TW202309096A - Anti-tnfr2 antibody and uses thereof - Google Patents

Abstract

The invention provides monoclonal antibodies and antigen-binding fragments thereof specific for TNFR2, and methods of using the same to treat cancer or autoimmune disorder, including combination therapy with antagonists of the PD-1/PD-L1 immune checkpoint.

Description

Anti-TNFR2 antibodies and uses thereof

References to Related Applications

This application claims the benefit of the filing date of U.S. Provisional Patent Application Serial No. 63/219,175, filed July 7, 2021, the entire contents of which U.S. Provisional Patent Application, including any drawings and sequence listing, are reproduced by Incorporated herein by reference. References to Sequence Listings

This application contains a Sequence Listing, which was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Created on June 28, 2022, this ASCII copy is named 131206-01120_SL.txt and is 95,799 bytes in size.

The present invention relates to anti-TNFR2 antibodies and uses thereof.

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

TNFR2 binds to TNFα ligands in a 3:3 trimerization pattern. The co-crystal structure of TNFR2 and TNFα has been solved and it has been shown that each TNFR2 molecule binds to two TNFα ligands. In addition, TNFα binds TNFR2 with a _Kd of 420 pM, about 20-fold weaker than its binding to TNFR1 ( _Kd = 19 nM). In general, TNF[alpha] binds preferentially to TNFR1, other things being equal.

In normal T cells, TNFα-TNFR2 interaction triggers cell survival signals via the NFkB signaling pathway. However, in autoimmune T cells, TNFα-TNFR2 interaction triggers apoptotic signaling via the caspase pathway.

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

Whereas TNFR1 is widely expressed, TNFR2 expression is largely restricted to immune cells and is predominantly overexpressed by tumor-infiltrating immunosuppressive CD4 ⁺ FoxP3 ⁺ regulatory T cells (Treg). Recent studies have shown that TNFR2 plays a key role in stimulating the activation and proliferation of Treg, a major checkpoint of the anti-tumor immune response (Chen and Oppenheim, Sci Signal 10:eaal2328, 2017). Activation of TNFR2 via its ligand TNFa leads to activation of NFkB signaling and expansion of TNFR2 ⁺ Treg. TNFR2 is also expressed in CD8 and CD4 Tconv cells and myeloid cells. Specifically, TNFR2 is expressed in exhausted CD8 T cells, similar to a clinically validated immune checkpoint.

T regulatory cells (Treg) are a small subset of T lymphocytes with different clinical applications. On the one hand, TNFR2 ⁺ Treg is highly immunosuppressive, and its inhibitory activity is stronger than that of highly suppressive CD103 ⁺ Treg ( J Immunol 179:154-161, 2007; J Immunol 180:6467-6471, 2008 ). Thus, TNFR2 ⁺ Tregs can be used in therapies that rely on the immunosuppressive activity of Tregs, for example in transplantation, allergy, asthma, infectious disease, graft versus host disease (GVHD) and autoimmunity. For example, depletion of CD4 ^{+ CD25high} Foxp3 ⁺ thymus-derived Tregs enhanced GVHD in an experimental GVHD mouse model (Cohen et al ., JEM 2002).

TNFR2 is also expressed in certain cancers, such as breast cancer, cervical cancer, colon cancer, and kidney cancer ( Front. Immunol. 9:1170, 2018), and may be involved in immune tolerance in these cancers. The ligand for TNFR2 (TNF[alpha]) promotes the survival and growth of these cancer cells. TNFR2 has been shown to participate in multiple tumor development processes by employing different signaling pathways in tumor cells. For example, nuclear factor-κB (NFκB) is involved in malignant transformation of TNFR2-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 the above-mentioned TNFR2 function. Therefore, in tumor immunity, inhibition of TNFR2 function suppresses Treg function and increases antitumor T cell responses.

Therefore, there is a need to develop therapeutic agents that allow the treatment of autoimmune disorders by enhancing the immunosuppressive function of Tregs by stimulating TNFR2 function on TNFR2 ⁺ Tregs, or inhibiting TNFR2 activation to treat diseases such as cancer.

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), which comprises the HCVR CDR1 sequence of SEQ ID NO: 1, the HCVR CDR2 sequence of SEQ ID NO: 2, and the HCVR CDR3 sequence of SEQ ID NO: 3; and (1b) light chain variable region (LCVR ), which comprises the LCVR CDR1 sequence of SEQ ID NO: 4, the LCVR CDR2 sequence of SEQ ID NO: 5, and the LCVR CDR3 sequence of SEQ ID NO: 6; or (2a) a heavy chain variable region (HCVR), which comprises SEQ ID NO: HCVR CDR1 sequence of ID NO: 14, HCVR CDR2 sequence of SEQ ID NO: 15 and HCVR CDR3 sequence of SEQ ID NO: 16; and (2b) light chain variable region (LCVR), which comprises SEQ ID NO: 17 LCVR CDR1 sequence, LCVR CDR2 sequence of SEQ ID NO: 18 and LCVR CDR3 sequence of SEQ ID NO: 19; or (3a) heavy chain variable region (HCVR), which comprises the HCVR CDR1 sequence of SEQ ID NO: 26, SEQ ID NO: The HCVR CDR2 sequence of ID NO: 27 and the HCVR CDR3 sequence of SEQ ID NO: 28; and (3b) the light chain variable region (LCVR), which comprises the LCVR CDR1 sequence of SEQ ID NO: 29, the LCVR CDR2 sequence and the LCVR CDR3 sequence of SEQ ID NO: 31; or (4a) heavy chain variable region (HCVR), it comprises the HCVR CDR1 sequence of SEQ ID NO: 39, the HCVR CDR2 sequence of SEQ ID NO: 40 and SEQ ID NO: The HCVR CDR3 sequence of ID NO: 41; and (4b) the light chain variable region (LCVR), which comprises the LCVR CDR1 sequence of SEQ ID NO: 42, the LCVR CDR2 sequence of SEQ ID NO: 43 and the LCVR CDR2 sequence of SEQ ID NO: 44 LCVR CDR3 sequence; or (5a) heavy chain variable region (HCVR), it comprises the HCVR CDR1 sequence of SEQ ID NO:51, the HCVR CDR2 sequence of SEQ ID NO:52 and the HCVR CDR3 sequence of SEQ ID NO:53; And (5b) light chain variable region (LCVR), it comprises 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 (6a) heavy chain A variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 63, the HCVR CDR2 sequence of SEQ ID NO: 64, and the HCVR CDR3 sequence of SEQ ID NO: 65; and (6b) the light chain variable region (LCVR ), which comprises 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 the isolated monoclonal antibody or antigen-binding fragment thereof, (1A) the HCVR sequence is SEQ ID NO: 7; and/or (1B) the LCVR sequence is SEQ ID NO: 8, or ( 2A) HCVR sequence is SEQ ID NO: 20; and/or (2B) LCVR sequence is SEQ ID NO: 21, or (3A) HCVR sequence is SEQ ID NO: 32; and/or (3B) LCVR sequence is SEQ ID NO: 33, or (4A) HCVR sequence is SEQ ID NO: 45; and/or (4B) LCVR sequence is SEQ ID NO: 46, or (5A) HCVR sequence is SEQ ID NO: 57; and/or (5B) ) LCVR sequence is SEQ ID NO: 58, or (6A) HCVR sequence is SEQ ID NO: 69; and/or (6B) LCVR sequence is SEQ ID NO: 70.

In certain embodiments, the monoclonal antibody has: (1a) the heavy chain sequence of SEQ ID NO: 9; and/or (1b) the light chain sequence of SEQ ID NO: 10, or (2a) 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.

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 Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, 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 monkey TNFR2 but does not substantially cross-react with mouse TNFR2.

In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention includes one or more point mutations in its amino acid sequence, and the one or more point mutations are designed to improve the developability of the antibody. For example, in certain embodiments, one or more point mutations render the antibody during its expression in a host cell, during its purification in manufacture and/or during its formation process and/or during its administration to an individual patient more stable. In certain embodiments, one or more point mutations render 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 issues (e.g., removed or reduced hydrophobicity and/or optimized charge) by substituting ( For example, one or more amino acids in one or more CDRs thereof).

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

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

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof enhances the binding between TNFα and TNFR2; enhances TNFα-mediated or costimulatory NFκB signaling (e.g., in TCR-activated CD8 and/or CD4 Tconv T cells); and/or promote the proliferation of TCR-activated effector T cells (such as CD8 and/or CD4 Tconv T cells) in the presence of Treg.

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

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

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

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

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is an agonist of TNFR2, or stimulates TNFR2 signaling (e.g., 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.

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

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

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof enhances the binding between TNFα and TNFR2; enhances TNFα-mediated or costimulatory NFκB signaling (e.g., in TCR-activated CD8 and/or CD4 Tconv T cells); and/or promote the proliferation of TCR-activated effector T cells (such as CD8 and/or CD4 Tconv T cells) in the presence of Treg.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof inhibits the binding between TNFα and TNFR2; inhibits TNFα-mediated or costimulatory NFκB signaling (e.g., in TCR-activated CD8 and/or CD4 Tconv T cells); and/or inhibit the proliferation of TCR-activated effector T cells (such as CD8 and/or CD4 Tconv T cells) in the presence of Treg.

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

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

Another aspect of the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody or antigen-binding fragment thereof is determined at an antigen comprising, consisting essentially of, or consisting of SEQ ID NO: 101 Where the isolated monoclonal antibody or antigen-binding fragment thereof specifically binds human TNFR2, 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 according to claim 19, (1) promotes the activation of CD4 ⁺ T cells in tumor infiltrating lymphocytes (TIL) instead of regulatory T cells (Treg) and proliferation ( for example, 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, an isolated monoclonal antibody or antigen-binding fragment thereof of the invention has a maximum tolerated dose (MTD) of about 150 mg/kg in cynomolgus monkeys.

Another aspect of the present invention provides 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 the present invention, wherein the patient (e.g., the patient's cancer) Having: (a) a higher level of TNFR2 expression compared to the mean level of TNFR2 expression in prostate cancer patients; optionally, the expression of TNFR2 is on effector T cells ( eg, CD4 ⁺ and/or CD8 ⁺ T cells), tumor-infiltrating CD8 ⁺ assessed in T cells and/or NK cells; and (b) higher CD8A expression levels compared to mean CD8A expression levels in AML patients.

In certain embodiments, the patient (eg, patient with cancer) has the higher expression level of TNFR2 in tumor infiltrating CD8A ⁺ (CD8α chain positive) T cells.

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

In certain embodiments, the cancer expresses higher than average levels of PD-L1.

In certain embodiments, the cancer is cervical cancer (e.g., cervical squamous cell carcinoma or endocervical adenocarcinoma), pleural mesothelioma, lung adenocarcinoma, or head and neck squamous cell carcinoma (HNSC, e.g., atypical sub- type (about 40% of which are HPV positive) and mesenchymal subtype (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 cemiplimab (cemiplimab), nivolumab ( Nivolumab, pembrolizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, teslelizumab Toripalimab, dostarlimab and INCMGA00012; (b) Antibodies specific for PD-L1 or antigen-binding fragments thereof, such as avelumab, Deva Durvalumab, atezolizumab, KN035 or CK-301, and/or (c) an antibody specific for PD-L2 or an antigen-binding fragment thereof.

In certain embodiments, the patient has relapsed or refractory cancer, and/or has been previously treated with (and optionally failed to respond to or relapsed 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.

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

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

Another aspect of the present 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, the isolated monoclonal antibody or antigen-binding fragment thereof The binding fragment 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 is not substantially An epitope consisting of or consisting of SEQ ID NO: 13 binds human TNFR2.

Another aspect of the present invention provides a method of treating cancer or autoimmune disorders (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 An amount of an isolated monoclonal antibody or antigen-binding fragment thereof of the invention.

In certain embodiments, the method is used to treat AID, wherein the method further comprises administering a second agent, such as a low dose of an anti-IL2 agent to treat chronic GVHD, or an anti-TNFα agent (such as adalimumab , infliximab (infliximab), etenercept (etenercept), golimumab (golimumab, etc.) for the treatment of rheumatoid arthritis, chronic plaque psoriasis, Crohn's disease, joint Adhesive spondylitis, psoriatic arthritis, polyarticular juvenile idiopathic arthritis, 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 the immune checkpoint is an antibody or antigen-binding fragment thereof specific for PD-1 or PD-L1.

In certain embodiments, the antibody is an anti-PD-1 antibody, such as simiprizumab, nivolumab, pembrolizumab, sparizumab, camrelizumab, sintilizumab monoclonal antibody, tislelizumab, toripalimab, dostalimumab and INCMGA00012.

In certain embodiments, the antibody is an anti-PD-L1 antibody, such as avelumab, durvalumab, atezolizumab, KN035 or CK-301.

In certain embodiments, the antagonist of the immune checkpoint is a (non-antibody) peptide inhibitor of PD-1/PD-L1, such as AUNP12; a small molecule inhibitor of PD-L1, such as CA-170, or a 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, an immuno-tumor agent, and/or an anti-neoplastic composition.

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

In certain embodiments, polynucleotides are codon-optimized for expression in human cells.

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

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

1. overview

TNFR2 has recently emerged as a promising therapeutic target in tumor immunology. TNFR2 expression on regulatory and effector T cells in the tumor microenvironment (TME) correlates with T cell exhaustion and resistance to immune checkpoint blockade. The invention described herein provides antibodies against human TNFR2, which are useful as anticancer agents. While not wishing to be bound by any particular theory, it is believed that co-stimulation of effector T cells with a target anti-TNFR2 antibody enhances the anti-tumor activity of effector T cells.

According to the invention described herein, mice were immunized with the recombinant extracellular domain (ECD) of human TNFR2 (rhTNFR2) to generate a series of different antibodies 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 to the monkey xenolog of rhTNFR2, a feature that would be beneficial in toxicity studies of human therapeutics in animals. Other desirable features include the ability of the subject antibodies to enhance the binding of human recombinant TNFa to TNFR2.

Two mouse antibodies, HFB3-1 and HFB3-14, with sub- or single-digit nanomolar binding affinities for human TNFR2 were initially selected for further characterization and humanization. Epitope mapping experiments showed that the two antibodies recognized different domains of TNFR2, with HFB3-1 binding to a region within the CRD2 domain and HFB3-14 binding within the CRD3 region. However, despite their different binding sites, both antibodies were selective for TNFR2, cross-reactive with cynomolgus and rhesus monkey xenologs, and enhanced binding of human recombinant TNFα to TNFR2, as well as stimulation of CD8 and conventional CD4 T cells (Tconv).

Several humanized variants of these mouse antibodies, including HFB3-1hz6 and HFB3-14hz1c, retained the binding and cross-reactivity characteristics of their respective parental antibodies. Humanized antibodies preferentially bind to TCR-activated primary CD8 and CD4 T cells compared to unstimulated T cells, and enhance CD3/CD28-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 NFκB signaling and induce upregulation of NFκB downstream target genes.

In addition, two humanized antibodies (HFB3-1hz6 and HFB3-14hz1c) displayed good developability characteristics and were stable under high temperature, low pH conditions and after several freeze/thaw cycles. Good plasma exposure of the lead antibody was also observed in the mouse model. In vivo efficacy evaluations of these antibodies in mouse tumor models as well as initial toxicity analysis were performed.

A third mouse monoclonal antibody, HFB3-18, was also identified which had a slightly lower (double-digit nM) binding affinity than the anti-mPD-1 monoclonal antibody but had the same ability to inhibit tumor growth in vivo (if not preferably), and produce its humanized form.

The functional characteristics of these antibodies as well as their favorable developability and pharmacokinetic characteristics support their development as a potential novel immunotherapy option for cancer patients, especially in certain cancer types and subtypes that exhibit high expression of TNFR2 and CD8A.

Detailed aspects of the invention are further and individually set forth in each section below. It should be understood, however, that any embodiment of the invention, including embodiments set forth only in examples or figures and embodiments set forth only in one section below, can be combined with any other embodiment of the invention. 2. Definition

The term "antibody" in its broadest sense encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies ( eg, bispecific antibodies). The term "antibody" can also broadly refer to a molecule comprising complementarity determining region (CDR) 1, CDR2 and CDR3 of the heavy chain and CDR1, CDR2 and CDR3 of the 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 various species (eg, mouse, human, cynomolgus monkey , etc. ).

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

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

A single chain Fv (scFv) as used herein 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 both a heavy chain and a light chain. In some of these embodiments, the heavy chain is a region of the antibody comprising three heavy chain CDRs and the light chain is a region of the antibody comprising three light chain CDRs.

As used herein, the term "heavy chain variable region (HCVR)" refers to a region comprising at least heavy chain CDR1 (CDR-H1), framework 2 (HFR2), CDR2 (CDR-H2), FR3 (HFR3) and CDR3 (CDR- H3) area. In some embodiments, the heavy chain variable region also comprises at least a portion ( e.g. , all) of FR1 (HFR1), which is N-terminal to CDR-H1, and/or at least a portion ( e.g., all) of FR4 (HFR4), which It 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 CH3. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an anti-IgG antibody comprising a gamma constant region, an anti-IgD antibody comprising a delta constant region, an anti-IgA antibody comprising an alpha constant region, an anti-IgE antibody comprising an epsilon constant region, and an anti-IgE antibody comprising a mu constant region Antibody system IgM antibody.

Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising the γ1 constant region), IgG2 (comprising the γ2 constant region), IgG3 (comprising the γ3 constant region), and IgG4 (comprising the γ4 constant region) antibodies; IgA antibodies include (but are not limited to) IgA1 (comprising the α1 constant region) and IgA2 (comprising the α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 (comprising the μ1 constant region) and IgM2 (comprising the 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 light chain CDR1 (CDR-L1), framework (FR) 2 (LFR2), CDR2 (CDR-L2), FR3 (LFR3) and CDR3 ( CDR-L3) region. In some embodiments, the light chain variable region also comprises at least a portion ( eg, all) of FR1 (LFR1 ) and/or at least a portion ( eg, all) of FR4 (LFR4).

The term "light chain constant region" as used herein refers to the region comprising the light chain constant domain _CL . 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 the 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 antigen, such as Fv, single chain Fv (scFv), Fab, Fab' and (Fab') ₂ . In certain embodiments, antibody fragments include Fab, Fab', F(ab') ₂ , _Fd , single chain Fv or scFv, disulfide-linked _Fv , V-NAR domains, IgNar, endobodies, _IgGΔCH2 , minibody, F(ab') ₃ , tetravalent antibody, trivalent antibody, diabody, single domain antibody, DVD-Ig, Fcab, _mAb2 , (scFv) ₂ or scFv-Fc.

The term "Fab" refers to an antibody fragment having a molecular mass of about 50,000 Daltons and having the activity of binding to an antigen. It comprises about half of the N-terminal side of the heavy chain and the entire light chain linked by a disulfide bridge. Fab can be obtained specifically by treating immunoglobulin with protease papain.

The term "F(ab') ₂ " denotes a fragment having about 100,000 daltons and the activity of binding to an antigen. This fragment is slightly larger than the two Fab fragments linked by 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 cleaving the disulfide bridges in the hinge region.

A single Fv chain "scFv" corresponds to a VH:VL polypeptide synthesized using genes encoding the VL and VH domains and a sequence encoding a peptide intended to bind these domains. The scFvs of the invention include CDRs maintained in proper conformation, eg, using genetic recombination techniques.

A dimer of "scFv" corresponds to two scFv molecules linked together by a peptide bond. This Fv chain is usually derived from the expression of a fusion gene comprising genes encoding VH and VL linked by a peptide-encoding linker sequence. Human scFv fragments may include CDR regions maintained in proper conformation, preferably by use of genetic recombination techniques.

A "dsFv" fragment is a VH-VL heterodimer stabilized by a disulfide bridge; it may be bivalent ( _dsFV2 ). Fragments of bivalent Sc(Fv) ₂ or multivalent antibodies can be formed spontaneously by association of monovalent scFv or by linking scFv fragments through peptide binding sequences.

The Fc fragment is underpinning the biological properties of the antibody, especially 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 with two antigen fixation sites. These fragments comprise a variable heavy domain VH linked to a variable light domain VL in the same VH-VL polypeptide chain. Using a binding sequence that is too short to match the two domains of the same chain, necessarily matches the two complementary domains of the other chain and thus creates two antigen fixation sites.

"Antibodies that bind to the same epitope as a reference antibody" can be determined by antibody competition assays. It refers to an antibody that blocks 50% or more of the binding of a reference antibody to its antigen in a competition assay, whereas the reference antibody blocks 50% or more of the binding of an 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 assays in which the tested antibodies prevent or inhibit specific binding of a reference antibody to a common antigen.

Various types of competitive binding assays can 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; solid phase direct labeling assay Sandwich assay; (see , e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct labeling of RIA using an ^I labeled solid phase (see , e.g., Morel et al ., 1988, Molec. Immunol. 25:7 -15); solid phase direct biotin-avidin EIA (see e.g. Cheung et al. , 1990, Virology 176:546-552); and directly labeled RIA (Moldenhauer et al ., 1990, Scand. J. Immunol. ).

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

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

The term "epitope" refers to that 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 specifically binding to an antibody. Epitopes may be contiguous or non-contiguous ( eg, in a polypeptide, amino acid residues that are not contiguous to 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 mimetic in that it comprises a three-dimensional structure similar to, but not or only included in, the epitope used to produce an antibody Some of the amino acid residues found. Epitopic determinants may include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphonyl or sulfonyl groups, and may have specific three-dimensional structural characteristics and/or specific charge characteristics.

