CN117980336A

CN117980336A - Anti-TNFR 2 antibodies and uses thereof

Info

Publication number: CN117980336A
Application number: CN202280060055.4A
Authority: CN
Inventors: N·A·A·贝尔特拉米内尔; J·沃特金斯; F·艾德里安; 章乾; A·劳厄; P·玛丽; L·施维泽; 魏硕; M·德林斯
Original assignee: Gaocheng Biomedical Hong Kong Co ltd
Current assignee: Gaocheng Biomedical Hong Kong Co ltd
Priority date: 2021-07-07
Filing date: 2022-07-06
Publication date: 2024-05-03

Abstract

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

Description

Anti-TNFR 2 antibodies and uses thereof

Citation of related application

The present application claims the benefit of the date of filing of U.S. provisional patent application No. 63/219,175 filed 7 at 2021, the entire contents of which, including any figures and sequence listing, are hereby incorporated by reference.

Background

Tumor necrosis factor receptor 2 (TNFR 2), also known as tumor necrosis factor receptor superfamily members 1B (TNFRSF 1B) and CD120B, is a 75kDa type I transmembrane protein containing an extracellular domain (ECD, residues 1-257), a transmembrane domain (TM, residues 258-287) with 4 cysteine-rich domains (CRD 1 to CRD 4) and an intracellular domain (ICD, residues 288-461) with a TRAF2 binding domain. TNFR2 shares relatively low sequence identity with another tnfα receptor, tumor necrosis factor receptor 1 (TNFR 1), and its homology between extracellular domains is only 28%.

TNFR2 binds to TNFα ligand in a 3:3 trimerization mode. The co-crystal structure of TNFR2 and tnfα has been resolved and binding of each TNFR2 molecule to two tnfα ligands has been shown. In addition, tnfα binds TNFR2 at K _d of 420pM, which is about 20-fold weaker than it binds TNFR1 (K _d =19 nM). Typically, tnfα preferentially binds to TNFR1 under otherwise identical conditions.

In normal T cells, tnfα -TNFR2 interactions trigger cell survival signaling via the NFkB signaling pathway. However, in autoimmune T cells, tnfα -TNFR2 interactions trigger apoptotic signals via the caspase pathway.

Human TNFR2 shows 62% amino acid sequence homology with mouse TNFR2, but it is 97% identical to rhesus TNFR 2.

Although TNFR1 is widely expressed, TNFR2 expression is primarily limited to immune cells and is primarily highly expressed by tumor-infiltrating immunosuppressive CD4 ⁺FoxP3⁺ regulatory T cells (tregs). Recent studies have shown that TNFR2 plays a key role in stimulating activation and proliferation of tregs, the primary checkpoint of the anti-tumor immune response (Chen and Oppenheim, SCI SIGNAL 10:eaal2328, 2017). Activation of TNFR2 via its ligand tnfα causes NFkB signaling activation and expansion of TNFR2 ⁺ Treg. TNFR2 is also expressed in CD8 and CD4 Tconv cells and bone marrow cells. In particular, TNFR2 is expressed in depleted CD 8T cells, similar to a clinically validated immune checkpoint.

T regulatory cells (tregs) are a small subset of T lymphocytes with different clinical applications. In one aspect, TNFR2 ⁺ tregs are highly immunosuppressive and have more potent inhibitory activity than the highly inhibitory CD103 ⁺ tregs (J Immunol 179:154-161,2007;J Immunol 180:6467-6471,2008). Thus, TNFR2 ⁺ tregs are useful in therapies that rely on immunosuppressive activity of tregs, such as for transplantation, allergy, asthma, infectious disease, graft Versus Host Disease (GVHD), and autoimmunity. For example, in the experimental GVHD mouse model, CD4 ⁺CD25^{High height}Foxp3⁺ thymic-derived Treg depletion can enhance GVHD (Cohen et al, JEM 2002).

TNFR2 is also expressed in certain cancers such as breast, cervical, colon and kidney cancers (front. Immunol.9:1170,2018) and may be involved in immune tolerance in these cancers. The ligand of TNFR2 (tnfα) promotes survival and growth of these cancer cells. TNFR2 has been shown to be involved in multiple tumor progression processes by employing different signaling pathways in tumor cells. For example, nuclear factor- κb (nfkb) is involved in the malignant transformation of TNFR 2-associated epithelial cells. 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 critical for TNFR2 function mentioned above. Thus, in tumor immunity, inhibiting TNFR2 function can inhibit Treg function and increase anti-tumor T cell responses.

Accordingly, there is a need to develop therapeutic agents that allow for the enhancement of the immunosuppressive function of tregs by stimulating TNFR2 function on TNFR2 ⁺ tregs to treat autoimmune disorders, or to inhibit TNFR2 activation to treat diseases such as cancer.

Disclosure of Invention

In one aspect, the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof is specific for human TNFR2, and wherein the monoclonal antibody comprises: (1a) A Heavy Chain Variable Region (HCVR) comprising a HCVR CDR1 sequence of SEQ ID No. 1, a HCVR CDR2 sequence of SEQ ID No. 2, and a HCVR CDR3 sequence of SEQ ID No. 3; and (1 b) 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 (2 a) 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 (2 b) 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 (3 a) a Heavy Chain Variable Region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO. 26, the HCVR CDR2 sequence of SEQ ID NO. 27 and the HCVR CDR3 sequence of SEQ ID NO. 28; and (3 b) a Light Chain Variable Region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO. 29, the LCVR CDR2 sequence of SEQ ID NO. 30 and the LCVR CDR3 sequence of SEQ ID NO. 31; or (4 a) a Heavy Chain Variable Region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO:39, the HCVR CDR2 sequence of SEQ ID NO:40 and the HCVR CDR3 sequence of SEQ ID NO: 41; and (4 b) a Light Chain Variable Region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO. 42, the LCVR CDR2 sequence of SEQ ID NO. 43 and the LCVR CDR3 sequence of SEQ ID NO. 44; or (5 a) 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 (5 b) a Light Chain Variable Region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO. 54, the LCVR CDR2 sequence of SEQ ID NO. 55 and the LCVR CDR3 sequence of SEQ ID NO. 56; or (6 a) a Heavy Chain Variable Region (HCVR) comprising a HCVR CDR1 sequence of SEQ ID No. 63, a HCVR CDR2 sequence of SEQ ID No. 64, and a HCVR CDR3 sequence of SEQ ID No. 65; and (6 b) 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.

In certain embodiments, in an 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.

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

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.

In certain embodiments, the antigen binding fragment thereof is a Fab, fab ', F (ab ') 2, fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, igNar, intracellular antibody, igG Δch2, minibody, F (ab ') 3, tetravalent antibody, trivalent antibody, diabody, single domain antibody, DVD-Ig, fcab, mAb2, (scFv) 2, or scFv-Fc.

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof cross-reacts with rhesus TNFR2, but does not substantially cross-react with mouse TNFR 2.

In certain embodiments, the monoclonal antibodies or antigen binding fragments thereof of the invention comprise one or more point mutations of their amino acid sequences designed to improve the developability of the antibodies. For example, in certain embodiments, one or more point mutations make the antibody more stable during its expression in a host cell, its purification during manufacture, and/or during the formation process, and/or during its administration to a subject patient. In certain embodiments, one or more point mutations make the antibody less likely to aggregate during the manufacturing and/or formulation process.

In certain embodiments, the invention provides therapeutic antibodies with minimized or reduced developability problems (such as removal or reduced hydrophobicity and/or optimized charge) by substituting one or more amino acids in their sequences (e.g., in one or more CDRs thereof).

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof does not substantially cross-react with TNFR 1.

In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof binds tnfα with a K _d of less than about 25nM, 20nM, 15nM, 10nM, 5nM, 2nM, or 1 nM.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof enhances binding between tnfα and TNFR 2; enhancing tnfa-mediated or co-stimulated nfkb signaling (e.g., in TCR-activated CD8 and/or CD4 Tconv T cells); and/or proliferation of effector T cells (e.g., CD8 and/or CD4 Tconv T cells) that promote TCR activation in the presence of tregs.

In certain embodiments, the isolated monoclonal antibody or antigen binding fragment thereof enhances tnfα -mediated CD25 expression on tregs.

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

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof promotes binding of tnfα to TNFR 2; inhibit tnfα binding to TNFR 2; or no apparent effect on tnfα binding to TNFR 2.

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 TNFR 2.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is an agonist of TNFR2, or stimulates TNFR2 signaling (such as in the presence of tnfα), wherein the agonist function is preferably Fc-independent.

In certain embodiments, the isolated monoclonal antibody or antigen binding fragment thereof activates CD4 ⁺ effector T cells, CD8 ⁺ effector T cells, other effector T cells, and/or NK cells in vitro.

In another aspect of the invention there is provided an isolated monoclonal antibody or antigen binding fragment thereof which competes with the isolated monoclonal antibody or antigen binding fragment thereof of any of the subject antibodies for binding to an epitope of SEQ ID NO. 13 and/or 101.

In another aspect the invention provides an isolated monoclonal antibody or antigen binding fragment thereof which specifically binds to an epitope of SEQ ID NO. 13 and/or 101.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof inhibits binding between tnfα and TNFR 2; inhibition of tnfa-mediated or co-stimulated nfkb signaling (e.g., in TCR-activated CD8 and/or CD4 Tconv T cells); and/or inhibit proliferation of TCR-activated effector T cells (e.g., CD8 and/or CD4 Tconv T cells) in the presence of tregs.

In certain embodiments, the isolated monoclonal antibody or antigen binding fragment thereof promotes Treg expansion.

In another aspect of the invention, an isolated monoclonal antibody or antigen-binding fragment thereof is provided that competes for binding to the same epitope as an isolated monoclonal antibody or antigen-binding fragment thereof of the invention.

In another aspect of the invention, an isolated monoclonal antibody or antigen-binding fragment thereof is provided, wherein the monoclonal antibody or antigen-binding fragment thereof specifically binds human TNFR2 at an epitope comprising, consisting essentially of, or consisting of SEQ ID NO. 101, optionally the isolated monoclonal antibody or antigen-binding fragment thereof does not bind human TNFR2 at an epitope consisting essentially of or consisting of SEQ ID NO. 13.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of claim 19, (1) promotes activation and proliferation of CD4 ⁺ T cells in tumor-infiltrating lymphocytes (TILs) other than regulatory T cells (tregs) (e.g., in an in vivo hTNFR2 knock-in MC38 mouse tumor model); and/or (2) promote NK cell activation in vitro and/or in vivo.

In certain embodiments, the isolated monoclonal antibodies or antigen binding fragments thereof of the invention have a Maximum Tolerated Dose (MTD) in cynomolgus monkeys of about 150 mg/kg.

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 of the invention, or an antigen-binding fragment thereof, wherein the patient (e.g., the patient's cancer) has: (a) Higher levels of TNFR2 expression compared to the average TNFR2 expression levels in prostate cancer patients; optionally, the 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 (b) a higher level of CD8A expression compared to the average CD8A expression level in AML patients.

In certain embodiments, the patient (e.g., the patient's cancer) has the higher expression level of TNFR2 in tumor-infiltrating CD8a ⁺ (CD 8a chain positive) T cells.

In certain embodiments, the patient has EBV ⁺ 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 type B clear cell (ccB subtype, or ccA/ccB unclassified subtype), cutaneous melanoma (e.g., cutaneous melanoma such as so-called triple wt subtypes lacking hotspot BRAF, N/H/K-RAS, or NF1 mutations; subtypes with BRAF hotspot mutations (including V600E, V K and V600R mutations and hotspot mutations at K601), subtypes with RAS hotspot mutations (including Q61R, Q61K, Q61L, Q H, 61_62qe > hk in NRAS, G12R/D/a and G13R/D in HRAS, G13D, G S and Q61K in HRAS, and G D, G R12 in KRAS) and Q12R and NF 1) and soft tissue sarcoma or any germ cell sarcoma.

In certain embodiments, the cancer expresses PD-L1 at a level greater than average.

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

In certain embodiments, the method further comprises administering to the patient: (a) An antibody or antigen binding fragment thereof specific for PD-1, such as cimetidine Li Shan antibody (cemiplimab), nivolumab, pembrolizumab (pembrolizumab), spabulab (spartalizumab), karilizumab (camrelizumab), singedi Li Shan antibody (sintilimab), tirelizumab (tislelizumab), teripep Li Shan antibody (toripalimab), rituximab (dostarlimab), and INCMGA00012; (b) An antibody or antigen-binding fragment thereof specific for PD-L1, such as avermectin (avelumab), dewarukast (durvalumab), alemtuzumab (atezolizumab), KN035 or CK-301, and/or (c) an antibody or antigen-binding fragment thereof specific for PD-L2.

In certain embodiments, the patient has recurrent or refractory cancer, and/or has been previously treated with (and optionally failed to respond to or relapse from) standard-of-care therapy.

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 3 weeks (Q3W), once every 4 weeks (Q4W), or once every 5 weeks (Q5W) (e.g., once every 4 weeks or Q4W).

In certain embodiments, the method comprises administering the isolated monoclonal antibody or antigen-binding fragment thereof to the patient at a dose of about 5mg, 15mg, 50mg, 100mg, or 150mg once every 4 weeks (Q4W) (e.g., intravenous administration over 60 minutes).

In certain embodiments, the method further comprises: (1) Selecting a patient having said higher TNFR2 expression level and CD8A expression level prior to the administering step; or (2) prior to the administering step, verifying that the patient has said higher TNFR2 expression level and CD8A expression level.

In another aspect, 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 that specifically binds to human TNFR 2at an epitope comprising, consisting essentially of, or consisting of SEQ ID NO:101, optionally, the isolated monoclonal antibody or antigen-binding fragment thereof does not bind to human TNFR 2at an epitope consisting essentially of or consisting of SEQ ID NO: 13.

Another aspect of the invention provides a method of treating cancer or an autoimmune disorder (AID, such as 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.

In certain embodiments, the methods are for treating AID, wherein the methods further comprise administering a second agent, such as a low dose of an anti-IL 2 agent, to treat chronic GVHD, or an anti-tnfα agent, such as adalimumab (adalimumab), infliximab (infliximab), etanercept (etenercept), golimumab (golimumab), and the like, to treat rheumatoid arthritis, chronic plaque psoriasis, crohn's disease, ankylosing spondylitis, psoriatic arthritis, juvenile idiopathic arthritis of polyarthritis, IBS, EAE, and non-infectious uveitis.

In certain embodiments, the method is for treating cancer, wherein the method further comprises administering an antagonist of an immune checkpoint.

In certain embodiments, the immune checkpoint is a PD-1/PD-L1 immune checkpoint.

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

In certain embodiments, the antibody is an anti-PD-1 antibody, such as a cimetidine Li Shan antibody, a nivolumab, a pembrolizumab, a spabulab, a carlizumab, a singdi Li Shan antibody, a tirelimumab, a terlipdine Li Shan antibody, a rituximab, and INCMGA00012.

In certain embodiments, the antibody is an anti-PD-L1 antibody, such as avermectin, dewaruzumab, alemtuzumab, KN035, or CK-301.

In certain embodiments, the antagonist of an immune checkpoint is a (non-antibody) peptide inhibitor of PD-1/PD-L1, such as AUNP; small molecule inhibitors of PD-L1, such as CA-170, or macrocyclic peptides, such as BMS-986189.

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.

In certain embodiments, the method further comprises administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, a tumor immunizing agent, and/or an anti-tumor composition.

In another aspect, the invention provides a polynucleotide encoding a heavy or light chain of the invention or an antigen binding portion thereof.

In certain embodiments, the polynucleotide is codon optimized for expression in human cells.

In another aspect, the invention provides a vector comprising a polynucleotide of the invention.

In certain embodiments, the vector is an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector).

Drawings

FIG. 1 shows the alignment of the VH and VL regions of human-mouse chimeric monoclonal antibodies HFB3-1, HFB3-3, HFB3-6, HFB3-14, HFB3-18, HFB3-19, HFB3-20, HFB3-21, HFB3-22, HFB3-23, HFB3-24, and HFB3-25, and their consensus sequences.

FIG. 2A shows the binding affinity of a selected human-mouse chimeric monoclonal antibody raised against the extracellular domain of recombinant human TNFR 2. EC ₅₀ and E _max values of test antibodies and isotype-matched negative control antibodies were measured against CHO cells expressing human TNFR2 (CHO. Hfb 3) or rhesus TNFR2 (CHO. Mkhfb 3).

FIG. 2B shows that different anti-TNFR 2 monoclonal antibodies can promote (HFB 3-1) or inhibit (HFB 3-18) the binding of TNFα to TNFR2, or have no effect on binding (HFB 3-6).

FIG. 3 shows that human-mouse chimeric monoclonal antibodies do not bind to the parental CHO cell line and do not bind to CHO cells expressing mouse TNFR2 (except for the marginal binding of HFB3-18 and HFB3-19 antibodies).

Fig. 4A shows the binding specificity of the human-mouse chimeric antibody to TNFR2 but not TNFR 1.

FIG. 4B shows the K _d、k_{Association with} and K _{Dissociation of} values of the human-mouse chimeric antibodies HFB3-1, HFB3-14, and HFB3-18 for His-tagged recombinant human TNFR 2.

Fig. 5 shows the expression of TNFR2 on T cell subtypes, particularly depleted CD 8T cells, in tumor-infiltrating lymphocytes.

Fig. 6 shows cell binding of anti-TNFR 2 chimeric monoclonal antibodies on TCR-activated (bottom panel) and non-TCR-activated (top panel) primary tregs, CD8 and CD4 Tconv. The HFB3 antibody preferentially recognizes primary T cells (TCR activation) that are co-stimulated by CD3/CD 28.

FIG. 7 shows that certain HFB3 antibodies of the invention (including HFB3-1, HFB3-14, HFB3-18, HFB3-23, HFB3-24, and HFB 3-25) trigger NF-. Kappa.B signaling and enhance the effect in the presence of a TNF. Alpha. Ligand.

FIG. 8 shows that co-stimulation of certain subject monoclonal antibodies (including HFB3-1, HFB3-14, HFB3-18, and HFB 3-25) and CD3/CD28 causes proliferation of CD8 and CD4 Tconv in a dose dependent manner.

FIG. 9 shows that an anti-TNFR 2 monoclonal antibody of the invention (e.g., HFB3-1hz6-hG1AA, which is a humanized form of HFB 3-1) facilitates cell proliferation on effector T cells (CD 8 and CD4 Tconv) in a dose-dependent manner in the presence of tregs.

Fig. 10 shows that the subject anti-TNFR 2 antibodies lack ADCC effect.

FIGS. 11A and 11B show the results of several features of the His-tagged extracellular domain (ECD) of TNFR2 (called HFB 2003), including the TNFα binding site, and epitope localization of monoclonal antibodies HFB3-1 and HFB3-14 and HFB3-18 (FIG. 11A) or HFB3-6 (FIG. 11B). These antibodies are mouse chimeric antibodies having a human IgG1 Fc region and are therefore also referred to as HFB3-1-hG1, HFB3-14-hG1, HFB3-18-hG1 or HFB3-6-hG1, respectively. FIG. 11B also includes epitope mapping data for reference antibodies SBT-1 and SBT-4 (references 1 and 2). The HFB3-1 antibodies bind 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. FIG. 11C shows more refined epitope mapping data for HFB 3-1; the potential HFB3-1hG1 epitope region (SEQ ID NO: 101) identified in 2 independent experiments is highlighted.

FIG. 11D provides a 3-D model showing the binding sites for HFB3-1, HFB3-14, HFB3-6, and HFB-3-18 on the TNFR2-TNFα complex.

