Antagonistic anti-tumor necrosis factor receptor superfamily polypeptide

Abstract

Antagonistic TNFR2 polypeptides, such as antibodies and antigen-binding fragments thereof, and the use of these polypeptides to inhibit the proliferation of regulatory T cells (T-regs) and / or myeloid-derived suppressor cells (MDSCs), expand the population of T effector cells or enhance the function of T effector cells, and suppress the proliferation or directly kill tumor cells, such as tumor cells expressing the TNFR2 antigen, are described. The polypeptides, such as antibodies and antigen-binding fragments thereof, are TNFR2 antagonists, e.g., dominant TNFR2 antagonists. The polypeptides can be used to suppress T-reg or MDSC-mediated inactivation of tumor-reactive T lymphocytes, expand the population of tumor-reactive cytotoxic T cells, and / or directly kill TNFR2+ tumor cells. The antagonistic TNFR2 polypeptides described herein can be used to treat a wide variety of cancers and infectious diseases. [Selected Figure] Figure 1

Description

Technical Field

[0001] Sequence Listing This application includes a Sequence Listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The name of the ASCII copy (created on August 20, 2019) is 00786 - 0083WO2_Sequence_Listing_08.20.19_ST25, and its size is 193,232 bytes. Technical Field The present invention relates to "antagonistic anti - tumor necrosis factor receptor superfamily polypeptide" ".

Background Art

[0002] The use of natural and genetically modified T lymphocytes is a prominent paradigm for improving various human pathologies. For example, conventional therapeutic platforms for treating cancer include surgical resection of tumor masses, radiotherapy, and administration of chemotherapeutic agents (Shewach, Chem. Rev., 109:2859 - 2861, 2009). However, in the past decade, there has been a resurgence in the application of adoptive immunotherapy to cancer treatment regimens. With the advent of chimeric antigen receptor (CAR - T) therapy, new methods have emerged for injecting autologous and allogeneic tumor - reactive T cells into patients (June, J. Clin. Invest., 117:1466 - 1476, 2007). CAR - T therapy utilizes the resources of the adaptive immune response to enhance cancer cell cytotoxicity and eradicate tumor masses. A common feature in adoptive immunotherapy is the use of T cells that demonstrate the ability to selectively enhance cytotoxicity against cells presenting different tumor antigens. Examples of this technology include the administration of tumor - infiltrating lymphocytes (Dudley et al., J. Immunother., 26:332 - 342, 2003), in addition to the administration of autologous or allogeneic T cells that have been genetically re - engineered to be reactive with tumor - specific antigens (Yee et al., PNAS., 99:16168 - 16173, 2002).

[0003] Despite the promise of T lymphocyte-based cancer immunotherapy, the development of this therapeutic platform is hampered by the natural properties of the immune system that suppress immune attacks initiated against self-cells. Cancer cells express class I major histocompatibility complex (MHC) proteins that distinguish these self-cells from foreign cells. To prevent fratricide of cells, regulatory T cells (T-reg cells) have evolved to suppress the activity of T cells that show reactivity to "self" MHC antigens. T-reg cells are a representative example of different classes of T cells that can be distinguished based on the unique surface protein presentation of T cells. The most well-understood populations of T-reg cells include CD4+, CD25+, FoxP3+ T-reg cells, and CD17+ T-reg cells. The exact mechanism by which these cells suppress autoreactive T cells is the subject of ongoing research, but it has been found that a particular class of T-reg cells can inhibit the production of the growth-promoting cytokine IL-2 in target T cells and, furthermore, can sequester IL-2 from autoreactive cells based on the affinity of CD25 (a subdomain of the IL-2 receptor) for IL-2 (Josefowicz et al., Ann. Rev. Immunol., 30:531-564, 2012).

[0004] T-reg cells play an important role in maintaining peripheral immune tolerance, but the same biochemical features that underlie the ability of these cells to regulate autoreactive T cells also have a fundamental impact on adoptive immunotherapy and the innate immune response by suppressing the activity of tumor-reactive T lymphocytes. By obtaining agents capable of inhibiting T-reg-mediated T cell suppression, in addition to significantly improving the scope and effectiveness of adoptive cancer immunotherapy, the ability of the immune system to eradicate pathogenic microorganisms that cause infectious diseases can be enhanced. Therefore, the development of chemical regulators of T-reg cell activity has become the subject of much pharmacological research.

[0005] There is a need for improved therapeutic agents for treating cell growth disorders, such as cancer, and a wide variety of infectious diseases. SUMMARY OF THE INVENTION

[0006] This specification describes antagonist tumor necrosis factor receptor superfamily polypeptides, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs. For example, it features antagonist tumor necrosis factor receptor 2 (TNFR2)-binding polypeptides, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs. Human TNFR2 contains four cysteine-rich domains (CRDs): CRD1 (amino acid residues 48-76 of SEQ ID NO: 7), CRD2 (amino acid residues 78-120 of SEQ ID NO: 7), CRD3 (amino acid residues 121-162 of SEQ ID NO: 7), and CRD4 (amino acid residues 162-202 of SEQ ID NO: 7). The antagonist TNFR2 polypeptides described herein include antagonist TNFR2 polypeptides that bind to one or more epitopes within CRD3 of TNFR2 and / or one or more epitopes within CRD4 of TNFR2, for example, antagonist TNFR2 polypeptides that bind to TNFR2 only within one or more epitopes of CRD3 and / or one or more epitopes of CRD4 without binding to TNFR2 within CRD1 and / or CRD2.

[0007] Antagonistic TNFR2 polypeptides described herein include IgG2 isotype antibodies and antigen-binding fragments thereof that specifically bind to TNFR2 at one or more of the epitopes described in detail above. The present disclosure is based in part on the surprising discovery that antibodies and antigen-binding fragments thereof exhibit significantly superior TNFR2 antagonist properties compared to other antibody isotypes when these molecules are in the form of the IgG2 isotype. Antagonistic TNFR2 polypeptides described herein also include antagonistic TNFR2 polypeptides having at least two TNFR2 binding sites (e.g., antigen-binding sites where TNFR2 is the "antigen") whose binding sites are spatially separated from each other by about 133 Å or more, because polypeptides that specifically bind to TNFR2 at one or more of the above-described epitopes but contain TNFR2 binding sites (e.g., antigen-binding sites) that are less than about 133 Å apart from each other, such as IgG1 antibodies and antigen-binding fragments thereof containing antigen-binding sites that are about 117 Å apart from each other, and IgG3 antibodies and antigen-binding fragments thereof containing antigen-binding sites that are 125 Å apart from each other, etc., are found herein to exhibit unexpectedly superior TNFR2 antagonist activity compared to such polypeptides.

[0008] Also featured are anti-TNFR2 polypeptides that take a single disulfide bond isoform, and pharmaceutical compositions containing them. For example, as the pharmaceutical compositions of the present disclosure, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or more of the polypeptides in the pharmaceutical composition are present as a single disulfide bond isoform, and pharmaceutical compositions containing an antagonist TNFR2-binding polypeptide are exemplified. An antagonist TNFR2-binding polypeptide that takes the human IgG2-A isoform exhibits a substantially superior TNFR2 antagonist effect as compared with TNFR2-binding polypeptides that take other human IgG2 isoforms, such as IgG2-B, IgG2-A / B1, and IgG2-A / B2. Therefore, by preparing a TNFR2 polypeptide that takes a single disulfide bond isoform as a pharmaceutical composition and administering it using the treatment methods described herein, a strong TNFR2 antagonist effect may be promoted.

[0009] The antagonist TNFR2 polypeptides of the present disclosure exhibit one or more advantageous biological properties, such as the ability to inhibit the proliferation of regulatory T cells (T-reg cells) and / or myeloid-derived suppressor cells (MDSC), and / or the ability to promote the death of regulatory T cells (T-reg cells) and / or myeloid-derived suppressor cells (MDSC). The antagonist TNFR2 polypeptides may be used to inhibit the proliferation of TNFR2-expressing cancer cells and cancer gene-expressing cancer cells, and / or to promote the death of TNFR2-expressing cancer cells and cancer gene-expressing cancer cells. Additionally or alternatively, the antagonist TNFR2 polypeptides may be administered to promote the mutual proliferation of T effector cells, such as cytotoxic CD8+ T cells. This mutual proliferation can occur, for example, by attenuating the proliferation and activity of T-reg cells or by directly proliferating T effector cells, such as cytotoxic CD8+ T cells. Therefore, the designation of the TNFR2 polypeptide as an antagonist means the ability of the TNFR2 polypeptide to attenuate the proliferation and activity of T-reg cells, MDSC, and / or TNFR2-expressing cancer cells, and, to clarify, does not indicate an antagonistic effect on the T effector cell response. The polypeptides described herein (e.g., single-chain polypeptides, antibodies, their antigen-binding fragments, and their constructs) may be used in the treatment of various pathological conditions, including cancer and infectious diseases.

[0010] In one aspect, the present disclosure features polypeptides, such as single-chain polypeptides, antibodies, their antigen-binding fragments, and their constructs, that specifically bind to human tumor necrosis factor receptor 2 (TNFR2) at an epitope within cysteine-rich domain (CRD) 3 (CRD3) and / or CRD4 and do not specifically bind to TNFR2 at an epitope defined by one or more amino acids within CRD1. The polypeptides (a) contain a human IgG2 hinge region lacking the cysteine residues at positions 232 and / or 233 of the amino acid sequence of the IgG2 hinge region, and / or (b) It contains antigen-binding sites that are separated from each other by a distance of at least about 133 Å.

[0011] Exemplary antagonist TNFR2 polypeptides (e.g., antibodies and antigen-binding fragments thereof) of the present disclosure having the above characteristics are described in Table 1 below. Table 1 provides a description of various antagonist TNFR2 antibodies and antigen-binding fragments thereof defined by their heavy-chain amino acid sequences and light-chain amino acid sequences. Antagonist TNFR2 antibodies and antigen-binding fragments thereof of the present disclosure include, in addition to antagonist TNFR2 antibodies and antigen-binding fragments thereof having the heavy chains and / or light chains shown in Table 1, antibodies and antigen-binding fragments thereof containing heavy chains and / or light chains having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) to the heavy chains and / or light chains shown in Table 1. Complementary determining regions are shown in bold.

[0012] Table 1. Exemplary antagonist TNFR2 antibodies of the present disclosure TIFF0007713880000001.tif248161TIFF0007713880000002.tif241163TIFF0007713880000003.tif247166TIFF0007713880000004.tif223151TIFF0007713880000005.tif223151TIFF0007713880000006.tif223151TIFF0007713880000007.tif223151TIFF0007713880000008.tif223151TIFF0007713880000009.tif223151TIFF0007713880000010.tif223151TIFF0007713880000011.tif223151TIFF0007713880000012.tif223151

[0013] For example, in some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 302.

[0014] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 303.

[0015] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 304.

[0016] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 305.

[0017] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 306.

[0018] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having the amino acid sequence of SEQ ID NO: 297.

[0019] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having the amino acid sequence of SEQ ID NO: 298.

[0020] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having the amino acid sequence of SEQ ID NO: 299.

[0021] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having the amino acid sequence of SEQ ID NO: 300.

[0022] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a light chain having the amino acid sequence of SEQ ID NO: 301.

[0023] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof containing a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 302, and a light chain having the amino acid sequence of SEQ ID NO: 297.

[0024] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 302, and a light chain having the amino acid sequence of SEQ ID NO: 298.

[0025] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 302, and a light chain having the amino acid sequence of SEQ ID NO: 299.

[0026] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 302, and a light chain having the amino acid sequence of SEQ ID NO: 300.

[0027] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 302, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 302, and a light chain having the amino acid sequence of SEQ ID NO: 301.

[0028] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 303, and a light chain having the amino acid sequence of SEQ ID NO: 297.

[0029] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 303, and a light chain having the amino acid sequence of SEQ ID NO: 298.

[0030] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 303, and a light chain having the amino acid sequence of SEQ ID NO: 299.

[0031] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof comprising a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence of SEQ ID NO: 303, and a light chain having the amino acid sequence of SEQ ID NO: 300.

[0032] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 303, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 303, and a light chain having the amino acid sequence of SEQ ID NO: 301.

[0033] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 304, and a light chain having the amino acid sequence of SEQ ID NO: 297.

[0034] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof containing a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 304, and a light chain having the amino acid sequence of SEQ ID NO: 298.

[0035] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof containing a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 304, and a light chain having the amino acid sequence of SEQ ID NO: 299.

[0036] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 304, and a light chain having the amino acid sequence of SEQ ID NO: 300.

[0037] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 304, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 304, and a light chain having the amino acid sequence of SEQ ID NO: 301.

[0038] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 305, and a light chain having the amino acid sequence of SEQ ID NO: 297.

[0039] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 305, and a light chain having the amino acid sequence of SEQ ID NO: 298.

[0040] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 305, and a light chain having the amino acid sequence of SEQ ID NO: 299.

[0041] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 305, and a light chain having the amino acid sequence of SEQ ID NO: 300.

[0042] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 305, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 305, and a light chain having the amino acid sequence of SEQ ID NO: 301.

[0043] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 306, and a light chain having the amino acid sequence of SEQ ID NO: 297.

[0044] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen - binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen - binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen - binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the antagonistic TNFR2 antibody or an antigen - binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 306, and a light chain having the amino acid sequence of SEQ ID NO: 298.

[0045] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 306, and a light chain having the amino acid sequence of SEQ ID NO: 299.

[0046] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 306, and a light chain having the amino acid sequence of SEQ ID NO: 300.

[0047] In some embodiments, the present disclosure features an antagonistic TNFR2 antibody or an antigen-binding fragment thereof that contains a heavy chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 306, and a light chain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In some embodiments, the antagonistic TNFR2 antibody or an antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 306, and a light chain having the amino acid sequence of SEQ ID NO: 301.

[0048] In some embodiments of the present disclosure, polypeptides, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, contain a human IgG2 hinge region lacking cysteine residues at positions 232 and / or 233 of the amino acid sequence of the IgG2 hinge region. For example, a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may contain a human IgG2 hinge region having an amino acid other than cysteine, such as a serine residue, at position 232 and / or 233 of the amino acid sequence of the IgG2 hinge region.

[0049] A polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may contain, for example, a human IgG2 hinge region having an amino acid substitution or deletion at one or both of cysteine residues 232 and 233. The amino acid substitution may be a conservative amino acid substitution, such as a C232S amino acid substitution and / or a C233S amino acid substitution.

[0050] In some embodiments, for example, when the IgG2 hinge region contains a serine residue at one or both of positions 232 and 233 of the IgG2 hinge amino acid sequence, the IgG2 hinge region has an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 291. For example, when the IgG2 hinge region contains serine residues at positions 232 and 233 of the IgG2 hinge amino acid sequence, the IgG2 hinge region can have an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 291. In some embodiments, for example, when the IgG2 hinge region contains serine residues at positions 232 and 233 of the IgG2 hinge amino acid sequence, the IgG2 hinge region has an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 291.

[0051] A polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may contain antigen-binding sites that are separated from each other by a distance of at least about 133 Å (e.g., a distance of about 133 Å to about 160 Å, e.g., a distance of about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å, 151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160 Å, etc.). In some embodiments, the antigen-binding sites are separated from each other by a distance of at least about 134 Å (e.g., a distance of about 134 Å to about 160 Å, e.g., a distance of about 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å, 151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160 Å, etc.). In some embodiments, the antigen-binding sites are separated from each other by a distance of at least about 139 Å (e.g., a distance of about 139 Å to about 160 Å, e.g., a distance of about 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å, 151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160 Å, etc.). In some embodiments, the antigen-binding sites are separated from each other by a distance of at least about 150 Å (e.g., a distance of about 150 Å to about 160 Å, e.g., a distance of about 150 Å, 151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160 Å, etc.).

[0052] For example, a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may contain antigen-binding sites that are separated from each other by a distance of about 133 Å to about 150 Å, e.g., a distance of about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, or 150 Å. In some embodiments, the antigen-binding sites are separated from each other by a distance of about 133 Å to about 145 Å, e.g., a distance of about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, or 145 Å. In some embodiments, the antigen-binding sites are separated from each other by a distance of about 133 Å to about 139 Å, e.g., a distance of about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, or 139 Å. In some embodiments, the antigen-binding sites are separated from each other by a distance of about 134 Å to about 139 Å, e.g., a distance of about 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, or 139 Å.

[0053] A polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may contain a complementarity-determining region (CDR) heavy chain 1 (CDR1) having the amino acid sequence GJTF(J)2Y (SEQ ID NO: 276) or GJTF(J)2YJ (SEQ ID NO: 277), where each J is independently a natural amino acid. In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) (a) a CDR-H2 having the amino acid sequence (J)3GSJ or (J)5GSJ, (b) a CDR-H3 having the amino acid sequence JRJDGJSJY(J)2FDJ (SEQ ID NO: 278) or JRJDGSY(J)2FD(J)3 (SEQ ID NO: 279), (c) a CDR-L1 having the amino acid sequence (J)9Y or (J)5Y, (d) a CDR-L2 having the amino acid sequence (J)6S or (J)2S, and / or (e) A CDR-L3 having the amino acid sequence (J)5Y(J)2T or (J)3Y(J)4T, further comprising, each J is independently a natural amino acid.

[0054] The polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may contain a CDR-H1 having the amino acid sequence Z 4 FZ 3 Z 5 SSZ 5 or Z 4 YZ 3 Z 5 TDZ 5 X, each Z 3 is independently an amino acid containing a polar and uncharged side chain at physiological pH, each Z 4 is independently glycine or alanine, each Z 5 is independently an amino acid containing a hydrophobic side chain, each X is independently leucine or isoleucine.

[0055] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is (a) having a CDR-H2 with the amino acid sequence SSGZ 4 Z 3 Y (SEQ ID NO: 263) or VDPEYZ 4 Z 3 T (SEQ ID NO: 264), (b) having a CDR-H2 with the amino acid sequence QZ 1 VZ 2 Z 4 YZ 3 SZ 5 WYZ 5 Z 2 Z 5 (SEQ ID NO: 265) or AZ 1 DZ 2 Z 4 Z 3 Z 5 SPZ5 Z 2 Z 5 CDR-H3 having WG (Accession No.: 266), (c) Amino acid sequence SASSSVYYMZ 5 (Accession No.: 267) or QNINKZ 5 CDR-L1 having (Accession No.: 268), (d) Amino acid sequence STSNLAZ 3 (Accession No.: 269), TYZ 3 or YTZ 3 CDR-L2 having, and / or, (e) Amino acid sequence QQRRNZ 5 PYZ 3 (Accession No.: 270) or CLQZ 5 VNLXZ 3 CDR-L3 having (Accession No.: 271), further comprising, each Z 1 is independently an amino acid containing a cationic side chain at physiological pH, each Z 2 is independently an amino acid containing an anionic side chain at physiological pH, each Z 3 is independently an amino acid containing a polar and uncharged side chain at physiological pH, each Z 4 is independently glycine or alanine, each Z 5 is independently an amino acid containing a hydrophobic side chain, each X is independently leucine or isoleucine.