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

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

"Chimeric antibody" as used herein refers to an antibody comprising at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey , etc. ) and at least one variable region from a second species (e.g., human, cynomolgus monkey, etc.) , chicken , etc. ) constant region. In some embodiments, chimeric antibodies comprise 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.

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

A "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 antibodies produced in humans, antibodies produced in non-human animals (such as ^XenoMouse® ) that contain human immunoglobulin genes, and antibodies selected using in vitro methods such as phage display , where the antibody repertoire is based on human immunoglobulin sequences.

A "host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, ^PER.C6® cells (Crucell), and 293 and CHO cells and 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 normally found in nature, or from at least some of the components from which it is normally derived. For example, a polypeptide is said to be "isolated" when it is separated from at least some of the cellular components from which it is produced. When a polypeptide is secreted by a cell after expression, a polypeptide is considered to be "isolated" when the supernatant containing the polypeptide is physically separated from the cell in which it was produced. Similarly, when a polynucleotide is not part of or arises from at least some of the larger polynucleotides normally found in nature (e.g., genomic DNA or mitochondrial DNA in the case of DNA polynucleotides) When cellular components are isolated ( eg, in the case of RNA polynucleotides), the polynucleotide is said to be "isolated." Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated" as long as the polynucleotide is not found in the vector in nature.

The terms "individual" and "patient" are used interchangeably herein and refer to mammals, such as humans. In some embodiments, methods of treating other non-human mammals are also provided, including but not limited to rodents, simians, cats, dogs, horses, cows, pigs, sheep, goats, Mammalian laboratory animals, mammalian farm animals, mammalian playground animals, and mammalian pets. In some instances, an "individual" or "patient" refers to a (human) individual or patient in need of treatment for a disease or condition.

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

"Tissue or cell sample" means a collection of similar cells obtained from the tissue of an individual or patient. The source of tissue or cell samples may be solid tissue such as from fresh, frozen and/or preserved organ or tissue samples or biopsies or aspirates; blood or any blood components; body fluids such as sputum, cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from any time during pregnancy or development of an individual. Tissue samples can also be primary or cultured cells or cell lines. Optionally, tissue or cell samples are obtained from diseased tissues/organs. A tissue sample may contain compounds not normally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

A "reference sample", "reference cell" or "reference tissue" as used herein refers to a sample, cell or tissue obtained from a source known or believed not to be suffering from the disease or disorder identified using the methods or compositions of the invention. In one embodiment, a reference sample, reference cell or reference tissue is obtained from a healthy part of the body of the same individual or patient from whom a composition or method of the invention is used to identify a disease or disorder. In one embodiment, a reference sample, reference cell or reference tissue is obtained from a healthy part of the body of at least one individual who is not the individual or patient for whom a disease or disorder is identified using a composition or method of the invention. In some embodiments, a reference sample, reference cell or reference tissue was previously obtained from a patient prior to or early in a disease or disorder.

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

"Disease associated with modulation of suppressive activity of T lymphocytes" means any disease (non-autoimmune) in which modulation of suppressive activity of T lymphocytes works, in particular by promoting the development or persistence of the disease. In particular, modulation of the suppressive activity of T lymphocytes has been shown to promote tumor development. Thus, the present invention is more particularly aimed at cancers in which inhibition of the 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 can be benign (also known as benign tumors), precancerous, or malignant. Cancer cells may be solid cancer cells ( ie, form a solid tumor) or leukemia cancer cells. The term "cancer growth" is used herein to refer to the proliferation or growth of one or more cells comprising a cancer with a corresponding increase in the size or extent of the cancer.

Examples of cancer 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 carcinoma, esophageal carcinoma, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, squamous lung carcinoma, peritoneal carcinoma, liver Cell 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, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, biliary tract cancer, gallbladder cancer, gastric cancer, melanoma and various types of head and neck cancer.

In certain embodiments, cancer as used herein includes blood 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, kidney cancer, ovarian cancer, liver cancer and prostate cancer).

A "chemotherapeutic agent" is a chemical compound that is useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and ^Cytoxan® (cyclophosphamide); alkyl sulfonates such as busulfan, proceton improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturdopa and uredopa; imine and methylmelamine, including hexamethylmelamine (altretamine), triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; Polyacetylenes (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycin (especially candocin 1 and candocin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; water pancratistatin; sarcodictyn; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, Estramustine, ifosfamide, methyldichloroethylamine, oxambucil hydrochloride, melphalan, novembichin, cholesteryl phenylacetate mustard ( phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, formustine (fotemustine), lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics ( such as calicheamicin, especially calicheamicin γ11 and calicheamicin ω11 (see , e.g., Agnew, Chem lntl. Ed. Engl, 33: 183-186 (1994)); dynemicins (dynemicin), including dynatomycin A; bisphosphonates, such as chloride Phosphonic acid; esperamicin; and neocarzinostatin chromophore and related chromoproteins (enediyne antibiotic chromophore), aclacinomysin, actinomycin , athramycin, azoserine, bleomycin, actinomycin C, carabicin, caminomycin, carzinophilin, chromophore Chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin ^{®doxorubicin} (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin) , pan-epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodomycin Rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin ); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, trimetrexate ); purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine , 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; Androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents, such as amines Aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglucuronolactone; aldophosphamide glycosides; levulinic acid; eniluracil (eniluracil); amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; Diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine (lonidainine); maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol ; nitroamine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazine; (procarbazine); ^PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; (tenuazonic acid); triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A) , roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine ("Ara-C");cyclophosphamide; thiote Paclitaxel; taxoids, e.g. , ^Taxol® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Cremophor-free, albumin-engineered nanoparticle formulations of ^Abraxane® paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois) and ^Taxotere® doxetaxel (Rhone-Poulenc Rorer, Antony, France); mechlorethamine; ^Gemzar® gemcitabine; 6-thioguanine; Purines; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide ; mitoxantrone; vincristine; ^NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; Amhotrexate; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment with irinotecan and 5-FU and leucovorin topoisomerase inhibitor RFS 2000; difluoromethanesulfonamide (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; Hydrofolate (LV); oxaliplatin, including oxaliplatin regimens (FOLFOX); inhibitors of PKC-α, Raf, H-Ras, EGFR ( eg, erlotinib (Tarceva ^® ) ) And VEGF-A that reduces cell proliferation and any pharmaceutically acceptable salt, acid or derivative of any of the above.

Other non-limiting exemplary chemotherapeutic agents include antihormonal agents used to modulate or inhibit the effects of hormones on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen ( tamoxifen) (including Nolvadex ^® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene ), LY117018, onapristone, and ^Fareston® toremifene; aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal gland, such as 4(5)-imidazole, amineglutamine Special, Megase ^® megestrol acetate (megestrol acetate), Aromasin ^® exemestane (exemestane), formestan (formestanie), fadrozole (fadrozole), Rivisor ^® vorozole (vorozole), Femara ^® letrole and ^Arimidex® anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides that inhibit, inter alia, signaling pathways involved in abnormal cell proliferation Ribozymes such as VEGF expression inhibitors ( e.g. , ^Angiozyme® ribozyme) and HER2 expression inhibitors; vaccines such as gene therapy Vaccines, such as ^Allovectin® vaccine, ^Leuvectin® vaccine and ^Vaxid® vaccine; ^Proleukin® rIL-2; ^Lurtotecan® topoisomerase 1 inhibitor; ^Abarelix® rmRH; and pharmaceutically acceptable salts and acids of any of the above or derivatives.

"Anti-angiogenic agent" or "angiogenesis inhibitor" refers to small molecular weight substances, polynucleotides (including , for example, inhibitory RNA (RNAi or siRNA) that directly or indirectly inhibit angiogenesis, angiogenesis, or undesirable vascular permeability. ), polypeptide, isolated protein, recombinant protein, antibody or a combination 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, an 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 ( ^Avastin® ) ) or directed against the VEGF-A receptor ( Antibodies such as KDR receptor or Flt-1 receptor); anti-PDGFR inhibitors such as Gleevec ^® (Imatinib Mesylate), small molecules that block VEGF receptor signaling ( such as PTK787/ZK2284 , SU6668, ^Sutent® /SU1 1248 (sunitinib malate), AMG706 or such small molecules as described in International Patent Application WO 2004/113304 ) . Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin, endostatin, and the like . See eg Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 ( eg Table 3 listing antiangiogenic therapies for malignant melanoma) ; Ferrara and Alitalo (1999) Nature Medicine 5(12): 1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 ( such as Table 2 listing known anti-angiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 ( eg Table 1 listing anti-angiogenic agents used in clinical trials).

"Growth inhibitory agent" as used herein refers to a compound or composition that inhibits the growth of cells (eg, cells expressing VEGF) in vitro or in vivo. Thus, a growth inhibitory agent can be one that significantly reduces the percentage of cells in S phase, eg, cells expressing VEGF. Examples of growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (phases other than S phase), such as agents that induce Gl arrest and M phase arrest. Classical M-phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors (eg, doxorubicin, pan-erubicin, daunorubicin, poside and bleomycin). Those agents that block G1 also spill over into S phase arrest, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, methyldichloroethylamine, cisplatin, amine Methotrexate, 5-fluorouracil and ara-C. Additional information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs", Murakami et al. (WB Saunders, Philadelphia, 1995), e.g. , p. 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from taxanes. Docetaxel (Taxotere ^® , Rhone-Poulenc Rorer) is derived from European yew and is a semi-synthetic analogue of paclitaxel (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 in the treatment of cancer comprising at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, agents such as chemotherapeutics, growth inhibitors, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, cancer immunotherapeutics (also known as tumor immunotherapeutics), cellular Apoptotic agents, anti-tubulin agents, and other agents for the treatment of cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists ( such as tyrosine kinase inhibitors), HER1/EGFR Inhibitors ( eg, erlotinib (Tarceva®), platelet-derived growth factor inhibitors ( eg , Gleevec® (imatinib mesylate)), COX-2 inhibitors ( eg, celecoxib) , interferon, CTLA4 inhibitors ( such as anti-CTLA antibody ipilimumab (YERVOY®)), PD-1 inhibitors ( such as anti-PD1 antibody, BMS-936558), PDL1 inhibitors ( such as anti-PDL1 antibody, MPDL3280A), PDL2 inhibitors ( eg, anti-PDL2 antibodies), VISTA inhibitors ( eg, anti-VISTA antibodies), cytokines, antagonists ( eg , neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3 , ErbB4, PDGFR-β, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4, VISTA or VEGF receptors, TRAIL/Apo2, and other biologically active and organic chemical agents, etc. The present invention also includes combinations thereof.

"Treatment" refers to therapeutic treatment, eg, where the goal is to slow down (lessen) the targeted pathological disorder or condition, and eg, where the goal is to inhibit recurrence of the disorder or disorder. "Treatment" encompasses any administration or application of a therapeutic agent for a disease (also referred to herein as a "disorder" or "disorder") in mammals, including humans, and includes inhibiting the disease or the progression of the disease, inhibiting or slowing down Disease or its progression, arrest of its development, partial or complete alleviation of disease, partial or complete alleviation of one or more symptoms of disease, or restoration or restoration of lost, lost or defective function; or stimulation of ineffective processes. The term "treating" also includes lessening the severity and/or reducing the incidence, extent or likelihood of any phenotypic characteristic. Those in need of treatment include those already with the disorder as well as those at risk of recurrence of the disorder or those in whom recurrence of the disorder is to be prevented or slowed.

The term "effective amount" or "therapeutically effective amount" refers to an amount of drug effective to treat a disease or condition in a subject. In some embodiments, an effective amount refers to an amount effective to achieve the desired therapeutic or prophylactic result at dosages and for periods of time required. A therapeutically effective amount of an antibody 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 effects where the therapeutically beneficial effects outweigh any toxic or detrimental effects of the subject antibody.

"Prophylactically effective amount" refers to the amount that can effectively achieve the desired prophylactic effect at the required dose and time period. Usually, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount due to the use of prophylactic doses prior to or early in the course of disease in an individual.

"Pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid or liquid filler, diluent conventional in the art for use together with a therapeutic agent comprising a "pharmaceutical composition" for administration to a subject , an encapsulating material, a formulation adjuvant or carrier. Pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed and are compatible with the other ingredients of the formulation. Pharmaceutically acceptable carriers are suitable for use in formulations. For example, if the therapeutic agent is to be administered orally, the carrier can be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally will not irritate the skin and will not cause injection site reactions.

An "article of manufacture" is any article of manufacture ( such as a package or container) or kit comprising at least one reagent, such as an agent for the treatment of a disease or condition, or a probe for the specific detection of a biomarker described herein. In some embodiments, an article of manufacture or kit is promoted, distributed, or sold as a unit for practicing the methods described herein. 3. Methods of treating cancer

The invention described herein provides anti-TNFR2 antibodies for use in methods of treatment of humans and other non-human mammals.

In pathological situations, Tregs can produce inappropriate immunosuppression, which can, for example, promote tumor growth. Tregs are associated with reduced anti-tumor immune responses, specifically by inappropriately suppressing the activity of effector T lymphocytes, thus promoting the development of various cancer types.

In some embodiments, there is provided a method of treating or preventing cancer comprising administering to an individual in need of such treatment an effective amount of any subject anti-TNFR2 antibody or antigen-binding fragment thereof.

In some embodiments, methods of treating cancer are provided, wherein the methods comprise administering to an individual having cancer any of the subject anti-TNFR2 antibodies or antigen-binding fragments thereof.

Cancers treatable by the methods/uses of the invention include those cancers in which regulated T lymphocytes exert their inhibitory activity, eg those cancers in which there are relatively large amounts of regulated T lymphocytes in the tumor tissue or in circulation. Modulating the expansion of T lymphocytes, which can be measured by the frequency of Tregs, generally correlates with increased Treg activation. The frequency of T lymphocytes can be modulated by any method known in the art, for example, by flow cytometric (FACS) analysis of intratumoral or circulating lymphocytes or by immunohistological staining of tumor tissue. evaluate.

Provided herein are non-limiting exemplary cancers that may be treated with any of the subject anti-TNFR2 antibodies or antigen-binding fragments thereof, including carcinomas, lymphomas, blastomas, sarcomas, and leukemias. 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 cancer or uterine cancer cancer, salivary gland cancer, kidney cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, biliary tract cancer, gallbladder cancer, gastric cancer, melanoma and various types 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) cancer), lymphoma or leukemia.

In certain embodiments, the cancer has a high TNFR2 index, defined as (a) total number of CD8 T cells in the tumor sample x TNFR2 expression on CD8 T cells; and (b) total number of Treg cells in the tumor sample x on Treg The ratio between the expression of TNFR2.

In a certain embodiment, the cancer has a TNFR2 index greater than 1, eg, 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 TILs (Tumor Infiltrating Lymphocytes), e.g., more than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% in the tumor % or more of T cells are CD8 T cells.

In a certain embodiment, the cancer has low expression levels of TNFR2 on tumor cells.

In certain embodiments, cancers are known to be sensitive 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 Tumor (Hodgkin's Lymphoma).

In a certain embodiment, the cancer has high expression levels of TNFR2 on exhausted T cells (eg, exhausted CD8 T cells) within the tumor. The cancer can be treated with combination therapy with, for example, an antagonist of the PD-1/PD-L1 pathway, such as any anti-PD-1 or anti-PD-L1 antibody, such as specifically described herein or known in the art.

In a certain embodiment, the method/use of the present invention can be used to treat cancers in which there are known high levels of regulatory T lymphocytes and/or these cancers/tumors are significantly associated with poor prognosis, including: chronic myelogenous leukemia (CML ), colon cancer, melanoma, uterine cancer, breast cancer, pancreatic cancer, stomach cancer, ovarian cancer, primary central nervous system lymphoma, multiple myeloma, prostate cancer, Hodgkin's lymphoma, or hepatocellular carcinoma.

In some embodiments, the cancer is a blood 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, kidney cancer, ovarian cancer, liver cancer, and prostate cancer. cancer).

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

In a certain embodiment, the cancer has high expression levels of TNFR2 and high expression levels of CD8A. In certain embodiments, high or higher TNFR2 expression levels correlate/compared to mean TNFR2 expression levels in prostate cancer patients; optionally, TNFR2 expression is on effector T cells (e.g., CD4+ and/or CD8+ T cells) , assessed in tumor infiltrating CD8+ T cells and/or NK cells; and/or high or higher CD8A expression levels correlate/compared to mean CD8A expression levels in AML patients.

In certain embodiments, the patient (eg, patient with cancer) has the higher expression level of TNFR2 in tumor infiltrating CD8A ⁺ (CD8α chain positive) T cells.

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

In certain embodiments, the patient has gastric adenocarcinoma, eg, 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 renal clear cell carcinoma of the kidney (KIRC). In certain embodiments, the patient has a KIRC.2, KIRC.3 or KIRC.4 subtype. In certain embodiments, the patient has a clear cell type B (ccB) subtype or a ccA (clear cell type A)/ccB unclassified subtype.

In certain embodiments, the patient has cutaneous melanoma.

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

In certain embodiments, the patient has a subtype with a BRAF hotspot mutation (eg, a V600E, V600K, or V600R mutation) or a hotspot mutation at K601.

In certain embodiments, the patient has a RAS hotspot mutation. In certain embodiments, the RAS hotspot mutation is an NRAS hotspot mutation, such as Q61R, Q61K, Q61L, Q61H, 61_62QE>HK, G12R/D/A, and G13R/D. In certain embodiments, the RAS hotspot mutation is a HRAS hotspot mutation, such as G13D, G13S, or Q61K. In certain embodiments, the RAS hotspot mutation is a KRAS hotspot mutation, such as G12D, G12R, or Q61R.

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

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

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

In certain embodiments, the patient has soft tissue sarcoma.

In certain embodiments, the cancer expresses higher than average levels of PD-L1.

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

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

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

In a certain embodiment, the method/use of the present invention can be used to treat recurrence of fibrosis from hepatitis C, since it has also been shown that increasing the frequency of regulatory T lymphocytes is a factor predicting the recurrence of this fibrosis.

In some embodiments, an anti-TNFR2 antibody of the invention may be used alone, or alternatively in combination with any other suitable compound known to treat a disease or condition.

Therefore, according to a specific embodiment of the present invention, an antibody system directed against TNFR2 and inhibiting the inhibitory activity of regulating T lymphocytes as previously defined is used in combination with a second therapeutic agent to treat diseases related to regulating the inhibitory activity of T lymphocytes, such as anticancer agent.

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

In such combination therapy, the antibody of the invention may be used before, after or concurrently with the second therapeutic agent. See other sections below for combination therapy. 4. Administration route and carrier

In various embodiments, the subject anti-TNFR2 monoclonal antibody can be administered subcutaneously or intravenously. For the sake of brevity, the "target anti-TNFR2 monoclonal antibody" refers to the mouse-human chimeric anti-TNFR2 antibody and its humanized variants of the present invention.

In some embodiments, the target anti-TNFR2 monoclonal antibody can be administered in vivo by a variety of routes, including (but not limited to) oral, intraarterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, Buccally, rectally, intraperitoneally, by inhalation, intradermally, topically, percutaneously and intrathecally or otherwise, for example by implantation.

In some embodiments, the subject anti-TNFR2 monoclonal antibody can be administered iv or sc .

A subject antibody composition can be formulated into preparations in solid, semi-solid, liquid or gaseous form; including but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and gaseous formulations. Sol.

In various embodiments, compositions comprising a subject anti-TNFR2 monoclonal antibody 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, 20th Edition (2003); Ansel et al ., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al. , Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers are available, which include vehicles, adjuvants, and diluents. In addition, various pharmaceutically acceptable auxiliary substances are also available, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

In various embodiments, the composition comprising the target anti-TNFR2 monoclonal antibody can be obtained by dissolving, suspending or emulsifying it in an aqueous or non-aqueous solvent (such as vegetable oil or other oil, synthetic fatty acid glycerides, esters of higher fatty acids) or propylene glycol); and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives for injection, including subcutaneous administration.

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

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

Also provided are pharmaceutical dosage packs comprising one or more containers each containing one or more dosages of a subject anti-TNFR2 monoclonal antibody. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising a subject anti-TNFR2 monoclonal antibody, with or without one or more other agents. In some embodiments, the unit dose is supplied in a single-use prefilled syringe for injection. In various embodiments, compositions contained in unit doses may comprise saline, sucrose, or the like; buffers, such as phosphate, or the like; and/or formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted upon addition of a suitable liquid, such as sterile water. In some embodiments, the composition includes one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, the compositions of the present invention comprise heparin and/or proteoglycans.

Pharmaceutical compositions are administered in amounts effective to treat or prevent the particular indication. A therapeutically effective amount will generally depend on the weight of the individual being treated, his or her physical or health condition, the extent of the disorder being treated, or the age of the individual being treated.

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

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

The target anti-TNFR2 monoclonal antibody composition can be administered to an individual as needed. In some embodiments, one or more effective doses of a subject anti-TNFR2 monoclonal antibody are administered to the individual. In various embodiments, an effective dose of a subject anti-TNFR2 monoclonal antibody is administered to the individual once a month, less than once a month (eg, every two months, every three months, or every six months). In other embodiments, an effective dose of a subject anti-TNFR2 monoclonal antibody is administered more than once a month (eg, every two weeks, every week, twice a week, three times a week, every day, or multiple times per day). The subject is administered at least one effective dose of a subject anti-TNFR2 monoclonal antibody. In some embodiments, the effective dose of a subject anti-TNFR2 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, a subject anti-TNFR2 monoclonal antibody is administered to an individual as needed to alleviate one or more symptoms of the disorder. 5. Combination therapy

The subject anti-TNFR2 monoclonal antibody of the present invention (including its functional fragments) can be combined with other biologically active substances or other treatment procedures and administered to individuals in need to treat diseases. For example, a subject anti-TNFR2 monoclonal antibody can be administered alone or with other treatment modalities. It can be provided before, substantially simultaneously with, or after other treatment modalities such as radiation therapy.