FIG. 12A shows that humanized variants of the chimeric monoclonal antibodies HFB3-1, HFB3-14, and HFB3-18 bind to CHO cells expressing human TNFR2 (CHO. HTNFR 2) but do not bind to the parent CHO cells.

FIG. 12B shows the binding affinity of selected humanized anti-TNFR 2 monoclonal antibodies. EC ₅₀ values of the test humanized and parent chimeric antibodies were measured against CHO cells expressing human TNFR2 (CHO. Hfb 3).

FIG. 13 shows the binding affinity of selected humanized anti-TNFR 2 monoclonal antibodies. EC ₅₀ values of the test humanized and parent chimeric antibodies were measured against CHO cells expressing rhesus TNFR2 (CHO. Mkhfb 3).

Fig. 14A shows that humanized anti-TNFR 2 antibodies bind to recombinant human and cynomolgus TNFR2 but not to recombinant human TNFR1 in an ELISA assay.

FIG. 14B shows the results of binding affinity of humanized variant and parent chimeric monoclonal antibodies HFB3-1 and HFB3-14 to recombinant human TNFR2 based on AHC (anti-human IgG Fc capture) biosensor measurements. The values are the average of two experiments obtained from two different days.

FIG. 14C shows the binding specificity of the exemplary humanized antibody HFB3-1hz6-hG1 to TNRF2 expressing/positive CHO cells (CHO.hTNFR2) compared to the parent CHO cells (Bmk: benchmark antibody 1).

FIG. 15 shows cell binding of humanized anti-TNFR 2 monoclonal antibodies to TCR activated CD 8T cells.

FIG. 16 shows the co-stimulatory effect of humanized anti-TNFR 2 monoclonal antibodies on proliferation of TCR-activated CD 4T cells.

Figure 17A shows that co-stimulation of Treg with certain humanized variant anti-TNFR 2 antibodies and tnfα causes nfkb downstream signaling.

FIG. 17B shows the activation of NFkB signaling in CD 8T cells using certain humanized variants of HFB3-1 antibodies with and without recombinant human TNFα. ", indicates statistical significance.

Fig. 18 shows that the subject humanized variant anti-TNFR 2 antibodies were stable upon storage.

FIG. 19 shows the Fc gamma R cross-linking dependence of the anti-TNFR 2 monoclonal antibodies HFB3-18 (but not HFB3-1 and HFB 3-14) on costimulatory primary T cells.

Figure 20 shows the confirmed co-stimulatory effect of selected humanized anti-TNFR 2 antibodies on proliferation of CD 8T cells in the presence or absence of tnfα.

FIG. 21A shows that the subject anti-TNFR 2 monoclonal antibodies co-stimulate 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 25ng/mL TNFα.

Figure 21B shows that humanized HFB3-1hz6 binds to peripheral CD4 and CD 8T cells (top panel) and stimulates T cell proliferation in vitro (bottom panel) in the presence of CD3/CD28 mediated TCR activation in a dose dependent manner.

FIG. 22 shows the ex vivo activation of isolated Natural Killer (NK) cells by humanized HFB3-1hz6-hG1 antibody and parent HFB3-1-hG1 antibody after stimulation with soluble IL-2 (10 ng/mL) and IL-15 (10 ng/mL). The top panel shows the time line of the experiment. CD107 a and TNFR2 expression was upregulated in a dose-dependent manner by HFB3-1hz6-hG1 and HFB3-1-hG1, but isotype control and anti-OX 40 antibodies (BMS) failed to trigger short term NK activation.

FIG. 23 shows the ex vivo activation of Natural Killer (NK) cells in the whole peripheral blood mononuclear cell fraction by HFB3-1hz6-hG1 and by the parent mouse HFB3-1-hG1 after stimulation with plate-bound anti-CD 3 (1. Mu.g/mL) and soluble anti-CD 28 (1. Mu.g/mL). The top panel shows the time line of the experiment. In CD3 ^-/CD56⁺ cells, CD 107. Alpha. Expression was up-regulated in a dose-dependent manner by HFB3-1hz6-hG1 and HFB3-1-hG1, but control anti-OX 40 antibody (MBS) failed to trigger short term NK activation.

Fig. 24A shows a time line of pharmacodynamic experiments in a mouse MC38 tumor model. 2 doses of HFB3-1-hG1 (doses of 0.1mg/kg, 1mg/kg and 10 mg/kg) or 10mg/kg isotype-matched control antibody (TT) were administered intraperitoneally 3 days apart.

Figure 24B shows the in vivo effect of antibody administration on total immune cell count in MC38 tumors. The administration of 10mg/kg HFB3-1-hG1 increased the absolute cell number of CD45 ⁺ cells. Based on one-way ANOVA test, p-value <0.05 (.

Fig. 24C shows the in vivo effect on cell count of different immune cells in MC38 tumors. The administration of 10mg/kg HFB3-1-hG1 increased the absolute cell numbers of CD8 ⁺, conventional CD4 ⁺ T and NK cells in the tumor microenvironment, but did not alter the number of T regulatory cells. Based on one-way ANOVA test, p values <0.05.

FIG. 25A shows the percentage of TNFR2 receptor occupied by the injected antibody HFB3-1-hG1 or 10mg/kg control antibody at doses of 0.1mg/kg, 1mg/kg and 10mg/kg on tumor infiltrating leukocytes. HFB3-1-hG1 produced drug receptor occupancy at a dose of only 10 mg/kg. Based on the one-way ANOVA test, p values <0.05 (, 0.01 (, or 0.001 ().

FIG. 25B shows the percentage of TNFR2 receptor occupied by the control antibody of either the 0.1mg/kg, 1mg/kg and 10mg/kg dose of the injected antibody HFB3-1-hG1 or 10mg/kg on selected peripheral blood cells. HFB3-1-hG1 at doses of 10mg/kg and 1mg/kg resulted in comparable drug receptor occupancy. Based on the one-way ANOVA test, p values <0.05 (, 0.01 (, or 0.001 ().

Fig. 26A shows the antibody concentration in the blood at day 4 of the experiment in fig. 24A. HFB3-1-hG1 was detected in the blood at doses of 10mg/kg and 1 mg/kg. Based on one-way ANOVA test, p-values <0.001 (/ x) or 0.0001 (/ x).

Fig. 26B shows soluble TNFR2 in the blood of day 4 of the experiment in fig. 24A. The administration of 10mg/kg and 1mg/kg HFB3-1-hG1 increased the amount of TNFR2 detectable in the blood. Based on one-way ANOVA test, p-values <0.001 (/ x) or 0.0001 (/ x).

FIGS. 27A and 27B show that humanized monoclonal antibodies (such as HFB3-1hz6 and HFB3-18hz 1) have similar therapeutic efficacy compared to rat anti-mPD-1 monoclonal antibodies.

FIG. 28 shows that humanized HFB3-1hz6 monoclonal antibody has therapeutic efficacy in MC38 tumor models, as does mouse anti-mPD-1 monoclonal antibody.

FIG. 29 shows that at two different doses (3 mg/kg and 10 mg/kg), humanized HFB3-1hz6 monoclonal antibodies inhibited tumor growth and increased the life of tumor bearing mice, and that combination treatment with HFB3-1hz6 and anti-mPD-1 antibodies better prolonged survival than treatment with anti-mPD-1 alone.

Fig. 30A shows that humanized HFB3-1hz6 monoclonal antibodies were eliminated from the body of cynomolgus monkeys over time (left panel), and anti-drug antibodies (ADA) were observed about 2 weeks after injection (right panel), which is common in non-human primates.

FIG. 30B shows that no cytokine elevation was observed after injection of HFB3-1hz6-hG1 at 15mg/kg, 50mg/kg or 150mg/kg compared to the reported data (dashed line) for CD3 XCD 20 bispecific IgG at.ltoreq.3 mg/kg.

FIG. 31 shows the cell count analysis after injection of HFB3-1hz6-hG1 at 15mg/kg, 50mg/kg or 150mg/kg, compared to the historical data range for normal monkeys (left and right lines in each figure).

FIG. 32 shows that humanized HFB3-1hz6 monoclonal antibodies have anti-tumor efficacy in a Hepa1-6 tumor model.

Fig. 33A-33C show kaplan-meyer survival curves (KAPLAN MEIER survivin cute) for patients with cutaneous melanoma (SKCM, fig. 33A), head and neck squamous cell carcinoma (HNSC, fig. 33B), and thymoma (THYM, fig. 33C) in a TCGA database based on TNFR2 levels. Higher TNFR2 expression is significantly associated with improved survival in melanoma and HNSC patients, but is not favored in THYM.

Fig. 34A and 34B show examples of TCGA bulk RNA analysis of patients with multiple cancers with solid tumors. Prostate cancer (PRAD) has low CD8A and TNFR2 expression and can be used as a negative control for increased TNFR2 expression in selected cancer types. AML has low CD8A (rather than TNFR 2) expression and can be used as a negative control for increased CD8A expression in selected cancer types.

Figure 35 shows TCGA stratification of cancer types with high TNFR2 expression (e.g., compared to TNFR2 expression in prostate cancer) and high CD8A expression (e.g., compared to CD8A expression in AML) based on the proportion of TNFR2 high/CD 8A high patient samples. ACC = adrenocortical carcinoma; BLCA = bladder urothelial cancer; BRCA = breast invasive cancer; CESC = cervical squamous cell carcinoma/endocervical adenocarcinoma; CHOL = biliary tract cancer; COAD = colon adenocarcinoma; EBV = Epstein-Barr Virus (Epstein-Barr Virus); esca=esophageal cancer; GBM = glioblastoma multiforme; HNSC = squamous cell carcinoma of the head and neck; KICH = kidney chromophobe; KIRC = kidney clear cell carcinoma; KIRP = renal papillary cell carcinoma; LGG = brain low glioma; LIHC = liver hepatocellular carcinoma; LUAD = lung adenocarcinoma; luc = squamous cell lung carcinoma; MESO = pleural mesothelioma; OV = ovarian serous cystic adenocarcinoma; 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 cancer; UCEC = endometrial cancer of the uterus; UCS = uterine carcinoma sarcoma; UVM = uveal melanoma.

Fig. 36 shows molecular subtype analysis of selected Renal Cell Carcinoma (RCC), cutaneous melanoma (SKCM), gastric adenocarcinoma/gastric carcinoma (STAD/GI), lung adenocarcinoma (LUAD) and head and neck squamous cell carcinoma (HNSC).

Detailed Description

1. Summary of the invention

TNFR2 has recently become a promising therapeutic target for tumor immunology. Regulatory and TNFR2 expression on effector T cells in the Tumor Microenvironment (TME) is associated with T cell depletion and resistance to immune checkpoint blockade. The invention described herein provides antibodies to human TNFR2 that are useful as anti-cancer agents. While not wishing to be bound by any particular theory, it is believed that co-stimulation of effector T cells with the subject anti-TNFR 2 antibodies enhances the anti-tumor activity of the effector T cells.

According to the invention described herein, mice are immunized with recombinant extracellular domains (ECD) of human TNFR2 (rhTNFR 2) to produce a range of different antibodies that are characterized for binding, cross-reactivity, selectivity, and functional activity. Antibodies were selected for their ability to induce proliferation of CD8 ⁺ and CD4 ⁺ effector T cells in the presence of Treg cells and for increased NFkB signaling. The selected antibodies also desirably exhibit cross-reactivity against monkey orthologs of rhTNFR2, which would be a feature of benefit in toxicity studies of human therapeutics in animals. Other desirable features include the ability of the subject antibodies to enhance binding of human recombinant tnfα to TNFR 2.

Two mouse antibodies HFB3-1 and HFB3-14, which have a sub-or one-digit nanomolar binding affinity for human TNFR2, were initially selected for further characterization and humanization. Epitope mapping experiments showed that these two antibodies recognized different domains of TNFR2, with HFB3-1 binding to regions within the domain of CRD2 and HFB3-14 binding within the CRD3 region. However, despite their different binding sites, both antibodies were selective for TNFR2, cross-reacted with cynomolgus and rhesus orthologs, and enhanced binding of human recombinant tnfα to TNFR2, as well as stimulated CD8 and conventional CD 4T cells (Tconv).

Several humanized variants of these mouse antibodies (including HFB3-1hz6 and HFB3-14hz1 c) retain the binding and cross-reactivity characteristics of their corresponding parent antibodies. Humanized antibodies preferentially bind to TCR-activated primary CD8 and CD 4T cells compared to non-stimulated T cells and enhance CD3/CD 28-induced T cell activation and proliferation. This co-stimulatory mechanism of action is independent of cross-linking and is consistent with the ability of the antibody to enhance nfkb signaling and induce up-regulation of the target gene downstream of nfkb.

In addition, the two humanized antibodies (HFB 3-1hz6 and HFB3-14hz1 c) exhibited good developability characteristics and were stable at high temperature, low pH conditions, and after several freeze/thaw cycles. Good plasma exposure of the lead antibodies was also observed in the mouse model. In vivo efficacy assessment and initial toxicity analysis of these antibodies in a mouse tumor model was performed.

A third mouse monoclonal antibody HFB3-18 was also identified, which has a slightly lower (two-digit nM) binding affinity than the anti-mPD-1 monoclonal antibody but the same ability to inhibit tumor growth in vivo (if not preferred), and produced a humanized version thereof.

The functional characteristics of these antibodies, as well as their advantageous developability and pharmacokinetic characteristics, support their development as potential novel immunotherapeutic options for cancer patients, particularly in certain cancer types and subtypes that exhibit high expression of TNFR2 and CD 8A.

Detailed aspects of the invention are further and individually set forth in the sections below. However, it should be understood that any of the embodiments of the present invention (including the embodiments set forth only in the examples or figures and the embodiments set forth only in part below) can be combined with any other embodiments of the present invention.

2. Definition of the definition

The term "antibody" is intended to cover a wide variety of antibody structures in its broadest sense, 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 comprising Complementarity Determining Regions (CDRs) 1, CDR2 and CDR3 of a heavy chain and CDR1, CDR2 and CDR3 of a light chain, wherein the molecule 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 multiple species (such as mouse, human, cynomolgus monkey, etc.).

However, in a narrower sense, "antibody" refers to a variety of monoclonal antibodies, including chimeric monoclonal antibodies, humanized monoclonal antibodies, and human monoclonal antibodies, particularly humanized monoclonal antibodies of the invention.

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 at least a portion of a heavy chain variable region and a heavy chain constant region, and at least one Light Chain (LC) comprising at least a portion of a light chain variable region and a light chain constant region. In some embodiments, the antibody comprises two heavy chains, wherein each heavy chain comprises at least a portion of a heavy chain variable region and a heavy chain constant region, and two light chains, wherein each light chain comprises at least a portion of a light chain variable region and a light chain constant region.

As used herein, single Chain FV (SCFV) or any other antibody comprising, for example, a single polypeptide chain comprising 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 a region of an antibody comprising three heavy chain CDRs and the light chain is a region of an antibody comprising three light chain CDRs.

The term "Heavy Chain Variable Region (HCVR)" as used herein refers to a region comprising at least heavy chain CDR1 (CDR-H1), framework 2 (HFR 2), CDR2 (CDR-H2), FR3 (HFR 3) and CDR3 (CDR-H3). In some embodiments, the heavy chain variable region further comprises at least a portion (e.g., all) of FR1 (HFR 1), which is the N-terminus of CDR-H1, and/or at least a portion (e.g., all) of FR4 (HFR 4), which is the C-terminus of CDR-H3.

The term "heavy chain constant region" as used herein refers to a region comprising at least three heavy chain constant domains CH1, CH2 and CH 3. 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 chain constant region corresponds to an antibody isotype. For example, the antibody comprising a gamma constant region is an IgG antibody, the antibody comprising a delta constant region is an IgD antibody, the antibody comprising an alpha constant region is an IgA antibody, the antibody comprising an epsilon constant region is an IgE antibody, and the antibody comprising a mu constant region is an IgM antibody.

Some isoforms may be further subdivided into subclasses. For example, igG antibodies include, but are not limited to, igG1 (comprising a γ1 constant region), igG2 (comprising a γ2 constant region), igG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region) antibodies; igA antibodies include, but are not limited to, igA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, igM1 (comprising a μ1 constant region) and IgM2 (comprising a μ2 constant region).

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

The term "Light Chain Variable Region (LCVR)" as used herein refers to a region comprising the light chain CDR1 (CDR-L1), framework (FR) 2 (LFR 2), CDR2 (CDR-L2), FR3 (LFR 3) and CDR3 (CDR-L3). In some embodiments, the light chain variable region further comprises at least a portion (e.g., all) of FR1 (LFR 1) and/or at least a portion (e.g., all) of FR4 (LFR 4).

The term "light chain constant region" as used herein refers to a region comprising light chain constant domain C _L. Non-limiting exemplary light chain constant regions include lambda and kappa.

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

The term "antibody fragment" or "antigen-binding portion" of an antibody includes, but is not limited to, fragments capable of binding an antigen, such as Fv, single chain Fv (scFv), fab ', and (Fab') ₂. In certain embodiments, the antibody fragment comprises a Fab, fab ', F (ab ') ₂、F_d, single chain Fv or scFv, disulfide-linked F _V, V-NAR domain, igNar, intracellular antibody, igG Δch ₂, miniantibody, F (ab ') ₃, tetravalent antibody, trivalent antibody, diabody, single domain antibody, DVD-Ig, fcab, mAb ₂、(scFv)₂, or scFv-Fc.

The term "Fab" refers to an antibody fragment having a molecular mass of about 50,000 daltons and has the activity of binding to an antigen. Which comprises about half of the heavy chain N-terminal side and the entire light chain linked by disulfide bridges. Fab is obtainable in particular by treating immunoglobulins with the protease papain.

The term "F (ab') ₂" denotes a fragment having about 100,000 daltons and activity of binding to an antigen. This fragment is slightly larger than the two Fab fragments linked via a disulfide bridge in the hinge region. These fragments are obtained by treating immunoglobulins with the protease pepsin. Fab fragments can be obtained from F (ab') 2 fragments by cleavage of the disulfide bridge of the hinge region.

A single Fv chain "scFv" corresponds to a VH: VL polypeptide synthesized using genes encoding VL and VH domains and sequences encoding peptides intended to bind to these domains. The scFv according to the invention comprises CDRs which are maintained in the appropriate conformation, for example using genetic recombination techniques.

The dimer of an "scFv" corresponds to two scFv molecules linked together by a peptide bond. This Fv chain is generally derived from the expression of a fusion gene comprising genes encoding VH and VL linked by a linker sequence encoding the peptide. The human scFv fragment may comprise CDR regions which are maintained in the appropriate conformation, preferably by using genetic recombination techniques.

The "dsFv" fragment is a VH-VL heterodimer stabilized by a disulfide bridge; it may be bivalent (dsFv ₂). Fragments of bivalent Sc (Fv) ₂ or multivalent antibodies can be produced by association of monovalent scFv spontaneously or by linking scFv fragments via peptide binding sequences.

The Fc fragment is a support for the biological properties of the antibody, particularly its ability to recognize or activate complement by immune effectors. It consists of a constant segment of the heavy chain beyond the hinge region.

The term "diabody" refers to a small antibody fragment having two antigen-fixing sites. These fragments comprise a variable heavy domain VH linked to a variable light domain VL in the same VH-VL polypeptide chain. Binding sequences that are too short to match two domains of the same strand are used, necessarily to match two complementary domains of the other strand and thus create two antigen fixing sites.

An "antibody that binds to the same epitope" as a reference antibody can be determined by an antibody competition assay. It refers to an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competition assay, whereas the reference antibody blocks 50% or more of the binding of the antibody to its antigen in a competition assay. The term "competition" when used in the context of antibodies competing for the same epitope means that competition between antibodies is determined by an assay in which the tested antibodies prevent or inhibit specific binding of a reference antibody to a common antigen.

