Anti-TNFR2 antibodies and uses thereof

Abstract

Provided are monoclonal antibodies and antigen-binding fragments thereof specific for TNFR2 and methods of using them to treat cancer or autoimmune disorders, including in combination with antagonists of the PD-1 / PD-L1 immune checkpoint.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 219,175 (filed July 7, 2021), the entire contents of which are incorporated by reference herein, including any drawings and sequence listings. [Background technology]

[0002] Tumor necrosis factor receptor 2 (TNFR2), also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) and CD120b, is a 75 kDa type I transmembrane protein that contains an extracellular domain (ECD, residues 1-257) with four cysteine-rich domains (CRD1-CRD4), a transmembrane domain (TM, residues 258-287), and an intracellular domain (ICD, residues 288-461) that contains a TRAF2-binding domain. TNFR2 shares relatively little sequence identity with the other TNFα receptor, tumor necrosis factor receptor 1 (TNFR1), with only 28% homology between their extracellular domains.

[0003] TNFR2 binds to TNFα ligands in a 3:3 trimerization manner. A co-crystal structure of TNFR2 with TNFα has been solved, showing that each TNFR2 molecule binds two TNFα ligands. Furthermore, TNFα has a K d It binds to TNFR2 at 1 k d = 19 nM). Not surprisingly, TNFα binds preferentially to TNFR1, all else being equal.

[0004] In normal T cells, TNFα-TNFR2 interaction induces cell survival signals via the NFkB signaling pathway, but in autoimmune T cells, TNFα-TNFR2 interaction induces apoptosis signals via the caspase pathway.

[0005] Human TNFR2 shows 62% amino acid sequence homology to mouse TNFR2, but is 97% identical to rhesus monkey TNFR2.

[0006] Whereas TNFR1 is ubiquitously expressed, TNFR2 expression is primarily restricted to immune cells, with tumor-infiltrating immunosuppressive CD4 + FoxP3 + It is predominantly and highly expressed by regulatory T cells (Tregs). Recent studies have shown that TNFR2 plays a key role in stimulating the activation and proliferation of Tregs, which are a major checkpoint in antitumor immune responses (Chen and Oppenheim, Sci Signal 10:eaal2328, 2017). Activation of TNFR2 via its ligand TNFα activates NFkB signaling, which then upregulates TNFR2. + Tregs expand. TNFR2 is also expressed on CD8 and CD4 Tconv cells and myeloid cells. In particular, TNFR2 is expressed on exhausted CD8 T cells, similar to clinically validated immune checkpoints.

[0007] Regulatory T cells (Tregs) are a small subset of T lymphocytes with diverse clinical applications. + Tregs are highly immunosuppressive and express the highly inhibitory CD103 + It has stronger suppressive activity than Treg (J Immunol 179:154-161, 2007; J Immunol 180:6467-6471, 2008). + Tregs can be used to treat conditions that depend on the immunosuppressive activity of Tregs, such as transplantation, allergy, asthma, infectious diseases, graft-versus-host disease (GVHD), and autoimmunity. For example, in an experimental GVHD mouse model, CD4 + CD25 high Foxp3 + Depletion of thymus-derived Tregs could enhance GVHD (Cohen et al., JEM 2002).

[0008] TNFR2 is also expressed in certain cancers (e.g., breast, cervical, colon, and kidney cancers) (Front. Immunol. 9:1170, 2018), and may be involved in immune tolerance in these cancers. The survival and growth of these cancer cells is promoted by the ligand of TNFR2 (TNFα). TNFR2 has been shown to be involved in various processes of tumor development by using different signal pathways in tumor cells. For example, nuclear factor-κB (NFκB) is involved in the malignant transformation of epithelial cells associated with TNFR2. AKT signaling has been shown to be another mediator of TNFR2 in carcinogenesis, tumor growth, and angiogenesis. Meanwhile, myosin light chain kinase (MLCK) and extracellular signal-regulated kinase (ERK) are also important for the function of TNFR2 mentioned above. Therefore, by inhibiting the function of TNFR2, it is possible to inhibit the function of Treg in cancer immunity and increase anti-tumor T cell responses.

[0009] Therefore, TNFR2 + There is a need to develop therapeutic reagents that can enhance the immunosuppressive function of Tregs to treat autoimmune disorders by stimulating the TNFR2 function of Tregs, or that can inhibit TNFR2 activation to treat diseases such as cancer. Summary of the Invention

[0010] In one embodiment, the present invention provides an isolated monoclonal antibody or antibody-binding fragment thereof, wherein the isolated monoclonal antibody or antibody-binding fragment thereof is specific to human TNFR2, the isolated monoclonal antibody or antibody-binding fragment thereof comprising: (1a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 1, the HCVR CDR2 sequence of SEQ ID NO: 2, and the HCVR CDR3 sequence of SEQ ID NO: 3; and (1b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 4, the LCVR CDR2 sequence of SEQ ID NO: 5, and the LCVR CDR3 sequence of SEQ ID NO: 6; or (2a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 14, the HCVR CDR2 sequence of SEQ ID NO: 15, and the HCVR CDR3 sequence of SEQ ID NO: 16; and (2b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 17, the LCVR CDR2 sequence of SEQ ID NO: 18, and the LCVR CDR3 sequence of SEQ ID NO: 19. or (3a) a heavy chain variable region (HCVR) comprising an HCVR CDR1 sequence of SEQ ID NO: 26, an HCVR CDR2 sequence of SEQ ID NO: 27, and an HCVR CDR3 sequence of SEQ ID NO: 28; and (3b) a light chain variable region (LCVR) comprising an LCVR CDR1 sequence of SEQ ID NO: 29, an LCVR CDR2 sequence of SEQ ID NO: 30, and an LCVR CDR3 sequence of SEQ ID NO: 31; or (4a) a heavy chain variable region (HCVR) comprising an HCVR CDR1 sequence of SEQ ID NO: 39, an HCVR CDR2 sequence of SEQ ID NO: 40, and an HCVR CDR3 sequence of SEQ ID NO: 41; and (4b) a light chain variable region (LCVR) comprising an LCVR CDR1 sequence of SEQ ID NO: 42, an LCVR CDR2 sequence of SEQ ID NO: 43, and an LCVR CDR3 sequence of SEQ ID NO: 44. or (5a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 51, the HCVR CDR2 sequence of SEQ ID NO: 52, and the HCVR CDR3 sequence of SEQ ID NO: 53; and (5b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 54.or (6a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 63, the HCVR CDR2 sequence of SEQ ID NO: 64, and the HCVR CDR3 sequence of SEQ ID NO: 65; and (6b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 66, the LCVR CDR2 sequence of SEQ ID NO: 67, and the LCVR CDR3 sequence of SEQ ID NO: 68.

[0011] In certain embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, (1A) the HCVR sequence is SEQ ID NO:7, and / or (1B) the LCVR sequence is SEQ ID NO:8, or (2A) the HCVR sequence is SEQ ID NO:20, and / or (2B) the LCVR sequence is SEQ ID NO:21, or (3A) the HCVR sequence is SEQ ID NO:32, and / or (3B) the LCVR sequence is SEQ ID NO:33, or (4A) the HCVR sequence is SEQ ID NO:45, and / or (4B) the LCVR sequence is SEQ ID NO:46, or (5A) the HCVR sequence is SEQ ID NO:57, and / or (5B) the LCVR sequence is SEQ ID NO:58, or (6A) the HCVR sequence is SEQ ID NO:69, and / or (6B) the LCVR sequence is SEQ ID NO:70.

[0012] In certain embodiments, the monoclonal antibody has (1a) the heavy chain sequence of SEQ ID NO:9 and / or (1b) the light chain sequence of SEQ ID NO:10, or (2a) the heavy chain sequence of SEQ ID NO:22 and / or (2b) the light chain sequence of SEQ ID NO:23, or (3a) the heavy chain sequence of SEQ ID NO:34 and / or (3b) the light chain sequence of SEQ ID NO:35, or (4a) the heavy chain sequence of SEQ ID NO:47 and / or (4b) the light chain sequence of SEQ ID NO:48, or (5a) the heavy chain sequence of SEQ ID NO:59 and / or (5b) the light chain sequence of SEQ ID NO:60, or (6a) the heavy chain sequence of SEQ ID NO:71 and / or (6b) the light chain sequence of SEQ ID NO:72.

[0013] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a human-mouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a resurfaced antibody.

[0014] In certain embodiments, the antigen-binding fragment is a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0015] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof cross-reacts with rhesus monkey TNFR2 but does not substantially cross-react with mouse TNFR2.

[0016] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention comprises one or more point mutations in its amino acid sequence that are designed to improve the developability of the antibody. For example, in certain embodiments, the one or more point mutations render the antibody more stable during expression in a host cell, purification during the manufacturing and / or formulation process, and / or administration to a subject patient. In certain embodiments, the one or more point mutations render the antibody less prone to aggregation during the manufacturing and / or formulation process.

[0017] In certain embodiments, the present invention provides therapeutic antibodies in which developability issues have been minimized or reduced by substituting one or more amino acids within their sequence (e.g., in one or more of their CDRs) (e.g., removing or reducing hydrophobicity and / or optimizing charge).

[0018] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof does not substantially cross-react with TNFR1.

[0019] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof has a K of less than about 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 2 nM, or less than 1 nM. d It binds to TNFα.

[0020] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof enhances binding of TNFα to TNFR2 in the presence of Tregs, enhances TNFα-mediated or costimulatory NFκB signaling (e.g., in TCR-activated CD8 and / or CD4 Tconv T cells), and / or promotes proliferation of TCR-activated effector T cells (e.g., CD8 and / or CD4 Tconv T cells).

[0021] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof enhances TNFα-mediated CD25 expression on Tregs.

[0022] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof binds to an epitope of SEQ ID NO:13 and / or 101.

[0023] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof enhances the binding of TNFα to TNFR2, inhibits the binding of TNFα to TNFR2, or has no apparent effect on the binding of TNFα to TNFR2.

[0024] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof does not block, inhibit, or otherwise substantially antagonize the binding of TNFα to TNFR2.

[0025] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is an agonist of TNFR2 or stimulates TNFR2 signaling (eg, in the presence of TNFα), and the agonist function is preferably Fc-independent.

[0026] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is capable of binding to CD4 + Effector T cells, CD8 + Activates effector T cells, other effector T cells, and / or NK cells.

[0027] Another aspect of the present invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that competes with any one of the subject antibodies, or antigen-binding fragment thereof, for binding to the epitope of SEQ ID NO: 13 and / or 101.

[0028] Another aspect of the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to an epitope of SEQ ID NO:13 and / or 101.

[0029] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof enhances binding of TNFα to TNFR2 in the presence of Tregs, enhances TNFα-mediated or costimulatory NFκB signaling (e.g., in TCR-activated CD8 and / or CD4 Tconv T cells), and / or promotes proliferation of TCR-activated effector T cells (e.g., CD8 and / or CD4 Tconv T cells).

[0030] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof inhibits binding of TNFα to TNFR2, inhibits TNFα-mediated or costimulatory NFκB signaling (e.g., in TCR-activated CD8 and / or CD4 Tconv T cells), and / or inhibits proliferation of TCR-activated effector T cells (e.g., CD8 and / or CD4 Tconv T cells) in the presence of Tregs.

[0031] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof promotes the expansion of Tregs.

[0032] Another aspect of the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that competes with the isolated monoclonal antibody, or antigen-binding fragment thereof, of the invention for binding to the same epitope.

[0033] Another aspect of the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof specifically binds to human TNFR2 at an epitope that comprises, consists essentially of, or consists of SEQ ID NO:101, and optionally, the isolated monoclonal antibody or antigen-binding fragment thereof does not bind to human TNFR2 at an epitope that consists essentially of, or consists of SEQ ID NO:13.

[0034] In certain embodiments, (1) CD4+ in tumor-infiltrating lymphocytes (TILs) (e.g., in an in vivo hTNFR2 knock-in MC38 mouse tumor model) + 20. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 19, which (1) promotes activation and proliferation of T cells but not regulatory T cells (Tregs), and / or (2) promotes activation of NK cells in vitro and / or in vivo.

[0035] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the invention has a maximum tolerated dose (MTD) of about 150 mg / kg in cynomolgus monkeys.

[0036] Another aspect of the invention provides a method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, wherein the patient (e.g., the patient's cancer) has (a) a level of TNFR2 expression that is higher than the average TNFR2 expression level in prostate cancer patients (optionally, the TNFR2 expression is expressed by effector T cells (e.g., CD4 + and / or CD8 + T cells), tumor-infiltrating CD8 + (b) have a level of CD8A expression that is higher than the average CD8A expression level in AML patients.

[0037] In certain embodiments, a patient (eg, a patient's cancer) has higher levels of TNFR2 expression in tumor-infiltrating CD8A+ (CD8 alpha chain positive) T cells.

[0038] In certain embodiments, the patient is +Gastric cancer (e.g., gastric adenocarcinoma, which tends to have high PD-L1 / CD274 expression), clear cell renal cell carcinoma, renal clear cell carcinoma (e.g., KIRC.2, KIRC.3, and KIRC.4 subtypes, or clear cell type B (ccB) subtype or ccA / ccB unclassified subtype), cutaneous melanoma (e.g., so-called triple wt subtype lacking hotspot BRAF, N / H / K-RAS, or NF1 mutations), subtypes with BRAF hotspot mutations (V600E , V600K, and V600R mutations, and a hotspot mutation in K601), subtypes with RAS hotspot mutations (including Q61R, Q61K, Q61L, Q61H, 61_62QE>HK, and G12R / D / A, G13R / D in NRAS, and G13D, G13S, and Q61K in HRAS, and G12D, G12R, and Q61R in KRAS), subtypes with any NF1 mutation, testicular germ cell tumor, or soft tissue sarcoma.

[0039] In certain embodiments, the cancer expresses PD-L1 at a higher than average level.

[0040] In certain embodiments, the cancer is cervical cancer (e.g., cervical squamous cell carcinoma or endocervical adenocarcinoma), pleural mesothelioma, lung adenocarcinoma, or head and neck squamous cell carcinoma (HNSC, e.g., atypical subtypes (about 40% of which are HPV positive) and mesenchymal subtypes (which tend to have high PD-L1 / CD274 expression)).

[0041] In certain embodiments, the method further comprises administering to the patient (a) an antibody or antigen-binding fragment thereof specific for PD-1 (e.g., cemiplimab, nivolumab, pembrolizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, and INCMGA00012), (b) an antibody or antigen-binding fragment thereof specific for PD-L1 (e.g., avelumab, durvalumab, atezolizumab, KN035, CK-301), and / or (c) an antibody or antigen-binding fragment thereof specific for PD-L2.

[0042] In certain embodiments, the patient has relapsed or refractory cancer, and / or has previously been treated with (and optionally failed to respond to or relapsed from) standard of care.

[0043] In certain embodiments, the method further comprises administering to the patient an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof once every three weeks (Q3W), once every four weeks (Q4W), or once every five weeks (Q5W) (e.g., once every four weeks or Q4W).

[0044] In certain embodiments, the method comprises administering to the patient (e.g., administered intravenously over 60 minutes) an isolated monoclonal antibody or antigen-binding fragment thereof once every four weeks (Q4W) at a dose of about 5 mg, about 15 mg, about 50 mg, about 100 mg, or about 150 mg.

[0045] In certain embodiments, the method further comprises (1) selecting a patient having said higher levels of TNFR2 expression and CD8A expression prior to the administration step, or (2) confirming that the patient has said higher levels of TNFR2 expression and CD8A expression prior to the administration step.

[0046] Another aspect of the present invention provides a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TNFR2 at an epitope that comprises, consists essentially of, or consists of SEQ ID NO:101, and optionally, the isolated monoclonal antibody or antigen-binding fragment thereof does not bind to human TNFR2 at an epitope that consists essentially of, or consists of SEQ ID NO:13.

[0047] Another aspect of the invention provides a method of treating cancer or an autoimmune disorder (AID, e.g., GVHD (graft-versus-host disease) and rheumatoid arthritis) in a patient in need thereof, the method comprising administering to the patient an effective amount of an isolated monoclonal antibody or antigen-binding fragment thereof of the invention.

[0048] In certain embodiments, the method is for treating AID and the method further comprises administering a second agent, e.g., a low dose anti-IL2 agent in treating chronic GVHD, or an anti-TNFα agent (e.g., adalimumab, infliximab, etanercept, golimumab, etc.) in treating rheumatoid arthritis, chronic plaque psoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, polyarticular juvenile rheumatoid arthritis, IBS, EAE, and non-infectious uveitis.

[0049] In certain embodiments, the method is for treating cancer, and the method further comprises administering an immune checkpoint antagonist.

[0050] In certain embodiments, the immune checkpoint is the PD-1 / PD-L1 immune checkpoint.

[0051] In certain embodiments, the immune checkpoint antagonist is an antibody or antigen-binding fragment thereof specific for PD-1 or PD-L1.

[0052] In certain embodiments, the antibody is an anti-PD-1 antibody, such as cemiplimab, nivolumab, pembrolizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, and INCMGA00012.

[0053] In certain embodiments, the antibody is an anti-PD-L1 antibody (e.g., avelumab, durvalumab, atezolizumab, KN035, or CK-301).

[0054] In certain embodiments, the immune checkpoint antagonist is a (non-antibody) peptide inhibitor of PD-1 / PD-L1 (e.g., AUNP12), a small molecule inhibitor of PD-L1 (e.g., CA-170), or a macrocyclic peptide (e.g., BMS-986189).

[0055] In certain embodiments, the cancer is breast cancer, colon cancer, cervical cancer, kidney cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., NSCLC), ovarian cancer, melanoma, skin cancer (e.g., squamous cell carcinoma or basal cell carcinoma), lymphoma, or leukemia. In certain embodiments, the cancer is melanoma.

[0056] In certain embodiments, the method further comprises administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, an immuno-oncology agent, and / or an anti-neoplastic composition.

[0057] Another aspect of the invention provides polynucleotides encoding the heavy or light chains or antigen-binding portions thereof of the invention.

[0058] In certain embodiments, the polynucleotide is codon optimized for expression in a human cell.

[0059] Another aspect of the present invention provides a vector comprising a polynucleotide of the present invention.