For the treatment of cancer, the subject anti-TNFR2 monoclonal antibody 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, a subject anti-TNFR2 monoclonal antibody that specifically binds to TNFR2 ("TNFR2-binding antagonist"), such as a TNFR2 antagonist antibody or antigen-binding fragment thereof, is combined with a second antagonist (e.g., immune checkpoint inhibitory agents, such as inhibitors of the PD-1 or PD-L1 pathway), are administered to individuals with diseases in which stimulating the immune system would be beneficial, such as cancer or infectious diseases. The two antagonists can be administered simultaneously or sequentially, eg, as described below for the combination of a subject anti-TNFR2 monoclonal antibody and a tumor immunological agent. One or more additional therapeutic agents ( eg, checkpoint modulators) can be added to the treatment with the subject anti-TNFR2 monoclonal antibody to treat cancer or autoimmune disease.

In certain embodiments, a subject anti-TNFR2 monoclonal antibody is administered to an individual, eg, an individual with cancer, simultaneously or sequentially with another therapy. For example, a subject anti-TNFR2 monoclonal antibody can be administered with one or more of radiation therapy, surgery, or chemotherapy, eg, targeted chemotherapy or immunotherapy.

In certain embodiments, methods of treating an individual with cancer comprise administering to the individual an anti-TNFR2 monoclonal antibody of the invention and one or more tumor immunological agents, such as immune checkpoint inhibitors.

Immunotherapy ( eg, therapy using tumor immunological agents) is effective in enhancing, stimulating and/or up-regulating an individual's immune response. In one aspect, co-administration of a subject anti-TNFR2 monoclonal antibody and a tumor immune agent (eg, a PD-1 inhibitor) has a synergistic effect in treating cancer, eg, in inhibiting tumor growth.

In one aspect, the subject anti-TNFR2 monoclonal antibody system is administered sequentially prior to the administration of the tumor immunotherapy agent. In one aspect, the target anti-TNFR2 monoclonal antibody system is administered simultaneously with a tumor immune agent (such as a PD-1 inhibitor). In another aspect, the target anti-TNFR2 monoclonal antibody system is administered sequentially after the administration of tumor immune agents (such as PD-1 inhibitors). The administration of the two doses can be , for example, separated by 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 once. or multiple weeks, or may be administered, e.g. , 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, after the first dose has been administered. Administration of the second dose is initiated on 5 days, 7 days, or one or more weeks.

In certain aspects, the subject anti-TNFR2 monoclonal antibody and the tumor immune agent ( eg, a PD-1 inhibitor) are administered to the patient at the same time, eg, infused into the patient at the same time, eg, within a period of 30 or 60 minutes. The target anti-TNFR2 monoclonal antibody can be co-formulated with tumor immune agents (such as PD-1 inhibitors).

Tumor immune agents include, for example, small molecule drugs, antibodies or fragments thereof, or other biological agents or small molecules. Examples of biological tumor immunological 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, monoclonal antibodies are humanized or human antibodies.

In one aspect, the tumor immunological agent is an agonist of (i) stimulatory (including co-stimulatory) molecules ( such as receptors or ligands) on immune cells (such as T cells), or (ii) inhibitory (including Antagonists of co-inhibitory) molecules ( such as receptors or ligands), both of which amplify antigen-specific T cell responses. In certain aspects, the tumor immunological agent is an agonist of (i) a stimulatory (including co-stimulatory) molecule ( eg, a receptor or a ligand) on cells involved in innate immunity (eg, NK cells), or (ii) ) antagonists of inhibitory (including co-inhibitory) molecules, such as receptors or ligands, and wherein the tumor immunological agent enhances innate immunity. Such tumor immune agents are generally referred to as immune checkpoint modulators, such as immune checkpoint inhibitors or immune checkpoint stimulators.

In certain embodiments, the tumor immune agent can 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, which The B7 membrane-bound ligand family includes 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 immune agent can be an agent that targets a member of the TNF membrane-bound ligand family or a co-stimulatory or co-inhibitory receptor ( eg, a member of the TNF receptor family) that specifically binds thereto. Exemplary TNF and TNFR family members that can be targeted by tumor immunological 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, Lymmotoxin α/TNPβ, TNFR2, TNFa, LTfiR, Lymmotoxin α 1β2, FAS, FASL, RELT, DR6, TROY, and NGFR. The tumor immune agent that can be used in combination with the target anti-TNFR2 monoclonal antibody to treat cancer can be targeting B7 family members, B7 receptor family members, TNF family members or TNFR family members (such as those family members described above) agents, such as antibodies.

In one aspect, the subject anti-TNFR2 monoclonal antibody is administered with one or more of: (i) an antagonist of a protein that inhibits T cell activation ( e.g., an immune checkpoint inhibitor), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM3, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7 -H4, 2B4, CD48, GARP, PDIH, LAIR1, TIM-1, TIM-4 and PSGL-1, and (ii) agonists of proteins that stimulate T cell activation, such as B7-1, B7- 2. CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, CD40L, DR3 and CD28H.

In one aspect, the tumor immunological agent is an agent that inhibits ( i.e., antagonizes) an interleukin that inhibits T cell activation, such as IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive interkines, or Agonists, such as the cytokines themselves, that stimulate T cell activation and stimulate an immune response to cytokines such as IL-2, IL-7, IL-12, IL-15, IL-21 and IFNα.

Other agents that can be combined with a subject anti-TNFR2 monoclonal antibody to stimulate the immune system ( eg, to treat cancer and infectious disease) include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, a subject anti-TNFR2 monoclonal antibody can be combined with an antagonist of KIR.

Other agents useful in 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 (WO1 1/70024, WO1 1/107553, WO11/131407, WO13/87699, WO13/119716, WO13/132044) or FPA008 (WO11/140249; WO13169264; WO14/036357).

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

Other agents that can be combined with a subject anti-TNFR2 monoclonal antibody include agents that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF secreting cell vaccines, CpG oligonucleotides, and imiquimod, or enhance tumor antigen presentation. Cellular immunogenic therapy ( eg anthracycline).

Other therapies that can be combined with a subject anti-TNFR2 monoclonal antibody include therapies that deplete or block Treg cells, such as agents that specifically bind to CD25.

Another therapy that can be combined with a subject anti-TNFR2 monoclonal antibody is therapy that inhibits metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase.

Another class of agents that may be used includes agents that inhibit adenosine formation or that inhibit adenosine A2A receptors.

Other therapies that can be combined with a subject anti-TNFR2 monoclonal antibody to treat cancer include therapies that reverse/prevent T cell anergy or exhaustion and therapies that trigger innate immune activation and/or inflammation at the tumor site.

A target anti-TNFR2 monoclonal antibody can be combined with more than one tumor immune agent (such as an immune checkpoint inhibitor), and can be combined, for example , with a combination approach targeting multiple elements of the immune pathway, such as one of the following or more: Therapies that enhance tumor antigen presentation ( e.g. dendritic cell vaccines, GM-CSF secreting cell vaccines, CpG oligonucleotides, imiquimod); e.g. by inhibiting CTLA-4 and/or PD1/PD- L1/PD-L2 pathway and/or depletion or blockade of Treg or other immunosuppressive cells to suppress negative immune regulation therapy; such as stimulation of CD-137, OX-40 and/or GITR pathway and/or stimulation of T cell effector function Therapies where agonists stimulate positive immune regulation; therapies that increase the frequency of anti-tumor T cells throughout the body; e.g. depletion or inhibition using CD25 antagonists ( e.g. daclizumab) or by ex vivo anti-CD25 bead depletion Therapy of Treg (such as Treg in tumor); Therapy affecting the function of suppressive myeloid cells in tumor; Therapy of enhancing the immunogenicity of tumor cells ( such as anthracycline); Adoptive T cell or NK cell transfer, including via Genetically modified cells, such as cells modified with chimeric antigen receptors (CAR-T therapy); inhibition of metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or monoxide Nitrogen synthase); therapy to reverse/prevent T cell anergy or exhaustion; therapy to trigger innate immune activation and/or inflammation at the tumor site; administration of immunostimulatory cytokines or blockade of immunosuppressive cytokines.

For example, a subject anti-TNFR2 monoclonal antibody can be used with the following agents: one or more agonists that bind positive co-stimulatory receptors; one or more antagonists (blockers) that attenuate signaling through inhibitory receptors; ), such as antagonists that overcome different immunosuppressive pathways within the tumor microenvironment ( such as blocking PD-L1/PD-1/PD-L2 interactions); one or more agents that increase systemic anti-tumor immune cells (such as T cells) Agents that frequency, deplete, or inhibit Treg ( e.g. , by inhibiting CD25); one or more agents that inhibit metabolic enzymes (e.g., IDO); one or more agents that reverse/prevent T cell anergy or exhaustion; and one or more agents that trigger tumor site Point innate immune activation and/or inflammatory agents.

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual a subject anti-TNFR2 monoclonal antibody and a tumor immunological agent, wherein the tumor immunological agent is CTLA-4 antagonists, eg, antagonistic CTLA-4 antibodies. Suitable CTLA-4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab.

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual a subject anti-TNFR2 monoclonal antibody and a tumor immunological agent, wherein the tumor immunological agent is PD-1 antagonists, such as antagonistic PD-1 antibodies. Suitable PD-1 antibodies include eg OPDIVO (nivolumab), KEYTRUDA (pembrolizumab) or MEDI-0680 (AMP-514; WO2012/145493). Tumor immune agents may also include pidilizumab (CT-011). Another approach to targeting the PD-1 receptor 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, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is a PD-L1 antagonist, such as an antagonistic PD-L1 antibody. Suitable PD-L1 antibodies include, for example, MPDL3280A (RG7446; WO2010/077634), durvalumab (MEDI4736), BMS-936559 (WO2007/005874), MSB0010718C (WO2013/79174) or rHigM12B7.

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is a LAG-3 antagonist, eg, an antagonistic LAG-3 antibody. Suitable LAG3 antibodies include eg BMS-986016 (WO10/19570, WO14/08218) or IMP-731 or IMP-321 (WO08/132601, WO09/44273).

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is a CD137 (4-1BB) agonist, such as an agonistic CD137 antibody. Suitable CD137 antibodies include for example urelumab or PF-05082566 (W012/32433).

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is a GITR agonist, such as an agonistic GITR antibody. Suitable GITR antibodies include eg the GITR antibodies disclosed in TRX-518 (WO06/105021, WO09/009116), MK-4166 (WO 11/028683) or WO2015/031667.

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is an OX40 agonist, such as an agonistic OX40 antibody. Suitable OX40 antibodies include eg MEDI-6383, MEDI-6469 or MOXR0916 (RG7888; WO06/029879).

In one embodiment, stimulation of the immune system in an individual suffering from a disease that would benefit ( eg, cancer or an infectious disease) is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is a CD40 agonist, such as an agonistic CD40 antibody. In certain embodiments, the tumor immunotherapy agent is a CD40 antagonist, such as an antagonistic CD40 antibody. Suitable CD40 antibodies include, for example, lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870, 893 or Chi Lob 7/4.

In one embodiment, stimulation of the immune system in an individual suffering from a disease that would benefit ( eg, cancer or an infectious disease) is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is a CD27 agonist, such as an agonistic CD27 antibody. Suitable CD27 antibodies include, for example, varlilumab (CDX-1127).

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is MGA271 (for B7H3) (WO1 1/109400).

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is a KIR antagonist, such as lirilumab.

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is an IDO antagonist. Suitable IDO antagonists include for example INCB-024360 (WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919 (WO09/73620, WO09/1156652, WO11/ 56652, WO 12/142237) or F001287.

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor immune The agent is a Toll receptor agonist, such as a TLR2/4 agonist ( such as BCG); a TLR7 agonist ( such as Hiltonol (Hiltonol) or imiquimod); a TLR7/8 agonist ( such as Resiquimod); or a TLR9 agonist ( eg CpG7909).

In one embodiment, an individual suffering from a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the individual an anti-TNFR2 monoclonal antibody of the invention and a tumor immunological agent, wherein the tumor The immune agent is a TGF-β inhibitor, such as GC1008, LY2157299, TEW7197 or IMC-TR1. 6. Exemplary Anti -TNFR2 Monoclonal Antibodies

The invention described herein provides monoclonal antibodies or antigen-binding fragments thereof specific for TNFR2.

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

For example, the epitopes of HFB3-1/HFB3-1-hG1 are depicted in Figures 11A-11C (SEQ ID NO: 13 is depicted in Figures 11A and 11B, and SEQ ID NO: 101 is depicted in Figure 11C).

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

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), which comprises the HCVR CDR1 sequence of SEQ ID NO: 1, the HCVR CDR2 sequence of SEQ ID NO: 2, and the HCVR CDR3 sequence of SEQ ID NO: 3; and (1b) light chain variable region (LCVR ), which comprises the LCVR CDR1 sequence of SEQ ID NO: 4, the LCVR CDR2 sequence of SEQ ID NO: 5, and the LCVR CDR3 sequence of SEQ ID NO: 6; or (2a) a heavy chain variable region (HCVR), which comprises SEQ ID NO: HCVR CDR1 sequence of ID NO: 14, HCVR CDR2 sequence of SEQ ID NO: 15 and HCVR CDR3 sequence of SEQ ID NO: 16; and (2b) light chain variable region (LCVR), which comprises SEQ ID NO: 17 LCVR CDR1 sequence, LCVR CDR2 sequence of SEQ ID NO: 18 and LCVR CDR3 sequence of SEQ ID NO: 19; or (3a) heavy chain variable region (HCVR), which comprises the HCVR CDR1 sequence of SEQ ID NO: 26, SEQ ID NO: The HCVR CDR2 sequence of ID NO: 27 and the HCVR CDR3 sequence of SEQ ID NO: 28; and (3b) the light chain variable region (LCVR), which comprises the LCVR CDR1 sequence of SEQ ID NO: 29, the LCVR CDR2 sequence and the LCVR CDR3 sequence of SEQ ID NO: 31; or (4a) heavy chain variable region (HCVR), it comprises the HCVR CDR1 sequence of SEQ ID NO: 39, the HCVR CDR2 sequence of SEQ ID NO: 40 and SEQ ID NO: The HCVR CDR3 sequence of ID NO: 41; and (4b) the light chain variable region (LCVR), which comprises the LCVR CDR1 sequence of SEQ ID NO: 42, the LCVR CDR2 sequence of SEQ ID NO: 43 and the LCVR CDR2 sequence of SEQ ID NO: 44 LCVR CDR3 sequence; or (5a) heavy chain variable region (HCVR), it comprises the HCVR CDR1 sequence of SEQ ID NO:51, the HCVR CDR2 sequence of SEQ ID NO:52 and the HCVR CDR3 sequence of SEQ ID NO:53; And (5b) light chain variable region (LCVR), it comprises 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 (6a) heavy chain A variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 63, the HCVR CDR2 sequence of SEQ ID NO: 64, and the HCVR CDR3 sequence of SEQ ID NO: 65; and (6b) the light chain variable region (LCVR ), which comprises 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 aspect of the invention described above, in some embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof: (1A) the HCVR sequence is SEQ ID NO: 7; and/or ( 1B) LCVR sequence of SEQ ID NO: 8, or (2A) HCVR sequence of SEQ ID NO: 20; and/or (2B) LCVR sequence of SEQ ID NO: 21, or (3A) HCVR sequence of SEQ ID NO: 32; and/or (3B) LCVR sequence is SEQ ID NO: 33, or (4A) HCVR sequence is SEQ ID NO: 45; and/or (4B) LCVR sequence is SEQ ID NO: 46, or (5A) HCVR The 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: (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) SEQ ID The light chain sequence of 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.

SCEDSTYTQLWNWVPECLS (SEQ ID NO: 13) SCEDSTYTQLWNWVPECLSC (SEQ ID NO: 101) HFB3-1hz6-hG1 ( 人類化單株抗體 )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係SEQ ID NO: 20，且LCVR係SEQ ID NO: 21。

HFB3-14-hG1 ( 小鼠單株抗體 )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係SEQ ID NO: 32，且LCVR係SEQ ID NO: 33。

CAPLRKCRPGFGVARPGTETSD(SEQ ID NO: 38) HFB3-14hz1c-hG1 ( 人類化單株抗體 )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係SEQ ID NO: 45，且LCVR係SEQ ID NO: 46。

HFB3-18-hG1 ( 小鼠單株抗體 )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係SEQ ID NO: 57，且LCVR係SEQ ID NO: 58。

HFB3-18hz1-hG1 ( 人類化單株抗體 )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係SEQ ID NO: 69，且LCVR係SEQ ID NO: 70。

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 line SEQ ID NO: 7, and LCVR line SEQ ID NO: 8 .

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.

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 .

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 line SEQ ID NO: 45, And the LCVR is SEQ ID NO: 46.

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 line SEQ ID NO: 57, and LCVR line SEQ ID NO: 58 .

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 line SEQ ID NO: 69, and LCVR line SEQ ID NO: 70 .

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

In some embodiments, the antigen-binding fragment thereof is Fab, Fab', F(ab') ₂ , _Fd , single chain Fv or scFv, disulfide-linked Fv _, V-NAR domain, IgNar, intrabody , IgGΔCH ₂ , minibody, 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 antibody or antigen-binding fragment thereof of the invention has an engineered Fc region that eliminates immune effector functions. For example, the engineered Fc region of a subject antibody may have a "LALA" double mutation (Leu234Ala and Leu235Ala), and thus have reduced effector function. These antibodies can be named G1AA due to the LALA double mutation on IgG1.

Other recombinant human IgG antibodies (hIgG) are known in the art that partially or completely do not bind Fcγ receptors (FcγRs) and complement protein C1q and thus have abolished immune effector functions, and are used in various therapeutic applications to reduce FcγR activation and Fc-mediated toxicity. Some of these Fc engineered antibodies/fragments partially achieved this goal, while others completely abolished FcyR activation and Fc-mediated toxicity. In certain embodiments, antibodies/fragments of the invention have an engineered hIgG Fc domain comprising hIgG1-P329G LALA or hIgG4-P329G SPLE (human IgG4 S228P/L235E variant of IgG4) mutations and completely eliminates FcγRs and C1q interaction, and does not affect FcRn interaction and Fc stability. The P329G Fc mutation disrupts the formation of proline and the sandwich motif of FcyRs. Since this motif is present at the interface of all IgG Fc/FcyR complexes, its disruption can be applied to all human and most other mammalian IgG subclasses to generate effector-silenced IgG molecules. Thus, in certain embodiments, the antibody/fragment of interest has either IgG subclass with the effector-silencing Fc mutation.

In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention is specific for human TNFR2, eg, does not substantially cross-react with TNFR1, and/or does not substantially cross-react with mouse TNFR2. In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the invention cross-react with monkey TNFR2 (eg, cynomolgus or rhesus monkey TNFR2).

In some embodiments, the dissociation constant (K _d ) of the monoclonal antibody or antigen-binding fragment thereof of the present invention to rhTNFR2 is ≤ 1 μM, ≤ 100 nM, ≤ 50 nM, ≤ 25 nM, ≤ 20 nM, ≤ 15 nM , ≤ 10 nM, ≤ 5 nM, ≤ 2 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 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, a monoclonal antibody or antigen-binding fragment thereof of the invention binds to a region within the CRD2 domain of TNFR2. In a certain embodiment, the monoclonal antibody or antigen-binding fragment thereof of the invention binds to an epitope bound by HFB3-1.

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

In a certain embodiment, the monoclonal antibody or antigen-binding fragment thereof of the invention binds to an epitope bound by HFB3-18.

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

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

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

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention does not substantially affect the binding of human recombinant TNFα to TNFR2.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention inhibits TNFα-mediated signal transduction (eg, NFκB signal transduction), and/or induces down-regulation of NFκB downstream target genes. However, in other embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention promotes TNFα-mediated signaling (eg, NFκB signaling), and/or induces up-regulation of NFκB downstream target genes.

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

In some embodiments, NFKB signaling is stimulated in Tregs. In some other embodiments, NFKB signaling is inhibited in Tregs.

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

In some embodiments, a monoclonal antibody or antigen-binding fragment thereof of the invention, particularly a humanized monoclonal antibody or antigen-binding fragment thereof, binds preferentially to (CD3 /CD28) TCR-activated primary CD8 and/or CD4 T cells.

In some embodiments, a monoclonal antibody or antigen-binding fragment thereof of the invention, particularly a humanized monoclonal antibody or antigen-binding fragment thereof, enhances CD3/CD28-induced activation and/or proliferation, such as CD3/CD28-induced primary CD8 and/or activation and/or proliferation of CD4 T cells, including activation and/or proliferation of primary CD8 and/or CD4 T cells in the presence of Treg.

In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the present invention, especially humanized monoclonal antibodies or antigen-binding fragments thereof, co-stimulate the activation and activation of primary CD8 and/or CD4 T cells in a cross-linking-independent manner. / or proliferate.

In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the present invention, especially humanized monoclonal antibodies or antigen-binding fragments thereof, co-stimulate the activation of primary CD8 and/or CD4 T cells in a cross-linking-dependent manner and/or or proliferate.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention enhances the binding between TNFα and TNFR2; enhances TNFα-mediated or co-stimulated NFκB signal transduction (for example, in TCR-activated CD8 and/or CD4 Tconv T cells); and/or promote the proliferation of TCR-activated effector T cells (such as CD8 and/or CD4 Tconv T cells) in the presence of Treg.