Various types of competitive binding assays may be used, for example: solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assay (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 labeling assay; a solid phase directly labeled sandwich; (see, e.g., harlow and Lane,1988,Antibodies,A Laboratory Manual,Cold Spring Harbor Press); RIA is directly labeled using an I ¹²⁵ -labeled solid phase (see, e.g., morel et al, 1988, molecular. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., cheung et al, 1990,Virology 176:546-552); and a directly labeled RIA (Moldenhauer et al, 1990, scand. J. Immunol.).

Typically, such assays involve the use of purified antigens bound to solid surfaces or cells bearing any of these (unlabeled test antigen binding protein and labeled reference antibody). Competitive inhibition is measured by determining the amount of label bound to a solid surface or cell in the presence of a test antibody. Typically, the test antibody is present in excess. Antibodies identified by competition assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to neighboring epitopes that are sufficiently close to the epitope to which the reference antibody binds to cause steric hindrance. In some embodiments, when the competing antibody is present in excess, it will inhibit the specific binding of at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% of the reference antibody to the common antigen. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or greater.

The term "antigen" refers to a molecule or portion of a molecule that is capable of being bound by a selective binding agent (such as an antibody or immunologically functional fragment thereof) and that is otherwise capable of being used in a mammal to produce an antibody that is capable of binding to the antigen. An antigen may have one or more epitopes capable of interacting with an antibody.

The term "epitope" is the portion of an antigenic molecule that is bound by a selective binding agent (such as an antibody or fragment thereof). The term includes any determinant capable of specific binding to an antibody. Epitopes can be contiguous or noncontiguous (e.g., amino acid residues in a polypeptide that are not contiguous with each other in the polypeptide sequence but are bound by an antigen binding protein in the context of the molecule). In some embodiments, an epitope may be mimotope in that it comprises a three-dimensional structure similar to the epitope used to generate the antibody, but does not include or only includes 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 groups, and may have specific three dimensional structural features and/or specific charge features.

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

In certain embodiments, if the antibody binds to the same region of the antigen, the antibody binds to the same epitope as the reference antibody, as determined by X-ray crystallography.

As used herein, "chimeric antibody" means an antibody comprising at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as human, cynomolgus monkey, chicken, etc.). In some embodiments, the chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, all variable regions of the chimeric antibody are from a first species and all constant regions of the chimeric antibody are from a second species.

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

"CDR-grafted antibody" as used herein 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.

"Human antibody" as used herein refers to an antibody produced in a human, in a non-human animal comprising human immunoglobulin genes (such as) Antibodies produced in (c) and antibodies selected using in vitro methods (such as phage display), wherein the antibody profile is based on human immunoglobulin sequences.

"Host cell" refers to a cell that may be or have been the recipient of a vector or an isolated polynucleotide. The host cell may be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells such as yeast; a plant cell; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to NSO cells,Cells (Crucell), 293 and CHO cells and derivatives thereof, such as 293-6E and DG44 cells, respectively.

The term "isolated" as used herein refers to a molecule that has been separated from at least some of the components typically found in nature, or from at least some of the components that typically produce it. For example, a polypeptide is said to be "isolated" when it is isolated from at least some of the cellular components from which it is produced. Physically separating a supernatant containing a polypeptide from the cell from which it is produced is considered to "isolate" the polypeptide when the polypeptide is secreted by the cell after expression. Similarly, a polynucleotide is said to be "isolated" when the polynucleotide is not part of a larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA in the case of DNA polynucleotides) that is typically found in nature or isolated from at least some cellular components that produce it (e.g., in the case of RNA polynucleotides). Thus, a DNA polynucleotide contained in a vector within a host cell may be referred to as "isolated" as long as the polynucleotide is not found in the vector in nature.

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 including, but not limited to, rodents, apes, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian playground animals, and mammalian pets are also provided. In some cases, "subject" or "patient" refers to a (human) subject or patient in need of treatment for a disease or disorder.

The term "sample" or "patient sample" as used herein refers to a material obtained or derived from a target subject containing, for example, cells and/or other molecular entities to be characterized and/or identified based on physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "disease sample" and variations thereof refers to any sample obtained from a target subject that would be expected or known to contain the cell and/or molecular entity to be characterized.

By "tissue or cell sample" is meant a collection of similar cells obtained from the tissue of a subject or patient. The source of the tissue or cell sample may be solid tissue such as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood component; body fluids such as sputum, cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from any time of gestation or development of a subject. The tissue sample may also be a primary or cultured cell or cell line. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ. The tissue sample may contain compounds that are not normally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

As used herein, "reference sample," "reference cell," or "reference tissue" refers to a sample, cell, or tissue obtained from a source known or considered not to suffer from a disease or disorder identified using the methods or compositions of the present invention. In one embodiment, the reference sample, reference cell or reference tissue is obtained from a healthy portion of the body of the same subject or patient that is identified as a disease or disorder using the compositions or methods of the invention. In one embodiment, the reference sample, reference cell or reference tissue is obtained from a healthy portion of the body of at least one individual who is not a subject or patient who is identified as a disease or disorder using the compositions or methods of the invention. In some embodiments, the reference sample, reference cells, or reference tissue is previously obtained from the patient prior to or early in the disease or disorder.

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

By "disease associated with the inhibitory activity of regulatory T lymphocytes" is meant any disease (non-autoimmune) in which the inhibitory activity of regulatory T lymphocytes plays a role, in particular by promoting the development or persistence of the disease. In particular, the inhibitory activity of regulatory T lymphocytes has been demonstrated to promote tumor progression. Thus, the present invention more specifically aims at cancers in which the inhibitory activity of T lymphocytes plays a role.

The term "cancer" is used herein to refer to a population of cells that exhibit abnormally high levels of proliferation and growth. Cancers may be benign (also known as benign tumors), precancerous, or malignant. The cancer cells may be solid cancer cells (i.e., forming a solid tumor) or leukemia cancer cells. The term "cancer growth" is used herein to refer to proliferation or growth of one or more cells comprising a cancer that results in a corresponding increase in the size or extent of the cancer.

Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. 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, squamous lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, biliary tract cancer, gall bladder cancer, stomach cancer, melanoma, and various types of head and neck cancer.

In certain embodiments, cancers as used herein include hematological cancers (such as AML and DLBCL) or solid tumors (such as breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer).

"Chemotherapeutic agents" are chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa (thiotepa) and(Cyclophosphamide); alkyl sulfonates such as busulfan (busulfan), imperoshu (improsulfan), and piposhu (piposulfan); aziridines such as benzodopa (benzodopa), carboquinone (carboquone), midadopa (meturedopa) and You Liduo bar (uredopa); ethyleneimine and methyl melamines, including altretamine (altretamine), triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and trimethylol melamine; polyacetyl (especially bullatacin and bullatacin ketone (bullatacinone)); camptothecins (including the synthetic analog topotecan); bryostatin (bryostatin); sponge statin (callystatin); CC-1065 (including adorinone (adozelesin), carbozelesin (carzelesin) and bizelesin (bizelesin) synthetic analogues thereof); nostoc (cryptophycin) (in particular, nostoc 1 and nostoc 8); dolastatin (dolastatin); duocarmycin (duocarmycin) (including synthetic analogs KW-2189 and CB1-TM 1); elstuporin (eleutherobin); a podocarpine (pancratistatin); the stoichiometriol (sarcodictyin); sponge chalone (spongistatin); nitrogen mustards, such as chlorambucil (chlorambucil), napthalen (chlornaphazine), chlorphosphamide (cholophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), methyldichloroethylamine, oxazatine hydrochloride, melphalan (melphalan), benazel (novembichin), benazel cholesterol (PHENESTERINE), prednimustine (prednimustine), triafosfamine (trofosfamide), uracil mustard; nitrosoureas such as carmustine (carmustine), chlorourectin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimnustine); antibiotics, e.g. enediyne antibiotics (e.g. calicheamicin, especially calicheamicin gamma ll and calicheamicin omega ll (see e.g. Agnew, chem lntl. Ed. Engl,33:183-186 (1994)); dactinomycin (dynemicin) including dactinomycin A, bisphosphonates, e.g. chlorophosphonates, epothilone (esperamicin), and neo-carcinomycin chromophores (neocarzinostatin chromophore) and related chromoprotein enediyne antibiotic chromophores), aclacinomycin (aclacinomysin), actinomycin (actinomycin), an aflatoxin (authramycin), diazoserine, bleomycin (bleomycin), actinomycin C, carmubicin (carabicin), carminomycin (caminomycin), carcinomycin (carzinophilin), chromomycins (chromomycinis), dactinomycin (dactinomycin), daunorubicin (daunorubicin), ditetracin (detorubicin), 6-diazo-5-oxo-L-norubicin,Doxorubicin (doxorubicin) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolido-doxorubicin, and deoxydoxorubicin (deoxydoxorubicin)), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), doxycycline (marcellomycin), mitomycin (e.g., mitomycin C), mycophenolic acid, noramycin (nogalamycin), olivamycin (olivomycin), pelomycin (peplomycin), pofeomycin (potfiromycin), puromycin (puromycin), triforine (quelamycin), rodomycin (rodorubicin), streptozocin (streptonigrin), streptozocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), bestatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as, for example, dimethylfolic acid (denopterin), methotrexate, pterin (pteropterin), trimellite (trimetricate); purine analogs, such as fludarabine (fludarabine), 6-mercaptopurine, thioxanthine (thiamiprine), thioguanine; pyrimidine analogs such as, for example, ancitabine (ancitabine), azacytidine (azacitidine), 6-azauridine (azauridine), carmofur (carmofur), cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine (enocitabine), fluorouridine; androgens, such as, for example, carbosterone (calusterone), drotasone propionate (dromostanolone propionate), cyclothiolane (epitiostanol), emasculane (mepitiostane), testosterone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements, such as folinic acid; acetoglucurolactone; aldehyde phosphoramide glycosides; aminolevulinic acid; enuracil (eniluracil); amsacrine (amsacrine); double Qu Buxi (bestrabucil); a birthday group (bisantrene); edatroxas (edatraxate); ground phosphoramide (defofamine); colchicine (demecolcine); deaquinone (diaziquone); epoxicillin (elfomithine); ammonium elegance (elliptinium acetate); epothilone (epothilone); eggshell (etoglucid); gallium nitrate; hydroxyurea; lentinan; lonidamine (lonidainine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocin); mitoguazone (mitoguazone); mitoxantrone (mitoxantrone); mo Pai darol (mopidanmol); diamine nitroacridine (nitraerine); penstatin (penstatin); egg ammonia nitrogen mustard (phenamet); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophylloic acid; 2-ethyl hydrazide; procarbazine (procarbazine); /(I)Polysaccharide complexes (JHS Natural Products, eugene, OR); raschig (razoxane); rhizomycin (rhizoxin); dorzolopyran (sizofiran); germanium spiroamine (spirogermanium); tenuazonic acid (tenuazonic acid); triiminoquinone (triaziquone); 2,2',2 "-trichlorotriethylamine; trichothecene (trichothecene) (especially T-2 toxin, wart A (verracurin A), cyclosporin a (roridin a), and serpentine (anguidine)); uratam (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannitol (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromine (pipobroman); ganciclovir (gacytosine); cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes (taxoids), for example,Paclitaxel (Bristol-Myers Squibb Oncology, prencton, N.J.),/>Albumin engineered paclitaxel nanoparticle formulations without cremophor (American Pharmaceutical Partners, schaumberg, illinois) and/>Docetaxel (doxetaxel) (Rhone-Poulenc Rorer, antony, france); chlorambucil; /(I)Gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin (oxaliplatin), and carboplatin; vinblastine (vinblastine); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (vincristine); /(I)Vinorelbine (vinorelbine); nux An Tuo (novantrone); teniposide (teniposide); edatroxas (edatrexate); daunomycin; aminopterin; hilder (xeloda); ibandronate (ibandronate); irinotecan (irinotecan) (Camptosar, CPT-11) (treatment regimen comprising irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid; capecitabine (capecitabine); combretastatin (combretastatin); leucovorin (LV); oxaliplatin, including oxaliplatin treatment regimen (FOLFOX); PKC-alpha, raf, H-Ras, EGFR (e.g., erlotinib/>) And an inhibitor of VEGF-Sub>A, and Sub>A pharmaceutically acceptable salt, acid or derivative of any of the foregoing.

Other non-limiting exemplary chemotherapeutic agents include anti-hormonal agents such as anti-estrogens and Selective Estrogen Receptor Modulators (SERMs) for modulating or inhibiting the action of hormones on cancer, including, for example, tamoxifen (tamoxifen) (includingTamoxifen), raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxy tamoxifen, trawoxifene (trioxifene), keoxifene (keoxifene), LY 117022, onapristone (onapristone) and/>Toremifene (toremifene); aromatase inhibitors that inhibit aromatase that regulates estrogen production in the adrenal gland, such as, for example, 4 (5) -imidazole, aminoglutethimide,/>Megestrol acetate (megestrol acetate),/>Exemestane (exemestane), formestane (formestanie), fatrozole (fadrozole),/>Vorozole,/>Letrozole (letrozole) andAnastrozole (anastrozole); and antiandrogens such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprorelin (leuprolide), and goserelin (goserelin); troxacitabine (1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly those that inhibit the expression of genes involved in signaling pathways of abnormal cell proliferation (e.g., PKC- α, ralf, and H-Ras); ribozymes, such as VEGF expression inhibitors (e.g./>Ribozymes) and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, e.g./>Vaccine,/>Vaccine and method for producing the sameA vaccine; /(I)rIL-2；/>Topoisomerase 1 inhibitors; rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

An "anti-angiogenic agent" or "angiogenesis inhibitor" refers to a small molecular weight substance that directly or indirectly inhibits angiogenesis, vasculogenesis, or undesired vascular permeability, a polynucleotide (including, for example, an inhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, a recombinant protein, an antibody, or a conjugate or fusion protein thereof. It is understood that anti-angiogenic agents include those agents that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, the anti-angiogenic agent is an antibody or other antagonist directed against an angiogenic agent, such as an antibody directed against VEGF-A (e.g., bevacizumab)) Or antibodies directed against Sub>A VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor); anti-PDGFR inhibitors such as/>(Imatinib mesylate (Imatinib Mesylate)), small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668,/>SU1 1248 (sunitinib malate (sunitinib malate)), AMG706 or small molecules such as those described in international patent application WO 2004/113304. Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin, endostatin, and the like. 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 listing anti-angiogenic therapies for malignant melanoma); ferrara and Alitalo (1999) Nature Medicine 5 (12): 1359-1364; tonini et al (2003) Oncogene 22:6549-6556 (e.g., table 2 listing known anti-angiogenic factors); and Sato (2003) int.J.Clin.Oncol.8:200-206 (e.g., table 1 listing anti-angiogenic agents for clinical trials).

As used herein, "growth inhibitory agent" refers to a compound or composition that inhibits the growth of cells (such as cells expressing VEGF) in vitro or in vivo. Thus, the growth inhibitory agent may be one that significantly reduces the percentage of cells in S phase (such as cells that express VEGF). Examples of growth inhibitors include, but are not limited to, agents that block cell cycle progression (in periods other than S-phase), such as agents that induce G1-phase and M-phase arrest. Classical M-phase blockers include vinca (vincristine and vinblastine), taxanes (taxane) and topoisomerase II inhibitors (such as doxorubicin, epirubicin, daunomycin, etoposide and bleomycin). Those agents that block G1 also spill over into S-phase blocks, e.g., DNA alkylating agents such as tamoxifen, prednisone (prednisone), dacarbazine, methyldichloroethylamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. For additional information see Mendelsohn and Israel editions, the Molecular Basis of Cancer, chapter 1, titled "CELL CYCLE regulation, oncogenes, and antineoplastic drugs", murakami et al (W.B. Saunders, philadelphia, 1995), for example page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs that are both derived from Taxus. Docetaxel @ sRhone-Poulenc Rorer) derived from Taxus baccata, a semisynthetic analog of Taxol (/ >)Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules of tubulin dimers and stabilize microtubules by preventing depolymerization, which can inhibit mitosis in cells.

The term "anti-neoplastic composition" refers to a composition useful for treating cancer comprising at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, for example, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, cancer immunotherapeutic agents (also known as tumor immunizing agents), apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies, anti-CD 20 antibodies, epidermal Growth Factor Receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1/EGFR inhibitors (e.g., erlotinib)Platelet-derived growth factor inhibitors (e.g./>)(Imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferon, CTLA4 inhibitors (e.g., anti-CTLA antibody ipilimumab)) PD-1 inhibitors (e.g., anti-PDl antibodies, BMS-936558), PDL1 inhibitors (e.g., anti-PDL 1 antibodies, MPDL 3280A), PDL2 inhibitors (e.g., anti-PDL 2 antibodies), VISTA inhibitors (e.g., anti-VISTA antibodies), 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, PDL2, CTLA4, VISTA or VEGF receptor, TRAIL/Apo2, and other bioactive and organic chemical agents, etc. The invention also includes combinations thereof.

"Treatment" refers to therapeutic treatment, for example, where the goal is to slow down (alleviate) a targeted pathological condition or disorder, and for example, where the goal is to inhibit recurrence of the condition or disorder. "treating" encompasses any administration or application of a therapeutic agent for a disease (also referred to herein as a "disorder" or "condition") in a mammal (including a human) and includes inhibiting the progression of the disease or disease, inhibiting or slowing the progression of the disease or its progression, blocking its progression, partially or fully alleviating the disease, partially or fully alleviating one or more symptoms of the disease, or restoring or repairing lost, lost or defective function; or to stimulate an ineffective process. The term "treating" also includes lessening the severity of any phenotypic feature and/or reducing the incidence, extent, or likelihood of such feature. Those subjects in need of treatment include those already with the disorder, those at risk of recurrence of the disorder, or those who want to prevent or slow down the recurrence of the disorder.

The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug that is effective to treat a disease or disorder in a subject. In some embodiments, an effective amount refers to an amount effective to achieve a desired therapeutic or prophylactic result at a desired dose and time period. The therapeutically effective amount of the antibodies of the invention may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the antagonist to elicit a desired response in the individual. A therapeutically effective amount encompasses any toxic or detrimental effect in which the therapeutically beneficial effect exceeds that of the subject antibody.

"Prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the desired dosage and for the desired period of time. Typically, but not necessarily, since a prophylactic dose is administered in a subject prior to or early in the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

By "pharmaceutically acceptable carrier" is meant a nontoxic solid, semisolid or liquid filler, diluent, encapsulating material, formulation aid or carrier conventional in the art for use with therapeutic agents that comprise "pharmaceutical compositions" for administration to a subject. The pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations employed and is compatible with other ingredients of the formulation. Pharmaceutically acceptable carriers are suitable for the formulation used. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier desirably does not irritate the skin and does not cause an injection site reaction.

An "article of manufacture" is any article of manufacture (e.g., package or container) or kit comprising at least one agent, e.g., an agent for treating a disease or disorder, or a probe for specifically detecting a biomarker described herein. In some embodiments, the article of manufacture or kit is promoted, distributed, or marketed as a unit for practicing the methods described herein.

3. Methods of treating cancer

The invention described herein provides anti-TNFR 2 antibodies for use in methods of treating humans and other non-human mammals.

In pathological situations, tregs may produce inappropriate immunosuppression, which may, for example, promote tumor growth. Tregs are associated with a reduction in anti-tumor immune responses, in particular by inappropriately inhibiting the activity of effector T lymphocytes, thus promoting the development of a variety of cancer types.