[0060] In certain embodiments, the vector is an expression vector (eg, a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector). [Brief description of the drawings]

[0061] [Figure 1] The sequence alignment and consensus sequence of the VH and VL regions of human-mouse chimeric monoclonal antibodies HFB3-1, -3, -6, -14, -18, -19, -20, -21, -22, -23, -24, and HFB3-25 are shown. [Figure 2A] Figure 1 shows the binding affinity of selected human-mouse chimeric monoclonal antibodies raised against the extracellular domain of recombinant human TNFR2. EC50 and Emax values ​​of test antibodies and isotype-matched negative control antibodies were measured against CHO cells expressing human TNFR2 (CHO.hHFB3) or rhesus TNFR2 (CHO.mkHFB3). [Figure 2B] It has been shown that different anti-TNFR2 monoclonal antibodies can enhance (HFB3-1) or inhibit (HFB3-18) the binding of TNFα to TNFR2, or have no effect on binding (HFB3-6). [Diagram 3] It is shown that the human-mouse chimeric monoclonal antibodies do not bind to the parent CHO cell line, nor to CHO cells expressing mouse TNFR2 (except for slight binding by the HFB3-18 and HFB3-19 antibodies). [Figure 4A] The binding specificity of the human-mouse chimeric antibody is shown, specifically against TNFR2 and not against TNFR1. [Figure 4B] The Kd, k on and k off values ​​of human-mouse chimeric antibodies HFB3-1, -14 and -18 against His-tagged recombinant human TNFR2 are shown. [Diagram 5] We show expression of TNFR2 in T cell subtypes in tumor-infiltrating lymphocytes, specifically in exhausted CD8 T cells. [Figure 6]Cell binding of anti-TNFR2 chimeric monoclonal antibodies to TCR-activated (lower panel) and non-activated (upper panel) primary Tregs, CD8, and CD4 Tregs is shown. Primary T cells activated by CD3 / CD28 costimulation (TCR activation) are preferentially recognized by the HFB3 antibody. [Figure 7] We demonstrate that certain HFB3 antibodies of the invention (including HFB3-1, -14, -18, -23, -24, and -25) induce NFκB signaling, an effect that can be enhanced in the presence of a TNFα ligand. [Figure 8] We show that co-stimulation with certain subject monoclonal antibodies (including HFB3-1, -14, -18, and -25) and CD3 / CD28 resulted in the proliferation of CD8 and CD4 Tconvs in a dose-dependent manner. [Figure 9] The results show that the anti-TNFR2 monoclonal antibodies of the present invention (e.g., the humanized version of HFB3-1, HFB3-1hz6-hG1AA) dose-dependently favored cell proliferation of effector T cells (CD8 and CD4 Tconv) in the presence of Tregs. [Figure 10] 1 illustrates the lack of ADCC effect of the subject anti-TNFR2 antibodies. [Figure 11A] Various characteristics of the His-tagged extracellular domain (ECD) of TNFR2 containing the TNFα binding site (designated HFB2003) are shown, as well as epitope mapping results for either monoclonal antibodies HFB3-1 and HFB3-14, and HFB3-18 (FIG. 11A) and HFB3-6 (FIG. 11B). These are mouse chimeric antibodies with a human IgG1 Fc region, and therefore are also designated HFB3-1-hG1, HFB3-14-hG1, HFB3-18-hG1, or HFB3-6-hG1, respectively. Also included in FIG. 11B are epitope mapping data for the benchmark antibodies SBT-1 and SBT-4 (benchmarks 1 and 2). The HFB3-1 antibody binds to the CRD2 region of the ECD, HFB3-14 and HFB3-6 bind to the CRD3 region of the ECD, and HFB3-18 binds to the CRD1 region of the ECD. [Figure 11B] Various characteristics of the His-tagged extracellular domain (ECD) of TNFR2 containing the TNFα binding site (designated HFB2003) are shown, as well as epitope mapping results for either monoclonal antibodies HFB3-1 and HFB3-14, and HFB3-18 (FIG. 11A) and HFB3-6 (FIG. 11B). These are mouse chimeric antibodies with a human IgG1 Fc region, and therefore are also designated HFB3-1-hG1, HFB3-14-hG1, HFB3-18-hG1, or HFB3-6-hG1, respectively. Also included in FIG. 11B are epitope mapping data for the benchmark antibodies SBT-1 and SBT-4 (benchmarks 1 and 2). The HFB3-1 antibody binds to the CRD2 region of the ECD, HFB3-14 and HFB3-6 bind to the CRD3 region of the ECD, and HFB3-18 binds to the CRD1 region of the ECD. [Figure 11C] 1 shows more refined epitope mapping data for HFB3-1. The potential HFB3-1hG1 epitope region (SEQ ID NO: 101) is highlighted and was confirmed in two independent experiments. [Figure 11D] 3D model showing the binding sites of HFB3-1, HFB3-14, HFB3-6, and HFB-3-18 in the TNFR2-TNFα complex. [Figure 12A] We show that humanized variants of chimeric monoclonal antibodies HFB3-1, -14, and -18 bind to CHO cells expressing human TNFR2 (CHO.hTNFR2) but not to the parental CHO cells. [Figure 12B] Figure 1 shows the binding affinity of selected humanized anti-TNFR2 monoclonal antibodies. EC50 values ​​for the test humanized and parental chimeric antibodies were measured against CHO cells expressing human TNFR2 (CHO.hHFB3). [Figure 13] Binding affinity of selected humanized anti-TNFR2 monoclonal antibodies is shown. EC50 values ​​for the test humanized and parental chimeric antibodies were measured against CHO cells expressing rhesus TNFR2 (CHO.mkHFB3). [Figure 14A] ELISA assays show that the humanized anti-TNFR2 antibodies bind to recombinant human TNFR2 and cynomolgus monkey TNFR2, but not to recombinant human TNFR1. [Figure 14B] The binding affinity results for recombinant human TNFR2 by humanized variants and parental chimeric monoclonal antibodies HFB3-1 and -14 are shown based on AHC (anti-human IgG Fc capture) biosensor measurements. Values ​​are the average of two experiments obtained on two different days. [Figure 14C] Binding specificity of exemplary humanized antibody HFB3-1hz6-hG1 to TNRF2 expressing / positive CHO cells (CHO.hTNFR2) compared to parental CHO cells (Bmk 1: Benchmark Antibody 1). [Figure 15] 1 shows cellular binding of a humanized anti-TNFR2 monoclonal antibody to TCR-activated CD8 T cells. [Figure 16] 1 shows the costimulatory effect of a humanized anti-TNFR2 monoclonal antibody to expand TCR-activated CD4 T cells. [Figure 17A] We show that costimulation of Tregs with certain humanized variant anti-TNFR2 antibodies and TNFα resulted in NFκB downstream signaling. [Figure 17B] Figure 1 shows activation of NFκB signaling in CD8 T cells using certain humanized variants of the HFB3-1 antibody with or without recombinant human TNFα. "*" indicates statistical significance. [Figure 18] 1 shows that the subject humanized variant anti-TNFR2 antibodies are stable under storage. [Figure 19] 1 shows the FcγR cross-linking dependency of anti-TNFR2 monoclonal antibody HFB3-18 (but not HFB3-1 and -14) on costimulating primary T cells. [Figure 20] 1 shows a confirmed costimulatory effect of selected humanized anti-TNFR2 antibodies on the proliferation of CD8 T cells either in the presence or absence of TNFα. [Figure 21A] We show that the subject anti-TNFR2 monoclonal antibodies costimulate downstream NFκB signaling ex vivo in humanized TNFR2 knock-in CD8 and CD4 Tconv cells in the presence of CD3 / CD28-mediated TCR activation and 25 ng / mL TNFα. [Figure 21B] Humanized HFB3-1hz6 binds to peripheral CD4 and CD8 T cells (upper panel) and stimulates T cell proliferation in vitro in a dose-dependent manner in the presence of CD3 / CD28-mediated TCR activation (lower panel). [Figure 22] Figure 1 shows ex vivo activation of isolated natural killer (NK) cells by humanized and parental HFB3-1hz6-hG1 antibodies after stimulation with soluble IL-2 (10 ng / mL) and IL-15 (10 ng / mL). The experimental timeline is shown in the upper panel. Expression of CD107α and TNFR2 was upregulated in a dose-dependent manner by HFB3-1hz6-hG1 and HFB3-1-hG1, whereas isotype control and anti-OX40 antibody (BMS) failed to induce short-term NK activation. [Diagram 23] Ex vivo activation of natural killer (NK) cells in total peripheral blood mononuclear cell fraction by HFB3-1hz6-hG1 and parental murine HFB3-1-hG1 after stimulation with plate-bound anti-CD3 (1 μg / mL) and soluble anti-CD28 (1 μg / mL). The experimental timeline is shown in the upper panel. Among CD3- / CD56+ cells, CD107α expression was upregulated by HFB3-1hz6-hG1 and HFB3-1-hG1 in a dose-dependent manner, whereas the control anti-OX40 antibody (MBS) failed to induce short-term NK activation. [Figure 24A] 1 shows the timeline of the pharmacodynamic study in the mouse MC38 tumor model. Two doses of HFB3-1-hG1 at dosages of 0.1 mg / kg, 1 mg / kg, and 10 mg / kg, or an isotype-matched control antibody (TT) at 10 mg / kg, were administered intraperitoneally, 3 days apart. [Figure 24B] Figure 1 shows the in vivo effect of antibody treatment on total immune cell counts in MC38 tumors. Treatment with 10 mg / kg HFB3-1-hG1 increased the absolute number of CD45+ cells. p-value <0.05 (*) based on one-way ANOVA test. [Figure 24C] Figure 1 shows the in vivo effect on cell counts of various immune cells in MC38 tumors. Treatment with 10 mg / kg HFB3-1-hG1 increased the absolute cell numbers of CD8+, conventional CD4+ T and NK cells in the tumor microenvironment, but did not change the number of regulatory T cells. *p-value < 0.05 based on one-way ANOVA test. [Figure 25A] Shown is the percentage of TNFR2 receptors occupied by injected antibody HFB3-1-hG1 (at doses of 0.1 mg / kg, 1 mg / kg, and 10 mg / kg) or control antibody (at dose of 10 mg / kg) in tumor-infiltrating leukocytes. Only the 10 mg / kg dose of HFB3-1-hG1 resulted in drug receptor occupancy. p-values ​​<0.05 (*), 0.01 (**), or 0.001 (***) based on one-way ANOVA test. [Figure 25B] The percentage of TNFR2 receptors occupied by infused antibody HFB3-1-hG1 (at doses of 0.1 mg / kg, 1 mg / kg, and 10 mg / kg) or control antibody (at dose of 10 mg / kg) in selected peripheral blood cells is shown. HFB3-1-hG1 at doses of 10 mg / kg and 1 mg / kg resulted in equivalent drug receptor occupancy. p-values ​​<0.05 (*), 0.01 (**), or 0.001 (***) based on one-way ANOVA test. [Figure 26A] Figure 24A shows the antibody concentration in blood on day 4 of the experiment. HFB3-1-hG1 at doses of 10 mg / kg and 1 mg / kg was detectable in blood. p value < 0.001 (***) or 0.0001 (****) based on one-way ANOVA test. [Figure 26B]Figure 24A shows soluble TNFR2 in blood on day 4 of the experiment. HFB3-1-hG1 administration at 10 mg / kg and 1 mg / kg increased the amount of TNFR2 detectable in blood. p-value <0.001 (***) or 0.0001 (****) based on one-way ANOVA test. [Figure 27A] Humanized monoclonal antibodies (e.g., HFB3-1hz6 and HFB3-18hz1) have been shown to have similar therapeutic efficacy compared to rat anti-mPD-1 monoclonal antibodies. [Figure 27B] Humanized monoclonal antibodies (e.g., HFB3-1hz6 and HFB3-18hz1) have been shown to have similar therapeutic efficacy compared to rat anti-mPD-1 monoclonal antibodies. [Figure 28] We show that the humanized HFB3-1hz6 monoclonal antibody has therapeutic efficacy in the MC38 tumor model, similar to the murine anti-mPD-1 monoclonal antibody. [Figure 29] We show that humanized HFB3-1hz6 monoclonal antibody inhibits tumor growth and extends the life span of tumor-bearing mice at two different doses (3 mg / kg and 10 mg / kg), and that combination treatment with HFB3-1hz6 and anti-mPD-1 antibody extends survival better than treatment with anti-mPD-1 alone. [Figure 30A] The figures show that the humanized HFB3-1hz6 monoclonal antibody was eliminated from the cynomolgus monkeys over time (left panel) and that anti-drug antibodies (ADA), which are common in non-human primates, were observed approximately 2 weeks after injection (right panel). [Figure 30B] This shows that no increase in cytokines was observed after injection of 15, 50, or 150 mg / kg HFB3-1hz6-hG1, compared to reported data from CD3xCD20 bispecific IgG at ≤3 mg / kg (dotted line). [Diagram 31]Cell count analysis following injection of 15, 50, or 150 mg / kg HFB3-1hz6-hG1 is shown compared to the range of historical data from normal monkeys (left and right lines in each panel). [Diagram 32] We show that the humanized HFB3-1hz6 monoclonal antibody has antitumor efficacy in the Hepa1-6 tumor model. [Figure 33A] Kaplan-Meier survival curves of cutaneous melanoma (SKCM) patients in the TCGA database based on TNFR2 levels. [Figure 33B] Kaplan-Meier survival curves of head and neck squamous cell carcinoma (HNSC) patients in the TCGA database based on TNFR2 levels. [Figure 33C] Kaplan-Meier survival curves of thymoma (THYM) patients in the TCGA database based on TNFR2 levels. Higher TNFR2 expression is significantly associated with improved survival in melanoma and HNSC patients, but not in THYM. [Figure 34A] Examples of TCGA bulk RNA analysis of patients with multiple cancers with solid tumors are shown. Prostate cancer (PRAD) has low expression of CD8A and TNFR2 and can be used as a negative control for increased TNFR2 expression in the cancer type of choice. AML has low expression of CD8A (but not TNFR2) and can be used as a negative control for increased CD8A expression in the cancer type of choice. [Figure 34B] Examples of TCGA bulk RNA analysis of patients with multiple cancers with solid tumors are shown. Prostate cancer (PRAD) has low expression of CD8A and TNFR2 and can be used as a negative control for increased TNFR2 expression in the cancer type of choice. AML has low expression of CD8A (but not TNFR2) and can be used as a negative control for increased CD8A expression in the cancer type of choice. [Diagram 35]TCGA ranking of cancer types with high TNFR2 expression (e.g., compared to prostate cancer) and high CD8A expression (e.g., compared to AML) based on the percentage of patient samples that are TNFR2-high / CD8A-high. ACC=adrenocortical carcinoma, BLCA=bladder urothelial carcinoma, BRCA=breast invasive carcinoma, CESC=cervical squamous cell carcinoma / endocervical adenocarcinoma, CHOL=cholangiocarcinoma, COAD=colon adenocarcinoma, EBV=Epstein-Barr virus, ESCA=esophageal carcinoma, GBM=glioblastoma multiforme, HNSC=head and neck squamous cell carcinoma, KICH=renal chromophobe, KIRC=renal clear cell carcinoma, KIRP=renal papillary cell carcinoma, LGG=brain low-grade glioma, LIHC=liver hepatocellular carcinoma, LUAD = lung adenocarcinoma, LUSC = lung squamous cell carcinoma, MESO = pleural mesothelioma, OV = ovarian serous cystadenocarcinoma, PAAD = pancreatic adenocarcinoma, PCPG = pheochromocytoma and paraganglioma, PD-L1 = programmed death ligand 1, PRAD = prostate adenocarcinoma, READ = rectal adenocarcinoma, SARC = sarcoma, SKCM = cutaneous melanoma, STAD = gastric adenocarcinoma, TGCT = testicular germ cell tumor, THCA = thyroid carcinoma, UCEC = uterine endometrial carcinoma, UCS = uterine carcinosarcoma, UVM = uveal melanoma. [Diagram 36] Molecular subtype analysis for selected renal cell carcinoma (RCC), cutaneous melanoma (SKCM), gastric adenocarcinoma / gastric cancer (STAD / GI), lung adenocarcinoma (LUAD), and head and neck squamous cell carcinoma (HNSC) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] 1. Overview TNFR2 has recently emerged as a promising therapeutic target in cancer immunology. TNFR2 expression on regulatory and effector T cells in the tumor microenvironment (TME) is associated with T cell exhaustion and resistance to immune checkpoint blockade. The invention described herein provides antibodies against human TNFR2 that can be used as anti-cancer agents. Without wishing to be bound by any particular theory, it is believed that co-stimulation of effector T cells with the subject anti-TNFR2 antibodies enhances the anti-tumor activity of effector T cells.

[0063] In accordance with the invention described herein, mice were immunized with the recombinant extracellular domain (ECD) of human TNFR2 (rhTNFR2) to generate a diverse set of antibodies that were characterized for binding, cross-reactivity, selectivity, and functional activity. The antibodies were then subjected to immunoreactivity against CD8 + and CD4 + The antibodies were selected for their ability to induce the proliferation of effector T cells, as well as for increased NFkB signaling. The selected antibodies also preferably exhibit cross-reactivity to the monkey orthologue of rhTNFR2, which may be a beneficial feature for toxicity testing of human therapeutics in animals. Further desirable features include the ability of the subject antibodies to enhance the binding of human recombinant TNFα to TNFR2.

[0064] Two murine antibodies, HFB3-1 and HFB3-14, with subnanomolar or single-digit nanomolar binding affinities for human TNFR2, were initially selected for further characterization and humanization. Epitope mapping experiments showed that these two antibodies recognize different domains of TNFR2, with HFB3-1 binding to a region within the CRD2 domain and HFB3-14 binding to the CRD3 region. However, despite the different binding sites, both antibodies are selective for TNFR2, cross-react with the orthologues of cynomolgus and rhesus monkeys, and stimulate CD8 and conventional CD4 T cells (Tconv) in addition to enhancing the binding of human recombinant TNFα to TNFR2.

[0065] Several humanized variants of these murine antibodies, including HFB3-1hz6 and HFB3-14hz1c, retained the binding and cross-reactivity profiles of their respective parental antibodies. The humanized antibodies preferentially bound to TCR-activated primary CD8 and CD4 T cells compared to unstimulated T cells and promoted CD3 / CD28-induced activation and proliferation of T cells. This costimulatory mechanism of action was independent of cross-linking and is consistent with the ability of the antibodies to enhance NFκB signaling and induce upregulation of NFκB downstream target genes.

[0066] Furthermore, both humanized antibodies (HFB3-1hz6 and HFB3-14hz1c) showed a good developability profile and were stable under high temperature and low pH conditions, as well as after several freeze-thaw cycles. Good plasma exposure of the lead antibodies was also observed in mouse models. In vivo efficacy evaluation and initial toxicity analysis of these antibodies in mouse tumor models are underway.

[0067] We also identified a third murine monoclonal antibody, HFB3-18, which has a slightly lower (double-digit nM) binding affinity than the anti-mPD-1 monoclonal antibody but is able to inhibit tumor growth in vivo as well or better, and created a humanized version of it.

[0068] The functional profile of these antibodies, together with their favorable developability and pharmacokinetic profiles, support their development as novel immunotherapeutic options for cancer patients, particularly in certain cancer types and subtypes that exhibit high expression of TNFR2 and CD8A.

[0069] Detailed aspects of the invention are described further and separately in each of the following sections, however, it should be understood that any one embodiment of the invention (including those described only in the examples or drawings and those described only in one section below) may be combined with any other embodiment(s) of the invention.

[0070] Detailed aspects of the invention are described further and separately in each of the following sections, however, it should be understood that any one embodiment of the invention (including those described only in the examples or drawings and those described only in one section below) may be combined with any other embodiment(s) of the invention.

[0071] 2.Definition The term "antibody" in its broadest sense encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). The term "antibody" may also broadly refer to a molecule that includes a heavy chain complementarity determining region (CDR)1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3, and that is capable of binding to an antigen. The term "antibody" also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species (e.g., mouse, human, cynomolgus monkey, etc.).

[0072] However, in the narrow sense, "antibody" refers to various monoclonal antibodies including chimeric monoclonal antibodies, humanized monoclonal antibodies, and human monoclonal antibodies, particularly the humanized monoclonal antibodies of the present invention.

[0073] In some embodiments, the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR). In some embodiments, the antibody comprises at least one heavy chain (HC) comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain (LC) comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the antibody comprises two heavy chains, each heavy chain comprising at least a portion of a heavy chain variable region and a heavy chain constant region, and two light chains, each light chain comprising at least a portion of a light chain variable region and a light chain constant region.

[0074] As used herein, a single chain Fv (scFv) or any other antibody that comprises, for example, a single polypeptide chain that comprises all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have a heavy chain and a light chain. In some such embodiments, the heavy chain is the region of the antibody that comprises the three heavy chain CDRs and the light chain is the region of the antibody that comprises the three light chain CDRs.

[0075] As used herein, the term "heavy chain variable region (HCVR)" refers to a region comprising at least heavy chain CDR1 (CDR-H1), framework 2 (HFR2), CDR2 (CDR-H2), FR3 (HFR3), and CDR3 (CDR-H3). In some embodiments, the heavy chain variable region also comprises at least a portion (e.g., the entirety) of FR1 (HFR1) that is N-terminal to CDR-H1, and / or at least a portion (e.g., the entirety) of FR4 (HFR4) that is C-terminal to CDR-H3.

[0076] As used herein, the term "heavy chain constant region" refers to a region that includes at least three heavy chain constant domains: CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy constant region corresponds to an antibody isotype. For example, an antibody that includes a gamma constant region is an IgG antibody, an antibody that includes a delta constant region is an IgD antibody, an antibody that includes an alpha constant region is an IgA antibody, an antibody that includes an epsilon constant region is an IgE antibody, and an antibody that includes a mu constant region is an IgM antibody.

[0077] Certain isotypes may be further subdivided into subclasses, for example, IgG antibodies include, but are not limited to, IgG1 antibodies (containing a gamma 1 constant region), IgG2 antibodies (containing a gamma 2 constant region), IgG3 antibodies (containing a gamma 3 constant region), and IgG4 antibodies (containing a gamma 4 constant region), IgA antibodies include, but are not limited to, IgAl (containing an alpha 1 constant region) and IgA2 (containing an alpha 2 constant region), and IgM antibodies include, but are not limited to, IgM1 (containing a mu 1 constant region) and IgM2 (containing a mu 2 constant region).