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

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof (including humanized monoclonal antibody or antigen-binding fragment thereof) of the present invention has good developability characteristics, including high temperature (such as 25°C or 40°C), low temperature Stable under pH conditions ( eg, pH 3.5 at about room temperature) and/or after several freeze/thaw cycles.

In certain embodiments, monoclonal antibodies or antigen-binding fragments thereof (including humanized monoclonal antibodies or antigen-binding fragments thereof) of the invention include one or more of amino acid sequences designed to improve antibody developability. point mutation. For example, Raybould et al. (Five computational developability guidelines for therapeutic antibody profiling, PNAS 116(10): 4025-4030, 2019) describe Therapeutic Antibody Profiler (TAP), which is a downloadable template for constructing variable domain sequences. Computational tools to source the model, test it against five developability guidelines, and report potential sequence instabilities and canonical forms. The authors further provide TAP which is freely available at opig.stats.ox.ac.uk/webapps/sabdab-sabpred/TAP.php.

There are still many hurdles to therapeutic mAb development besides achieving the desired affinity for the antigen. These barriers include inherent immunogenicity, chemical conformational instability, self-association, high viscosity, multispecificity, and poor performance. For example, high levels of hydrophobicity, especially in the highly variable complementarity determining regions (CDRs), have repeatedly been implicated in aggregation, viscosity and multispecificity. The asymmetry in the net charge of the heavy and light chain variable domains was also correlated with self-association and viscosity at high concentrations. Positively and negatively charged patches in the CDR are associated with high clearance rates and poor performance levels. Product heterogeneity (eg, via oxidation, isomerization, or glycosylation) often arises from specific sequence motifs that are susceptible to post-translational or co-translational modification. Computational tools can be used to facilitate the identification of sequence instabilities. Warszawski et al. (Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces. PLoS Comput Bio l 15(8): e1007207. https://doi.org/10.1371/journal.pcbi.1007207) also A method for optimizing antibody affinity and stability by automated design of the variable light chain-heavy chain interface is described. Other methods can be used to identify potential developability issues of candidate antibodies, and in preferred embodiments of the invention, these issues can be addressed by introducing one or more point mutations into candidate antibodies by conventional means to generate the optimal product of the invention. Optimized therapeutic antibodies. 7. Humanized Antibody

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 therapeutic agents.

Antibodies can be humanized by any standard method. Non-limiting exemplary humanization methods include, for example , U.S. Patent No. 5,530,101; U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; U.S. Patent No. 6,180,370; (1986); Riechmann et al. , Nature 332: 323-27 (1988); Verhoeyen et al. , Science 239: 1534-36 (1988); and the methods described in US Publication No. US 2009/0136500. 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 the corresponding position in a human framework region. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 of the framework regions of the non-human variable region One, at least 11, at least 12, at least 15 or at least 20 amino acids are 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 from the framework regions of different human immunoglobulin genes. That is, in some of these embodiments, one or more non-human amino acids may be substituted with corresponding amino acids in the human framework regions of the first human antibody or encoded by the first human immunoglobulin gene, one or more One or more non-human amino acids can be substituted by the corresponding amino acids in the human framework region of the second human antibody or encoded by the second human immunoglobulin gene, and one or more non-human amino acids can be replaced by the human amino acid of the third human antibody. Corresponding amino acid substitutions in the framework regions or are encoded by third human immunoglobulin genes, etc. Additionally, in some embodiments, all corresponding human amino acids used to substitute for a single framework region (eg, FR2) need not be from the same human framework. However, in some embodiments, all 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, antibodies are humanized by replacing one or more intact framework regions with corresponding human framework regions. In some embodiments, the human framework region with the highest level of homology to the non-human framework region being replaced is selected. In some embodiments, the humanized antibody is a CDR-grafted antibody.

In some embodiments, after CDR grafting, one or more framework amino acids are changed back to the corresponding amino acids in the mouse framework regions. In some embodiments, the "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 Or 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 less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or more are made to the framework region of the antibody following CDR grafting Few, 4 or less, 3 or less, 2 or less, 1 or 0 back mutations.

In some embodiments, a humanized antibody also 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. Human antibodies can be produced 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 US Patent No. 5,545,807; US Patent No. 6,713,610; US Patent No. 6,673,986; US Patent No. 6,162,963; ; US Patent No. 5,874,299; and US Patent No. 5,545,806.

Non-limiting exemplary methods also include the use of phage display libraries to produce 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 region

In some embodiments, the humanized, chimeric or human antibodies described herein comprise one or more human constant regions. In some embodiments, the human heavy chain constant region has an isotype selected from IgA, IgG and IgD. In some embodiments, the human light chain constant region has an isotype selected from kappa and lambda. In some embodiments, the antibodies described herein comprise a human IgG constant region, eg, human IgGl, IgG2, IgG3 or IgG4. In some embodiments, the antibody or Fc fusion partner comprises a C237S mutation, eg, in the IgG1 constant region. In some embodiments, the antibodies described herein comprise a human IgG2 heavy chain constant region. In some of these embodiments, the IgG2 constant region comprises the P331S mutation, as described in US Patent No. 6,900,292. In some embodiments, the antibodies described herein comprise a human IgG4 heavy chain constant region. In some of these embodiments, the antibodies described herein comprise the S241P mutation in the human IgG4 constant region. See, eg, Angal et al. , Mol. Immunol. 30(1):105-108 (1993). In some embodiments, an antibody described herein comprises a human IgG4 constant region and a human kappa light chain.

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

In some embodiments, an effector function is not desirable. For example, in some embodiments, effector functions may not be desirable in the treatment of inflammatory and/or autoimmune disorders. In some of these embodiments, a 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 affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the antigen and/or epitope to which the antibody binds and the ligand that binds the constant region of the antibody. 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, some polypeptides are also purified using hydrophobic interaction chromatography (HIC) (eg, butyl or phenyl columns). Many 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 molecule encoding the antibody of the present invention

The invention also provides nucleic acid molecules comprising a polynucleotide encoding one or more chains of the antibodies described herein. In some embodiments, a nucleic acid molecule comprises a polynucleotide encoding a heavy or light chain of an antibody described herein. In some embodiments, a 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 of these embodiments, the heavy and light chains are represented by one nucleic acid molecule, or are represented by two separate polypeptides by two separate nucleic acid molecules. In some embodiments, such as when the antibody is a scFv, a single polynucleotide encodes a single polypeptide comprising a heavy chain and a light chain linked together.

In some embodiments, a polynucleotide encoding a heavy or light chain of an antibody described herein comprises a nucleotide sequence encoding a leader sequence that is N-terminal to the heavy or light chain when translated. 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 host cell of choice (eg, a mammalian cell). 10. Carrier

Vectors are provided comprising polynucleotides encoding the heavy and/or light chains of the antibodies described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like . In some embodiments, the vector 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 represented by the vector as two separate polypeptides. In some embodiments, the heavy and light chains are represented as part of a single polypeptide, such as in the case of an antibody 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 (eg, similar molar amounts or similar mass amounts). In some embodiments, the first vector and the second vector are transfected into the host cell with a molar or mass ratio between 5:1 and 1:5. In some embodiments, a heavy chain-encoding vector and a light chain-encoding vector are used in a mass ratio between 1:1 and 1:5. In some embodiments, the vector encoding the heavy chain and the vector encoding the light chain are used in a mass ratio of 1:2.

In some embodiments, a vector is selected that is optimized for expression of the polypeptide in CHO or CHO-derived cells or NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al. , Biotechnol. Prog. 20:880-889 (2004). In some embodiments, the vector is selected for in vivo expression of the antibody of interest in an animal, including a human. In some of these embodiments, 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, eg, in PCT Publication No. WO 2006/076288. 11. Host cells

In various embodiments, the heavy and/or light chains of the antibodies described herein can be in prokaryotic cells (such as bacterial cells); or in eukaryotic cells (such as fungal cells such as yeast), plant cells, insect cells and expressed in mammalian cells. This representation can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides 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; PER.C6® cells ( Crucell); and NSO cells. In some embodiments, the heavy and/or light chains of the antibodies described herein can be expressed in yeast. See , eg, U.S. Publication No. US 2006/0270045 Al. In some embodiments, particular eukaryotic host cells are selected based on their ability to make desired post-translational modifications to the heavy and/or light chains of TNFR2 antibodies. For example, in some embodiments, a polypeptide produced by CHO cells has a higher level of sialylation than the same polypeptide produced in 293 cells.

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

In some embodiments, one or more polypeptides can be produced in vivo according to any suitable method in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides. Example Example 1 Monoclonal Antibodies Specific to Human and Monkey TNFR2

To generate monoclonal antibodies specific for human TNFR2 that are cross-reactive with the monkey xenolog TNFR2, mice were immunized with the recombinant extracellular domain (ECD) of human TNFR2 (rhTNFR2) using standard procedures to generate a series of different human-mouse chimeric monoclonal antibody.

At least 25 such monoclonal antibodies were generated, the VH and VL sequences of the selected antibodies were compared, and a consensus sequence was 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 human and monkey TNFR2 expressed by CHO cells (CHO.hHFB3 and CHO.mkHFB3 cells, respectively). Briefly, approximately 40,000 CHO.hHFB3 or CHO.mkHFB3 cells were seeded in tissue culture wells, and serial 1:3 dilutions of each test antibody (starting (highest) concentration at approximately 66 nM antibody) were added into each cell type and incubate for approximately 1 hour. Antibody binding to cells was detected by using a 17 nM anti-human Fc antibody labeled with AF647 (Alexsa Fluor ^® 647 fluorescent dye). An isotype-matched negative control antibody was also used in this assay. Data, including _EC50 values and _Emax for each antibody, are compiled in Figure 2A.

Eleven (11) tested antibodies showed sub- or single-digit nM levels of affinity ( _EC50 ) for hTNFR2 expressed on CHO cells. These antibodies also showed cross-reactivity to the rhesus xenolog of TNFR2 expressed on CHO cells, and the binding affinities had substantially the same tendency as hTNFR2 binding. See Figure 2A.

Interestingly, some antibodies (such as HFB3-1 and HFB3-14) promote the binding of TNFα to TNFR2, others (such as HFB3-18) inhibit the binding of TNFα to TNFR2, and some antibodies (such as HFB3-6) appear The above has no effect on the binding of TNFα and TNFR2. See Figure 2B. Specifically, the binding of 25 ng/mL TNF[alpha] to CHO.hHFB3 cells was measured after pre-incubation of CHO cells with the respective antibodies for approximately 1 hour. Increasing concentrations of antibody were then plotted against the percentage of cells bound to TNF[alpha] (labeled HFB2003L).

The same experiment was also set up to test the ability of the test antibody to bind to CHO cells expressing mouse TNFR2 and to the parental CHO cell line (which may or may not express hamster TNFR2). Two monoclonal antibodies (HFB3-18 and HFB3-19) exhibited marginal levels of mouse xenolog binding, whereas the other antibodies had no detectable levels of mouse TNFR2 binding. As a positive control, the use of the HM102 monoclonal antibody specific for mouse TNFR2 showed positive binding to CHO cells expressing mouse TNFR2, whereas an isotype-matched control antibody did not bind (Figure 3).

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

Binding specificity to human TNFR2 (to the relevant TNFR1 receptor) was also verified using recombinant human TNFR2 and TNFR1 proteins.

Briefly, tissue culture plates were coated overnight at 4°C with 0.1 μg/mL of His-tagged recombinant human TNFR2 or TNFR1. Coated plates were then incubated on ice with 1:3 serial dilutions of each test antibody for approximately 1 hour at a starting (maximum) concentration of approximately 66 nM antibody. Antibody binding to cells was detected by using a 1 :5000 dilution of HRP-labeled anti-human Fc antibody and TMB substrate. An isotype-matched negative control antibody F3, as well as MR2-1 positive control antibodies specific for rhTNFR2 and positive control antibodies specific for rhTNFR1 were also used in this analysis. Data, including _EC50 values for each antibody, are compiled in Figure 4A.

Six of the eleven tested antibodies (i.e. HFB3-1, HFB3-14, HFB3-21, HFB3-23, HFB3-24 and HFB3-25) showed sub-nM affinity (EC ₅₀ ), while the other 4 antibodies (HFB-3, HFB-6, HFB-19 and HFB-22) showed single-digit nM affinities for the same antigen. HFB3-18 showed relatively weakest binding to monomeric rhTNFR2 with double-digit nM affinities. However, none of the 11 antibodies showed any detectable level of binding to the His-tagged TNFRl receptor, confirming the binding specificity to TNFR2.

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. AHC biosensors enable the kinetic characterization of macromolecular interactions between human Fc-containing proteins ( eg, targeted antibodies) and target analytes ( eg, recombinant human TNFR2). Immobilization of human Fc-containing proteins is achieved via factory-immobilized anti-human Fc-specific antibodies whose high affinity for the human Fc domain provides the stable baseline required for demanding kinetic applications . In this particular experiment, the test antibody (humanized\) was loaded at a concentration of 20 μg/mL in assay buffer (PBS, pH 7.4, 0.1% BSA, 0.1% Tween20). The analyte was 500 nM, 167 nM or 55.7 nM of His-tagged recombinant human TNFR2. The capture assay was run at 25° C. The K _d of the tested antibodies were 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 recognized different domains of TNFR2. One structural feature shared by most members of the TNFR superfamily is that they contain about two to four cysteine-rich domains (CRDs). HFB3-1-hG1 binds to a region within the CRD2 domain (Figure 11C), whereas HFB3-18-hG1 binds to a conformational epitope within CDR1. HFB3-6-hG1 binds to a region within CRD3, and HFB3-14-hG1 also binds to an epitope within the CRD3 region that is smaller than that 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 Figure 1 ID. Example 2 Expression of TNFR2 in T cell subtypes

This experiment demonstrates that TNFR2 is predominantly expressed on Treg and CD4 ⁺ and CD8 ⁺ T cells in various 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 in tumor samples and the mean relative expression of TNRF2 among T cell subtypes were determined using RNA-seq analysis Level (level 2-8). The results are compiled in Figure 5.

In every tumor sample analyzed (including BCC or basal cell carcinoma, SCC or squamous cell carcinoma, melanoma, and NSCLC or non-small cell lung cancer), TNFR2 was predominantly and most frequently found in Treg as well as CD4 ⁺ and CD8 ⁺ T in cells. In addition, the highest relative performance levels were also found in Tregs. See Figure 5 left. Data suggest that TNFR2 is an attractive target for cancer therapy.

Additional expression analysis of TNFR2 in SCC cancer samples was also performed in conjunction with the expression of several immune checkpoint genes such as PD-1, TIM3, CTLA4, and 4-1BB. The expression of TNFR2 was found to be consistent with the expression of these immune checkpoint genes in exhausted CD8+ T cells (Fig. is beneficial. Example 3 The combination of anti -TNFR2 monoclonal antibody and primary Treg , CD8 and CD4 Tconv cells

Given the expression pattern of TNFR2 on T cell subtypes (see Example 2), this experiment demonstrates that the target anti-TNFR2 monoclonal antibody can bind to primary T cell subtypes preferentially over activated T cells.

Briefly, flat-bottomed 96-well plates were coated overnight at 4°C with 10 nM of anti-CD3 antibody. At the same time, T cell subtypes including Treg, CD8 or CD4 habituated T cells (Tconv) were isolated from human PBMCs. The isolated T cell subtypes were seeded at a density of approximately 50,000 cells/well in the presence of 6.6 nM anti-CD28 antibody to co-stimulate primary T cells for approximately 3 days. The stimulated primary T cells were then treated for 1 hour on ice with 1:3 serial dilutions of the anti-TNFR2 human-mouse chimeric monoclonal antibody of the present invention at different concentrations, up to a maximum concentration of 66 nM. Bound chimeric antibody was detected by adding 17 nM 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 Figure 6 shows that CD4 Tconv is the most abundant T cell subtype, accounting for about 30% of the total hPBMC, followed by 10% of CD8 T cells and about 1% of Treg. However, non-TCR-activated primary T cells could not detect binding to the target anti-TNFR2 antibody, except for the relatively low level of binding seen in primary Treg. In summary, receptor occupancy Emax was highest in Treg, followed by CD8, and again by CD4 Tconv. Given the relatively low abundance of Treg compared to CD8 and CD4 Tconv, the expression of TNFR2 on Treg was much higher than that on CD8 and CD4 T cells on a per cell basis.

However, in TCR-activated T cells, a significant 5-6-fold increase in the binding of some anti-TNFR2 antibodies was observed in Treg, while substantially higher binding was also observed in CD8 and CD4 Tconv (Figure 6 lower panel) .

Among the antibodies tested, HFB3-1, HFB3-6, HFB3-24, HFB3-25, and SBT1 (positive control) exhibited high affinities at sub-nM levels, while HFB3-14 and HFB3-19 exhibited single-digit nM affinities . HFB3-18, HFB3-21 and HFB3-22 had double digit nM affinities. Example 4 The binding of certain anti -TNFR2 monoclonal antibodies to primary CD8 and CD4 Tconv cells co-stimulates NFκB signaling

This experiment demonstrates that the anti-TNFR2 monoclonal antibody of the invention co-stimulates TNFα-mediated NFκB signaling as confirmed by QPCR quantification of NFκB signaling pathway genes.

Briefly, CD4 Tconv (CD4 ⁺ CD25 ⁻ ) or CD8 ⁺ T cells were isolated from hPBMCs using standard techniques and commercially available kits. Isolated T cells were incubated for approximately 24 hours with 10 μg/mL (66 nM) of each test mAb of the invention or appropriate positive or negative controls and 25 ng/mL (1.5 nM) of TNFα. Stimulated T cells were then harvested and their mRNA isolated, reverse transcribed, and QPCR analyzed for selected NFKB signaling pathway genes (eg, CD25, Foxp3, NFKB2, ReIB, and LTA). The expression levels of these genes in the presence or absence of co-stimulation by the target antibodies were compared in the bar graph of FIG. 7 . Results are presented as fold change compared to unstimulated control (1×).

The results showed that certain targeted antibodies (including HFB3-1, HFB3-14, HFB3-23, HFB3-24 and HFB3-25) induced NFκB signaling. It should be noted that HFB3-1 and HFB3-14, but not HFB3-18, occasionally induce NFκB signaling, especially in NFκB2, RelB and LTA. Costimulatory Effect of Example 5 Anti -TNFR2 Monoclonal Antibody Correlates with Proliferation of Isolated Primary CD8 and CD4 Tconv Cells

In this experiment, flat-bottom 96-well plates were coated overnight at 4°C with 10 nM anti-CD3 monoclonal antibody and 20 nM or 100 nM anti-TNFR2 antibody of interest. Simultaneously, CD8 and CD4 Tconv cells were isolated from hPBMC as described, and T cell proliferation was followed with 2 μΜ of CTV (CellTrace ^™ Violet Cell Proliferation Kit from Invitrogen). The CellTrace™ Violet Dye readily diffuses into cells, where the dye is cleaved by intracellular esterases to produce a highly fluorescent compound that is then covalently bound to an intracellular amine, resulting in a stable, well-retained fluorescent stain that Staining can be fixed with aldehyde fixative. Excess unbound reagent diffuses passively into the extracellular medium where it can be quenched and washed away with complete medium.

Labeled T cells were then plated in coated 96-well plates at a density of approximately 50,000 cells/well in the presence of 6.6 nM anti-CD28 antibody for co-stimulation for approximately 3 days. Cells were then fixed for FACS analysis of fluorescent signals.

The data in Figure 8 show that certain targeted anti-TNFR2 antibodies co-stimulate CD8 and CD4 Tconv proliferation even at a lower concentration of 20 nM. The benchmark positive control antibodies SBT-1 and SBT-4 also co-stimulated T cell proliferation under the same conditions, but to a lower extent than HFB3-1, HFB3-14, HFB3-18 and HFB3-25.

Other experiments showed that this co-stimulation of primary T cell proliferation could be dependent on FcγR cross-linking of certain monoclonal antibodies (e.g. HFB3-18) but not discernible for others (e.g. HFB3-1 and HFB3-14) Cross-link dependence.

Specifically, CD8 and CD4 Tconv were isolated from donor KP59095 and activated by CD3/CD28 TCR and targeted anti-TNFR2 monoclonal antibodies HFB3-1, HFB3 in the presence or absence of 25 ng/mL recombinant human TNFα (rhTNFα) -14 or HFB3-18 stimulated isolated primary T cells. Anti-TNFR2 antibody binding plates are either supplied with soluble antibodies present in the binding mixture.

All three plate-bound anti-TNFR2 antibodies (HFB3-1, HFB3-14 and HFB3-18) stimulated CD8 T cell proliferation in the presence of 25 ng/mL rhTNFα (see Figure 19 lower left panel). However, only soluble HFB3-1 and HFB3-14 (but not soluble HFB3-18) could stimulate CD8 T cell proliferation (Fig. Required, but probably not, for HFB3-1- and HFB3-14-mediated proliferation of CD8 T cells ( i.e., crosslink-independent).

Similar results were also obtained for CD4 Tconv proliferation under similar conditions (data not shown). Example 6 In the presence of Treg, anti -TNFR2 monoclonal antibody is beneficial to cell proliferation on Teff cell terminal (CD8 and CD4 Tconv)

This experiment confirmed that in the presence of Treg, the target anti-TNFR2 monoclonal antibody can co-stimulate the proliferation of Teff cells (CD8 and CD4 Tconv) together with CD3/CD28-mediated TCR activation.

