IgG1 controls bound to Jurkat-NFκB cells.

[0321] To make the system more sensitive, Jurkat-NFκB cells were stimulated with coated anti-CD3 during the assay. When no CD3 stimulation was applied, all signals except OX40L were very weak (data not shown). Both OX40L and anti-CD28 triggered strong signals. Figure 5A , 23A 23B). In contrast, GBR 830, MAB1, and MAB10 triggered signals similar to the IgG control even at the highest concentrations. Figure 5B , 23A 23B). MAB10 does not induce agonistic activity in Jurkat-NFcations B-OX40 cells, therefore it does not induce agonistic activity in the OX40-OX-40L co-stimulatory pathway. Figure 23A and 23B Similar results were obtained for GBR830 and MAB1, while IMG-007 induced luminescence 3-fold higher than the untreated control. OX40L, used as a positive control, induced luminescence 8-fold higher than the untreated control (untreated OKT3-coated). IgG1 was used as a negative control. Both the MEDI0562 (humanized 9B12) OX40 agonist and the 7H11 antibody, which showed consistent agonistic activity in T-cell-based proliferation assays, triggered signals more than twice that of the IgG control. Figure 5A Similarly, the A26 antibody also triggered a signal at least 2 times higher than the background, thus indicating T cell activating activity.

[0322] The assay was also performed using coated antibodies. Signaling induced by the positive control OX40L was weaker but still present. However, similar to soluble conditions, all antibodies except GBR 830 and MAB1 triggered signals higher than the IgG1 control (Figure 6). Therefore, GBR 830 and MAB1 did not induce OX40 signaling in assays using coated antibodies, compared to other OX40 antibodies tested.

[0323] These data show that in the Jurkat-OX40-NFκB signal transduction assay, GBR 830, affinity-matured MAB1, and Fc-modified MAB10 antibodies, when used in solution or as coating agents, cannot induce signal transduction via OX40.

[0324] In summary, in human T-cell-based assays using coated antibodies, GBR 830 and MAB1 showed no OX40 signaling (agonistic activity) in a total of 18 and 13 human T-cell donors, respectively. Therefore, the affinity-matured MAB1 antibody showed a significant increase in affinity compared to the parental clone GBR 830 in vitro, with no detectable agonistic activity. In contrast, the positive control humanized 7H11 antibody showed agonistic activity in 17 of the 18 human T-cell donors. Similarly, the OX40 agonist antibody MEDI0562 (humanized 9B12) showed agonistic activity in 9 of the 11 donors. Anti-OX40 antibodies described as antagonists, such as A26, A10, or 112V8 (KHK4083 / AMG451), also revealed agonistic activity with a significant proportion of human T-cell donors. Therefore, coated antibody assays can detect the agonistic activity of OX40L and 7H11 anti-OX40 antibodies, as well as other anti-OX40 antibodies.

[0325] In Jurkat-based signal transduction assays, GBR 830, MAB1, and MAB10 did not induce significant signals, while control antibodies 7H11 and MEDI0562 induced signals at least twice that of the IgG control. In this assay, the A26 "antagonist" antibody also revealed agonistic effects. Figure 23A As shown in Figure B, the anti-OX40 antibody IMG-007 also induces agonistic activity in Jurkat cells.

[0326] These data indicate that the more than 10-fold increase in affinity of the MAB1 antibody compared to the parental GBR 830 antibody does not lead to an increase in the agonist activity of OX40 signaling. Therefore, there is no observable trade-off between the MAB1 and MAB10 antibodies in terms of better antibody binding and increased agonist activity. The increased affinity and lack of detectable agonist activity in vitro are unique and unexpected properties of MAB1 and MAB10 anti-OX40 antibodies.

[0327] Example 3: In vitro characterization of the pharmacological properties of MAB1 Materials and methods PBMC separation Human peripheral blood mononuclear cells (PBMCs) were collected from apheresis blood component filters using Ficoll gradient separation. Briefly, the blood was diluted 1 / 2 with PBS, and the cell suspension was layered in 50 ml Sepmate tubes (StemmCell Technologies) pre-filled with Ficoll. After centrifugation at 1200 g for 10 min with the brake engaged, the supernatant containing the mononuclear cells was collected and transferred to the tube. The PBMCs were washed three times with PBS by centrifugation at 350 g for 10 min and used immediately or frozen in CryoStor CS10 medium.

[0328] Activated T cell proliferation T cell isolation and purity assessment were performed according to Miltenyi's instructions for the Human Pan T Cell Isolation Kit. In short, human PBMCs were counted and resuspended in PBS + 0.5% BSA + 2 mM EDTA (40 μL / 100ml). 7 In a separation buffer containing (total cells). Add 10 μL of Pan T cell biotin-antibody Cocktail / 10 7 The total cells were mixed with the cell suspension and incubated at 4°C for 5 minutes. After incubation, 30 μL of separation buffer was added (per 10 cells). 7 (Total cells), and added 20 μL of Pan T cell microbeads Crocktail. The cells were incubated at 4°C for 10 min and then added to an LS column that had previously been washed with 3 mL of separation buffer. After further washing the column with 3 mL of separation buffer, all flow-through containing the purified enriched T cell population was collected and used for subsequent assays.

[0329] Human CD3+ T cell purity was assessed by flow cytometry. In short, 100,000 isolated CD3+ T cells were seeded into 96-well U-bottom cells and washed once. The cells were then resuspended in flow cytometry buffer containing the following anti-human antibodies in the presence of Fc blocking: CD3 APC-eF780 (clone SK7), CD4 PE-Cy7 (clone OKT4), CD8 AF700 (clone SK-1), CD33 PE-Cy5 (clone HIM3-4), CD56 PE-eF610 (clone CMSSB), CD19 AF488 (HIB19), CD45RA APC (clone HI100), and CCR7 PE (clone 3D12). After 20 min in the refrigerator, cells were washed with flow cytometry buffer and resuspended in DAPI solution. Cells were then obtained using a Cytoflex-S (Coulter) instrument with the following gating strategy: CD45, CD56, CD3, CD4, and CD8-specific antibodies were used to assess single live cells expressing CD45, CD3, CD4, and CD8 by flow cytometry. The fraction of CD3+CD56- T cells and the relative fractions of CD4 and CD8 T cells were quantified in single live cells. Proliferation assays were performed using only cell isolates with more than 90% CD3+ T cells.

[0330] T cell activation was initiated on the day of T cell isolation. Six-well plates were pre-coated with anti-human CD3 Ab OKT3 (4 μg / mL in PBS) at 4°C. The next day, the wells were washed with sterile PBS, and the isolated T cells were incubated at 5 x 10⁻⁶ ppm in complete RPMI medium supplemented with sCD28 (1 μg / mL). 6 Cells were seeded at a density of 1 / mL in OKT3-coated wells. The cells were then incubated at 37°C for 30 hours.

[0331] Assessment of OX40 expression in activated T cells and differentiated Treg populations Prior to ADCC assays using activated T cells, the surface expression of OX40 and other activation markers in the cells were tested. Briefly, 100,000 activated T cells were collected, washed once, and then resuspended in 50 μL of flow cytometry buffer containing the following anti-human antibodies: CD3 APC-eF780, CD4 PE-Cy7, CD8 BV421, CD25 AF700, CD69SB600, CD107a AF488, and CD134 PE. After incubation at 4°C for 20 min, the cells were washed once, resuspended in Sytox Blue solution, and obtained using a Cytoflex-S cytometer.

[0332] Proliferation assay On the day of T cell activation, 96-well plates were pre-coated with OX40L (5 μg / mL in PBS) at 4°C for 36 hours. After incubating T cells with OKT3 and sCD28 for 30 hours, activated T cells were recovered and cultured in 5 x 10⁻⁶ wells. 6 Cells were seeded in 96-well U-bottom plates, each well containing 200 μL of complete RPMI and serially diluted GBR 830, MAB1, KHK4083, and control Ab (starting at 100 ug / mL, 8 1 / 5 dilutions), and incubated at 37°C for 1 hour. Cells were then transferred to OX40L coated plates and incubated at 37°C for 3 days. 18 hours before the end of the assay, 0.5 Cu of thymidine solution was added to each well. At the end of incubation, thymidine incorporation was measured using a MicroBetaTrilux counter (PerkinElmer). The percentage of proliferation inhibition was calculated using the following formula: %damage = 100 - (value) 100 / (maximum value) The percentage of proliferation inhibition for each condition was then plotted using GraphPad Prism software. After logarithmic transformation, a nonlinear regression was applied to all datasets using a log(agonist) versus response regression model to determine the EC50 values.

[0333] Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by OX40 antibody-mediated killing of activated T cells and In vitro Treg differentiation T cell isolation and activation were performed on PBMCs at 5 x 10 7 Cells / mL were resuspended in flow cytometry buffer and enrichment cocktail was added at 50 μl / mL. Cells were incubated at RT for 10 min, then RapidSpheres were added at 40 μL / mL, mixed, and incubated at RT for 5 min. The final volume of the suspension was adjusted to 10 mL with flow cytometry buffer and incubated on a magnet for 3 min. The suspension was poured into a new tube and washed once. T cells were then loaded at 1 x 10⁻⁶ cells / mL. 6 Cells / mL plated in a solution containing CD3 / CD28 dynabeads (1 x 10⁻⁶ cells / mL). 6 The solution was added to a complete RPMI solution (pills / mL) and incubated at 37°C for 40 hours.

[0334] CD4+ T cell isolation and in vitro differentiation: One day prior to selection, 6-well plates were coated with anti-human CD3 Ab (5 μg / mL OKT3 in PBS) at 37°C for 18 hours. PBMCs were then divided into groups of 5 x 10⁻⁶ cells / well. 7Cells were resuspended at 1 / mL in flow cytometry buffer, and anti-CD45RO biotinylated Ab and separation cocktail were added to the suspension at 50 μl / mL. Cells were incubated at RT for 5 min, then RapidSpheres were added to the suspension at 40 μL / mL, mixed, and incubated at RT for 10 min. The final volume of the suspension was adjusted to 10 mL with flow cytometry buffer and placed in a magnet for 5 min. The suspension was poured into a new tube, and the cells were washed once. CD4+ T cells were 1 x 10⁻⁶ cells / mL. 6 Cells were plated in complete RPMI containing sCD28 (1 μg / mL), IL2 (100 IU / mL), rapamycin (0.1 μg / mL), TGF-β (0.005 μg / mL), and retinoic acid (0.01 mM). Cells were incubated in an incubator for 4 days, then recovered and plated again in new OKT3-pre-coated wells for 3 days.

[0335] NK cell isolation: PBMCs were separated at 5 x 10 7 Cells / mL were resuspended in flow cytometry buffer, and an enrichment cocktail was added at a concentration of 50 μL / mL. Cells were incubated at RT for 10 min, and magnetic particles were added at a concentration of 100 μL / mL. The mixture was then incubated at RT for 5 min. The final volume of the suspension was adjusted to 10 mL with flow cytometry buffer and placed in the magnet for 2.5 min. The suspension was poured into a new tube and washed once. NK cells were added at a concentration of 1 x 10⁻⁶. 6 Cells were plated in complete RPMI at 1 / mL and incubated at 37°C for 24 hours. On the second day, 100 IU / mL IL2 was added, and the cells were incubated for another 24 hours.