In some embodiments, methods for treating or preventing cancer are provided, comprising administering to a subject in need of such treatment an effective amount of any subject anti-TNFR 2 antibody, or antigen-binding fragment thereof.

In some embodiments, methods of treating cancer are provided, wherein the methods comprise administering any subject anti-TNFR 2 antibody, or antigen-binding fragment thereof, to a subject having cancer.

Cancers treatable by the methods/uses of the invention include those in which regulatory T lymphocytes exert their inhibitory activity, such as those in which relatively large numbers of regulatory T lymphocytes are present in tumor tissue or circulation. Expansion of regulatory T lymphocytes, which can be measured by the frequency of tregs, is generally associated with increased Treg activation. The frequency of regulatory T lymphocytes can be assessed by any method known in the art, for example by flow cytometry (FACS) analysis of intratumoral lymphocytes or circulating lymphocytes, or by immunohistological staining of tumor tissue.

Non-limiting exemplary cancers that can be treated with any of the subject anti-TNFR 2 antibodies or antigen-binding fragments thereof, including carcinoma, lymphoma, blastoma, sarcoma, and leukemia 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, squamous lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic cancer, brain cancer, endometrial cancer, testicular cancer, biliary tract cancer, gall bladder cancer, gastric cancer, melanoma, and various types of head and neck cancer.

In certain embodiments, the cancer is melanoma, breast cancer, colon cancer, cervical cancer, kidney 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.

In certain embodiments, the cancer has a high TNFR2 index, defined as (a) total CD 8T cell count x TNFR2 expression on CD 8T cells in the tumor sample; and (b) the total Treg cell number in the tumor sample x TNFR2 expression on Treg.

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

In a certain embodiment, the cancer has a TNFR2 index of about 0.5 to about 1.

In certain embodiments, the cancer has a high proportion of CD8 TIL (tumor infiltrating lymphocytes), such as more than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more of the T cells in the tumor are CD 8T cells.

In certain embodiments, the cancer has a low TNFR2 expression level on tumor cells.

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

In certain embodiments, the cancer has a high TNFR2 expression level on tumor-depleted T cells (such as depleted CD 8T cells). Such cancers may be treated with combination therapies having, for example, antagonists of the PD-1/PD-L1 pathway, such as any of the anti-PD-1 or anti-PD-L1 antibodies (e.g., as specifically set forth herein or known in the art).

In certain embodiments, the methods/uses of the invention are useful for treating cancers in which known high levels of regulatory T lymphocytes are present and/or which are clearly associated with a poor prognosis, comprising: chronic Myelogenous Leukemia (CML), colon cancer, melanoma, uterine cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, primary central nervous system lymphoma, multiple myeloma, prostate cancer, hodgkin's lymphoma, or hepatocellular carcinoma.

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

In some embodiments, the cancer is BCC, SCC, melanoma, colorectal cancer, or NSCLC.

In certain embodiments, the cancer has a high TNFR2 expression level and a CD8A expression level. In certain embodiments, a high or higher expression level of TNFR2 is correlated/compared with an average expression level of TNFR2 in a prostate cancer patient; 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 a high or higher CD8A expression level is correlated/compared with the average CD8A expression level in AML patients.

In certain embodiments, the patient has EBV ⁺ gastric cancer.

In certain embodiments, the patient has gastric adenocarcinoma, such as gastric adenocarcinoma with increased/high PD-L1/CD274 expression.

In certain embodiments, the patient has clear cell Renal Cell Carcinoma (RCC).

In certain embodiments, the patient has kidney clear cell carcinoma (KIRC). In certain embodiments, the patient has a kirc.2, kirc.3, or kirc.4 subtype. In certain embodiments, the patient has a type B clear cell (ccB) subtype or a ccA (type a clear cell)/ccB unclassified subtype.

In certain embodiments, the patient has cutaneous melanoma.

In certain embodiments, the patient has cutaneous melanoma (SKCM) of the skin.

In certain embodiments, the patient has a subtype with a BRAF hotspot mutation (such as the V600E, V K or V600R mutation) or a hotspot mutation at K601.

In certain embodiments, the patient has a RAS hot spot mutation. In certain embodiments, the RAS hotspot mutation is an NRAS hotspot mutation, such as Q61R, Q61K, Q61L, Q H, 61_62QE > HK, G12R/D/A, and G13R/D. In certain embodiments, the RAS hotspot mutation is an HRAS hotspot mutation, such as G13D, G S or Q61K. In certain embodiments, the RAS hotspot mutation is a KRAS hotspot mutation, such as G12D, G R or Q61R.

In certain embodiments, the patient has a subtype with any NF1 mutation.

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

In certain embodiments, the patient has a testicular germ cell tumor.

In certain embodiments, the patient has soft tissue sarcoma.

In certain embodiments, the cancer is cervical cancer (e.g., cervical squamous cell carcinoma or cervical adenocarcinoma), pleural mesothelioma, lung adenocarcinoma, or head and neck squamous cell carcinoma (HNSC).

In certain embodiments, the patient has a HNSC subtype, such as atypical subtype. In certain embodiments, the atypical subtype HNSC is further HPV positive.

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

In certain embodiments, the methods/uses of the invention are useful for treating recurrence of fibrosis resulting from hepatitis C, as it has also been demonstrated that increasing the frequency of regulatory T lymphocytes is a factor in predicting recurrence of such fibrosis.

In some embodiments, the anti-TNFR 2 antibodies of the present invention can be used alone, or alternatively, in combination with any other suitable compound known to be capable of treating a disease or indication.

Thus, according to a specific embodiment of the invention, an antibody directed against TNFR2 and inhibiting the inhibitory activity of regulatory T lymphocytes as defined previously is used in combination with a second therapeutic agent for the treatment of a disease associated with the inhibitory activity of regulatory T lymphocytes, for example an anticancer agent.

That is, when the use is to treat cancer, the antibodies can be used in combination with known therapies (such as, for example, surgery, radiation therapy, chemotherapy, or combinations thereof) directed against the cancer. For example, the antibodies may be used in combination with adoptive immunotherapy consisting of the injection of one or more effector lymphocytes against tumor antigens, in particular EBV antigens. According to some aspects, other anti-cancer agents used in combination with the antibodies to TNFR2 of the invention in cancer therapy include anti-angiogenic agents. According to certain aspects, the antibody may be co-administered with a cytokine (e.g., a cytokine that stimulates an anti-tumor immune response).

In such combination therapies, the antibodies of the invention may be used before, after, or simultaneously with the second therapeutic agent. See further section below for combination therapies.

4. Route of administration and vector

In various embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered subcutaneously or intravenously. For the sake of brevity, the "subject anti-TNFR 2 monoclonal antibodies" refer to the mouse-human chimeric anti-TNFR 2 antibodies of the invention and humanized variants thereof.

In some embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered in vivo by a variety of routes including, but not limited to, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, by inhalation, intradermal, topical, transdermal and intrathecal or otherwise, for example by implantation.

In some embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered intravenously (i.v.) or subcutaneously (s.c.).

The subject antibody compositions may be formulated as a formulation in solid, semi-solid, liquid, or gaseous form; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols.

In various embodiments, compositions comprising subject anti-TNFR 2 monoclonal antibodies are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, e.g., gennaro, remington: THE SCIENCE AND PRACTICE of PHARMACY WITH FACTS AND Comparisons: drugfacts Plus, 20 th edition (2003); ansel et al Pharmaceutical Dosage Forms and Drug DELIVERY SYSTEMS, 7 th edition, lippencott WILLIAMS AND WILKINS (2004); kibbe et al, handbook of Pharmaceutical Excipients, 3 rd edition, pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers are available, including vehicles, adjuvants and diluents. In addition, a variety of pharmaceutically acceptable auxiliary substances are available, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizing agents, wetting agents and the like. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

In various embodiments, a composition comprising the subject anti-TNFR 2 monoclonal antibody can be prepared by dissolving, suspending, or emulsifying it in an aqueous or non-aqueous solvent (such as a vegetable or other oil, a synthetic fatty glyceride, an ester of a higher fatty acid, or propylene glycol); and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives, for injection, including subcutaneous administration.

In various embodiments, the compositions may be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.

In various embodiments, the compositions may also be formulated as sustained release microcapsules such as containing biodegradable or non-biodegradable polymers. Non-limiting exemplary biodegradable formulations include polylactic-co-glycolic acid (PLGA) polymers. Non-limiting exemplary biodegradable formulations include polyglycerin fatty acid esters. Some methods of making such formulations are described, for example, in EP 1125584 Al.

Also provided are pharmaceutical dosage packages comprising one or more containers each containing one or more doses of the subject anti-TNFR 2 monoclonal antibody. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising the subject anti-TNFR 2 monoclonal antibody, with or without one or more additional agents. In some embodiments, such unit doses are supplied in single use, pre-filled syringes. In various embodiments, the composition contained in the unit dose may comprise saline, sucrose, or the like; buffers, such as phosphates or the like; and/or formulated in a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that is reconstituted upon addition of an appropriate liquid (e.g., sterile water). In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including (but not limited to) sucrose and arginine. In some embodiments, the compositions of the invention comprise heparin and/or proteoglycans.

The pharmaceutical composition is administered in an amount effective to treat or prevent the particular indication. The therapeutically effective amount will generally depend on the weight of the subject being treated, his or her body or health condition, the breadth of the condition being treated, or the age of the subject being treated.

In some embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered in an amount ranging from about 50 μg/kg body weight to about 50mg/kg body weight per dose. In some embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered in an amount ranging from about 100 μg/kg body weight to about 50mg/kg body weight per dose. In some embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered in an amount ranging from about 100 μg/kg body weight to about 20mg/kg body weight per dose. In some embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered in an amount ranging from about 0.5mg/kg body weight to about 20mg/kg body weight per dose.

In some embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered in an amount ranging from about 10mg to about 1,000mg per dose. In some embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered in an amount ranging from about 20mg to about 500mg per dose. In some embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered in an amount ranging from about 20mg to about 300mg per dose. In some embodiments, the subject anti-TNFR 2 monoclonal antibodies can be administered in an amount ranging from about 20mg to about 200mg per dose.

The subject anti-TNFR 2 monoclonal antibody compositions can be administered to a subject as desired. In some embodiments, the subject is administered one or more effective doses of the subject anti-TNFR 2 monoclonal antibody. In various embodiments, an effective dose of the subject anti-TNFR 2 monoclonal antibody is administered to the subject once a month, less than once a month (e.g., every two months, every three months, or every six months). In other embodiments, an effective dose of the subject anti-TNFR 2 monoclonal antibody is administered more than once a month (e.g., every two weeks, weekly, twice weekly, thrice weekly, daily, or multiple times daily). At least one effective dose of the subject anti-TNFR 2 monoclonal antibody is administered to the subject. In some embodiments, an effective dose of the subject anti-TNFR 2 monoclonal antibody can be administered multiple times, including for a period of at least one month, at least six months, or at least one year. In some embodiments, the subject anti-TNFR 2 monoclonal antibodies are administered to a subject as needed to alleviate one or more symptoms of the disorder.

5. Combination therapy

The subject anti-TNFR 2 monoclonal antibodies (including functional fragments thereof) of the present invention can be administered to a subject in need thereof in combination with other biologically active substances or other therapeutic procedures to treat a disease. For example, the subject anti-TNFR 2 monoclonal antibodies can be administered alone or with other therapeutic modalities. Which may be provided before, substantially simultaneously with, or after other modes of treatment, such as radiation therapy.

To treat cancer, the subject anti-TNFR 2 monoclonal antibodies can be administered in combination with one or more anti-cancer agents, such as immune checkpoint inhibitors, chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, or anti-tumor compositions.

In certain embodiments, the subject anti-TNFR 2 monoclonal antibodies specifically bind to TNFR2 ("TNFR 2-binding antagonists"), e.g., TNFR2 antagonist antibodies or antigen-binding fragments thereof, in combination with a second antagonist (such as an immune checkpoint inhibitor, e.g., an inhibitor of the PD-1 or PD-L1 pathway) to a subject suffering from a disease (e.g., cancer or infectious disease) in which stimulation of the immune system would be beneficial. The two antagonists may be administered simultaneously or sequentially, for example as described below for the combination of the subject anti-TNFR 2 monoclonal antibody with a tumor immunizing agent. One or more other therapeutic agents (e.g., checkpoint modulator) may be added to treatment with the subject anti-TNFR 2 monoclonal antibodies to treat cancer or autoimmune disease.

In certain embodiments, the subject anti-TNFR 2 monoclonal antibodies are administered to a subject, e.g., a subject having cancer, concurrently or consecutively with another treatment. For example, the subject anti-TNFR 2 monoclonal antibodies can be administered with one or more of the following: radiation therapy, surgery or chemotherapy, such as targeted chemotherapy or immunotherapy.

In certain embodiments, a method of treating an individual having cancer comprises administering to the individual an anti-TNFR 2 monoclonal antibody of the present invention and one or more tumor immunizing agents, e.g., an immune checkpoint inhibitor.

Immunotherapy (e.g., therapy with a tumor immunizing agent) can be effective to enhance, stimulate, and/or up-regulate an immune response in a subject. In one aspect, the administration of the subject anti-TNFR 2 monoclonal antibodies together with a tumor immunizing agent (such as a PD-1 inhibitor) has a synergistic effect in treating cancer, e.g., in inhibiting tumor growth.

In one aspect, the subject anti-TNFR 2 monoclonal antibodies are administered sequentially prior to administration of the tumor immunizing agent. In one aspect, the subject anti-TNFR 2 monoclonal antibodies are administered concurrently with a tumor immunizing agent (such as a PD-1 inhibitor). In another aspect, the subject anti-TNFR 2 monoclonal antibodies are administered sequentially after administration of the tumor immunizing agent (e.g., PD-1 inhibitor). The administration of the two doses may begin, for example, at intervals of 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 one or more weeks, or the administration of the second dose 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 one or more weeks after the first dose has been administered.

In certain aspects, the subject anti-TNFR 2 monoclonal antibody and a tumor immunizing agent (e.g., a PD-1 inhibitor) are administered to the patient simultaneously, e.g., infused to the patient simultaneously, e.g., over a period of 30 or 60 minutes. The subject anti-TNFR 2 monoclonal antibodies can be co-formulated with a tumor immunizing agent, such as a PD-1 inhibitor.

Tumor immunizing agents include, for example, small molecule drugs, antibodies or fragments thereof, or other biological agents or small molecules. Examples of biological tumor immunizing agents include, but are not limited to, antibodies, antibody fragments, vaccines, and cytokines. In one aspect, the antibody is a monoclonal antibody. In certain aspects, the monoclonal antibody is a humanized or human antibody.

In one aspect, the tumor immunizing agent is an agonist of (i) a stimulatory (including co-stimulatory) molecule (e.g., a receptor or ligand) or (ii) an antagonist of an inhibitory (including co-inhibitory) molecule (e.g., a receptor or ligand) on an immune cell (e.g., a T cell), all of which amplify an antigen-specific T cell response. In certain aspects, the tumor immunizing agent is an agonist of (i) a stimulatory (including co-stimulatory) molecule (e.g., a receptor or ligand) or (ii) an antagonist of an inhibitory (including co-inhibitory) molecule (e.g., a receptor or ligand) on a cell (e.g., an NK cell) involved in innate immunity, and wherein the tumor immunizing agent enhances innate immunity. Such tumor immunity agents are often referred to as immune checkpoint modulators, e.g., immune checkpoint inhibitors or immune checkpoint stimulators.

In certain embodiments, the tumor immunizing agent may be an agent that targets (or specifically binds to) a member of the B7 membrane bound ligand family or specifically binds to a co-stimulatory or co-inhibitory receptor of a B7 family member, 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. The tumor immunizing agent may be an agent that targets a member of the TNF membrane-binding ligand family or a co-stimulatory or co-inhibitory receptor (e.g., TNF receptor family member) that specifically binds thereto. Exemplary TNF and TNFR family members that can be targeted by tumor immunizing agents 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、 lymphotoxins α/t n p beta, TNFR2, TNFa, LTfiR, lymphotoxins a1 beta 2, FAS, FASL, RELT, DR6, TROY, and NGFR. Tumor immunizing agents that may be used in combination with the subject anti-TNFR 2 monoclonal antibodies to treat cancer may be agents that target a B7 family member, a B7 receptor family member, a TNF family member, or a TNFR family member (such as those described above), e.g., antibodies.

In one aspect, the subject anti-TNFR 2 monoclonal antibodies are administered with one or more of the following: (i) Antagonists of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L2, LAG-3, TIM3, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PDIH, LAIR1, TIM-4, and PSGL-1, and (ii) agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD 137), 4-1BBL, ICOS, ICOS-L, OX, OX40L, GITR, GITRL, CD, CD27, CD40L, DR3, and CD28H.

In one aspect, the tumor immunizing agent is an agent that inhibits (i.e., antagonizes) cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-beta, VEGF, and other immunosuppressive cytokines), or an agonist of a cytokine that stimulates T cell activation and stimulates an immune response (e.g., cytokine itself), such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFN alpha.

Other agents that may be combined with the subject anti-TNFR 2 monoclonal antibodies to stimulate the immune system (e.g., to treat 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-TNFR 2 monoclonal antibodies can be combined with antagonists of KIR.

Other agents for combination therapy include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-IR antagonists, such as CSF-IR antagonist antibodies, including RG7155 (WOl 1/70024, WOl 1/107553, WO11/131407, W013/87699, W013/119716, WO 13/132044) or FPA008 (WOl 1/140249; W013168664; WO 14/036357).

Tumor immunizing agents also include agents that inhibit TGF-beta signaling.

Other agents that may be combined with the subject anti-TNFR 2 monoclonal antibodies include agents that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF secreting cell vaccines, cpG oligonucleotides, and imiquimod (imiquimod), or therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines).

Other therapies that may be combined with the subject anti-TNFR 2 monoclonal antibodies include therapies that deplete or block Treg cells, such as agents that specifically bind to CD 25.

Another therapy that may be combined with the subject anti-TNFR 2 monoclonal antibodies is a therapy that inhibits metabolic enzymes such as Indoleamine Dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase.

Another class of agents that may be used include agents that inhibit adenosine formation or inhibit the adenosine A2A receptor.

Other therapies that may be combined with the subject anti-TNFR 2 monoclonal antibodies to treat cancer include therapies that reverse/prevent T cell anergy or depletion and therapies that trigger innate immune activation and/or inflammation of the tumor site.

The subject anti-TNFR 2 monoclonal antibodies can be combined with more than one tumor immunizing agent (such as an immune checkpoint inhibitor) and can be combined with, for example, a combination method that targets multiple elements of the immune pathway, such as one or more of the following: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF secreting cell vaccines, cpG oligonucleotides, imiquimod); therapies that suppress negative immune regulation, for example, by inhibiting CTLA-4 and/or PD 1/PD-L2 pathways and/or depleting or blocking tregs or other immunosuppressive cells; therapies that stimulate positive immune regulation, for example, with agonists that stimulate CD-137, OX-40, and/or GITR pathways and/or that stimulate T cell effector functions; a therapy that increases systemic anti-tumor T cell frequency; therapies that deplete or inhibit tregs, such as tregs in tumors, for example, using CD25 antagonists (e.g., daclizumab) or by ex vivo anti-CD 25 bead depletion; therapies that affect the function of inhibitory bone marrow cells in tumors; 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 such as Indoleamine Dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies to reverse/prevent T cell anergy or depletion; therapies that trigger innate immune activation and/or inflammation of tumor sites; administration of an immunostimulatory cytokine or blocking an immunosuppressive cytokine.