Briefly, CD3 ⁺ T cells (including CD8 and CD4 Tconv effector T cells) and Treg were isolated from human PBMC, and were co-stimulated by CD3/CD28-mediated TCR activation and target anti-TNFR2 monoclonal antibody for about 4 days, substantially above as described above. Proliferation of total CD4 ⁺ T cells and CD8 ⁺ T cells in the presence of Tregs was determined using the CellTrace ^™ Violet Cell Proliferation Kit from Invitrogen (CTV). CD4 ⁺ T cells Activation of CD8 ⁺ T cells was also determined by measuring the percentage of CD25 ⁺ T cells in the respective T cell populations.

The results in Figure 9 show that the anti-TNFR2 monoclonal antibodies of the invention ( e.g. HFB3-1hz6-hG1AA, which is a humanized form of HFB3-1, see below) still favor effector T cells (CD8 and Cell proliferation on CD4 Tconv). Example 7 Anti -TNFR2 monoclonal antibody has negligible ADCC effect on HH lymphoma cells

This experiment confirmed that the target anti-TNFR2 monoclonal antibodies had negligible ADCC effect on T-cell lymphoma, which indicated that these antibodies are suitable as T-cell co-stimulators.

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 one mechanism by which antibodies can be used to limit and suppress infection as part of the humoral immune response. ADCC requires effector cells, a class known as natural killer (NK) cells that normally interact with IgG antibodies.

In this experiment, Jurkat.CD16V/NFAT/luc cells were used as effector cells and HH lymphoma cell line as target cells. The ratio of effector cells to target cells was about 6:1. The co-cultured effector cells and target cells were incubated at 0 nM, 0.0066 nM, 0.66 nM or Incubate overnight at a concentration of 66 nM. The antibody moganulizumab was used as a positive control for ADCC.

The results in Figure 10 show that the ADCC effect of the positive control antibody, mogamizumab, on target cells was at least 120 times stronger than that of any of the tested anti-TNFR2 monoclonal antibodies. The data demonstrate that the subject anti-TNFR2 antibody is suitable as a T cell co-stimulator due to its low/no ADCC effect on T cells. Example 8 Binding of humanized anti -TNFR2 monoclonal antibody to TNFR2

Generated multiple humanized monoclonal antibodies against HFB3-1, HFB3-14 and HFB3-18, including at least 20 against HFB3-1, 16 against HFB3-14 and one against HFB3-18 (this is attributed to The selected human germline is highly similar to the parental HFB3-18 monoclonal antibody coding sequence). The ability of the humanized monoclonal antibodies to bind human TNFR2 expressed on CHO cells was determined essentially as described in Example 1.

Figure 12A shows that humanized HFB3-1hz6, HFB3-14hz1c and HFB3-18hz1 bind to CHO cells expressing human TNFR2 (CHO.hTNFR), but not to parental CHO cells. Figure 12B shows that at least seven humanized HFB3-1 antibodies (i.e., HFB3-1hz6, HFB3-1hz8, HFB3-1hz9, HFB3-1hz10, HFB3-1hz11, HFB3-1hz12, and HFB3-1hz14) and at least eight humanized HFB3 -14 antibodies (i.e., HFB3-14hz1c, HFB3-14hz2c, HFB3-14hz3c, HFB3-14hz4c, HFB3-14hz6c, HFB3-14hz7c, HFB3-14hz12c, and HFB3-14hz14c) remain approximately identical to the respective parental chimeric antibodies (if not Better) level of binding affinity to CHO cells expressing TNFR2 (CHO.hHFB3).

Similar experiments were repeated using instead CHO cells expressing the rhesus monkey xenolog of TNFR2 (CHO.mkHFB3). Figure 13 shows that the general trend of binding to CHO cells expressing monkey TNFR2 matches that of CHO.hTNFR2. However, slightly unstable binding was observed for two humanized variants based on HFB3-14, namely HFB3-14hz2c and HFB3-14hz3c.

The binding of humanized anti-TNFR2 antibodies is specific for TNFR2 and not specific for TNFR1. ELISA analysis in Figure 14A confirmed that humanized monoclonal antibodies HFB3-1hz6, HFB3-14hz1c and HFB3-18hz1 bind to recombinant human and cynomolgus TNFR2 (hTNFR2-His and cynoTNFR2-His, respectively) without recognizing recombinant human TNFR1 (hTNFR1-His). In addition, the binding EC50 of these humanized anti-TNFR2 antibodies to recombinant human and cynomolgus TNFR2 ranged from sub to single digit nM.

The binding affinity of the humanized variants to human TNFR2 was also verified using recombinant human TNFR2 protein and an AHC biosensor. Anti-human IgG Fc capture (AHC) biosensors enable kinetic characterization of macromolecular interactions between human Fc-containing proteins ( eg, target antibodies) and target analytes ( eg, recombinant human TNFR2). Immobilization of human Fc-containing proteins is achieved via factory-immobilized anti-human Fc-specific antibodies whose high affinity for the human Fc domain provides the stable baseline required for demanding kinetic applications . In this particular experiment, test antibodies (humanized versus parental chimeric antibodies) were loaded at a concentration of 20 μg/mL in assay buffer (PBS (pH 7.4), 0.1% BSA, 0.1% Tween20). The analyte was 500 nM, 167 nM or 55.7 nM of His-tagged recombinant human TNFR2. Run capture assays at 25 °C.

As shown in Figure 14B, there were no major differences in distinguishing the humanized variants from their respective chimeric parental antibodies based on affinity for recombinant human TNFR2.

Example 3 shows that chimeric anti-TNFR2 antibodies bind to TCR activated T cells. Essentially the same experiment was run on the humanized variants and the results are shown in FIG. 15 .

Specifically, based on binding to TCR-activated CD8 cells, most humanized HFB3-1 antibodies display sub-nM levels of affinity and do not appear to bind the two variants of TCR-activated CD8 cells (HFB3-1hz5 and HFB3-1hz7 )except. Meanwhile, all humanized HFB3-14 variants exhibited single-digit nM affinities for TCR-activated CD8 T cells. There were no major differences distinguishing the different variants. It should be noted that positive control antibodies SBT-2 and SBT-3 are not good binders of primary CD8 cells. Example 9 Co-stimulatory effect of humanized anti -TNFR2 monoclonal antibody on the proliferation of TCR- activated CD4 and CD8 T cells

Example 5 shows that the co-stimulatory effect of chimeric anti-TNFR2 monoclonal antibodies proliferates isolated human primary CD8 and CD4 Tconv cells. This experiment demonstrates that humanized variants of HFB3-1 and HFB3-14 are identical in TCR-activated CD4 T cells.

In particular, Figure 16 shows that the humanized variants HFB3-1hz5, HFB3-1hz6, HFB3-1hz8, HFB3-1hz10, HFB3-1hz11 and HFB3-1hz12 strongly stimulate TCR activation based on the CTV proliferation assay (see above) CD4 T cells, each to a greater extent than the parental HFB3-1 chimeric antibody. The same process was repeated for HFB3-14hz1c and HFB3-14hz3c variants.

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

Confirmed co-stimulatory data for HFB3-1hz6-hG1, HFB3-14hz1c-hG1 and HFB3-18hz1-hG1 were also obtained to show that these variants have the ability to proliferate TCR-activated CD8 T cells (activated by CD3/CD28 stimulation) co-stimulatory effect. In particular, both the parental chimeric antibody and selected humanized variants enhanced CD8 T cell proliferation stimulated by CD3/CD28 TCR activation. In addition, cooperation of TNF[alpha] (right panel) further enhanced anti-TNFR2 antibody-mediated CD8 proliferation. See Figure 20. Example 10 Certain Humanized Anti -TNFR2 Monoclonal Antibodies Induce NFκB Signaling in Tregs

Example 4 shows that binding of certain chimeric anti-TNFR2 monoclonal antibodies to primary CD8 and CD4 Tconv cells co-stimulates NFKB signaling. Similar experiments here showed that certain humanized variant anti-TNFR2 antibodies induce NFKB signaling in Tregs.

In particular, Figure 17A shows that co-stimulation of Tregs to produce LTA, TNF, and NFKB downstream signaling in TNF AIP3 using certain humanized variant anti-TNFR2 antibodies and TNFα. The variants HFB3-1hz6, HFB3-1hz9, HFB3-1hz10 and HFB3-1hz11 promoted NFKB signaling to a greater extent than the parental chimeric antibody HFB3-1. Meanwhile, the variants HFB3-14hz1c, HFB3-14hz2c, HFB3-14hz3c and HFB3-14hz4c (especially HFB3-14hz1c and HFB3-14hz3c) also promoted NFκB signaling to a greater extent than the parental chimeric antibody HFB3-14. Additionally, Figure 17B shows activation of NFKB signaling in CD8 T cells using a humanized variant of the HFB3-1 antibody with and without human recombinant TNFα. Example 11 anti- TNFR2 antibody system stable

To confirm that the subject humanized anti-TNFR2 antibodies are stable upon storage and thus suitable for further development as therapeutics, various developability assays were run on selected humanized antibodies.

In the first experiment, selected target humanized antibodies were stored in PBS (pH 7.4) at 25°C or 40°C, and the stability of each antibody was determined on day 7 and day 14. The results in Figure 18 show that, except for one variant, HFB3-14hz4c-hG1AA, all antibodies tested were stable under the conditions tested.

In a second experiment, the same antibody was tested for stability at low pH conditions (100 mM AcH, pH 3.5, 25° C.) for 0 hours, 3 hours and 6 hours. The results in Figure 18 again show that, except for one variant, HFB3-14hz4c-hG1AA, all antibodies tested are stable under the conditions tested.

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

Similar experiments were repeated for HFB3-1hz6-hG1, HFB3-14hz1c-hG1 and HFB3-18hz1-hG1. 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 suggest that the subject variant humanized anti-TNFR2 monoclonal antibodies do not present major developability issues and are suitable for use as therapeutic antibodies. Example 12 Anti- TNFR2 antibodies in humanized TNFR2 knock-in (KI) mice and their effects on T cells

To better demonstrate the therapeutic efficacy of the target anti-TNFR2 antibody, humanized TNFR2 knock-in (KI) mice were generated on the C57BL/6 mouse background through a commercial service (Biocytogen, Wakefield, MA).

In the first series of experiments, the association of selected humanized anti-TNFR2 antibodies with KI mouse CD3 T cells (TNFR2 KI CD3 T cells ) in vitro combination. The results showed that 1 μg/mL KI mouse CD3 activated splenocytes better than 0.2 μg/mL CD3. Human TNFR2 expression on KI CD3 ⁺ T cells can be detected, which expression/detection can be enhanced by TNFα and under mild (0.2 μg/mL) CD3 stimulation. In addition, a single dose of 200 nM of each of the six anti-TNFR2 antibodies ( ie HFB3-1, HFB3-14 and HFB3-18 and their humanized variants HFB3-1hz6, HFB3-14hz1c and HFB3-18hz1) did not A discernible difference in TNFR2 binding was shown, which may be due to saturating levels of binding. Data not shown.

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

The ability of the target anti-TNFR2 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 not as pronounced as that from human T cells, HFB3-1-hG1 and its humanized variant HFB3-1hz6-hG1 did induce a greater response than the other antibodies ( See Figure 21). It should be noted that induction of signals from the HFB3-18 series is expected to be lacking.

The pharmacokinetic (PK) characteristics of the humanized anti-TNFR2 monoclonal antibodies (HFB3-1hz6-hG1, HFB3-141c-hG1 and HFB3-18hz1-hG1) in C57BL/6 mice were examined. All three humanized monoclonal antibodies exhibited a T _1/2 as expected for a well-performing antibody. See below. T1/2 Elimination period 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 days to 8.6 days Example 13 Effect of humanized HFB3-1hz6-hG1 on natural killer (NK) cell activation in vitro

This experiment demonstrates that the subject humanized HFB3-1hz6-hG1 antibody co-stimulates natural killer (NK) cells in the presence of NK cell activation by IL-2/IL-15 or via CD3/CD28.

In one experiment, NK cells were isolated from peripheral blood mononuclear cells (PBMC) supplied by two human patients using an NK cell isolation kit (Miltenyi Biotec). NK cells were first stimulated for 24 hours by soluble IL-2 (10 ng/mL) and IL-15 (10 ng/mL), and then treated with isotype control antibodies, mouse HFB3-1-hG1, humanized HFB3-1 - hz6-hG1 or anti-OX40 control antibody (BMS) (22 nM, 66 nM or 200 nM, respectively) for 16 hours. At the end of the experiment, the expression of CD107α representing NK cell degranulation and activation and the expression of TNFR2 on the surface of NK cells 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-OX40 antibodies were unable to promote short-term activation of NK (40 hours since stimulation with IL-2/IL-15), which may be due to insufficient OX40 expression.

In another experiment, two human patient-donated whole PBMCs were co-stimulated for 48 hours with plate-bound anti-CD3 (1 μg/mL) and soluble anti-CD28 (1 μg/mL) and then treated with isotype control antibodies, Mouse HFB3-1-hG1, humanized HFB3-1-hz6-hG1 or anti-OX40 antibody (BMS) (22 nM, 66 nM or 200 nM, respectively) were treated for 16 hours. CD107α expression was determined for CD3 negative/CD56 positive ( ie NK cells). See Figure 23.

Similarly, HFB3-1-hG1 and HFB3-1-hz6-hG1 significantly increased CD107α expression in a dose-dependent manner, indicating that these antibodies can promote NK cell activation in whole PBMCs. Under prolonged activation (64 hours since anti-CD3/CD28 stimulation), anti-OX40 antibodies were able to activate NK cells.

These data show that both humanized HFB3-1-hz6-hG1 and parental mouse HFB3-1-hG1 can promote NK cell activation. Example 14 Pharmacodynamics of humanized HFB3-1hz6-hG1 in MC38 tumor model

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

After only 2 doses of treatment on Day 0 and Day 3, there was no significant difference in tumor weight between treatments (Fig. 24B upper left panel). Administration of HFB3-1-hG1 at 10 mg/kg increased the absolute number of CD45+ cells present in the tumor (Figure 24B lower left panel), but the percentage of CD45+ in viable tumor cells was not significantly increased (Figure 24B lower right panel). Treatment with HFB3-1-hG1 at 10 mg/kg also increased the absolute cell numbers of CD8+, habitual CD4+ T and NK cells in the tumor microenvironment, but did not change the number of T regulatory cells ( FIG. 24C ). Administration of other lower doses of HFB3-1-hG1 did not produce any observable effects.

The TNFR2 receptor occupancy of CD8 T cells, customary CD4 T cells, T regulatory cells and NK cells in tumors and peripheral blood was determined. In tumors, only the 10 mg/kg dose of HFB3-1-hG1 produced drug receptor occupancy on T cells in tumors; no occupancy was observed for 1 mg/kg and 0.1 mg/kg doses (see Figure 25A). However, at 1 mg/kg and 10 mg/kg, receptor occupancy was observed in tumor NK cells. In peripheral blood, 10 mg/kg and 1 mg/kg doses of HFB3-1-hG1 produced comparable drug receptor occupancy, and no significant occupancy was observed at the 0.1 mg/kg dose.

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

In conclusion, the data on the short-term treatment of mice with HFB3-1-hG1 highly suggest that HFB3-1-hG1 has the ability to stimulate the activation and proliferation of immune cells, efficiently binds to the TNFR2 receptor on immune cells, and has good in vivo activity in the blood. Potential for retention. Synergistic anti-tumor efficacy of Example 15 and anti -PD-1 antibody

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

Specifically, 8-week-old humanized TNFR2 KI mice were inoculated with approximately 5×10 ⁵ MC38 tumor cells (derived from C57BL6 murine colon adenocarcinoma)/mouse. After about 7 days, on day 0, the average tumor size in mice reached about 89 mm ³ (between 74-98 mm ³ ). Mice were then randomized into 5 experimental groups (n=8/group) to be administered one of the following: (1) isotype-matched control (TT-hG1AA); (2) anti-mPD-1 (RMP- 1-14); (3) HFB3-1hz6-hG1; (4) HFB3-14hz1c-hG1; and (5) HFB3-18hz1-hG1. The antibody was injected intraperitoneally ( ip ) at a dose of approximately 10 mg/kg on days 0, 3, 6, 9, 12, 15 and 18, for a total of 7 doses (Q3D ,×7). The tumor volume of the experimental group was measured during the course of the study. On or about day 21, the mean tumor volume of the isotype control group reached >2000 mm ³ and the experiment was terminated and all mice sacrificed. Tumor volume over time for each group is plotted in Figure 27A and Figure 27B. By day 21, statistical significance of tumor growth inhibition (TGI) was achieved in the group of mice receiving HFB3-1hz6, HFB3-18hz1 and anti-PD-1 (RMP-14) ( FIG. 27B ).

The results showed that the humanized antibodies HFB3-1hz6 and -hG1 and HFB3-18hz1-hG1 inhibited tumor growth as potently (if not better) than the anti-mPD-1 antibody, while the other humanized antibody was equally effective but to a lesser extent Low. No difference in apparent body weight was observed among the different experimental mouse groups.

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

In addition, in the MC38 tumor model, HFB3-1hz6-hG1 and anti-PD-1 antibody synergistically inhibit tumor growth and prolong the lifespan of mice. Specifically, humanized TNRF2 KI mice were inoculated with MC38 cancer cells on day -7. From day 0, mice were injected intraperitoneally with isotype control, HFB3-1hz6-hG1 or anti-mPD-1 antibody alone or in combination every 3 days (n=8/group). Treatment with 3 mg/kg and 10 mg/kg HFB3-1hz6-hG1 every 3 days for a total of 7 doses (Q3d × 7) and anti-PD-1 10 mg/kg every 3 days ( RMP-14) treatment with a total of 4 doses (Q3d × 4) significantly inhibited tumor growth and prolonged the lifespan of mice. Additionally, combination treatment with HFB3-1hz6-hG1 (10 mg/kg, Q3d × 7) and anti-PD-1 antibody (10 mg/kg, Q3d × 4) resulted in better survival than treatment with anti-PD-1 antibody alone. See Figure 29. Data were analyzed using ANOVA comparing treatment groups to isotype controls. Anti-tumor efficacy of HFB3-1hz6-hG1 in example 16 hepatoma syngeneic mouse model

In the Hepa1-6 syngeneic mouse model, ^when tumor volumes reached approximately 100 mm, treatment with an isotype control antibody, anti-mPD-1 at 10 mg/kg, or HFB3-1hz6-hG1 at doses of 0.3-10 mg/kg mice. HFB3-1hz6-hG1 can effectively inhibit tumor growth. At a dose of 10 mg/kg, HFB3-1hz6-hG1 controlled tumor growth even more effectively than anti-mPD-1 (see Figure 32). Toxicological Assessment of Anti -TNFR2 Antibodies in Example 17 Non-Human Primates

The toxicology of humanized anti-TNFR2 antibodies was examined using a non-human primate model. Single doses of 15 mg/kg (low), 50 mg/kg (medium) and 150 mg/kg (high) of humanized HFB3-1hz6-hG1 monoclonal antibody were injected into two cynomolgus monkeys/group, and then in different Plasma was collected at time points up to 336 hr (day 14).

Toxicokinetic analysis of HFB3-1hz6-hG1 showed that the antibody was eliminated over time. Compared with the reported data for CD3×CD20 bispecific IgG at ≤ 3 mg/kg (dotted line), no Interleukins IL-6, IL-2, IFN-γ and TNF-α increased (Figure 30).

After injection of 15 mg/kg, 50 mg/kg or 150 mg/kg of HFB3-1hz6-hG1, compared with the historical data range of normal monkeys, no differences in white blood cells, red blood cells, platelets, neutrophils and lymphocytes were found. Abnormal quantity (Figure 31).

Toxicological evaluations to date have shown no discernable toxic effects from treatment of non-human primate subjects with HFB3-1hz6-hG1 at doses up to 150 mg/kg.

In a dose ranging study (DRF) of multiple doses of HFB3-1hz6-hG1 administered to cynomolgus monkeys, IL-2, IL-4, IL-5, TNFα, and IFN-γ in monkeys were repeatedly dosed. No change up to 150 mg/kg. In monkeys at the end of the 4-week dose, changes in IL-6 levels were observed in 10 mg/kg and 150 mg/kg male animals. A dose-dependent decrease in neutrophil and platelet counts was observed in monkeys 2 weeks after HFB3-1hz6-hG1 administration. Diarrhea (liquid or loose stools) was frequently observed in monkeys following weekly dosing of HFB3-1hz6-hG1.

Based on the above observations, the drug half-life following a single 1 mg/kg dose injection in humans is expected to be 23 days, and the antibody would be suitable for dosing at 1 mg/kg every four weeks. Example 18 Selection of indications 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-TNFR2 antibodies of the invention arises from the stimulation of tumor-infiltrating T cells and NK cells by TNFR2, thereby activating NK cells and enhancing CD8+ T cell-mediated antitumor activity. tumor response. This example provides evidence that tumor types that would potentially benefit from treatment with anti-TNFR2 antibodies of the invention include tumors expressing high TNFR2 and high CD8A.

In the bulk RNA analysis of cancer using the TCGA database, the CD8A cut-off value was based on the CD8A level of acute myeloid leukemia (AML), which is presumed to be mainly composed of myeloid cells and low or no CD8+ T cells. TNFR2 cut-off values are based on TNFR2 levels in prostate cancer, which is assumed to be an immune desert. See Figures 34A-34B.

The grading of 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 cancer (including cervical squamous cell carcinoma, pleural mesothelioma, lung adenocarcinoma and head and neck cancer) squamous cell carcinoma) has been identified as the top TNFR2/CD8A high cancer.

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

Renal cell carcinoma (RCC), cutaneous melanoma of the skin (SKCM), gastric adenocarcinoma/gastric carcinoma (STAD/GI), lung adenocarcinoma (LUAD), and head and neck squamous cell carcinoma (HNSC) were further identified using publicly available data. ) TNFR2 and CD8 scores of molecular subtypes ( FIG. 36 ).