[0078] As used herein, the term "heavy chain" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence, and with or without the C-terminal lysine.

[0079] As used herein, the term "light chain variable region (LCVR)" refers to a region comprising light chain CDR1 (CDR-L1), framework (FR) 2 (LFR2), CDR2 (CDR-L2), FR3 (LFR3), and CDR3 (CDR-L3). In some embodiments, the light chain variable region also comprises at least a portion (e.g., the entirety) of FR1 (LFR1) and / or at least a portion (e.g., the entirety) of FR4 (LFR4).

[0080] As used herein, the term "light chain constant region" refers to a light chain constant domain C L Non-limiting exemplary light chain constant regions include lambda and kappa.

[0081] As used herein, the term "light chain" refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. As used herein, the term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0082] The term "antibody fragment" or "antigen-binding portion (of an antibody)" includes, but is not limited to, fragments capable of binding to an antigen, such as Fv, single chain Fv (scFv), Fab, Fab', and (Fab')2. In certain embodiments, antibody fragments include Fab, Fab', F(ab')2, F d , single chain Fv or scFv, disulfide bond F v, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0083] The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 daltons and has antigen-binding activity. It contains approximately the N-terminal half of the heavy chain and the entire light chain connected by disulfide bridges. Fab can be obtained, in particular, by treating immunoglobulins with the protease papain.

[0084] The term "F(ab')2" refers to a fragment of approximately 100,000 daltons and antigen-binding activity. This fragment is slightly larger than two Fab fragments that are connected via disulfide bridges in the hinge region. These fragments can be obtained by treating immunoglobulins with the protease pepsin. Fab fragments can be obtained from F(ab')2 fragments by cleaving the disulfide bonds in the hinge region.

[0085] A single Fv chain "scFv" corresponds to a VH:VL polypeptide synthesized using genes encoding the VL and VH domains and a sequence encoding a peptide intended to bind these domains. An scFv according to the invention contains the CDRs maintained in the proper conformation, e.g., using recombinant genetic techniques.

[0086] A "scFv" dimer corresponds to two scFv molecules linked together by a peptide bond. The Fv chains are often the result of expression of a fusion gene comprising VH and VL encoding genes connected by a peptide-encoding linker sequence. Human scFv fragments can contain the CDR regions maintained in the proper conformation, preferably by the use of genetic recombination techniques.

[0087] A "dsFv" fragment is a VH-VL heterodimer stabilized by disulfide bonds, which may be bivalent (dsFV2). Divalent Sc(Fv)2 or multivalent antibody fragments may form spontaneously by association of monovalent scFvs or may be generated by linking scFv fragments through peptide bond sequences.

[0088] The Fc fragment retains the biological properties of the antibody, in particular its ability to be recognized by immune effectors or to activate complement. It consists of the constant fragment of the heavy chain beyond the hinge region.

[0089] The term "diabody" refers to small antibody fragments with two antigen-fixing sites. These fragments contain a variable heavy domain VH connected to a variable light domain VL in the same VH-VL polypeptide chain. If a binding sequence is used that is too short for the two domains in the same chain to match, By necessity matching two complementary domains on separate chains, two antigen-binding sites are created.

[0090] An "antibody that binds to the same epitope" as a reference antibody can be determined by an antibody competition assay. This refers to an antibody that blocks the binding of a reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. The term "competition", when used in the context of antibodies competing for the same epitope, means that competition between the antibodies is determined by an assay in which the antibody being tested prevents or inhibits the specific binding of the reference antibody to a common antigen.

[0091] Many types of competitive binding assays can be used, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid-phase direct label assays; solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); 125 solid-phase direct label RIA using (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552); and direct label RIA (see, Moldenhauer et al., 1990, Scand. J. Immunol.) can be used.

[0092] Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either an unlabeled test antigen-binding protein and a labeled reference antibody. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes to which the reference antibody binds in sufficient proximity to create steric hindrance. In some embodiments, the competing antibody, when present in excess, inhibits specific binding of the reference antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

[0093] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent (e.g., an antibody or an immunologically functional fragment thereof) and which can be used in a mammal to generate antibodies capable of binding to that antigen. An antigen can have one or more epitopes capable of interacting with an antibody.

[0094] The term "epitope" refers to a portion of an antigen molecule that is bound by a selective binding agent (e.g., an antibody or fragment thereof). The term includes any determinant capable of specific binding to an antibody. Epitopes may be contiguous or non-contiguous (e.g., in the case of a polypeptide, the amino acid residues are not contiguous to one another within the polypeptide sequence, but are bound by the antigen binding protein within the context of the molecule). In some embodiments, an epitope may be mimetic in that it contains a three dimensional structure similar to the epitope used to generate the antibody, but contains none or only some of the amino acid residues found in the epitope used to generate the antibody. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and may have three dimensional structural characteristics and / or specific charge characteristics.

[0095] In some embodiments, an "epitope" is defined by the method used to determine it. For example, in some embodiments, an antibody binds to the same epitope as a reference antibody if it binds to the same region of the antigen as quantified by hydrogen-deuterium exchange (HDX).

[0096] In certain embodiments, an antibody binds to the same epitope as a reference antibody if it binds to the same region of the antigen as determined by x-ray crystallography.

[0097] As used herein, a "chimeric antibody" refers to an antibody that comprises at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (e.g., human, cynomolgus monkey, chicken, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, all of the variable regions of the chimeric antibody are from a first species and all of the constant regions of the chimeric antibody are from a second species.

[0098] As used herein, a "humanized antibody" refers to an antibody in which at least one amino acid in the framework region of a non-human variable region (e.g., mouse, rat, cynomolgus monkey, chicken, etc.) is replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or a fragment thereof. In some embodiments, a humanized antibody fragment is a Fab, scFv, (Fab')2, etc.

[0099] As used herein, "CDR-grafted antibody" refers to a humanized antibody in which one or more complementarity determining regions (CDRs) of a first (non-human) species have been grafted onto the framework regions (FRs) of a second (human) species.

[0100] As used herein, "human antibody" refers to antibodies generated in humans, antibodies generated in non-human animals containing human immunoglobulin genes (e.g., XenoMouse®), and antibodies selected using in vitro methods (e.g., phage display) where the antibody repertoire is based on human immunoglobulin sequences.

[0101] "Host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells (e.g., primate or non-primate animal cells), fungal cells (e.g., yeast), plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives (e.g., 293-6E cells and DG44 cells, respectively).

[0102] As used herein, the term "isolated" refers to a molecule that is separated from at least some of the components typically found in nature or from at least some of the components typically produced. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell that produced it. If the polypeptide is secreted by the cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide that is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA) or when it is separated from at least some of the components of the cell that produced it, e.g., in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated" as long as the polynucleotide is not found within that vector in nature.

[0103] The terms "subject" and "patient" are used interchangeably herein and refer to a mammal, such as a human. In some embodiments, methods of treating other non-human mammals are also provided, including, but not limited to, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. In some cases, "subject" or "patient" refers to a (human) subject or patient in need of treatment for a disease or disorder.

[0104] As used herein, the term "sample" or "patient sample" refers to a substance obtained or derived from a subject of interest that contains cells and / or other molecular entities to be characterized and / or identified, e.g., based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase "disease sample" and variations thereof refer to any sample obtained from a subject of interest that is expected or known to contain the cells and / or molecular entities to be characterized.

[0105] "Tissue or cell sample" refers to a collection of similar cells obtained from the tissue of a subject or patient. The source of the tissue or cell sample can be solid tissue from fresh, frozen, and / or archived organ or tissue samples or biopsies or aspirates; blood or any blood constituent; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or tissue fluid; cells from any time point in the subject's gestation or development. The tissue sample can also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue or organ. The tissue sample can contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0106] As used herein, a "reference sample", "reference cell" or "reference tissue" refers to a sample, cell or tissue obtained from a source that is known or believed to be free of the disease or condition for which the method or composition of the present invention is being used to identify. In one embodiment, the reference sample, reference cell or reference tissue is obtained from a healthy part of the body of the same subject or patient for whom the composition or method of the present invention is being used to identify the disease or condition. In one embodiment, the reference sample, reference cell or reference tissue is obtained from a healthy part of the body of at least one individual who is not the subject or patient for whom the composition or method of the present invention is being used to identify the disease or condition. In some embodiments, the reference sample, reference cell or reference tissue is previously obtained from the patient before the onset of the disease or condition or at an early stage of the disease or condition.

[0107] A "disorder" or "disease" is any condition that would benefit from treatment with one or more Gal-9 antagonists of the present invention. This includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question. Non-limiting examples of disorders that may be treated herein include cancer.

[0108] "Diseases related to the suppressive activity of regulatory T lymphocytes" refers to any disease (not autoimmune) in which the suppressive activity of regulatory T lymphocytes is involved, particularly by promoting the onset or persistence of the disease.In particular, it has been demonstrated that the suppressive activity of regulatory T lymphocytes promotes the development of tumors.Therefore, the present invention is more particularly aimed at cancers in which the suppressive activity of T lymphocytes is involved.

[0109] The term "cancer" is used herein to refer to a group of cells that exhibits abnormally high proliferation and growth. Cancers can be benign (also referred to as benign tumors), pre-malignant, or malignant. Cancer cells can be solid cancer cells (i.e., cells that form solid tumors) or leukemic cancer cells. The term "cancer growth" is used herein to refer to the proliferation or growth by the cell or cells that make up the cancer that results in an increase in the size or extent of the corresponding cancer.

[0110] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific, non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain tumor, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancer.

[0111] In certain embodiments, cancer, as used herein, includes hematological cancers (e.g., AML and DLBCL), or solid tumors (e.g., breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer).

[0112] A "chemotherapeutic agent" is a chemical compound that may be useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and Cytoxan® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturdopa, and uredopa; ethylenimines and methylamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethyleneethyphosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189, and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongiostatins; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (such as the calicheamicins, especially calicheamicin gamma 1l and calicheamicin omegall (e.g., Agnew, Chem see Intl. Ed. Engl, 33:183-186 (1994)); dynemicins (including dynemicin A); bisphosphonates, e.g., clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enadiyne antibiotic chromophores, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin® doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxo rubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogues, e.g., denopterin, methotrexate. , pteropterin, trimetrexate; purine analogues, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenergics, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g. For example, furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diazicon; elfomitine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin; rosoxantrone;Podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, veracrine A, roridin A, and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as Taxol® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Abraxane® cremophor-free albumin engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and Taxotere® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; Gemzar® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (camptosar, CPT- 11) (including treatment regimens of irinotecan with 5-FU and leucovorin); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin (including oxaliplatin treatment regimens (FOLFOX)); inhibitors of PKC-alpha, Raf, H-Ras, EGFR, e.g., erlotinib (Tarceva®) which reduces cell proliferation, and VEGF-A inhibitors, as well as pharmacologic acceptable salts, acids, or derivatives of any of the above;

[0113] Further non-limiting exemplary chemotherapeutic agents include anti-hormonal agents that act to regulate or inhibit hormone action on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs) (e.g., tamoxifen (including Nolvadex® tamoxifen), raloxifene, droxifene, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY117018, onapristone, and Fareston® toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, Megase® megestrol acetate, Aromasin® exemestane, formestanie, fadrozole, Rivisor® vorozole, Femara® letrozole, and Arimidex® anastase inhibitors. lozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as, for example, PKC-alpha, Ralf, H-Ras; ribozymes, such as VEGF expression inhibitors (e.g., Angiozyme® ribozyme) and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, such as, for example, Allovectin® vaccine, Leuvectin® vaccine, and Vaxid® vaccine; Proleukin® rIL-2; Lurtotecan® topoisomerase 1 inhibitor; Abarelix® rmRH; and pharma- ceutically acceptable salts, acids, or derivatives of any of the above.

[0114] "Anti-angiogenic agents" or "angiogenesis inhibitors" refer to low molecular weight substances, polynucleotides (including, for example, inhibitory RNA (RNAi or siRNA)), polypeptides, isolated proteins, recombinant proteins, antibodies, or conjugates or fusion proteins thereof that either directly or indirectly inhibit angiogenesis, vasculogenesis, or undesirable vascular permeability. It is understood that angiogenesis inhibitors include agents that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, antiangiogenic agents are antibodies or other antagonists against angiogenic agents, such as antibodies against VEGF-A (e.g., bevacizumab (Avastin®)) or VEGF-A receptors (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors, such as Gleevec® (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, Sutent® / SU11248 (sunitinib malate), AMG706, or, for example, those described in International Patent Application WO2004 / 113304). Antiangiogenic agents also include native angiogenesis inhibitors (e.g., angiostatin, endostatin, etc.). See, e.g., Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3, which lists antiangiogenic therapies in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 2, which lists known antiangiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1, which lists antiangiogenic agents used in clinical trials).

[0115] As used herein, a "growth inhibitor" refers to a compound or composition that inhibits the growth of cells (e.g., cells expressing VEGF) in vitro or in vivo. Thus, a growth inhibitor can be one that significantly reduces the percentage of cells in S phase (e.g., cells expressing VEGF). Examples of growth inhibitors include, but are not limited to, agents that block cell cycle progression (at a stage other than S phase) (e.g., agents that induce G1 arrest and M phase arrest). Classical M phase inhibitors include vincas (vincristine and vinblastine), taxanes, topoisomerase II inhibitors (e.g., doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin). Agents that arrest G1, such as DNA alkylating agents (e.g., tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C), also extend the arrest to S phase. Further information can be found, for example, on page 13, in Mendelsohn and Israel, eds, The Molecular Basis of Cancer, Chapter 1, "Cell cycle regulation, oncogenes, and antitineoplastic drugs" (Murakami et al.) (WB Saunders, Philadelphia, 1995). The taxanes (paclitaxel and docetaxel) are anticancer drugs, both derived from the yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer) is derived from the European yew and is a semisynthetic analog of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, thereby inhibiting mitosis in cells.

[0116] The term "anti-neoplastic composition" refers to a composition useful for the treatment of cancer that includes at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, cancer immunotherapeutic agents (also referred to as cancer immunotherapy agents), apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (Tarceva®)), platelet-derived growth factor inhibitors (e.g., Gleevec® (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, CTLA4 inhibitors (e.g., anti-CTLA4 inhibitors), and anti-tumor agents. These include the antibody ipilimumab (YERVOY®), PD-1 inhibitors (e.g., anti-PD1 antibody, BMS-936558), PDL1 inhibitors (e.g., anti-PDL1 antibody, MPDL3280A), PDL2 inhibitors (e.g., anti-PDL2 antibody), VISTA inhibitors (e.g., anti-VISTA antibody), cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4, VISTA, or VEGF receptor(s), TRAIL / Apo2, and other bioactive and organic chemical agents. Combinations of these are also included in the present invention.

[0117] "Treatment" refers to therapeutic treatments, for example, where the objective is to slow (alleviate) the targeted pathological condition or disorder, and therapeutic treatments, for example, where the objective is to inhibit the recurrence of the pathological condition or disorder. "Treatment" encompasses any administration or application of a therapeutic agent to a disease (also referred to herein as a "disorder" or "condition") in a mammal (including a human), and includes inhibiting the disease or progression of the disease, suppressing or slowing the disease or its progression, arresting its onset, partially or completely alleviating the disease, partially or completely alleviating one or more symptoms of the disease, restoring or repairing a lost, defective, or defective function, or stimulating an inefficient process. The term "treatment" also includes reducing the severity of any phenotypic characteristic and / or reducing the incidence, extent, or likelihood of the characteristic. Those in need of treatment include those who already have the disorder, as well as those at risk of recurrence of the disorder or those in whom recurrence of the disorder should be prevented or delayed.

[0118] The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a subject. In some embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an antibody of the invention may vary depending on factors such as the individual's disease state, age, sex, weight, and the ability of the antagonist to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or adverse effects of the subject antibody are outweighed by the therapeutically beneficial effects.

[0119] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0120] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art for use with a therapeutic agent that together constitutes a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used. For example, if the therapeutic agent is administered orally, the carrier can be a gel capsule. If the therapeutic agent is administered subcutaneously, the carrier is ideally non-irritating to the skin and does not cause injection site reactions.

[0121] An "article of manufacture" refers to any manufacture (e.g., package or container) or kit that contains at least one reagent (e.g., a pharmaceutical agent for the treatment of a disease or disorder) or probe for specifically detecting a biomarker described herein. In some embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0122] 3. How to treat cancer The invention described herein provides anti-TNFR2 antibodies for use in methods of treating humans and other non-human mammals.

[0123] In pathological situations, Tregs can cause inappropriate immune suppression, thereby promoting, for example, tumor growth. Tregs have been implicated in reducing antitumor immune responses and promoting the development of numerous cancer types, particularly by inappropriately inhibiting the activity of effector T lymphocytes.

[0124] In some embodiments, methods are provided for treating or preventing cancer, comprising administering an effective amount of any of the subject anti-TNFR2 antibodies, or antigen-binding fragments thereof, to a subject in need of such treatment.

[0125] In some embodiments, methods of treating cancer are provided comprising administering to a subject with cancer any of the subject anti-TNFR2 antibodies, or antigen-binding fragments thereof.

[0126] Cancers treatable by the method / use of the present invention include cancers in which regulatory T lymphocytes exert their suppressive activity, such as cancers in which a relatively large amount of regulatory T lymphocytes are present in tumor tissue or circulation.The proliferation of regulatory T lymphocytes (which can be measured by the frequency of Treg) generally correlates with the increased activation of Treg.The frequency of regulatory T lymphocytes can be evaluated by any method known in the art, such as by flow cytometry (FACS) analysis of intratumoral or circulating lymphocytes, or by immunohistological staining of tumor tissue.

[0127] Non-limiting exemplary cancers (including carcinomas, lymphomas, germinomas, sarcomas, and leukemias) that can be treated using any of the subject anti-TNFR2 antibodies or antigen-binding fragments thereof are provided herein.More specific non-limiting examples of such cancers include melanoma, cervical cancer, squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain tumor, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, stomach cancer, melanoma, and various types of head and neck cancer.

[0128] In certain embodiments, the cancer is melanoma, breast cancer, colon cancer, cervical cancer, renal cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (NSCLC), ovarian cancer, skin cancer (e.g., squamous cell carcinoma or basal cell carcinoma), lymphoma, or leukemia.

[0129] In certain embodiments, the cancer has a high TNFR2 index, which is defined as the ratio of (a) the total number of CD8 T cells in the tumor sample × TNFR2 expression on CD8 T cells, and (b) the total number of Treg cells in the tumor sample × TNFR2 expression on Tregs.

[0130] In certain embodiments, the cancer has a TNFR2 index of more than 1, such as more than 1.5, more than 2, more than 3, more than 4, or more than 5. For example, representative TNFR2 indexes in certain cancers include 4.57 in melanoma, 1.67 in breast cancer, 1.05 in NSCLC, 1.03 in SCC, 0.78 in BCC, and 0.46 in HCC.

[0131] In certain embodiments, the cancer has a TNFR2 index of about 0.5 to about 1.

[0132] In certain embodiments, the cancer has a high percentage of CD8 TILs (tumor infiltrating lymphocytes), e.g., greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60% or more of the T cells in the tumor are CD8 T cells.

[0133] In certain embodiments, the cancer has low levels of TNFR2 expression in tumor cells.

[0134] In certain embodiments, the cancer is known to be susceptible to immunotherapy (e.g., inflammatory), such as melanoma, NSCLC, renal cell carcinoma, gastric cancer, colorectal cancer, urothelial carcinoma, HCC, head and neck cancer, and Hodgkin's lymphoma.

[0135] In certain embodiments, the cancer has high levels of TNFR2 expression in intratumoral exhausted T cells (e.g., exhausted CD8 T cells). Such cancers can be treated, for example, by combination therapy with an antagonist of the PD-1 / PD-L1 pathway (e.g., any anti-PD-1 or anti-PD-L1 antibody, such as those specifically described herein or known in the art).

[0136] In certain embodiments, the methods / uses of the invention can be used to treat cancers where high levels of regulatory T lymphocytes are known and / or where the cancer / tumor is associated with a significantly poor prognosis, including chronic myeloid leukemia (CML), colon cancer, melanoma, uterine cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, primary lymphoma of the central nervous system, multiple myeloma, prostate cancer, Hodgkin's lymphoma, or hepatocellular carcinoma.