[0336] Assessment of OX40 expression in activated T cells and differentiated Treg populations Prior to ADCC assays using in vitro differentiated Tregs, the surface expression of OX40 and other activation markers in the cells was tested. For activated T cells, 100,000 activated T cells were collected, washed once, and then resuspended in 50 μL of flow cytometry buffer containing the following anti-human antibodies: CD3 APC-eF780, CD4 PE-Cy7, CD8 BV421, CD25AF700, CD69 SB600, CD107a AF488, and CD134 PE. o After incubation at C for 20 min, the cells were washed once, resuspended in Sytox Blue solution, and obtained using a Cytoflex-S cell analyzer.

[0337] For activated T cells, the following gating strategy was used: after staining with live cell dyes, CD3, CD4, CD8 and CD134-specific antibodies, single live activated CD3+ cells expressing CD4 or CD8 were gated, and the fraction of CD134-positive cells was assessed.

[0338] For in vitro differentiated Tregs, 100,000 cells were collected and resuspended in PBS buffer in the presence of Fc blocking and fixable live / dead staining (L / D yellow), and incubated at 4°C for 20 min. After incubation, the cells were washed and resuspended in 50 μL of flow cytometry buffer containing the following anti-human antibodies: CD8 AF700, CD4 PE-eF610, CD25 SB645, CD127APC, CD69 SB600, CD107a AF488, and CD134 PE. After incubation at 4°C for 20 min, the cells were washed once and resuspended in 80 μL of fixation / permeabilization buffer at 4°C for 20 min. After washing with permeabilization buffer, the cells were stained with anti-human FoxP3 PE-Cy7 at 4°C for 30 min. After washing 2x with permeabilization buffer, the cells were resuspended in flow cytometry buffer and obtained using a Cytoflex-S cytometer.

[0339] For in vitro differentiated Tregs, the following gating strategy was used: after staining with live cell dyes and specific antibodies against CD3, CD4, CD127, CD25, FOXP3, and CD134, single live cells co-expressing CD4 and CD25 were gated, and the expression of CD4 was evaluated. Pos CD25 Pos CD127 Low FOXP3 Pos The fraction of CD134-positive cells in the cell.

[0340] ADCC-mediated killing of activated T cells induced by OX40 antibody Collect activated T cells and remove dynabeads. Count the cells and incubate at 1x10⁻⁶ cells in the presence of eF670 2 μM. 6 Cells / mL were resuspended in PBS and incubated at 37°C for 15 min. After washing, the cells were sputtered at 0.2 x 10⁻⁶ mL. 6 Cells were resuspended at 50 μL / well in 96-well U-bottom plates. Serial dilutions of 1:5 were performed, and GBR 830, MAB1, KHK4083, and allotype controls were added at 20 nM. NK cells were collected, washed, and plated at 1 x 10⁻⁶ cells / well. 6Cells were resuspended at 1 / mL and dispensed into wells. After incubation for 4.5 hours, the plate was washed once, and the cells were resuspended in fixable L / D NIR dye and incubated at 4°C for 30 min. The cells were then washed and resuspended in flow cytometry buffer and acquired on a Cytoflex-S cytometer. For analysis, the number of viable cells was extrapolated for each condition using FlowJo software (BD). For the % of ADCC, the following formula was used:

[0341] OX40 antibody-induced ADCC-mediated killing of in vitro differentiated Tregs Collect and wash in vitro differentiated Tregs. Count the cells and incubate at 1x10⁻⁶ cells in the presence of eF670 2 μM. 6 Cells / mL were resuspended in PBS and incubated at 37°C for 15 min. After washing, the cells were sputtered at 0.2 x 10⁻⁶ mL. 6 Cells were resuspended at 50 μL / well in 96-well U-bottom plates. Serial dilutions of 1:5 were performed, and GBR 830, MAB1, KHK4083, and allotype controls were added at 20 nM. NK cells were collected, washed, and plated at 1 x 10⁻⁶ cells / well. 6 Cells were resuspended at 1 / mL and dispensed. After incubation for 4.5 hours, the plate was washed once, and the cells were resuspended in fixable L / D NIR dye and incubated at 4°C for 30 min. The cells were then washed and resuspended in flow cytometry buffer and harvested on a Cytoflex-S cytometer.

[0342] For the analysis, the number of viable cells was extrapolated for each condition using FlowJo software (BD). The % for ADCC was calculated using the following formula:

[0343] result T cell proliferation assay and purity First, the potency of GBR 830 and MAB1 in inhibiting the proliferation of activated and purified T cells exposed to OX40 ligands was tested. Their potency was compared with that of antibody KHK4083. Inhibition of T cell proliferation was assessed by thymidine incorporation.

[0344] T cells were separated from healthy donor PBMCs using negative immunomagnetic selection, and the purity of the T cell fractions was assessed by flow cytometry using the aforementioned gating strategy. Proliferation assays were performed using isolated T cell fractions with over 90% CD56-CD3+ cellularity in single viable cells.

[0345] The inhibition of T cell proliferation induced by antagonistic antibodies GBR 830, MAB1, and KHK4083 was evaluated. To assess the potency of OX40 antagonistic antibodies in inhibiting T cell activation, T cells purified from PBMCs were stimulated by antibody-triggered TCR / CD3 and CD28 responses and exposed to OX40 ligands in the presence of increasing concentrations of anti-OX40 antibody. TCR / CD3 and CD28 triggered OX40 expression on the T cell surface, followed by incubation at 37°C for 1 hour with an OX40-blocking antibody. Activated T cells were then exposed to immobilized OX40 ligands for 3 days, and their proliferation was measured by thymidine incorporation.

[0346] exist Figure 10 This study provides data on the inhibition of T cell proliferation induced by GBR 830, MAB1, and KHK4083 in T cell proliferation assays. Data from five independent experiments using five different PBMC donors showed that MAB1 was 33-fold more effective than GBR 830 in inhibiting T cell proliferation (inhibiting EC50 by 0.17+ / -0.01 and 5.67+ / -2.1 nM, respectively; Tukey-HSD test, p=0.0014), and equally effective as KHK4083 (inhibiting EC50 by 0.17+ / -0.01 and 0.15+ / -0.01 nM, respectively; Tukey-HSD test, p>0.05). Table 11 provides a summary of the EC50 values ​​for T cell proliferation inhibition by MAB1, GBR 830, and KHK4083, as well as a statistical analysis of T cell proliferation inhibition, including the half-maximal effective concentration (EC50) of GBR 830, MAB1, and KHK4083 inducing T cell proliferation inhibition in the thymidine proliferation assay, and a comparison of p-values ​​between MAB1 and GBR 830 and KHK4083 (AMG451).

[0347] MAB1 T cell inhibition was compared with GBR 830 and KHK4083 (AMG451). The EC50 values ​​obtained from five independent experiments using T cells from five healthy donors were analyzed using the Tukey-HSD test. MAB1 unexpectedly induced statistically significantly greater T cell proliferation inhibition than GBR 830, approximately 30-fold higher.

[0348] Table 11: Statistical analysis of T cell proliferation inhibition

[0349] Values ​​are the mean (±SD) from independent experiments. EC50 = the half-maximal effective concentration of T cell proliferation inhibition in five independent experiments using T cells from five healthy donors.

[0350] ADCC measurement Next, the efficacy of GBR 830 and MAB1 in killing T cells via in vitro ADCC was evaluated and compared with KHK4083. First, the expression level of OX40 on the surface of activated T cells and in vitro differentiated Tregs was determined, and the efficacy of OX40 antibodies in in vitro inducing the killing of these T cell populations via ADCC was measured.

[0351] Quantitative analysis of OX40 expression on the surface of activated T cells and in vitro differentiated Tregs T cells isolated from PBMCs via immunomagnetic selection and activated for 40 hours via TCR and CD28 triggering were used as the source of activated T cells in this assay. Tregs were differentiated in vitro from immunomagnetically selected CD4 memory T cells, which were activated via TCR / CD28 triggering and exposed to TGF-β, IL-2, retinoic acid, and rapamycin. OX40 (CD134) expression on the surface of activated T cells and Treg cell populations was measured by flow cytometry using the aforementioned gating strategy. Consistent and robust upregulation of OX40 expression was observed on the surface of over 63% of activated CD8+ T cells and 82% of activated CD4+ T cells (data not shown). OX40 expression on the surface of in vitro differentiated Tregs was assessed using a comparable protocol with the same gating strategy. OX40 was upregulated on the surface of over 87% of Tregs (data not shown).

[0352] ADCC-mediated killing of activated T cells induced by OX40 antibody To assess whether OX40 antagonistic antibodies induce cytotoxicity in OX40-expressing T cells, purified activated T cells and Tregs were co-cultured with activated human NK cells in the presence of increased concentrations of GBR830, MAB1, baseline, and control antibodies. The fraction of T cells killed by ADCC was assessed by flow cytometry after 4.5 hours.

[0353] Figure 11 The study demonstrated T-cell killing induced by ADCC from GBR 830, MAB1, and KHK4083. Data from two independent experiments using six different PBMC donors showed that MAB1 and KHK4083 were more effective than GBR 830 in killing activated T cells via ADCC. However, MAB1 induced 3-fold less ADCC than the KHK4083 antibody. Therefore, both KHK4083 and MAB1 showed higher T-cell killing efficacy than GBR 830, while KHK4083 also showed higher T-cell killing efficacy than MAB1. The relatively reduced T-cell killing potential of MAB1 compared to KHK4083 suggests superior safety.

[0354] Table 12 provides a summary of the half-maximal effective concentrations (EC50) for T cell killing via ADCC induced by GBR 830, MAB1, and KHK4083, along with a statistical analysis of ADCC killing. All values ​​are mean (±SD) from different experiments. EC50 = half-maximal effective concentration for T cell killing via ADCC from 15 independent experiments using T cells and NK cells from five healthy donors.

[0355] Table 12.

[0356] OX40 antibody-induced ADCC-mediated killing of in vitro differentiated Tregs Isolated human NK cells and in vitro differentiated human Tregs were incubated in the presence of GBR 830, MAB1, KHK4083, and an isotype control antibody. A representative example of ADCC-mediated killing of in vitro differentiated Tregs induced by OX40 antibody is shown in… Figure 12 Data from five independent experiments using five different PBMC donors showed that MAB1 and KHK4083 induced more ADCC-mediated Treg killing compared to GBR 830. However, KHK4083 also showed statistically higher ADCC potency than MAB1 (Tukey-HSD test, p=0.015). Therefore, both KHK4083 and MAB1 showed higher ADCC-mediated T cell killing potency than GBR 830, and KHK4083 also showed higher T cell killing potency than MAB1.

[0357] Table 13 provides a summary of the half-maximal effective concentrations (EC50) of ADCC-induced T cell killing in in vitro differentiated Tregs induced by GBR 830, MAB1, and KHK4083, along with a statistical analysis of ADCC killing. All values ​​are mean (±SD) from different experiments. EC50 = half-maximal effective concentration of ADCC-induced T cell killing from 15 independent experiments using T cells and NK cells from five healthy donors.