[0056] A polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may contain a CDR-H1 having an amino acid sequence of GFTFSSY (SEQ ID NO: 23), GYTFTDYX (SEQ ID NO: 257), or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) compared to these sequences, wherein each X is independently leucine or isoleucine, and optionally, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is (a) a CDR-H2 having an amino acid sequence of SSGGSY (SEQ ID NO: 24), VDPEYGST (SEQ ID NO: 258), or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) compared to these sequences, (b) a CDR-H3 having an amino acid sequence of QRVDGYSSYWYFDV (SEQ ID NO: 25), ARDDGSYSPFDYWG (SEQ ID NO: 259), ARDDGSYSPFDY (SEQ ID NO: 296), or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) compared to these sequences, (c) a CDR-L1 having an amino acid sequence of SASSSVYYMY (SEQ ID NO: 26), QNINKY (SEQ ID NO: 260), or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) compared to these sequences, (d) a CDR-L2 having an amino acid sequence of STSNLAS (SEQ ID NO: 27), TYS, YTS, or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) compared to SEQ ID NO: 27, and / or (e) a CDR-L3 having an amino acid sequence of QQRRNYPYT (SEQ ID NO: 28), CLQYVNLXT (SEQ ID NO: 261), or an amino acid sequence having up to two amino acid substitutions (e.g., conservative amino acid substitutions) compared to these sequences, and further comprises.

[0057] In some embodiments, a polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises the following CDRs: (a) CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 23); (b) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO: 24); and (c) CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ ID NO: 25); The heavy chain comprises one or more of: A polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may comprise, for example, the following CDRs: (a) CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257); (b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258); and (c) CDR-H3 having the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259); and Each X is independently leucine or isoleucine.

[0058] In some embodiments, CDR-H1 has the amino acid sequence GYTFTDYL (SEQ ID NO: 274). In some embodiments, CDR-H1 has the amino acid sequence GYTFTDYI (SEQ ID NO: 275). In some embodiments, CDR-H1 has the amino acid sequence GYTFTDVI (SEQ ID NO: 293). In some embodiments, CDR-H1 has the amino acid sequence GYTFTDYS (SEQ ID NO: 294).

[0059] Additionally or alternatively, the polypeptide (e.g., single chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) may comprise, for example, the following CDRs: (a) CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID NO: 26); (b) A CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO: 27), and (c) A CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO: 28), may contain a light chain having one or more of them. In some embodiments, the antibody or its antigen-binding fragment is the following CDRs, (a) A CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260), (b) A CDR-L2 having the amino acid sequence TYS or YTS, and (c) A CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261), contains a light chain having one or more of them, where each X is independently leucine or isoleucine.

[0060] In some embodiments, CDR-L2 has the amino acid sequence TYS. In some embodiments, CDR-L2 has the amino acid sequence YTS. CDR-L3 may have the amino acid sequence CLQYVNLLT (SEQ ID NO: 272). In some embodiments, CDR-L3 has the amino acid sequence CLQYVNLIT (SEQ ID NO: 273).

[0061] The polypeptide (e.g., single-chain polypeptide, antibody, its antigen-binding fragment, or its construct) is (a) A CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 23), (b) A CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO: 24), and (c) A CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ ID NO: 25), may contain three heavy-chain CDRs including (d) A CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID NO: 26), (e) A CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO: 27), and (f) A CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO: 28), may further contain three light chain CDRs including .

[0062] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is (a) A CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257), such as GYTFTDYL (SEQ ID NO: 274) or GYTFTDYI (SEQ ID NO: 275), (b) A CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258), and (c) A CDR-H3 having the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259), contains three heavy chain CDRs including (d) A CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260), (e) A CDR-L2 having the amino acid sequence TYS or YTS, and (f) A CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261), such as CLQYVNLLT (SEQ ID NO: 272) or CLQYVNLIT (SEQ ID NO: 273), further contains three light chain CDRs including Each X is independently leucine or isoleucine.

[0063] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) includes a framework region having the amino acid sequence LLIR (SEQ ID NO: 262) bound to the N-terminus of CDR-L2 and / or a framework region having the amino acid sequence TLE bound to the C-terminus of CDR-L2.

[0064] A polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may have a heavy-chain variable domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the heavy-chain variable domain has an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the heavy-chain variable domain has an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 2.

[0065] Additionally or alternatively, a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may have a light-chain variable domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the light-chain variable domain has an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the light-chain variable domain has an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO: 4.

[0066] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has a K of less than about 100 nM D therein (e.g., a K of about 10 pM to about 99 nM D , e.g., a K of about 20 pM to about 80 nM, about 30 pM to about 70 nM, about 40 pM to about 60 nM, about 50 pM to about 50 nM, about 60 pM to about 40 nM, about 70 pM to about 30 nM, about 80 pM to about 20 nM, about 90 pM to about 10 nM, or about 100 pM to about 1 nM, etc. of K DIt specifically binds to a peptide having any one of the amino acid sequences of SEQ ID NOs: 11, 19, 20, and 34 to 117, and does not specifically bind to a peptide containing amino acids 56 to 60 (KCSPG) of SEQ ID NO: 7. The polypeptide (for example, a single-chain polypeptide, an antibody, its antigen-binding fragment, or a construct thereof) is, for example, among other values, about 1 pM, 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM, 150 pM, 155 pM, 160 pM, 165 pM, 170 pM, 175 pM, 180 pM, 185 pM, 190 pM, 195 pM, 200 pM, 205 pM, 210 pM, 215 pM, 220 pM, 225 pM, 230 pM, 235 pM, 240 pM, 245 pM, 250 pM, 255 pM, 260 pM, 265 pM, 270 pM, 275 pM, 280 pM, 285 pM, 290 pM, 295 pM, 300 pM, 305 pM, 310 pM, 315 pM, 320 pM, 325 pM, 330 pM, 335 pM, 340 pM, 345 pM, 350 pM, 355 pM, 360 pM, 365 pM, 370 pM, 375 pM, 380 pM, 385 pM, 390 pM, 395 pM, 400 pM, 405 pM, 410 pM, 415 pM, 420 pM, 425 pM, 430 pM, 435 pM, 440 pM, 445 pM, 450 pM, 455 pM, 460 pM, 465 pM, 470 pM, 475 pM, 480 pM, 485 pM, 490 pM, 495 pM, 500 pM, 505 pM, 510 pM, 515 pM, 520 pM, 525 pM, 530 pM, 535 pM, 540 pM, 545 pM, 550 pM, 555 pM, 560 pM, 565 pM, 570 pM, 575 pM, 580 pM, 585 pM, 590 pM, 595 pM, 600 pM, 605 pM, 610 pM, 615 pM, 620 pM, 625 pM, 630 pM, 635 pM, 640 pM, 645 pM, 650 pM, 655 pM, 660 pM, 665 pM, 670 pM, 675 pM, 680 pM, 685 pM, 690 pM, 695 pM, 700 pM, 705 pM,710 pM, 715 pM, 720 pM, 725 pM, 730 pM, 735 pM, 740 pM, 745 pM, 750 pM, 755 pM, 760 pM, 765 pM, 770 pM, 775 pM, 780 pM, 785 pM, 790 pM, 795 pM, 800 pM, 805 pM, 810 pM, 815 pM, 820 pM, 825 pM, 830 pM, 835 pM, 840 pM, 845 pM, 850 pM, 855 pM, 860 pM, 865 pM, 870 pM, 875 pM, 880 pM, 885 pM, 890 pM, 895 pM, 900 pM, 905 pM, 910 pM, 915 pM, 920 pM, 925 pM, 930 pM, 935 pM, 940 pM, 945 pM, 950 pM, 955 pM, 960 pM, 965 pM, 970 pM, 975 pM, 980 pM, 985 pM, 990 pM, 995 pM, 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 96 nM, 97 nM, 98 nM, or 99 nM of K, D and can bind to a peptide having any one amino acid sequence of SEQ ID NOs: 11, 19, 20, and 34-117.

[0067] The polypeptide (e.g., a single-chain polypeptide, an antibody, its antigen-binding fragment, or its construct) is (a) amino acids 142-146 (KCRPG) of SEQ ID NO: 7, (b) amino acids 142-149 (KCRPGFGV) of SEQ ID NO: 7, (c) amino acids 137-144 (CAPLRKCR) of SEQ ID NO: 7, (d) amino acids 150-190 (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI) of SEQ ID NO: 7, (e) amino acids 161-169 (CKPCAPGTF) of SEQ ID NO: 7, (f) Amino acids 75 - 128 of SEQ ID NO:7 (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL) (optionally, the epitope is within amino acids 80 - 86 (DSTYTQL), 91 - 98 (PECLSCGS), or 116 - 123 (RICTCRPG) of SEQ ID NO:7), (g) Amino acids 174 - 184 of SEQ ID NO:7 (SSTDICRPHQI), (h) Amino acids 126 - 140 of SEQ ID NO:7 (CALSKQEGCRLCAPL), and / or (i) Amino acids 156 - 165 of SEQ ID NO:7 (TSDVVCKPCA), can specifically bind to TNFR2 in the epitopes within.

[0068] In some embodiments, the polypeptide (e.g., a single - chain polypeptide, an antibody, an antigen - binding fragment thereof, or a construct thereof) specifically binds to TNFR2 in two or more of the above - mentioned epitopes (e.g., in 2, 3, 4, 5, 6, 7, 8, 9, 10, or more epitopes within the above - mentioned amino acid ranges).

[0069] In some embodiments, the polypeptide (e.g., a single - chain polypeptide, an antibody, an antigen - binding fragment thereof, or a construct thereof) has a K D e.g., a K of about 1 nM or less, such as D and specifically binds to TNFR2. For example, the polypeptide (e.g., a single - chain polypeptide, an antibody, an antigen - binding fragment thereof, or a construct thereof) has a K of about 1 pM to about 10 nM D, for example, among other values, approximately 1 pM, 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM, 150 pM, 155 pM, 160 pM, 165 pM, 170 pM, 175 pM, 180 pM, 185 pM, 190 pM, 195 pM, 200 pM, 205 pM, 210 pM, 215 pM, 220 pM, 225 pM, 230 pM, 235 pM, 240 pM, 245 pM, 250 pM, 255 pM, 260 pM, 265 pM, 270 pM, 275 pM, 280 pM, 285 pM, 290 pM, 295 pM, 300 pM, 305 pM, 310 pM, 315 pM, 320 pM, 325 pM, 330 pM, 335 pM, 340 pM, 345 pM, 350 pM, 355 pM, 360 pM, 365 pM, 370 pM, 375 pM, 380 pM, 385 pM, 390 pM, 395 pM, 400 pM, 405 pM, 410 pM, 415 pM, 420 pM, 425 pM, 430 pM, 435 pM, 440 pM, 445 pM, 450 pM, 455 pM, 460 pM, 465 pM, 470 pM, 475 pM, 480 pM, 485 pM, 490 pM, 495 pM, 500 pM, 505 pM, 510 pM, 515 pM, 520 pM, 525 pM, 530 pM, 535 pM, 540 pM, 545 pM, 550 pM, 555 pM, 560 pM, 565 pM, 570 pM, 575 pM, 580 pM, 585 pM, 590 pM, 595 pM, 600 pM, 605 pM, 610 pM, 615 pM, 620 pM, 625 pM, 630 pM, 635 pM, 640 pM, 645 pM, 650 pM, 655 pM, 660 pM, 665 pM, 670 pM, 675 pM, 680 pM, 685 pM, 690 pM, 695 pM, 700 pM, 705 pM, 710 pM, 715 pM, 720 pM, 725 pM, 730 pM, 735 pM, 740 pM, 745 pM, 750 pM, 755 pM, 760 pM, 765 pM, 770 pM, 775 pM, 780 pM, 785 pM, 790 pM, 795 pM, 800 pM, 805 pM, 810 pM, 815 pM, 820 pM, 825 pM, 830 pM,K such as 835 pM, 840 pM, 845 pM, 850 pM, 855 pM, 860 pM, 865 pM, 870 pM, 875 pM, 880 pM, 885 pM, 890 pM, 895 pM, 900 pM, 905 pM, 910 pM, 915 pM, 920 pM, 925 pM, 930 pM, 935 pM, 940 pM, 945 pM, 950 pM, 955 pM, 960 pM, 965 pM, 970 pM, 975 pM, 980 pM, 985 pM, 990 pM, 995 pM, 1 nM, 5 nM, or 10 nM, D and can specifically bind to TNFR2. In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has a K of about 621 pM D and specifically binds to TNFR2. In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has a K of about 44 pM D and specifically binds to TNFR2.

[0070] The polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has a k of at least about 10 4 M -1 s -1 and can specifically bind to TNFR2 to form an antibody-antigen complex. For example, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has a k of about 1 × 10 on M 4 s -1 to -1 about 1 × 10 8 M -1 s -1 and can specifically bind to TNFR2. For example, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has a k of about 1 × 10 on M 4 s -1 , 2 × 10 -1 M 4 s -1 , 3 × 10 -1 M 4 s -1 , 4 × 10 -1 M 4 s -1 , 5 × 10 -1 M 4 s -1 s-1 , 6×10 4 M -1 s -1 , 7×10 4 M -1 s -1 , 8×10 4 M -1 s -1 , 9×10 4 M -1 s -1 , 1×10 5 M -1 s -1 , 2×10 5 M -1 s -1 , 3×10 5 M -1 s -1 , 4×10 5 M -1 s -1 , 5×10 5 M -1 s -1 , 6×10 5 M -1 s -1 , 7×10 5 M -1 s -1 , 8×10 5 M -1 s -1 , 9×10 5 M -1 s -1 , 1×10 6 M -1 s -1 , 2×10 6 M -1 s -1 , 3×10 6 M -1 s -1 , 4×10 6 M -1 s -1 , 5×10 6 M -1 s -1 , 6×10 6 M -1 s -1 , 7×10 6 M -1 s -1 , 8×10 6 M -1 s -1 , 9×10 6 M-1 s -1 、 1×10 7 M -1 s -1 、 2×10 7 M -1 s -1 、 3×10 7 M -1 s -1 、 4×10 7 M -1 s -1 、 5×10 7 M -1 s -1 、 6×10 7 M -1 s -1 、 7×10 7 M -1 s -1 、 8×10 7 M -1 s -1 、 9×10 7 M -1 s -1 、 or 1×10 8 M -1 s -1 of k on can specifically bind to TNFR2 to form an antibody-antigen complex. In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has a k of about 4.9×10 6 M -1 s -1 of k on and specifically binds to TNFR2 to form an antibody-antigen complex. In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) has a k of about 3.6×10 5 M -1 s -1 of k on and specifically binds to TNFR2 to form an antibody-antigen complex.

[0071] A polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) specifically binds to TNFR2 and, for example, has a K of about 10 -3 s -1 or less, for example, about 10 off 、 for example, about 10-6 s -1 ~ about 10 -3 s -1 such as K off (for example, about 1×10 -6 s -1 , 2×10 -6 s -1 , 3×10 -6 s -1 , 4×10 -6 s -1 , 5×10 -6 s -1 , 6×10 -6 s -1 , 7×10 -6 s -1 , 8×10 -6 s -1 , 9×10 -6 s -1 , 1×10 -5 s -1 , 2×10 -5 s -1 , 3×10 -5 s -1 , 4×10 -5 s -1 , 5×10 -5 s -1 , 6×10 -5 s -1 , 7×10 -5 s -1 , 8×10 -5 s -1 , 9×10 -5 s -1 , 1×10 -4 s -1 , 2×10 -4 s -1 , 3×10 -4 s -1 , 4×10 -4 s -1 , 5×10 -4 s -1 , 6×10 -4 s -1 , 7×10 -4 s -1 , 8×10 -4 s -1 , 9×10 -4 s -1 , or 1×10 -3 s -1 of K off) can form an antibody-antigen complex that dissociates. In some embodiments, the antibody-antigen complex dissociates at about 2.2×10 -4 s -1 with a K off for dissociation.

[0072] The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can, for example, bind to cells expressing TNFR2, such as T-reg cells (e.g., CD25 HiIt can inhibit TNFR2 signaling within, for example, T-reg cells expressing it, myeloid-derived suppressor cells (MDSC), and / or TNFR2+ cancer cells. In some embodiments, a single-chain polypeptide, antibody, or antigen-binding fragment thereof can be evaluated, for example, by observing a decrease in the expression of one or more of the above genes, or by using other methods well-known in the art for evaluating gene activation. When evaluated, it can reduce or inhibit the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3. For example, an antagonistic TNFR2 single-chain polypeptide, antibody, antigen-binding fragment thereof, and constructs thereof can reduce the expression or post-translational modification (e.g., phosphorylation) of one or more of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, or cIAP2 / BIRC3 compared to the expression or post-translational modification (e.g., phosphorylation) of one or more of these proteins isolated from a sample not treated with the antagonistic TNFR2 single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof described herein, by, for example, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Exemplary assays that can be used to measure expression levels and phosphorylation status are well-known in the art and include, for example, Western blot assays for measuring protein content and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments for measuring mRNA content.In a preferred embodiment, the anti-TNFR2 polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, and constructs thereof) is a dominant TNFR2 antagonist, and as a result, it can inhibit TNFR2 activation even in the presence of a TNFR2 agonist (e.g., TNFα or Bacillus Calmette-Guerin (BCG), etc.) or a growth-promoting factor such as IL-2.

[0073] The antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein have the following properties: (a) Inhibition of the proliferation of T-reg cells and / or direct killing of T-reg cells (e.g., by binding to TNFR2 on the surface of T-reg cells and inactivating TNFR2, thereby reducing the amount of T-reg cells in a cell population by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% compared to a cell population not exposed to the polypeptide), (b) Inhibition of the proliferation of MDSC and / or direct killing of MDSC (e.g., by binding to TNFR2 on the surface of MDSC and inactivating TNFR2, thereby reducing the amount of MDSC in a cell population by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% compared to a cell population not exposed to the polypeptide), (c) Promotion of the proliferation of T effector cells, such as CD8+ T cells, etc. (e.g., thereby increasing the amount of CD8+ effector T cells in the cell population by about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5-fold, 5.1-fold, 5.2-fold, 5.3-fold, 5.4-fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6-fold, 6.1-fold, 6.2-fold, 6.3-fold, 6.4-fold, 6.5-fold, 6.6-fold, 6.7-fold, 6.8-fold, 6.9-fold, 7-fold, 7.1-fold, 7.2-fold, 7.3-fold, 7.4-fold, 7.5-fold, 7.6-fold, 7.7-fold, 7.8-fold, 7.9-fold, 8-fold, 8.1-fold, 8.2-fold, 8.3-fold, 8.4-fold, 8.5-fold, 8.6-fold, 8.7-fold, 8.8-fold, 8.9-fold, 9-fold, 9.1-fold, 9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold, 9.6-fold, 9.7-fold, 9.8-fold, 9.9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more as compared to the cell population not exposed to the polypeptide), and / or (d) Inhibition of the proliferation of TNFR2-expressing cancer cells (e.g., Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic cancer cells or lymphoma cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.) and / or direct killing of TNFR2-expressing cancer cells (e.g., thereby reducing the amount of TNFR2-expressing cancer cells in the cell population by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% as compared to the cell population not exposed to the polypeptide), One or more or all of them may be shown.