Notably, in each cancer type tested, there were subtypes with high percentages of cancers within subtypes that exhibited the hallmark high CD8A and high TNFR2 expression pattern (e.g., approximately 60% STAD/ GI - EBV+ cancers have this hallmark high expression), while other subtypes (such as ESCC subtype and HM-SNV subtype) have almost no CD8A ^hi TNFR2 ^hi expression.

Thus, the tested cancer subtypes with the hallmark high CD8A and high TNFR2 expression patterns are prime candidates for beneficial treatment by the subject antibodies of the 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.Stroma (PD-L1/CD274 tends to be higher).

In patients with colorectal cancer, those with defective mismatch repair (dMMR)/microsatellite instability-high ( MSI-H) tumors were significantly more sensitive to immune checkpoint inhibitors (ICIs), and the former group of patients obtained more immune benefit from immunotherapy than the latter group of patients.

Since MSI score data for all cancer indications are not directly available, applicants used the parameter mutation count as a surrogate for MSI score and investigated whether TNFR2 ^hi and CD8A ^hi tumors that could be treated by the target antibody were enriched in MSI versus MSS. For this purpose, mutation counts >250 were considered MSI, while mutation counts <250 were considered MSS. Data (not shown here) show that TNFR2 ^hi and CD8A ^hi expression patterns in COAD data (from The Cancer Genome Atlas (TCGA)-CRC Colon Adenocarcinoma (COAD) cohort) with mutation counts >250 versus <250 and Not strongly enriched. In MSI (all but 4% CD8A ^hi ), TNFR2 ^hi was in roughly equal proportion to TNFR2 ^lo . The ratio in MSS (all CD8A ^lo ) becomes 45% vs. 55%. Example 19 : Dosing studies of HFB3-1hz6-hG1 in humans

In part by the minimum expected biological effect levels from in vitro cytokine release assays, the minimum pharmacologically active doses in human TNFR2 knock-in (hTNFR2 KI) mice bearing MC38 tumors, and human and non-human primate The starting dose of HFB3-1hz6-hG1 in this study was 5 mg administered every 4 weeks (Q4W) as a 60-minute IV infusion, as informed by data on the highest dose without serious toxicity in animals.

The dose was escalated to 150 mg to determine the maximum tolerated dose. Dosing for EBV+ gastric cancer, clear cell renal cell carcinoma, cutaneous melanoma, soft tissue sarcoma, testicular germ cell tumor, and PD-L1+ cancer (including cervical cancer, pleural mesothelioma, lung adenocarcinoma, head and neck squamous cell carcinoma) expand. Additional patient cohorts will be enrolled based on Phase I antitumor activity/efficacy.

none

Figure 1 shows 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. Sequence alignment of VH and VL regions of HFB3-24 and HFB3-25, and their consensus sequences. Figure 2A shows the binding affinities of selected human-mouse chimeric monoclonal antibodies raised against the extracellular domain of recombinant human TNFR2. _EC50 and _Emax values of test antibodies and isotype-matched negative control antibodies were measured against CHO cells expressing human TNFR2 (CHO.hHFB3) or rhesus monkey TNFR2 (CHO.mkHFB3). Figure 2B shows that different anti-TNFR2 monoclonal antibodies can promote (HFB3-1) or inhibit (HFB3-18) the binding of TNFα to TNFR2, or have no effect on the binding (HFB3-6). Figure 3 shows that the human-mouse chimeric monoclonal antibody does not bind to the parental CHO cell line and does not bind to CHO cells expressing mouse TNFR2 (except for marginal binding of the HFB3-18 and HFB3-19 antibodies). Figure 4A shows the binding specificity of human-mouse chimeric antibodies specific for TNFR2 but not TNFRl. Figure 4B shows the K _d , k _association and k _dissociation values of human-mouse chimeric antibodies HFB3-1, HFB3-14 and HFB3-18 on His-tagged recombinant human TNFR2. Figure 5 shows the expression of TNFR2 on T cell subsets in tumor infiltrating lymphocytes, especially exhausted CD8 T cells. Figure 6 shows cell binding of anti-TNFR2 chimeric monoclonal antibody on TCR activated (bottom panel) and non-TCR activated (top panel) primary Treg, CD8 and CD4 Tconv. HFB3 antibody can preferentially recognize primary T cells activated by CD3/CD28 co-stimulation (TCR activation). Figure 7 shows that certain HFB3 antibodies of the invention (including HFB3-1, HFB3-14, HFB3-18, HFB3-23, HFB3-24, and HFB3-25) trigger NFκB signaling and in the presence of TNFα ligands can enhance this effect. Figure 8 shows that co-stimulation of certain target monoclonal antibodies (including HFB3-1, HFB3-14, HFB3-18 and HFB3-25) and CD3/CD28 caused CD8 and CD4 Tconv to proliferate in a dose-dependent manner. Figure 9 shows that the anti-TNFR2 monoclonal antibody of the present invention (such as HFB3-1hz6-hG1AA, which is a humanized form of HFB3-1) favors effector T cells (CD8 and CD4 Tconv) in the presence of Treg in a dose-dependent manner of cell proliferation. Figure 10 shows the lack of ADCC effect of the subject anti-TNFR2 antibodies. Figures 11A and 11B show various features of the His-tagged extracellular domain (ECD) of TNFR2 (referred to as HFB2003), including the TNFα binding site, and monoclonal antibodies HFB3-1 and HFB3-14 and HFB3- 18 (FIG. 11A) or HFB3-6 (FIG. 11B) epitope mapping results. These antibodies are mouse chimeric antibodies with the Fc region of human IgG1 and are therefore also referred to as HFB3-1-hG1, HFB3-14-hG1, HFB3-18-hG1 or HFB3-6-hG1, respectively. Figure 1 IB also includes epitope mapping data for benchmark antibodies SBT-1 and SBT-4 (Benchmarks 1 and 2). The HFB3-1 antibody binds to the CRD2 region of the ECD, HFB3-14 and HFB3-6 bind to the CRD3 region of the ECD, and HFB3-18 binds to the CRD1 region of the ECD. Figure 11C shows more refined epitope mapping data for HFB3-1; a potential HFB3-1 hG1 epitope region (SEQ ID NO: 101 ) identified in 2 independent experiments is highlighted. Figure 1 ID provides a 3-D model showing the binding sites of HFB3-1, HFB3-14, HFB3-6 and HFB-3-18 on the TNFR2-TNFα complex. Figure 12A shows that humanized variants of chimeric monoclonal antibodies HFB3-1, HFB3-14, and HFB3-18 bind to CHO cells expressing human TNFR2 (CHO.hTNFR2), but not to parental CHO cells. Figure 12B shows the binding affinities of selected humanized anti-TNFR2 mAbs. The _EC50 values of the test humanized antibody and the parental chimeric antibody were measured against CHO cells expressing human TNFR2 (CHO.hHFB3). Figure 13 shows the binding affinities of selected humanized anti-TNFR2 monoclonal antibodies. The _EC50 values of the humanized antibody and the parental chimeric antibody were measured against CHO cells expressing rhesus monkey TNFR2 (CHO.mkHFB3). Figure 14A shows that humanized anti-TNFR2 antibodies bind recombinant human and cynomolgus TNFR2, but not recombinant human TNFRl, in an ELISA assay. Figure 14B shows the results of the binding affinities of humanized variants and parental chimeric monoclonal antibodies HFB3-1 and HFB3-14 to recombinant human TNFR2 based on AHC (anti-human IgG Fc capture) biosensor measurements. Values are the mean of two experiments obtained on two different days. Figure 14C shows the binding specificity of an exemplary humanized antibody HFB3-1hz6-hG1 to TNRF2 expressing/positive CHO cells (CHO.hTNFR2) compared to parental CHO cells (Bmk 1: Benchmark Antibody 1). Figure 15 shows the cellular binding of humanized anti-TNFR2 monoclonal antibody to TCR-activated CD8 T cells. Figure 16 shows the co-stimulatory effect of humanized anti-TNFR2 monoclonal antibody on the proliferation of TCR-activated CD4 T cells. Figure 17A shows that co-stimulation of Treg with certain humanized variant anti-TNFR2 antibodies and TNF[alpha] elicits NF[kappa]B downstream signaling. Figure 17B shows activation of NFKB signaling in CD8 T cells using a certain humanized variant of the HFB3-1 antibody with and without recombinant human TNFα. "*" indicates statistical significance. Figure 18 shows that the subject humanized variant anti-TNFR2 antibodies are stable upon storage. Figure 19 shows the FcγR cross-linking dependence of anti-TNFR2 monoclonal antibody HFB3-18 (but not HFB3-1 and HFB3-14) on costimulatory primary T cells. Figure 20 shows the confirmed co-stimulatory effect of selected humanized anti-TNFR2 antibodies on the proliferation of CD8 T cells in the presence or absence of TNFα. Figure 21A shows that target anti-TNFR2 mAbs co-stimulate downstream NFκB signaling ex vivo in the presence of CD3/CD28-mediated TCR activation in humanized TNFR2 knock-in CD8 and CD4 Tconv cells in the presence of 25 ng/mL TNFα. Figure 21B shows that humanized HFB3-1hz6 binds to peripheral CD4 and CD8 T cells (top panel) and stimulates T cell proliferation in vitro (bottom panel) in a dose-dependent manner in the presence of CD3/CD28-mediated TCR activation. Figure 22 shows that after stimulation with soluble IL-2 (10 ng/mL) and IL-15 (10 ng/mL), humanized HFB3-1hz6-hG1 antibody and parental HFB3-1-hG1 antibody against isolated natural killer In vitro activation of (NK) cells. The top image shows the timeline of the experiment. CD107α and TNFR2 expression were upregulated by HFB3-1hz6-hG1 and HFB3-1-hG1 in a dose-dependent manner, but isotype control and anti-OX40 antibody (BMS) failed to trigger short-term NK activation. Figure 23 shows the response of HFB3-1hz6-hG1 and parental mouse HFB3-1-hG1 to whole peripheral blood mononuclear cells after stimulation with plate-bound anti-CD3 (1 µg/mL) and soluble anti-CD28 (1 µg/mL). Ex vivo activation of natural killer (NK) cells in cellular fractions. The top image shows the timeline of the experiment. In CD3 ⁻ /CD56 ⁺ cells, CD107α expression was upregulated by HFB3-1hz6-hG1 and HFB3-1-hG1 in a dose-dependent manner, but control anti-OX40 antibody (MBS) failed to trigger short-term NK activation. Figure 24A shows a timeline of pharmacodynamic experiments in the mouse MC38 tumor model. Two doses of HFB3-1-hG1 (0.1 mg/kg, 1 mg/kg and 10 mg/kg doses) or 10 mg/kg of an isotype-matched control antibody (TT) were administered intraperitoneally at 3 days interval. Figure 24B shows the in vivo effect of antibody administration on total immune cell counts in MC38 tumors. Administration of HFB3-1-hG1 at 10 mg/kg increased the absolute cell number of CD45 ⁺ cells. Based on one-way ANOVA test, p -value <0.05 (*). Figure 24C shows the in vivo effect on cell counts of different immune cells in MC38 tumors. Administration of HFB3-1-hG1 at 10 mg/kg increased the absolute cell numbers of CD8 ⁺ , habitual CD4 ⁺ T and NK cells in the tumor microenvironment, but did not change the number of T regulatory cells. *p-value < 0.05 based on one-way ANOVA test. Figure 25A shows the percentage of TNFR2 receptors on tumor infiltrating leukocytes occupied by 0.1 mg/kg, 1 mg/kg and 10 mg/kg doses of injected antibody HFB3-1-hG1 or 10 mg/kg control antibody. Only the 10 mg/kg dose of HFB3-1-hG1 produced drug receptor occupancy. p-value < 0.05 (*), 0.01 (**) or 0.001 (***) based on one-way ANOVA test. Figure 25B shows the percentage of TNFR2 receptors occupied by 0.1 mg/kg, 1 mg/kg and 10 mg/kg doses of injected antibody HFB3-1-hG1 or 10 mg/kg control antibody on selected peripheral blood cells. HFB3-1-hG1 at doses of 10 mg/kg and 1 mg/kg produced comparable drug receptor occupancy. p-value < 0.05 (*), 0.01 (**) or 0.001 (***) based on one-way ANOVA test. Figure 26A shows the antibody concentration in blood on day 4 of the experiment in Figure 24A. HFB3-1-hG1 was detectable in blood at doses of 10 mg/kg and 1 mg/kg. p -value < 0.001 (***) or 0.0001 (****) based on one-way ANOVA test. Figure 26B shows soluble TNFR2 in blood on day 4 of the experiment in Figure 24A. Administration of HFB3-1-hG1 at 10 mg/kg and 1 mg/kg increased the amount of TNFR2 detectable in blood. p-value < 0.001 (***) or 0.0001 (****) based on one-way ANOVA test. Figures 27A and 27B show that humanized mAbs such as HFB3-1hz6 and HFB3-18hz1 have similar therapeutic potency compared to rat anti-mPD-1 mAbs. Figure 28 shows that the humanized HFB3-1hz6 mAb has therapeutic efficacy in the MC38 tumor model, as does the mouse anti-mPD-1 mAb. Figure 29 shows that at two different doses (3 mg/kg and 10 mg/kg), the humanized HFB3-1hz6 monoclonal antibody inhibited tumor growth and increased the lifespan of tumor-bearing mice, compared with anti-mPD alone Combination treatment with HFB3-1hz6 and anti-mPD-1 antibody prolonged survival better than treatment with -1. Figure 30A shows that the humanized HFB3-1hz6 monoclonal antibody was eliminated from the body of cynomolgus monkeys over time (left panel), and anti-drug antibodies (ADA) were observed at about 2 weeks after injection (right panel), which was observed in non- Common among human primates. Figure 30B shows that after injection of 15 mg/kg, 50 mg/kg, or 150 mg/kg of HFB3-1hz6-hG1, compared with the reported data (dashed line) of ≤ 3 mg/kg of CD3×CD20 bispecific IgG Ratio, no increase in cytokines was observed. Figure 31 shows the cell counts after injection of 15 mg/kg, 50 mg/kg or 150 mg/kg of HFB3-1hz6-hG1 compared to the range of historical data in normal monkeys (left and right lines in each figure) analyze. Figure 32 shows that the humanized HFB3-1hz6 monoclonal antibody has anti-tumor efficacy in the Hepa1-6 tumor model. Figures 33A-33C show the TCGA database based on TNFR2 levels, cutaneous melanoma (SKCM, Figure 33A), head and neck squamous cell carcinoma (HNSC, Figure 33B) and thymoma (THYM, Figure 33C) patients. Kaplan Meier survival curve. Higher TNFR2 expression was significantly associated with improved survival in melanoma and HNSC patients, but not favorably in THYM. Figures 34A and 34B show examples of TCGA bulk RNA analysis of patients with various cancers of 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 (but not TNFR2) expression and can be used as a negative control for increased CD8A expression in selected cancer types. Figure 35 shows TCGA grading of cancer types with high TNFR2 expression (compared eg to TNFR2 expression in prostate cancer) and high CD8A expression (eg compared to CD8A expression in AML) based on the ratio of TNFR2high/CD8Ahigh patient samples . ACC = adrenocortical carcinoma; BLCA = urothelial carcinoma of the bladder; BRCA = invasive carcinoma of the breast; CESC = squamous cell carcinoma/endocervical adenocarcinoma of the cervix; CHOL = biliary tract carcinoma; COAD = adenocarcinoma of the colon; EBV = Epstein-Barr Virus; ESCA = Esophageal Cancer; GBM = Glioblastoma Multiforme; HNSC = Squamous Cell Carcinoma of the Head and Neck; KICH = Kidney Chromophobe Cells; KIRP = renal papillary cell carcinoma of the kidney; LGG = low-grade glioma of the brain; LIHC = hepatocellular carcinoma of the liver; LUAD = adenocarcinoma of the lung; LUSC = squamous cell carcinoma of the lung; MESO = mesothelioma of the pleura; OV = Serous cystadenocarcinoma of the ovary; PAAD = pancreatic adenocarcinoma; PCPG = pheochromocytoma and paraganglioma; PD-L1 = programmed death ligand 1; PRAD = prostate adenocarcinoma; READ = rectal adenocarcinoma; SARC = sarcoma; SKCM = cutaneous melanoma of the skin; STAD = gastric adenocarcinoma; TGCT = testicular germ cell tumor; THCA = thyroid cancer; . Figure 36 shows molecular subgroups of selected renal cell carcinoma (RCC), cutaneous cutaneous melanoma (SKCM), gastric adenocarcinoma/gastric carcinoma (STAD/GI), lung adenocarcinoma (LUAD) and head and neck squamous cell carcinoma (HNSC). type analysis.