[0358] Table 13

[0359] In assays evaluating the proliferation of purified T cells triggered by TCR / CD3 and CD28 in the presence of fixed purified OX40L, MAB1 has been shown to be as effective as KHK4083 in blocking the proliferation of primary human T cells in vitro. Figure 10 ).

[0360] Furthermore, in both assay settings, MAB1 was statistically significantly more potent in inhibiting T cell proliferation than GBR 830. While MAB1 also showed increased ADCC-mediated T cell killing compared to GBR 830, it was less potent than KHK4083 (…). Figure 11 Compared to HKH4083, MAB1 also exhibited less ADCC-mediated T cell killing. Finally, under the same assay conditions, MAB1 also showed a lower in vitro ability to kill differentiated Tregs than HKH4083. Figure 12 In summary, these results indicate that MAB1, in addition to demonstrating greater potency in blocking T cell proliferation, can also preserve Tregs, suggesting a more favorable outcome in autoimmune diseases, where blocking T cells while maintaining Treg activity is crucial for restoring immune tolerance.

[0361] In summary, GBR 830 and MAB1 exhibited a lack of agonistic activity, while the benchmark KHK4083 showed residual agonistic activity. In vitro, MAB1 demonstrated stronger inhibitory potency against T cell proliferation than GBR 830. Furthermore, compared to KHK4083, MAB1 also showed reduced potency against exhausted activated T cells and the unique potential to limit Treg exhaustion. These and other pharmacological properties support the potential efficacy of MAB1 in the field of OX40 therapy for autoimmune diseases.

[0362] Example 4: Characterization of MAB1 binding Materials and methods MAB1's Fc Engineering YTE mutations (M252Y, S254T, and T256E according to EU designations) were introduced into the Fc domain of the MAB1 heavy chain to obtain MAB10, which contains the heavy chain amino acid sequence of SEQ ID NO: 73 that pairs with the MAB1 light chain amino acid sequence (SEQ ID NO: 10). Figure 21 Therefore, MAB1 and MAB10 have the same heavy chain variable region sequence; the only sequence difference between the two antibodies is the “YTE” mutation in the heavy chain constant region of MAB10. MAB1 and MAB10 have the same light chain sequence.

[0363] Affinity to OX40 determined Surface plasmon resonance (SPR) analysis was used at different temperatures and pH levels, and the affinity of MAB10 antibody, as well as AMG451 (nocarotelimab or KHK4083), GBR830, and MAB1 antibodies for human OX40 protein was determined using a single-cycle kinetic (SCK) approach.

[0364] Measurements were performed on a Biacore 8K+ instrument (Cytiva Life Sciences) using Biacore 8K+ control software, and analysis was performed using Biacore Insight evaluation software (v3.0). In short, goat anti-human IgG (Fc-specific) antibody (JIR, catalog number: 109-005-098) was immobilized in two flow cells with approximately 13,000–14,000 resonance units (RUs) on a Series S CM5 sensor chip (Cytiva Life Sciences, catalog number: BR100530). Triple samples of AMG451 huIgG1 unfucosylated (FlowEighteen38, batch number: 230900054), GBR830 (FlowEighteen38, batch number: 230900056), MAB1 (FlowEighteen38, batch number: 231000022), MAB10 (FlowEighteen38, batch number: 230900169), and human isotype control (BioLegend, catalog number: 403502) antibodies were diluted to 5 nM and injected into flow cell 2 at 25 or 37 °C. Human OX40 (Acro Biosystems, catalog number: OX0-H5224) protein was injected into both flow cells at twice-dilutions from 100 to 6.25 nM at 25 or 37 °C, and dissociation rates were measured for 600 seconds. After each binding event, baseline levels were restored using regeneration solution on both flow paths. HBS-EP + pH 7.4 solution was used as the run buffer. Data were analyzed using the SCK assessment method in Biacore Insight evaluation software, and kinetic parameters were determined using a 1:1 binding fit model.

[0365] Table 14 reports the kinetic parameters generated at pH 7.4 and 25°C. Each measurement is the average of three identical replicates.

[0366] SPR epitope grouping determination Epitopebinning experiments were performed on a Biacore 8K+ instrument using Biacore 8K+ control software and analyzed using Biacore Insight evaluation software (v3.0). Epitopebinning sandwich assays were used. Briefly, in flow cell 2, MAB10, MAB1, GBR830, and AMG451 antibodies were immobilized at approximately 10,000–16,000 resonance units (RUs) on a Series S CM5 sensor chip using an amine conjugation kit. As a control, an anti-His human isotype control antibody was also immobilized. Antigens were injected into flow cells 1 and 2, followed by each test antibody in both flow cells. Baseline levels were restored using 10 mM glycine-HCl pH 1.5 buffer as a regeneration solution and HBS-EP+ pH 7.4 solution as a run buffer. Epitopebinning extension analysis data were performed using Biacore Insight evaluation software.

[0367] Affinity assay for Fcγ receptor Using a multicycle kinetics (MCK) approach, surface plasmon resonance (SPR) analysis was employed to determine the affinity of the MAB10 antibody, as well as the AMG451, GBR830, and MAB1 antibodies, for several Fcγ receptors.

[0368] Measurements were performed on a Biacore 8K+ instrument (Cytiva Life Sciences) using Biacore 8K+ control software, and analysis was performed using Biacore Insight evaluation software (v3.0). In short, mouse anti-tetrameric His antibody (Qiagen, catalog number: 34670) was immobilized at approximately 6,000-7,000 resonance units (RUs) onto a Series S CM5 sensor chip (Cytiva Life Sciences, catalog number: BR100012) in two flow cells using an amine conjugation kit (Cytiva Life Sciences). Human CD64, CD32b, CD32a (167 His), CD32a (167 Arg), CD16b (NA2 allotype), CD16a (F176V), and CD16a (176F) proteins (Sino Biological, Cat.nr. 10256-H08H, 10259-H08H, 10374-H08H1, 10374-H08H, 11046-H08H, 10389-H08H1, and 10389-H08H) were injected into flow cell 2. Antibodies against AMG451 huIgG1 unfucosylated (FlowEighteen38, batch number: 230900054), GBR830 (FlowEighteen38, batch number: 230900056), MAB1 (FlowEighteen38, batch number: 231000022), MAB10 (FlowEighteen38, batch number: 230900169), and human isotype control (BioLegend, catalog number: 403502) were prepared as eleven-step, two-fold dilution solutions (5,000–4.88 nM) and injected into two flow cells. Dissociation rates were measured over 150 seconds. After each binding event, baseline levels were restored using regeneration solution on both flow paths. HBS-EP+ pH 7.4 solution was used as the run buffer. Data were analyzed using the MCK assessment method in Biacore Insight assessment software, and kinetic parameters were determined using 1:1 binding and steady-state affinity methods.

[0369] The kinetic parameters are reported in Table 15. Each measurement is the average of three identical replicates.

[0370] Affinity determination for FcRn The affinity of the MAB10 antibody, as well as the AMG451, GBR830, and MAB1 antibodies for the FcRn receptor was determined using a multi-cycle kinetic (MCK) approach and surface plasmon resonance (SPR) analysis.

[0371] Measurements were performed on the Biacore 8K+ instrument (Cytiva Life Sciences) using the Biacore 8K+ control software and analyzed using the Biacore Insight evaluation software (v3.0). In short, in flow cell 2, the AMG451 huIgG1 unfucosylated (FlowEighteen38, batch number: 230900054), GBR830 (FlowEighteen38, batch number: 230900056), MAB1 (FlowEighteen38, batch number: 231000022), Mab10 (FlowEighteen38, batch number: 230900169), and human isotype control (BioLegend, catalog number: 403502) antibodies were immobilized on a Series S CM5 sensor chip (Cytiva Life Sciences, catalog number: BR100530) with approximately ≈180 resonance units (abbreviated as RU). Human FcRn protein (Acro Biosystems, catalog number: FCN-H52W7) was prepared into a 9-step double-dilution solution (500–1.95 nM) and injected into two flow cells. The dissociation rate was measured for 90 seconds. After each binding event, the baseline level was restored using regeneration solution on both flow paths. HBS-EP + pH 7.4 or 6.0 solution was used as the run buffer. Data were analyzed using the MCK assessment method in Biacore Insight assessment software, and kinetic parameters were determined using 1:1 binding and steady-state affinity methods.

[0372] Sensor maps showing the binding and dissociation of huFcRn protein at pH 6.0 are shown. Figure 13A In -C, the kinetic parameters are reported in Table 16. Each measurement is the average of three identical replicates.

[0373] result Affinity to OX40 determined Table 14 (below) shows the surface plasmon resonance (SPR) results, indicating that all anti-OX40 antibodies bind to the recombinant human OX40 receptor. In particular, MAB10, MAB1, and AMG451 exhibit nanomolar affinities similar to human OX40, while GBR830 shows the lowest affinity at a KD of 59.3 nM. The table also provides the mean ± SD of the binding constant (ka), dissociation constant (kd), and binding affinity (KD) for each molecule to human OX40, as determined by SPR analysis. Each measurement is the average of three identical replicates.

[0374] GBR830 has undergone an affinity maturation process, which produced MAB1 with an 8-fold increase in binding affinity.

[0375] MAB10 and MAB1, which share the same binding arm (e.g., the same variable region), show highly similar binding constants (ka), dissociation constants (kd), and binding affinity (KD), indicating that adding the YTE mutation to MAB10 does not affect binding to the human OX40 receptor.

[0376] Table 14. Overview of kinetic parameters of recombinant human OX40 protein by GBR830, MAB1, MAB10 and AMG451 at pH 7.4 and 25°C.

[0377]

[0378] SPR grouping determination The binding levels (RU) of each test antibody to the antigen captured by the immobilized antibody were extracted from the binding curves using Biacore Insight assessment software. No binding response was defined as RU < 10, indicating epitope overlap, while a binding response was defined as RU > 10, indicating the presence of different epitope bins. MAB10 was confirmed to show epitope overlap with MAB1 and GBR830, as all these antibodies were grouped together. Furthermore, no binding with GBR830 or MAB1 was observed when MAB10 captured OX40, confirming epitope overlap for these molecules. Additionally, no binding was observed between the human isotype control and the captured huOX40.

[0379] In contrast, AMG451 does not fall into the same group as MAB1, MAB10, or GBR830. Therefore, the epitope target of AMG451 is different from that of MAB1, MAB10, or GBR830.

[0380] Affinity assay for Fcγ receptor The surface plasmon resonance (SPR) results from Table 15 show that MAB10 binds significantly weaker to all Fcγ receptors compared to GBR830 and MAB1 antibodies, indicating reduced effector function.

[0381] A comparison between MAB1 and MAB10 demonstrated that the YTE mutation resulted in lower binding affinity for CD16a variants, CD16b, and CD32a (167H). MAB10 exhibited a similar spectrum to the remaining antibodies that only target the CD64 protein. No binding of MAB10 was observed for CD32a (167R) and CD32b proteins. Furthermore, data from MAB11 (MAB9 (SEQ ID NO: 74) with “LS” modification in the Fc heavy chain) showed that this Fc modification did not affect Fcγ receptor binding.

[0382] Table 15 shows the binding affinity (KD) of each molecule to human FcγRI, FcγRIIIa176F, FcγRIIIa176V, FcγRIIIb, FcγRIIa167R, FcγRIIa167 and FcγRIIb, as determined by surface plasmon resonance analysis.