[0074] For example, using the antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., the total amount of T-reg cells or cancer cells in a patient (e.g., a human patient, etc.) or in a sample (e.g., a sample isolated from a patient, e.g., a human patient undergoing treatment for cancer or an infectious disease described herein) may be decreased by comparing with a patient not undergoing treatment with the polypeptide or a sample not treated with the polypeptide, respectively.

[0075] In some embodiments, the antagonist TNFR2 polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) decreases the expression of TNFR2 by T-reg cells or cancer cells (e.g., TNFR2+ cancer cells, such as Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T-cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic cancer cells or lymphoma cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, or upper gastrointestinal cancer cells, etc.), and / or decreases the secretion of soluble TNFR2 by one or more of the above cells.

[0076] Using the antagonist TNFR2 polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof), the proliferation of T-reg cells in a patient (e.g., a human patient) or in a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infectious disease described herein) may be inhibited or suppressed, or the total amount of T-reg cells may be decreased.

[0077] Using the antagonist TNFR2 polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof), the proliferation of T-reg cells expressing TNFR2 (e.g., activated T-reg cells expressing CD25 Hi ), and / or cancer cells can be inhibited or suppressed, and / or the T-reg cells expressing TNFR2 and / or cancer cells can be directly killed. For example, the cancer cells can be selected from the group consisting of Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T-cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic cancer cells or lymphoma cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, or upper gastrointestinal cancer cells. Without being limited by mechanism, binding to TNFR2 on cancer cells may inhibit or suppress the proliferation of cancer cells, and / or directly kill cancer cells, for example, by promoting apoptosis of cancer cells.

[0078] The antagonist TNFR2 polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) bind to TNFR2 on the surface of cells expressing all or part of all proteins and small molecules selected from the group consisting of MDSCs (e.g., B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1, CD2, CD31 (PECAM-1), CD43, CD44, complement component C5a R1, F4 / 80 (EMR1), Fcγ RIII (CD16), Fcγ RII (CD32), Fcγ RIIA (CD32a), Fcγ RIIB (CD32b), Fcγ RIIB / C (CD32b / c), Fcγ RIIC (CD32c), Fcγ RIIIA (CD16A), Fcγ RIIIB (CD16b), galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1RI, IL-4Rα, IL-6Rα, integrin α4 (CD49d), integrin αL (CD11a), integrin αM (CD11b), M-CSFR, MGL1 (CD301a), MGL1 / 2 (CD301a / b), MGL2 (CD301b), nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), siglec-3 (CD33), transferrin receptor (TfR), VEGFR1 (Flt-1), and VEGFR2 (KDR or Flk-1)). Specifically, MDSCs do not express proteins selected from the group consisting of B7-2 (CD86), B7-H4, CD11c, CD14, CD21, CD23 (FcεRII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6). Binding to TNFR2 on MDSCs may inhibit or suppress the proliferation of MDSCs and / or directly kill MDSCs, for example, by promoting apoptosis of MDSCs. The polypeptides described herein, e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., may not require TNFα to inhibit the proliferation of T-reg cells, cancer cells (e.g., TNFR2-expressing cancer cells), and / or MDSCs.

[0079] In some embodiments, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, inhibit the proliferation of T-reg cells and / or directly kill T-reg cells at a higher titer in patients with cancer compared to subjects without cancer. In some embodiments, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, inhibit the proliferation of T-reg cells and / or directly kill T-reg cells at a higher titer in the tumor microenvironment compared to a site without cancer cells in a patient with cancer, such as a site far from the tumor.

[0080] For example, in some embodiments, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, inhibit the proliferation of T-reg cells and / or directly kill T-reg cells at a higher titer in the tumor microenvironment than at a site without cancer cells in a patient with cancer, such as a site far from the tumor, or compared to a subject without cancer. For example, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, have an IC 50 50, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more-fold lower for inhibiting the proliferation of T-reg cells in the tumor microenvironment than 50can be shown. The polypeptides described herein, such as single-chain polypeptides, antibodies, their antigen-binding fragments, and their constructs, etc., in the tumor microenvironment containing T cell lymphoma cells (such as Hodgkin lymphoma cells or cutaneous non-Hodgkin lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells, can inhibit the proliferation of T-reg cells at a higher titer in a site that does not contain such cancer cells in a patient suffering from one or more of the above cancers, such as a site far from the tumor, etc., or compared to a subject without cancer.

[0081] In some embodiments, the polypeptides described herein, such as single-chain polypeptides, antibodies, their antigen-binding fragments, and their constructs, etc., inhibit or suppress the proliferation of MDSCs and / or directly kill MDSCs at a higher titer in a patient suffering from cancer compared to a subject without cancer. In some embodiments, the polypeptides described herein, such as single-chain polypeptides, antibodies, their antigen-binding fragments, and their constructs, etc., inhibit or suppress the proliferation of MDSCs and / or directly kill MDSCs at a higher titer in the tumor microenvironment compared to a site that does not contain cancer cells in a patient suffering from cancer, such as a site far from the tumor, etc., or compared to a subject without cancer.

[0082] For example, the antagonistic TNFR2 polypeptides (such as single-chain polypeptides, antibodies, their antigen-binding fragments, and their constructs) described herein can bind to TNFR2 on the surface of MDSCs present within the tumor microenvironment and can inhibit or suppress the proliferation of MDSCs or promote the apoptosis of MDSCs at a higher titer in the tumor microenvironment compared to a site that does not contain cancer cells in a patient suffering from cancer, such as a site far from the tumor, etc., or compared to a subject without cancer. For example, the polypeptides described herein, such as single-chain polypeptides, antibodies, their antigen-binding fragments, and their constructs, etc., are the IC of the polypeptide for inhibiting the proliferation of MDSCs at a site that does not contain cancer cells. 50Thus, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more, lower multiples of an IC for inhibiting the proliferation of MDSC in the tumor microenvironment 50 can be shown. The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., in the tumor microenvironment containing T cell lymphoma cells (e.g., Hodgkin lymphoma cells or cutaneous non-Hodgkin lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells, can inhibit the proliferation of MDSC, or promote the apoptosis of MDSC, at a higher titer than in a site not containing such cancer cells, such as a site far from the tumor, etc., in a patient suffering from one or more of the above cancers, or compared to a subject without cancer.

[0083] In some embodiments, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., can proliferate T effector cells, such as CD8+ cytotoxic T cells, etc., at a higher titer in a patient suffering from cancer compared to a subject without cancer. In some embodiments, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., can proliferate T effector cells, such as CD8+ cytotoxic T cells, etc., at a higher titer in the tumor microenvironment compared to a site not containing cancer cells, such as a site far from the tumor, etc., in a patient suffering from cancer, or compared to a subject without cancer.

[0084] For example, in some embodiments, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, are present at a higher titer in the tumor microenvironment than in sites of a patient with cancer that do not contain cancer cells, such as sites distant from the tumor, or compared to a subject without cancer, and directly expand T effector cells, such as CD8+ cytotoxic T cells. For example, the polypeptides described herein have an EC 50 for expanding T effector cells in a cancer patient that is, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more lower than the EC for expanding T effector cells in a subject without cancer. 50 The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can directly expand T effector cells, such as CD8+ cytotoxic T cells, at a higher titer in the tumor microenvironment of a tumor containing T cell lymphoma cells (such as Hodgkin lymphoma cells or cutaneous non-Hodgkin lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells, than in sites of a patient with one or more of the above cancers or a subject without cancer that do not contain such cancer cells, such as sites distant from the tumor. In some embodiments, the T effector cells (such as CD8+ cytotoxic T cells) specifically react with an antigen present on one or more types of cancer cells, such as Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells.

[0085] In some embodiments, the polypeptide is a human IgG2 isotype antibody or an antigen-binding fragment thereof. Additionally or alternatively, the polypeptide is selected from the group consisting of a monoclonal antibody or an antigen-binding fragment thereof, a polyclonal antibody or an antigen-binding fragment thereof, a human antibody or an antigen-binding fragment thereof, a humanized antibody or an antigen-binding fragment thereof, a primatized antibody or an antigen-binding fragment thereof, a bispecific antibody or an antigen-binding fragment thereof, a multispecific antibody or an antigen-binding fragment thereof, a dual variable immunoglobulin domain, a monovalent antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, an Fv fragment, a Fab fragment, an F(ab’)2 molecule, and a tandem scFv (taFv), or an antigen-binding fragment thereof. In some embodiments, the antibody or an antigen-binding fragment thereof contains two or more CDRs covalently linked to each other, for example, by an amide bond, a thioether bond, a carbon-carbon bond, or a disulfide bridge, or by a linker, such as a linker described herein. In some embodiments, the antibody or an antigen-binding fragment thereof is a human antibody, a humanized antibody, or a chimeric antibody, or an antigen-binding fragment thereof.

[0086] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is conjugated to a therapeutic agent, such as a cytotoxic agent (e.g., a cytotoxic agent described herein).

[0087] Any of the antagonist TNFR2 antibodies of the above-described embodiments may be a bispecific antibody, for example, a bispecific monoclonal antibody, in which one arm of the antibody specifically binds to TNFR2 and the other specifically binds to an immune checkpoint protein, such as, among others, PD-1, PD-L1, or CTLA-4 described herein. The arm of the bispecific antibody that specifically binds to TNFR2 may specifically bind to an epitope of human TNFR2 defined by one or more amino acids within CRD3 and / or an epitope defined by one or more amino acids within CRD4. In some embodiments, the arm of the bispecific antibody that specifically binds to TNFR2 is (a) Amino acids 142-146 (KCRPG) of SEQ ID NO: 7, (b) Amino acids 142-149 (KCRPGFGV) of SEQ ID NO: 7, (c) Amino acids 137-144 (CAPLRKCR) of SEQ ID NO: 7, (d) Amino acids 150-190 (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI) of SEQ ID NO: 7, (e) Amino acids 161-169 (CKPCAPGTF) of SEQ ID NO: 7, (f) Amino acids 75-128 (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL) of SEQ ID NO: 7 (optionally, the epitope is within amino acids 80-86 (DSTYTQL), 91-98 (PECLSCGS), or 116-123 (RICTCRPG) of SEQ ID NO: 7), (g) Amino acids 174-184 (SSTDICRPHQI) of SEQ ID NO: 7, (h) Amino acids 126-140 (CALSKQEGCRLCAPL) of SEQ ID NO: 7, and, (i) Amino acids 156-165 (TSDVVCKPCA) of SEQ ID NO: 7, specifically binds to an epitope of human TNFR2 selected from

[0088] In some embodiments, the bispecific antibody contains one arm that specifically binds to TNFR2, such as an epitope of the above-mentioned human TNFR2, and one arm that specifically binds to an immune checkpoint protein that specifically binds to PD-1. In some embodiments, the arm of the bispecific antibody that specifically binds to PD-1 can specifically bind to an epitope(s) on PD-1, the same as in the case of nivolumab, pembrolizumab, avelumab, durvalumab, or atezolizumab. For example, the arm of the bispecific antibody that specifically binds to PD-1, when evaluated using, for example, the competitive binding assay described herein or those well-known in the art, such as competitive ELISA, can competitively inhibit the binding of PD-1 to nivolumab, pembrolizumab, avelumab, durvalumab, and / or atezolizumab.

[0089] In some embodiments, the bispecific antibody contains one arm that specifically binds to TNFR2, such as an epitope of the above-mentioned human TNFR2, and one arm that specifically binds to PD-L1. In some embodiments, the arm of the bispecific antibody that specifically binds to PD-L1 can specifically bind to an epitope(s) on PD-L1, the same as in the case of atezolizumab or avelumab. For example, the arm of the bispecific antibody that specifically binds to PD-L1, when evaluated using, for example, the competitive binding assay described herein or those well-known in the art, such as competitive ELISA, can competitively inhibit the binding of PD-L1 to atezolizumab and / or avelumab.

[0090] In some embodiments, the bispecific antibody contains one arm that specifically binds to TNFR2, such as an epitope of the human TNFR2 described above, and one arm that specifically binds to CTLA-4. In some embodiments, the arm of the bispecific antibody that specifically binds to CTLA-4 can specifically bind to the epitope(s) on CTLA-4, which is the same as that in the case of ipilimumab or tremelimumab. For example, when the arm of the bispecific antibody that specifically binds to CTLA-4 is evaluated using, for example, the competitive binding assay described herein or those well known in the art, such as competitive ELISA, it can competitively inhibit the binding of CTLA-4 to ipilimumab and / or tremelimumab.

[0091] A second aspect features a construct containing a first polypeptide domain and a second polypeptide domain. The first polypeptide domain and the second polypeptide domain are each independently an antigen-binding fragment of any of the first aspect or its embodiments. The first polypeptide domain and the second polypeptide domain may be linked to each other, for example, by a covalent linker, such as a linker containing an amide bond or a disulfide bond (e.g., an amide bond or a disulfide bond).

[0092] A third aspect features a polynucleotide encoding the polypeptide of the first aspect (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof), and / or a construct of any of the second aspect or its embodiments.

[0093] A fourth aspect features a vector encoding the polynucleotide of the third aspect. The vector may be an expression vector, for example, a eukaryotic expression vector or the like. In some embodiments, the vector is a viral vector, such as an adenovirus (e.g., adenoviruses of serotypes 1-57, such as adenoviruses of serotypes 2, 5, 11, 12, 24, 26, 34, 35, 40, 48, 49, 50, 52, or Pan9), a retrovirus (e.g., a γ-retrovirus or a lentivirus), a poxvirus, an adeno-associated virus, a baculovirus, a herpes simplex virus, or a vaccinia virus (e.g., a modified vaccinia Ankara virus) and the like.

[0094] A fifth aspect features an isolated host cell containing the polynucleotide of the third aspect and / or the vector of the fourth aspect. The host cell may be a prokaryotic cell or a eukaryotic cell, for example, a mammalian cell (e.g., a Chinese hamster ovary (CHO) cell) or the like. The host cell may be, for example, a host cell described in Dinnis and James, Biotechnology and Bioengineering 91:180-189, 2005 (the disclosure of which is incorporated herein by reference).

[0095] A sixth aspect features a pharmaceutical composition containing a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) that specifically binds to human TNFR2 and exhibits an antagonistic effect on TNFR2 activity upon binding. The polypeptide may be, for example, an antibody or an antigen-binding fragment thereof according to any of the first aspect or its embodiments. Additionally or alternatively, the antibody or its antigen-binding fragment may specifically bind to human TNFR2 at an epitope within CRD3 and / or CRD4 and not bind to TNFR2 at an epitope defined by one or more amino acids within CRD1, and at least 10% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform, such as an IgG2-A disulfide bond isoform or an IgG2-B disulfide bond isoform. In some embodiments, about 10% to about 99.999% of the antibody or its antigen-binding fragment in the pharmaceutical composition, for example, about 11% to about 99.9%, about 12% to about 99.9%, about 13% to about 99.9%, about 14% to about 99.9%, about 15% to about 99%, about 16% to about 99.9%, about 17% to about 99.9%, about 18% to about 99.9%, about 19% to about 99.9%, about 20% to about 99.9%, about 21% to about 99.9%, about 22% to about 99.9%, about 23% to about 99.9%, about 24% to about 99.9%, about 25% to about 99.9%, about 26% to about 99.9%, about 27% to about 99.9%, about 28% to about 99.9%, about 29% to about 99.9%, about 30% to about 99.9%, about 31% to about 99.9%, about 32% to about 99.9%, about 33% to about 99.9%, about 34% to about 99.9%, about 35% to about 99.9%, about 36% to about 99.9%, about 37% to about 99.9%, about 38% to about 99.9%, about 39% to about 99.9%, about 40% to about 99.9%, about 41% to about 99.9%, about 42% to about 99.9%, about 43% to about 99.9%, about 44% to about 99.9%, about 45% to about 99.9%, about 46% to about 99.9%, about 47% to about 99.9%, about 48% to about 99.9%, about 49% to about 99.9%, about 50% to about 99.9%, about 51% to about 99.9%, about 52% to about 99.9%, about 53% to about 99.9%, about 54% to about 99.9%, about 55% to about 99.9%, about 56% to about 99.9%, about 57% to about 99.9%, about 58% to about 99.9%, about 59% to about 99.9%, about 60% to about 99.9%, about 61% to about 99.9%, about 62% to about 99.9%, about 63% to about 99.9%, about 64% to about 99.9%, about 65% to about 99.9%, about 66% to about 99.9%, about 67% to about 99.9%, about 68% to about 99.9%, about 69% to about 99.9%, about 70% to about 99.9%, about 71% to about 99.9%, about 72% to about 99.9%, about 73% to about 99.9%, about 74% to about 99.9%, about 75% to about 99.9%, about 76% to about 99.9%, about 77% to about 99.9%, about 78% to about 99.9%, about 79% to about 99.9%, about 80% to about 99.9%, about 81% to about 99.9%, about 82% to about 99.9%, about 83% to about 99.9%, about 84% to about 99.9%, about 85% to about 99.9%, about 86% to about 99.9%, about 87% to about 99.9%, about 88% to about 99.9%, about 89% to about 99.9%, about 90% to about 99.9%, about 91% to about 99.9%, about 92% to about 99.9%, about 93% to about 99.9%, about 94% to about 99.9%, about 95% to about 99.9%, about 96% to about 99.9%, about 97% to about 99.9%, about 98% to about 99.9%, or about 99% to about 99.99%, etc., are present as a single disulfide bond isoform.

[0096] In some embodiments, at least about 10% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 15% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 20% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 25% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 30% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 35% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 40% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 45% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 50% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 60% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 65% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 70% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 75% of the antibody or its antigen-binding fragment in the pharmaceutical composition is present as a single disulfide bond isoform.In some embodiments, at least about 80% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 85% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 90% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 95% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 96% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 97% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 98% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 99% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond isoform. In some embodiments, at least about 99.9% of the antibody or antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond isoform.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof, when subjected to gel electrophoresis analysis performed under non-reducing conditions, yields only a single detectable band.