[0137] In some embodiments, the cancer is a hematological cancer (e.g., AML and DLBCL), or a solid tumor (e.g., breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer).

[0138] In some embodiments, the cancer is BCC, SCC, melanoma, colon cancer, or NSCLC.

[0139] In certain embodiments, the cancer has high levels of TNFR2 expression and CD8A expression. In certain embodiments, the high or higher levels of TNFR2 expression are relative to / compared to the average TNFR2 expression level in prostate cancer patients, and optionally TNFR2 expression is assessed in effector T cells (e.g., CD4+ and / or CD8+ T cells), tumor-infiltrating CD8+ T cells, and / or NK cells, and / or the high or higher levels of CD8A expression are relative to / compared to the average CD8A expression level in AML patients.

[0140] In certain embodiments, the patient (e.g., the patient's cancer) is characterized by tumor-infiltrating CD8A + (CD8 α-chain positive) T cells have higher levels of TNFR2 expression.

[0141] In certain embodiments, the patient is + I have stomach cancer.

[0142] In certain embodiments, the patient has gastric adenocarcinoma (e.g., gastric adenocarcinoma with increased / high PD-L1 / CD274 expression).

[0143] In certain embodiments, the patient has clear cell renal cell carcinoma (RCC).

[0144] In certain embodiments, the patient has renal clear cell carcinoma (KIRC). In certain embodiments, the patient has KIRC.2, KIRC.3, or KIRC.4 subtype. In certain embodiments, the patient has clear cell type B (ccB) subtype, or ccA (clear cell type A) / ccB unclassified subtype.

[0145] In certain embodiments, the patient has cutaneous melanoma.

[0146] In certain embodiments, the patient has cutaneous melanoma (SKCM).

[0147] In certain embodiments, the patent has a subtype with a BRAF hotspot mutation (eg, a V600E, V600K, or V600R mutation) or a hotspot mutation in K601.

[0148] In certain embodiments, the patient has a RAS hotspot mutation. In certain embodiments, the RAS hotspot mutation is an NRAS hotspot mutation (e.g., Q61R, Q61K, Q61L, Q61H, 61_62QE>HK, G12R / D / A, and G13R / D). In certain embodiments, the RAS hotspot mutation is an HRAS hotspot mutation (e.g., G13D, G13S, or Q61K). In certain embodiments, the RAS hotspot mutation is a KRAS hotspot mutation (e.g., G12D, G12R, or Q61R).

[0149] In certain embodiments, the patient has a subtype involving any NF1 mutation.

[0150] In certain embodiments, the patient has the triple wt subtype of SKCM lacking hotspot BRAF, N / H / K-RAS, or NF1 mutations.

[0151] In certain embodiments, the patient has testicular germ cell tumor.

[0152] In certain embodiments, the patient has a soft tissue sarcoma.

[0153] In certain embodiments, the cancer expresses PD-L1 at a higher than average level.

[0154] In certain embodiments, the cancer is cervical cancer (eg, cervical squamous cell carcinoma or endocervical adenocarcinoma), pleural mesothelioma, lung adenocarcinoma, or head and neck squamous cell carcinoma (HNSC).

[0155] In certain embodiments, the patient has an HNSC subtype (e.g., an atypical subtype). In certain embodiments, the atypical subtype HNSC is further HPV positive.

[0156] In certain embodiments, the patient has HNSC mesenchymal subtype. In certain embodiments, the mesenchymal subtype has high PD-L1 / CD274 expression.

[0157] In certain embodiments, the method / use of the present invention can be used to treat recurrence of fibrosis due to Hepatitis C, since it has also been demonstrated that an increased frequency of regulatory T lymphocytes is a predictor of recurrence of such fibrosis.

[0158] In some embodiments, the anti-TNFR2 antibodies of the present invention may be used alone or in combination with other suitable compounds known to be capable of treating a disease or indication.

[0159] Thus, according to a particular embodiment of the invention, an antibody directed against TNFR2 and inhibiting the suppressor activity of regulatory T lymphocytes as defined above is used in combination with a second therapeutic agent (e.g., an anti-cancer agent) for treating a disease associated with the suppressor activity of regulatory T lymphocytes.

[0160] That is, when the use is the treatment of cancer, the antibody can be used in combination with known therapies against cancer, such as surgery, radiotherapy, chemotherapy, or a combination thereof. For example, the antibody can be used in combination with adoptive immunotherapy consisting of one or more injections of effector lymphocytes against tumor antigens (particularly EBV antigens). According to some embodiments, other anti-cancer agents used in combination with the antibody against TNFR2 according to the present invention for cancer therapy include anti-angiogenic agents. According to certain embodiments, the antibody can be co-administered with (cytokines, for example, cytokines that stimulate anti-tumor immune responses).

[0161] In such combination therapy, the antibodies of the invention can be used before, after, or simultaneously with the second therapeutic agent, see further section below on combination therapy.

[0162] 4. Route of Administration and Carriers In various embodiments, the subject anti-TNFR2 monoclonal antibodies can be administered subcutaneously or intravenously. For ease of reference, "the subject anti-TNFR2 monoclonal antibodies" refers to the mouse-human chimeric anti-TNFR2 antibodies of the invention and humanized variants thereof.

[0163] In some embodiments, the subject anti-TNFR2 monoclonal antibodies can be administered in vivo by a variety of routes, including, but not limited to, oral, intraarterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, inhalation, intradermal, topical, transdermal, and intrathecal, or by other methods (e.g., implantation).

[0164] In some embodiments, the subject anti-TNFR2 monoclonal antibodies can be administered intravenously or subcutaneously.

[0165] The subject antibody compositions can be formulated into solid, semi-solid, liquid, or gaseous form preparations, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injectable solutions, inhalants, and aerosols.

[0166] In various embodiments, compositions comprising the subject anti-TNFR2 monoclonal antibodies are provided in formulations with a wide variety of pharma- ceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). A variety of pharma-ceutically acceptable carriers, including vehicles, adjuvants, and diluents, can be used. In addition, a variety of pharma-ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, can also be used. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0167] In various embodiments, the subject TNFR2 monoclonal antibodies can be formulated for injection (including subcutaneous administration) by dissolving, suspending, or emulsifying in an aqueous or non-aqueous solvent, such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol, together with solubilizing agents, isotonicity agents, suspending agents, emulsifying agents, stabilizers, and preservatives as desired.

[0168] In various embodiments, the compositions can be formulated for inhalation using pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0169] The composition can also be formulated in various embodiments into sustained release microcapsules, for example, using biodegradable or non-biodegradable polymers.Non-limiting exemplary biodegradable formulations include polylactic-co-glycolic acid (PLGA) polymers.Non-limiting exemplary non-biodegradable formulations include polyglycerol fatty acid esters.Certain methods for making such formulations are described, for example, in EP1125584A1.

[0170] Also provided are pharmaceutical dosage packs comprising one or more containers, each containing one or more doses of a subject anti-TNFR2 monoclonal antibody. In some embodiments, unit dosages are provided, the unit dosages comprising a predetermined amount of a composition comprising a subject anti-TNFR2 monoclonal antibody, with or without one or more additional agents. In some embodiments, such unit dosages are provided in disposable pre-filled injection syringes. In various embodiments, the composition contained in the unit dosage can include saline, sucrose, or the like, a buffer (e.g., phosphate), and / or can be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition can be provided as a lyophilized powder that can be reconstituted by adding an appropriate liquid (e.g., sterile water). In some embodiments, the composition includes one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, the compositions of the present invention include heparin and / or proteoglycan.

[0171] The pharmaceutical compositions are administered in an amount effective for treating or preventing a particular indication. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the condition being treated, or the age of the subject being treated.

[0172] In some embodiments, a subject anti-TNFR2 monoclonal antibody can be administered in an amount ranging from about 50 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, a subject anti-TNFR2 monoclonal antibody can be administered in an amount ranging from about 100 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, a subject anti-TNFR2 monoclonal antibody can be administered in an amount ranging from about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, a subject anti-TNFR2 monoclonal antibody can be administered in an amount ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose.

[0173] In some embodiments, a subject anti-TNFR2 monoclonal antibody can be administered in an amount ranging from about 10 mg to about 1,000 mg per dose. In some embodiments, a subject anti-TNFR2 monoclonal antibody can be administered in an amount ranging from about 20 mg to about 500 mg per dose. In some embodiments, a subject anti-TNFR2 monoclonal antibody can be administered in an amount ranging from about 20 mg to about 300 mg per dose. In some embodiments, a subject anti-TNFR2 monoclonal antibody can be administered in an amount ranging from about 20 mg to about 200 mg per dose.

[0174] The subject anti-TNFR2 monoclonal antibody compositions can be administered to a subject as needed. In some embodiments, an effective dose of a subject anti-TNFR2 monoclonal antibody is administered to a subject one or more times. In various embodiments, an effective dose of a subject anti-TNFR2 monoclonal antibody is administered to a subject once a month, less than once a month, for example, every two months, every three months, or every six months. In other embodiments, an effective dose of a subject anti-TNFR2 monoclonal antibody is administered multiple times a month, for example, every two weeks, every week, twice a week, three times a week, daily, or multiple times a day. An effective dose of a subject anti-TNFR2 monoclonal antibody is administered to a subject at least once. In some embodiments, an effective dose of a subject anti-TNFR2 monoclonal antibody can be administered multiple times (including over a period of at least one month, at least six months, or at least one year). In some embodiments, a subject anti-TNFR2 monoclonal antibody is administered to a subject as needed to alleviate one or more symptoms of a condition.

[0175] 5. Combination Therapy The subject anti-TNFR2 monoclonal antibody (including its functional fragment) of the present invention can be administered to a subject in need thereof in combination with other biologically active substances or other therapeutic procedures for the treatment of disease.For example, the subject anti-TNFR2 monoclonal antibody can be administered alone or together with other therapeutic modalities.They can be provided before, substantially simultaneously with, or after other therapeutic modalities (e.g., radiation therapy).

[0176] In treating cancer, the subject anti-TNFR2 monoclonal antibodies can be administered in combination with one or more anti-cancer agents (e.g., immune checkpoint inhibitors, chemotherapeutic agents, growth inhibitory agents, anti-angiogenic agents, or anti-neoplastic compositions).

[0177] In certain embodiments, the subject anti-TNFR2 monoclonal antibodies that specifically bind to TNFR2 ("TNFR2-binding antagonists"), e.g., TNFR2 antagonist antibodies or antigen-binding fragments thereof, are administered together with a second antagonist, e.g., an immune checkpoint inhibitor (e.g., an inhibitor of the PD-1 or PD-L1 pathway), to a subject with a disease in which stimulation of the immune system would be beneficial (e.g., cancer or an infectious disease). The two antagonists can be administered simultaneously or sequentially, e.g., as described below for the combination of the subject anti-TNFR2 monoclonal antibodies with cancer immunotherapy. To treat cancer or autoimmune disease, treatment with the subject anti-TNFR2 monoclonal antibodies can be supplemented with one or more additional therapeutic agents, e.g., checkpoint modulators.

[0178] In certain embodiments, the subject anti-TNFR2 monoclonal antibodies are administered to a subject (e.g., a subject with cancer) simultaneously or sequentially with another treatment.For example, the subject anti-TNFR2 monoclonal antibodies can be administered with one or more of radiation therapy, surgery, or chemotherapy, such as targeted chemotherapy or immunotherapy.

[0179] In certain embodiments, a method of treating a subject with cancer comprises administering to the subject an anti-TNFR2 monoclonal antibody of the invention and one or more immuno-cancer agents (e.g., immune checkpoint inhibitors).

[0180] Immunotherapy (e.g., cancer immunotherapy) is effective in enhancing, stimulating, and / or upregulating immune response in a subject. In one embodiment, the administration of the subject anti-TNFR2 monoclonal antibody and cancer immunotherapy (e.g., PD-1 inhibitor) has a synergistic effect in treating cancer, e.g., inhibiting tumor growth.

[0181] In one embodiment, the subject anti-TNFR2 monoclonal antibodies are administered sequentially prior to administration of an immuno-cancer agent. In one embodiment, the subject anti-TNFR2 monoclonal antibodies are administered simultaneously with an immuno-cancer agent (e.g., a PD-1 inhibitor). In one embodiment, the subject anti-TNFR2 monoclonal antibodies are administered sequentially after administration of an immuno-cancer agent (e.g., a PD-1 inhibitor). Administration of the two agents may begin, for example, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 5 days, 7 days, or a week or more apart, or administration of the second agent may begin, for example, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 5 days, 7 days, or a week after administration of the first agent.

[0182] In certain embodiments, a subject anti-TNFR2 monoclonal antibody and an immuno-cancer agent (e.g., a PD-1 inhibitor) are administered simultaneously, e.g., infused simultaneously, e.g., over a period of 30 minutes or 60 minutes, into a patient. A subject anti-TNFR2 monoclonal antibody can be co-formulated with an immuno-cancer agent (e.g., a PD-1 inhibitor).

[0183] The cancer immunotherapy agent may, for example, be a small molecule drug, an antibody or its fragment, or other biologic or small molecule. Examples of biological cancer immunotherapy agents include, but are not limited to, antibodies, antibody fragments, vaccines, and cytokines. In one embodiment, the antibody is a monoclonal antibody. In certain embodiments, the monoclonal antibody is a humanized or human antibody.

[0184] In one embodiment, the cancer immunotherapy agent is an agonist of (i) stimulatory (including costimulatory) molecules (e.g., receptors or ligands) on immune cells, e.g., T cells, or an antagonist of (ii) inhibitory (including co-inhibitory) molecules (e.g., receptors or ligands), both of which lead to the amplification of antigen-specific T cell responses.In certain embodiments, the cancer immunotherapy agent is an agonist of (i) stimulatory (including costimulatory) molecules (e.g., receptors or ligands) on cells involved in innate immunity (e.g., NK cells), or an antagonist of (ii) inhibitory (including co-inhibitory) molecules (e.g., receptors or ligands), and the cancer immunotherapy agent enhances innate immunity.Such cancer immunotherapy agents are often referred to as immune checkpoint modulators, e.g., immune checkpoint inhibitors or immune checkpoint stimulators.

[0185] In certain embodiments, the cancer immunotherapy agent may be an agent that targets (or specifically binds) a member of the B7 family of membrane-bound ligands (including B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5, and B7-H6), or a costimulatory or co-inhibitory receptor that specifically binds to a member of the B7 family. The cancer immunotherapy agent may be an agent that targets (or specifically binds) a member of the TNF family of membrane-bound ligands (e.g., a member of the TNF receptor family). Exemplary TNF and TNFR family members that may be targeted by an immuno-oncology agent include CD40 and CD40L, OX-40, OX-40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fnl4, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTfiR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin alpha / ΤΝΡβ, TNFR2, TNFa, LTfiR, lymphotoxin a 1β2, FAS, FASL, RELT, DR6, TROY, and NGFR. The cancer immunotherapy agent that can be used in combination with the subject anti-TNFR2 monoclonal antibody to treat cancer can be an agent, such as an antibody, that targets B7 family members, B7 receptor family members, TNF family members, or TNFR family members (such as those described above).

[0186] In one embodiment, a subject anti-TNFR2 monoclonal antibody is capable of binding to: (i) proteins that inhibit T cell activation (e.g., CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM3, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PDIH, LAIRl, TIM-1, TIM-4, and PSGL-1, and (ii) an antagonist of PSGL-1, and PSGL-1 (e.g., an immune checkpoint inhibitor), and (ii) an agonist of a protein that stimulates T cell activation (e.g., B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, CD40L, DR3, and CD28H).

[0187] In one embodiment, the cancer immunotherapy agent is an agent that inhibits (i.e., an antagonist of) cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive cytokines), or an agonist (e.g., the cytokine itself) of cytokines that stimulate T cell activation (e.g., IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα) and stimulate the immune response.

[0188] Other agents that can be combined with the subject anti-TNFR2 monoclonal antibodies to stimulate the immune system, for example, for the treatment of cancer and infectious diseases, include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, the subject anti-TNFR2 monoclonal antibodies can be combined with antagonists of KIR.

[0189] Still other agents for combination therapy include agents that inhibit or deplete macrophages or monocytes, including, but not limited to, CSF-IR antagonists, e.g., CSF-IR antagonist antibodies, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA008 (WO11 / 140249; WO13169264; WO14 / 036357).

[0190] Immuno-oncology agents also include agents that inhibit TGF-β signaling.

[0191] Additional agents that can be combined with the subject anti-TNFR2 monoclonal antibodies include agents that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, and Imiquimod) or therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines).

[0192] Yet other therapies that can be combined with the subject anti-TNFR2 monoclonal antibodies include therapies that deplete or block Treg cells (eg, agents that specifically bind to CD25).

[0193] Additional therapies that can be combined with the subject anti-TNFR2 monoclonal antibodies include those that inhibit metabolic enzymes, such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase.

[0194] Another class of agents that may be used includes agents that inhibit the formation of adenosine or agents that inhibit the adenosine A2A receptor.

[0195] Other therapies that can be combined with the subject anti-TNFR2 monoclonal antibodies to treat cancer include therapies that reverse / prevent T cell allergy or exhaustion, and therapies that induce innate immune activation and / or inflammation at the tumor site.

[0196] The subject anti-TNFR2 monoclonal antibodies can be combined with multiple cancer immunological agents (e.g., immune checkpoint inhibitors), e.g., combinatorial approaches that target multiple elements of immune pathways, such as one or more of the following: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, Imiquimod); therapies that inhibit negative immune regulation, e.g., by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or by depleting or blocking Tregs or other immune suppressive cells; therapies that stimulate positive immune regulation, e.g., using agonists that stimulate the CD-137, OX-40, and / or GITR pathways and / or stimulate T cell effector function; therapies that systemically increase the frequency of anti-tumor T cells; e.g., using antagonists of CD25 (e.g., daclizumab) or ex vivo. Therapies that deplete or inhibit Tregs (e.g., Tregs within the tumor) by in vivo anti-CD25 bead depletion; therapies that affect the function of suppressor myeloid cells within the tumor; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell transfer, including genetically modified cells (e.g., cells modified with chimeric antigen receptor (CAR-T therapy)); therapies that inhibit metabolic enzymes (e.g., indoleamine dioxygenase (IDO), dioxygenase, arginase, nitric oxide synthase); therapies that restore / prevent T cell anergy or exhaustion; therapies that induce innate immune activation and / or inflammation at the tumor site; administration of immunostimulatory cytokines or blockade of immunosuppressive cytokines.

[0197] For example, the subject anti-TNFR2 monoclonal antibodies can be used in conjunction with one or more agonistic agents that ligate positive costimulatory receptors; one or more antagonists (blocking agents) that attenuate signaling through inhibitory receptors (e.g., antagonists that overcome unique immunosuppressive pathways within the tumor microenvironment) (e.g., blockade of PD-L1 / PD-1 / PD-L2 interaction); one or more agents that systemically increase the frequency of anti-tumor immune cells (e.g., T cells) and deplete or inhibit Tregs (e.g., by inhibiting CD25); one or more agents that inhibit metabolic enzymes (e.g., IDO); one or more agents that restore / prevent T cell anergy or exhaustion; and one or more agents that induce innate immune activation and / or inflammation at the tumor site.

[0198] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system (e.g., cancer or an infectious disease) is treated by administering to the subject a subject anti-TNFR2 monoclonal antibody and an immuno-oncology agent, where the immuno-oncology agent is a CTLA-4 antagonist (e.g., an antagonistic CTLA-4 antibody). Suitable CTLA-4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab.

[0199] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system (e.g., cancer or infectious disease) is treated by administering to the subject a subject anti-TNFR2 monoclonal antibody and an immuno-oncology agent, where the immuno-oncology agent is a PD-1 antagonist (e.g., an antagonistic PD-1 antibody). Suitable PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO2012 / 145493). Immuno-oncology agents also include pidilizumab (CT-011). Another approach to targeting the PD-1 receptor is a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224.

[0200] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system (e.g., cancer or an infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the invention and an immuno-oncology agent, where the immuno-oncology agent is a PD-L1 antagonist (e.g., an antagonistic PD-L1 antibody). Suitable PD-L1 antibodies include, for example, MPDL3280A (RG7446; WO2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), MSB0010718C (WO2013 / 79174), or rHigM12B7.