[0383] Table 15. Overview of binding affinity (KD) of GBR830, MAB1, MAB11 and MAB10 to a group of Fcγ receptors.

[0384]

[0385] Affinity determination for FcRn From Figure 13A -C and the surface plasmon resonance (SPR) results in Table 16 show that, compared with GBR830 ( Figure 13B ) and MAB1 ( Figure 13C Compared to ) antibodies, MAB10 ( Figure 13A The YTE mutation binds to human FcRn with higher affinity, indicating a potentially longer half-life, suggesting that the effect of the YTE mutation increases the binding constant of MAB10 by 8.8 times compared to MAB1.

[0386] Table 16 shows the binding affinity (KD) of each molecule to human FcRn from surface plasmon resonance analysis. Each measurement is the average of three identical replication conditions.

[0387] Table 16. Overview of the binding affinity (KD) of GBR830, MAB1 and MAB10 to human FcRn.

[0388]

[0389] All anti-OX40 antibodies bound to the recombinant human OX40 receptor. MAB10, MAB1, and AMG451 showed nanomolar affinities similar to human OX40, while GBR830 showed the lowest affinity with a KD of 59.3 nM. Compared to GBR830, MAB1 and MAB10 showed an 8-fold increase in binding affinity to human OX40.

[0390] MAB10 and MAB1 share the same variable domain and exhibit highly similar binding constants (ka), dissociation constants (kd), and binding affinity (KD), indicating that adding the YTE mutation to MAB10 does not adversely affect binding to the human OX40 receptor.

[0391] Compared to GBR830 and MAB1, the YTE mutation in MAB10 leads to a reduced binding affinity of MAB10 to the Fcγ receptor, indicating a decrease in Fc-mediated effector functions of MAB10, such as ADCC and ADCP.

[0392] The YTE mutation also significantly affected FcRn binding of MAB10, as an 8.8-fold higher affinity was measured in MAB10 compared to GBR830 and MAB1. Without being bound by theory, this increased affinity could indicate a potentially longer half-life.

[0393] Example 5: In vitro characterization of the pharmacological properties of MAB10 Materials and methods OX40 / OX40L-induced cytokine release in T cell assays To assess the potential of MAB10 to inhibit T cell activation and subsequent cytokine release in the presence of OX40L, functional characterization was performed using a T cell activation assay.

[0394] Human PBMCs were harvested from the erythrocyte sedimentation rate (ESR) buffycoat layer from Biopler (Switzerland) blood transfusion center using a Ficoll density gradient and incubated overnight at 37°C and 5% CO2 in preheated complete RPMI medium (RPMI supplemented with 10% non-heat-inactivated fetal bovine serum, 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1% sodium pyruvate, and 1% penicillin / streptomycin) at 1 million cells / ml. In parallel, 6-well plates (TPP, catalog number: 92006) were coated with 1 μg / ml recombinant OKT3 (human anti-CD3 antibody diluted in PBS) and incubated overnight at 4°C. The next day, T cells were separated from resting PBMCs and distributed at 100,000 cells per well on OKT3-coated plates (washed three times in PBS) supplemented with 1 μg / ml soluble CD28 (sCD28). T cells were incubated at 37°C and 5% CO2 for 30 h. Additionally, 96-well flat-bottomed plates (TPP, catalog number: 92096) were pre-coated with recombinant OKT3 (1 μg / ml) and recombinant human OX40L (5 μg / ml) diluted in PBS and incubated overnight at 4°C. On the day of assay, the coated OKT3 / OX40L plates were washed three times in PBS, and 100,000 activated T cells were labeled with 5 μM eFluor450 cell proliferation dye solution (ThermoFisher Scientific, catalog number: 65-0842-85) and seeded at 100,000 cells / well. Shortly after T cell seeding, serially diluted GBR830, MAB1, MAB10, and AMG451, along with control antibodies, were seeded starting at 200 nM and diluted 3-fold. The plates were then incubated at 37°C and 5% CO2 for 4 days. After incubation, T cell assay readings were assessed by measuring cytokine levels in the supernatant. Plates were centrifuged at 350 g for 3 min, and the supernatant was maintained at -80°C until cytokine release was assessed. Cytokines were quantified according to the manufacturer's instructions using the Meiso Scale Discovery U-PLEX TH1 / TH2 combination kit (MeasScale Discovery, K15010K; IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, TNF-α, IL-31, IL-17α, IL-21, and IL-22). Quantitative values ​​were plotted using Prism software (GraphPad).

[0395] Based on that data, a subset of the cytokine quantification results was plotted using Prism software (GraphPad): IFN-γ ( Figure 14A ), TNF-α ( Figure 14B ), IL-5 ( Figure 14C ) and IL-13 ( Figure 14D ), IL-2 ( Figure 14E ), IL-4 ( Figure 14F ), IL-31 Figure 14G ), IL-17α ( Figure 14H ), IL-21 Figure 14I ) and IL-22 ( Figure 14J The half-maximum effective concentration (EC50) was calculated from the dose-response curve using a four-parameter logistic regression model with a variable slope. Based on the goodness-of-fit (R²)... 2 Values ​​>0.7), EC50 values ​​outside the range, or values ​​where the curve has not reached a plateau were excluded. Statistical analysis of the log-transformed EC50 values ​​for the treatment groups was performed using paired one-way ANOVA (donors as paired factors), followed by post-hoc Tukey HSD comparisons. Figure 15A -J provides the EC50 value: IFN-γ ( Figure 15A ), TNF-α ( Figure 15B ), IL-5 ( Figure 15C ), IL-13 Figure 15D ), IL-2 ( Figure 15E ), IL-4 ( Figure 15F ), IL-31 Figure 15G ), IL-17α ( Figure 15H ), IL-21 Figure 15I ) and IL-22 ( Figure 15J The mean EC50+ / - standard deviation is reported in pM in Table 17.

[0396] OX40 internalization determination Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were harvested from the erythrocyte sedimentation rate (ESR) brown layer (BC) using Ficoll gradient separation. EasySep was used according to the manufacturer's instructions. TM Human T-cell isolation kit (#17951, Stemm Cell Technologies) was used to isolate T cells from PBMCs. In short, before proceeding with isolation using the Big Easy Magnet protocol, PBMCs were rinsed with cold EasySep buffer at 5 x 10⁻⁶ ppm. 7 Resuspend the isolated T cells at 10⁻⁶ cells / mL. 6 Cells / mL were resuspended in complete culture medium and treated with Dynabeads human T-activator CD3 / CD28 (10 6 Activation was performed for 2 days using beads / mL to obtain activated T cells.

[0397] On the day of the experiment, Dynabeads were removed from the activated T cell culture medium. The cells were then cultured at 0.8 x 10⁻⁶ cells / mL. 6 Cells were resuspended at 50 μl / well and seeded into uncoated 96-well flat-bottomed black μClear cells (0.4 x 10⁻⁶ cells / well). 5 Cells / well). The plate was held at RT for 1 hour to allow cell sedimentation. Assay molecules were labeled with Fab-Fluor reagent at a 2x concentration by incubating the antibody with Fabfluor dye in internalization medium at a 1:3 molar ratio at 37°C and 5% CO2 for 15 minutes. 50 μL of labeled antibody was added to the top of the cells to achieve a final concentration of 8 μg / mL (1x) in the wells. Control wells containing untreated (no Ab conditions) or only T cells and Fab-Fluor reagent (Fabfluor conditions) were added for background assessment. The plate was immediately placed in the Incucyte live cell analysis system, and images were acquired by scanning four fields per hour for 24 hours using 20X magnification in the phase and red channels of the non-adherent cell module.

[0398] The analysis process included setting 79.22 μm. 2 Minimum area selection was used to detect T cells (referred to as total cells in the formula). T cells showing a red mean intensity (RCU) greater than 0.358 indicated positive internalization of OX40 (referred to as red cells in the formula). The threshold for RCU was determined based on signals obtained from allotype controls and experimental controls. The percentage of internalization was obtained from the raw data Excel file using the following formula: %internalization = (red cells / total cells) × 100 The %OX40 internalization during the incubation period was plotted using GraphPad Prism software (version 10).

[0399] Cross-reactivity with non-human OX40 ELISA: Human (hu), cynomolgus monkey (cy), mouse (mo), rat (ra), and rabbit (rb) OX40 protein were diluted at 2 μg / mL in phosphate-buffered saline (PBS) (pH 7.4) and analyzed using MaxSorp. TMHigh protein-binding capacity 96-well plates were coated overnight at 4°C with 100 μL of the antibody. The next day, the plates were washed three times with PBS-Tween 0.05%, pH 7.4 (PBS-T) using an automated plate washer, and blocked for 2 h at room temperature with 250 μL / well of 4% skim milk / PBS solution. After blocking, the plates were washed three times with PBS-T. Next, 100 μL of 11 serially diluted MAB10 antibody (ranging from 50 nM to 0.001 nM) in PBS containing 1% skim milk was added to the wells, and the plates were incubated at room temperature for 1 h. After washing five times with PBS-T, 100 μL of anti-human IgG peroxidase-conjugated antibody diluted 1:5,000 in PBS containing 1% skim milk was added for detection. The plates were incubated at room temperature for 1 h, then washed five times with PBS-T and three times with PBS (pH 7.4). Signal development was performed by adding 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) to each well. Development was allowed for 5 min, and the reaction was stopped by adding 100 μL of H₂SO₄ solution. The optical density (OD) at 450 nm was measured using a microplate spectrophotometer as the level of MAB10 binding to immobilized OX40 protein. Titration curves of MAB10 antibody against OX40 protein in humans, cynomolgus monkeys, mice, rats, and rabbits were obtained using GraphPad Prism 7 software and an (agonist) versus response-variable slope (four-parameter) fitting model. Three identical replicates were performed, and the data were averaged.

[0400] SPR: The affinity of the MAB10 antibody for human (hu), cynomolgus monkey (cy), mouse (mo), rat (ra), and rabbit (rb) OX40 proteins was determined using the single-period kinetic (SCK) method and surface plasmon resonance (SPR).

[0401] Measurements were performed on a Biacore 8K+ instrument using Biacore 8K+ control software, and analysis was performed using BiacoreInsight evaluation software (v3.0). In short, goat anti-human IgG (Fc-specific) antibody was immobilized on a Series S CM5 sensor chip with approximately 11,000–14,000 resonance units (RUs) using an amine conjugation kit in two flow cells. Triple samples of MAB10 and human isotype control antibody were diluted to 5 nM or 20 nM to determine the affinity for hu / cy / mo / raOX40 and rbOX40, respectively, and injected into flow cell 2.

[0402] OX40 protein was injected into two flow cells at two-fold dilutions: 100–6.25 nM (hu / mo / ra OX40), 500–31.25 nM (cyOX40), and 1000–62.5 nM (rbOX40). Dissociation rate measurements were performed over a 600-second period. After each binding event, baseline levels were restored using 10 mM glycine-HCl pH 1.5 as the regeneration solution for both flow paths. HBS-EP+ pH 7.4 was used as the run buffer. Data were analyzed using the SCK assessment method in Biacore Insight software, and kinetic parameters were determined using a 1:1 binding fit model. A threshold of 10 RU was established as the cutoff value for detecting significant binding activity. Interactions resulting in a response of less than 10 RU were considered non-binding (NB).