[0098] In some embodiments, the single disulfide bond isoform of the antibody or antigen-binding fragment is IgG2-A, as described herein. In some embodiments, the single disulfide bond isoform of the antibody or antigen-binding fragment is IgG2-B, as described herein.

[0099] Additionally or alternatively, the pharmaceutical composition may contain a construct of the second aspect or any of its embodiments, a polynucleotide of the third aspect or any of its embodiments, a vector of the fourth aspect or any of its embodiments, and / or a host cell of the fifth aspect or any of its embodiments. The pharmaceutical composition may further contain a pharmaceutically acceptable carrier or excipient.

[0100] In some embodiments, the polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) is included in the pharmaceutical composition in an amount of about 0.001 mg / ml to about 100 mg / ml, such as an amount of about 0.01 mg / ml to about 10 mg / ml.

[0101] The pharmaceutical composition may further contain another therapeutic agent, for example, an immunotherapeutic agent and the like. In some embodiments, the immunotherapeutic agent is an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-L2 agent, a TNF-α cross-linking agent, a TRAIL cross-linking agent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, an anti-4-1BB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAILR1 agent, an anti-TRAILR2 agent, an anti-TWEAK agent, an anti-TWEAKR agent, an anti-cell surface lymphocyte protein agent, an anti-BRAF agent, an anti-MEK agent, an anti-CD33 agent, an anti-CD20 agent, an anti-HLA-DR agent, an anti-HLA class I agent, an anti-CD52 agent, an anti-A33 agent, an anti-GD3 agent, an anti-PSMA agent, an anti-Ceacan1 agent, an anti-Galedin9 agent, an anti-HVEM agent, an anti-VISTA agent, an anti-B7 H4 agent, an anti-HHLA2 agent, an anti-CD155 agent, an anti-CD80 agent, an anti-BTLA agent, an anti-CD160 agent, an anti-CD28 agent, an anti-CD226 agent, an anti-CEACAM1 agent, an anti-TIM3 agent, an anti-TIGIT agent, an anti-CD96 agent, an anti-CD70 agent, an anti-CD27 agent, an anti-LIGHT agent, an anti-CD137 agent, an anti-DR4 agent, an anti-CR5 agent, an anti-TNFRS agent, an anti-TNFR1 agent, an anti-FAS agent, an anti-CD95 agent, an anti-TRAIL agent, an anti-DR6 agent, an anti-EDAR agent, an anti-NGFR agent, an anti-OPG agent, an anti-RANKL agent, an anti-LTβ receptor agent, an anti-BCMA agent, an anti-TACI agent, an anti-BAFFR agent, an anti-EDAR2 agent, an anti-TROY agent, and an anti-RELT agent, and is selected from the group consisting of. For example, the immunotherapeutic agent may be an anti-CTLA-4 agent, an anti-PD-1 agent, or an anti-PD-L1 agent.

[0102] In some embodiments, the immunotherapeutic agent is an anti-CTLA-4 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-PD-L2 antibody or an antigen-binding fragment thereof, a TNF-α bridging antibody or an antigen-binding fragment thereof, a TRAIL bridging antibody or an antigen-binding fragment thereof, an anti-CD27 antibody or an antigen-binding fragment thereof, an anti-CD30 antibody or an antigen-binding fragment thereof, an anti-CD40 antibody or an antigen-binding fragment thereof, an anti-4-1BB antibody or an antigen-binding fragment thereof, an anti-GITR antibody or an antigen-binding fragment thereof, an anti-OX40 antibody or an antigen-binding fragment thereof, an anti-TRAILR1 antibody or an antigen-binding fragment thereof, an anti-TRAILR2 antibody or an antigen-binding fragment thereof, an anti-TWEAK antibody or an antigen-binding fragment thereof, an anti-TWEAKR antibody or an antigen-binding fragment thereof, an anti-cell surface lymphocyte protein antibody or an antigen-binding fragment thereof, an anti-BRAF antibody or an antigen-binding fragment thereof, an anti-MEK antibody or an antigen-binding fragment thereof, an anti-CD33 antibody or an antigen-binding fragment thereof, an anti-CD20 antibody or an antigen-binding fragment thereof, an anti-HLA-DR antibody or an antigen-binding fragment thereof, an anti-HLA class I antibody or an antigen-binding fragment thereof, an anti-CD52 antibody or an antigen-binding fragment thereof, an anti-A33 antibody or an antigen-binding fragment thereof, an anti-GD3 antibody or an antigen-binding fragment thereof, an anti-PSMA antibody or an antigen-binding fragment thereof, an anti-Ceacan1 antibody or an antigen-binding fragment thereof, an anti-Galedin9 antibody or an antigen-binding fragment thereof, an anti-HVEM antibody or an antigen-binding fragment thereof, an anti-VISTA antibody or an antigen-binding fragment thereof, an anti-B7Selected from the group consisting of an H4 antibody or an antigen-binding fragment thereof, an anti-HHLA2 antibody or an antigen-binding fragment thereof, an anti-CD155 antibody or an antigen-binding fragment thereof, an anti-CD80 antibody or an antigen-binding fragment thereof, an anti-BTLA antibody or an antigen-binding fragment thereof, an anti-CD160 antibody or an antigen-binding fragment thereof, an anti-CD28 antibody or an antigen-binding fragment thereof, an anti-CD226 antibody or an antigen-binding fragment thereof, an anti-CEACAM1 antibody or an antigen-binding fragment thereof, an anti-TIM3 antibody or an antigen-binding fragment thereof, an anti-TIGIT antibody or an antigen-binding fragment thereof, an anti-CD96 antibody or an antigen-binding fragment thereof, an anti-CD70 antibody or an antigen-binding fragment thereof, an anti-CD27 antibody or an antigen-binding fragment thereof, an anti-LIGHT antibody or an antigen-binding fragment thereof, an anti-CD137 antibody or an antigen-binding fragment thereof, an anti-DR4 antibody or an antigen-binding fragment thereof, an anti-CR5 antibody or an antigen-binding fragment thereof, an anti-TNFRS antibody or an antigen-binding fragment thereof, an anti-TNFR1 antibody or an antigen-binding fragment thereof, an anti-FAS antibody or an antigen-binding fragment thereof, an anti-CD95 antibody or an antigen-binding fragment thereof, an anti-TRAIL antibody or an antigen-binding fragment thereof, an anti-DR6 antibody or an antigen-binding fragment thereof, an anti-EDAR antibody or an antigen-binding fragment thereof, an anti-NGFR antibody or an antigen-binding fragment thereof, an anti-OPG antibody or an antigen-binding fragment thereof, an anti-RANKL antibody or an antigen-binding fragment thereof, an anti-LTβ receptor antibody or an antigen-binding fragment thereof, an anti-BCMA antibody or an antigen-binding fragment thereof, an anti-TACI antibody or an antigen-binding fragment thereof, an anti-BAFFR antibody or an antigen-binding fragment thereof, an anti-EDAR2 antibody or an antigen-binding fragment thereof, an anti-TROY antibody or an antigen-binding fragment thereof, and an anti-RELT antibody or an antigen-binding fragment thereof. For example, the immunotherapeutic agent may be an anti-CTLA-4 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, or an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0103] In some embodiments, the pharmaceutical composition contains an anti-CTLA-4 antibody or an antigen-binding fragment thereof, such as ipilimumab or tremelimumab. Additionally or alternatively, the pharmaceutical composition may contain an anti-PD-1 antibody or an antigen-binding fragment thereof, such as nivolumab, pembrolizumab, avelumab, durvalumab, or atezolizumab.

[0104] In some embodiments, the immunotherapeutic agent is an anti-cell surface lymphocyte protein antibody or an antigen-binding fragment thereof, such as, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD139, CD140, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167, CD168, CD169, CD170, CD171, CD172, CD173, CD174, CD175, CD176,An antibody or an antigen-binding fragment thereof that binds to one or more of CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD187, CD188, CD189, CD190, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CD198, CD199, CD200, CD201, CD202, CD203, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CD211, CD212, CD213, CD214, CD215, CD216, CD217, CD218, CD219, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235, CD236, CD237, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD287, CD288, CD289, CD290, CD291, CD292, CD293, CD294, CD295, CD296, CD297, CD298, CD299, CD300, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, and / or CD320, etc.

[0105] In some embodiments, the immunotherapeutic agent is an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to a chemokine or lymphokine, such as a chemokine or lymphokine involved in tumor growth. For example, the immunotherapeutic agent may be an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to one or more or all of CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5 to inhibit its activity. In some embodiments, the immunotherapeutic agent is an agent (e.g., a polypeptide, an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof) that binds to one or more or all of CCL3, CCL4, CCL8, and CCL22 to inhibit its activity.

[0106] The immunotherapeutic agent may be able to specifically bind to one or more of the immunological targets described in Table 1 of Mahoney et al., Cancer Immunotherapy, 14:561-584 (2015) (the entire disclosure of which is incorporated herein by reference). For example, the immunotherapeutic agent may be an antibody or an antigen-binding fragment thereof that specifically binds to one or more of agents such as OX40L, TL1A, CD40L, LIGHT, BTLA, LAG3, TIM3, Singlecs, ICOS, B7-H3, B7-H4, VISTA, TMIGD2, BTNL2, CD48, KIR, LIR, LIR antibody, ILT, NKG2D, NKG2A, MICA, MICB, CD244, CSF1R, IDO, TGFβ, CD39, CD73, CXCR4, CXCL12, SIRPA, CD47, VEGF, or neuropilin.

[0107] In some embodiments, the immunotherapeutic agent is targretin, interferon-α, clobetasol, pegylated interferon (e.g., PEGASYS®), prednisone, romidepsin, bexarotene, methotrexate, triamcinolone cream, an anti-chemokine, vorinostat, gabapentin, an antibody against a lymphocyte cell surface receptor and / or lymphokine, an antibody against a surface cancer protein, and / or a small molecule therapeutic such as vorinostat.

[0108] In some embodiments, the pharmaceutical composition contains a bispecific antibody, e.g., a bispecific monoclonal antibody, in which one arm of the antibody specifically binds to TNFR2 and the other specifically binds to an immune checkpoint protein, e.g., inter alia, PD-1, PD-L1, or CTLA-4 as described herein. The arm of the bispecific antibody that specifically binds to TNFR2 can specifically bind to an epitope of human TNFR2 defined by one or more amino acids within CRD3 and / or an epitope defined by one or more amino acids within CRD4, e.g., an epitope on human TNFR2 as described above and herein, since it gives rise to an antagonist (dominant antagonist) phenotype.

[0109] In some embodiments, the bispecific antibody contains one arm that specifically binds to TNFR2, e.g., an epitope of human TNFR2 as described above, and one arm that specifically binds to an immune checkpoint protein that specifically binds to PD-1. In some embodiments, the arm of the bispecific antibody that specifically binds to PD-1 can specifically bind to an epitope(s) on PD-1, in the same manner as nivolumab, pembrolizumab, avelumab, durvalumab, or atezolizumab. For example, the arm of the bispecific antibody that specifically binds to PD-1 can competitively inhibit the binding of PD-1 to nivolumab, pembrolizumab, avelumab, durvalumab, and / or atezolizumab when evaluated using, e.g., the competitive binding assay described herein or those well known in the art, e.g., competitive ELISA.

[0110] In some embodiments, the bispecific antibody contains one arm that specifically binds to TNFR2, such as an epitope of the human TNFR2 described above, and one arm that specifically binds to PD-L1. In some embodiments, the arm of the bispecific antibody that specifically binds to PD-L1 can specifically bind to the epitope(s) on PD-L1, the same as in the case of atezolizumab or avelumab. For example, the arm of the bispecific antibody that specifically binds to PD-L1 can competitively inhibit the binding of PD-L1 to atezolizumab and / or avelumab when, for example, using the competitive binding assay described herein or those well known in the art, such as competitive ELISA.

[0111] In some embodiments, the bispecific antibody contains one arm that specifically binds to TNFR2, such as an epitope of the human TNFR2 described above, and one arm that specifically binds to CTLA-4. In some embodiments, the arm of the bispecific antibody that specifically binds to CTLA-4 can specifically bind to the epitope(s) on CTLA-4, the same as in the case of ipilimumab or tremelimumab. For example, the arm of the bispecific antibody that specifically binds to CTLA-4 can competitively inhibit the binding of CTLA-4 to ipilimumab and / or tremelimumab when evaluated using, for example, the competitive binding assay described herein or those well known in the art, such as competitive ELISA.

[0112] In some embodiments, another therapeutic agent in the pharmaceutical composition is a chimeric antigen receptor (CAR-T) agent, a chemotherapeutic agent, a small molecule anti-cancer agent, or a cancer vaccine.

[0113] In some embodiments, another therapeutic agent in the pharmaceutical composition is a chimeric antigen receptor (CAR-T) agent, such as T cells modified to express a T cell receptor that specifically binds to one or more antigens expressed on the surface of cancer cells. The antibodies or antigen-binding fragments thereof, single-chain polypeptides, constructs, polynucleotides, vectors, or host cells (e.g., TNFR2 antagonist antibodies or antigen-binding fragments thereof) described herein can be formulated for co-administration with a CAR-T agent, for example, by mixing the antibody or antigen-binding fragment thereof, single-chain polypeptide, construct, polynucleotide, vector, or host cell with the CAR-T agent. In some embodiments, the antibody or antigen-binding fragment thereof, single-chain polypeptide, construct, polynucleotide, vector, or host cell is formulated for administration separately from a chemotherapeutic agent, for example, for continuous administration.

[0114] In some embodiments, another therapeutic agent in the pharmaceutical composition is a chemotherapeutic agent, such as a chemotherapeutic agent described herein. The antibodies or antigen-binding fragments thereof, single-chain polypeptides, constructs, polynucleotides, vectors, or host cells (e.g., TNFR2 antagonist antibodies or antigen-binding fragments thereof) described herein can be formulated for co-administration with a chemotherapeutic agent, for example, by mixing the antibody or antigen-binding fragment thereof, single-chain polypeptide, construct, polynucleotide, vector, or host cell with the chemotherapeutic agent. In some embodiments, the antibody or antigen-binding fragment thereof, single-chain polypeptide, construct, polynucleotide, vector, or host cell is formulated for administration separately from the chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is directly conjugated to the antibody or antigen-binding fragment thereof, single-chain polypeptide, construct, polynucleotide, vector, or host cell, for example, using conjugation techniques described herein or well-known in the art.

[0115] In some embodiments, another therapeutic agent is a small molecule anti-cancer agent, such as a small molecule described in Imai et al., Nature Reviews Cancer 6:714-727 (2006), the disclosure of which is incorporated herein by reference.

[0116] In some embodiments, another therapeutic agent is a cancer vaccine, such as a vaccine described in Palucka et al., Journal of Immunology 186:1325-1331 (2011), the disclosure of which is incorporated herein by reference.

[0117] A seventh aspect features a method for producing the polypeptide of the first aspect (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof), and / or the construct of the second aspect or any of its embodiments. The method may include expressing a polynucleotide encoding the polypeptide or construct in a host cell (e.g., a host cell described herein), and then recovering the polypeptide from the host cell culture medium.

[0118] An eighth aspect features a method for suppressing or inhibiting an immune response involving T-reg cells in a mammal (e.g., a human) by administering to the mammal the polypeptide of the first aspect or any of its embodiments (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof), the construct of the second aspect or any of its embodiments, the polynucleotide of the third aspect or any of its embodiments, the vector of the fourth aspect or any of its embodiments, the host cell of the fifth aspect or any of its embodiments, and / or the pharmaceutical composition of the sixth aspect or any of its embodiments.

[0119] A ninth aspect features a method for treating a cell proliferation disorder in a mammal (e.g., a human) by administering to the mammal a polypeptide of the first aspect or any of its embodiments (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof), a construct of the second aspect or any of its embodiments, a polynucleotide of the third aspect or any of its embodiments, a vector of the fourth aspect or any of its embodiments, a host cell of the fifth aspect or any of its embodiments, and / or a pharmaceutical composition of the sixth aspect or any of its embodiments.

[0120] The cell proliferation disorder may be, for example, cancer, such as cancer selected from the group consisting of leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, heart cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and throat cancer. In some embodiments, the cancer is selected from the group consisting of Hodgkin lymphoma, cutaneous non-Hodgkin lymphoma, T-cell lymphoma, ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, skin cancer, lung cancer, liver cancer, endometrial cancer, hematopoietic or lymphatic cancer, central nervous system cancer, breast cancer, pancreatic cancer, gastric cancer, esophageal cancer, and upper gastrointestinal cancer. In some embodiments, the cancer is selected from the group consisting of T-cell lymphoma, ovarian cancer, and colon cancer.

[0121] In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, Ewing sarcoma family, osteosarcoma, and malignant fibrous histiocytoma, central nervous system fetal tumor, central nervous system germ cell tumor, craniopharyngioma, epithelioma, bronchial tumor, Burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative tumor, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, epithelioma, esophageal cancer, sensory neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, pediatric brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumor, pancreatic neuroendocrine tumor, Wilms tumor, and other pediatric kidney tumors, Langerhans cell histiocytosis, small cell lung cancer, cutaneous T cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, nasal and paranasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low-grade ovarian cancer, pancreatic neuroendocrine tumor, papillomatosis, paraganglioma, paranasal and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, renal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, rhabdomyosarcoma, Sézary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia.

[0122] A tenth aspect is characterized by a method for treating an infectious disease in a mammal (e.g., a human) by administering to the mammal a polypeptide of the first aspect or any of its embodiments (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof), a construct of the second aspect or any of its embodiments, a polynucleotide of the third aspect or any of its embodiments, a vector of the fourth aspect or any of its embodiments, a host cell of the fifth aspect or any of its embodiments, and / or a pharmaceutical composition of the sixth aspect or any of its embodiments. The infectious disease can be caused, for example, by a virus, a bacterium, a fungus, and / or a parasite.