         
           <![CDATA[ <110> HIFIBIO (HK) LIMITED)]]>
           <![CDATA[ <120> Anti-TNFR2 antibody and its use]]>
           <![CDATA[ <140> TW 111125395]]>
           <![CDATA[ <141> 2022-07-06]]>
           <![CDATA[ <150> US 63/219175]]>
           <![CDATA[ <151> 2021-07-07]]>
           <![CDATA[ <160> 101 ]]>
           <![CDATA[ <170> PatentIn version 3.5]]>
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           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 5]]>
          Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala
          1 5 10
           <![CDATA[ <210> 6]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 6]]>
          Gln Gln Trp Ser Ser Asn Pro Pro Thr
          1 5
           <![CDATA[ <210> 7]]>
           <![CDATA[ <400> 7]]>
          000
           <![CDATA[ <210> 8]]>
           <![CDATA[ <400> 8]]>
          000
           <![CDATA[ <210> 9]]>
           <![CDATA[ <211> 448]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 9]]>
          Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Ile Ser Cys Lys Ala Ser Ser Tyr Ser Phe Thr Asp Tyr
                      20 25 30
          Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile
                  35 40 45
          Gly Ile Ile Phe Pro Lys Tyr Gly Thr Thr Ser Tyr Asn Gln Lys Phe
              50 55 60
          Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Thr Asp Gly Gly Thr Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr
                      100 105 110
          Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
                  115 120 125
          Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
              130 135 140
          Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
          145 150 155 160
          Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
                          165 170 175
          Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser
                      180 185 190
          Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
                  195 200 205
          Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
              210 215 220
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
          225 230 235 240
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                          245 250 255
          Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
                      260 265 270
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
                  275 280 285
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
              290 295 300
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
          305 310 315 320
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                          325 330 335
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                      340 345 350
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
                  355 360 365
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
              370 375 380
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
          385 390 395 400
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                          405 410 415
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                      420 425 430
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                  435 440 445
           <![CDATA[ <210> 10]]>
           <![CDATA[ <211> 213]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 10]]>
          Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly
          1 5 10 15
          Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met
                      20 25 30
          Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
                  35 40 45
          Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
              50 55 60
          Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
          65 70 75 80
          Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr
                          85 90 95
          Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
                      100 105 110
          Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
                  115 120 125
          Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
              130 135 140
          Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
          145 150 155 160
          Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
                          165 170 175
          Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
                      180 185 190
          Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
                  195 200 205
          Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 11]]>
           <![CDATA[ <211> 1347]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 11]]>
          gaatttcagc tgcagcagtc tggccccgag ctggttaagc ctggcgcctc tgtgaagatc 60
          agctgcaagg ccagcagcta cagcttcacc gactacaaca tgaactgggt caagcagagc 120
          aacggcaaga gcctggaatg gatcggcatc atcttcccta agtacggcac caccagctac 180
          aaccagaagt tcaagggcaa agccaacactg accgtggacc agagcagcag cacagcctac 240
          atgcagctca acagcctgac cagcgaggac agcgccgtgt actactgtgc tacagatggc 300
          ggcacctggt acttcgatgt gtggggcact ggcaccaccg tgacagttag ttctgcgtcg 360
          accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tggggggcaca 420
          gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480
          tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540
          tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600
          tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660
          tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720
          gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780
          acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840
          gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900
          taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960
          aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020
          aaagggcagc cccgagaacc acagggtgtac accctgcccc catcccggga ggagatgacc 1080
          aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140
          gagtggggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200
          tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1260
          gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320
          agcctctccc tgtccccggg taaatga 1347
           <![CDATA[ <210> 12]]>
           <![CDATA[ <211> 642]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 12]]>
          cagattgtgc tgacacagtc tcccgctctg atgagcgcta gccctggcga gaaagtgacc 60
          atgacatgta gcgccagcag cagcgtgacc tacatgtact ggtatcagca gaagcccaga 120
          agcagcccca agccttggat ctacctgacc agcaatctgg ccagcggagt gcctgccaga 180
          ttttctggct ctggcagcgg cacaagctac agcctgacaa tcagcagcat ggaagccgag 240
          gatgccgcca cctactactg ccagcagtgg tccagcaatc ctcctacatt tggctccggc 300
          accaagctgg aaatcaagcg tacggtggct gcaccatctg tcttcatctt cccgccatct 360
          gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420
          agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480
          agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540
          agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600
          agctcgcccg tcacaaagag cttcaacagg ggagagtgtt ag 642
           <![CDATA[ <210> 13]]>
           <![CDATA[ <211> 19]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 13]]>
          Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu
          1 5 10 15
          Cys Leu Ser
           <![CDATA[ <210> 14]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 14]]>
          Ser Tyr Ser Phe Thr Asp Tyr Asn
          1 5
           <![CDATA[ <210> 15]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 15]]>
          Ile Phe Pro Lys Tyr Gly Thr Thr Ser Tyr Ala Gln Lys Leu Gln Gly
          1 5 10 15
           <![CDATA[ <210> 16]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 16]]>
          Ala Thr Asp Gly Gly Thr Trp Tyr Phe Asp Val
          1 5 10
           <![CDATA[ <210> 17]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 17]]>
          Ser Ser Val Thr Tyr
          1 5
           <![CDATA[ <210> 18]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 18]]>
          Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ser
          1 5 10
           <![CDATA[ <210> 19]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 19]]>
          Gln Gln Trp Ser Ser Asn Pro Pro Thr
          1 5
           <![CDATA[ <210> 20]]>
           <![CDATA[ <400> 20]]>
          000
           <![CDATA[ <210> 21]]>
           <![CDATA[ <400> 21]]>
          000
           <![CDATA[ <210> 22]]>
           <![CDATA[ <211> 448]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 22]]>
          Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Ser Tyr Ser Phe Thr Asp Tyr
                      20 25 30
          Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Met
                  35 40 45
          Gly Ile Ile Phe Pro Lys Tyr Gly Thr Thr Ser Tyr Ala Gln Lys Leu
              50 55 60
          Gln Gly Arg Val Thr Leu Thr Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
          65 70 75 80
          Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Thr Asp Gly Gly Thr Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr
                      100 105 110
          Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
                  115 120 125
          Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
              130 135 140
          Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
          145 150 155 160
          Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
                          165 170 175
          Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser
                      180 185 190
          Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
                  195 200 205
          Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
              210 215 220
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
          225 230 235 240
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                          245 250 255
          Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
                      260 265 270
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
                  275 280 285
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
              290 295 300
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
          305 310 315 320
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                          325 330 335
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                      340 345 350
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
                  355 360 365
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
              370 375 380
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
          385 390 395 400
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                          405 410 415
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                      420 425 430
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                  435 440 445
           <![CDATA[ <210> 23]]>
           <![CDATA[ <211> 213]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 23]]>
          Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Thr Tyr Met
                      20 25 30
          Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr
                  35 40 45
          Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
              50 55 60
          Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
          65 70 75 80
          Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr
                          85 90 95
          Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
                      100 105 110
          Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
                  115 120 125
          Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
              130 135 140
          Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
          145 150 155 160
          Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
                          165 170 175
          Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
                      180 185 190
          Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
                  195 200 205
          Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 24]]>
           <![CDATA[ <211> 1347]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 24]]>
          caggttcagc tggttcagtc tggcgccgag ctgaaaaaac ctggcgcctc tgtgaaggtg 60
          tcctgcaagg ccagcagcta cagcttcacc gactacaaca tgaactgggt ccgacaggcc 120
          cctggccagt ctcttgagtg gatgggcatc atcttcccta agtacggcac caccagctac 180
          gcccagaaac tgcagggaag agtgaccctg accacgaca ccagcacaag caccgcctac 240
          atggaactgc ggagcctgag atccgatgac accgccgtgt actactgtgc cacagatggc 300
          ggcacctggt acttcgatgt gtggggcact ggcaccaccg tgacagtctc ttctgcgtcg 360
          accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tggggggcaca 420
          gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480
          tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540
          tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600
          tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660
          tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720
          gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780
          acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840
          gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900
          taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960
          aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020
          aaagggcagc cccgagaacc acagggtgtac accctgcccc catcccggga ggagatgacc 1080
          aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140
          gagtggggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200
          tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1260
          gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320
          agcctctccc tgtccccggg taaatga 1347
           <![CDATA[ <210> 25]]>
           <![CDATA[ <211> 642]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 25]]>
          gacatccagc tgacccagtc tccaagcttt ctgagcgcca gcgtgggcga cagagtgacc 60
          attacatgta gagccagcag cagcgtgacc tatatgtact ggtatcagca gaagcccggc 120
          aaggccccta agccttggat ctacctgacc agcaatctgg ccagcggcgt gccaagcaga 180
          ttttctggct ctggcagcgg caccgagtac accctgacca tatctagcct gcagcctgag 240
          gatgccgcca cctactattg ccagcagtgg tccagcaatc ctcctacctt tggctccggc 300
          accaagctgg aaatcaagcg tacggtggct gcaccatctg tcttcatctt cccgccatct 360
          gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420
          agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480
          agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540
          agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600
          agctcgcccg tcacaaagag cttcaacagg ggagagtgtt ag 642
           <![CDATA[ <210> 26]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 2]]>6
          Gly Tyr Thr Phe Thr Asp Tyr Tyr
          1 5
           <![CDATA[ <210> 27]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence (Artificia]]>l Sequence)
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 27]]>
          Ile Asn Pro Asn Asp Gly Gly Thr Thr Tyr Ser Gln Lys Phe Lys Gly
          1 5 10 15
           <![CDATA[ <210> 28]]>
           <![CDATA[ <211> 18]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 28]]>
          Ala Arg Glu Gly Asn Tyr Tyr Ala Tyr Asp Val Arg Val Trp Tyr Phe
          1 5 10 15
          Asp Val
           <![CDATA[ <210> 29]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 29]]>
          Gln Asp Ile Ile Thr Tyr
          1 5
           <![CDATA[ <210> 30]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> People]]>Artificial Sequence
           <![CDATA[ <22]]>0>]]&gt;
           <br/> &lt;![CDATA[ <221&gt;Source]]>
           <br/> <![CDATA[ &lt;223>]]&gt; <![CDATA[/comment=“Description of artificial sequence: synthetic peptide”
           <![CDATA[ <400> 30]]>
          Ser Thr Ser Ser Leu Asn Ser Gly Val Pro Ser
          1 5 10
           <![CDATA[ <210> 31]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 31]]>
          Gln Gln Tyr Ser Glu Leu Pro Tyr Thr
          1 5
           <![CDATA[ <210> 32]]>
           <![CDATA[ <400> 32]]>
          000
           <![CDATA[ <210> 33]]>
           <![CDATA[ <400> 33]]>
          000
           <![CDATA[ <210> 34]]>
           <![CDATA[ <211> 455]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 34]]>
          Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
          1 5 10 15
          Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
                      20 25 30
          Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
                  35 40 45
          Gly Asp Ile Asn Pro Asn Asp Gly Gly Thr Thr Tyr Ser Gln Lys Phe
              50 55 60
          Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
                          85 90 95
          Ala Arg Glu Gly Asn Tyr Tyr Ala Tyr Asp Val Arg Val Trp Tyr Phe
                      100 105 110
          Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
                  115 120 125
          Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
              130 135 140
          Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
          145 150 155 160
          Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
                          165 170 175
          Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
                      180 185 190
          Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
                  195 200 205
          Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
              210 215 220
          Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
          225 230 235 240
          Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
                          245 250 255
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
                      260 265 270
          Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
                  275 280 285
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
              290 295 300
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
          305 310 315 320
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
                          325 330 335
          Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
                      340 345 350
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
                  355 360 365
          Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
              370 375 380
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
          385 390 395 400
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
                          405 410 415
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
                      420 425 430
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
                  435 440 445
          Leu Ser Leu Ser Pro Gly Lys
              450 455
           <![CDATA[ <210> 35]]>
           <![CDATA[ <211> 214]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 35]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
          1 5 10 15
          Glu Thr Val Thr Ile Thr Cys Arg Ser Ser Glu Asn Ile Tyr Ser Asn
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
                  35 40 45
          Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
          65 70 75 80
          Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Trp
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
                      100 105 110
          Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
                  115 120 125
          Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
              130 135 140
          Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
          145 150 155 160
          Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
                          165 170 175
          Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
                      180 185 190
          Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
                  195 200 205
          Phe Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 36]]>
           <![CDATA[ <211> 1368]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 36]]>
          gaagttcagc tgcagcagtc tggacccgag ctggttaagc ctggcgcctc tgtcagaatc 60
          agctgcaagg ccagcggcta caccttcacc gactactaca tgaactgggt caagcagagc 120
          cacggcaaga gcctggaatg gatcggcgac atcaaccccca atgatggcgg caccacctac 180
          agccagaagt tcaagggcaa agccaacactg accgtggaca agagcagcag caccgcctac 240
          atggaactga gaagcctgac cagcgaggac agcgccgtgt acttttgtgc cagagagggc 300
          aactactacg cctacgacgt ccgcgtgtgg tacttcgatg tgtggggcac aggcaccacc 360
          gtgacagtta gttctgcgtc gaccaagggc ccatcggtct tccccctggc accctcctcc 420
          aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480
          ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540
          gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 600
          ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660
          aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720
          gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 780
          atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840
          gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900
          gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960
          tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1020
          gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1080
          ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140
          tatcccagcg acatcgccgt ggagtggggag agcaatgggc agccggagaa caactacaag 1200
          accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg 1260
          gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320
          cacaaccact acacgcagaa gagcctctcc ctgtccccgg gtaaatga 1368
           <![CDATA[ <210> 37]]>
           <![CDATA[ <211> 645]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 37]]>
          gacatccaga tgacacagtc tccagccagc ctgtccgtgt ctgtgggaga gacagtgacc 60
          atcacctgtc ggagcagcga gaacatctac agcaacctgg cctggtatca gcagaagcag 120
          ggcaagtctc ctcagctgct ggtgtacgcc gccaccaatc ttgctgatgg cgtgcccagc 180
          agattttccg gctctggctc tggcacacag tacagcctga agatcaacag cctgcagagc 240
          gaggacttcg gcagctacta ctgccagcac ttttggggca ccccttggac atttggcgga 300
          ggcaccaagc tggaaatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360
          tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420
          cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
          gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540
          ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600
          ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645
           <![CDATA[ <210> 38]]>
           <![CDATA[ <211> 22]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 38]]>
          Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro
          1 5 10 15
          Gly Thr Glu Thr Ser Asp
                      20
           <![CDATA[ <210> 39]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 39]]>
          Gly Tyr Thr Phe Thr Asp Tyr Tyr
          1 5
           <![CDATA[ <210> 40]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 40]]>
          Ile Asn Pro Asn Asp Gly Gly Thr Thr Tyr Ala Gln Lys Phe Gln Gly
          1 5 10 15
           <![CDATA[ <210> 41]]>
           <![CDATA[ <211> 18]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 41]]>
          Ala Arg Glu Gly Asn Tyr Tyr Ala Tyr Asp Val Arg Val Trp Tyr Phe
          1 5 10 15
          Asp Val
           <![CDATA[ <210> 42]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <40]]>0> 42]]>
           <br/> <![CDATA[Gln Asp Ile Ile Thr Tyr
          1 5
           <![CDATA[ <210> 43]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 43]]>
          Ser Thr Ser Ser Leu Asn Ser Gly Val Pro Ser
          1 5 10
           <![CDATA[ <210> 44]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 44]]>
          Gln Gln Tyr Ser Glu Leu Pro Tyr Thr
          1 5
           <![CDATA[ <210> 45]]>
           <![CDATA[ <400> 45]]>
          000
           <![CDATA[ <210> 46]]>
           <![CDATA[ <400> 46]]>
          000
           <![CDATA[ <210> 47]]>
           <![CDATA[ <211> 455]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 47]]>
          Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
                      20 25 30
          Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Asp Ile Asn Pro Asn Asp Gly Gly Thr Thr Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
          65 70 75 80
          Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
                          85 90 95
          Ala Arg Glu Gly Asn Tyr Tyr Ala Tyr Asp Val Arg Val Trp Tyr Phe
                      100 105 110
          Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
                  115 120 125
          Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
              130 135 140
          Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
          145 150 155 160
          Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
                          165 170 175
          Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
                      180 185 190
          Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
                  195 200 205
          Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
              210 215 220
          Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
          225 230 235 240
          Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
                          245 250 255
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
                      260 265 270
          Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
                  275 280 285
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
              290 295 300
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
          305 310 315 320
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
                          325 330 335
          Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
                      340 345 350
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
                  355 360 365
          Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
              370 375 380
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
          385 390 395 400
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
                          405 410 415
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
                      420 425 430
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
                  435 440 445
          Leu Ser Leu Ser Pro Gly Lys
              450 455
           <![CDATA[ <210> 48]]>
           <![CDATA[ <211> 214]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 48]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Gln Asp Ile Ile Thr Tyr
                      20 25 30
          Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile
                  35 40 45
          Tyr Ser Thr Ser Ser Leu Asn Ser Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Glu Leu Pro Tyr
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Val Glu Leu Lys Arg Thr Val Ala Ala
                      100 105 110
          Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
                  115 120 125
          Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
              130 135 140
          Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
          145 150 155 160
          Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
                          165 170 175
          Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
                      180 185 190
          Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
                  195 200 205
          Phe Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 49]]>
           <![CDATA[ <211> 1368]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 49]]>
          caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60
          tcctgcaagg ccagcggcta cacctttacc gactactaca tgaactgggt ccgacaggcc 120
          cctggacagg gacttgaatg gatgggcgac atcaacccca acgacggcgg cacaacatac 180
          gcccagaaat tccagggcag agtgaccatc accgccgacg agtctacaag caccgcctac 240
          atggaactga gcagcctgag aagcgaggat accgccgtgt acttctgtgc cagagagggc 300
          aactactacg cctacgacgt ccgcgtgtgg tacttcgatg tttggggcca gggcaccacc 360
          gtgacagtct cttctgcgtc gaccaagggc ccatcggtct tccccctggc accctcctcc 420
          aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480
          ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540
          gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 600
          ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660
          aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720
          gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 780
          atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840
          gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900
          gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960
          tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1020
          gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1080
          ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140
          tatcccagcg acatcgccgt ggagtggggag agcaatgggc agccggagaa caactacaag 1200
          accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg 1260
          gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320
          cacaaccact acacgcagaa gagcctctcc ctgtccccgg gtaaatga 1368
           <![CDATA[ <210> 50]]>
           <![CDATA[ <211> 645]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 50]]>
          gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60
          attacatgtg gcgccagcca ggacatcatc acctacctga actggtatca gcagaaaccc 120
          ggcaaggccg tgaagctgct gatctacagc accagcagcc tgaatagcgg cgtgcccagc 180
          agattttctg gcagcggctc tggcaccgac ttcaccctga ccatatctag cctgcagcct 240
          gaggacttcg ccacctacta ctgccagcag tacagcgagc tgccctacac atttggcgga 300
          ggcaccaagg tggaactgaa gcgtacggtt gctgccccctt ccgtgttcat cttccccacct 360
          tccgacgagc agctgaagtc tggcacagcc tctgtcgtgt gcctgctgaa caacttctac 420
          cctcgggaag ccaaggtgca gtggaaggtg gacaatgccc tgcagtccgg caactcccaa 480
          gagtctgtga ccgagcagga ctccaaggac agcacctaca gcctgtcctc cacactgacc 540
          ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccatcagggc 600
          ctgtctagcc ctgtgaccaa gtctttcaac cggggcgagt gttag 645
           <![CDATA[ <210> 51]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 51]]>
          Gly Phe Thr Phe Ser Asp Ala Trp
          1 5
           <![CDATA[ <210> 52]]>
           <![CDATA[ <211> 18]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 52]]>
          Val Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu Ser Val
          1 5 10 15
          Lys Gly
           <![CDATA[ <210> 53]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 53]]>
          Thr Arg Ser Val Gly Gly Tyr Gly Thr Thr Tyr Trp Tyr Phe Asp Val
          1 5 10 15
           <![CDATA[ <210> 54]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 54]]>
          Gln Asn Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr
          1 5 10
           <![CDATA[ <210> 55]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 55]]>
          Gly Ala Ser Thr Arg Glu Ser Gly Val Pro Asp
          1 5 10
           <![CDATA[ <210> 56]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 56]]>
          Gln Ser Glu His Ser Tyr Pro Tyr Thr
          1 5
           <![CDATA[ <210> 57]]>
           <![CDATA[ <400> 57]]>
          000
           <![CDATA[ <210> 58]]>
           <![CDATA[ <400> 58]]>
          000
           <![CDATA[ <210> 59]]>
           <![CDATA[ <211> 455]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 59]]>
          Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
                      20 25 30
          Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Glu Val Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu
              50 55 60
          Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
          65 70 75 80
          Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr
                          85 90 95
          Tyr Cys Thr Arg Ser Val Gly Gly Tyr Gly Thr Thr Tyr Trp Tyr Phe
                      100 105 110
          Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
                  115 120 125
          Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
              130 135 140
          Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
          145 150 155 160
          Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
                          165 170 175
          Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
                      180 185 190
          Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
                  195 200 205
          Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
              210 215 220
          Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
          225 230 235 240
          Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
                          245 250 255
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
                      260 265 270
          Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
                  275 280 285
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
              290 295 300
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
          305 310 315 320
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
                          325 330 335
          Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
                      340 345 350
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
                  355 360 365
          Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
              370 375 380
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
          385 390 395 400
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
                          405 410 415
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
                      420 425 430
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
                  435 440 445
          Leu Ser Leu Ser Pro Gly Lys
              450 455
           <![CDATA[ <210> 60]]>
           <![CDATA[ <211> 220]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 60]]>
          Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly
          1 5 10 15
          Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Asn Leu Leu Asn Ser
                      20 25 30
          Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Phe Gly Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Ser
                          85 90 95
          Glu His Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
                      100 105 110
          Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
                  115 120 125
          Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
              130 135 140
          Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
          145 150 155 160
          Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
                          165 170 175
          Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
                      180 185 190
          Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
                  195 200 205
          Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
              210 215 220
           <![CDATA[ <210> 61]]>
           <![CDATA[ <211> 1368]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 61]]>
          gaagtgaagc tggaagaatc tggcggcgga ctggttcagc ctggcggatc tatgaagctg 60
          agctgtgccg ccagcggctt caccttttct gacgcctgga tggactgggt ccgacagtct 120
          cctgagaaag gcctggaatg ggttgccgaa gtgcggaaca aggccaacaa ccacgccacc 180
          tactacgccg agtctgtgaa gggcagattc accatcagcc gggacgacag caagagcagc 240
          gtgtacctgc agatgaacag cctgagagcc gaggacaccg gcatctacta ctgcacaaga 300
          agcgttggcg gctacggcac cacctactgg tactttgatg tgtggggcac cggcaccaca 360
          gtgaccgtta gttctgcgtc gaccaagggc ccatcggtct tccccctggc accctcctcc 420
          aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480
          ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540
          gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 600
          ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660
          aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720
          gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 780
          atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840
          gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900
          gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960
          tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1020
          gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1080
          ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140
          tatcccagcg acatcgccgt ggagtggggag agcaatgggc agccggagaa caactacaag 1200
          accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg 1260
          gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320
          cacaaccact acacgcagaa gagcctctcc ctgtccccgg gtaaatga 1368
           <![CDATA[ <210> 62]]>
           <![CDATA[ <211> 663]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 62]]>
          gacatcgtga tgacacagag ccctagcagc ctgtctgtgt ctgccggcga gaaagtgacc 60
          atgagctgca agagcagcca gaacctgctg aacagcggca accagaagaa ctacctggcc 120
          tggtatcagc agaagcccgg ccagcctcct aagctgctga tctttggagc cagcaccaga 180
          gaaagcggcg tgcccgatag atttacaggc tctggcagcg gcaccgactt caccctgaca 240
          atcagttctg tgcaggccga ggacctggcc gtgtactact gtcagagcga gcacagctac 300
          ccctacacct ttggcggcgg aacaaagctg gaaatcaagc gtacggtggc tgcaccatct 360
          gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420
          ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480
          caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540
          ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600
          gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660
          tag 663
           <![CDATA[ <210> 63]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 63]]>
          Gly Phe Thr Phe Ser Asp Ala Trp
          1 5
           <![CDATA[ <210> 64]]>
           <![CDATA[ <211> 18]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 64]]>
          Val Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Ala Ser Val
          1 5 10 15
          Lys Gly
           <![CDATA[ <210> 65]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 65]]>
          Thr Arg Ser Val Gly Gly Tyr Gly Thr Thr Tyr Trp Tyr Phe Asp Val
          1 5 10 15
           <![CDATA[ <210> 66]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <22]]>3> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <br/>
           <br/> &lt;![CDATA[ <400&gt;66]]>
           <br/> <![CDATA[Gln Asn Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr
          1 5 10
           <![CDATA[ <210> 67]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 67]]>
          Gly Ala Ser Thr Arg Glu Ser Gly Val Pro Asp
          1 5 10
           <![CDATA[ <210> 68]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 68]]>
          Gln Ser Glu His Ser Tyr Pro Tyr Thr
          1 5
           <![CDATA[ <210> 69]]>
           <![CDATA[ <400> 69]]>
          000
           <![CDATA[ <210> 70]]>
           <![CDATA[ <400> 70]]>
          000
           <![CDATA[ <210> 71]]>
           <![CDATA[ <211> 455]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 71]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
                      20 25 30
          Trp Met Asp Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Gly Glu Val Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Ala
              50 55 60
          Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Thr Arg Ser Val Gly Gly Tyr Gly Thr Thr Tyr Trp Tyr Phe
                      100 105 110
          Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
                  115 120 125
          Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
              130 135 140
          Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
          145 150 155 160
          Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
                          165 170 175
          Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
                      180 185 190
          Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
                  195 200 205
          Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
              210 215 220
          Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
          225 230 235 240
          Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
                          245 250 255
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
                      260 265 270
          Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
                  275 280 285
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
              290 295 300
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
          305 310 315 320
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
                          325 330 335
          Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
                      340 345 350
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
                  355 360 365
          Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
              370 375 380
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
          385 390 395 400
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
                          405 410 415
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
                      420 425 430
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
                  435 440 445
          Leu Ser Leu Ser Pro Gly Lys
              450 455
           <![CDATA[ <210> 72]]>
           <![CDATA[ <211> 220]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 72]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Leu Leu Asn Ser
                      20 25 30
          Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Phe Gly Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Ser
                          85 90 95
          Glu His Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
                      100 105 110
          Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
                  115 120 125
          Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
              130 135 140
          Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
          145 150 155 160
          Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
                          165 170 175
          Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
                      180 185 190
          Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
                  195 200 205
          Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
              210 215 220
           <![CDATA[ <210> 73]]>
           <![CDATA[ <211> 1368]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 73]]>
          gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgaagctg 60
          tcttgtgccg ccagcggctt caccttttcc gacgcttgga tggactgggt ccgacaggcc 120
          tctggcaaag gccttgagtg ggttggagaa gtgcggaaca aggccaacaa ccacgccacc 180
          tactatgccg cctctgtgaa gggcagattc accatcagcc gggacgacag caagaacacc 240
          gcctacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgcaccaga 300
          tctgttggcg gctacggcac cacctactgg tactttgatg tgtggggcca gggcaccacc 360
          gtgacagttt cttctgcgtc gaccaagggc ccatcggtct tccccctggc accctcctcc 420
          aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480
          ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540
          gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 600
          ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660
          aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 720
          gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 780
          atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 840
          gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 900
          gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 960
          tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1020
          gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1080
          ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140
          tatcccagcg acatcgccgt ggagtggggag agcaatgggc agccggagaa caactacaag 1200
          accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg 1260
          gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1320
          cacaaccact acacgcagaa gagcctctcc ctgtccccgg gtaaatga 1368
           <![CDATA[ <210> 74]]>
           <![CDATA[ <211> 663]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Polynucleotide"]]>
           <![CDATA[ <400> 74]]>
          gacatcgtga tgacacagag ccctgatagc ctggccgtgt ctctgggaga gagagccacc 60
          atcaactgca agagcagcca gaacctgctg aacagcggca accagaagaa ctacctggcc 120
          tggtatcagc agaagcccgg ccagcctcct aagctgctga tctttggagc cagcaccaga 180
          gaaagcggcg tgcccgatag attttctggc agcggctctg gcaccgactt caccctgaca 240
          attagctccc tgcaggccga ggatgtggcc gtgtactact gtcagagcga gcacagctac 300
          ccctacacct ttggccaggg caccaagctg gaaatcaagc gtacggtggc tgcaccatct 360
          gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420
          ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480
          caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540
          ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600
          gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660
          tag 663
           <![CDATA[ <210> 75]]>
           <![CDATA[ <211> 38]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 75]]>
          Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu
          1 5 10 15
          Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro
                      20 25 30
          Gly Thr Glu Thr Ser Asp
                  35
           <![CDATA[ <210> 76]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 76]]>
          Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Ile Ser Cys Lys Ala Ser Ser Tyr Ser Phe Thr Asp Tyr
                      20 25 30
          Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile
                  35 40 45
          Gly Ile Ile Phe Pro Lys Tyr Gly Thr Thr Ser Tyr Asn Gln Lys Phe
              50 55 60
          Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Thr Asp Gly Gly Thr Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr
                      100 105 110
          Thr Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 77]]>
           <![CDATA[ <211> 117]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 77]]>
          Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Ser Phe Thr Asp Tyr
                      20 25 30
          Asn Met Asn Trp Val Lys Gln Ile Asn Gly Lys Ser Leu Glu Trp Ile
                  35 40 45
          Gly Ile Ile Tyr Pro Ile Tyr Gly Thr Thr Ser Tyr Asn Gln Lys Phe
              50 55 60
          Arg Gly Lys Ala Thr Leu Thr Val Asp Leu Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Asp Arg Ser Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr
                      100 105 110
          Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 78]]>
           <![CDATA[ <211> 117]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 78]]>
          Glu Phe Gln Leu Gln Gln Ser Gly Pro Lys Leu Val Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr
                      20 25 30
          Ser Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile
                  35 40 45
          Gly Ile Ile Asn Pro Asn Tyr Gly Ser Thr Ser Tyr Asn Gln Lys Phe
              50 55 60
          Lys Val Lys Ala Thr Leu Thr Val Asp His Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Gln Val Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Ser Ser Thr Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr
                      100 105 110
          Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 79]]>
           <![CDATA[ <211> 125]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 79]]>
          Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
          1 5 10 15
          Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
                      20 25 30
          Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
                  35 40 45
          Gly Asp Ile Asn Pro Asn Asp Gly Gly Thr Thr Tyr Ser Gln Lys Phe
              50 55 60
          Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
                          85 90 95
          Ala Arg Glu Gly Asn Tyr Tyr Ala Tyr Asp Val Arg Val Trp Tyr Phe
                      100 105 110
          Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 80]]>
           <![CDATA[ <211> 117]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 80]]>
          Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
          1 5 10 15
          Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser
                      20 25 30
          Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile
                  35 40 45
          Gly Glu Ile Asp Pro Glu Ala Gly Gly Thr Ala Tyr Asn Gln Lys Phe
              50 55 60
          Lys Gly Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Thr Arg Glu Asp Tyr Asp Trp Phe Thr Tyr Trp Gly Gln Gly Thr Leu
                      100 105 110
          Val Thr Val Ser Ala
                  115
           <![CDATA[ <210> 81]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 81]]>
          Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
                      20 25 30
          Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
                  35 40 45
          Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Ile Lys Tyr Ser Gln Lys Phe
              50 55 60
          Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Gln Leu Ser Ser Leu Thr Tyr Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Ser Tyr Tyr Asp Tyr Asp Gly Gly Phe Ala Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ala
                  115 120
           <![CDATA[ <210> 82]]>
           <![CDATA[ <211> 117]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 82]]>
          Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
          1 5 10 15
          Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asp Ile Lys Asp Asp
                      20 25 30
          Phe Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
                  35 40 45
          Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Ser Lys Phe
              50 55 60
          Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Ser Asn Thr Ala Tyr
          65 70 75 80
          Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ser Thr Leu Leu Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr
                      100 105 110
          Leu Thr Val Ser Ser
                  115
           <![CDATA[ <210> 83]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 83]]>
          Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
          1 5 10 15
          Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala
                      20 25 30
          Gly Met Gln Trp Val Gln Lys Met Pro Gly Lys Gly Phe Lys Trp Ile
                  35 40 45
          Gly Trp Ile Asn Thr His Ser Gly Glu Pro Lys Tyr Ala Glu Asp Phe
              50 55 60
          Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
                          85 90 95
          Ala Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
                      100 105 110
          Ser Ser
           <![CDATA[ <210> 84]]>
           <![CDATA[ <211> 125]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 84]]>
          Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
                      20 25 30
          Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Glu Val Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu
              50 55 60
          Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
          65 70 75 80
          Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr
                          85 90 95
          Tyr Cys Thr Arg Ser Val Gly Gly Tyr Gly Thr Thr Tyr Trp Tyr Phe
                      100 105 110
          Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 85]]>
           <![CDATA[ <211> 116]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence (Artifi]]>cial Sequence)
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 85]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Ser Tyr
                      20 25 30
          Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
                  35 40 45
          Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr
          65 70 75 80
          Leu Gln Met Ser His Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Asp Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
                      100 105 110
          Thr Val Ser Ser
                  115
           <![CDATA[ <210> 86]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 86]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Asp Tyr
                      20 25 30
          Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe
          65 70 75 80
          Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Val Arg Glu Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val
                      100 105 110
          Ser Ser
           <![CDATA[ <210> 87]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 87]]>
          Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
          1 5 10 15
          Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly
                      20 25 30
          Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
                  35 40 45
          Met Gly Tyr Ile Ser Ser Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu
              50 55 60
          Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
          65 70 75 80
          Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Glu Ile Thr Thr Val Val Tyr Tyr Val Met Asp Asn Trp Gly
                      100 105 110
          Gln Gly Thr Ser Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 88]]>
           <![CDATA[ <211> 106]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 88]]>
          Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly
          1 5 10 15
          Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met
                      20 25 30
          Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
                  35 40 45
          Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
              50 55 60
          Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
          65 70 75 80
          Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr
                          85 90 95
          Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
                      100 105
           <![CDATA[ <210> 89]]>
           <![CDATA[ <211> 106]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 89]]>
          Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly
          1 5 10 15
          Glu Lys Val Thr Met Ile Cys Ser Ala Ser Ser Ser Val Arg Tyr Met
                      20 25 30
          Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
                  35 40 45
          Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
              50 55 60
          Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
          65 70 75 80
          Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr
                          85 90 95
          Phe Gly Pro Gly Thr Lys Leu Glu Leu Lys
                      100 105
           <![CDATA[ <210> 90]]>
           <![CDATA[ <211> 105]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 90]]>
          Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly
          1 5 10 15
          Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
                      20 25 30
          Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
                  35 40 45
          Leu Thr Ser Asp Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
              50 55 60
          Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
          65 70 75 80
          Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr
                          85 90 95
          Phe Gly Gly Gly Thr Lys Leu Glu Ile
                      100 105
           <![CDATA[ <210> 91]]>
           <![CDATA[ <211> 106]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 91]]>
          Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
          1 5 10 15
          Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
                      20 25 30
          His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr
                  35 40 45
          Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
              50 55 60
          Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu
          65 70 75 80
          Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Pro Thr
                          85 90 95
          Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
                      100 105
           <![CDATA[ <210> 92]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 92]]>
          Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
          1 5 10 15
          Glu Lys Val Thr Leu Thr Cys Ser Ala Ser Ser Gly Val Ser Ser Ser
                      20 25 30
          Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Val Trp
                  35 40 45
          Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
              50 55 60
          Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Ser Met Glu
          65 70 75 80
          Ala Glu Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Tyr Pro
                          85 90 95
          Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
                      100 105
           <![CDATA[ <210> 93]]>
           <![CDATA[ <211> 106]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence (Artificial Sequence]]>)
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 93]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
          1 5 10 15
          Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr
                      20 25 30
          Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile
                  35 40 45
          His Tyr Thr Ser Ile Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
          65 70 75 80
          Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr
                          85 90 95
          Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
                      100 105
           <![CDATA[ <210> 94]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 94]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
          1 5 10 15
          Glu Thr Val Thr Ile Thr Cys Arg Ser Ser Glu Asn Ile Tyr Ser Asn
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
                  35 40 45
          Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
          65 70 75 80
          Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Trp
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
                      100 105
           <![CDATA[ <210> 95]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 95]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Arg Gln Gly Arg Ser Pro Gln Leu Leu Val
                  35 40 45
          Tyr His Ala Lys Thr Leu Thr Glu Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg
                      100 105
           <![CDATA[ <210> 96]]>
           <![CDATA[ <211> 113]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 96]]>
          Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly
          1 5 10 15
          Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Asn Leu Leu Asn Ser
                      20 25 30
          Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Phe Gly Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Ser
                          85 90 95
          Glu His Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
                      100 105 110
          Lys
           <![CDATA[ <210> 97]]>
           <![CDATA[ <211> 112]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 97]]>
          Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
          1 5 10 15
          Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
                      20 25 30
          Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
                  35 40 45
          Pro Lys Leu Leu Ile Tyr Lys Leu Ser Asn Arg Phe Ser Gly Val Pro
              50 55 60
          Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
          65 70 75 80
          Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
                          85 90 95
          Thr His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
                      100 105 110
           <![CDATA[ <210> 98]]>
           <![CDATA[ <211> 112]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 98]]>
          Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
          1 5 10 15
          Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
                      20 25 30
          Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
                  35 40 45
          Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
              50 55 60
          Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
          65 70 75 80
          Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
                          85 90 95
          Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
                      100 105 110
           <![CDATA[ <210> 99]]>
           <![CDATA[ <211> 111]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 99]]>
          Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly
          1 5 10 15
          Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
                      20 25 30
          Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser
                  35 40 45
          Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro
              50 55 60
          Asp Arg Phe Ser Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
          65 70 75 80
          Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn
                          85 90 95
          Leu Glu Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
                      100 105 110
           <![CDATA[ <210> 100]]>
           <![CDATA[ <211> 241]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 100]]>
          Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser
          1 5 10 15
          Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys
                      20 25 30
          Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr
                  35 40 45
          Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu
              50 55 60
          Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser
          65 70 75 80
          Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys
                          85 90 95
          Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys
                      100 105 110
          Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala
                  115 120 125
          Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro
              130 135 140
          Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His
          145 150 155 160
          Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala
                          165 170 175
          Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val
                      180 185 190
          His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr
                  195 200 205
          Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly
              210 215 220
          Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp His His His His His His
          225 230 235 240
          His
           <![CDATA[ <210> 101]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> Source]]>
           <![CDATA[ <223> /Comment = "Description of Artificial Sequence: Synthetic Peptide"]]>
           <![CDATA[ <400> 101]]>
          Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu
          1 5 10 15
          Cys Leu Ser Cys
          