[0403] The kinetic parameters are reported in Table 18. Each measurement represents the average of three identical repeated conditions.

[0404] Jurkat-NFκB-OX40 reports signal transduction inhibition and receptor occupancy in cells. The previously described Jurkat-NF The B-OX40 cell line was used to characterize the relationship between the binding of MAB10, GBR830, and MAB1 to OX40 on the surface of T cells and their ability to inhibit the OX40-OX40L co-stimulatory pathway.

[0405] To evaluate Jurkat-NF Inhibition of the OX40-OX40L co-stimulatory pathway in B-OX40 cells was achieved by coating sterile 96-well μCLEAR microplates overnight at 4°C with OKT3 (5 μg / mL) diluted in PBS. On the day of the experiment, the coated plates were washed three times with PBS, and Jurkat-NF... B-OX40 cells were used at 0.1 x 10⁻⁶. 6Cells / well (25 μL) were plated in OKT3 pre-coated plates. Anti-OX40 antibody was prepared three times concentrated to reach a final concentration of 200 nM in the wells, followed by 11 consecutive 1 / 3 dilutions to obtain a minimum concentration of 0.003 nM. IgG1 control antibody was added at the highest dose (200 nM). Three different final concentrations (5, 0.3, and 0.07 μg / mL) of OX40L or culture medium only (condition without OX40L) were added to the top of the cells (25 μL), along with 25 μL of each antibody. Control wells were included to calculate the percentage inhibition of OX40-OX40L signaling. The condition “OX40LOKT3” (in the calculation formula) represents the highest achievable co-stimulation of the OX40-OX40L pathway and is used as the maximum value to calculate the percentage inhibition. The condition of seeding cells on top of wells coated with OKT3 but without the addition of OX40L and anti-OX40 antibody (stated as "OKT3 only" in the formula) represents Jurkat-NF in the absence of OX40L co-stimulation. Background activation of B-OX40 cells. The luminescence under OKT3 conditions alone was subtracted from the sample signal to obtain specific signals of inhibition of the OX40-OX40L pathway induced by different anti-OX40 antibody treatments.

[0406] The plate was incubated at 37°C and 5% CO2 for 5 hours. At the end of the incubation period, 75 μL of Bio-Glo solution was added to the wells and incubated for 15 minutes. The luminescence was measured using a Synergy Neo microplate reader with the following settings: readout type - endpoint; integration time - 0.2 seconds; emission - well; optical position - top; gain 135; readout height - 6.00 mm.

[0407] To evaluate the binding of the antibody to OX40 (receptor occupancy), Jurkat-NF was used. B-OX40 cells were used at 0.1 x 10⁻⁶. 6Cells were plated in 96-well U-type plates (25 μL each). Anti-OX40 antibody was prepared three times concentrated to a final concentration of 200 nM in each well using 11 consecutive 1 / 3 dilutions. Three different final concentrations (5, 0.3, and 0.07 μg / mL) of OX40 or culture medium were added to the top of the cells (25 μL), and 25 μL of each treatment was added. Culture medium was added to the control wells. Two binding time points were assessed, t=0 h and t=5 h. For t=0 h, the plate was incubated directly on ice for 30 min, followed by FACS staining. For t=5 h, the plate was incubated at 37 °C and 5% CO2 for 5 h, followed by FACS staining. FACS staining was performed on ice. After incubation with the test antibody, cells were washed twice with FACS buffer containing 0.05% sodium azide to avoid internalization. Cells were resuspended in PE-labeled anti-human IgG Fc and incubated at 4°C for 30 min, then washed twice with FACS buffer containing 0.05% sodium azide. Cells were resuspended in DAPI solution (0.1 μg / mL) and harvested using a Cytoflex-S flow cytometer.

[0408] Use the following formula to calculate the % suppression in Excel:

[0409] The receptor occupancy (RO) percentage is calculated as follows:

[0410] Max is defined as the saturation dose of the molecule, which corresponds to the second (highest) concentration at which OX40L reaches a plateau at 5 μg / mL and t=5h.

[0411] Maximum inhibition was determined from previously calculated parameters using JMP software (version 17), and EC50 values ​​were calculated using a four-parameter logistic regression model with variable slopes in GraphPad Prism software (version 10). Unit-point combined nonlinear regression models from GraphPad Prism (version 10) were applied to the receptor occupancy dataset to determine Kd.

[0412] By applying the “agonist vs. response - find any EC” nonlinear regression model to the receptor occupancy dataset, EC20, EC40, EC60, and EC80 of receptor occupancy were determined.

[0413] In meta-analyses of individual experiments, values ​​were set to NQ according to the following exclusion criteria: ECXX was considered NQ when the inhibition window was less than 10%, the dose-response curve R² < 0.7 was poorly fitted, the ECXX value was outside the test concentration range, and the maximum inhibition % was less than 10%. Kd quantification was considered NQ when the dose-response curve R² < 0.7 was poorly fitted and the Kd value was outside the test concentration range.

[0414] Off-target assessment The Retrogenix cell microarray technology platform was used to screen for specific off-target binding interactions with MAB10. For specific interactions with MAB10, 6105 human plasma membrane proteins, secretory proteins, and 400 heterodimers were screened.

[0415] result OX40 / OX40L-induced cytokine release in T cell assays The release of human IFN-γ, TNF-α, IL-5, IL-13, IL-2, IL-4, IL-31, IL-17a, IL-21, and IL-22 was quantified in a T cell proliferation assay using OX40L as a co-stimulatory factor. All anti-OX40 antibodies inhibited the release of all four cytokines. Figure 14A -J). MAB10 and MAB1 induce statistically comparable inhibitory potency, and have higher inhibitory potency than GBR830, such as Figure 15A -J and Table 17 show that the relevant EC50 of MAB10 is between 4.2 and 16.4 nM.

[0416] MAB10 exhibits a potent effect in reducing OX40L-induced cytokine production. Figure 14A -J and Figure 15A -J). The ability of anti-OX40 antibody and benchmark antibody AMG451 to inhibit OX40 / OX40L-induced cytokine release was evaluated in a T cell proliferation assay. IFN-γ ( Figure 14A ), TNF-α ( Figure 14B ), IL-5 ( Figure 14C ), IL-13 Figure 14D ), IL-2 ( Figure 14E ), IL-4 ( Figure 14F ), IL-31 Figure 14G ), IL-17α ( Figure 14H ), IL-21 Figure 14I ) and IL-22 ( Figure 14JCytokine release. GBR830, MAB1, MAB10, AMG451, and control IgG1 show cytokine release at different concentrations in pg / mL. Each curve is a nonlinear logic 4PL model with a variable slope, and the sign represents the mean + / - SEM of eight individual T cell donors from two independent experiments.

[0417] For GBR830, MAB1, MAB10, and AMG451, the results show the effect of targeting IFN-γ ( Figure 15A ), TNF-α ( Figure 15B ), IL-5 ( Figure 15C ), IL-13 Figure 15D ), IL-2 ( Figure 14E ), IL-4 ( Figure 14F ), IL-31 Figure 14G ), IL-17α ( Figure 14H ), IL-21 Figure 14I ) and IL-22 ( Figure 14J The half-maximum effective concentration (EC50) value in nM. Each symbol represents the EC50 value of a single donor from eight individual T cell donors tested in two independent experiments. Based on goodness-of-fit (R²) 2 Values ​​>0.7, EC50 values ​​outside the range, or values ​​where the curve has not reached a plateau should be excluded. Paired one-way ANOVA was used, followed by Tukey post-hoc comparisons to compare EC50 values ​​(ns p>0.05, *p<0.05, **0.01). <p<0.05,***p<0.01)。

[0418] Table 17. Average + / -SD of the half-maximal effective concentration (EC50) values ​​in nM for GBR830, MAB1, MAB10 and AMG451, and their fold differences relative to GBR830.

[0419]

[0420] Comparisons among GBR830, MAB1, and MAB10 confirmed that, compared to GBR830, the enhanced affinity of MAB1 and MAB10 binders for OX40 resulted in a significant increase in T cell proliferation inhibition potency and broader inhibition of cytokine release, including Th1, Th2, Th17, and Th22 cytokines. Furthermore, the incorporation of the YTE mutation into MAB10 did not affect potency, and both MAB10 and MAB1 exhibited similar EC50 values ​​in this assay. For all cytokines, the fold differences between the GBR830 antibody and MAB1 and MAB10 ranged from 2.4 / 2 to 18.5 / 17.2 fold, respectively.

[0421] OX40 internalization determination Internalization of OX40 induced by the binding of MAB10, MAB1, GBR830, AMG451, and IMG-007, as well as positive and negative controls (SP34), were assessed by in vivo imaging using activated T cells. MAB10 did not induce OX40 internalization in activated T cells. Figure 24 MAB1, GBR830, AMG451, and IMG-007 also did not induce OX40 target internalization, as the internalization rate was less than 10% at 8 μg / mL. Negative controls (IgG1, Fabfluor alone, or untreated conditions (without the antibody)) confirmed no nonspecific internalization during the assay. In contrast, the positive control SP34 (an antibody targeting CD3 and known to induce OX40 internalization) showed up to 50% internalization on activated T cells. Therefore, the increased affinity of MAB1 for the OX40 target and the Fc region modification in MAB10 does not affect the rate of OX40 internalization in T cells after incubation with either MAB10 or MAB1.

[0422] Cross-reactivity with non-human OX40 The binding of the MAB10 antibody to recombinant OX40 proteins from different species was tested using ELISA. MAB10 was demonstrated to bind to human, cynomolgus monkey, and rabbit OX40 proteins. The calculated EC50 values ​​for human OX40 were 0.0485 ± 0.0017, for cynomolgus monkey OX40 it was 0.1792 ± 0.0139, and for rabbit OX40 it was 0.3793 ± 0.0173. The calculated EC50 values ​​for human proteins were 4 or 8-fold compared to their orthologs in cynomolgus monkeys and rabbits. In contrast, no binding of MAB10 to rat or mouse OX40 proteins was observed.

[0423] The SPR cross-reactivity results are shown in Table 18. MAB10 bound to human, cynomolgus monkey, and rabbit OX40. MAB10 exhibited the highest affinity for human OX40 (KD = 6.7 ± 0.4 nM), followed by cynomolgus monkey (KD = 268 nM) and rabbit OX40 (KD = 3305 nM). No binding (NB) to mouse or rat OX40 was detected under the experimental conditions tested. Notably, the KD values ​​of MAB10 binding to ox40 obtained in this experiment were consistent with the results obtained by ELISA.