[0123] In some embodiments, the infectious disease is hepatitis C virus, yellow fever virus, Kadam virus, Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, calicivirus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, louping ill virus, Negishi virus, Meaban virus, Saumarez Reef virus, Turure virus, Aroa virus, dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israeli fowl meningitis virus, Untanaya virus, Tembusu virus, Zika virus, Banj virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus, yellow fever virus, Enderbury virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, cell fusion agent virus, Ippy virus, Lassa virus, lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus, Guanarito virus, Funin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, Chapare virus, Lujo virus, hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus,Caused by a virus selected from the group consisting of Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Marburg virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O'nyong'nyong virus, and chikungunya virus, variola virus, monkeypox virus, vaccinia virus, herpes simplex virus, human herpes virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus, Kaposi's sarcoma-associated herpes virus (KSHV), influenza virus, severe acute respiratory syndrome (SARS) virus, rabies virus, vesicular stomatitis virus (VSV), human respiratory syncytial virus (RSV), Newcastle disease virus, Hendra virus, Nipah virus, measles virus, rinderpest virus, canine distemper virus, Sendai virus, human parainfluenza virus (e.g., 1, 2, 3, and 4), rhinovirus, mumps virus, poliovirus, human enterovirus (A, B, C, and D), hepatitis A virus, coxsackievirus, hepatitis B virus, human papillomavirus, adeno-associated virus, astrovirus, JC virus, BK virus, SV40 virus, Norwalk virus, rotavirus, human immunodeficiency virus (HIV), human T-lymphotropic virus types I and II.,

[0124] In some embodiments, the infectious disease is caused by bacteria belonging to a genus selected from the group consisting of Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriacece, Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordatella, Legionella, Pasturella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus.

[0125] In some embodiments, the infectious disease is caused by fungi selected from the group consisting of Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, and Absidia.

[0126] In some embodiments, the infectious disease is caused by a parasite selected from the group consisting of Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. Exemplary parasites include richuris trichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, and Dracunculus medinensis, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyes, Paragonimus westermani, Taenia solium, Taenia saginata, Hymenolepis nana, and Echinococcus granulosus.

[0127] In some embodiments of the eighth, ninth, and / or tenth aspects, the method further comprises administering an immunotherapeutic agent to a human. The immunotherapeutic agent may be selected from the group consisting of, for example, an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-L2 agent, a TNF-α crosslinking agent, a TRAIL crosslinking agent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, an anti-4-1BB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAILR1 agent, an anti-TRAILR2 agent, an anti-TWEAK agent, an anti-TWEAKR agent, an anti-cell surface lymphocyte protein agent, an anti-BRAF agent, an anti-MEK agent, an anti-CD33 agent, an anti-CD20 agent, an anti-HLA-DR agent, an anti-HLA class I agent, an anti-CD52 agent, an anti-A33 agent, an anti-GD3 agent, an anti-PSMA agent, an anti-Ceacan1 agent, an anti-Galedin9 agent, an anti-HVEM agent, an anti-VISTA agent, an anti-B7 H4 agent, an anti-HHLA2 agent, an anti-CD155 agent, an anti-CD80 agent, an anti-BTLA agent, an anti-CD160 agent, an anti-CD28 agent, an anti-CD226 agent, an anti-CEACAM1 agent, an anti-TIM3 agent, an anti-TIGIT agent, an anti-CD96 agent, an anti-CD70 agent, an anti-CD27 agent, an anti-LIGHT agent, an anti-CD137 agent, an anti-DR4 agent, an anti-CR5 agent, an anti-TNFRS agent, an anti-TNFR1 agent, an anti-FAS agent, an anti-CD95 agent, an anti-TRAIL agent, an anti-DR6 agent, an anti-EDAR agent, an anti-NGFR agent, an anti-OPG agent, an anti-RANKL agent, an anti-LTβ receptor agent, an anti-BCMA agent, an anti-TACI agent, an anti-BAFFR agent, an anti-EDAR2 agent, an anti-TROY agent, and an anti-RELT agent, such as, for example, an anti-CTLA-4 agent, an anti-PD-1 agent, and / or an anti-PD-L1 agent.

[0128] Immunotherapeutic agents administered to humans include, for example, anti-CTLA-4 antibodies or antigen-binding fragments thereof, anti-PD-1 antibodies or antigen-binding fragments thereof, anti-PD-L1 antibodies or antigen-binding fragments thereof, anti-PD-L2 antibodies or antigen-binding fragments thereof, TNF-α bridging antibodies or antigen-binding fragments thereof, TRAIL bridging antibodies or antigen-binding fragments thereof, anti-CD27 antibodies or antigen-binding fragments thereof, anti-CD30 antibodies or antigen-binding fragments thereof, anti-CD40 antibodies or antigen-binding fragments thereof, anti-4-1BB antibodies or antigen-binding fragments thereof, anti-GITR antibodies or antigen-binding fragments thereof, anti-OX40 antibodies or antigen-binding fragments thereof, anti-TRAILR1 antibodies or antigen-binding fragments thereof, anti-TRAILR2 antibodies or antigen-binding fragments thereof, anti-TWEAK antibodies or antigen-binding fragments thereof, anti-TWEAKR antibodies or antigen-binding fragments thereof, anti-cell surface lymphocyte protein antibodies or antigen-binding fragments thereof, anti-BRAF antibodies or antigen-binding fragments thereof, anti-MEK antibodies or antigen-binding fragments thereof, anti-CD33 antibodies or antigen-binding fragments thereof, anti-CD20 antibodies or antigen-binding fragments thereof, anti-HLA-DR antibodies or antigen-binding fragments thereof, anti-HLA class I antibodies or antigen-binding fragments thereof, anti-CD52 antibodies or antigen-binding fragments thereof, anti-A33 antibodies or antigen-binding fragments thereof, anti-GD3 antibodies or antigen-binding fragments thereof, anti-PSMA antibodies or antigen-binding fragments thereof, anti-Ceacan1 antibodies or antigen-binding fragments thereof, anti-Galedin9 antibodies or antigen-binding fragments thereof, anti-HVEM antibodies or antigen-binding fragments thereof, anti-VISTA antibodies or antigen-binding fragments thereof, anti-B7An H4 antibody or an antigen-binding fragment thereof, an anti-HHLA2 antibody or an antigen-binding fragment thereof, an anti-CD155 antibody or an antigen-binding fragment thereof, an anti-CD80 antibody or an antigen-binding fragment thereof, an anti-BTLA antibody or an antigen-binding fragment thereof, an anti-CD160 antibody or an antigen-binding fragment thereof, an anti-CD28 antibody or an antigen-binding fragment thereof, an anti-CD226 antibody or an antigen-binding fragment thereof, an anti-CEACAM1 antibody or an antigen-binding fragment thereof, an anti-TIM3 antibody or an antigen-binding fragment thereof, an anti-TIGIT antibody or an antigen-binding fragment thereof, an anti-CD96 antibody or an antigen-binding fragment thereof, an anti-CD70 antibody or an antigen-binding fragment thereof, an anti-CD27 antibody or an antigen-binding fragment thereof, an anti-LIGHT antibody or an antigen-binding fragment thereof, an anti-CD137 antibody or an antigen-binding fragment thereof, an anti-DR4 antibody or an antigen-binding fragment thereof, an anti-CR5 antibody or an antigen-binding fragment thereof, an anti-TNFRS antibody or an antigen-binding fragment thereof, an anti-TNFR1 antibody or an antigen-binding fragment thereof, an anti-FAS antibody or an antigen-binding fragment thereof, an anti-CD95 antibody or an antigen-binding fragment thereof, an anti-TRAIL antibody or an antigen-binding fragment thereof, an anti-DR6 antibody or an antigen-binding fragment thereof, an anti-EDAR antibody or an antigen-binding fragment thereof, an anti-NGFR antibody or an antigen-binding fragment thereof, an anti-OPG antibody or an antigen-binding fragment thereof, an anti-RANKL antibody or an antigen-binding fragment thereof, an anti-LTβ receptor antibody or an antigen-binding fragment thereof, an anti-BCMA antibody or an antigen-binding fragment thereof, an anti-TACI antibody or an antigen-binding fragment thereof, an anti-BAFFR antibody or an antigen-binding fragment thereof, an anti-EDAR2 antibody or an antigen-binding fragment thereof, an anti-TROY antibody or an antigen-binding fragment thereof, and an anti-RELT antibody or an antigen-binding fragment thereof may be selected. In some embodiments, the immunotherapeutic agent administered to a human is an anti-CTLA-4 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, or an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0129] In some embodiments of the eighth, ninth, and / or tenth aspects, the method comprises administering to a mammal (e.g., a human) an anti-CTLA-4 antibody or an antigen-binding fragment thereof, such as ipilimumab or tremelimumab. Additionally or alternatively, the method may comprise administering to a human an anti-PD-1 antibody or an antigen-binding fragment thereof, such as nivolumab, pembrolizumab, avelumab, durvalumab, or atezolizumab.

[0130] In some embodiments of the eighth, ninth, and / or tenth aspects, the method is an anti-cell surface lymphocyte protein antibody or an antigen-binding fragment thereof, such as CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD139, CD140, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167, CD168, CD169, CD170, CD171, CD172, CD173administering to a mammal (e.g., a human) an antibody or an antigen-binding fragment thereof that binds to one or more of CD174, CD175, CD176, CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD187, CD188, CD189, CD190, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CD198, CD199, CD200, CD201, CD202, CD203, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CD211, CD212, CD213, CD214, CD215, CD216, CD217, CD218, CD219, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235, CD236, CD237, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD287, CD288, CD289, CD290, CD291, CD292, CD293, CD294, CD295, CD296, CD297, CD298, CD299, CD300, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, and / or CD320, etc.

[0131] In some embodiments of the eighth, ninth, and / or tenth aspects, the method comprises administering to a mammal (e.g., a human) an agent (e.g., a polypeptide, antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct thereof) that binds to a chemokine or lymphokine, such as a chemokine or lymphokine involved in tumor growth. For example, the immunotherapeutic agent may be an agent (e.g., a polypeptide, antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct thereof) that binds to one or more or all of CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5 and inhibits its activity. In some embodiments, the immunotherapeutic agent is an agent (e.g., a polypeptide, antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct thereof) that binds to one or more or all of CCL3, CCL4, CCL8, and CCL22 and inhibits its activity.

[0132] In some embodiments of the eighth, ninth, and / or tenth aspects, the method comprises administering to a mammal (e.g., a human) an immunotherapeutic agent that can specifically bind to one or more of the immunological targets described in Table 1 of Mahoney et al., Cancer Immunotherapy, 14:561-584 (2015) (the entire disclosure of which is incorporated herein by reference). For example, the immunotherapeutic agent may be an antibody or antigen-binding fragment thereof that specifically binds to one or more of agents such as OX40L, TL1A, CD40L, LIGHT, BTLA, LAG3, TIM3, Singlecs, ICOS, B7-H3, B7-H4, VISTA, TMIGD2, BTNL2, CD48, KIR, LIR, LIR antibody, ILT, NKG2D, NKG2A, MICA, MICB, CD244, CSF1R, IDO, TGFβ, CD39, CD73, CXCR4, CXCL12, SIRPA, CD47, VEGF, or neuropilin.

[0133] In some embodiments of the eighth, ninth, and / or tenth aspects, the method comprises administering to a mammal (e.g., a human) an immunotherapeutic agent selected from the group consisting of tagretin, interferon-α, clobetasol, pegylated interferon (e.g., PEGASYS®), prednisone, romidepsin, bexarotene, methotrexate, triamcinolone cream, anti-chemokine, vorinostat, gabapentin, an antibody against a lymphocyte cell surface receptor and / or lymphokine, an antibody against a surface cancer protein, and a small molecule therapeutic agent such as vorinostat.

[0134] In some embodiments, the method comprises administering to a mammal (e.g., a human) a CAR-T agent, a chemotherapeutic agent, a small molecule anti-cancer agent, or a cancer vaccine, such as the CAR-T agents, chemotherapeutic agents, small molecule anti-cancer agents, or cancer vaccines described above and herein.

[0135] In some embodiments, a polypeptide that specifically binds to TNFR2, such as a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct, etc., is administered to a mammal (e.g., a human) in an amount of about 0.001 mg / kg to about 100 mg / kg, such as an amount of about 0.01 mg / kg to about 10 mg / kg.

[0136] The eleventh aspect features a kit containing a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) of the first aspect or any of its embodiments, a construct of the second aspect or any of its embodiments, a polynucleotide of the third aspect or any of its embodiments, a vector of the fourth aspect or any of its embodiments, a host cell of the fifth aspect or any of its embodiments, and / or a pharmaceutical composition of the sixth aspect or any of its embodiments.

[0137] In some embodiments, the kit contains instructions for transfecting a vector into a host cell. Additionally or alternatively, the kit may contain instructions for expressing a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) in a host cell. The kit may include reagents that can be used to express a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) in a host cell. In some embodiments, the kit includes instructions for administering a drug to a mammal (e.g., a human), such as a human patient, suffering from a cell growth disorder and / or an infectious disease described herein. In some embodiments, the kit contains instructions for making or using a drug.

[0138] Definitions As used herein, the term "about" means a value that is up to 10% more or less than the recited value. For example, the term "about 5 nM" refers to a range of 4.5 nM to 5.5 nM.

[0139] As used herein, the term "antibody" (Ab) means an immunoglobulin molecule that specifically binds to a particular antigen or immunologically reacts with a particular antigen, and includes chimeric antibodies, humanized antibodies, primatized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, trispecific antibodies, and tetravalent antibodies, diabodies, triabodies, and tetravalent antibodies), and antigen-binding fragments of antibodies including, but not limited to, for example, Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments. Further, unless otherwise specified, the term "monoclonal antibody" (mAb) means both an intact molecule that can specifically bind to a target protein and an antibody fragment (e.g., Fab fragment and F(ab')2 fragment, etc.) that can specifically bind to the target protein. Fab fragments and F(ab')2 fragments lack the Fc fragment of the intact antibody and, compared to the intact antibody, can be excreted more rapidly from the circulating blood of an animal and exhibit lower non-specific tissue binding (see Wahl et al., J. Nucl. Med. 24:316, 1983, which is incorporated herein by reference).

[0140] As used herein, the term "antigen-binding fragment" means one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of the full-length antibody. The antibody fragment can be Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments included in the "antigen-binding fragment" of an antibody include (i) V L domain, V H domain, C L domain, and C H(i) A Fab fragment, which is a monovalent fragment composed of 1 domain; (ii) An F(ab’)2 fragment, which is a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) A V H domain and a C H domain; an Fd fragment composed of 1 domain; (iv) A V L domain and a V H domain; an Fv fragment composed of a V H domain and a V L domain; a dAb containing a V H domain and a V H domain; a dAb fragment composed of a V L domain or a V L domain; (viii) An isolated complementarity-determining region (CDR); and (ix) Combinations of two or more isolated CDRs optionally linked by a synthetic linker, but not limited thereto. Further, even if the two domains of the Fv fragment, V H and V L are encoded by separate genes, using recombinant methods, the V H region and the V L region pair can be linked by a linker capable of generating a single protein chain of a monovalent molecule (known as single-chain Fv (scFv), see, for example, Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988), and the V H and V L and V HThey can be linked. These antibody fragments may be obtained using conventional techniques well known to those skilled in the art, and the fragments may be screened for utility in the same manner as for intact antibodies. Antigen-binding fragments may be prepared using recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, chemical peptide synthesis methods well known in the art.

[0141] As used herein, the terms "anti-tumor necrosis factor receptor 2 antibody," "TNFR2 antibody," "anti-TNFR2 antibody moiety," and / or "anti-TNFR2 antibody fragment," etc. include at least a part of an immunoglobulin molecule, but not limited to, for example, at least one complementarity-determining region (CDR) of a heavy chain or a light chain or its ligand-binding portion that can specifically bind to TNFR2, a heavy chain variable region or a light chain variable region, a heavy chain constant region or a light chain constant region, or any part thereof, etc., and include any protein or peptide-containing molecule. For example, two or more parts of an immunoglobulin molecule may be covalently bonded to each other by, for example, an amide bond, a thioether bond, a carbon-carbon bond, a disulfide bridge, or a linker, such as a linker described herein or well known in the art. TNFR2 antibodies also include antibody-like protein scaffolds, such as the tenth fibronectin type III domain ( 10 Fn3) containing BC, DE, and FG structural loops whose structure and solvent exposure are similar to those of antibody CDRs. 10 The tertiary structure of the Fn3 domain is similar to the tertiary structure of the variable region of the IgG heavy chain, and those skilled in the art can, for example, 10 replace the residues of the BC, DE, and FG loops of Fn3 with residues derived from the CDR-H1, CDR-H2, or CDR-H3 regions of a TNFR2 monoclonal antibody, thereby grafting the CDRs of the TNFR2 monoclonal antibody onto the fibronectin scaffold.

[0142] As used herein, the terms "antagonist TNFR2 antibody" and "antagonistic TNFR2 antibody" mean a TNFR2 antibody capable of inhibiting or suppressing TNFR2 activation, attenuating one or more signal transduction pathways involving TNFR2, and / or inhibiting or suppressing at least one activity involving TNFR2 activation. For example, an antagonistic TNFR2 antibody can inhibit or suppress the growth and proliferation of regulatory T cells. An antagonistic TNFR2 antibody can inhibit or suppress TNFR2 activation by inhibiting the binding of TNFR2 to TNFα. In this manner, an antagonistic TNFR2 antibody can result in the suppression of TNFR2 activity by inhibiting the trimerization of TNFR2 that may otherwise be induced by interaction with TNFα.

[0143] As used herein, the term "bispecific antibody" means an antibody (e.g., a monoclonal antibody, often a human antibody or a humanized antibody) having binding specificities for at least two different antigens. For example, one of the binding specificities may be directed to TNFR2 and the other may be for any other antigen, such as a cell surface protein, receptor, receptor subunit, tissue-specific antigen, virus-derived protein, envelope protein encoded by a virus, bacterium-derived protein, or bacterial surface protein.

[0144] As used herein, the term "chemotherapeutic agent" means any chemical agent having therapeutic utility in the treatment of cancer, such as the cancers described herein. Chemotherapeutic agents include both chemical agents and biological agents. These agents can function to inhibit the cellular activities on which cancer cells depend to continue to survive. Categories of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones, hormone analogs, and antitumor agents. Exemplary chemotherapeutic agents suitable for use with the compositions and methods described herein include Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison’s Principles of Internal medicine, 14 th edition; Perry et al., Chemotherapeutic, Chapter 17 in Abeloff, Clinical Oncology 2 nd ed., 2000; Baltzer L. and Berkery R. (eds): Oncology Pocket Guide to Chemotherapeutic, 2 nd ed. St. Luois, mosby-Year Book, 1995; Fischer D.S., Knobf M.F., Durivage H.J. (eds): The Cancer Chemotherapeutic Handbook, 4 th ed. St. Luois, Mosby-Year Handbook (in the case of chemotherapeutic agents, the respective disclosures of which are incorporated herein by reference) are included but not limited to the chemotherapeutic agents described therein.

[0145] As used herein, the term "chimeric" antibody means an antibody having a variable domain sequence (e.g., a CDR sequence) derived from an immunoglobulin of a particular source organism, e.g., a rat or mouse, and a constant region derived from an immunoglobulin of a different organism, e.g., particularly a human, another primate, a pig, a goat, a rabbit, a hamster, a cat, a dog, a guinea pig, a member of the bovine family (e.g., particularly a cow, a bison, a water buffalo, a yak, and a zebu), a female bovine, a sheep, a horse, or a bison. Methods for making chimeric antibodies are well known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al, 1986, BioTechniques 4:214-221; Gillies et al, 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference.