For example, the subject anti-TNFR 2 monoclonal antibodies can be used with the following agents: one or more agonists that bind to positive co-stimulatory receptors; one or more antagonists (blockers) that attenuate signaling via inhibitory receptors, e.g., antagonists that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., block PD-L1/PD-L2 interactions); one or more agents that increase the frequency of systemic anti-tumor immune cells (such as T cells), deplete or inhibit tregs (e.g., by inhibiting CD 25); one or more agents that inhibit a metabolic enzyme (such as IDO); one or more agents that reverse/prevent T cell anergy or depletion; and one or more agents that trigger innate immune activation and/or inflammation of the tumor site.

In one embodiment, a subject having a disease (e.g., cancer or infectious disease) that can benefit from stimulating the immune system is treated by administering to the subject a subject anti-TNFR 2 monoclonal antibody and a tumor immunizing agent, wherein the tumor immunizing agent is a CTLA-4 antagonist, such as an antagonistic CTLA-4 antibody. Suitable CTLA-4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab (tremelimumab).

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-TNFR 2 monoclonal antibody and a tumor immunizing agent, wherein the tumor immunizing agent is a PD-1 antagonist, such as an antagonistic PD-1 antibody. Suitable PD-1 antibodies include, for example, OPDIVO (Nawuzumab), KEYTRUDA (Palimumab) or MEDI-0680 (AMP-514; WO 2012/145493). The tumor immunizing agent may further include Pidilizumab (CT-011). Another approach to target PD-1 receptors is a recombinant protein consisting of the extracellular domain of PD-L2 fused to the Fc portion of IgG1 (B7-DC), called AMP-224.

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-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is a PD-L1 antagonist, such as an antagonistic PD-L1 antibody. Suitable PD-L1 antibodies include, for example, MPDL3280A (RG 7446; WO 2010/077634), dewaruzumab (MEDI 4736), BMS-936559 (WO 2007/005874), MSB0010718C (WO 2013/79174) or rHigM B7.

In one embodiment, a subject suffering from 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-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is a LAG-3 antagonist, such as an antagonistic LAG-3 antibody. Suitable LAG3 antibodies include, for example, BMS-986016 (WO 10/19570, WO 14/08218) or IMP-731 or IMP-321 (WO 08/132601, WO 09/44273).

In one embodiment, a subject suffering from 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-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is a CD137 (4-1 BB) agonist, such as an agonistic CD137 antibody. Suitable antibodies to CD137 include, for example, wu Ruilu mab (urelumab) or PF-05082566 (W012/32433).

In one embodiment, a subject having a disease (e.g., cancer or infectious disease) that can benefit from stimulating the immune system is treated by administering an anti-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent to the subject, wherein the tumor immunizing agent is a GITR agonist, such as an agonistic GITR antibody. Suitable antibodies to GITR include, for example, the antibodies to GITR disclosed in TRX-518 (WO 06/105021, WO 09/009116), MK-4166 (WO 11/028683), or WO 2015/031667.

In one embodiment, a subject having a disease that can benefit from stimulation of the immune system (e.g., cancer or infectious disease) is treated by administering to the subject an anti-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is an OX40 agonist, such as an agonistic OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG 7888; WO 06/029879).

In one embodiment, a subject suffering from 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-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is a CD40 agonist, such as an agonistic CD40 antibody. In certain embodiments, the tumor immunizing agent is a CD40 antagonist, such as an antagonistic CD40 antibody. Suitable CD40 antibodies include, for example, lu Katuo Mumab (lucatumumab, HCD 122), daclizumab (dacetuzumab, SGN-40), CP-870,893 or Chi Lob 7/4.

In one embodiment, a subject suffering from 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-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is a CD27 agonist, such as an agonistic CD27 antibody. Suitable antibodies to CD27 include, for example, valdecomab (varlilumab, CDX-1127).

In one embodiment, a subject suffering from a disease that can benefit from stimulation of the immune system (e.g., cancer or infectious disease) is treated by administering to the subject an anti-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is MGA271 (against B7H 3) (WOl 1/109400).

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-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is a KIR antagonist, such as Li Ruilu mab (lirilumab).

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-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO 2006/122150, WO07/75598, WO08/36653, WO 08/36642), indomod (indoximod), NLG-919 (WO 09/73620, WO09/1156652, WOl/56652, WO 12/142237) or F001287.

In one embodiment, a subject having a disease that can benefit from stimulation of the immune system (e.g., cancer or infectious disease) is treated by administering to the subject an anti-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is a Toll-like receptor agonist, such as a TLR2/4 agonist (e.g., bacillus calmette-guerin); TLR7 agonists (e.g., hiltonol (Hiltonol) or imiquimod); TLR7/8 agonists (e.g., resiquimod (Resiquimod)); or TLR9 agonists (e.g., cpG 7909).

In one embodiment, a subject suffering from 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-TNFR 2 monoclonal antibody of the invention and a tumor immunizing agent, wherein the tumor immunizing agent is a TGF- β inhibitor, such as GC1008, LY2157299, TEW7197, or IMC-TR1.

6. Exemplary anti-TNFR 2 monoclonal antibodies

The invention described herein provides monoclonal antibodies or antigen binding fragments thereof that are specific for TNFR 2.

Accordingly, one aspect of the invention provides an isolated monoclonal antibody or antigen binding fragment thereof which competes with any of the isolated monoclonal antibodies or antigen binding fragments thereof described herein for binding to an epitope of SEQ ID NO 13/101 or 38, or to an epitope bound by HFB 3-18.

For example, the epitope of HFB3-1/HFB3-1-hG1 is depicted in FIGS. 11A-11C (SEQ ID NO:13 is depicted in FIGS. 11A and 11B, and SEQ ID NO:101 is depicted in FIG. 11C).

A related aspect of the invention provides an isolated monoclonal antibody, or antigen binding fragment thereof, which specifically binds to an epitope of SEQ ID NO 13/101 or 38 or an epitope bound by HFB 3-18.

Another related aspect of the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof is specific for human TNFR2, and wherein the monoclonal antibody comprises: (1a) A Heavy Chain Variable Region (HCVR) comprising a HCVR CDR1 sequence of SEQ ID No. 1, a HCVR CDR2 sequence of SEQ ID No. 2, and a HCVR CDR3 sequence of SEQ ID No. 3; and (1 b) 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 (2 a) 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 (2 b) 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 (3 a) a Heavy Chain Variable Region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO. 26, the HCVR CDR2 sequence of SEQ ID NO. 27 and the HCVR CDR3 sequence of SEQ ID NO. 28; and (3 b) a Light Chain Variable Region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO. 29, the LCVR CDR2 sequence of SEQ ID NO. 30 and the LCVR CDR3 sequence of SEQ ID NO. 31; or (4 a) a Heavy Chain Variable Region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO:39, the HCVR CDR2 sequence of SEQ ID NO:40 and the HCVR CDR3 sequence of SEQ ID NO: 41; and (4 b) a Light Chain Variable Region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO. 42, the LCVR CDR2 sequence of SEQ ID NO. 43 and the LCVR CDR3 sequence of SEQ ID NO. 44; or (5 a) 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 (5 b) a Light Chain Variable Region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO. 54, the LCVR CDR2 sequence of SEQ ID NO. 55 and the LCVR CDR3 sequence of SEQ ID NO. 56; or (6 a) a Heavy Chain Variable Region (HCVR) comprising a HCVR CDR1 sequence of SEQ ID No. 63, a HCVR CDR2 sequence of SEQ ID No. 64, and a HCVR CDR3 sequence of SEQ ID No. 65; and (6 b) 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.

For any of the aspects of the invention described above, in some embodiments, in the isolated monoclonal antibody or antigen binding fragment thereof: (1A) HCVR has the sequence of 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.

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

Some sequences of the antibodies of the invention are provided 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)

HCVR is SEQ ID NO. 7 and LCVR is SEQ ID NO. 8.

HC：

LC：

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)

HCVR is SEQ ID NO. 20 and LCVR is SEQ ID NO. 21.

HC：

LC：

/>

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)

HCVR is SEQ ID NO. 32 and LCVR is SEQ ID NO. 33.

HC：

LC：

/>

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)

HCVR is SEQ ID NO. 45 and LCVR is SEQ ID NO. 46.

HC：

LC：

/>

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)

HCVR is SEQ ID NO:57 and LCVR is SEQ ID NO:58.

HC：

LC：

/>

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)

HCVR is SEQ ID NO:69 and LCVR is SEQ ID NO:70.

HC：

LC：

/>

In some embodiments, the monoclonal antibodies or antigen binding fragments thereof of the invention are human-mouse chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, or resurfaced antibodies.

In some embodiments, the antigen binding fragment thereof is a Fab, fab ', F (ab ') ₂、F_d, single chain Fv or scFv, disulfide linked F _v, V-NAR domain, igNar, intracellular antibody, igG Δch ₂, miniantibody, F (ab ') ₃, tetravalent antibody, trivalent antibody, diabody, single domain antibody, DVD-Ig, fcab, mAb ₂、(scFv)₂, or scFv-Fc.

In some embodiments, the monoclonal antibodies or antigen binding fragments thereof of the invention have an engineered Fc region that abrogates immune effector function. For example, the engineered Fc region of the subject antibodies may have "LALA" double mutations (Leu 234Ala and Leu235 Ala), and thus have reduced effector function. Such antibodies may be designated G1AA by having double LALA mutations on IgG 1.

Other recombinant human IgG antibodies (hIgG) are known in the art to be partially or completely non-binding to fcγreceptor (fcγr) and complement protein C1q and thus have an abrogating immune effector function, and are used in a variety of therapeutic applications to reduce fcγr activation and Fc-mediated toxicity. Some such Fc engineered antibodies/fragments partially achieve this goal, while others completely eliminate fcγr activation and Fc-mediated toxicity. In certain embodiments, the antibodies/fragments of the invention have an engineered hIgG Fc domain comprising an hIgG1-P329G LALA or hIgG4-P329G SPLE (human IgG4S228P/L235E variant of IgG 4) mutation and completely eliminate FcγR and C1q interactions and do not affect FcRn interactions and Fc stability. The P329G Fc mutation disrupts the formation of the sandwich motif of proline and fcγr. Because this motif is present in the interface of all IgG Fc/fcγr complexes, its disruption can be applied to all human and most other mammalian IgG subclasses to produce effector-silenced IgG molecules. Thus, in certain embodiments, the subject antibodies/fragments have any IgG subclass that contains such effector silencing Fc mutations.

In certain embodiments, the monoclonal antibodies of the invention, or antigen-binding fragments thereof, are specific for human TNFR2, e.g., do not substantially cross-react with TNFR1, and/or do not substantially cross-react with mouse TNFR 2. In certain embodiments, the monoclonal antibodies of the invention, or antigen binding fragments thereof, cross-react with monkey TNFR2 (such as cynomolgus monkey or rhesus monkey TNFR 2).

In some embodiments, the monoclonal antibodies of the invention, or antigen binding fragments thereof, have a dissociation constant (K _d) for rhTNFR2 of.ltoreq.1. Mu.m,.ltoreq.100 nM,.ltoreq.50 nM,.ltoreq.25 nM,.ltoreq.20 nM,.ltoreq.15 nM,.ltoreq.10 nM,.ltoreq.5 nM,.ltoreq.2 nM,.ltoreq.1 nM,.ltoreq.0.01 nM or.ltoreq.0.001 nM (e.g., 10 ^-8 M or less, e.g., 10 ^-8 M to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M).

In some embodiments, the monoclonal antibodies of the invention, or antigen binding fragments thereof, bind to a region within the CRD2 domain of TNFR 2. In a certain embodiment, the monoclonal antibody or antigen binding fragment thereof of the invention binds to an epitope bound by HFB 3-1.

In some embodiments, the monoclonal antibodies of the invention, or antigen binding fragments thereof, bind to a region within the CRD3 domain of TNFR 2. In a certain embodiment, the monoclonal antibody or antigen binding fragment thereof of the invention binds to an epitope bound by HFB 3-14.

In a certain embodiment, the monoclonal antibody or antigen binding fragment thereof of the invention binds to an epitope bound by HFB 3-18.

In a certain embodiment, the monoclonal antibody or antigen binding fragment thereof of the invention binds to an epitope of SEQ ID NO. 13/101 or 38.

In some embodiments, the monoclonal antibodies of the invention, or antigen-binding fragments thereof, enhance binding of human recombinant tnfα to TNFR 2.

In some embodiments, the monoclonal antibodies of the invention, or antigen-binding fragments thereof, block binding of human recombinant tnfα to TNFR 2.

In some embodiments, the monoclonal antibodies of the invention, or antigen-binding fragments thereof, do not substantially affect binding of human recombinant tnfα to TNFR 2.

In some embodiments, the monoclonal antibodies of the invention, or antigen binding fragments thereof, inhibit tnfα -mediated signaling (such as nfkb signaling), and/or induce down-regulation of a target gene downstream of nfkb. However, in other embodiments, the monoclonal antibodies of the invention, or antigen binding fragments thereof, promote tnfα -mediated signaling (such as nfkb signaling), and/or induce up-regulation of a target gene downstream of nfkb.

In some embodiments, nfkb signaling is stimulated in effector T cells (such as CD8 and/or CD4 Tconv T cells). In some other embodiments, nfkb signaling is inhibited in effector T cells (such as CD8 and/or CD4 Tconv T cells).

In some embodiments, nfkb signaling is stimulated in Treg. In some other embodiments, nfkb signaling is inhibited in Treg.

In some embodiments, the monoclonal antibodies of the invention, or antigen-binding fragments thereof, stimulate CD8 and/or conventional CD 4T cell proliferation, optionally with or without co-stimulation of CD3/CD28, and/or optionally with or without tnfα co-stimulation.

In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof, particularly humanized monoclonal antibodies or antigen-binding fragments thereof, of the invention preferentially bind to (CD 3/CD 28) TCR-activated primary CD8 and/or CD 4T cells compared to non-stimulated primary CD8 and/or CD 4T cells.

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/CD 28-induced activation and/or proliferation, such as CD3/CD 28-induced activation and/or proliferation of primary CD8 and/or CD 4T cells, including activation and/or proliferation of primary CD8 and/or CD 4T cells in the presence of tregs.

In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention, particularly humanized monoclonal antibodies or antigen-binding fragments thereof, co-stimulate activation and/or proliferation of primary CD8 and/or CD 4T cells in a cross-linked independent manner.

In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention, particularly humanized monoclonal antibodies or antigen-binding fragments thereof, co-stimulate activation and/or proliferation of primary CD8 and/or CD 4T cells in a cross-linking dependent manner.

In some embodiments, the monoclonal antibodies of the invention, or antigen-binding fragments thereof, enhance binding between tnfα and TNFR 2; enhancing tnfa-mediated or co-stimulated nfkb signaling (e.g., in TCR-activated CD8 and/or CD4 Tconv T cells); and/or proliferation of effector T cells (e.g., CD8 and/or CD4 Tconv T cells) that promote TCR activation in the presence of tregs.

In some embodiments, the monoclonal antibodies of the invention, or antigen-binding fragments thereof, enhance tnfα -mediated CD25 expression on tregs.

In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention (including humanized monoclonal antibodies or antigen-binding fragments thereof) have good developability characteristics, including stability at high temperatures (e.g., 25 ℃ or 40 ℃), low pH conditions (e.g., pH3.5 at about room temperature), and/or after several cycles of freezing/thawing.

In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention (including humanized monoclonal antibodies or antigen-binding fragments thereof) comprise one or more point mutations in the amino acid sequence designed to improve the developability of the antibodies. For example, raybould et al (F ive computational developability guidelines for therapeutic antibody profiling,PNAS116(10):4025-4030,2019) describe a therapeutic antibody profiler (T AP), which is a computational tool that builds a downloadable homology model of variable domain sequences, tests it for five developability guidelines, and reports potential sequence instability and canonical forms. The present authors further provide TAPs that are freely available on the opig.stats.ox.ac.uk/webapps/sabdab-sabpre d/TAP.php.

There are a number of obstacles to therapeutic mAb development beyond achieving the desired affinity for an antigen. These include inherent immunogenicity, chemical and conformational instability, self-association, high viscosity, multi-specificity and poor expression. For example, high levels of hydrophobicity, especially in highly variable Complementarity Determining Regions (CDRs), have been repeatedly involved in aggregation, viscosity and multi-specificity. The asymmetry of the net charge of the heavy and light chain variable domains is also related to self-association and viscosity at high concentrations. Patches of positive and negative charges in CDRs are associated with high clearance and poor expression levels. Product heterogeneity (e.g., via oxidation, isomerization, or glycosylation) is typically derived from a particular sequence motif that is amenable to post-translational or co-translational modification. Computational tools can be used to facilitate identification of sequence instability. Warszawski et al (Optimizing antibody affinity and stability by t he automated design of the variable light-heavy chain interfaces.PL oS Comput Biol 15(8):e1007207.https://doi.org/10.1371/journal.pcbi.1007207) also describe methods for optimizing antibody affinity and stability by automated design of variable light chain-heavy chain interfaces. Other methods may be used to identify potential developability issues with candidate antibodies, and in preferred embodiments of the invention, one or more point mutations may be introduced into candidate antibodies via conventional methods to address such issues to produce optimized therapeutic antibodies of the invention.

7. Humanized antibodies

In some embodiments, the antibodies of the invention are humanized antibodies. Humanized antibodies are useful as therapeutic molecules because humanized antibodies reduce or eliminate human immune responses to non-human antibodies, such as human anti-mouse antibody (HAMA) responses, which can generate immune responses to antibody therapeutics, and reduce the effectiveness of the therapeutics.

Antibodies can be humanized by any standard method. Non-limiting exemplary methods of humanization include, for example, U.S. Pat. nos. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. nos. 5,693,761; U.S. Pat. nos. 5,693,762; U.S. Pat. nos. 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 the method described in U.S. publication No. US 2009/0135500. All documents are incorporated by reference.

Humanized antibodies are antibodies in which at least one amino acid in the framework region of a non-human variable region has been replaced with an amino acid at a 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 regions of the non-human variable region have been replaced with amino acids at one or more corresponding positions in one or more human framework regions.

In some embodiments, some of the corresponding human amino acids used for substitution are framework regions from different human immunoglobulin genes. That is, in some such embodiments, one or more non-human amino acids may be replaced by a corresponding amino acid of a human framework region of a first human antibody or encoded by a first human immunoglobulin gene, one or more non-human amino acids may be replaced by a corresponding amino acid of a human framework region of a second human antibody or encoded by a second human immunoglobulin gene, one or more non-human amino acids may be replaced by a corresponding amino acid of a human framework region of a third human antibody or encoded by a third human immunoglobulin gene, and so forth. In addition, in some embodiments, all of the corresponding human amino acids used to replace a single framework region (e.g., FR 2) need not be from the same human framework. However, in some embodiments, all of the corresponding human amino acids used for substitution are from the same human antibody or are encoded by the same human immunoglobulin gene.

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

In some embodiments, after CDR grafting, one or more framework amino acids are changed back to the corresponding amino acids in the mouse framework region. In some embodiments, such "back mutations" are made to retain one or more mouse framework amino acids that appear to contribute to the structure of one or more CDRs and/or that may be involved in antigen contact and/or appear to be involved in the overall structural integrity of the antibody. In some embodiments, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or 0 back mutations are made to the framework regions of the antibody after CDR grafting.

In some embodiments, the humanized antibody further comprises a human heavy chain constant region and/or a human light chain constant region.