[0424] Table 18: SPR Cross-Reactive Binding Affinity

[0425] Jurkat-NFκB-OX40 reporter cell receptor occupancy assessment and signal transduction inhibition MAB10 consistently and dose-dependently inhibits NFκB signaling mediated by the binding of OX40L to OX40. Figure 25A -C). Figure 25A The reading is 0.07 μg / mL OX40L. Figure 25B The concentration is 0.3 μg / mL OX40L. Figure 25C The inhibitory potency was shown at 5 μg / mL OX40L. Compared to lower concentrations of OX40L, the inhibitory potency depended on the concentration of OX40L used, as indicated by the increased EC50 at a saturation dose of 5 μg / mL OX40L. MAB10 showed a mean EC50 (inhibition) of 4.59 nM at 5 μg / mL OX40L, compared to 0.79 nM at 0.07 μg / mL OX40L. The inhibition observed with MAB1 was similar to that observed with MAB10. However, GBR830 also inhibited the OX40-OX40L pathway, with a higher EC50 than both MAB1 and MAB10. GBR830 was also more affected by the OX40L concentration, as this molecule only achieved 53% maximum inhibition at a saturation dose of OX40L, while MAB10 had a mean maximum inhibition of 95% under the same conditions.

[0426] The binding of the molecule to the OX40 target decreases with increasing OX40L concentration, meaning that higher concentrations of the indicated antibody are required to achieve the same binding at higher doses of OX40L. Figure 26A -D). Figure 26A It shows no OX40L. Figure 26B The reading is 0.07 μg / mL OX40L. Figure 26C The concentration is shown as 0.3 μg / mL OX40L. Figure 26D The concentration of OX40L was 5 μg / mL. The receptor occupancy of GBR830 was most affected by the concentration of OX40L, while MAB1 and MAB10 were only slightly affected by 5 μg / mL OX40L.

[0427] Data shows that MAB10 can inhibit the binding of OX40L and OX40, and it is superior to GBR830 in doing so. Figure 25B -C is shown.

[0428] Off-target assessment For specific interactions with MAB10, 6105 human plasma membrane proteins, secretory proteins, and 400 heterodimers were screened. MAB10 showed significant specific interactions with its primary target, TNFRSF4 (OX40), on both fixed and live-cell microarrays; no off-target binding was observed. Furthermore, it interacted with FCGR3A (Fcγ receptor IIIa) alone and as part of a heterodimer containing FCER1G or CD247 on fixed-cell microarrays, and with FCGR2A and TSLP on live-cell microarrays. The interaction with the Fcγ receptor is likely mediated by the Fc domain. In subsequent flow cytometry studies, MAB10 exhibited strong binding to TNFRSF4 (OX40) but no significant interaction with TSLP (data not shown).

[0429] Example 6: In vitro characterization of MAB10 antibody-dependent cytotoxicity Materials and methods Antibody-dependent cell cytotoxicity assay To assess the potential of MAB10-induced antibody-dependent cell cytotoxicity (ADCC), a short-term NK-mediated killing assay was performed on activated T cells and regulatory T cells expressing OX40.

[0430] Human PBMCs were harvested from the erythrocyte sedimentation rate (ESR) amber layer using Ficoll density gradient isolation. When using autologous cells for assays: naïve T cells were isolated from the PBMCs to differentiate into regulatory T cells using ImmunoCult Human Treg Differentiation Additive (StemmCell Technologies, catalog number: #10977) and ImmunoCult-XF T cell expansion medium (StemmCell Technologies, catalog number: #10981); PBMCs from the same donor were frozen to isolate T cells and NK cells for further activation steps performed during the experiment. Naïve T cells were isolated from the PBMCs using the EasySep Human Naïve CD4+ T Cell Isolation Kit (StemmCell Technologies, catalog number: #19555) for 7 days, following the manufacturer's instructions. Two days prior to the assay, the remaining autologous PBMCs were thawed in pre-warmed complete RPMI medium (RPMI supplemented with 10% heat-inactivated fetal bovine serum, 1% glutamine, 1% non-essential amino acids, 1% sodium pyruvate, and 1% penicillin / streptomycin), and T cells and NK cells were isolated according to the manufacturer's operating procedures. The isolated T cells were resuspended in complete medium at 1 million cells / ml and treated with Dynabeads. TMActivation was performed for 2 days at 1 million beads / ml. Isolated NK cells were maintained in culture at 2 million cells / mL for 2 days in the presence of 100 units / mL recombinant human IL-2. On the day of assay, 10,000 target cells (activated T cells or regulatory T cells) were co-cultured with 50,000 NK cells in 384-well flat-bottomed plates (ThermoFisher Scientific, catalog number: 242764) to achieve an effector-target ratio of 5:1. Shortly after target and effector cell seeding, plates were seeded starting at 20 nM and serially diluted 5-fold with GBR830, MAB1, MAB10, and AMG451, as well as the control antibody. The plates were then incubated at 37°C and 5% CO2 for 4 days. After incubation, target cell killing was evaluated by measuring lactate dehydrogenase (LDH) release (Promega, catalog number: G1780) in the supernatant. The ADCC percentage was calculated using the following formula: ADCC (%) = (sample - no treatment) / (target only + lysis buffer - no treatment) × 100, where the "no treatment" condition is spontaneous LDH release induced by the target and effector cells in the absence of treatment, and the "target only + lysis buffer" condition is the maximum LDH release expected in the assay. Based on these data, the percentage of ADCC was plotted using Prism software (GraphPad). Maximum killing was determined using JMP (SAS) software, and the half-maximum effective concentration (MCC) or EC50 value was calculated from the dose-response curve using a four-parameter logistic regression model with variable slope. The treatment groups were compared in statistical analysis of the maximum ADCC values ​​for unconverted cells using paired one-way ANOVA (donor as paired factor), followed by post-hoc Tukey HSD comparisons. Maximum ADCC values ​​are presented in... Figure 4 In A and B, the mean maximum ADCC and EC50 + / - standard deviation are reported as percentages or pM in Tables 19, 20, 21 and 22.

[0431] result All molecules were tested in the ADCC assay to assess their potential to induce the killing of activated and regulatory T cells expressing OX40. Figure 16A -B、 Figure 17A -B and Figure 18A -B shows that all anti-OX40 antibodies induced various levels of ADCC in both cell types. Indicators such as maximum ADCC and EC50 values ​​are summarized in Tables 19 to 22.

[0432] On activated T cells, MAB10 induced a significantly lower ADCC than AMG451, which exhibited a 5-fold killing ability (EC50 0.02 nM and 60.29% of maximum ADCC). GBR830, MAB1, and MAB10 showed similar effects in eliminating activated T cells, with comparable sub-nanomolar EC50 values ​​and approximately 34.87–45.62% of maximum ADCC values.

[0433] On regulatory T cells, MAB10 induced significantly lower ADCC compared to AMG451. The enhanced affinity for OX40 in MAB1 resulted in an 8-fold increase in cytotoxic activity against regulatory T cells compared to GBR830. The incorporation of the YTE mutation in MAB10 enhanced its safety, as evidenced by statistically lower maximum cytotoxic activity against regulatory T cells compared to GBR830 and MAB1, with corresponding EC50 values ​​of 2.31 nM compared to 2.5 and 0.28 nM, and a 1.6 to 2.3-fold reduction in maximum ADCC. In contrast, AMG451 demonstrated 46-fold greater potency than MAB10 in eliminating regulatory T cells via ADCC (EC50 0.05 nM and 85.75% of maximum ADCC).

[0434] MAB10 induced significantly lower ADCC on activated or regulatory T cells than AMG451. Anti-OX40 antibody and the baseline antibody AMG451 induced activated T cells (… Figure 16A ) or regulatory T cells ( Figure 16B The killing ability was assessed in the ADCC assay at an effector-target ratio of 5:1 and measured by LDH release after 4.5 hours. At the endpoint, LDH release was assessed from the co-culture supernatant, and the percentage of ADCC was determined. The percentage of ADCC for GBR830, MAB1, MAB10, AMG451, and control IgG1 at different concentrations is shown. Each curve is a nonlinear logic 4PL model with a variable slope, and the sign represents 8 (from 3 to 4 independent experiments). Figure 16A ) or 6 ( Figure 16B The average value of individual donors + / -SD.

[0435] exist Figure 17A -B shows the maximum ADCC of GBR830, MAB1, MAB10, and AMG451. Figure 17A The maximum ADCC of activated T cells is shown. Figure 17BThe maximum ADCC of regulatory T cells is shown. Each symbol represents a value from one of 6 to 8 individual donors tested in 3 to 4 independent experiments. Maximum ADCC values ​​were compared using paired one-way ANOVA followed by Tukey post-hoc comparisons (ns p>0.05, *p<0.05, **0.01). <p<0.05,***p<0.01)。

[0436] Figure 18A -B shows the half-maximum effective concentrations (EC50) of GBR830, MAB1, MAB10, and AMG451. Figure 18A The EC50 of the indicated antibody against ADCC in activated T cells is shown. Figure 18B The EC50 of ADCC for the indicated antibody against regulatory T cells is shown. Each symbol represents a value from one of 8 to 6 individual donors tested in 3 to 4 independent experiments. EC50 values ​​of ADCC were compared using paired one-way ANOVA followed by Tukey post-hoc comparisons (ns p>0.05, *p<0.05, **0.01). <p<0.05,***p<0.01)。

[0437] Table 19 shows the maximum ADCC values ​​of GBR830, MAB1, MAB10, and AMG451 on activated T cells, and their fold differences relative to MAB10.

[0438]

[0439] Table 20 shows the maximum ADCC values ​​of GBR830, MAB1, MAB10, and AMG451 on regulatory T cells, and their fold differences relative to MAB10.

[0440]

[0441] Table 21 shows the mean + / - SD of EC50 values ​​of GBR830, MAB1, MAB10, and AMG451 on activated T cells, as well as the fold difference relative to MAB10.

[0442]

[0443] Table 22 shows the mean + / - SD of EC50 values ​​of GBR830, MAB1, MAB10, and AMG451 on regulatory T cells, as well as the fold differences relative to MAB10.

[0444]

[0445] In summary, in Fc-mediated assays such as antibody-dependent cell cytotoxicity assays, MAB10 induced significantly lower ADCC than AMG451 on both OX40-expressing activated and regulatory T cells. GBR830, MAB1, and MAB10 showed similar effects in eliminating activated T cells. Compared to GBR830, the enhanced affinity for OX40 in MAB1 resulted in increased cytotoxic activity against regulatory T cells, and the incorporation of the YTE mutation in MAB10 demonstrated statistically lower maximum cytotoxic activity against regulatory T cells relative to GBR830 and MAB1, which could enhance its safety in future human studies.

[0446] Example 7: In vivo non-human primate studies Materials and methods MAB10 was prepared at concentrations of 1.5, 5, and 10 mg / mL in buffer (5 mM histidine, 4% sucrose, 0.01% polysorbate 20, pH 6.0). A single subcutaneous administration was administered to cynomolgus monkeys. Samples were collected at predetermined intervals 90 days after administration.

[0447] The animals were assigned to the following groups:

[0448] a Based on the most recent weight measurement.

[0449] Group 1 animals (control) received solvent control (5 mM histidine, 4% sucrose, 0.01% polysorbate 20, pH 6.0).

[0450] The cynomolgus monkey was chosen as the animal model for this study because MAB10 is a fully humanized anti-OX40 monoclonal antibody that cross-reacts only in non-human primates and not in other laboratory species. Furthermore, the cynomolgus monkey is a non-rodent species accepted by regulatory authorities for non-clinical toxicity testing. The total number of animals used in this study was considered the minimum required to adequately characterize the effect of the test substance. The subcutaneous route of exposure was chosen because this is the expected route of human exposure.