      

Claims (45)

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) heavy chain variable region (HCVR), which comprises the HCVR CDR1 sequence of SEQ ID NO: 1, the HCVR CDR2 sequence of SEQ ID NO: 2, and the HCVR CDR3 sequence of SEQ ID NO: 3; and (1b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 4, the LCVR CDR2 sequence of SEQ ID NO: 5, and the LCVR CDR3 sequence of SEQ ID NO: 6; or (2a) heavy chain variable region (HCVR), which comprises the HCVR CDR1 sequence of SEQ ID NO: 14, the HCVR CDR2 sequence of SEQ ID NO: 15, and the HCVR CDR3 sequence of SEQ ID NO: 16; and (2b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 17, the LCVR CDR2 sequence of SEQ ID NO: 18, and the LCVR CDR3 sequence of SEQ ID NO: 19; or (3a) heavy chain variable region (HCVR), which comprises 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 (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 (4a) heavy chain variable region (HCVR), which comprises 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 (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 (5a) heavy chain variable region (HCVR), which comprises the HCVR CDR1 sequence of SEQ ID NO: 51, the HCVR CDR2 sequence of SEQ ID NO: 52, and the HCVR CDR3 sequence of SEQ ID NO: 53; and (5b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 54, the LCVR CDR2 sequence of SEQ ID NO: 55, and the LCVR CDR3 sequence of SEQ ID NO: 56; or (6a) heavy chain variable region (HCVR), which comprises the HCVR CDR1 sequence of SEQ ID NO: 63, the HCVR CDR2 sequence of SEQ ID NO: 64, and the HCVR CDR3 sequence of SEQ ID NO: 65; and (6b) light chain variable region (LCVR), which comprises 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. The isolated monoclonal antibody or antigen-binding fragment thereof according to 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. 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. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 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 Antibody. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antigen-binding fragment thereof is Fab, Fab', F(ab') ₂ , _Fd , single-chain Fv or scFv , disulfide-linked F _v , V-NAR domain, IgNar, intrabody, IgGΔCH ₂ , minibody, F(ab') ₃ , tetravalent antibody, trivalent antibody, diabody, single domain antibody, DVD-Ig, Fcab, _mAb2 , (scFv) ₂ or scFv-Fc. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the monoclonal antibody or antigen-binding fragment thereof cross-reacts with rhesus monkey TNFR2, but does not substantially cross-react with mouse TNFR2 reaction. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the monoclonal antibody or antigen-binding fragment thereof does not substantially cross-react with TNFR1. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the monoclonal antibody or antigen-binding fragment thereof is in an amount of less than about 25 nM, 20 nM, 15 nM, 10 nM, 5 A _Kd of nM, 2 nM or 1 nM binds TNF[alpha]. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which enhances the binding between TNFα and TNFR2; enhances TNFα-mediated or co-stimulated NFκB signal transduction (for example, after TCR activation CD8 and/or CD4 Tconv T cells); and/or promote the proliferation of TCR-activated effector T cells (such as CD8 and/or CD4 Tconv T cells) in the presence of Treg. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which enhances TNFα-mediated CD25 expression on Treg. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, which binds to the epitope of SEQ ID NO: 13 and/or 101. An isolated monoclonal antibody or antigen-binding fragment thereof that competes for binding to SEQ ID NO: 13 and/or 101 with the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 epitope. An isolated monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to the epitope of SEQ ID NO: 13 and/or 101. The isolated monoclonal antibody or antigen-binding fragment thereof as claimed in claim 12 or 13, which enhances the binding between TNFα and TNFR2; enhances TNFα-mediated or co-stimulated NFκB signal transduction (such as in TCR-activated CD8 and/or CD4 Tconv T cells); and/or promote the proliferation of TCR-activated effector T cells (such as CD8 and/or CD4 Tconv T cells) in the presence of Treg. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which inhibits the binding between TNFα and TNFR2; inhibits TNFα-mediated or co-stimulated NFκB signal transduction (for example, in TCR activation CD8 and/or CD4 Tconv T cells); and/or inhibit the proliferation of TCR-activated effector T cells (such as CD8 and/or CD4 Tconv T cells) in the presence of Treg. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which promotes Treg expansion. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which promotes the activation of natural killer cells. 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 according to any one of claims 1 to 8 and 15 to 16. 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. The isolated monoclonal antibody or antigen-binding fragment thereof as claimed in claim 19, which (1) promotes activation and proliferation of CD4 ⁺ T cells in tumor-infiltrating lymphocytes (TIL) rather than regulatory T cells (Treg) ( for example, in live In vivo hTNFR2 knock-in MC38 mouse tumor model); and/or (2) promote NK cell activation in vitro and/or in vivo . The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, which has a maximum tolerated dose (MTD) of about 150 mg/kg in cynomolgus monkeys. 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 according to any one of claims 1 to 21, wherein the patient ( such as The cancer in the patient) has: (a) a higher expression level of TNFR2 compared to the average expression level of TNFR2 in prostate cancer patients; optionally, the expression of TNFR2 is in effector T cells ( such as CD4 ⁺ and/or CD8 ⁺ T cells; cells), tumor infiltrating CD8 ⁺ T cells and/or NK cells; and (b) higher CD8A expression levels compared to the mean CD8A expression levels in AML patients. The method of claim 22, wherein the patient ( eg, the patient with the cancer) has the higher expression level of TNFR2 in tumor-infiltrating CD8A ⁺ (CD8α chain positive) T cells. The method of claim 22 or 23, wherein the patient suffers from EBV ⁺ gastric cancer ( such as gastric adenocarcinoma), clear cell renal cell carcinoma, clear cell renal cell carcinoma of the kidney, skin melanoma ( such as cutaneous melanoma of the skin), testicular germ cell tumor or soft tissue sarcoma. The method of claim 22 or 23, wherein the cancer exhibits "high" levels of PD-L1. The method according to claim 25, wherein the cancer is cervical cancer ( such as cervical squamous cell carcinoma or endocervical adenocarcinoma), pleural mesothelioma, lung adenocarcinoma or head and neck squamous cell carcinoma. The method of any one of claims 22 to 26, further comprising administering to the patient: (a) Antibodies specific for PD-1 or their antigen-binding fragments, such as cemiplimab, nivolumab, pembrolizumab, sparizumab (spartalizumab), camrelizumab, sintilimab, tislelizumab, toripalimab, dostalimab ( dostarlimab) and INCMGA00012; (b) Antibodies specific for PD-L1 or antigen-binding fragments thereof, such as avelumab, durvalumab, atezolizumab, KN035 or CK-301 , and/or (c) Antibodies or antigen-binding fragments thereof specific to PD-L2. The method of any one of claims 22 to 27, wherein the patient has relapsed or refractory cancer, and/or has previously been treated with standard of care treatment (and, as the case may be, fails to respond to standard of care treatment or is spontaneous its recurrence). The method of any one of claims 22 to 28, comprising administering to the patient once every 3 weeks (Q3W), once every 4 weeks (Q4W) or once every 5 weeks (Q5W) ( eg, once every 4 weeks or Q4W) The effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof is administered. The method according to claim 29, comprising administering the isolated monoclonal antibody or its antigen-binding agent to the patient once every 4 weeks (Q4W) at a dose of about 5 mg, 15 mg, 50 mg, 100 mg or 150 mg Fragments ( eg administered intravenously over 60 minutes). The method according to any one of claims 22 to 30, further comprising: (1) Prior to the administration step, select patients with the higher TNFR2 expression level and CD8A expression level; or (2) Prior to the administration step, verify that the patient has the higher TNFR2 expression level and CD8A expression level. 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 an antigen-binding fragment thereof, the isolated monoclonal antibody or an antigen-binding fragment thereof comprising SEQ ID NO: 101. Specifically binding to human TNFR2 at an epitope consisting essentially of or consisting of it, as the case may be, the isolated monoclonal antibody or antigen-binding fragment thereof is not substantially composed of SEQ ID NO: 13 Consists of or consists of an epitope that binds human TNFR2. 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 according to any one of claims 1-21. The method according to claim 33, which is used for treating cancer, wherein the method further comprises administering an antagonist of an immune checkpoint. The method according to claim 34, wherein the immune checkpoint is a PD-1/PD-L1 immune checkpoint. The method according to claim 34 or 35, wherein the antagonist of the immune checkpoint is an antibody or an antigen-binding fragment thereof specific to PD-1 or PD-L1. The method of claim 36, wherein the antibody is an anti-PD-1 antibody, such as simiprizumab, nivolumab, pembrolizumab, sparizumab, camrelizumab, Dilimumab, Tislelizumab, Toripalimab, Dostalimumab and INCMGA00012. The method according to claim 37, wherein the antibody is an anti-PD-L1 antibody, such as avelumab, durvalumab, atezolizumab, KN035 or CK-301. 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 AUNP12; a small molecule inhibitor of PD-L1, such as CA- 170, or macrocyclic peptides, such as BMS-986189. The method according to any one of claims 34 to 39, wherein the cancer is melanoma, breast cancer, colon cancer, cervical cancer, kidney cancer, liver cancer (such as hepatocellular carcinoma), lung cancer (NSCLC), ovarian cancer, skin cancer (such as squamous cell carcinoma or basal cell carcinoma), lymphoma or leukemia. The method according to any one of claims 22 to 40, further comprising administering chemotherapeutic agents, anti-angiogenic agents, growth inhibitors, tumor immune agents and/or anti-tumor compositions to the patient. A polynucleotide encoding the heavy chain or light chain or antigen-binding portion thereof according to any one of claims 1 to 21. The polynucleotide according to claim 42, which is codon-optimized for expression in human cells. A vector comprising the polynucleotide according to claim 42 or 43. The vector according to claim 44, which is an expression vector ( such as a mammalian, yeast, insect or bacterial expression vector).

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