[0201] In one embodiment, a subject having a disease that may benefit from stimulating the immune system (e.g., cancer or infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the present invention and an immuno-oncology agent, where the immuno-oncology agent is a LAG-3 antagonist (e.g., an antagonistic LAG-3 antibody). Suitable LAG3 antibodies include, for example, BMS-986016 (WO10 / 19570, WO14 / 08218), or IMP-731 or IMP-321 (WO08 / 132601, WO09 / 44273).

[0202] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system (e.g., cancer or an infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the present invention and an immuno-oncology agent, where the immuno-oncology agent is a CD137 (4-1BB) agonist (e.g., an agonistic CD137 antibody). Suitable CD137 antibodies include, for example, urelumab or PF-05082566 (W012 / 32433).

[0203] In one embodiment, a subject having a disease that may benefit from stimulating the immune system (e.g., cancer or infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the present invention and an immuno-oncology agent, where the immuno-oncology agent is a GITR agonist (e.g., an agonistic GITR antibody). Suitable GITR antibodies include, for example, TRX-518 (WO06 / 105021, WO09 / 009116), MK-4166 (WO11 / 028683), or the GITR antibodies disclosed in WO2015 / 031667.

[0204] In one embodiment, a subject with a disease that may benefit from stimulating the immune system (e.g., cancer or infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the present invention and an immuno-oncology agent, wherein the immuno-oncology agent is an OX40 agonist (e.g., an agonistic OX40 antibody). Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469, or MOXR0916 (RG7888; WO06 / 029879).

[0205] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system (e.g., cancer or infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the present invention and an immuno-oncology agent, where the immuno-oncology agent is a CD40 agonist (e.g., an agonistic CD40 antibody). In certain embodiments, the immuno-oncology agent is a CD40 antagonist (e.g., an antagonistic CD40 antibody). Suitable CD40 antibodies include, for example, lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893, or Chi Lob7 / 4.

[0206] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system (e.g., cancer or an infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the present invention and an immuno-oncology agent, where the immuno-oncology agent is a CD27 agonist (e.g., an agonistic CD27 antibody). Suitable CD27 antibodies include, for example, valilumab (CDX-1127).

[0207] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system (e.g., cancer or an infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the invention and an immuno-oncology agent, where the immuno-oncology agent is MGA271 (directed against B7H3) (WO1 1 / 109400).

[0208] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system (e.g., cancer or an infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the invention and an immuno-oncology agent, where the immuno-oncology agent is a KIR antagonist (e.g., lirilumab).

[0209] In one embodiment, a subject with a disease that may benefit from stimulating the immune system (e.g., cancer or infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the present invention and an immuno-cancer agent, wherein the immuno-cancer agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006 / 122150, WO07 / 75598, WO08 / 36653, WO08 / 36642), indoximod, NLG-919 (WO09 / 73620, WO09 / 1156652, WO1 1 / 56652, WO12 / 142237), or F001287.

[0210] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system, e.g., cancer or an infectious disease, is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the invention and an immuno-cancer agent, where the immuno-cancer agent is a Toll-like receptor agonist, e.g., a TLR2 / 4 agonist (e.g., Bacillus Calmette-Guerin); a TLR7 agonist (e.g., Hiltonol or Imiquimod); a TLR7 / 8 agonist (e.g., Resiquimod); or a TLR9 agonist (e.g., CpG7909).

[0211] In one embodiment, a subject having a disease that may benefit from stimulation of the immune system (e.g., cancer or an infectious disease) is treated by administering to the subject an anti-TNFR2 monoclonal antibody of the invention and an immuno-cancer agent, where the immuno-cancer agent is a TGF-β inhibitor, e.g., GC1008, LY2157299, TEW7197, or IMC-TR1.

[0212] 6. Exemplary Anti-TNFR2 Monoclonal Antibodies The invention described herein provides monoclonal antibodies, or antigen-binding fragments thereof, specific for TNFR2.

[0213] Thus, one aspect of the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that competes with any of the isolated monoclonal antibodies or antigen-binding fragments thereof described herein for binding to the epitope of SEQ ID NO: 13 / 101 or 38, or to the epitope bound by HFB3-18.

[0214] For example, the epitope of HFB3-1 / HFB3-1-hG1 is shown in Figures 11A-11C (SEQ ID NO: 13 in Figures 11A and 11B, and SEQ ID NO: 101 in Figure 11C).

[0215] A related aspect of the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to an epitope of SEQ ID NO: 13 / 101 or 38, or an epitope bound by HFB3-18.

[0216] Another related aspect of the invention provides an isolated monoclonal antibody or antibody-binding fragment thereof, the isolated monoclonal antibody or antibody-binding fragment thereof is specific to human TNFR2, the isolated monoclonal antibody or antibody-binding fragment thereof comprising: (1a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 1, the HCVR CDR2 sequence of SEQ ID NO: 2, and the HCVR CDR3 sequence of SEQ ID NO: 3; and (1b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 4, the LCVR CDR2 sequence of SEQ ID NO: 5, and the LCVR CDR3 sequence of SEQ ID NO: 6; or (2a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 14, the HCVR CDR2 sequence of SEQ ID NO: 15, and the HCVR CDR3 sequence of SEQ ID NO: 16; and (2b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 17, the LCVR CDR2 sequence of SEQ ID NO: 18, and the LCVR CDR3 sequence of SEQ ID NO: 19. or (3a) a heavy chain variable region (HCVR) comprising an HCVR CDR1 sequence of SEQ ID NO: 26, an HCVR CDR2 sequence of SEQ ID NO: 27, and an HCVR CDR3 sequence of SEQ ID NO: 28; and (3b) a light chain variable region (LCVR) comprising an LCVR CDR1 sequence of SEQ ID NO: 29, an LCVR CDR2 sequence of SEQ ID NO: 30, and an LCVR CDR3 sequence of SEQ ID NO: 31; or (4a) a heavy chain variable region (HCVR) comprising an HCVR CDR1 sequence of SEQ ID NO: 39, an HCVR CDR2 sequence of SEQ ID NO: 40, and an HCVR CDR3 sequence of SEQ ID NO: 41; and (4b) a light chain variable region (LCVR) comprising an LCVR CDR1 sequence of SEQ ID NO: 42, an LCVR CDR2 sequence of SEQ ID NO: 43, and an LCVR CDR3 sequence of SEQ ID NO: 44. or (5a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 51, the HCVR CDR2 sequence of SEQ ID NO: 52, and the HCVR CDR3 sequence of SEQ ID NO: 53; and (5b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 54.or (6a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 63, the HCVR CDR2 sequence of SEQ ID NO: 64, and the HCVR CDR3 sequence of SEQ ID NO: 65; and (6b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 66, the LCVR CDR2 sequence of SEQ ID NO: 67, and the LCVR CDR3 sequence of SEQ ID NO: 68.

[0217] Regarding any of the above aspects of the invention, in some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, (1A) the HCVR sequence is SEQ ID NO:7, and / or (1B) the LCVR sequence is SEQ ID NO:8, or (2A) the HCVR sequence is SEQ ID NO:20, and / or (2B) the LCVR sequence is SEQ ID NO:21, or (3A) the HCVR sequence is SEQ ID NO:32, and / or (3B) the LCVR sequence is SEQ ID NO:33, or (4A) the HCVR sequence is SEQ ID NO:45, and / or (4B) the LCVR sequence is SEQ ID NO:46, or (5A) the HCVR sequence is SEQ ID NO:57, and / or (5B) the LCVR sequence is SEQ ID NO:58, or (6A) the HCVR sequence is SEQ ID NO:69, and / or (6B) the LCVR sequence is SEQ ID NO:70.

[0218] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof has (1a) the heavy chain sequence of SEQ ID NO:9 and / or (1b) the light chain sequence of SEQ ID NO:10, or (2a) the heavy chain sequence of SEQ ID NO:22 and / or (2b) the light chain sequence of SEQ ID NO:23, or (3a) the heavy chain sequence of SEQ ID NO:34 and / or (3b) the light chain sequence of SEQ ID NO:35, or (4a) the heavy chain sequence of SEQ ID NO:47 and / or (4b) the light chain sequence of SEQ ID NO:48, or (5a) the heavy chain sequence of SEQ ID NO:59 and / or (5b) the light chain sequence of SEQ ID NO:60, or (6a) the heavy chain sequence of SEQ ID NO:71 and / or (6b) the light chain sequence of SEQ ID NO:72.

[0219] A portion of the sequence of the antibody of the present invention is shown below. HFB3-1-hG1 (mouse monoclonal antibody) CDR-H1: SYSFTDYN (SEQ ID NO: 1) CDR-H2: IFPKYGTTSYNQKFKG (SEQ ID NO: 2) CDR-H3: ATDGGTWYFDV (SEQ ID NO: 3) CDR-L1: SSVTY (SEQ ID NO: 4) CDR-L2: LTSNLASGVPA (SEQ ID NO: 5) CDR-L3: QQWSSNPPT (SEQ ID NO: 6) The HCVR is SEQ ID NO:7 and the LCVR is SEQ ID NO:8. HC:EFQLQQSGPELVKPGASVKISCKASSYSFTDYNMNWVKQSNGKSLEWIGIIFPKYGTTSYNQKFKGKATLTVDQSSSTAYMQLNSLTSEDSAVYYCATDGGTWYFDVWGTG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) LC:QIVLTQSPALMSASPGEKVTMTCSASSSVTYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10) (SEQ ID NO:12) SCEDSTYTQLWNWVPECLS (SEQ ID NO: 13) SCEDSTYTQLWNWVPECLSC (SEQ ID NO: 101) HFB3-1hz6-hG1 (humanized monoclonal antibody) CDR-H1: SYSFTDYN (SEQ ID NO: 14) CDR-H2: IFPKYGTTSYAQKLQG (SEQ ID NO: 15) CDR-H3: ATDGGTWYFDV (SEQ ID NO: 16) CDR-L1: SSVTY (SEQ ID NO: 17) CDR-L2: LTSNLASGVPS (SEQ ID NO: 18) CDR-L3: QQWSSNPPT (SEQ ID NO: 19) The HCVR is sequence number 20 and the LCVR is sequence number 21. HC:QVQLVQSGAELKKPGASVKVSCKASSYSFTDYNMNWVRQAPGQSLEWMGIIFPKYGTTSYAQKLQGRVTLTTDTSTSTAYMELRSLRSDDTAVYYCATDGGTWYFDVWGTG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22) LC:DIQLTQSPSFLSASVGDRVTITCRASSSVTYMYWYQQKPGKAPKPWIYLTSNLASGVPSRFSGSGSGTEYTLTISSLQPEDAATYYCQQWSSNPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23) (SEQ ID NO:25) HFB3-14-hG1 (mouse monoclonal antibody) CDR-H1: GYTFTDYY (SEQ ID NO: 26) CDR-H2: INPNDGGTTYSQKFKG (SEQ ID NO: 27) CDR-H3: AREGNYYAYDVRVWYFDV (SEQ ID NO: 28) CDR-L1: QDIITY (SEQ ID NO: 29) CDR-L2: STSSLNSGVPS (SEQ ID NO: 30) CDR-L3: QQYSELPYT (SEQ ID NO: 31) The HCVR is sequence number 32 and the LCVR is sequence number 33. HC:EVQLQQSGPELVKPGASVRISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNDGGTTYSQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYFCAREGNYYAYDVRVWYFD VWGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34) LC:DIQMTQSPASLSVSVGETVTITCRSSENIYSNLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 35) (SEQ ID NO:37) CAPLRKCRPGFGVARPGTETSD (SEQ ID NO: 38) HFB3-14hz1c-hG1 (humanized monoclonal antibody) CDR-H1: GYTFTDYY (SEQ ID NO: 39) CDR-H2: INPNDGGTTYAQKFQG (SEQ ID NO: 40) CDR-H3: AREGNYYAYDVRVWYFDV (SEQ ID NO: 41) CDR-L1: QDIITY (SEQ ID NO: 42) CDR-L2: STSSLNSGVPS (SEQ ID NO: 43) CDR-L3: QQYSELPYT (SEQ ID NO: 44) The HCVR is sequence number 45 and the LCVR is sequence number 46. HC:QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGDINPNDGGTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCAREGNYYAYDVRVWYFD VWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47) LC:DIQMTQSPSSLSASVGDRVTITCGASQDIITYLNWYQQKPGKAVKLLIYSTSSLNSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSELPYTFGGGTKVELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 48) (SEQ ID NO:50) HFB3-18-hG1 (mouse monoclonal antibody) CDR-H1: GFTFSDAW (SEQ ID NO:51) CDR-H2: VRNKANNHATYYAESVKG (SEQ ID NO: 52) CDR-H3: TRSVGGYGTTYWYFDV (SEQ ID NO: 53) CDR-L1: QNLLNSGNQKNY (SEQ ID NO:54) CDR-L2: GASTRESGVPD (SEQ ID NO: 55) CDR-L3: QSEHSYPYT (SEQ ID NO: 56) The HCVR is sequence number 57 and the LCVR is sequence number 58. HC:EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEVRNKANNHATYYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCTRSVGGYGTTYWYFD VWGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 59) LC: DIVMTQSPSSLSVSAGEKVTMSCKSSQNLLNSGNQKNYLAWYQQKPGQPPKLLIFGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQSEHSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 60) (SEQ ID NO:62) HFB3-18hz1-hG1 (humanized monoclonal antibody) CDR-H1: GFTFSDAW (SEQ ID NO: 63) CDR-H2: VRNKANNHATYYAASVKG (SEQ ID NO: 64) CDR-H3: TRSVGGYGTTYWYFDV (SEQ ID NO: 65) CDR-L1: QNLLNSGNQKNY (SEQ ID NO: 66) CDR-L2: GASTRESGVPD (SEQ ID NO: 67) CDR-L3: QSEHSYPYT (SEQ ID NO: 68) The HCVR is sequence number 69 and the LCVR is sequence number 70. HC:EVQLVESGGGLVQPGGSLKLSCAASGFTFSDAWMDWVRQASGKGLEWVGEVRNKANNHATYYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRSVGGYGTTYWYFD VWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71) LC: DIVMTQSPDSLAVSLGERATINCKSSQNLLNSGNQKNYLAWYQQKPGQPPKLLIFGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQSEHSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 72) (SEQ ID NO:74) RPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSD (SEQ ID NO: 75)

[0220] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention is a human-mouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a resurfaced antibody.

[0221] In some embodiments, the antigen-binding fragment is a Fab, Fab', F(ab'), F d , single chain Fv or scFv, disulfide-linked Fv , V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0222] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention have an engineered Fc region that abolishes immune effector function. For example, the engineered Fc region of the subject antibodies may have a "LALA" double mutation (Leu234Ala and Leu235Ala) and therefore have reduced effector function.

[0223] Such antibodies are sometimes designated G1AA because they have a LALA double mutation in IgG1.

[0224] Other recombinant human IgG antibodies (hIgG) with partial or complete lack of binding to Fcγ receptors (FcγR) and the complement protein C1q, and thus abolished immune effector functions, are known in the art and are used in various therapeutic applications to reduce FcγR activation and Fc-mediated toxicity. Certain such Fc engineered antibodies / fragments partially achieve this goal, while others completely abolish FcγR activation and Fc-mediated toxicity. In certain embodiments, the antibodies / fragments of the invention have engineered hIgG Fc domains containing hIgG1-P329G LALA or hIgG4-P329G SPLE (human IgG4 S228P / L235E variant of IgG4) mutations, in which FcγR and C1q interactions are completely abolished and FcRn interactions and Fc stability are unaffected. The P329G Fc mutation disrupts the formation of a proline sandwich motif with FcγR. Since this motif is present at the interface of all IgG Fc / FcγR complexes, its disruption can be applied to all human and most other mammalian IgG subclasses to generate effector-silent IgG molecules. Thus, in certain embodiments, the subject antibodies / fragments have any one IgG subclass with such effector-silent Fc mutations.

[0225] In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention are specific for human TNFR2, e.g., do not substantially cross-react with TNFR1 and / or do not substantially cross-react with mouse TNFR2. In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention cross-react with monkey TNFR2 (e.g., cynomolgus or rhesus TNFR2).

[0226] In some embodiments, the dissociation constant (K d) is 1 μM or less, 100 nM or less, 50 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M).

[0227] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention binds to a region within the CRD2 domain of TNFR2. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention binds to an epitope bound by HFB3-1.

[0228] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention binds to a region within the CRD3 domain of TNFR2. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention binds to an epitope bound by HFB3-14.

[0229] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention binds to an epitope bound by HFB3-18.

[0230] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention binds to an epitope of SEQ ID NO: 13 / 101 or 38.

[0231] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention enhances binding of human recombinant TNFα to TNFR2.

[0232] In some embodiments, the monoclonal antibodies or antigen-binding fragments of the invention block the binding of human recombinant TNFα to TNFR2.

[0233] In some embodiments, the monoclonal antibodies or antigen-binding fragments of the invention do not substantially affect the binding of human recombinant TNFα to TNFR2.

[0234] In some embodiments, the monoclonal antibodies or antigen-binding fragments of the invention inhibit TNFα-mediated signaling (e.g., NFκB signaling) and / or induce downregulation of NFκB downstream target genes, although in other embodiments, the monoclonal antibodies or antigen-binding fragments of the invention promote TNFα-mediated signaling (e.g., NFκB signaling) and / or induce upregulation of NFκB downstream target genes.

[0235] In some embodiments, NFκB signaling is stimulated in effector T cells (e.g., CD8 and / or CD4 Tconv T cells). In some other embodiments, NFκB signaling is inhibited in effector T cells (e.g., CD8 and / or CD4 Tconv T cells).

[0236] In some embodiments, NFκB signaling is stimulated in Tregs. In some other embodiments, NFκB signaling is inhibited in Tregs.

[0237] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention stimulate the proliferation of CD8 and / or normal CD4 T cells, optionally with or without costimulation with CD3 / CD28 and / or optionally with or without costimulation with TNFα.

[0238] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention, particularly humanized monoclonal antibodies or antigen-binding fragments thereof, preferentially bind to (CD3 / CD28)TCR-activated primary CD8 and / or CD4 T cells compared to unstimulated primary CD8 and / or CD4 T cells.

[0239] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention, particularly humanized monoclonal antibodies or antigen-binding fragments thereof, enhance CD3 / CD28-induced activation and / or proliferation (e.g., CD3 / CD28-induced activation and / or proliferation of primary CD8 and / or CD4 T cells, including activation and / or proliferation of primary CD8 and / or CD4 T cells in the presence of Tregs).

[0240] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention, particularly humanized monoclonal antibodies or antigen-binding fragments thereof, costimulate the activation and / or proliferation of primary CD8 and / or CD4 T cells in a cross-linking-independent manner.

[0241] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention, particularly humanized monoclonal antibodies or antigen-binding fragments thereof, costimulate the activation and / or proliferation of primary CD8 and / or CD4 T cells in a cross-linking dependent manner.

[0242] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention enhance binding of TNFα to TNFR2 in the presence of Tregs, enhance TNFα-mediated or costimulatory NFκB signaling (e.g., in TCR-activated CD8 and / or CD4 Tconv T cells), and / or promote proliferation of TCR-activated effector T cells (e.g., CD8 and / or CD4 Tconv T cells).

[0243] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention enhances TNFα-mediated CD25 expression on Tregs.

[0244] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof (including humanized monoclonal antibodies or antigen-binding fragments thereof) of the present invention have a good developability profile, including being stable under elevated temperatures (e.g., 25° C. or 40° C.), under low pH conditions (e.g., pH 3.5 at about room temperature), and / or after several rounds of freeze / thaw cycles.

[0245] In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof (including humanized monoclonal antibodies or antigen-binding fragments thereof) of the present invention contain one or more point mutations in the amino acid sequence that are designed to improve the developability of the antibody. For example, Raybould et al. (Five computational developability guidelines for therapeutic antibody profiling, PNAS 116(10):4025-4030,019) describe the Therapeutic Antibody Profiler (TAP), a computational tool that builds downloadable homology models of variable domain sequences, tests them against five developability guidelines, and reports the prevailing sequence trends and canonical forms. The authors further provide TAP for free use at opig.stats.ox.ac.uk / webapps / sabdab-sabpred / TAP.php.