[0451] Non-compartmental model analysis Non-compartmental modeling was used for parameter estimation using Phoenix pharmacokinetic software (version 8.3). For MAB10 in serum, an extravascular model was used for parameter estimation. All parameters were derived from individual MAB10 concentrations in serum on day 1. Parameters were estimated using the nominal dose level relative to the dose administration and the nominal sampling time. Serum concentration values ​​obtained at the time point prior to administration were used as the concentrations at time 0 on day 1. Concentration values ​​reported below the limit of quantitation (<375 ng / mL) were considered as 0.

[0452] In practice, at least the last three observed concentration values are used to identify the terminal elimination phase of each concentration-versus-time curve. Log-linear regression of the unweighted concentration data is used to determine the slope of the terminal elimination phase. When RSQadj ≥ 0.9, the parameters dependent on the determination of the terminal elimination phase are reported.

[0453] result In Figure 19 In vivo pharmacokinetic data from cynomolgus monkeys (n = 3) administered 20 mg / kg MAB10 subcutaneously are provided. The data represent the change in the mean concentration (SD) of MAB10 over time. The dotted line represents the LLOQ of 0.375 μg / mL; concentrations <LLOQ were plotted as 1 / 2 LLOQ. Single subcutaneous administration of MAB10 to cynomolgus monkeys at 3, 10, and 20 mg / kg was well tolerated locally and did not cause any systemic toxicity. MAB10 also exhibited an extended half-life of 26 days (622 hours) in cynomolgus monkeys.

[0454] Numbered embodiments The embodiments disclosed herein include embodiments P1 to P54 as provided in the numbered embodiments of the present disclosure: Embodiment P1. An antibody that binds to OX40 or an antigen-binding fragment thereof, comprising three heavy-chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) of a heavy-chain variable domain (VH) comprising the amino acid sequence shown in SEQ ID NO: 7 and three light-chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2, and CDR-L3) of a light-chain variable domain (VL) comprising the amino acid sequence shown in SEQ ID NO: 8, optionally wherein the antibody or antigen-binding fragment thereof is an isolated antibody or antigen-binding fragment.

[0455] Embodiment P2. An antibody that binds to OX40 or an antigen-binding fragment thereof, comprising three heavy-chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) of a heavy-chain variable domain (VH) comprising the amino acid sequence shown in SEQ ID NO: 7 and three light-chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2, and CDR-L3) of a light-chain variable domain (VL) comprising the amino acid sequence shown in SEQ ID NOs: 12, 15, 18, 21, 24, 27, 30, or 33; optionally wherein the antibody or antigen-binding fragment thereof is an isolated antibody or antigen-binding fragment.

[0456] Embodiment P3. The antibody according to Embodiment P1 or P2, wherein: a. The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to Kabat. b. The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to Chothia; c. The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to IMGT; d. The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to AbM; e. The CDR-H1, CDR-H2, and CDR-H3, as well as CDR-L1, CDR-L2, and CDR-L3, are defined according to Contact; or f. The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to Honneger (AHo).

[0457] Implementation method P4. An antibody or antigen-binding fragment thereof that binds to OX40, comprising: a. A heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3; and b. A light chain variable region comprising a CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, a CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and a CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 38 or 71, wherein X1 is F, T, W, or M; wherein X2 is G, I, V, L, or E; wherein X3 is A, D, E, L, H, T, or F; and wherein X4 is W, P, F, Y, or T, optionally wherein the antibody or its antigen-binding fragment is an isolated antibody or antigen-binding fragment.

[0458] Implementation P5. The antibody or antigen-binding fragment thereof according to Implementation P4, wherein the CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 38.

[0459] Implementation method P6. The antibody or antigen-binding fragment thereof according to implementation method P4 or P5, wherein X1 is F or T, X2 is L, G or E, X3 is A, and X4 is W.

[0460] Embodiment P7. An antibody or antigen-binding fragment thereof according to any one of Embodiments P4-P6, wherein CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6, 11, 14, 17, 20, 23, 26 or 29.

[0461] Implementation method P8. An antibody or antigen-binding fragment thereof that binds to OX40, comprising: a. A heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3; and b. Light chain variable regions comprising CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6, 11, 14, 17, 20, 23, 26, 29, or 32.

[0462] Implementation method P9. An antibody or antigen-binding fragment thereof that binds to OX40, comprising: (a) Heavy chain variable region, which contains: Heavy chain CDR1, comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 59 or SEQ ID NO: 65; Heavy chain CDR2, comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60 or SEQ ID NO: 66; and Heavy chain CDR3, comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 55, SEQ ID NO: 61 or SEQ ID NO: 67; and (b) Contains a light chain variable region, which includes: The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 56, SEQ ID NO: 62 or SEQ ID NO: 68; The light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 57, amino acid sequence AT, or amino acid sequence of SEQ ID NO: 69; and The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 58 or SEQ ID NO: 70.

[0463] Embodiment P10. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 1, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 2, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 3, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0464] Embodiment P11. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 41, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 42, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 3, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0465] Embodiment P12. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 47, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 48, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 3, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0466] Embodiment P13. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 53, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 54, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 55, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 56, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 57, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 58.

[0467] Embodiment P14. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 59, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 60, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 61, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 62, the light chain CDR2 comprises the amino acid sequence AT, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0468] Embodiment P15. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 65, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 66, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 67, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 68, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 69, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 70.

[0469] Implementation P16. An antibody or antigen-binding fragment thereof according to any one of Implementations P1-P15, wherein the OX40 is human OX40.

[0470] Embodiment P17. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P16, wherein the antibody or antigen-binding fragment thereof is an antagonist of OX40.

[0471] Implementation P18. An antibody or antigen-binding fragment thereof according to any one of Implementations P1-P17, wherein the antibody or antigen-binding fragment thereof is a humanized antibody.

[0472] Embodiment P19. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P18, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7.

[0473] Embodiment P20. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P19, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

[0474] Embodiment P21. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P20, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

[0475] Embodiment P22. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P19, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 12, 15, 18, 21, 24, 27, 30 or 33.

[0476] Implementation P23. An antibody or antigen-binding fragment thereof according to any one of Implementations P1-P22, comprising a heavy chain Fc region containing amino acid modifications that increase the serum half-life of the antibody or antigen-binding fragment thereof.

[0477] Implementation method P24. The antibody or antigen-binding fragment thereof according to any one of implementation methods P1-P23 comprises an IgG1 heavy chain.

[0478] Implementation P25. An antibody or antigen-binding fragment thereof according to any one of Implementations P1-P24, wherein the amino acid modification of the Fc region comprises mutations M252Y, S254T and T256E numbered according to the EU numbering system.

[0479] Embodiment P26. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P25, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 9.

[0480] Embodiment P27. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P25, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 73.

[0481] Embodiment P28. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P21 or P23-P27, wherein the antibody or antigen-binding fragment thereof comprises a light chain containing the amino acid sequence of SEQ ID NO: 10.

[0482] Embodiment P29. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1-P21, P23-P25, P27 or P28, wherein the antibody or antigen-binding fragment thereof comprises a light chain containing the amino acid sequence of SEQ ID NO: 10 and a heavy chain containing the amino acid sequence of SEQ ID NO: 73.

[0483] Embodiment P30. An antibody or antigen-binding fragment thereof according to any one of Embodiments P1, P3-P8, P16-P19, P22-P24 or P26, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 31 or 34.

[0484] Implementation P31. An antibody or antigen-binding fragment thereof according to any one of Implementation P1, P3-P8, P16-P19, P22-P24 or P27, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 73 and a light chain containing the amino acid sequence of SEQ ID NO: 13, 16, 19, 22, 25, 28, 31 or 34.

[0485] Implementation method P32. An antibody or antigen-binding fragment thereof according to any one of implementation methods P1-P31, wherein the antibody has increased inhibition of T cell proliferation compared with terazolizumab.

[0486] Implementation P33. An isolated nucleic acid comprising a nucleotide sequence encoding the heavy chain complementarity-determining region of any one of Implementations P1-P3, the heavy chain variable region of any one of Implementations P4-P22, or the heavy chain of any one of Implementations P23-P27 or P29-P31.

[0487] Embodiment P34. An isolated nucleic acid comprising a nucleotide sequence encoding a light chain complementarity-determining region as described in any one of Embodiments P1-P3, a light chain variable region as described in any one of Embodiments P4-P22, or a light chain as described in any one of Embodiments P28-P31.

[0488] Implementation method P35. An expression vector comprising the nucleic acid described in implementation method P33.

[0489] Implementation method P36. An expression vector comprising the nucleic acid described in implementation method P34.

[0490] Implementation P37. The expression vector according to Implementation P36 further comprises the nucleic acid described in Implementation P33.

[0491] Implementation P38. Host cells comprising the expression vector described in Implementation P35.

[0492] Implementation P39. Host cells comprising the expression vector described in Implementation P36.

[0493] Implementation P40. Host cells comprising the expression vector described in Implementation P37.

[0494] Implementation P41. A host cell comprising the expression vector described in Implementation P35 and the expression vector described in Implementation P36.

[0495] Implementation P42. A method for producing an antibody or antigen-binding fragment of human OX40, the method comprising: (a) The host cells described in embodiment P40 or embodiment P41 are cultured under conditions that cause the host cells to express one or more polypeptides containing a heavy chain or heavy chain variable region and a light chain or light chain variable region, thereby producing the antibody or an antigen-binding fragment of the antibody; and (b) Purify the antibody or the antigen-binding fragment of the antibody.

[0496] Implementation P43. A pharmaceutical composition comprising an antibody or antigen-binding fragment as described in any one of Implementations P1-P32, and a pharmaceutically acceptable carrier.

[0497] Implementation P44. A method for treating an OX40-mediated disorder in a subject, comprising administering to the subject a therapeutically effective amount of any one of Implementations P1-P32, an antibody or its antigen-binding fragment thereof, or a pharmaceutical composition as described in Implementation P42.

[0498] Implementation P45. A method for reducing or inhibiting T cell proliferation in a subject in need, comprising administering to the subject a therapeutically effective amount of any one of Implementations P1-P32, or an antigen-binding fragment thereof, or a pharmaceutical composition as described in Implementation P43.

[0499] Implementation P46. The method according to implementation P45, wherein the subject suffers from an OX40-mediated disorder.

[0500] Implementation method P47. The method according to any one of claims P45-P46, wherein the subject is a human.

[0501] Implementation P48. The method according to any one of Implementation P44, P46 or P47, wherein the OX40-mediated disorder is arthritis, rheumatoid arthritis, psoriatic arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peroni disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, nodular prurigo, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory disorders of the central and peripheral nervous systems, such as multiple sclerosis, lupus (such as systemic lupus erythematosus or lupus nephritis). And Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrotic alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, chronic spontaneous urticaria (CSU), chronic inducible urticaria (CIU), Wegener's granulomatosis, pancreatitis, trauma (surgery), graft-versus-host disease (GVHD), transplant rejection, cardiovascular diseases (including ischemic diseases such as myocardial infarction and atherosclerosis), intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypoacidity, hidradenitis suppurativa, alopecia areata, and neuromyelitis optica.