[0146] As used herein, the term "complementary determining region" (CDR) means the hypervariable regions present in both the light chain variable domain and the heavy chain variable domain. The more highly conserved portions of the variable domain are called the framework regions (FR). As is understood in the art, the amino acid positions that represent the hypervariable regions of an antibody can vary depending on context and the various definitions well known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria while being outside the hypervariable region under a different set of criteria. One or more of these positions can also be present within an extended hypervariable region. The antibodies described herein may contain modifications within these hybrid hypervariable positions. The variable domains of the native heavy and light chains each contain four framework regions that predominantly assume a β-sheet structure, joined by three CDRs that form loops that connect to the β-sheet structure and in some cases by three CDRs that form part of the β-sheet structure. The CDRs within each molecular chain are maintained in proximity to each other in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 by the FR regions and, together with the CDRs of the other antibody chain, contribute to the formation of the target binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987), which is incorporated herein by reference). As used herein, the numbering of immunoglobulin amino acid residues is done according to the Kabat et al immunoglobulin amino acid residue numbering system, unless otherwise specified.

[0147] As used herein, the terms "conservative mutation", "conservative substitution", or "conservative amino acid substitution" mean the substitution of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized in Table 2 below for each of the 20 natural amino acids.

[0148] TIFF0007713880000013.tif127167TIFF0007713880000014.tif90159

[0149] From this table, it will be understood that conservative amino acid families include, for example, (i) G, A, V, L, I, P, and M, (ii) D and E, (iii) C, S, and T, (iv) H, K, and R, (v) N and Q, and (vi) F, Y, and W. Therefore, a conservative mutation or conservative substitution is a conservative mutation or substitution in which one amino acid is substituted with a member of the same amino acid family (e.g., substitution of Ser with Thr, or substitution of Lys with Arg).

[0150] In this specification, an amino acid substitution may be represented using (AA1)(N)(AA2), where "AA1" represents the amino acid that is normally present at a particular position within the amino acid sequence, "N" represents the residue number within the amino acid sequence at which the substitution occurs, and "AA2" represents the amino acid that is present within the amino acid sequence after the substitution has occurred. For example, the notation "C232S" with respect to an antibody hinge region, such as the IgG2 antibody hinge region, means the substitution of the native cysteine residue with a serine residue at amino acid residue 232 of the designated hinge amino acid sequence. Similarly, the notation "C233S" with respect to an antibody hinge region, such as the IgG2 antibody hinge region, means the substitution of the native cysteine residue with a serine residue at amino acid residue 233 of the designated hinge amino acid sequence.

[0151] As used herein, the term "conjugate" means a compound formed by the chemical bonding of a reactive functional group of one molecule to a suitable reactive functional group of another molecule.

[0152] As used herein with respect to a TNFR2 antagonist, the term "construct" means a fusion protein containing a first polypeptide domain linked to a second polypeptide domain. The polypeptide domains may each independently be, for example, an antagonistic TNFR2 single-chain polypeptide as described herein. The first polypeptide domain may be linked to the second polypeptide domain, for example, by a linker, such as, inter alia, a peptide linker or a disulfide bridge. Exemplary linkers that may be used to link the polypeptide domains of an antagonistic TNFR2 construct include, but are not limited to, those described in Leriche et al., Bioorg. Med. Chem., 20:571-582 (2012), the disclosure of which is incorporated herein by reference in its entirety.

[0153] As used herein, the term "derivatized antibody" means an antibody modified by a chemical reaction such that a residue is cleaved or a non-native compound moiety is added to the isolated antibody. Derivatized antibodies can be obtained by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by adding well-known chemical protecting / blocking groups, proteolytic cleavage, or conjugation to a cell ligand or other protein. Without limitation, any of a variety of chemical modifications may be performed using established techniques, using well-known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicaamycin, etc. In addition, for example, one or more non-natural amino acids may be included in the derivative using the amber suppression method (see, e.g., U.S. Patent No. 6,964,859, incorporated herein by reference).

[0154] As used herein, the term "diabody" refers to a V domain and a V domain linked by a linker that is too short (e.g., a linker composed of 5 amino acids) to allow intramolecular binding of the VH and VL domains on the same polypeptide chain, where each polypeptide chain contains a V domain and a V domain, meaning a bivalent antibody containing two polypeptide chains. This structure forces each domain to pair with a complementary domain on a different polypeptide chain so that a homodimeric structure is formed. Therefore, the term "triabody" refers to a trivalent antibody containing three polypeptide chains, where each polypeptide chain contains one VH domain and one VL domain linked by a very short linker (e.g., a linker composed of 1 to 2 amino acids) that allows intramolecular binding of the VH and VL domains within the same polypeptide chain. Peptides constructed in this way usually trimerize so that the VH and VL domains of adjacent polypeptide chains are spatially close to each other to allow proper folding for folding into their native structure (see Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993, which is incorporated herein by reference). H domain and a V L domain, respectively, of a bivalent antibody containing two polypeptide chains. This structure forces each domain to pair with a complementary domain on a different polypeptide chain so that a homodimeric structure is formed. Therefore, the term "triabody" refers to a trivalent antibody containing three polypeptide chains, where each polypeptide chain contains one VH domain and one VL domain linked by a very short linker (e.g., a linker composed of 1 to 2 amino acids) that allows intramolecular binding of the VH and VL domains within the same polypeptide chain. Peptides constructed in this way usually trimerize so that the VH and VL domains of adjacent polypeptide chains are spatially close to each other to allow proper folding for folding into their native structure (see Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993, which is incorporated herein by reference).

[0155] As used herein, a "disulfide bond isoform" of an antibody or an antigen-binding fragment thereof is a form of the antibody or an antigen-binding fragment thereof having a specific internal disulfide bond pattern. A disulfide bond isoform is a structural isomer of any antibody or an antigen-binding fragment thereof that does not differ from each other in amino acid sequence but exhibits different disulfide bond connectivity. For example, with respect to a human IgG2 antibody or a variant thereof, the antibody can exist as one of four possible disulfide bond isoforms designated herein as isoform IgG2-A, IgG2-B, IgG2-A / B1, and IgG2-A / B2. The connectivity of the disulfide bonds within each of these isoforms is illustrated in FIGS. 13A-13D.

[0156] As used herein, a "dominant antagonist" of TNFR2 is an antagonist (e.g., an antagonistic polypeptide such as a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof, etc.) that can inhibit TNFR2 activation even in the presence of a TNFR2 agonist such as TNFα or IL-2. For example, the IC 50 of the antagonist as measured in the same assay in the absence of a TNFR2 agonist such as TNFα or IL-2, compared with the IC 50 of the antagonist in the presence of a TNFR2 agonist (e.g., TNFα) or IL-2 is less than 200% (e.g., less than 200%, less than 100%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, or less). When the TNFR2 antagonist is higher, the TNFR2 antagonist is a dominant antagonist. Inhibition of TNFR2 activation can be evaluated, for example, by measuring inhibition of NFκB signaling (e.g., by monitoring a decrease in the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3 using a conventional gene expression assay) in addition to measuring inhibition of the proliferation of TNFR2+ cells such as T-reg cells, cancer cells expressing TNFR2, or myeloid-derived suppressor cells.

[0157] As used herein, "Dual Variable Domain Immunoglobulin" ("DVD-Ig") refers to an antibody in which the target-binding variable domains of two monoclonal antibodies are joined via a linker to create a tetravalent dual-targeting single agent (Gu et al., Meth. Enzymol., 502:25-41, 2012 (incorporated herein by reference)). Suitable linkers for use in the light chains of the DVDs described herein include the linkers identified in Table 2.1 on page 30 of Gu et al.: short K chain linker ADAAP (SEQ ID NO: 118) (mouse) and TVAAP (SEQ ID NO: 119) (human); long κ chain linker ADAAPTVSIFP (SEQ ID NO: 120) (mouse) and TVAAPSVFIFPP (SEQ ID NO: 121) (human); short λ chain linker QPKAAP (SEQ ID NO: 122) (human); long λ chain linker QPKAAPSVTLFPP (SEQ ID NO: 123) (human); GS-short linker GGSGG (SEQ ID NO: 124), GS-medium linker GGSGGGGSG (SEQ ID NO: 125), and GS-long linker GGSGGGGSGGGGS (SEQ ID NO: 126) (all GS linkers are mouse and human). Suitable linkers for use in the heavy chains of the DVDs include the linkers identified in Table 2.1 on page 30 of Gu & Ghayur, 2012, Methods in Enzymology 502:25-41 (incorporated herein by reference): short linker AKTTAP (SEQ ID NO: 127) (mouse) and ASTKGP (SEQ ID NO: 128) (human); long linker AKTTAPSVYPLAP (SEQ ID NO: 129) (mouse) and ASTKGPSVFPLAP (SEQ ID NO: 130) (human); GS-short linker GGGGSG (SEQ ID NO: 131), GS-medium linker GGGGSGGGGS (SEQ ID NO: 26), and GS-long linker GGGGSGGGGSGGGG (SEQ ID NO: 133) (all GS linkers are mouse and human).

[0158] As used herein, the term "endogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally present within a particular organism (e.g., a human) or at a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell).

[0159] As used herein, the term "epitope" means a portion of an antigen that is recognized and bound by a polypeptide, such as an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct, described herein. In the context of a protein antigen (e.g., TNFR2 such as human TNFR2 shown in SEQ ID NO:7, or TNFR2 of a non-human mammal such as a non-human mammal described herein), an epitope may be a continuous epitope that is a single contiguous segment of one or more amino acids covalently linked to each other by peptide bonds, where all constituent amino acids are bound to a polypeptide (e.g., an antibody, an antigen-binding fragment thereof, a single-chain polypeptide, or a construct thereof). Exemplary assays for measuring the binding of an antagonist TNFR2 polypeptide to a specific amino acid within an antigen are described in Example 1 below. A continuous epitope may be composed of, for example, 1, 5, 10, 15, 20, or more amino acids internal to an antigen, such as the TNFR2 protein described herein (e.g., human TNFR2 shown in SEQ ID NO:7). For example, a continuous epitope may be composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more amino acids. Examples of continuous epitopes on TNFR2 to which the antagonist polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein bind include one or more contiguous residues or all residues of the SSTDICRPHQI motif (SEQ ID NO:288), one or more contiguous residues or all residues of the CALSKQEGCRLCAPL motif (SEQ ID NO:289), and one or more contiguous residues or all residues of the TSDVVCKPCA motif (SEQ ID NO:290), as well as corresponding regions on the TNFR2 proteins of non-human mammals (e.g., bison, cows, and other non-human mammals described herein).In some embodiments, the epitope may be a discontinuous epitope containing the amino acids of two or more segments that are each separated from each other by one or more intervening amino acid residues in the amino acid sequence of the antigen. The discontinuous epitope may be composed of, for example, the amino acid residues of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such segments, such as the SSTDICRPHQI motif (SEQ ID NO: 288), the CALSKQEGCRLCAPL motif (SEQ ID NO: 289), and the TSDVVCKPCA motif (SEQ ID NO: 290) within human TNFR2, and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) segments containing amino acids derived from one or more corresponding regions on the TNFR2 protein of non-human mammals (e.g., bison, cows, and other non-human mammals described herein). Despite this separation by intervening amino acids, the segments that make up the discontinuous epitope may be spatially close to each other in the three-dimensional structure of the antigen. Exemplary discontinuous epitopes on TNFR2 to which the antagonist polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein bind include epitopes containing the following elements: (i) one or more or all of the residues of the SSTDICRPHQI motif (SEQ ID NO: 288), (ii) one or more or all of the residues of the CALSKQEGCRLCAPL motif (SEQ ID NO: 289), and (iii) one or more or all of the residues of the TSDVVCKPCA motif (SEQ ID NO: 290). Another example of a discontinuous epitope on TNFR2 to which the antagonist polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein bind includes epitopes containing elements (i) and (ii) above, epitopes containing elements (i) and (iii) above, and epitopes containing elements (ii) and (iii) above.

[0160] As used herein, the term "foreign" refers to a molecule (e.g., polypeptide, nucleic acid, or cofactor) that does not naturally occur within a particular organism (e.g., a human) or at a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell). Foreign substances include foreign substances introduced into an organism from an external source or culture substances extracted from such foreign substances.

[0161] As used herein, the terms "framework region" or "FW region" include amino acid residues adjacent to the CDRs. FW region residues may be present, for example, among others, within human antibodies, rodent-derived antibodies (e.g., mouse antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), single-chain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies.

[0162] As used herein, the term "fusion protein" means a protein linked to another molecule by a covalent bond. A fusion protein can be chemically synthesized, for example, using an amide bond formation reaction between the N-terminus of one protein and the C-terminus of another protein. Alternatively, a fusion protein containing a protein covalently linked to another protein can be recombinantly expressed in a cell (e.g., a eukaryotic cell or a prokaryotic cell) by expression of a polynucleotide encoding the fusion protein from a vector or the genome of the cell. A fusion protein may contain a protein covalently linked to a linker (and further that linker covalently linked to another molecule). Examples of linkers that can be used in the formation of fusion proteins include peptide-containing linkers, such as peptide-containing linkers containing natural or unnatural amino acids. In some embodiments, D-amino acid residues are not present in native proteins and thus may desirably be included in the linker as they confer resistance to degradation by endogenous proteases. Linkers can be prepared using various strategies well known in the art and depending on the reactive components of the linker, and the linker can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0163] As used herein, the term "heterospecific antibody" means a monoclonal antibody having binding specificities for at least two different antigens, preferably a human antibody or a humanized antibody. Conventionally, the recombinant production of heterospecific antibodies has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537, 1983). Similar methods are disclosed, for example, in WO93 / 08829, U.S. Patent Nos. 6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985, 5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549, 4,676,980, WO91 / 00360, WO92 / 00373, EP03089, Traunecker et al., EMBO J. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986) (incorporated herein by reference). Heterospecific antibodies may contain Fc mutations that enforce correct molecular chain pairing in multispecific antibodies, as described in Klein et al, mAbs 4(6):653-663, 2012 (incorporated herein by reference).

[0164] As used herein, the term "hinge region" means the domain of an antibody or an antigen-binding fragment thereof (e.g., an IgG2 antibody or an antigen-binding fragment thereof) that is located between the antigen-binding site(s) of the antibody or antigen-binding fragment thereof, e.g., the Fab region of the antibody or antigen-binding fragment thereof, and the portion of the antibody or antigen-binding fragment thereof that determines the isotype of the antibody or antigen-binding fragment thereof, e.g., the Fc region of the antibody or antigen-binding fragment thereof. For example, in the context of a monoclonal antibody, the hinge region is a polypeptide that is located approximately in the middle of each heavy chain and links the CH1 domain to the CH2 and CH3 domains. The hinge region of an antibody or antigen-binding fragment thereof can effect intermolecular linkages of the antibody or antigen-binding fragment thereof. For example, in a monoclonal antibody, cysteine residues within the hinge region effect a defined covalent linkage between the heavy chains by forming interchain disulfide bonds. The amino acid sequence of wild-type human IgG2 is ERKCCVECPPCP (SEQ ID NO: 292). As used herein, antibody hinge regions are numbered according to the Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987) (the disclosure of which is incorporated herein by reference) numbering system. For example, using the Kabat et al numbering scheme, the wild-type human IgG2 hinge region set forth in SEQ ID NO: 292 is numbered from residue 226 to residue 243, such that the N-terminal glutamic acid residue of SEQ ID NO: 292 is residue 226 and the C-terminal proline residue of SEQ ID NO: 292 is residue 243. Throughout the present disclosure, variant IgG2 hinge regions, e.g., the variant set forth in SEQ ID NO: 291 (ERKCCVECPPCP), are numbered according to the Kabat et al convention, unless expressly stated to the contrary.

[0165] As used herein, the term "human antibody" has slightly small sequence changes or mutations, but proteins (e.g., CDR, framework, C L 、C H domains (e.g., C H 1, C H 2, C H 3), hinge, (V L 、V HIt means an antibody in which substantially all parts of ()) are substantially non-immunogenic in humans. Human antibodies can be produced using human cells (e.g., by recombinant expression), or using non-human animal cells or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. Further, when the human antibody is a single-chain antibody, the human antibody may contain a linker peptide that does not exist in natural human antibodies. For example, Fv may contain a linker peptide that links the variable region of the heavy chain and the variable region of the light chain, such as 2 to about 8 glycines or other amino acid residues. Such linker peptides are considered to be of human origin. Human antibodies can be produced using various methods well known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111, and PCT Publications WO1998 / 46645, WO1998 / 50433, WO1998 / 24893, WO1998 / 16654, WO1996 / 34096, WO1996 / 33735, and WO1991 / 10741, which are incorporated herein by reference. Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. See, for example, PCT Publications WO98 / 24893, WO92 / 01047, WO96 / 34096, WO96 / 33735, U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, and 5,939,598, which are incorporated herein by reference.

[0166] As used herein, the term "humanized" antibody means an antibody in the form of a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab’, F(ab’)2, or other target-binding subdomains of an antibody, etc.) that contains minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, usually two, variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. All or substantially all of the FR regions may also be those of a human immunoglobulin sequence. A humanized antibody may also contain the immunoglobulin constant region (Fc), usually at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are well known in the art. See, e.g., Riechmann et al., Nature 332:323-7, 1988; U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370 to Queen et al., EP 239400, PCT Publication WO 91 / 09967, U.S. Pat. No. 5,225,539, EP 592106, and EP 519596, which are incorporated herein by reference.

[0167] As used herein, the term "hydrophobic side chain" means an amino acid side chain that exhibits relatively low solubility in water, e.g., due to the steric or electrical properties of the chemical moiety present within the side chain. Examples of amino acids containing hydrophobic side chains include, in addition to amino acids containing unsaturated aliphatic hydrocarbons, such as alanine, valine, leucine, isoleucine, proline, and methionine, amino acids containing an electrostatically neutral aromatic ring system at physiological pH, such as tryptophan, phenylalanine, and tyrosine.