[0246] There are many barriers to the development of therapeutic mAbs beyond achieving the desired affinity for the antigen. These barriers include intrinsic immunogenicity, chemical and conformational instability, self-association, high viscosity, multispecificity, and poor expression. For example, high levels of hydrophobicity are repeatedly implicated in aggregation, viscosity, and multispecificity, especially in the highly variable complementarity determining regions (CDRs). Asymmetry in the net charge of the heavy and light chain variable domains also correlates with self-association and viscosity at high concentrations. Patches of positive and negative charges in the CDRs are associated with high clearance rates and low expression levels. Product heterogeneity (e.g., oxidation, isomerization, or glycosylation) is often due to certain sequence motifs that are prone to post-translational or co-translational modifications. Computational tools are available that facilitate the identification of sequence propensities. Warszawski et al. (Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces. PLoS Comput Biol 15(8):e1007207. https: / / doi.org / 10.1371 / journal.pcbi.1007207) also describe a method for optimizing antibody affinity and stability by automated design of the variable light-heavy chain interface. Additional methods are available for identifying potential developability problems of a candidate antibody, and in a preferred embodiment of the invention, one or more point mutations can be introduced into the candidate antibody via conventional methods to address such problems and lead to an optimized therapeutic antibody of the invention.

[0247] 7. Humanized antibodies In some embodiments, the antibodies of the invention are humanized antibodies, which are useful as therapeutic molecules because they reduce or eliminate the human immune response to non-human antibodies (e.g., the human anti-mouse antibody (HAMA) response), which can result in an immune response to antibody therapeutics and reduce the effectiveness of the therapeutics.

[0248] Antibodies can be humanized by any standard method. Non-limiting exemplary methods of humanization include, for example, those described in U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-27 (1988); Verhoeyen et al., Science 239:1534-36 (1988); and U.S. Publication No. US2009 / 0136500. All of these are incorporated by reference.

[0249] A humanized antibody is an antibody in which at least one amino acid in a framework region of a non-human variable region is replaced with an amino acid from the corresponding position in a human framework region. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 15, or at least 20 amino acids in the framework region of a non-human variable region are replaced with an amino acid from one or more corresponding positions in one or more human framework regions.

[0250] In some embodiments, some of the corresponding human amino acids used for substitutions are derived from framework regions of different human immunoglobulin genes. That is, in some such embodiments, one or more of the non-human amino acids may be replaced with corresponding amino acids from a human framework region of a first human antibody, encoded by a first human immunoglobulin gene, one or more of the non-human amino acids may be replaced with corresponding amino acids from a human framework region of a second human antibody, encoded by a second human immunoglobulin gene, one or more of the non-human amino acids may be replaced with corresponding amino acids from a human framework region of a third human antibody, encoded by a third human immunoglobulin gene, etc. Furthermore, in some embodiments, all of the corresponding human amino acids used for substitutions within a single framework region, e.g., FR2, need not be from the same human framework. However, in some embodiments, all of the corresponding human amino acids used for substitutions are from the same human antibody or encoded by the same human immunoglobulin gene.

[0251] In some embodiments, antibodies are humanized by replacing one or more entire framework regions with the corresponding human framework regions. In some embodiments, the human framework region having the highest level of homology to the non-human framework region being replaced is selected. In some embodiments, such humanized antibodies are CDR-grafted antibodies.

[0252] In some embodiments, following CDR grafting, one or more framework amino acids are returned to the corresponding amino acid in the murine framework region. Such "backmutations" are made in some embodiments to retain one or more murine framework amino acids that may contribute to the structure of one or more CDRs and / or may be involved in antigen contact and / or may be involved in the overall structural integrity of the antibody. In some embodiments, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, no more than 1, or no backmutations are made to the framework regions of the antibody following CDR grafting.

[0253] In some embodiments, a humanized antibody also comprises a human heavy chain constant region and / or a human light chain constant region.

[0254] 8. Human antibodies In some embodiments, the antibodies of the present invention are human antibodies. Human antibodies can be produced by any suitable method. Non-limiting exemplary methods include producing human antibodies in transgenic mice that contain human immunoglobulin loci. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-55 (1993); Jakobovits et al, Nature 362:255-8 (1993); Onberg et al, Nature 368:856-9 (1994); and U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.

[0255] Non-limiting exemplary methods include producing human antibodies using phage display libraries. See, e.g., Hoogenboom et al., J. Mol. Biol. 227:381-8 (1992); Marks et al., J. Mol. Biol. 222:581-97 (1991); and PCT Publication No. WO 99 / 10494.

[0256] Antibody constant region In some embodiments, the humanized, chimeric, or human antibodies described herein comprise one or more human constant regions. In some embodiments, the human heavy chain constant region is an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is an isotype selected from K and λ. In some embodiments, the antibodies described herein comprise a human IgG constant region, e.g., human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or Fc fusion partner comprises a C237S mutation, e.g., in the IgG1 constant region. In some embodiments, the antibodies described herein comprise a human IgG2 heavy chain constant region. In some such embodiments, the IgG2 constant region comprises a P331S mutation as described in U.S. Pat. No. 6,900,292. In some embodiments, the antibodies described herein comprise a human IgG4 heavy chain constant region. In some such embodiments, the antibodies described herein comprise a S241P mutation in the human IgG4 constant region. See, e.g., Angal et al. Mol. Immunol. 30(1):105-108 (1993). In some embodiments, the antibodies described herein comprise a human IgG4 constant region and a human kappa light chain.

[0257] The choice of heavy chain constant region may determine whether the antibody has effector function in vivo. Such effector function may, in some embodiments, include antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), resulting in the killing of cells to which the antibody binds. Typically, antibodies that contain human IgG1 or IgG3 heavy chains have effector function.

[0258] In some embodiments, effector function is undesirable. For example, in some embodiments, effector function may be undesirable in the treatment of inflammatory and / or autoimmune diseases. In some such embodiments, a human IgG4 or IgG2 heavy chain constant region is selected or engineered. In some embodiments, the IgG4 constant region comprises a S241P mutation.

[0259] Any of the antibodies described herein can be purified by any suitable method, including but not limited to the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the antigen and / or epitope to which the antibody binds, as well as ligands that bind to the antibody constant region. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to bind to the constant region and purify the antibody.

[0260] In some embodiments, hydrophobic interaction chromatography (HIC), such as a butyl or phenyl column, is also used to purify some polypeptides. Many methods of purifying polypeptides are known in the art.

[0261] Alternatively, in some embodiments, the antibodies described herein are produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0262] 9. Nucleic acid molecules encoding antibodies of the invention The present invention also provides nucleic acid molecules comprising a polynucleotide encoding one or more chains of an antibody described herein. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an antibody described herein. In some embodiments, the nucleic acid molecule comprises both a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain of an antibody described herein. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain and a second nucleic acid molecule comprises a second polynucleotide encoding a light chain.

[0263] In some such embodiments, the heavy and light chains are expressed as two separate polypeptides, either from one nucleic acid molecule, or from two separate nucleic acid molecules, in some embodiments, for example when the antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both the heavy and light chains joined together.

[0264] In some embodiments, a polynucleotide encoding a heavy or light chain of an antibody described herein comprises a nucleotide sequence encoding a leader sequence located at the N-terminus of the heavy or light chain when translated. As explained above, the leader sequence may be the native heavy or light chain leader sequence or another heterologous leader sequence.

[0265] The nucleic acid molecule can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell (e.g., a mammalian cell).

[0266] 10. Vector Vectors are provided that include polynucleotides encoding the heavy and / or light chains of the antibodies described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, the vector includes a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, for example, when the antibody is an scFv.

[0267] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into the host cell in similar amounts (e.g., similar molar amounts or similar masses). In some embodiments, a molar or mass ratio of the first vector to the second vector of 5:1 to 1:5 is transfected into the host cell. In some embodiments, a mass ratio of 1:1 to 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[0268] In some embodiments, a vector is selected that is optimized for the expression of a polypeptide in CHO or CHO-derived cells, or NSO cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol.Prog.20:880-889 (2004). In some embodiments, a vector is selected for the in vivo expression of the subject antibody in animals, including humans. In some such embodiments, the expression of the polypeptide(s) is under the control of a promoter(s) that functions in a tissue-specific manner. For example, liver-specific promoters are described, for example, in PCT Publication No. WO 2006 / 076288.

[0269] 11.Host cells In various embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in prokaryotic cells (e.g., bacterial cells), or eukaryotic cells (e.g., fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells). Such expression can be carried out, for example, according to procedures known in the art.

[0270] Exemplary eukaryotic cells that can be used to express a polypeptide include, but are not limited to, COS cells (including COS 7 cells), 293 cells (including 293-6E cells), CHO cells (including CHO-S cells and DG44 cells), PER.C6® cells (Crucell), and NSO cells.

[0271] In some embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in yeast. See, e.g., U.S. Publication No. US2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make the desired post-translational modifications to the heavy and / or light chains of the TNFR2 antibody. For example, in some embodiments, CHO cells produce polypeptides that have higher levels of sialylation than the same polypeptides produced in 293 cells.

[0272] Introduction of one or more nucleic acids into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, gene transfer, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into a desired host cell according to any suitable method.

[0273] In some embodiments, one or more polypeptides can be produced in vivo, in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptide, according to any suitable method. EXAMPLES

[0274] Example 1: Monoclonal antibodies specific for human and monkey TNFR2 To generate monoclonal antibodies specific for human TNFR2 with cross-reactivity to the monkey ortholog TNFR2, we immunized mice with the recombinant extracellular domain (ECD) of human TNFR2 (rhTNFR2) using standard procedures to generate a diverse panel of human-mouse chimeric monoclonal antibodies.

[0275] At least 25 such monoclonal antibodies were generated and the VH and VL sequences of selected antibodies were aligned to obtain a consensus sequence as shown in Figure 1. The H-CDR3 and L-CDR3 regions are indicated by the boxed sequences.

[0276] These monoclonal antibodies were then tested for their ability to bind to human and monkey TNFR2 expressed by CHO cells (CHO.hHFB3 and CHO.mkHFB3 cells, respectively). Briefly, approximately 40,000 CHO.hHFB3 or CHO.mkHFB3 cells were seeded into tissue culture wells, and serial 1:3 dilutions of each test antibody with a starting (highest) antibody concentration of approximately 66 nM were added to each cell type and incubated for approximately 1 hour. Antibody bound to the cells was detected using 17 nM anti-human Fc antibody labeled with AF647 (Alexsa Fluor® 647 fluorescent dye). An isotype-matched negative control antibody was also used in the assay. Data for each antibody (EC 50 Value and E max (including ) are summarized in Figure 2A.

[0277] Eleven of the antibodies tested had sub-nM or single-digit nM affinity (EC 50 ) These antibodies also showed cross-reactivity to the orthologous rhesus monkey TNFR2 expressed in CHO cells, with essentially the same trend in binding affinity compared to hTNFR2 binding. See Figure 2A.

[0278] Interestingly, some antibodies (e.g., HFB3-1 and -14) promoted the binding of TNFα to TNFR2, others (e.g., HFB3-18) inhibited the binding of TNFα to TNFR2, and others (e.g., HFB3-6) did not appear to affect the binding of TNFα to TNFR2. See Figure 2B. Specifically, the binding of 25 ng / mL TNFα to CHO.hHFB3 cells was measured after preincubating CHO cells with each antibody for about 1 hour. The percentage of cells that bound TNFα (designated HFB2003L) was then plotted against increasing concentrations of antibody.

[0279] The same experiment was also set up to test the binding ability of the test antibodies to CHO cells expressing mouse TNFR2 and the parental CHO cell line (which may or may not express hamster TNFR2). Two monoclonal antibodies (HFB3-18 and HFB3-19) showed a slight level of binding to the mouse orthologue, while all other antibodies had no detectable level of binding to mouse TNFR2. As a positive control, HM102 monoclonal antibody specific for mouse TNFR2 was used and showed positive binding to CHO cells expressing mouse TNFR2, whereas the isotype-matched control antibody did not bind (Figure 3).

[0280] No binding was observed in the parental CHO cell line (Figure 3).

[0281] Binding specificity for human TNFR2 (vs the related TNFR1 receptor) was also confirmed using recombinant human TNFR2 and TNFR1 proteins.

[0282] Briefly, tissue culture plates were coated with 0.1 μg / mL His-tagged recombinant human TNFR2 or TNFR1 overnight at 4°C. The coated plates were then incubated for approximately 1 hour on ice with serial 1:3 dilutions of each test antibody with a starting (highest) antibody concentration of approximately 66 nM. Cell-bound antibody was detected using a 1:5000 dilution of HRP-conjugated anti-human Fc antibody and TMB substrate. An isotype-matched negative control antibody F3, a MR2-1 positive control antibody specific for rhTNFR2, and a positive control antibody specific for rhTNFR1 were also used in the assay. Data for each antibody (EC 50 The results are summarized in Figure 4A.

[0283] Six of the eleven antibodies tested, namely HFB3-1, -14, -21, -23, -24, and -25, had sub-nM affinity (EC 50), whereas an additional four antibodies (HFB-3, -6, -19, and -22) showed single-digit nM affinity for the same antigen. HFB3-18 showed relatively weak binding to monomeric rhTNFR2, with a double-digit nM affinity. However, none of the eleven antibodies showed detectable levels of binding to His-tagged TNFR1 receptor, indicating binding specificity for TNFR2.

[0284] Anti-human IgG Fc Capture (AHC) biosensor was used to confirm the binding affinity of human-mouse chimeric antibodies HFB3-1, 14, and 18 to recombinant human TNFR2 protein. The AHC biosensor allows for the kinetic characterization of macromolecular interactions between human Fc-containing proteins (e.g., the subject antibodies) and target analytes (e.g., recombinant human TNFR2). Immobilization of human Fc-containing proteins is achieved by a factory-immobilized anti-human Fc-specific antibody, whose high affinity for the human Fc domain provides a stable baseline required for demanding kinetic applications. In this particular experiment, the test antibody (humanized) was loaded at a concentration of 20 μg / mL in assay buffer (PBS, pH 7.4, 0.1% BSA, 0.1% Tween 20). The analyte was His-tagged recombinant human TNFR2 at 500, 167, or 55.7 nM. The capture assay was performed at 25°C. The K of the tested antibodies was 0.01 μg / mL. d was in the nM range (see Figure 4B).

[0285] Epitope mapping experiments of the HFB3-1-hG1, HFB3-14-hG1, HFB3-6-hG1, and HFB3-18-hG1 antibodies showed that these antibodies recognize different domains of TNFR2. One structural feature shared by most members of the TNFR superfamily is the inclusion of approximately two to four cysteine-rich domains (CRDs). HFB3-1-hG1 binds to a region within the CRD2 domain (Figure 11C), while HFB3-18-hG1 binds to a conformational epitope within CDR1. HFB3-6-hG1 binds to a region within CRD3, and HFB3-14-hG1 also binds to an epitope within the CRD3 region (although smaller than that of HFB3-6-hG1) (see Figure 11B). The location of the epitopes in a 3D model of the TNFR2-TNFα complex can be visualized in Figure 11D.

[0286] Example 2: TNFR2 expression in T cell subtypes This study demonstrated that TNFR2 mediates the expression of Treg and CD4 + and CD8 + Demonstrated to be predominantly expressed on T cells.

[0287] T cell subtypes (Treg and CD4 + and CD8 + We isolated T cell subtypes (including T cells) from various tumor samples and used RNA-seq analysis to quantify the relative percentages of T cell subtypes in the tumor samples and the average relative expression levels of TNRF2 (on a scale of 2 to 8) in T cell subtypes. The results are summarized in Figure 5.

[0288] In each tumor sample analyzed, including BCC (basal cell carcinoma), SCC (squamous cell carcinoma), melanoma, and NSCLC (non-small cell lung cancer), TNFR2 was found to mediate Treg and CD4 + and CD8 + It was predominantly and most frequently found in T cells. Moreover, the highest relative expression level was also found in Tregs. See Figure 5, left panel. This data suggests that TNFR2 is an attractive target for cancer therapy.

[0289] Additional TNFR2 expression analysis was also performed in SCC cancer samples along with the expression of several immune checkpoint genes (e.g., PD-1, TIM3, CTLA4, and 4-1BB). In exhausted CD8+ T cells, the expression of TNFR2 was found to be consistent with the expression of these immune checkpoint genes (Figure 5, right panel). This suggests that combination therapy with anti-TNFR2 antibodies and inhibitors of these immune checkpoint genes may be therapeutically beneficial.

[0290] Example 3: Binding of anti-TNFR2 monoclonal antibodies to primary Treg, CD8, and CD4 Tconv cells Given the expression pattern of TNFR2 in T cell subtypes (see Example 2), this experiment demonstrates that the subject anti-TNFR2 monoclonal antibodies can bind to primary T cell subtypes and preferentially bind to activated T cells.

[0291] Briefly, flat-bottom 96-well plates were coated with 10 nM anti-CD3 antibody overnight at 4°C. Meanwhile, Treg, T cell subtypes, including CD8 or CD4 conventional T cells (Tconv), were isolated from human PBMCs. Primary T cells were co-stimulated for approximately 3 days by seeding the isolated T cell subtypes at a density of approximately 50,000 cells / well in the presence of 6.6 nM anti-CD28 antibody. The stimulated primary T cells were then treated with 1:3 serial dilutions of the anti-TNFR2 human-mouse chimeric monoclonal antibody of the present invention at various concentrations (the highest concentration was 66 nM) for 1 h on ice. Bound chimeric antibody was detected by adding 17 nM anti-hFc antibody labeled with AF647 dye and incubating on ice for 1 h, followed by FACS analysis to detect AF647 signals.

[0292] The top panel of Figure 6 shows that CD4 Tconvs are the most abundant T cell subtype, accounting for approximately 30% of total hPBMCs, followed by CD8 T cells at 10% and Tregs at approximately 1%. However, non-TCR-activated primary T cells did not bind the subject anti-TNFR2 antibodies at detectable levels, except for relatively low levels of binding in primary Tregs. Overall, receptor occupancy Emax was highest in Tregs, followed by CD8 and CD4 Tconvs. Given the relatively low abundance of Tregs compared to CD8 and CD4 Tconvs, the expression of TNFR2 in Tregs is much higher than that in CD8 and CD4 T cells on a cell-by-cell basis.

[0293] However, in TCR-activated T cells, a dramatic 5- to 6-fold increase in binding was observed for several anti-TNFR2 antibodies in Tregs, and substantially higher binding was also observed in CD8 and CD4 T cells (Figure 6, lower panel).

[0294] Of the antibodies tested, HFB3-1, -6, -24, -25, and SBT1 (positive control) showed high affinity at sub-nM levels, whereas HFB3-14 and -19 showed single-digit nM affinities, and HFB3-18, -21, and -22 had double-digit nM affinities.

[0295] Example 4: Binding of certain anti-TNFR2 monoclonal antibodies to primary CD8 and CD4 Tconv cells costimulated NFκB signaling This experiment demonstrates that anti-TNFR2 monoclonal antibodies of the invention costimulate TNFα-mediated NFκB signaling as demonstrated by QPCR quantification of NFκB signaling pathway genes.

[0296] Briefly, CD4 Tconv (CD4 + CD25 - ) or CD8 +T cells were isolated from hPBMCs. The isolated T cells were incubated with 10 μg / mL (66 nM) of various test monoclonal antibodies of the invention or appropriate positive or negative controls and 25 ng / mL (1.5 nM) of TNFα for approximately 24 hours. Stimulated T cells were then harvested, their mRNA isolated, reverse transcribed, and subjected to QPCR analysis of NFκB signaling pathway genes (e.g., CD25, Foxp3, NFκB2, RelB, and LTA). The expression levels of these genes in the presence and absence of costimulation with the subject antibodies are compared in the bar graphs of FIG. 7. Results are presented as fold change compared to the unstimulated control (1×).

[0297] The results showed that certain of the subject antibodies (including HFB3-1, -14, -23, -24, and -25) induced NFκB signaling. Notably, HFB3-1 and -14 (but not HFB3-18) occasionally induced NFκB signaling, particularly in NFκB2, RelB, and LTA.