[0502] Implementation P49. The method according to any one of Implementation P44, P46 or P47, wherein the OX40-mediated disorder is atopic dermatitis, nodular prurigo, alopecia areata, chronic spontaneous urticaria (CSU) and chronic inducible urticaria (CIU), asthma, hidradenitis suppurativa, lupus nephritis, systemic lupus erythematosus, pemphigus vulgaris, psoriatic arthritis, vasculitis, Hashimoto's thyroiditis, systemic sclerosis, scleroderma, scleroderma, chronic pruritus of unknown cause, ankylosing spondylitis, Sjögren's syndrome, psoriasis or vitiligo.

[0503] Implementation P50. The method according to any one of implementations P44, P46 or P47, wherein the OX40-mediated barrier is atopic dermatitis.

[0504] Implementation P51. The method according to any one of Implementation P44, P46 or P47, wherein the OX40-mediated barrier is chronic spontaneous urticaria.

[0505] Implementation P52. The method according to any one of implementations P44, P46 or P47, wherein the OX40-mediated barrier is chronic induced urticaria.

[0506] Implementation P52. The method according to any one of implementations P44, P46 or P47, wherein the OX40-mediated barrier is asthma.

[0507] Implementation P53. The method according to any one of Implementation P44, P46 or P47, wherein the OX40-mediated disorder is rheumatoid arthritis or systemic lupus erythematosus.

[0508] Implementation P54. The method according to any one of Implementation P44, P46 or P47, wherein the OX40-mediated disorder is alopecia areata, scleroderma or hidradenitis suppurativa.

[0509] By incorporating via reference All publications and patents (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.) cited throughout this specification, whether above or below, are incorporated herein by reference in their entirety for all purposes. Where the material incorporated by reference contradicts or is inconsistent with this specification, the specification shall supersede any such material.

[0510] Equivalence The invention may be implemented in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the foregoing embodiments are to be considered illustrative in all respects and not limiting of the invention as described herein. Accordingly, the scope of the invention is defined by the appended claims rather than by the foregoing description, and all modifications within the meaning and scope of equivalents of the claims are intended to be included therein.

[0511] sequence list

[0512]

[0513]

[0514]

Claims

1. An antibody or antigen-binding fragment thereof that binds to OX40, comprising: a) Three heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2 and CDR-H3) of the heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7, and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2 and CDR-L3) of the light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 8; b) Three heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2 and CDR-H3) of the heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7, and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2 and CDR-L3) of the light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 12; c) Three heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2 and CDR-H3) of the heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7, and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2 and CDR-L3) of the light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 15; d) Heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) containing the heavy chain variable domain (VH) of the amino acid sequence of SEQ ID NO: 7, and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2, and CDR-L3) containing the light chain variable domain (VL) of the amino acid sequence of SEQ ID NO: 18; e) Heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2 and CDR-H3) of the heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7, and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2 and CDR-L3) of the light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 21; f) Heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2 and CDR-H3) of the heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7, and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2 and CDR-L3) of the light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 24; g) Heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2 and CDR-H3) of the heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7, and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2 and CDR-L3) of the light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 27; h) Heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2 and CDR-H3) of the heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7, and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2 and CDR-L3) of the light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 30; i) Heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2 and CDR-H3) of the heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7, and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2 and CDR-L3) of the light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO:

33.

2. An antibody or antigen-binding fragment thereof that binds to OX40, comprising three heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2 and CDR-H3) comprising a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7 and three light chain complementarity-determining regions (CDRs) (CDR-L1, CDR-L2 and CDR-L3) comprising a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO:

8.

3. The antibody or antigen-binding fragment according to any one of claims 1 or 2, wherein: a) The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to Kabat. b) The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to Chothia; c) The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to IMGT; d) The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to AbM; e) The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to Contact; or f) The CDR-H1, CDR-H2, and CDR-H3, as well as the CDR-L1, CDR-L2, and CDR-L3, are defined according to Honneger (AHo).

4. An antibody or antigen-binding fragment thereof that binds to OX40, comprising: a) A heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3; and b) A light chain variable region comprising CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 38 or 71, wherein X1 is F, T, W, or M; wherein X2 is G, I, V, L, or E; wherein X3 is A, D, E, L, H, T, or F; and wherein X4 is W, P, F, Y, or T.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:

38.

6. The antibody or antigen-binding fragment thereof according to claim 4 or 5, wherein X1 is F or T, X2 is L, G or E, X3 is A, and X4 is W.

7. The antibody or antigen-binding fragment thereof according to any one of claims 4-6, wherein CDR-L3 comprises the amino acid sequence of SEQ ID NO: 6, 11, 14, 17, 20, 23, 26 or 29.

8. An antibody or antigen-binding fragment thereof that binds to OX40, comprising: a) A heavy chain variable region comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3; and b) Light chain variable regions, including CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6, 11, 14, 17, 20, 23, 26, 29, or 32.

9. An antibody or antigen-binding fragment thereof that binds to OX40, comprising: (a) Heavy chain variable region, which contains: Heavy chain CDR1, comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 59 or SEQ ID NO: 65; Heavy chain CDR2, comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60 or SEQ ID NO: 66; and Heavy chain CDR3, comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 55, SEQ ID NO: 61 or SEQ ID NO: 67; and (b) Contains a light chain variable region, which includes: The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 56, SEQ ID NO: 62 or SEQ ID NO: 68; The light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 57, amino acid sequence AT, or amino acid sequence of SEQ ID NO: 69; and The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 58 or SEQ ID NO:

70.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 1, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 2, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 3, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

6.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 41, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 42, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 3, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

6.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 47, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 48, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 3, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 4, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

6.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 53, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 54, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 55, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 56, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 57, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

58.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 59, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 60, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 61, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 62, the light chain CDR2 comprises the amino acid sequence AT, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

6.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 65, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 66, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 67, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 68, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 69, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

70.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1-15, wherein the OX40 is human OX40.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof is an antagonist of OX40.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1-17, wherein the antibody or antigen-binding fragment thereof is a humanized antibody and / or the CDRs are located between human or humanized frame sequences.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1-18, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

7.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1-20, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1, 3-8 or 16-19, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence selected from SEQ ID NO: 12, 15, 18, 21, 24, 27, 30 or 33.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain Fc region comprising amino acid modifications that increase the serum half-life of the antibody or antigen-binding fragment thereof.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1-23, wherein the antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1-24, wherein the antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain Fc region comprising mutations M252Y, S254T and T256E numbered according to the EU numbering system.

26. The antibody or antigen-binding fragment thereof according to any one of claims 1-25, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO:

9.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1-25, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO:

73.

28. The antibody or antigen-binding fragment thereof according to any one of claims 1-21 or 23-27, wherein the antibody or antigen-binding fragment thereof comprises a light chain containing the amino acid sequence of SEQ ID NO:

10.

29. The antibody or antigen-binding fragment thereof according to any one of claims 1-21, 23-25, 27 or 28, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 10 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

73.

30. The antibody or antigen-binding fragment thereof according to any one of claims 1, 3-8, 16-19, 22-24 or 26, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 19, 22, 25, 28, 31 or 34.

31. The antibody or antigen-binding fragment thereof according to any one of claims 1, 3-8, 16-19, 22-24 or 27, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 73 and a light chain comprising the amino acid sequence of SEQ ID NO: 13, 16, 19, 22, 25, 28, 31 or 34.

32. The antibody or antigen-binding fragment thereof according to any one of claims 1-31, wherein the antibody has increased inhibition of T cell proliferation compared to terazolizumab.

33. An isolated nucleic acid comprising a nucleotide sequence encoding the heavy chain complementarity-determining region of any one of claims 1-3, the heavy chain variable region of any one of claims 4-22, or the heavy chain of any one of claims 23-27 or 29-31.

34. An isolated nucleic acid comprising a nucleotide sequence encoding the complementarity-determining region of the light chain as described in any one of claims 1-3, the variable region of the light chain as described in any one of claims 4-22, or the light chain as described in any one of claims 28-31.

35. An expression vector comprising the nucleic acid of claim 33.

36. An expression vector comprising the nucleic acid of claim 34.

37. The expression vector according to claim 36, further comprising the nucleic acid according to claim 33.

38. A host cell comprising the expression vector of claim 35.

39. A host cell comprising the expression vector of claim 36.

40. A host cell comprising the expression vector of claim 37.

41. A host cell comprising the expression vector of claim 35 and the expression vector of claim 36.

42. A method for producing an antibody or antigen-binding fragment thereof that binds to human OX40, the method comprising: (a) culturing the host cells of claim 40 or claim 41 under conditions that cause the host cells to express one or more polypeptides comprising a heavy chain or a heavy chain variable region and a light chain or a light chain variable region, thereby producing the antibody or an antigen-binding fragment of the antibody; and (b) Purify the antibody or the antigen-binding fragment of the antibody.

43. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1-32, and a pharmaceutically acceptable carrier.

44. A method of treating an OX40-mediated disorder in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-32, or a pharmaceutical composition as described in claim 42.

45. A method for reducing or inhibiting T cell proliferation in a subject in need, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-32, or the pharmaceutical composition of claim 43.

46. ​​The method of claim 45, wherein the subject suffers from an OX40-mediated disorder.

47. The method according to any one of claims 45-46, wherein the subject is a human being.

48. The method according to any one of claims 44, 46, or 47, wherein the OX40-mediated disorder is arthritis, rheumatoid arthritis, psoriatic arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peroni disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, nodular prurigo, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory diseases of the central and peripheral nervous systems, such as multiple sclerosis, lupus (such as systemic lupus erythematosus or lupus nephritis). Inflammation) and Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrotic alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, chronic spontaneous urticaria (CSU), chronic inducible urticaria (CIU), Wegener's granulomatosis, pancreatitis, trauma (surgery), graft-versus-host disease (GVHD), transplant rejection, cardiovascular diseases including ischemic diseases such as myocardial infarction, as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypoacidity, hidradenitis suppurativa, alopecia areata, and neuromyelitis optica.

49. The method according to any one of claims 44, 46 or 47, wherein the OX40-mediated disorder is atopic dermatitis, nodular prurigo, alopecia areata, chronic spontaneous urticaria (CSU) and chronic inducible urticaria (CIU), asthma, hidradenitis suppurativa, lupus nephritis, systemic lupus erythematosus, pemphigus vulgaris, psoriatic arthritis, vasculitis, Hashimoto's thyroiditis, systemic sclerosis, scleroderma, scleroderma, chronic pruritus of unknown cause, ankylosing spondylitis, Sjögren's syndrome, psoriasis or vitiligo.

50. The method according to any one of claims 44, 46 or 47, wherein the OX40-mediated barrier is atopic dermatitis.

51. The method according to any one of claims 44, 46 or 47, wherein the OX40-mediated disorder is chronic spontaneous urticaria.

52. The method according to any one of claims 44, 46 or 47, wherein the OX40-mediated barrier is chronic induced urticaria.

53. The method according to any one of claims 44, 46 or 47, wherein the OX40-mediated disorder is asthma.

54. The method according to any one of claims 44, 46 or 47, wherein the OX40-mediated disorder is rheumatoid arthritis or systemic lupus erythematosus.

55. The method according to any one of claims 44, 46 or 47, wherein the OX40-mediated disorder is alopecia areata, scleroderma or hidradenitis suppurativa.

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