8. Human antibodies

In some embodiments, the antibodies of the invention are human antibodies. The human antibodies may be made by any suitable method. Non-limiting exemplary methods include making human antibodies in transgenic mice comprising 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 5,545,807; U.S. patent No. 6,713,610; U.S. patent No. 6,673,986; U.S. patent No. 6,162,963; U.S. patent No. 5,545,807; U.S. patent No. 6,300,129; U.S. patent No. 6,255,458; U.S. patent No. 5,877,397; U.S. patent No. 5,874,299; and U.S. Pat. No. 5,545,806.

Non-limiting exemplary methods also include the use of phage display libraries to make human antibodies. 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.

Antibody constant regions

In some embodiments, a humanized, chimeric, or human antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region has an isotype selected from IgA, igG, and IgD. In some embodiments, the human light chain constant region has 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 an 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. patent 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 an S241P mutation in a 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.

The choice of heavy chain constant region can determine whether an antibody will have an in vivo effector function. In some embodiments, such effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), and can kill cells to which the antibody binds. Typically, antibodies comprising human IgG1 or IgG3 heavy chains have effector functions.

In some embodiments, effector function is not desirable. For example, in some embodiments, effector function may be undesirable in treating inflammatory disorders and/or autoimmune disorders. In some of these embodiments, the human IgG4 or IgG2 heavy chain constant region is selected or engineered. In some embodiments, the IgG4 constant region comprises the S241P mutation.

Any of the antibodies described herein can be purified by any suitable method. Such methods include, but are not limited to, the use of an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include antigens and/or epitopes to which antibodies bind and ligands that bind to antibody constant regions. For example, protein a, protein G, protein a/G, or antibody affinity columns can be used to bind constant regions and purify antibodies.

In some embodiments, hydrophobic Interaction Chromatography (HIC) (e.g., butyl or phenyl column) is also used to purify some polypeptides. Numerous methods of purifying polypeptides are known in the art.

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).

9. Nucleic acid molecules encoding antibodies of the invention

The invention also provides nucleic acid molecules comprising polynucleotides encoding one or more strands of the antibodies described herein. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding the heavy or light chain of an antibody described herein. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain of an antibody described herein and a polynucleotide encoding a light chain of an antibody described herein. In some embodiments, the first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain and the second nucleic acid molecule comprises a second polynucleotide encoding a light chain.

In some such embodiments, the heavy and light chains are expressed by one nucleic acid molecule, or by two separate nucleic acid molecules as two separate polypeptides. In some embodiments, such as when the antibody is an scFv, the single polynucleotide encodes a single polypeptide comprising a heavy chain and a light chain linked together.

In some embodiments, a polynucleotide encoding a heavy chain or a light chain of an antibody described herein comprises a nucleotide sequence encoding a leader sequence that is located at the N-terminus of the heavy chain or light chain upon translation. As discussed above, the leader sequence may be a native heavy or light chain leader sequence, or may be another heterologous leader sequence.

Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell (such as a mammalian cell).

10. Carrier body

Vectors are provided comprising polynucleotides encoding the heavy and/or light chains of 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 comprises 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 as two separate polypeptides by the vector. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.

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 (such as similar molar amounts or similar mass amounts). In some embodiments, the first vector and the second vector are transfected into the host cell at a molar ratio or mass ratio between 5:1 and 1:5. In some embodiments, a vector encoding a heavy chain and a vector encoding a light chain are used in a mass ratio of between 1:1 and 1:5. In some embodiments, a 1:2 mass ratio of vector encoding a heavy chain and vector encoding a light chain is used.

In some embodiments, a vector optimized for expression of the polypeptide in CHO or CHO-derived cells or NSO cells is selected. 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 in vivo expression of the subject antibody in an animal (including a human). In some such embodiments, the one or more polypeptides are expressed under the control of one or more promoters that function in a tissue-specific manner. For example, liver-specific promoters are described, for example, in PCT publication No. WO 2006/076288.

11. Host cells

In various embodiments, the heavy and/or light chains of an antibody described herein can be in a prokaryotic cell (such as a bacterial cell); or in eukaryotic cells (such as fungal cells, such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells useful for expressing the polypeptide include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; cells (Crucell); and NSO cells. In some embodiments, the heavy and/or light chains of an antibody described herein can be expressed in yeast. See, for example, U.S. publication No. US2006/0270045 Al. 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 chain of the TNFR2 antibody. For example, in some embodiments, CHO cells produce polypeptides that have higher sialylation levels than the same polypeptide produced in 293 cells.

The introduction of one or more nucleic acids into a desired host cell may be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, and the like. Non-limiting exemplary methods are described, for example, in Sambrook et al Molecular Cloning, A Laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory Press (2001). The nucleic acid may be transiently or stably transfected into the desired host cell according to any suitable method.

In some embodiments, one or more polypeptides may be produced in vivo in an animal engineered or transfected with one or more nucleic acid molecules encoding the polypeptides according to any suitable method.

Examples

Example 1 monoclonal antibodies specific for human and monkey TNFR2

To generate monoclonal antibodies specific for human TNFR2 that are cross-reactive with the monkey ortholog TNFR2, mice were immunized with the recombinant extracellular domain (ECD) of human TNFR2 (rhTNFR 2) using standard procedures to generate a series of different human-mouse chimeric monoclonal antibodies.

At least 25 such monoclonal antibodies were generated, VH and VL sequences of selected antibodies were aligned, and consensus sequences were obtained, as shown in fig. 1. The H-CDR3 and L-CDR3 regions are marked by boxed sequences.

These monoclonal antibodies were then tested for their ability to bind to human and monkey TNFR2 expressed by CHO cells (CHO. Hfb3 and CHO. Mkhfb3 cells, respectively). Briefly, about 40,000 cho.hfb3 or cho.mkhfb3 cells were seeded into tissue culture wells, and a continuous 1:3 dilution of each test antibody (starting (highest) concentration of about 66nM antibody) was added to each cell type and incubated for about 1 hour. By using a composition consisting of AF647 (ALEXSA647 Fluorescent dye) labeled 17nM anti-human Fc antibody to detect antibodies bound to cells. Isotype-matched negative control antibodies were also used in this assay. The data (including EC ₅₀ values and E _max for each antibody) are compiled in fig. 2A.

Eleven (11) test antibodies showed affinity (EC ₅₀) to the nM level of the hTNFR2 sub-or mono-numbers expressed on CHO cells. These antibodies also showed cross-reactivity against rhesus orthologs of TNFR2 expressed on CHO cells and binding affinity had substantially the same trend as hTNFR2 binding. See fig. 2A.

Interestingly, some antibodies (such as HFB3-1 and HFB 3-14) promoted binding of TNFα to TNFR2, other antibodies (such as HFB 3-18) inhibited binding of TNFα to TNFR2, and other antibodies (such as HFB 3-6) apparently had no effect on binding of TNFα to TNFR 2. See fig. 2B. Specifically, after pre-incubating CHO cells with the corresponding antibodies for about 1 hour, binding of 25ng/mL tnfα to cho.hfb3 cells was measured. The percentage of cells binding to tnfα (labeled HFB 2003L) was then plotted against increasing concentrations of antibody.

The same experiment was also set up to test the ability of the antibodies to bind to CHO cells expressing mouse TNFR2 and parent CHO cell lines (which may or may not express hamster TNFR 2). Two monoclonal antibodies (HFB 3-18 and HFB 3-19) exhibited marginal levels of mouse ortholog binding, while the other antibodies did not have detectable levels of mouse TNFR2 binding. As a positive control, HM102 monoclonal antibody specific for mouse TNFR2 was used to show positive binding to CHO cells expressing mouse TNFR2, whereas isotype matched control antibodies did not bind (fig. 3).

No binding was observed for the parental CHO cell line (fig. 3).

Recombinant human TNFR2 and TNFR1 proteins were also used to verify binding specificity for human TNFR2 (compare the relevant TNFR1 receptor).

Briefly, tissue culture plates were coated overnight at 4℃with 0.1. Mu.g/mL of His-tagged recombinant human TNFR2 or TNFR 1. The coated plates were then incubated with 1:3 serial dilutions of each test antibody on ice for about 1 hour at an initial (highest) concentration of about 66nM antibody. Antibodies bound to cells were detected by using a 1:5000 dilution of HRP-labeled anti-human Fc antibody and TMB substrate. An isotype-matched negative control antibody F3 was also used in this assay, as well as an MR2-1 positive control antibody specific for rhTNFR2 and a positive control antibody specific for rhTNFR 1. The data (including EC ₅₀ values for each antibody) are compiled in fig. 4A.

6 Of the 11 antibodies tested (i.e., HFB3-1, HFB3-14, HFB3-21, HFB3-23, HFB3-24, and HFB 3-25) showed sub-nM affinity (EC ₅₀) for His-tagged monomer rhTNFR2, while the other 4 antibodies (HFB-3, HFB-6, HFB-19, and HFB-22) showed one-digit nM affinity for the same antigen. HFB3-18 showed the relatively weakest binding to monomeric rhTNFR2, with a two digit nM affinity. However, none of the 11 antibodies showed any detectable level of binding to His-tagged TNFR1 receptor, confirming binding specificity to TNFR 2.

The binding affinity of human-mouse chimeric antibodies HFB3-1, HFB3-14, and HFB3-18 to recombinant human TNFR2 protein was verified using an anti-human IgG Fc capture (AHC) biosensor. The AHC biosensor enables kinetic characterization of macromolecular interactions between a human Fc-containing protein (e.g., a subject antibody) and a target analyte (e.g., recombinant human TNFR 2). Immobilization of human Fc-containing proteins is achieved via factory-immobilized anti-human Fc-specific antibodies that provide a stable baseline for the high affinity of human Fc domains required for demanding kinetic applications. In this particular experiment, the test antibody (humanized \ loaded at a concentration of 20 μg/mL in assay buffer (PBS, pH 7.4, 0.1% BSA, 0.1% tween 20) was performed at 25 ℃ for a capture assay with His-tagged recombinant human tnfr2 with an analyte of 500nM, 167nM or 55.7nM, K _d of the tested antibodies was in the nM range (see fig. 4B).

Epitope mapping experiments of HFB3-1-hG1, HFB3-14-hG1, HFB3-6-hG1 and HFB3-18-hG1 antibodies showed that these antibodies recognize different domains of TNFR 2. One structural feature shared by most members of the TNFR superfamily is that it contains about two to four cysteine-rich domains (CRDs). HFB3-1-hG1 binds to a region within the domain of CRD2 (FIG. 11C), whereas HFB3-18-hG1 binds to a conformational epitope within CDR 1. HFB3-6-hG1 binds to a region within CRD3, and HFB3-14-hG1 also binds to an epitope within the CRD3 region that is smaller than the epitope of HFB3-6-hG1 (see FIG. 11B). The position of its epitope on the 3D model of the TNFR2-tnfα complex can be visualized in fig. 11D.

EXAMPLE 2 expression of TNFR2 in T cell subtypes

This experiment demonstrates that TNFR2 is expressed primarily on Treg and CD4 ⁺ and CD8 ⁺ T cells in a variety of cancer types.

T cell subtypes (including Treg and CD4 ⁺ and CD8 ⁺ T cells) were isolated from multiple tumor samples, and the relative percentages of T cell subtypes and the average relative expression levels of TNRF2 in the T cell subtypes (grade 2-8) in the tumor samples were determined using RNA sequence analysis. The results are compiled in fig. 5.

In each tumor sample analyzed (including BCC or basal cell carcinoma, SCC or squamous cell carcinoma, melanoma, and NSCLC or non-small cell lung carcinoma), TNFR2 was predominantly and most frequently found in Treg as well as CD4 ⁺ and CD8 ⁺ T cells. In addition, the highest relative expression levels were also found in tregs. See left view of fig. 5. The data indicate that TNFR2 is an attractive target for cancer therapy.

Other expression assays for TNFR2 in SCC cancer samples were also performed in conjunction with expression of several immune checkpoint genes (such as PD-1, TIM3, CTLA4, and 4-1 BB). Expression of TNFR2 was found to be consistent with expression of these immune checkpoint genes in depleted cd8+ T cells (right panel of fig. 5), indicating that combination therapies using anti-TNFR 2 antibodies and these immune checkpoint gene inhibitors are therapeutically beneficial.

EXAMPLE 3 binding of anti-TNFR 2 monoclonal antibodies to primary Treg, CD8 and CD4 Tconv cells

In view of the pattern of expression of TNFR2 on T cell subtypes (see example 2), this experiment demonstrates that the subject anti-TNFR 2 monoclonal antibodies can bind to primary T cell subtypes in preference to activated T cells.

Briefly, flat bottom 96-well plates were coated overnight at 4 ℃ with 10nM of anti-CD 3 antibody. Meanwhile, T cell subtypes, including Treg, CD8 or CD4 conventional T cells (Tconv), were isolated from human PBMCs. The isolated T cell subtype was seeded at a density of about 50,000 cells/well in the presence of 6.6nM of anti-CD 28 antibody to co-stimulate primary T cells for about 3 days. Stimulated primary T cells were then treated with 1:3 serial dilutions of the anti-TNFR 2 human-mouse chimeric monoclonal antibody of the invention at different concentrations for 1 hour on ice, with a maximum concentration of 66nM. Bound chimeric antibody was detected by adding 17nM of anti-hFc antibody labeled with AF647 dye and incubating on ice for 1 hour, followed by detection of AF647 signal by FACS analysis.

The top panel of fig. 6 shows that CD4 Tconv is the most abundant T cell subtype, accounting for about 30% of the total hPBMC, followed by 10% CD 8T cells and about 1% Treg. However, non-TCR-activated primary T cells were unable to detect binding to the subject anti-TNFR 2 antibodies, except for the relatively low levels of binding that occurred in primary tregs. In summary, receptor occupancy Emax is highest in tregs, followed by CD8, again CD4 Tconv. Given the relatively low abundance of tregs compared to CD8 and CD4 Tconv, the expression of TNFR2 on tregs was much higher than on CD8 and CD 4T cells on a per cell basis.

However, in TCR-activated T cells, a significant 5-6 fold increase in binding of some anti-TNFR 2 antibodies was observed in tregs, while substantially higher binding was also observed in CD8 and CD4 Tconv (fig. 6, bottom panel).

In the antibodies tested, HFB3-1, HFB3-6, HFB3-24, HFB3-25 and SBT1 (positive control) exhibited high affinity at sub-nM levels, while HFB3-14 and HFB3-19 exhibited one-digit nM affinity. HFB3-18, HFB3-21 and HFB3-22 have a two digit nM affinity.

EXAMPLE 4 binding of certain anti-TNFR 2 monoclonal antibodies to primary CD8 and CD4 Tconv cells costimulatory NFkB signaling

This experiment demonstrates that the anti-TNFR 2 monoclonal antibodies of the present invention co-stimulate tnfα -mediated nfkb signaling as demonstrated by QPCR quantification of nfkb signaling pathway genes.

Briefly, CD4 Tconv (CD 4 ⁺CD25^-) or CD8 ⁺ T cells were isolated from hPBMC using standard techniques and commercially available kits. Isolated T cells were incubated with 10. Mu.g/mL (66 nM) of the various test monoclonal antibodies of the invention or appropriate positive or negative controls and 25ng/mL (1.5 nM) of TNF. Alpha. For about 24 hours. The stimulated T cells are then harvested and their mRNA isolated, reverse transcribed, and QPCR analyzed for selected nfkb signaling pathway genes (such as CD25, foxp3, nfkb 2, relB, and LTA). The expression levels of these genes in the presence or absence of co-stimulation of the subject antibodies are compared in the bar graph of fig. 7. Results are presented as fold change compared to no-stimulus control (1×).

The results show that certain subject antibodies (including HFB3-1, HFB3-14, HFB3-23, HFB3-24, and HFB 3-25) induce NF-. Kappa.B signaling. It should be noted that HFB3-1 and HFB3-14, but not HFB3-18, from time to time induce NFkB signaling, especially in NFkB 2, relB and LTA.

Example 5 Co-stimulatory Effect of anti-TNFR 2 monoclonal antibodies with proliferation of isolated primary CD8 and CD4 Tconv cells

In this experiment, flat bottom 96-well plates were coated overnight at 4 ℃ with 10nM anti-CD 3 monoclonal antibody and 20nM or 100nM subject anti-TNFR 2 antibody. At the same time, CD8 and CD4 Tconv cells were isolated from hpbmcs as described and T cell proliferation was followed with 2 μm CTV (CELLTRACE ^TM uv cell proliferation kit from Invitrogen). CELLTRACE ^TM violet dyes readily diffuse into cells where they are cleaved by intracellular esterases to produce highly fluorescent compounds which are then covalently bound to intracellular amines, resulting in stable, well-retained fluorescent staining which can be immobilized with aldehyde fixatives. The excess unconjugated reagent passively diffuses to the extracellular medium, wherein the unconjugated reagent can be quenched and washed out with complete medium.

The labeled T cells were then seeded in coated 96-well plates at a density of about 50,000 cells/well in the presence of 6.6nM anti-CD 28 antibody for co-stimulation for about 3 days. Cells were then fixed for FACS analysis of fluorescence signals.

The data in fig. 8 show that certain subject anti-TNFR 2 antibodies co-stimulate CD8 and CD4 Tconv proliferation even at lower 20nM concentrations. The baseline positive control antibodies SBT-1 and SBT-4 also co-stimulated T cell proliferation under the same conditions, but to a lesser extent than HFB3-1, HFB3-14, HFB3-18, and HFB3-25.

Other experiments showed that this co-stimulation of primary T cell proliferation may rely on fcγr cross-linking of certain monoclonal antibodies (such as HFB 3-18) without discernible cross-linking dependence on other antibodies (such as HFB3-1 and HFB 3-14).

In particular, CD8 and CD4 Tconv were isolated from donor KP59095 and isolated primary T cells were stimulated by CD3/CD28 TCR activation and the subject anti-TNFR 2 monoclonal antibodies HFB3-1, HFB3-14, or HFB3-18 in the presence or absence of 25ng/mL recombinant human TNFα (rhTNFα). anti-TNFR 2 antibodies bind to the plate or are supplied as soluble antibodies present in the binding mixture.

All three plate-bound anti-TNFR 2 antibodies (HFB 3-1, HFB3-14, and HFB 3-18) stimulated CD 8T cell proliferation in the presence of 25ng/mL rhTNFα (see FIG. 19, bottom left panel). However, only soluble HFB3-1 and HFB3-14 (but not soluble HFB 3-18) were able to stimulate CD 8T cell proliferation (FIG. 19, bottom right panel), indicating that FcγR cross-linking may be necessary for HFB3-18 mediated CD 8T cell proliferation, but may not be necessary for HFB3-1 and HFB3-14 mediated CD 8T cell proliferation (i.e., cross-linking independence).

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

Example 6 anti-TNFR 2 monoclonal antibodies in the Presence of Treg favor cell proliferation at the Teff cell ends (CD 8 and CD4 Tconv)

This experiment demonstrates that the subject anti-TNFR 2 monoclonal antibodies co-stimulate Teff cell proliferation (CD 8 and CD4 Tconv) in the presence of tregs along with CD3/CD28 mediated TCR activation.

Briefly, CD3 ⁺ T cells (including CD8 and CD4 Tconv effector T cells) as well as tregs were isolated from human PBMCs and co-stimulated with the subject anti-TNFR 2 monoclonal antibody by CD3/CD 28-mediated TCR activation for about 4 days, substantially as described above. Proliferation of total CD4 ⁺ T cells and CD8 ⁺ T cells in the presence of tregs was determined using the CELLTRACE ^TM uv cell proliferation kit from Invitrogen (CTV). Activation of CD4 ⁺ T cells CD8 ⁺ T cells was also determined by measuring the percentage of CD25 ⁺ T cells in the corresponding T cell population.