[0168] As used herein, the term "immunotherapeutic agent" refers to a compound, such as an antibody, antigen-binding fragment, single-chain polypeptide, or construct described herein, that specifically binds to an immune checkpoint protein (e.g., an immune checkpoint receptor or immune checkpoint ligand) and exerts an antagonistic effect on the receptor or ligand, thereby reducing or inhibiting the signal transduction of the receptor or ligand that could otherwise lead to downregulation of the immune response. Immunotherapeutic agents include compounds, such as antibodies, antigen-binding fragments, single-chain polypeptides, and constructs, that specifically bind to receptors expressed on the surface of hematopoietic cells, such as lymphocytes (e.g., T cells), and can suppress the signal transduction induced by receptors or ligands that could otherwise cause immune tolerance to endogenous ("self") antigens, such as tumor-associated antigens. An immunotherapeutic agent can reduce the signal transduction induced by a receptor or ligand by, for example, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% compared to the signal transduction induced by the receptor or ligand in the absence of the immunotherapeutic agent. Exemplary assays that can be used to measure the degree of signal transduction of a receptor or ligand include, for example, enzyme immunoassay (ELISA) techniques for measuring changes in protein expression involving a specific signal transduction pathway, as well as polymerase chain reaction (PCR)-based techniques useful for measuring changes in gene expression involving a specific signal transduction pathway, such as quantitative PCR experiments, reverse transcription PCR experiments, and real-time PCR experiments. Exemplary methods that can be used to confirm whether a drug is an "immunotherapeutic agent" include the assays described in Mahoney et al., Cancer Immunotherapy, 14:561-584 (2015) (the entire disclosure of which is incorporated herein by reference).Examples of immunotherapeutic agents include, for example, antibodies or antigen-binding fragments thereof that specifically bind to one or more of OX40L, TL1A, CD40L, LIGHT, BTLA, LAG3, TIM3, Singlecs, ICOS, B7-H3, B7-H4, VISTA, TMIGD2, BTNL2, CD48, KIR, LIR, LIR antibodies, ILT, NKG2D, NKG2A, MICA, MICB, CD244, CSF1R, IDO, TGFβ, CD39, CD73, CXCR4, CXCL12, SIRPA, CD47, VEGF, and neuropilin. Another example of an immunotherapeutic agent includes targretin, interferon-α, clobetasol, peginterferon (e.g., PEGASYS®), prednisone, romidepsin, bexarotene, methotrexate, triamcinolone cream, anti-chemokine, vorinostat, gabapentin, antibodies against lymphocyte cell surface receptors and / or lymphokines, antibodies against surface cancer proteins, and / or small molecule therapeutic agents such as vorinostat. Specific examples of immunotherapeutic agents that can be used with the compositions and methods described herein include anti-PD-1 antibodies and antigen-binding fragments thereof, such as nivolumab, pembrolizumab, avelumab, durvalumab, and atezolizumab, in addition to anti-PD-L1 antibodies and antigen-binding fragments thereof, such as atezolizumab and avelumab, and anti-CTLA-4 antibodies and antigen-binding fragments thereof, such as ipilimumab or tremelimumab.

[0169] As used herein, the term "monoclonal antibody" means an antibody derived from a single clone, including any eukaryotic cell clone, prokaryotic cell clone, or phage clone, and does not refer to the method of making a monoclonal antibody.

[0170] As used herein, the term "multispecific antibody" means an antibody that exhibits affinity for two or more target antigens. A multispecific antibody can have a structure similar to a complete immunoglobulin molecule and can include an Fc region, for example, an IgG Fc region. Examples of such structures include, but are not limited to, IgG-Fv, IgG-(scFv)2, DVD-Ig, (scFv)2-(scFv)2-Fc, and (scFv)2-Fc-(scFv)2. In the case of IgG-(scFv)2, the scFv can be attached to either the N-terminus or the C-terminus of either the heavy chain or the light chain. Exemplary multispecific molecules that include an Fc region and can incorporate an anti-TNFR2 antibody or an antigen-binding fragment thereof are discussed by Kontermann, 2012, mAbs 4(2):182-197, Yazaki et al, 2013, Protein Engineering, Design & Selection 26(3):187-193, and Grote et al, 2012, in Proetzel & Ebersbach (eds.), Antibody Methods and Protocols, Methods in Molecular Biology vol.901, chapter 16:247-263 (incorporated herein by reference). In some embodiments, the antibody fragment may be a component of a multispecific molecule that does not include an Fc region and is based on a fragment of IgG or DVD or scFv. Exemplary multispecific molecules that lack an Fc region and can incorporate an antibody or antibody fragment are described by Hudson and Souriau, 2003, Nature Medicine 9:129-134 (incorporated herein by reference), including scFv dimers (diabodies), trimers (triabodies), and tetramers (tetrabodies), Fab dimers (conjugates with an adhesive polypeptide or protein domain), and Fab trimers (chemically conjugated).

[0171] As used herein, the term "myeloid-derived suppressor cell" or "MDSC" refers to cells of the immune system that regulate the activities of various effector cells and antigen-presenting cells, such as, among others, T cells, NK cells, dendritic cells, and macrophages. Myeloid-derived suppressor cells are distinguished by their gene expression profiles and express all or some of the proteins and small molecules selected from the group consisting of B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1, CD2, CD31 (PECAM-1), CD43, CD44, complement component C5a R1, F4 / 80 (EMR1), Fcγ RIII (CD16), Fcγ RII (CD32), Fcγ RIIA (CD32a), Fcγ RIIB (CD32b), Fcγ RIIB / C (CD32b / c), Fcγ RIIC (CD32c), Fcγ RIIIA (CD16A), Fcγ RIIIB (CD16b), galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1RI, IL-4Rα, IL-6Rα, integrin α4 (CD49d), integrin αL (CD11a), integrin αM (CD11b), M-CSFR, MGL1 (CD301a), MGL1 / 2 (CD301a / b), MGL2 (CD301b), nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), siglec-3 (CD33), transferrin receptor (TfR), VEGFR1 (Flt-1), and VEGFR2 (KDR or Flk-1). Specifically, MDSCs do not express the proteins selected from the group consisting of B7-2 (CD86), B7-H4, CD11c, CD14, CD21, CD23 (FcεRII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6).

[0172] As used herein, the terms "neutral TNFR2 polypeptide" and "phenotypically neutral TNFR2 polypeptide" mean a polypeptide (e.g., a single-chain polypeptide, an antibody, or an antibody fragment, etc.) that binds to TNFR2 but does not exert an antagonistic or agonistic effect on TNFR2 activation. For example, when a polypeptide binds to TNFR2 and is evaluated by, for example, measuring the proliferation of TNFR2-expressing cells (e.g., T-reg cells, TNFR2+ cancer cells, and / or MDSCs) and / or measuring the expression of one or more NFκB target genes, such as CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and / or cIAP2 / BIRC3, etc., and does not enhance or suppress TNFR2 activation, then that TNFR2 polypeptide is a neutral TNFR2 polypeptide.

[0173] As used herein, the term "non-natural constant region" means an antibody constant region derived from a source different from the antibody variable region, or an antibody constant region that is a synthetic polypeptide made by humans and has an amino acid sequence different from the natural antibody constant region sequence. For example, an antibody containing a non-natural constant region may have a variable region derived from a non-human source (e.g., mouse, rat, or rabbit) and a constant region derived from a human source (e.g., a human antibody constant region), or, in particular, a constant region derived from another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, a member of the bovine family (e.g., in particular, cow, bison, swine, elk, and yak, etc.), female cow, sheep, horse, or bison.

[0174] As used herein, the term "percent (%) sequence identity" means the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence, after the sequences have been aligned (e.g., gaps may be introduced into one or both of the candidate and reference sequences as appropriate to obtain maximum percent sequence identity, and non-homologous sequences may be disregarded for comparison purposes) to obtain the maximum percent sequence identity. Alignment for the purpose of measuring percent sequence identity can be performed in a variety of ways well known to those of skill in the art, using, for example, generally available computer software such as BLAST, ALIGN, or Megalign (DNASTAR). Those of skill in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to obtain the maximum alignment over the full length of the sequences being compared. For example, when a reference sequence is aligned to a candidate sequence for comparison, the candidate sequence can be shown to exhibit 50% to 100% sequence identity to the full length of the candidate sequence or to a selected portion of the contiguous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes can be, for example, at least 30% (e.g., 30%, 40, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. If an amino acid residue at a certain position within the candidate sequence is the same as that at the corresponding position within the reference sequence, the molecules are identical at that position.

[0175] As used herein, the term "primateized antibody" means an antibody that includes the framework region of an antibody derived from a primate and other regions of an antibody from a non-primate source, such as CDRs and / or constant regions. Methods for producing primateized antibodies are well known in the art. See, for example, U.S. Pat. Nos. 5,658,570, 5,681,722, and 5,693,780, which are incorporated herein by reference. For example, a primateized antibody or antigen-binding fragment thereof described herein can be produced by inserting the CDRs of a non-primate antibody or antigen-binding fragment thereof into an antibody or antigen-binding fragment thereof that contains one or more framework regions of a primate.

[0176] As used herein, the term "proliferation" with respect to a population of cells, such as a population of TNFR2+ cells (e.g., T-reg cells, MDSC, or TNFR2+ cancer cells), means mitosis and cytokinesis to produce multiple cells. Cell proliferation can be evidenced, for example, by an increase in the amount of cells (e.g., TNFR2+ cells) in a cell sample over any period of time, such as over 1 hour or multiple hours, 1 day or multiple days, or 1 week or multiple weeks. One of ordinary skill in the art can monitor cell proliferation using various well-known techniques, such as visible microscopy, hemocytometry, flow cytometry, fluorescence-activated cell sorting, and other assays well-known in the art. In the present disclosure, cell proliferation is considered to be "inhibited" when the rate of proliferation of a population of cells, such as a population of TNFR2+ cells contacted with an antagonistic TNFR2 polypeptide described herein, is decreased compared to the rate of proliferation of a population of control cells, such as a population of TNFR2+ cells not contacted with an antagonistic TNFR2 polypeptide. A decrease in the rate of proliferation can be evidenced, for example, by a decrease in the amount of the target cells in the sample over any period of time, such as a decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or more in the amount of the target cells in the sample over any period of time. Additionally or alternatively, inhibition of cell proliferation can be evidenced by the result that the rate at which the target cells (e.g., TNFR2+ cells contacted with an antagonistic TNFR2 polypeptide described herein) divide is decreased compared to the rate at which control cells (e.g., TNFR2+ cells not contacted with an antagonistic TNFR2 polypeptide) divide, such as by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or more.

[0177] As used herein, the term "functionally linked" with respect to polynucleotide fragments is intended to mean that two polynucleotide fragments are linked such that the amino acid sequences encoded by the two polynucleotide fragments are maintained in-frame.

[0178] As used herein, the term "pharmacokinetic profile" means the absorption, distribution, metabolism, and clearance of a drug over time after administration of the drug to a patient.

[0179] As used herein, a "retrograde antagonist" of TNFR2 is an antagonist (e.g., an antagonistic polypeptide such as a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) that significantly inhibits TNFR2 activation to a lower degree in the presence of a TNFR2 agonist (e.g., TNFα or IL-2) compared to the degree of inhibition of the same antagonist measured in the absence of a TNFR2 agonist (e.g., TNFα or IL-2). For example, the IC of the antagonist measured in the same assay in the absence of a TNFR2 agonist (e.g., TNFα or IL-2) 50 Compared to, the IC of the antagonist in the presence of a TNFR2 agonist (e.g., TNFα or Bacillus Calmette-Guerin (BCG)) or IL-2 50When it becomes, for example, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more-fold higher, the TNFR2 antagonist is a reverse antagonist. Inhibition of TNFR2 activation can be evaluated by measuring inhibition of NFκB signaling (e.g., by monitoring a decrease in the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3 using a conventional gene expression assay), in addition to measuring inhibition of proliferation of, for example, TNFR2+ cells such as T-reg cells, cancer cells expressing TNFR2, or myeloid-derived suppressor cells.

[0180] As used herein, the term "control sequence" includes a promoter, an enhancer, and other expression control elements (e.g., a polyadenylation signal) that control the transcription or translation of an antibody chain gene. Such control sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990), which is incorporated herein by reference.

[0181] As used herein, the term "scFv" means a single-chain Fv antibody in which the variable domains of the heavy and light chains of an antibody are linked to form a single molecular chain. The scFv fragment contains a single polypeptide chain comprising the variable regions of the antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and / or CDR-L3) and the variable region of the antibody heavy chain (VH) (e.g., CDR-H1, CDR-H2, and / or CDR-H3) separated by a linker. The linker that links the VL and VH regions of the scFv fragment may be a peptide linker composed of proteinaceous constituent amino acids. Alternative linkers may be used to enhance the resistance of the scFv fragment to proteolysis (e.g., linkers containing D-amino acids), to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers such as polyethylene glycol-containing linkers, or polypeptides containing repeats of glycine and serine residues), to improve the biophysical stability of the molecule (e.g., linkers containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites). scFv molecules are well known in the art and are described, for example, in U.S. Patent 5,892,019, Flo et al., (Gene 77:51, 1989); Bird et al., (Science 242:423, 1988); Pantoliano et al., (Biochemistry 30:10117, 1991); Milenic et al., (Cancer Research 51:6363, 1991); and Takkinen et al., (Protein Engineering 4:837, 1991). The VL and VH domains of the scFv molecule may be derived from one or more antibody molecules. Those skilled in the art will also understand that the variable regions of the scFv molecules described herein may be modified such that the amino acid sequence varies from the antibody molecule from which the variable region is derived.For example, in one embodiment, nucleotide substitutions or amino acid substitutions that result in conservative substitutions or conservative mutations of amino acid residues may be made (e.g., in CDR residues and / or framework residues). Alternatively or in addition, techniques recognized in the art may be used to introduce mutations into CDR amino acid residues to optimize antigen binding. For scFv fragments, see, for example, WO2011 / 084714, which is incorporated herein by reference.

[0182] As used herein, the phrase "specifically binds" means, for example, a binding reaction that determines the presence of an antigen within a heterogeneous population of proteins and other biomolecules that is specifically recognized by an antibody or an antigen-binding fragment thereof. An antibody or an antigen-binding fragment thereof that specifically binds to an antigen binds to the antigen with a K D of less than 100 nM. For example, an antibody or an antigen-binding fragment thereof that specifically binds to an antigen binds to the antigen with a K D of up to 100 nM (e.g., 1 pM to 100 nM). An antibody or an antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or its epitope has a K D greater than 100 nM (e.g., greater than 500 nm, greater than 1 μM, greater than 100 μM, greater than 500 μM, or greater than 1 mM) for that particular antigen or its epitope. Various immunoassay formats may be used to select antibodies that specifically immunoreact with a particular protein or carbohydrate. For example, a solid-phase ELISA immunoassay is routinely used to select antibodies that specifically immunoreact with a protein or carbohydrate. For a description of immunoassay formats and conditions that can be used to measure specific immunoreactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999).

[0183] As used herein, the terms "subject" and "patient" mean an organism undergoing treatment for a particular disease or condition (e.g., cancer or an infectious disease, etc.) described herein. Examples of subjects and patients include mammals undergoing treatment for a disease or condition, such as a cell proliferation disorder such as cancer, or an infectious disease, such as, inter alia, humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovine family (e.g., inter alia, cows, bison, water buffalo, elk, and yaks, etc.), female cows, sheep, horses, and bison, etc.

[0184] As used herein, the term "transfection" means any of a variety of techniques commonly used to introduce foreign DNA into a prokaryotic host cell or a eukaryotic host cell, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0185] As used herein, the terms "treat" or "treatment" mean a therapeutic treatment aimed at preventing or delaying (mitigating) an undesirable physiological change or disorder, such as a cell proliferation disorder such as cancer or the progression of an infectious disease. Advantageous or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the degree of disease, stabilization (i.e., not worsening) of the disease, delay or slowing of disease progression, improvement or temporary alleviation of the condition, and remission (partial or complete). Subjects and patients in need of treatment include, in addition to subjects and patients already having symptoms or disorders, subjects and patients having a tendency to have symptoms or disorders, or subjects and patients in whom prevention of symptoms or disorders is carried out.

[0186] As used herein, the term "tumor microenvironment" refers to cancer cells that form a tumor, as well as populations of non-cancer cells, populations of non-cancer molecules, and / or blood vessels within the tumor or adjacent to or surrounding the cancer cells.

[0187] As used herein, the terms "tumor necrosis factor receptor superfamily", "TNFR superfamily", or "TNFRS" refer to a group of type I transmembrane proteins having an amino-terminal extracellular domain characterized by a carboxy-terminal intracellular domain and a common cysteine-rich domain (CRD). The TNFR superfamily includes receptors that mediate cell signaling as a result of binding to one or more ligands of the TNF superfamily. The TNFR superfamily can be classified into two subgroups: receptors containing an intracellular death domain and receptors lacking this domain. The death domain is an 80-amino acid motif that propagates the apoptosis signaling cascade following activation of the receptor. Exemplary TNFR superfamily members containing an intracellular death domain include TNFR1, while TNFR2 represents a TNFR superfamily protein lacking this domain. Members of the TNFR superfamily include TNFR1, TNFR2, RANK, CD30, CD40, lymphotoxin β receptor (LT-βR), OX40, Fas receptor, decoy receptor 3 (DCR3), CD27, 4-1BB, death receptor 4 (DR4), death receptor 5 (DR5), decoy receptor 1 (DCR1), decoy receptor 2 (DCR2), osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, herpesvirus entry mediator, nerve growth factor receptor, B cell maturation antigen, glucocorticoid-induced TNFR-related, TROY, death receptor 6 (DR6), death receptor 3 (DR3), and ectodysplasin A2 receptor.

[0188] As used herein, the terms "tumor necrosis factor receptor 2 signaling", "TNFR2 signaling", "TNFR2 signal transduction", etc. are used interchangeably and refer to the cellular events that typically occur upon activation of TNFR2 on the surface of TNFR2+ cells, such as T-reg cells, MDSCs, or TNFR2+ cancer cells, by an endogenous TNFR2 ligand, such as TNFα. TNFR2 signaling can be evidenced by an increase in the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3. As used herein, when a cell is contacted with an agent, such as a TNFR2 antagonist polypeptide described herein, TNFR2 signaling is considered to be "inhibited" if the expression (and / or post-translational modification, if such modification is required for activation of the encoded protein) of one or more or all of the above genes is decreased in TNFR2+ cells compared to TNFR2+ cells not contacted with the agent (e.g., the TNFR2 antagonist polypeptide). For example, the expression or post-translational modification (e.g., phosphorylation) of one or more of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, or cIAP2 / BIRC3 in TNFR2+ cells contacted with an antagonist TNFR2 polypeptide is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower compared to the expression or post-translational modification (e.g., phosphorylation) of one or more of these genes in TNFR2+ cells not contacted with the antagonist TNFR2 polypeptide, then TNFR2 signaling is considered to be "inhibited".Exemplary assays that can be used to measure expression levels and phosphorylation status are well known in the art and include, for example, Western blot assays for measuring protein content and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments for measuring mRNA content.