[0298] Example 5: The costimulatory effect of anti-TNFR2 monoclonal antibodies correlates with the proliferation of isolated primary CD8 and CD4 Tconv cells In this experiment, flat-bottom 96-well plates were coated overnight at 4° C. with 10 nM anti-CD3 monoclonal antibody and 20 or 100 nM of the subject anti-TNFR2 antibody. Meanwhile, CD8 and CD4 Tconv cells were isolated from hPBMCs as described above and labeled with 2 μM CTV (Invitrogen's CellTrace™ Violet Cell Proliferation Kit) to track T cell proliferation. CellTrace™ Violet dye readily diffuses into cells and is cleaved by intracellular esterases to produce highly fluorescent compounds that then covalently bind to intracellular amines, resulting in a stable and well-retained fluorescent stain that can be fixed with aldehyde fixative. Excess unbound reagent passively diffuses into the extracellular medium where it can be quenched and washed away with complete medium.

[0299] The labeled T cells were then seeded at a density of approximately 50,000 cells / well in the presence of 6.6 nM anti-CD28 antibody in coated 96-well plates and co-stimulated for approximately 3 days, after which the cells were fixed for FACS analysis of the fluorescent signal.

[0300] The data in Figure 8 show that certain subject anti-TNFR2 antibodies costimulate CD8 and CD4 Tconv proliferation even at the lower 20 nM concentration. The benchmark positive control antibodies SBT-1 and -4 also costimulated T cell proliferation under the same conditions, but to a lesser extent than HFB3-1, -14, -18, and -25.

[0301] Additional experiments demonstrated that such costimulation of primary T cell proliferation may be dependent on FcγR cross-linking for certain monoclonal antibodies (e.g., HFB3-18), whereas no appreciable cross-linking dependency was observed for other antibodies (e.g., HFB3-1 and -14).

[0302] Specifically, CD8 and CD4 Tconvs were isolated from donor KP59095, and isolated primary T cells were stimulated by CD3 / CD28 TCR activation and the subject anti-TNFR2 monoclonal antibodies HFB3-1, -14, or -18 in the presence or absence of 25 ng / mL recombinant human TNFα (rhTNFα). Anti-TNFR2 antibodies were supplied either plate-bound or as soluble antibodies present in the binding mixture.

[0303] In the presence of 25 ng / mL rhTNFα, all three plate-bound anti-TNFR2 antibodies (HFB3-1, -14, and -18) stimulated CD8 T cell proliferation (see FIG. 19, bottom left panel). However, only soluble HFB3-1 and HFB3-14 (but not soluble HFB3-18) were able to stimulate CD8 T cell proliferation (FIG. 19, bottom right panel). This suggests that FcγR cross-linking may be required for HFB3-18-mediated CD8 T cell proliferation, but not required (i.e., cross-linking-dependent) for HFB3-1- and HFB3-14-mediated CD8 T cell proliferation.

[0304] Similar results were obtained under similar conditions for proliferation of CD4 Tconv (data not shown).

[0305] Example 6: Anti-TNFR2 monoclonal antibodies favor cell proliferation at the Teff cell end (CD8 and CD4 Tconv) in the presence of Tregs This experiment demonstrates that the subject anti-TNFR2 monoclonal antibodies are able to costimulate proliferation of Teff cells (CD8 and CD4 Tconv) in the presence of Tregs along with CD3 / CD28-mediated TCR activation.

[0306] In brief, CD3 + T cells (including CD8 and CD4 Tconv effector T cells, as well as Tregs) were isolated from human PBMCs and subjected to CD3 / CD28-mediated TCR activation and costimulation with the subject anti-TNFR2 monoclonal antibodies for approximately 4 days, essentially as described above. Total CD4 Tregs in the presence of Tregs were isolated using Invitrogen's CellTrace™ Violet Cell Proliferation Kit (CTV). + T cells and CD8 + T cell proliferation was quantified. + By measuring the percentage of T cells, CD4 + T cells, CD8 + T cell activation was also quantified.

[0307] The results in Figure 9 show that the anti-TNFR2 monoclonal antibodies of the present invention (e.g., the humanized version of HFB3-1, HFB3-1hz6-hG1AA (see below)) favor cell proliferation of effector T cells (CD8 and CD4 Tconv) in the presence of Tregs.

[0308] Example 7: Anti-TNFR2 monoclonal antibodies had negligible ADCC effect on HH lymphoma cells This experiment demonstrates that the subject anti-TNFR2 monoclonal antibodies exhibit negligible ADCC effects against T cell lymphoma, suggesting that such antibodies may be suitable for use as T cell costimulators.

[0309] Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cell-mediated immune defense in which effector cells of the immune system actively lyse target cells to which specific antibodies have bound membrane surface antigens. This is one mechanism by which antibodies, as part of the humoral immune response, can act to limit and contain infection. ADCC requires effector cells, classically known to be natural killer (NK) cells that typically interact with IgG antibodies.

[0310] In this experiment, Jurkat.CD16V / NFAT / luc cells were used as effector cells and HH lymphoma cells were used as target cells. The effector:target cell ratio was about 6:1. Co-cultured effector and target cells were incubated overnight in the presence of the subject anti-TNFR2 monoclonal antibodies (e.g., HFB3-1, -14, or -18) or an isotype-matched control (hIgG1) at concentrations of 0, 0.0066, 0.66, or 66 nM. Moganulizumab antibody was used as a positive control in ADCC.

[0311] The results in Figure 10 show that the positive control antibody moganulizumab exhibited at least 120-fold more potent ADCC effect on target cells than any of the anti-TNFR2 monoclonal antibodies tested. This data demonstrates that the subject anti-TNFR2 antibodies have low / non-existent ADCC effect on T cells, making them suitable for use as T cell costimulators.

[0312] Example 8: Binding of humanized anti-TNFR2 monoclonal antibodies to TNFR2 Several humanized monoclonal antibodies against HFB3-1, -14, and -18 were generated, at least 20 for HFB3-1, 16 for HFB3-14, and 1 for HFB3-18 (due to the high similarity of the selected human germline to the parent HFB3-18 monoclonal antibody coding sequence). The binding ability of these humanized monoclonal antibodies to human TNFR2 expressed in CHO cells was quantified essentially as described in Example 1.

[0313] FIG. 12A shows that humanized HFB3-1hz6, HFB3-14hz1c, and HFB3-18hz1 bound to human TNFR2-expressing CHO cells (CHO.hTNFR) but not to parental CHO cells. Figure 12B shows that at least seven humanized HFB3-1 antibodies, namely, HFB3-1hz6, -1hz8, -1hz9, -1hz10, -1hz11, -1hz12, - and 1hz14, and at least eight humanized HFB3-14 antibodies, namely, HFB3-14hz1c, -14hz2c, -14hz3c, -14hz4c, -14hz6c, -14hz7c, -14hz12c, and -14hz14c, retained levels of binding affinity to CHO cell-expressed TNFR2 (CHO.hHFB3) that were similar to or greater than those of their respective parent chimeric antibodies.

[0314] The same experiment was repeated using CHO cells expressing the rhesus monkey orthologue of TNFR2 instead (CHO.mkHFB3). Figure 13 shows that the general trend of binding to CHO cells expressing monkey TNFR2 was consistent with that to CHO.hTNFR2. However, some erratic binding was observed for two of the humanized variants based on HFB3-14, namely HFB3-14hz2c and -14hz3c.

[0315] The binding of the humanized anti-TNFR2 antibodies is specific to TNFR2 and does not bind to TNFR1. The ELISA assay in FIG. 14A showed that the humanized monoclonal antibodies HFB3-1hz6, HFB3-14hz1c, and HFB3-18hz1 bind to recombinant human and cynomolgus TNFR2 (hTNFR2-His and cynoTNFR2-His, respectively) without recognizing recombinant human TNFR1 (hTNFR1-His). Furthermore, the binding EC50 of these humanized anti-TNFR2 antibodies to recombinant human and cynomolgus TNFR2 ranged from sub-nM to single-digit nM.

[0316] The binding affinity of the humanized variants to human TNFR2 was confirmed using recombinant human TNFR2 protein and an AHC biosensor. The anti-human IgG Fc capture (AHC) biosensor allows for the kinetic characterization of macromolecular interactions between human Fc-containing proteins (e.g., the subject antibodies) and target analytes (e.g., recombinant human TNFR2). Immobilization of the human Fc-containing protein is achieved by a factory-immobilized anti-human Fc-specific antibody, whose high affinity for the human Fc domain provides a stable baseline required for demanding kinetic applications. In this particular experiment, the test antibodies (humanized vs. parental chimeric antibodies) were loaded at a concentration of 20 μg / mL in assay buffer (PBS, pH 7.4, 0.1% BSA, 0,1% Tween 20). The analyte was His-tagged recombinant human TNFR2 at 500, 167, or 55.7 nM. The capture assay was performed at 25°C.

[0317] As shown in Figure 14B, no significant differences were observed between the humanized variants and their respective chimeric parent antibodies in terms of affinity for recombinant human TNFR2.

[0318] Example 3 shows that chimeric anti-TNFR2 antibodies bind to TCR-activated T cells. Essentially the same experiment was performed on the humanized variants, and the results are shown in FIG.

[0319] Specifically, in terms of binding to TCR-activated CD8 cells, most of the humanized HFB3-1 antibodies showed sub-nM affinity, with the exception of two variants (HFB3-1hz5 and -1hz7), which appeared not to bind to TCR-activated CD8 cells. On the other hand, all humanized HFB3-14 variants showed single-digit nM affinity to TCR-activated CD8 T cells. No significant differences were observed to distinguish the various variants. Of note, the positive control antibodies SBT-2 and -3 did not bind well to primary CD8 cells.

[0320] Example 9: Costimulatory effect of humanized anti-TNFR2 monoclonal antibodies on the proliferation of TCR-activated CD4 and CD8 T cells Example 5 shows that the costimulatory effect of chimeric anti-TNFR2 monoclonal antibodies leads to the proliferation of isolated human primary CD8 and CD4 Tconv cells. This experiment demonstrates the same in TCR-activated CD4 T cells using humanized variants of HFB3-1 and HFB3-14.

[0321] Specifically, Figure 16 shows that the humanized variants HFB3-1hz5, -1hz6, -1hz8, -1hz10, -1hz11, and -1hz12 each potently stimulated TCR-activated CD4 T cells to a greater extent than the parental HFB3-1 chimeric antibody based on the CTV proliferation assay (see above). The same was repeated for the variants HFB3-14hz1c and -14hz3c.

[0322] Similarly, CD25 +T cell activation based on the percentage of the T cell population was also determined for the above variants.

[0323] Confirmatory costimulatory data was also obtained for HFB3-1hz6-hG1, -14hz1c-hG1, and -18hz1-hG1, showing that these variants have costimulatory effects to expand TCR-activated CD8 T cells (activated by CD3 / CD28 stimulation). Specifically, both the parental chimeric antibody and selected humanized variants enhanced the proliferation of CD8 T cells stimulated by CD3 / CD28 TCR activation. Furthermore, the synergistic effect of TNFα (right panel) further enhanced anti-TNFR2 antibody-mediated CD8 proliferation. See Figure 20.

[0324] Example 10: Certain humanized anti-TNFR2 monoclonal antibodies induced NFκB signaling in Tregs Example 4 showed that binding of certain chimeric anti-TNFR2 monoclonal antibodies to primary CD8 and CD4 Tconv cells costimulated NFκB signaling. Similar experiments showed that certain humanized variant anti-TNFR2 antibodies induced NFκB signaling in Tregs.

[0325] Specifically, FIG. 17A shows that co-stimulation of Tregs with certain humanized variant anti-TNFR2 antibodies and TNFα resulted in NFκB downstream signaling in LTA, TNF, and TNF AIP3. Variants HFB3-1hz6, -1hz9, -1hz10, and -1hz11 promoted NFκB signaling to a greater extent than the parent chimeric antibody HFB3-1. Meanwhile, variants HFB3-14hz1c, -14hz2c, -14hz3c, and -14hz4c (especially HFB3-14hz1c and -14hz3c) also promoted NFκB signaling to a greater extent than the parent chimeric antibody HFB3-14. Furthermore, FIG. 17B shows the activation of NFκB signaling in CD8 T cells using certain humanized variants of the HFB3-1 antibody with or without human recombinant TNFα.

[0326] Example 11: Anti-TNFR2 antibodies are stable To confirm that the subject humanized anti-human TNFR2 antibodies are stable under storage and therefore suitable for further development as therapeutic agents, various developability assays were performed on selected humanized antibodies.

[0327] In a first experiment, selected subject humanized antibodies were stored in PBS (pH 7.4) at 25° C. or 40° C. and the stability of the various antibodies was measured on days 7 and 14. The results in FIG. 18 show that all tested antibodies were stable under the conditions tested, except for one variant, HFB3-14hz4c-hG1AA.

[0328] In a second experiment, the same antibodies were tested for stability under low pH conditions (100 mM AcH, pH 3.5, 25° C.) for 0, 3, and 6 hours. The results in FIG. 18 again showed that all tested antibodies were stable under the conditions tested, except for one variant, HFB3-14hz4c-hG1AA.

[0329] In a third experiment, the same antibodies were subjected to 1, 2, or 3 freeze-thaw cycles. The results in Figure 18 again showed that all tested antibodies, except for two variants (HFB3-1hz6-hG1AA and HFB3-1hz10-hG1AA), were stable under the test conditions.

[0330] Similar experiments were repeated for HFB3-1hz6-hG1, -14hz1c-hG1, and -18hz1-hG1. All three variants were largely stable in the three studies outlined above, except that HFB3-1hz6-hG1 and -18hz1-hG1 began to degrade after 14 days.

[0331] Taken together, this data suggests that these subject variant humanized anti-TNFR2 monoclonal antibodies do not pose significant developability issues and are suitable for use as therapeutic antibodies.

[0332] Example 12: Anti-TNFR2 antibodies and their effects on T cells in humanized TNFR2 knock-in (KI) mice To more fully demonstrate the therapeutic efficacy of the subject anti-TNFR2 antibodies, humanized TNFR2 knock-in (KI) mice were generated in a C57BL / 6 mouse background using a commercial service (Biocytogen, Wakefield, Mass.).

[0333] In a first series of experiments, ex vivo binding between selected humanized anti-TNFR2 antibodies and CD3 T cells from KI mice (TNFR2 KI CD3 T cells) was analyzed under costimulation with 1 μg / mL CD28 and 0.2 or 1 μg / mL CD3. The results showed that 1 μg / mL CD3 activated splenocytes from KI mice better than 0.2 μg / mL CD3. Human TNFR2 expression was significantly higher in KI CD3 T cells than in KI CD3 T cells. + It can be detected in T cells, and its expression / detection can be enhanced by TNFα and under mild (0.2 μg / mL) CD3 stimulation. Furthermore, a single dose of 200 nM of each of the six anti-TNFR2 antibodies (i.e., HFB3-1, -14, and -18, and their humanized variants -1hz6, -14hz1c, and -18hz1) showed no discernible difference in TNFR2 binding. This may be due to saturation levels of binding. Data not shown.

[0334] The same ex vivo binding experiments were repeated on CD8 T cells isolated from tnfr2 KI mice. Here, binding of anti-TNFR2 monoclonal antibodies (chimeric and humanized versions) to TNFR2 could be observed under strong CD3 (1 μg / mL) stimulation, whereas TNFα enhanced TNFR2 binding under mild CD3 (0.2 μg / mL) stimulation. Data not shown.

[0335] We next examined the ability of the subject anti-TNFR2 antibodies (chimeric and humanized) to costimulate downstream NFκB signals in TNFR2 KI CD8 and CD4 Tconc cells ex vivo in the presence of CD3 / CD28-mediated TCR activation and in the presence of TNFα.

[0336] Although the signal response from hTNFR2 knock-in (KI) mouse T cells was not as pronounced as that from human T cells, HFB3-1-hG1 and its humanized variant HFB3-1hz6-hG1 induced a greater response than the other antibodies (see FIG. 21). Notably, the lack of signal induction from the HFB3-18 series is expected.

[0337] The pharmacokinetic (PK) profiles of the subject humanized anti-TNFR2 monoclonal antibodies (HFB3-1hz6-hG1, HFB3-141c-hG1, and HFB3-18hz1-hG1) were investigated in C57BL / 6 mice. All three humanized monoclonal antibodies demonstrated Tregs consistent with expectations for a well-behaved antibody. 1 / 2 See below. [Table 1]

[0338] Example 13: Effect of humanized HFB3-1hz6-hG1 on ex vivo natural killer (NK) cell activation This experiment demonstrates that the humanized HFB3-1hz6-hG1 antibody costimulates natural killer (NK) cells in the presence of IL-2 / IL-15 or CD3 / CD28-mediated NK cell activation.

[0339] In one experiment, NK cells were isolated from peripheral blood mononuclear cells (PBMCs) provided by two human patients using an NK cell isolation kit (Miltenyi Biotec). NK cells were first stimulated with soluble IL-2 (10 ng / mL) and IL-15 (10 ng / mL) for 24 h, and then treated with isotype control antibody, mouse HFB3-1-hG1, humanized HFB3-1-hz6-hG1, or anti-OX40 control antibody (BMS) at 22 nM, 66 nM, or 200 nM, respectively, for 16 h. At the end of the experiment, NK cell surface CD107α expression and TNFR2 expression, which represent NK cell degranulation and activation, were measured by FACS.

[0340] Both mouse HFB3-1-hG1 and humanized HFB3-1-hz6-hG1 significantly increased NK cell activation in a dose-dependent manner. Anti-OX40 antibody was unable to promote short-term NK activation (40 hours after IL-2 / IL-15 stimulation), likely due to insufficient expression of OX40.

[0341] In another experiment, total PBMCs provided by two human patients were co-stimulated with plate-bound anti-CD3 (1 μg / mL) and soluble anti-CD28 (1 μg / mL) for 48 hours, and then treated with isotype control antibody, mouse HFB3-1-hG1, humanized HFB3-1-hz6-hG1, or anti-OX40 antibody (BMS) at 22 nM, 66 nM, or 200 nM, respectively, for 16 hours. CD107α expression was measured for CD3 negative / CD56 positive (i.e., NK cells). See FIG. 23.

[0342] Similarly, HFB3-1-hG1 and HFB3-1-hz6-hG1 significantly increased the expression of CD107α in a dose-dependent manner, indicating that these antibodies could promote the activation of NK cells in whole PBMCs. Under long-term activation (64 hours after anti-CD3 / CD28 stimulation), anti-OX40 antibody could activate NK cells.

[0343] These data indicate that both humanized HFB3-1-hz6-hG1 and parental murine HFB3-1-hG1 can promote the activation of NK cells.

[0344] Example 14: Pharmacodynamics of humanized HFB3-1hz6-hG1 in the MC38 tumor model The pharmacodynamics of HFB-1-hG1 was investigated using the MC38 colon cancer tumor model in humanized TNFR2-KI mice (see FIG. 24A). Briefly, 8-week-old humanized TNFR2 KI mice were inoculated with approximately 5×105 MC38 tumor cells per mouse in the right anterior flank. Mice were randomized, and 7 days later (day 0), mice (n=5 per group) were intraperitoneally injected with 10 mg / kg, 1 mg / kg, or 0.1 mg / kg HFB3-1-hG1, or 10 mg / kg isotype control antibody. The same treatment was administered again on day 3. On day 4, mice were euthanized and pharmacodynamic readouts were performed on tumor and blood samples. FACS was used to sort tumor-infiltrating leukocytes and peripheral leukocytes, and receptor occupancy by antibodies was quantified.

[0345] After only two doses on days 0 and 3, there was still no significant difference in tumor weight between treatments (Figure 24B, upper left panel). Administration of 10 mg / kg HFB3-1-hG1 increased the absolute number of CD45+ cells present in the tumor (Figure 24B, lower left panel), but did not significantly increase the percentage of CD45+ among live tumor cells (Figure 24B, lower right panel). Treatment with 10 mg / kg HFB3-1-hG1 also increased the absolute cell numbers of CD8+, conventional CD4+ T and NK cells in the tumor microenvironment, but did not change the number of regulatory T cells (Figure 24C). Administration of other low doses of HFB3-1-hG1 did not produce any observable effect.

[0346] TNFR2 receptor occupancy was determined for CD8 T cells, conventional CD4 T cells, regulatory T cells, and NK cells in tumors and peripheral blood. In tumors, only the 10 mg / kg dose of HFB3-1-hG1 led to drug receptor occupancy in T cells within the tumor, with no occupancy observed at doses of 1 and 0.1 mg / kg (see FIG. 25A). However, receptor occupancy was observed in tumor NK cells at 1 mg / kg and 10 mg / kg. In peripheral blood, HFB3-1-hG1 at doses of 10 mg / kg and 1 mg / kg led to equivalent drug receptor occupancy, with no significant occupancy observed at the 0.1 mg / kg dose.