The results in FIG. 9 show that the anti-TNFR 2 monoclonal antibodies of the invention (e.g., HFB3-1hz6-hG1AA, which is a humanized form of HFB3-1, see below) are beneficial for cell proliferation on effector T cells (CD 8 and CD4 Tconv) even in the presence of tregs.

Example 7 anti-TNFR 2 monoclonal antibodies have negligible ADCC effect on HH lymphoma cells

This experiment demonstrates that the subject anti-TNFR 2 monoclonal antibodies have negligible ADCC effect on T cell lymphomas, indicating that such antibodies are suitable for use as T cell costimulators.

Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cell-mediated immune defense whereby effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by specific antibodies. It is a mechanism by which antibodies can be used to limit and inhibit infection as part of the humoral immune response. ADCC requires effector cells, a class of which is known as Natural Killer (NK) cells that typically interact with IgG antibodies.

In this experiment, jurkat. CD16V/NFAT/luc cells were used as effector cells, while HH lymphoma cells were the target cells. The ratio of effector cells to target cells was about 6:1. The co-cultured effector and target cells are incubated overnight in the presence of the subject anti-TNFR 2 monoclonal antibody (e.g., HFB3-1, HFB3-14, or HFB 3-18) or an isotype matched control (hIgG 1) at a concentration of 0nM, 0.0066nM, 0.66nM, or 66 nM. The antibody mogambir monoclonal antibody (moganulizumab) was used as a positive control for ADCC.

The results in fig. 10 show that the positive control antibody moghatti mab was at least 120-fold more potent in ADCC against target cells than either of the anti-TNFR 2 monoclonal antibodies tested. The data demonstrate that the subject anti-TNFR 2 antibodies are suitable for use as T cell costimulators due to their low/no ADCC effect on T cells.

EXAMPLE 8 binding of humanized anti-TNFR 2 monoclonal antibody to TNFR2

Various humanized monoclonal antibodies were generated against HFB3-1, HFB3-14 and HFB3-18, including at least 20 against HFB3-1, 16 against HFB3-14 and one against HFB3-18 (due to the selected human germline being highly similar to the parent HFB3-18 monoclonal antibody coding sequence). The ability of these humanized monoclonal antibodies to bind human TNFR2 expressed on CHO cells was assayed essentially as described in example 1.

FIG. 12A shows that humanized HFB3-1hz6, HFB3-14hz1c, and HFB3-18hz1 bind to CHO cells expressing human TNFR2 (CHO. HTNFR) but do not bind to parent CHO cells. FIG. 12B shows that at least 7 humanized HFB3-1 antibodies (i.e., HFB3-1hz6, HFB3-1hz8, HFB3-1hz9, HFB3-1hz10, HFB3-1hz11, HFB3-1hz12 and HFB3-1hz 14) and at least 8 humanized HFB3-14 antibodies (i.e., HFB3-14hz1c, HFB3-14hz2c, HFB3-14hz3c, HFB3-14hz4c, HFB3-14hz6c, HFB3-14hz7c, HFB3-14hz12c and HFB3-14hz14 c) retain substantially the same (if not the preferred) level of binding affinity for cells expressing TNFR2 (CHO. HHFB 3) as the corresponding parent chimeric antibodies.

Similar experiments were repeated using CHO cells expressing TNFR2 ortholog of rhesus (CHO. Mkhfb 3) instead. FIG. 13 shows that the general trend of binding to CHO cells expressing monkey TNFR2 matched CHO.hTNFR2. However, slightly unstable binding was observed for the two humanized variants based on HFB3-14 (i.e., HFB3-14hz2c and HFB3-14hz3 c).

Binding of the humanized anti-TNFR 2 antibody is specific for TNFR2 and not specific for TNFR 1. ELISA assays in FIG. 14A demonstrated that humanized monoclonal antibodies HFB3-1hz6, HFB3-14hz1c and HFB3-18hz1 bind to recombinant human and cynomolgus TNFR2 (hTNFR 2-His and cynoTNFR-His, respectively) without recognizing recombinant human TNFR1 (hTNFR 1-His). In addition, the EC50 for binding of these humanized anti-TNFR 2 antibodies to recombinant human and cynomolgus TNFR2 is in the range of one-digit nM.

The binding affinity of humanized variants to human TNFR2 was also verified using recombinant human TNFR2 protein and an AHC biosensor. An anti-human IgG Fc capture (AHC) biosensor enables kinetic characterization of macromolecular interactions between human Fc-containing proteins (e.g., subject antibodies) and target analytes (e.g., recombinant human TNFR 2). Immobilization of human Fc-containing proteins is achieved by factory-immobilized anti-human Fc-specific antibodies that provide a stable baseline for the high affinity of human Fc domains required for demanding kinetic applications. In this particular experiment, the test antibody (humanized vs parent chimeric antibody) was loaded in assay buffer (PBS (pH 7.4), 0.1% BSA, 0.1% Tween 20) at a concentration of 20. Mu.g/mL. The analytes were 500nM, 167nM or 55.7nM of His-tagged recombinant human TNFR2. The capture assay was run at 25 ℃.

As shown in fig. 14B, there was no major difference in distinguishing humanized variants from their corresponding chimeric parent antibodies based on affinity for recombinant human TNFR 2.

Example 3 shows that chimeric anti-TNFR 2 antibodies bind to TCR-activated T cells. Substantially the same experiment was run on the humanized variants and the results are shown in fig. 15.

In particular, most humanized HFB3-1 antibodies exhibit sub-nM levels of affinity based on binding to TCR-activated CD8 cells, except for two variants (HFB 3-1hz5 and HFB3-1hz 7) that do not appear to bind to TCR-activated CD8 cells. At the same time, all humanized HFB3-14 variants exhibited one-digit nM affinity for TCR-activated CD 8T cells. There is no major difference in distinguishing between the different variants. It should be noted that the positive control antibodies SBT-2 and SBT-3 are not good binders to primary CD8 cells.

Example 9 Co-stimulatory Effect of humanized anti-TNFR 2 monoclonal antibodies on proliferation of TCR-activated CD4 and CD 8T cells

Example 5 shows that the costimulatory effect of chimeric anti-TNFR 2 monoclonal antibodies proliferated isolated human primary CD8 and CD4 Tconv cells. This experiment demonstrates that humanized variants using HFB3-1 and HFB3-14 are identical in TCR-activated CD 4T cells.

In particular, FIG. 16 shows that humanized variants HFB3-1hz5, HFB3-1hz6, HFB3-1hz8, HFB3-1hz10, HFB3-1hz11 and HFB3-1hz12 strongly stimulated TCR-activated CD 4T cells, each to a greater extent than the parent HFB3-1 chimeric antibody, based on the CTV proliferation assay (see above). The same procedure was repeated for HFB3-14hz1c and HFB3-14hz3c variants.

Likewise, T cell activation based on the percentage of CD25 ⁺ T cell population was also confirmed for the above variants.

Confirmed costimulation data for HFB3-1hz6-hG1, HFB3-14hz1c-hG1 and HFB3-18hz1-hG1 were also obtained to show that these variants have a costimulatory effect for proliferation of TCR activated CD 8T cells (activated by CD3/CD28 stimulation). In particular, both the parent chimeric antibody and the selected humanized variant enhance proliferation of CD 8T cells stimulated by CD3/CD28 TCR activation. In addition, the cooperation of tnfα (right panel) further enhanced anti-TNFR 2 antibody-mediated CD8 proliferation. See fig. 20.

Example 10 certain humanized anti-TNFR 2 monoclonal antibodies induce nfκB signaling in tregs

Example 4 shows that binding of certain chimeric anti-TNFR 2 monoclonal antibodies to primary CD8 and CD4Tconv cells co-stimulates nfkb signaling. Similar experiments herein demonstrate that certain humanized variant anti-TNFR 2 antibodies induce nfkb signaling in tregs.

In particular, figure 17A shows that the co-stimulation of tregs with certain humanized variant anti-TNFR 2 antibodies and tnfa produced nfkb downstream signaling in LTA, TNF and TNF AIP 3. Variants HFB3-1hz6, HFB3-1hz9, HFB3-1hz10 and HFB3-1hz11 promote NFκB signaling to a greater extent than the parent chimeric antibody HFB 3-1. At the same time, variants HFB3-14hz1c, HFB3-14hz2c, HFB3-14hz3c and HFB3-14hz4c (in particular HFB3-14hz1c and HFB3-14hz3 c) also promote NF-. Kappa.B signaling to a greater extent than the parent chimeric antibody HFB 3-14. In addition, FIG. 17B shows activation of NF-. Kappa.B signaling in CD 8T cells with and without human recombinant TNFα using a humanized variant of the HFB3-1 antibody.

Example 11 anti-TNFR 2 antibody is stable

To confirm that the subject humanized anti-TNFR 2 antibodies are stable upon storage and thus suitable for further development as therapeutic agents, a variety of developability assays were run on selected humanized antibodies.

In a first experiment, selected subject humanized antibodies were stored in PBS (pH 7.4) at 25℃or 40℃and the stability of each antibody was determined on days 7 and 14. The results in FIG. 18 show that all antibodies tested were stable under the conditions tested, except for the 1 variant HFB3-14hz4c-hG1 AA.

In a second experiment, the same antibodies were tested for stability at low pH (100 mM AcH, pH3.5, 25 ℃) for 0 hours, 3 hours and 6 hours. The results in FIG. 18 again show that all antibodies tested were stable under the conditions tested, except for the 1 variant HFB3-14hz4c-hG1 AA.

In a third experiment, the same antibody was subjected to 1, 2 or 3 freeze-thaw cycles. The results in FIG. 18 again show that all antibodies tested were stable under the conditions tested, except for the 2 variants (HFB 3-1hz6-hG1AA and HFB3-1hz10-hG1 AA).

Similar experiments were repeated for HFB3-1hz6-hG1, HFB3-14hz1c-hG1 and HFB3-18hz1-hG 1. All three variants were generally stable under the three tests outlined above, except that HFB3-1hz6-hG1 and HFB3-18hz1-hG1 began to degrade after 14 days.

Taken together, the data indicate that these subject variant humanized anti-TNFR 2 monoclonal antibodies do not have major developability issues and are suitable for use as therapeutic antibodies.

Example 12 anti-TNFR 2 antibodies in humanized TNFR2 knock-in (KI) mice and their effects on T cells

To better demonstrate the therapeutic efficacy of the subject anti-TNFR 2 antibodies, humanized TNFR2 knock-in (KI) mice were generated by commercial service (Biocytogen, wakefield, MA) in the context of C57BL/6 mice.

In a first series of experiments, ex vivo binding between selected humanized anti-TNFR 2 antibodies and KI mouse CD 3T cells (TNFR 2 KI CD 3T cells) under co-stimulation of 1 μg/mL CD28 and 0.2 μg/mL or 1 μg/mL CD3 was analyzed. The results showed that 1 μg/mL KI mouse CD3 activated spleen cells better than 0.2 μg/mL CD3. Expression of human TNFR2 on KI CD3 ⁺ T cells can be detected and the expression/detection can be enhanced by TNFα and under mild (0.2 μg/mL) CD3 stimulation. In addition, a single dose of 200nM of each of the 6 anti-TNFR 2 antibodies (i.e., HFB3-1, HFB3-14, and HFB3-18, as well as humanized variants thereof, HFB3-1hz6, HFB3-14hz1c, and HFB3-18hz 1) did not show a discernable difference in TNFR2 binding, possibly due to saturated binding levels. The data are not shown.

The same ex vivo binding experiments were also repeated on CD 8T cells isolated from TNFR2 KI mice. Here, binding of anti-TNFR 2 monoclonal antibodies (chimeric and humanized versions thereof) to TNFR2 can be observed under strong CD3 (1 μg/mL) stimulation. At the same time, TNFα enhanced TNFR2 binding under mild CD3 (0.2 μg/mL) stimulation. The data are not shown.

The ability of the subject anti-TNFR 2 antibodies (chimeric and humanized) to co-stimulate downstream nfkb signaling in TNFR2 KI CD8 and CD4 Tconc cells ex vivo in the presence of TCR activation via CD3/CD28 and in the presence of tnfα was then examined.

Although the signaling response from hTNFR2 knock-in (KI) mouse T cells was less pronounced than that from human T cells, HFB3-1-hG1 and its humanized variant HFB3-1hz6-hG1 did induce a greater response than other antibodies (see FIG. 21). It should be noted that the lack of signal induction from the HFB3-18 series is expected.

The Pharmacokinetic (PK) profile of the subject humanized anti-TNFR 2 monoclonal antibodies (HFB 3-1hz6-hG1, HFB3-141C-hG1 and HFB3-18hz1-hG 1) in C57BL/6 mice was examined. All three humanized monoclonal antibodies exhibited T _1/2 consistent with the well-behaved antibody expectations. See below.

	T1/2	Period of elimination
			HFB3-1hz6-hG1	4.9 Days	6.1 Days to infinity
HFB3-141c-hG1	13.0 Days	5.7 Days to infinity
			HFB3-18hz1-hG1	10.6 Days	3.5 To 8.6 days

Example 13 Effect of humanized HFB3-1hz6-hG1 on Natural Killer (NK) cell ex vivo activation

This experiment shows that the subject humanized HFB3-1hz6-hG1 antibody costimulates Natural Killer (NK) cells in the presence of NK cell activation by IL-2/IL-15 or via CD3/CD 28.

In one experiment, NK cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) supplied from two human patients using NK cell isolation kit (Miltenyi Biotec). NK cells were first stimulated by soluble IL-2 (10 ng/mL) and IL-15 (10 ng/mL) for 24 hours and then treated with isotype control antibody, mouse HFB3-1-hG1, humanized HFB3-1-hz6-hG1, or anti-OX 40 control antibody (BMS) (22 nM, 66nM, or 200nM, respectively) for 16 hours. At the end of the experiment, CD107 a expression and TNFR2 expression on NK cell surface, representing NK cell degranulation and activation, were measured by FACS.

Both mouse HFB3-1-hG1 and humanized HFB3-1-hz6-hG1 significantly increased NK cell activation in a dose dependent manner. anti-OX 40 antibodies failed to promote NK short-term activation (40 hours since IL-2/IL-15 stimulation), which may be due to inadequate OX40 expression.

In another experiment, whole PBMC from two human patients were co-stimulated with plate-bound anti-CD 3 (1. Mu.g/mL) and soluble anti-CD 28 (1. Mu.g/mL) for 48 hours and then treated with isotype control antibody, mouse HFB3-1-hG1, humanized HFB3-1-hz6-hG1 or anti-OX 40 antibody (BMS) (22 nM, 66nM or 200nM, respectively) for 16 hours. CD107 a expression was determined against CD3 negative/CD 56 positive (i.e. NK cells). See fig. 23.

Similarly, HFB3-1-hG1 and HFB3-1-hz6-hG1 significantly increased CD 107. Alpha. Expression in a dose-dependent manner, indicating that these antibodies can promote NK cell activation in whole PBMC. Under prolonged activation (64 hours since anti-CD 3/CD28 stimulation), anti-OX 40 antibodies were able to activate NK cells.

These data indicate that both humanized HFB3-1-hz6-hG1 and the parental mouse HFB3-1-hG1 promote NK cell activation.

EXAMPLE 14 pharmacodynamics of humanized HFB3-1hz6-hG1 in the MC38 tumor model

The pharmacodynamics of HFB-1-hG1 was examined in humanized TNFR2-KI mice using MC38 colorectal carcinoma tumor model (see FIG. 24A). Briefly, about 5×105 MC38 tumor cells/mouse were inoculated into the right anterior flank of 8-week-old humanized TNFR2KI mice. After randomizing the mice and 7 days, mice (n=5 per group) were intraperitoneally injected with 10mg/kg, 1mg/kg, or 0.1mg/kg of HFB3-1-hG1 or 10mg/kg isotype control antibody on day 0. The same treatment was again administered at D3. On day 4, mice were euthanized and pharmacodynamic readings were performed on tumor and blood samples. FACS was used to sort tumor infiltrating leukocytes and peripheral leukocytes and to determine antibody occupancy of the receptor.

There was no significant difference in tumor weight between treatments after only 2 doses of treatment on day 0 and day 3 (upper left panel of fig. 24B). Administration of 10mg/kg of HFB3-1-hG1 increased the absolute number of CD45+ cells present in the tumor (lower left panel of FIG. 24B), but did not significantly increase the percentage of CD45+ in live tumor cells (lower right panel of FIG. 24B). Treatment with 10mg/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 alter the number of T regulatory cells (FIG. 24C). Administration of other lower doses of HFB3-1-hG1 did not produce any observable effect.

TNFR2 receptor occupancy by CD 8T cells, conventional CD 4T cells, T regulatory cells and NK cells in tumors and peripheral blood was determined. In tumors, only 10mg/kg of HFB3-1-hG1 produces drug receptor occupancy on T cells in the tumor; no occupancy was observed for the 1mg/kg and 0.1mg/kg doses (see FIG. 25A). However, receptor occupancy was observed in tumor NK cells at 1mg/kg and 10 mg/kg. HFB3-1-hG1 at doses of 10mg/kg and 1mg/kg produced comparable drug receptor occupancy in peripheral blood, and no significant occupancy was observed at the 0.1mg/kg dose.

The pharmacokinetics of HFB3-1-hG1 was determined at the end of the experiment. HFB3-1-hG1 administration was detectable in blood at 1mg/kg and 10mg/kg doses on day 4. It should be noted that the retention of HFB3-1-hG1 at the dose of 10mg/kg was much higher than the level of isotype control at the same dose (see FIG. 26A). Interestingly, administration of 10mg/kg and 1mg/kg HFB3-1-hG1 also increased the amount of TNFR2 detectable in the blood (see FIG. 26B). It is speculated that TNFR2 in blood is attributed to receptor shedding.

In summary, data on short-term treatment of mice with HFB3-1-hG1 highly demonstrate that HFB3-1-hG1 has the potential to stimulate activation and proliferation of immune cells, to bind efficiently to TNFR2 receptors on immune cells, and to be well retained in the blood.

Example 15 synergistic anti-tumor efficacy with anti-PD-1 antibodies

The anti-tumor efficacy of the subject humanized anti-TNFR 2 monoclonal antibodies was demonstrated in a widely used mouse colorectal cancer model in the context of humanized TNFR2 KI mice.

In particular, 8 week old humanized TNFR2 KI mice were vaccinated with about 5X 10 ⁵ MC38 tumor cells (which were derived from C57BL6 murine colon adenocarcinoma)/mouse. After about 7 days, on day 0, the average tumor size in the mice reached about 89mm ³ (between 74-98mm ³). Mice were then randomized into 5 experimental groups (n=8/group) to administer one of the following: (1) an isotype-matched control (TT-hG 1 AA); (2) anti-mPD-1 (RMP-1-14); (3) HFB3-1hz6-hG1; (4) HFB3-14hz1c-hG1; and (5) HFB3-18hz1-hG1. Antibodies were injected intraperitoneally (i.p.) at a dose of about 10mg/kg on days 0,3, 6, 9, 12, 15, and 18 for a total of 7 doses (Q3D, ×7). Tumor volumes of the experimental groups were measured during the course of the study. On day 21 or about day 21, the average tumor volume of the isotype control group reached >2000mm ³, and the experiment was terminated and all mice were sacrificed. Tumor volumes over time for each group are plotted in fig. 27A and 27B. By day 21, statistical significance of Tumor Growth Inhibition (TGI) was achieved in groups of mice receiving HFB3-1hz6, HFB3-18hz1 and anti-PD-1 (RMP-14) (FIG. 27B).

The results show that the humanized antibodies HFB3-1hz6 and-hG 1 and HFB3-18hz1-hG1 inhibit tumor growth as strongly (if not preferentially) as the anti-mPD-1 antibody, while the other humanized antibody is equally effective but to a lesser extent. No apparent weight differences were observed in the different experimental groups of mice.