[0347] The pharmacokinetics of HFB3-1-hG1 was quantified at the end of the experiment. HFB3-1-hG1 administration at doses of 1 and 10 mg / kg was detectable in the blood on day 4. Notably, HFB3-1-hG1 at 10 mg / kg was retained at a much higher level than the isotype control at the same dose (see FIG. 26A). Interestingly, administration of HFB3-1-hG1 at 10 mg / kg and 1 mg / kg also increased the amount of TNFR2 detectable in the blood (see FIG. 26B). TNFR2 in the blood is likely due to receptor shedding.

[0348] Overall, the data from short-term treatment of mice with HFB3-1-hG1 strongly suggest that HFB3-1-hG1 can stimulate immune cell activation and proliferation, effectively bind to the TNFR2 receptor on immune cells, and has good retention in the blood in vivo.

[0349] Example 15: Synergistic antitumor efficacy with anti-PD-1 antibody The anti-tumor efficacy of a humanized anti-TNFR2 monoclonal antibody was demonstrated in a widely used mouse colon cancer model in a humanized TNFR2 KI mouse background.

[0350] Specifically, 8-week-old humanized TNFR2 KI mice were injected with approximately 5 × 10 5MC38 tumor cells (derived from a C57BL6 mouse colon adenocarcinoma) were inoculated into the mice. Approximately 7 days later, on day 0, the average tumor size in the mice was approximately 89 mm 3 (74~98mm 3 ) were reached. Mice were then randomized into five experimental groups (n=8 / group) and administered one of the following: (1) isotype-matched control (TT-hG1AA), (2) anti-mPD-1 (RMP-1-14), (3) HFB3-1hz6-hG1, (4) HFB3-14hz1c-hG1, and (5) HFB3-18hz1-hG1. Antibodies were injected intraperitoneally at a dose of approximately 10 mg / kg on days 0, 3, 6, 9, 12, 15, and 18 for a total of seven doses (Q3D, ×7). Tumor volumes were measured for the experimental groups during the study. Around day 21, the mean tumor volume of the isotype control group was 2000 mm 3 Thus, the experiment was terminated and all mice were sacrificed. The tumor volumes over time in the various groups are plotted in Figure 27A and Figure 27B. By day 21, statistical significance of tumor growth inhibition (TGI) was achieved in the groups of mice treated with HFB3-1hz6, HFB3-18hz1, and anti-PD-1 (RMP-14) (Figure 27B).

[0351] The results showed that the humanized antibodies HFB3-1hz6 and -hG1, as well as HFB3-18hz1-hG1, inhibited tumor growth as potently (or more potently) as the anti-mPD-1 antibody, while the other humanized antibodies were similarly effective, albeit to a slightly lesser extent. No obvious differences in body weight were observed between the different groups of experimental mice.

[0352] Similar results were obtained in a separate experiment (4 mice per group) using only anti-mPD-1 and HFB3-1hz6-hG1 and isotype control, injected intraperitoneally at 10 mg / kg Q3d×3 (once every 3 days for a total of 3 doses). On day 6 (last dose of antibody), there was a statistically significant difference in tumor volume between the isotype control, anti-mPD-1, and HFB3-1hz6-hG1 groups (based on 2-way ANOVA test). See Figure 28.

[0353] Furthermore, HFB3-1hz6-hG1 and anti-PD-1 antibodies synergistically suppressed tumor growth and extended mouse life span in an MC38 tumor model. Specifically, humanized TNRF2 KI mice were inoculated with MC38 cancer cells on day -7. Starting on day 0, mice were intraperitoneally injected with isotype control, HFB3-1hz6-hG1, or anti-mPD-1 antibodies, either alone or in combination, every 3 days (n=8 per group). Treatment with 3 and 10 mg / kg HFB3-1hz6-hG1 once every 3 days for a total of 7 doses (Q3d×7) and 10 mg / kg anti-PD-1 (RMP-14) once every 3 days for a total of 4 doses (Q3d×4) significantly inhibited tumor growth and extended mouse life span compared to treatment with isotype control. Furthermore, combined treatment with both HFB3-1hz6-hG1 (10 mg / kg, Q3dx7) and anti-PD-1 antibody (10 mg / kg, Q3dx4) resulted in improved survival compared to treatment with anti-PD-1 antibody alone. See Figure 29. Data were analyzed using ANOVA comparing treatment groups and isotype control.

[0354] Example 16: Antitumor efficacy of HFB3-1hz6-hG1 in hepatocellular carcinoma mouse model In the Hepa1-6 syngeneic mouse model, the tumor volume was approximately 100 mm 3 Once tumor growth was reached, mice were treated with isotype control antibody, 10 mg / kg anti-mPD-1, or HFB3-1hz6-hG1 at doses ranging from 0.3 to 10 mg / kg. HFB3-1hz6-hG1 was more effective at suppressing tumor growth. At a dose of 10 mg / kg, HFB3-1hz6-hG1 was more effective at controlling tumor growth than anti-mPD-1 (see Figure 32).

[0355] Example 17: Toxicological evaluation of anti-TNFR2 antibodies in non-human primates The toxicity of humanized anti-TNFR2 antibodies was investigated in a non-human primate model. Two cynomolgus monkeys per group were injected with a single dose of 15 mg / kg (low), 50 mg / kg (medium), and 150 mg / kg (high) of humanized HFB3-1hz6-hG1 monoclonal antibody, and plasma was collected at different time points up to 336 hours (day 14).

[0356] Toxicokinetic analysis of HFB3-1hz6-hG1 showed that the antibody cleared over time. No increase in the cytokines IL-6, IL-2, IFN-γ, and TNF-α was observed after injection of 15, 50, or 150 mg / kg HFB3-1hz6-hG1, compared to the data reported for CD3xCD20 bispecific IgG at 3 mg / kg or less (dotted line) (Figure 30).

[0357] Following injection of 15, 50, or 150 mg / kg HFB3-1hz6-hG1, no abnormalities were observed in white blood cell, red blood cell, platelet, neutrophil, and lymphocyte counts compared to the range of previous data obtained from normal monkeys (Figure 31).

[0358] Toxicological evaluations to date have not demonstrated any discernible toxic effects from treatment of non-human primate subjects with HFB3-1hz6-hG1 at doses up to 150 mg / kg.

[0359] In a dose-ranging study (DRF) in which multiple doses of HFB3-1hz6-hG1 were administered to cynomolgus monkeys, no changes in IL-2, IL-4, IL-5, TNFα, or IFN-γ were observed in monkeys receiving repeated doses up to 150 mg / kg. Changes in IL-6 levels were observed in male animals at 10 mg / kg and 150 mg / kg at the end of four weekly doses in monkeys. Dose-dependent decreases in neutrophil and platelet counts were observed 2 weeks after administration of HFB3-1hz6-hG1 in monkeys. Diarrhea (liquid or loose stools) was frequently observed after weekly administration of HFB3-1hz6-hG1 in monkeys.

[0360] Based on the above observations, the drug half-life in humans after a single injection of a 1 mg / kg dose is predicted to be 23 days, making this antibody suitable for administration at 1 mg / kg every 4 weeks.

[0361] Example 18: Selection of indications based on TNFR2 expression Without wishing to be bound by any particular theory, it is believed that the anti-tumor efficacy of the anti-TNFR2 antibodies of the present invention results from stimulating TNFR2 in tumor-infiltrating T cells and NK cells, thereby activating NK cells and enhancing CD8+ T cell-mediated anti-tumor responses. In this example, we provide evidence that tumor types that may benefit from treatment with the anti-TNFR2 antibodies of the present invention include tumors with high TNFR2 and high CD8A expression.

[0362] In bulk RNA analysis of cancers using the TCGA database, the CD8A cutoff is based on CD8A levels in acute myeloid leukemia (AML), which is presumed to be composed primarily of myeloid cells and has few or no CD8+ T cells. The TNFR2 cutoff value is based on TNFR2 levels in prostate cancer, which is presumed to be an immune desert. See Figures 34A-34B.

[0363] The ranking of cancer types based on TNFR2 / CD8A levels is shown in Figure 35. EBV+ gastric adenocarcinoma / carcinoma, clear cell renal cell carcinoma, cutaneous melanoma, testicular germ cell tumor, soft tissue sarcoma, and high PD-L1 cancers (including cervical squamous cell carcinoma, pleural mesothelioma, lung adenocarcinoma, and head and neck squamous cell carcinoma) were identified as the top high TNFR2 / high CD8A cancers.

[0364] Survival analysis of cancer patients using the TCGA database showed that at the median gene expression cutoff, high TNFR2 expression was significantly associated with better survival in cutaneous melanoma and head and neck squamous cell carcinoma (Figures 33A and 33B), and showed a trend toward better survival in lung adenocarcinoma (data not shown). No significant trends were observed for cervical squamous cell carcinoma / endocervical adenocarcinoma, renal clear cell carcinoma, testicular germ cell tumor, sarcoma, gastric adenocarcinoma, or mesothelioma.

[0365] TNFR2 and CD8 scoring was further determined for molecular subtypes of renal cell carcinoma (RCC), cutaneous melanoma (SKCM), gastric adenocarcinoma / gastric cancer (STAD / GI), lung adenocarcinoma (LUAD), and head and neck squamous cell carcinoma (HNSC) using published data (Figure 36).

[0366] Within each cancer type tested, there are subtypes with a high percentage of cancers that display the characteristic high CD8A and high TNFR2 expression pattern (e.g., approximately 60% of STAD / GI-EBV+ cancers have this characteristic high expression), while other subtypes (e.g., ESCC and HM-SNV subtypes have low CD8A and TNFR2 expression). hi TNFR2 hi It is clear that there are some cases in which the expression of the IL-1 gene is substantially absent.

[0367] Thus, the cancer subtypes tested that have characteristic high CD8A and high TNFR2 expression patterns are prime candidates for beneficial treatment with the antibodies of the present subject matter, including KIRC.2, KIRC.3, KIRC.4, SKCM.Triple_WT, SKCM.BRAF_hotspot_mutants (and possibly SKCM.RAS_hotspot_mutants and SKCM.NF1_any_mutants), LUAD.6 and LUAD.5, HNSC.Atypical (40% HPV positive) and HNSC.mesenchymal (tendency towards relatively high PD-L1 / CD274).

[0368] In colorectal cancer patients, mismatch repair deficient (dMMR) / microsatellite instability high (MSI-H) tumors are significantly more sensitive to immune checkpoint inhibitors (ICIs) than microsatellite stable (MSS) / microsatellite instability low (MSI-L) tumors, with the former patient group deriving more clinical benefit from immunotherapy than the latter.

[0369] Since MSI score data is not directly accessible for all cancer indications, applicants use the parameter mutation count as a surrogate for MSI score and to estimate the TNFR2 mutation counts treatable by the subject antibodies. hi and CD8A hi Tumors were examined for MSI or MSS enrichment. For this purpose, a mutation count of >250 was considered MSI and a mutation count of <250 was considered MSS. Data (not shown) showed that the TNFR2 hi and CD8A hi The expression pattern of TNFR2 was not strongly enriched in mutation counts of <250 versus >250. hi and TNFR2 lo was observed (4% CD8A hi In MSS, the split was 45% vs. 55% (all CD8A lo ).

[0370] Example 19: Administration study of HFB3-1hz6-hG1 in humans Based in part on data from the minimal expected biological effect level based on in vitro cytokine release assays, the minimal pharmacologically active dose in human TNFR2 knock-in (hTNFR2 KI) mice bearing MC38 tumors, and the highest dose without severe toxicity in humans and non-human primates, the starting dose of HFB3-1hz6-hG1 in this study will be 5 mg administered as a 60-minute intravenous infusion every 4 weeks (Q4W).

[0371] Dose escalation will be performed up to 150 mg to determine the maximum tolerated dose. Dose expansion will be performed in EBV+ gastric cancer, clear cell renal cell carcinoma, cutaneous melanoma, soft tissue sarcoma, testicular germ cell tumors, and PD-L1+ cancers (including cervical cancer, pleural mesothelioma, lung adenocarcinoma, and head and neck squamous cell carcinoma). Based on Phase I antitumor activity / efficacy, additional patient cohorts will be enrolled. [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12]

Table 13

Table 14

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof is specific for human TNFR2, and the monoclonal antibody is (1a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 1, the HCVR CDR2 sequence of SEQ ID NO: 2, and the HCVR CDR3 sequence of SEQ ID NO: 3, said heavy chain variable region, and (1b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 4, the LCVR CDR2 sequence of SEQ ID NO: 5, and the LCVR CDR3 sequence of SEQ ID NO: 6, said light chain variable region, or (2a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 14, the HCVR CDR2 sequence of SEQ ID NO: 15, and the HCVR CDR3 sequence of SEQ ID NO: 16, said heavy chain variable region, and (2b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 17, the LCVR CDR2 sequence of SEQ ID NO: 18, and the LCVR CDR3 sequence of SEQ ID NO: 19, said light chain variable region, said isolated monoclonal antibody or antibody-binding fragment thereof.

2. (1A) said HCVR sequence is SEQ ID NO: 7, and / or (1B) said LCVR sequence is SEQ ID NO: 8, or (2A) said HCVR sequence is SEQ ID NO: 20, and / or (2B) said LCVR sequence is SEQ ID NO: 21, The isolated monoclonal antibody or antibody-binding fragment thereof according to claim 1.

3. The monoclonal antibody is (1a) the heavy chain sequence of SEQ ID NO: 9, and / or (1b) the light chain sequence of SEQ ID NO: 10, or (2a) the heavy chain sequence of SEQ ID NO: 22, and / or (2b) the light chain sequence of SEQ ID NO: 23, having the isolated monoclonal antibody or antibody-binding fragment thereof according to claim 1 or 2.

4. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, which is a mouse antibody, a human-mouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a reshaped antibody.

5. wherein the antigen-binding fragment is Fab, Fab', F(ab') ２ , F ｄ , single-chain Fv or scFv, disulfide-bonded F ｖ , V-NAR domain, IgNar, intrabody, IgGΔCH ２ , minibody, F(ab') ３ , tetrabody, tribody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb ２ , (scFv) ２ , or scFv-Fc, the isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1.

6. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof cross-reacts with cynomolgus monkey TNFR2 but does not substantially cross-react with mouse TNFR2.

7. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof does not substantially cross-react with TNFR1.

8. The monoclonal antibody or antigen-binding fragment thereof binds to TNFα with a K of less than about 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 2 nM, or less than 1 nM ｄ The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, which binds to TNFα with ｄ .

9. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, which enhances the binding of TNFα to TNFR2, enhances TNFα-mediated or co-stimulatory NFκB signaling, and / or promotes the proliferation of TCR-activated effector T cells in the presence of Treg.

10. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, which enhances TNFα-mediated CD25 expression in Treg.

11. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, which binds to the epitope of SEQ ID NO: 13 and / or 101.

12. An isolated monoclonal antibody or antigen-binding fragment thereof that competes with the isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 with respect to binding to the epitope of SEQ ID NO: 13 and / or 101.

13. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to the epitope of SEQ ID NO: 13 and / or 101.

14. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 12 or 13, which enhances the binding of TNFα to TNFR2, enhances TNFα-mediated or co-stimulatory NFκB signaling, and / or promotes the proliferation of TCR-activated effector T cells in the presence of Treg.

15. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, which promotes the proliferation of Treg.

16. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, which promotes the activation of natural killer cells.

17. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the maximum tolerated dose (MTD) in cynomolgus monkeys is about 150 mg / kg. A pharmaceutical composition comprising the isolated monoclonal antibody according to claim 1, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition for performing cancer treatment in a patient in need thereof, comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the patient has (a) a level of TNFR2 expression higher than the average TNFR2 expression level in prostate cancer patients (optionally, the TNFR2 expression is evaluated in effector T cells, tumor-infiltrating CD8 ＋ T cells, and / or NK cells), and (b) having a CD8A expression level higher than the average CD8A expression level in AML patients A pharmaceutical composition.

20. wherein the patient has a higher level of TNFR2 expression than the above in tumor-infiltrating CD8A ＋ ＋ (CD8α chain-positive) T cells, the pharmaceutical composition according to claim 19.

21. wherein the patient has EBV ＋ The pharmaceutical composition according to claim 19 or 20, wherein the patient has gastric cancer, clear cell renal cell carcinoma, renal clear cell carcinoma, cutaneous melanoma, testicular germ cell tumor, or soft tissue sarcoma.

22. The pharmaceutical composition according to claim 19 or 20, wherein the cancer expresses a "high" level of PD-L1.

23. The pharmaceutical composition according to claim 22, wherein the cancer is cervical cancer, pleural mesothelioma, lung adenocarcinoma, or head and neck squamous cell carcinoma.

24. administering to the patient (a) an antibody specific for PD-1 or an antigen-binding fragment thereof, (b) an antibody specific for PD-L1 or an antigen-binding fragment thereof, and / or (c) an antibody specific for PD-L2 or an antigen-binding fragment thereof The pharmaceutical composition according to claim 19, for administration in combination with.

25. An antibody specific for PD-1 or an antigen-binding fragment thereof is selected from the group consisting of semiprimab, nivolumab, pembrolizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, and INCMGA00012, and / or An antibody specific for PD-L1 or an antigen-binding fragment thereof is selected from the group consisting of avelumab, durvalumab, atezolizumab, KN035, or CK-301, The pharmaceutical composition according to claim 24.

26. The pharmaceutical composition according to claim 19, wherein the patient has recurrent or refractory cancer and / or has been treated with standard therapy in the past.

27. The pharmaceutical composition according to claim 19, for administration to the patient once every 3 weeks (Q3W), once every 4 weeks (Q4W), or once every 5 weeks (Q5W).

28. The pharmaceutical composition according to claim 27, for administration to the patient at a dose of about 5 mg, about 15 mg, about 50 mg, about 100 mg, or about 150 mg once every 4 weeks (Q4W).

29. A pharmaceutical composition for treating cancer or autoimmune disorders in a patient in need thereof, comprising the isolated monoclonal antibody according to claim 1 or an antigen-binding fragment thereof.

30. The pharmaceutical composition according to claim 29 for treating cancer for administering an antagonist of an immune checkpoint.

31. The pharmaceutical composition according to claim 30, wherein the immune checkpoint is a PD-1 / PD-L1 immune checkpoint.

32. The pharmaceutical composition according to claim 30 or 31, wherein the antagonist of the immune checkpoint is an antibody specific for PD-1 or PD-L1 or an antigen-binding fragment thereof.

33. The pharmaceutical composition according to claim 32, wherein the antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

34. The pharmaceutical composition according to claim 33, wherein the anti-PD-1 antibody is semiprimab, nivolumab, pembrolizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, or INCMGA00012, and the anti-PD-L1 antibody is avelumab, durvalumab, atezolizumab, KN035, or CK-301.

35. The pharmaceutical composition according to claim 30, wherein the antagonist of the immune checkpoint is a (non-antibody) peptide inhibitor of PD-1 / PD-L1, a small molecule inhibitor of PD-L1, or a macrocyclic peptide.

36. The pharmaceutical composition according to claim 35, wherein the peptide inhibitor is AUNP12, the small molecule inhibitor of PD-L1 is CA-170, and the macrocyclic peptide is BMS-986189.

37. The pharmaceutical composition according to claim 29, wherein the cancer is melanoma, breast cancer, colon cancer, cervical cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, skin cancer, lymphoma, or leukemia.

38. The pharmaceutical composition according to claim 37, wherein the cancer is hepatocellular carcinoma, non-small cell lung cancer (NSCLC), squamous cell carcinoma, or basal cell carcinoma.

39. The pharmaceutical composition according to claim 29 for administration in combination with a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitor, a cancer immunotherapeutic agent, and / or an anti-neoplastic composition.

40. A polynucleotide encoding the heavy chain or light chain according to claim 1 or an antigen-binding portion thereof.

41. The polynucleotide according to claim 40, which is codon-optimized for expression in human cells.

42. A vector comprising the polynucleotide according to claim 40 or 41.

43. The vector according to claim 42, which is an expression vector.

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