Similar results were obtained in another experiment using only anti-mPD-1 and HFB3-1hz6-hG1 and isotype control (4 mice/group), Q3d 3 (once every three days, total three doses, intraperitoneal injection of 10 mg/kg). On day 6 (last dose of antibody), tumor volumes between isotype control group and anti-mPD-1 group and HFB3-1hz6-hG1 group were statistically significantly different (based on the 2-factor ANOVA test). See fig. 28.

In addition, HFB3-1hz6-hG1 and anti-PD-1 antibodies synergistically inhibited tumor growth and prolonged mice life in MC38 tumor models. Specifically, on day-7, humanized TNRF2 KI mice were vaccinated with MC38 cancer cells. Mice were intraperitoneally injected with isotype control, HFB3-1hz6-hG1, or anti-mPD-1 antibody (n=8/group) alone or in combination every 3 days from day 0. A total of 7 doses (Q3dX7) per 3 days and a total of 4 doses (Q3dX4) per 3 days of treatment with 3mg/kg and 10mg/kg HFB3-1hz6-hG1 (RMP-14) significantly inhibited tumor growth and prolonged mice life compared to isotype control treatment. In addition, combination therapy with HFB3-1hz6-hG1 (10 mg/kg, Q3 d.times.7) and anti-PD-1 antibody (10 mg/kg, Q3 d.times.4) resulted in survival superior to treatment with anti-PD-1 antibody alone. See fig. 29. Data were analyzed using ANOVA comparing treatment groups to isotype control.

EXAMPLE 16 antitumor efficacy of HFB3-1hz6-hG1 in a hepatoma isogenic mouse model

In the Hepa1-6 syngeneic mouse model, mice were treated with isotype control antibody, 10mg/kg of anti-mPD-1 or 0.3-10mg/kg of HFB3-1hz6-hG1 at a dose when the tumor volume reached about 100mm ³. HFB3-1hz6-hG1 can effectively inhibit tumor growth. HFB3-1hz6-hG1 even more effectively controlled tumor growth than anti-mPD-1 at the dose of 10mg/kg (see FIG. 32).

Example 17 toxicology assessment of anti-TNFR 2 antibodies in non-human primate

The toxicology of humanized anti-TNFR 2 antibodies was examined using a non-human primate model. Two cynomolgus monkeys/group were injected with single doses of 15mg/kg (low), 50mg/kg (medium) and 150mg/kg (high) of humanized HFB3-1hz6-hG1 monoclonal antibody, and plasma was collected at different time points up to 336 hours (day 14).

Toxicological kinetic analysis of HFB3-1hz6-hG1 showed that the antibody was eliminated over time. After injection of 15mg/kg, 50mg/kg or 150mg/kg HFB3-1hz6-hG1, no increase in cytokines IL-6, IL-2, IFN-gamma and TNF-alpha was observed compared to the reported data for CD3 XCD 20 bispecific IgG +.3 mg/kg (dashed line) (FIG. 30).

After injection of 15mg/kg, 50mg/kg or 150mg/kg HFB3-1hz6-hG1, no abnormalities in the numbers of leukocytes, erythrocytes, platelets, neutrophils and lymphocytes were found compared to the historical data range for normal monkeys (FIG. 31).

Toxicology assessment has not heretofore shown a discernible toxic effect from treatment of non-human primate subjects with HFB3-1hz6-hG1 in doses up to 150 mg/kg.

In a dose range discovery study (DRF) where multiple doses of HFB3-1hz6-hG1 were administered to cynomolgus monkeys, IL-2, IL-4, IL-5, TNF alpha and IFN gamma in the monkeys did not change upon repeated administration up to 150 mg/kg. At the end of the 4 week dose in monkeys, a change in IL-6 levels was observed in 10mg/kg and 150mg/kg male animals. Dose-dependent reduction of neutrophil and platelet counts was observed in monkeys 2 weeks after HFB3-1hz6-hG1 administration. Diarrhea (liquid or loose stool) was frequently observed in monkeys after weekly administration of HFB3-1hz6-hG 1.

Based on the above observations, the drug half-life following a single 1mg/kg dose injection in humans was predicted to be 23 days, and the antibody would be suitable for administration at 1mg/kg every four weeks.

Example 18 indication selection based on TNFR2 expression

While not wishing to be bound by any particular theory, it is believed that the anti-tumor efficacy of the anti-TNFR 2 antibodies of the present invention results from the stimulation of tumor infiltrating T cells and NK cells by TNFR2, thereby activating NK cells and enhancing cd8+ T cell mediated anti-tumor responses. This example provides the following evidence: types of tumors that would likely benefit from treatment with the anti-TNFR 2 antibodies of the invention include tumors that express high TNFR2 and high CD 8A.

In a large number of RNA analyses of cancers using the TCGA database, the CD8A cut-off is based on the CD8A level of Acute Myelogenous Leukemia (AML), which is presumed to consist mainly of bone marrow cells and low or no cd8+ T cells. The TNFR2 cutoff value is based on TNFR2 levels in prostate cancer, which is assumed to be immune deserts. See fig. 34A-34B.

The grading of the cancer types according to TNFR2/CD8A levels is shown in FIG. 35. Ebv+ gastric adenocarcinoma/gastric cancer, clear cell renal cell carcinoma, cutaneous melanoma, testicular germ cell tumor, soft tissue sarcoma, and PD-L1 high cancers (including cervical squamous cell carcinoma, pleural mesothelioma, lung adenocarcinoma, and head and neck squamous cell carcinoma) have been identified as top-grade high TNFR 2/high CD8A cancers.

Survival analysis of cancer patients using TCGA database showed that high TNFR2 expression was significantly correlated with better survival in cutaneous melanoma and head and neck squamous cell carcinoma (fig. 33A and 33B) and trend of better survival in lung adenocarcinoma (data not shown) at median gene expression cut-off. No significant trend was observed for cervical squamous cell carcinoma/endocervical adenocarcinoma, renal clear cell carcinoma, testicular germ cell tumor, sarcoma, gastric adenocarcinoma, and mesothelioma.

Using publicly available data, TNFR2 and CD8 scores for molecular subtypes of Renal Cell Carcinoma (RCC), cutaneous melanoma (SKCM), gastric adenocarcinoma/gastric carcinoma (STAD/GI), lung adenocarcinoma (LUAD), and head and neck squamous cell carcinoma (HNSC) were further determined (fig. 36).

It is apparent that in each cancer type tested, there are subtypes that have a high percentage of cancers within the subtype that exhibit a marked high CD8A and high TNFR2 expression pattern (e.g., about 60% of STAD/GI-ebv+ cancers have this marked high expression), while the other subtypes (e.g., ESCC subtype and HM-SNV subtype) have little CD8A ^hiTNFR2^hi expression.

Thus, the cancer subtypes tested with a high CD8A and high TNFR2 expression pattern that are indicative are the primary candidates for beneficial treatment by the subject antibodies of the present invention, including kirc.2, kirc.3, kirc.4, skcm, triple WT, skcm.braf_hotspot_mutant (possibly also skcm.ras_hotspot_mutant and skcm.nf1_any_mutant), luad.6 and luad.5, hnsc. Atypical (40% HPV positive) and hnsc. Interstitial (PD-L1/CD 274 tends to be higher).

In colorectal cancer patients, tumors with defective mismatch repair (dMMR)/high microsatellite instability (MSI-H) are significantly more sensitive to Immune Checkpoint Inhibitors (ICI) than those with microsatellite stability (MSS)/low microsatellite instability (MSI-L), and the former group of patients receive more immune benefit from immunotherapy than the latter group of patients.

Because MSI score data for all cancer indications is not directly available, applicants used parametric mutation counts as a surrogate for MSI scores and studied whether TNFR2 ^hi and CD8a ^hi tumors treatable by the subject antibody were enriched in MSI versus MSS. For this purpose, mutation count >250 is considered MSI, while mutation count <250 is considered MSS. The data (not shown here) show that TNFR2 ^hi and CD8a ^hi expression patterns are not strongly enriched in the COAD data (mutation count >250 vs <250 from The Cancer Genome Atlas (TCGA) -CRC colon adenocarcinoma (COAD) group). In MSI (all except 4% CD8a ^hi), the ratio of TNFR2 ^hi to TNFR2 ^lo is approximately equal. The ratio in MSS (all CD8A ^lo) became 45% versus 55%.

Example 19: drug administration study of HFB3-1hz6-hG1 in humans

The initial dose of HFB3-1hz6-hG1 in this study was 5mg administered every 4 weeks (Q4W) by intravenous infusion over 60 minutes, as informed in part by data from the minimum expected level of biological effects determined based on in vitro cytokine release, the minimum pharmacologically active dose in human TNFR2 knock-in (hTNFR 2 KI) mice bearing MC38 tumors, and the highest non-severe toxic dose in human and non-human primates.

The dose was escalated to 150mg to determine the maximum tolerated dose. Dose expansion was performed on ebv+ gastric cancer, clear cell renal cell carcinoma, cutaneous melanoma, soft tissue sarcoma, testicular germ cell tumor, and PD-l1+ cancers (including cervical cancer, pleural mesothelioma, lung adenocarcinoma, head and neck squamous cell carcinoma). Other patient groups were enrolled based on phase I antitumor activity/efficacy.

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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 wherein the monoclonal antibody comprises:

(1a) A Heavy Chain Variable Region (HCVR) comprising a HCVR CDR1 sequence of SEQ ID No.1, a HCVR CDR2 sequence of SEQ ID No. 2, and a HCVR CDR3 sequence of SEQ ID No. 3; and, a step of, in the first embodiment,

(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 (b)

(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, a step of, in the first embodiment,

(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 (b)

(3A) A Heavy Chain Variable Region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO. 26, the HCVR CDR2 sequence of SEQ ID NO. 27 and the HCVR CDR3 sequence of SEQ ID NO. 28; and, a step of, in the first embodiment,

(3B) A Light Chain Variable Region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO. 29, the LCVR CDR2 sequence of SEQ ID NO. 30 and the LCVR CDR3 sequence of SEQ ID NO. 31; or (b)

(4A) A Heavy Chain Variable Region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO:39, the HCVR CDR2 sequence of SEQ ID NO:40 and the HCVR CDR3 sequence of SEQ ID NO: 41; and, a step of, in the first embodiment,

(4B) A Light Chain Variable Region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO. 42, the LCVR CDR2 sequence of SEQ ID NO. 43 and the LCVR CDR3 sequence of SEQ ID NO. 44; or (b)

(5A) A Heavy Chain Variable Region (HCVR) comprising a HCVR CDR1 sequence of SEQ ID No. 51, a HCVR CDR2 sequence of SEQ ID No. 52, and a HCVR CDR3 sequence of SEQ ID No. 53; and, a step of, in the first embodiment,

(5B) A Light Chain Variable Region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO:54, the LCVR CDR2 sequence of SEQ ID NO:55 and the LCVR CDR3 sequence of SEQ ID NO: 56; or (b)

(6A) A Heavy Chain Variable Region (HCVR) comprising a HCVR CDR1 sequence of SEQ ID No. 63, a HCVR CDR2 sequence of SEQ ID No. 64, and a HCVR CDR3 sequence of SEQ ID No. 65; and, a step of, in the first embodiment,

(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.

2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein:

(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.

3. The isolated monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2, wherein 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.

4. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-3, which is a mouse antibody, a human-mouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a resurfaced antibody.

5. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antigen-binding fragment thereof is Fab, fab ', F (ab ') ₂、F_d, single chain Fv or scFv, disulfide linked F _v, V-NAR domain, igNar, intracellular antibody, igG Δch ₂, miniantibody, F (ab ') ₃, tetravalent antibody, trivalent antibody, diabody, single domain antibody, DVD-Ig, fcab, mAb ₂、(scFv)₂, or scFv-Fc.

6. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the monoclonal antibody or antigen-binding fragment thereof cross-reacts with rhesus TNFR2 but does not substantially cross-react with mouse TNFR 2.

7. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the monoclonal antibody or antigen-binding fragment thereof does not substantially cross-react with TNFR 1.

8. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the monoclonal antibody or antigen-binding fragment thereof binds tnfa with a K _d of less than about 25nM, 20nM, 15nM, 10nM, 5nM, 2nM, or 1 nM.

9. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-8, which enhances binding between tnfa and TNFR 2; enhancing tnfa-mediated or co-stimulated nfkb signaling (e.g., in TCR-activated CD8 and/or CD4 Tconv T cells); and/or proliferation of effector T cells (e.g., CD8 and/or CD4 Tconv T cells) that promote TCR activation in the presence of tregs.

10. The isolated monoclonal antibody or antigen binding fragment thereof of any one of claims 1-9, which enhances tnfa-mediated CD25 expression on tregs.

11. The isolated monoclonal antibody or antigen binding fragment thereof of any one of claims 1-10, which binds to an epitope of SEQ ID No. 13 and/or 101.

12. An isolated monoclonal antibody or antigen binding fragment thereof which competes with the isolated monoclonal antibody or antigen binding fragment thereof of any one of claims 1-11 for 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 said epitope of SEQ ID No. 13 and/or 101.

14. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 12 or 13, which enhances binding between tnfa and TNFR 2; enhancing tnfa-mediated or co-stimulated nfkb signaling (e.g., in TCR-activated CD8 and/or CD4 Tconv T cells); and/or proliferation of effector T cells (e.g., CD8 and/or CD4 Tconv T cells) that promote TCR activation in the presence of tregs.

15. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-8, which inhibits binding between tnfa and TNFR 2; inhibition of tnfa-mediated or co-stimulated nfkb signaling (e.g., in TCR-activated CD8 and/or CD4Tconv T cells); and/or inhibit proliferation of TCR-activated effector T cells (e.g., CD8 and/or CD4Tconv T cells) in the presence of tregs.

16. The isolated monoclonal antibody or antigen binding fragment thereof of any one of claims 1-8, which promotes Treg expansion.

17. The isolated monoclonal antibody or antigen binding fragment thereof of any one of claims 1-8, which promotes natural killer cell activation.

18. An isolated monoclonal antibody or antigen binding fragment thereof that competes for binding to the same epitope as the isolated monoclonal antibody or antigen binding fragment thereof of any one of claims 1-8 and 15-16.

19. An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof specifically binds human TNFR2 at an epitope comprising, consisting essentially of, or consisting of SEQ ID No. 101, optionally, the isolated monoclonal antibody or antigen-binding fragment thereof does not bind human TNFR2 at an epitope consisting essentially of or consisting of SEQ ID No. 13.

20. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 19, which (1) promotes activation and proliferation of CD4 ⁺ T cells in tumor-infiltrating lymphocytes (TILs) other than regulatory T cells (tregs) (e.g., in an in vivo hTNFR2 knock-in MC38 mouse tumor model); and/or (2) promote NK cell activation in vitro and/or in vivo.

21. The isolated monoclonal antibody or antigen binding fragment thereof of any one of claims 1-20, having a Maximum Tolerated Dose (MTD) in cynomolgus monkeys of about 150 mg/kg.

22. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-21, wherein the patient (e.g., the cancer of the patient) has:

(a) Higher levels of TNFR2 expression compared to the average TNFR2 expression levels in prostate cancer patients; optionally, the 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

(B) A higher level of CD8A expression compared to the average CD8A expression level in AML patients.

23. The method of claim 22, wherein the patient (e.g., the cancer of the patient) has the higher expression level of TNFR2 in tumor infiltrating CD8a ⁺ (CD 8a chain positive) T cells.

24. A method according to claim 22 or 23 wherein the patient has EBV ⁺ gastric cancer (e.g. gastric adenocarcinoma), clear cell renal cell carcinoma, renal clear cell carcinoma, cutaneous melanoma (e.g. cutaneous melanoma), testicular germ cell tumor or soft tissue sarcoma.

25. The method of claim 22 or 23, wherein the cancer expresses "high" levels of PD-L1.

26. The method of claim 25, wherein 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.

27. The method of any one of claims 22-26, further comprising administering to the patient:

(a) Antibodies or antigen binding fragments thereof specific for PD-1, such as cimetidine Li Shan antibody, nivolumab, pembrolizumab, spabulab, carlizumab, singdi Li Shan antibody, tirelimumab, terlipne Li Shan antibody, ritalimumab, and INCMGA00012;

(b) Antibodies or antigen-binding fragments thereof specific for PD-L1, such as Avermectin, dewaruzumab, abstrauzumab, KN035 or CK-301, and/or

(C) An antibody or antigen-binding fragment thereof specific for PD-L2.

28. The method of any one of claims 22-27, wherein the patient has recurrent or refractory cancer, and/or has been previously treated with (and optionally failed to respond to or recur from) standard-of-care therapy.

29. The method of any one of claims 22-28, comprising administering the effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof to the patient once every 3 weeks (Q3W), once every 4 weeks (Q4W), or once every 5 weeks (Q5W) (e.g., once every 4 weeks or Q4W).

30. The method of claim 29, comprising administering the isolated monoclonal antibody or antigen-binding fragment thereof to the patient at a dose of about 5mg, 15mg, 50mg, 100mg, or 150mg once every 4 weeks (Q4W) (e.g., intravenous administration over 60 minutes).

31. The method of any one of claims 22-30, the method further comprising:

(1) Selecting a patient having said higher TNFR2 expression level and CD8A expression level prior to said administering step; or (b)

(2) Prior to the administering step, the patient is validated for the higher TNFR2 expression level and CD8A expression level.

32. 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 that specifically binds to human TNFR2 at an epitope comprising, consisting essentially of, or consisting of SEQ ID No. 101, optionally, the isolated monoclonal antibody or antigen-binding fragment thereof does not bind to human TNFR2 at an epitope consisting essentially of or consisting of SEQ ID No. 13.

33. A method of treating cancer or an autoimmune disorder in a patient in need thereof, the method comprising administering to the patient an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-21.

34. The method of claim 33, for treating cancer, wherein the method further comprises administering an antagonist of an immune checkpoint.

35. The method of claim 34, wherein the immune checkpoint is a PD-1/PD-L1 immune checkpoint.

36. The method of claim 34 or 35, wherein the antagonist of the immune checkpoint is an antibody or antigen-binding fragment thereof specific for PD-1 or PD-L1.

37. The method of claim 36, wherein the antibody is an anti-PD-1 antibody, such as a cimetidine Li Shan antibody, a nivolumab, a pembrolizumab, a spabulab, a carlizumab, a singdi Li Shan antibody, a tirelimumab, a terlipressin Li Shan antibody, a rituximab, and INCMGA00012.

38. The method of claim 37, wherein the antibody is an anti-PD-L1 antibody, such as avermectin, dewaruzumab, alemtuzumab, KN035, or CK-301.

39. The method of claim 34 or 35, wherein the antagonist of the immune checkpoint is a (non-antibody) peptide inhibitor of PD-1/PD-L1, such as AUNP; small molecule inhibitors of PD-L1, such as CA-170, or macrocyclic peptides, such as BMS-986189.

40. The method of any one of claims 34-39, wherein the cancer is melanoma, breast cancer, colon cancer, cervical cancer, kidney 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.

41. The method of any one of claims 22-40, further comprising administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, a tumor immunizing agent, and/or an anti-tumor composition.

42. A polynucleotide encoding the heavy or light chain or antigen binding portion thereof of any one of claims 1-21.

43. The polynucleotide of claim 42, wherein said polynucleotide is codon optimized for expression in a human cell.

44. A vector comprising the polynucleotide of claim 42 or 43.

45. The vector of claim 44, which is an expression vector (e.g., mammalian, yeast, insect, or bacterial expression vector).