[0189] As used herein, the term "variable region CDR" includes amino acids within the CDR, i.e., complementarity determining region, identified using sequence- or structure-based methods. As used herein, the term "CDR" or "complementary determining region" means the non-contiguous antigen-binding sites present within the variable regions of both the heavy chain polypeptide and the light chain polypeptide. These specific regions are described in Kabat et al., J. Biol. Chem. 252:6609-6616, 1977 and Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; Chothia et al., (J. Mol. Biol. 196:901-917, 1987), as well as MacCallum et al., (J. Mol. Biol. 262:732-745, 1996), and the definitions include overlaps or subsets of amino acid residues when compared to each other. The term "CDR" may be, for example, the CDR defined by Kabat based on sequence comparison.

[0190] As used herein, the term "vector" includes nucleic acid vectors such as DNA vectors (e.g., plasmids), RNA vectors, viruses or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO1994 / 11026 (incorporated herein by reference). The expression vectors described herein contain, in addition to the polynucleotide sequence, for example, other sequence elements used to express the protein, and / or other sequence elements used to introduce these polynucleotide sequences into the genome of mammalian cells. Specific vectors that can be used to express the antibodies and antibody fragments described herein include plasmids containing control sequences such as promoter regions and enhancer regions that induce gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that increase the translation rate of these genes, or polynucleotide sequences that improve the stability or nuclear export of the mRNA produced by gene transcription. Examples of these sequence elements include 5' untranslated regions and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites for inducing efficient transcription of the genes carried on the expression vector. The expression vectors described herein may also contain a polynucleotide encoding a marker for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0191] As used herein, the term "VH" means the variable region of an immunoglobulin heavy chain of an antibody that includes the heavy chain of an Fv, scFv, or Fab. Reference to "VL" means the variable region of an immunoglobulin light chain that includes the light chain of an Fv, scFv, dsFv, or Fab. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins having the same structural features. While an antibody exhibits binding specificity for a particular target, an immunoglobulin includes both an antibody and other antibody-like molecules lacking target specificity. Native antibodies and immunoglobulins are typically heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a native antibody has a variable domain (VH) at the amino terminus followed by a number of constant domains. Each light chain of a native antibody has a variable domain (VL) at the amino terminus and a constant domain at the carboxy terminus.

Brief Description of the Drawings

[0192]

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Modes for Carrying Out the Invention

[0193] The antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, inhibit the activation of TNFR2 on TNFR2-expressing cells. This inhibition can occur, for example, by binding to TNFR2 (e.g., on the outer surface of T-reg cells, cancer cells expressing TNFR2, or myeloid-derived suppressor cells (MDSC)) and preventing the receptor from adopting a three-dimensional structure suitable for binding its associated ligand, TNFα. TNFα enhances TNFR2 signaling by aggregating trimers of the TNFR2 protein. This trimerization event brings individual TNFR2 proteins into proximity, initiating TNFR2 signaling via the MAPK / NFκB / TRAF2 / 3 pathway and ultimately causing cell proliferation and avoidance of apoptosis. The antagonist TNFR2 polypeptides described herein can counteract this interaction, for example, by binding to the receptor and preventing receptor trimerization. For example, one mechanism by which this counteraction can occur is by formation of an antiparallel TNFR2 dimer, an inactive conformational form of the receptor.

[0194] The TNFR2 polypeptides described herein specifically bind to epitopes within TNFR2 that promote the antagonist action of the receptor and various advantageous downstream biological activities. Human TNFR2 contains four cysteine-rich domains (CRDs): CRD1 (amino acid residues 48-76 of SEQ ID NO: 7), CRD2 (amino acid residues 78-120 of SEQ ID NO: 7), CRD3 (amino acid residues 121-162 of SEQ ID NO: 7), and CRD4 (amino acid residues 162-202 of SEQ ID NO: 7). The antagonist TNFR2 polypeptides described herein specifically bind to TNFR2 at one or more epitopes within CRD3 and / or CRD4. In some embodiments, the antagonist TNFR2 polypeptide does not bind to epitopes within CRD1 and / or CRD2. For example, the polypeptides of the present disclosure (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof) comprise the following residues, (a) Amino acids 142 - 146 (KCRPG) of SEQ ID NO: 7, (b) Amino acids 142 - 149 (KCRPGFGV) of SEQ ID NO: 7, (c) Amino acids 137 - 144 (CAPLRKCR) of SEQ ID NO: 7, (d) Amino acids 150 - 190 (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI) of SEQ ID NO: 7, (e) Amino acids 161 - 169 (CKPCAPGTF) of SEQ ID NO: 7, (f) Amino acids 75 - 128 (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL) of SEQ ID NO: 7 (optionally, the epitope is within amino acids 80 - 86 (DSTYTQL), 91 - 98 (PECLSCGS), or 116 - 123 (RICTCRPG) of SEQ ID NO: 7), (g) Amino acids 174 - 184 (SSTDICRPHQI) of SEQ ID NO: 7, (h) Amino acids 126 - 140 (CALSKQEGCRLCAPL) of SEQ ID NO: 7, and / or (i) Amino acids 156 - 165 (TSDVVCKPCA) of SEQ ID NO: 7, one or more internal epitopes among or equivalent epitopes within TNFR2 of a non - human mammal, such as the non - human mammals described herein that can bind to human TNFR2.

[0195] This disclosure is based in part on the discovery that anti - TNFR2 polypeptides exhibit substantially improved TNFR2 antagonist activity when these molecules are in the form of the IgG2 isotype. As described in the following examples, this class of TNFR2 polypeptides has been found at present to exhibit surprisingly superior ability to inhibit TNFR2 signaling, attenuate the proliferation of T - reg cells and cancer cells, and enhance the proliferation of effector T cells, compared to other isotypes of TNFR2 - binding polypeptides.

[0196] Another discovery underlying the present disclosure is that an antagonistic TNFR2 polypeptide containing antigen-binding sites that are spatially separated from each other by about 133 Å or more exhibits unexpectedly superior TNFR2 antagonist activity as compared to a polypeptide that specifically binds to TNFR2 at one or more of the above epitopes and contains antigen-binding sites that are less than about 133 Å apart from each other. Examples of such polypeptides include IgG1 antibodies and antigen-binding fragments thereof containing antigen-binding sites that are about 117 Å apart from each other, and IgG3 antibodies and antigen-binding fragments thereof containing antigen-binding sites that are 125 Å apart from each other.

[0197] The antagonist TNFR2 polypeptides of the present disclosure can be formulated into pharmaceutical compositions. The polypeptide contained in the pharmaceutical composition preferably takes a single disulfide bond isoform. For example, as the pharmaceutical composition of the present disclosure, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or more of the polypeptides in the pharmaceutical composition are present as a single disulfide bond isoform, and pharmaceutical compositions containing antagonist TNFR2 polypeptides can be mentioned. The antagonist TNFR2 polypeptides of the present disclosure advantageously take the IgG2-A disulfide bond isoform, which has surprisingly been found to promote a substantially stronger level of TNFR2 antagonist activity compared to other IgG2 disulfide bond isoforms, such as the IgG2-B isoform, the IgG2-A / B1 isoform, and the IgG2-A / B2 isoform. These isoforms are illustrated in FIGS. 13A to 13D. For example, the polypeptide of the present disclosure may be modified to mainly take the IgG2-A isoform by introducing mutations that inhibit the formation of other disulfide bond isoforms into the IgG2 hinge region. Exemplary mutations within the amino acid sequence of the human IgG2 hinge region that promote the formation of the IgG2-A isoform, excluding the remaining above-mentioned isoforms, include deletions and / or substitutions of cysteine residues at positions 232 and 233 of the wild-type human IgG2 hinge amino acid sequence, as described in SEQ ID NO: 291. For example, to modify an IgG2 antibody or an antigen-binding fragment thereof to mainly take the IgG2-A isoform, conservative amino acid substitutions may be introduced into cysteine residues 232 and / or 233 of SEQ ID NO: 291. An exemplary IgG2 hinge region that is mainly present as the IgG2-A isoform has the amino acid sequence of SEQ ID NO: 292 and contains C232S substitution and C233S substitution compared to SEQ ID NO: 291.

[0198] The following biological activities, (a) For example, inhibition of the proliferation of T-reg cells, and / or direct killing of T-reg cells, by binding to TNFR2 on the surface of T-reg cells and inactivating TNFR2, (b) For example, inhibition of the proliferation of MDSC, and / or direct killing of MDSC, by binding to TNFR2 on the surface of MDSC and inactivating TNFR2, (c) Promotion of the proliferation of T effector cells, such as CD8+ T cells, etc., and / or (d) Inhibition of the proliferation of TNFR2-expressing cancer cells (such as Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic cancer cells or lymphoma cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper digestive tract cancer cells, etc.) and / or direct killing of TNFR2-expressing cancer cells, are examples of an antagonistic TNFR2 phenotype exhibited to a greater degree by the polypeptides of the present disclosure as compared to a TNFR2-binding polypeptide that (i) exhibits an isotype other than IgG2, (ii) contains antigen-binding sites less than 133 Å apart from each other, and / or (iii) does not predominantly exist as a single disulfide bond isoform (such as the IgG2-A isoform).

[0199] In the following sections, in addition to exemplary properties of the antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., an explanation regarding its use in a treatment method is provided.

[0200] Antagonistic TNFR2 polypeptide IgG2 isotype antibodies promote optimal TNFR2 antagonist activity As described above and herein, optimal TNFR2 antagonist activity in human TNFR2 antagonist antibodies, humanized TNFR2 antagonist antibodies, and chimeric TNFR2 antagonist antibodies, and antigen-binding fragments thereof, is obtained when the antibody or antibody fragment exhibits the human IgG2 isotype, and particularly when the antibody or antibody fragment exhibits the IgG2-A disulfide bond isoform. The disulfide bond patterns in the various isoforms of human IgG2 antibodies are shown in FIGS. 13A-13D. As shown in FIG. 13A, the IgG2-A isoform exhibits disulfide bonds between the cysteine residues C221, C222, C225, and C228 present on each heavy chain, in addition to the disulfide bond between the cysteine residue C133 of the heavy chain and C214 of the light chain.

[0201] To stabilize the IgG2-A disulfide-linked isoform, mutations may be introduced into the IgG2 hinge region so that the formation of disulfide bonds between cysteine residues present as unbound thiols within the IgG2-A isoform is prevented or suppressed. Examples of such mutations are amino acid substitutions or deletions at residues C232 and C233 of the human IgG2 hinge region. By removing one or both of these residues and, optionally, replacing these residues with amino acids that cannot form disulfide bonds, the disulfide bond pattern in the population of IgG2 isoforms can be biased towards the IgG2-A isoform. Examples of amino acid substitutions that can be used to obtain a population of IgG2-A isoform antibodies include conservative amino acid substitutions such as the C232S amino acid substitution and the C233S amino acid substitution. Due to the similar molecular volume and molecular polarity of cysteine and serine, the C232S substitution and the C233S substitution are characterized by the advantageous effect of inhibiting the formation of disulfide bonds at positions 232 and / or 233 of the IgG2 hinge region while retaining the steric and electronegativity characteristics of the native cysteine residues. By introducing the C232S substitution and / or the C233S substitution into a TNFR2 antibody or a fragment thereof, a population of TNFR2 antagonist antibodies or fragments that exhibit the IgG2-A isoform can be obtained. Methods for introducing amino acid substitutions and deletions into an antibody or an antigen-binding fragment thereof include mutagenesis techniques described herein and well known in the art.

[0202] Spacing between antigen-binding sites The antagonist TNFR2 polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof) may contain antigen-binding sites (i.e., antigen-binding arms) that are separated from each other by at least about 133 Å, which is the distance observed between the antigen-binding arms of human IgG2 isotype antibodies. As described in the following examples, it has been discovered that this spacing results in antibodies having optimal TNFR2 antagonist properties. TNFR2 antagonist polypeptides of the present disclosure include, for example, those having antigen-binding arms separated by a distance of about 133 Å to about 160 Å, such as, for example, a distance of about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å, 151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160 Å. For example, a polypeptide (e.g., single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) may contain antigen-binding sites that are separated from each other by a distance of about 133 Å to about 150 Å, such as, for example, a distance of about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, or 150 Å. In some embodiments, the antigen-binding is separated by a distance of about 133 Å to about 145 Å, such as, for example, a distance of about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, or 145 Å. In some embodiments, the antigen-binding is separated by a distance of about 133 Å to about 139 Å, such as, for example, a distance of about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, or 139 Å. In some embodiments, the antigen-binding is separated by a distance of about 134 Å to about 139 Å, such as, for example, a distance of about 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, or 139 Å.

[0203] The TNFR2 antagonist polypeptides described herein may have, for example, 2, 3, 4, 5, or more antigen-binding arms separated by the distances specified above. Examples of antibody fragments having two or more antigen-binding arms include, but are not limited to, diabodies, triabodies, F(ab’)2 molecules, and tandem scFv (taFv) molecules. Methods for making these antibody fragments include peptide synthesis techniques and recombinant protein expression techniques described herein and well-known in the art.

[0204] There are various methods for measuring the distance between the antigen-binding arms of an antibody or antibody fragment. For example, the distance between the antigen-binding arms of an antibody can be measured by analyzing the three-dimensional structure of the antibody or antibody fragment using computer software, such as by using PYMOL® and other molecular imaging software. Data obtained from X-ray crystallography experiments and nuclear magnetic resonance (NMR) techniques well-known in the art can be used to calculate the three-dimensional structure of polypeptides, such as antibodies and antibody fragments. Examples of X-ray crystallography and NMR methods that can be used to obtain three-dimensional polypeptide structures are described, for example, in Eigenbrot et al., Journal of Molecular Biology, 229:969-995, 1993; and Huang et al., Science, 317:1930-1934, 2007 (the entire disclosures of each are incorporated herein by reference).

[0205] Homogeneity of a population of TNFR2 antagonist polypeptides A pharmaceutical composition can be prepared in which the TNFR2 antagonist polypeptide (e.g., an antibody, its antigen-binding fragment, a single-chain polypeptide, or a construct thereof) described in this specification exists as a single disulfide bond isoform. For example, at least 10% or more of the polypeptides in the pharmaceutical composition may exist as a single disulfide bond isoform (e.g., the IgG2-A isoform). This can be achieved, for example, by preventing or suppressing the formation of disulfide bonds that can generate IgG2 isoforms other than IgG2-A by causing amino acid substitutions or deletions in one or both of cysteine residues 232 and 233 (see, for example, FIGS. 13A to 13D). Examples of the pharmaceutical compositions of the present disclosure include pharmaceutical compositions in which about 10% to about 99.999% of the antagonist TNFR2 polypeptides in the pharmaceutical composition exist as a single disulfide bond isoform, such as the IgG2-A isoform. For example, the pharmaceutical compositions of the present disclosure include, for example, pharmaceutical compositions containing an antagonist TNFR2 polypeptide in which 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or more of the polypeptides in the pharmaceutical composition exist as a single disulfide bond isoform.

[0206] Techniques for measuring the relative amounts of various disulfide bond isoforms contained in a sample of an antagonist TNFR2 polypeptide include liquid chromatography techniques well known in the art and described herein, such as the liquid chromatography techniques exemplified in Wypych et al., The Journal of Biological Chemistry, 283:16194-16205, 2008 (the entire disclosure of which is incorporated herein by reference).

[0207] Effect on the TNFR2 / MAPK / TRAF2 / 3 signal transduction cascade The anti-TNFR2 polypeptides described herein (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) can interact with TNFR2 to inhibit its activity. Therefore, the anti-TNFR2 polypeptides described herein do not promote TNFR2 signaling but can selectively neutralize the TNFα-TNFR2 interaction. This neutralization is particularly important in therapeutic applications, such as cancer immunotherapy, since TNFR2 activation upon binding to TNFα causes the propagation of the MAPK and TRAF2 / 3 signaling cascades, and the activation of NFκB-mediated transcription of genes involved in T-reg cell proliferation and the avoidance of apoptosis (Faustman, et al., Nat. Rev. Drug Disc., 9:482-493, 2010). The TNFR2 polypeptides described herein (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) bind to TNFR2 at one or more specific epitopes that prevent the receptor from forming trimers with adjacent TNFR2 proteins. This trimerization activates intracellular signaling by TNFR2, which, for example, promotes the proliferation of TNFR2+ cells, such as T-reg cells, MDSCs, and / or TNFR2+ cancer cells. Advantageously, the TNFR2 antagonist polypeptides described herein bind to TNFR2 at specific epitopes so as to stabilize TNFR2 in an antiparallel dimer structure where the TNFα binding site is sterically inaccessible. This binding prevents TNFα from aggregating TNFR2 trimer formation, which would otherwise induce TNFR2 signaling. Therefore, the polypeptides described herein can be used to inhibit the growth and proliferation of TNFR2+ cells, such as T-reg cells, MDSCs, and TNFR2+ cancer cells. For example, the inhibition of the proliferation of T-reg and MDSC allows for the proliferation of T effector cells, which can initiate an immune response against, for example, cancer cells or foreign pathogens.Therefore, in order to enhance the effect of the immune response in a subject (e.g., an immune response against cancer cells or pathogenic microorganisms), an antagonistic TNFR2 polypeptide described herein may be administered to a mammalian subject, such as a human, having a cell growth disorder or an infection.

[0208] Effect on T-reg cell proliferation Using an antagonistic TNFR2 polypeptide described herein, such as a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof, etc., the activity of T-reg cells generally involved in T cell-mediated cytotoxicity against autologous cells, such as the attack of tumor cells by T lymphocytes, etc., may be attenuated. This attenuation can be achieved, for example, by the ability of the antagonistic TNFR2 polypeptide described herein to inhibit the proliferation of T-reg cells and / or directly kill T-reg cells. Therefore, in order to extend the duration of an adaptive immune response, such as a response against cancer cells or pathogenic microorganisms, etc., an antagonistic TNFR2 polypeptide may be administered to a mammalian subject, such as a human, etc. (e.g., by any of the various administration routes described herein). In this method, for example, an antagonistic TNFR2 polypeptide described herein, such as a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof, etc., may be synergized with existing techniques for enhancing T lymphocyte-based therapies against cancer and infections. For example, by administering the TNFR2 antagonist described herein to suppress the activity of T-reg cells, the cytotoxic effect of tumor-reactive T cells may be enhanced. The TNFR2 antagonist may also be synergized with existing strategies for enhancing the survival rate of tumor-reactive T cells, such as lymphocyte depletion therapy and growth factor therapy, etc., thereby extending the duration of the anti-tumor reactivity in vivo.

[0209] Inhibition of T-reg proliferation enhances the activity of CD8+ T lymphocytes capable of initiating an attack against pathogenic microorganisms. Thus, antagonist TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, etc., may also be used to treat a wide variety of infectious diseases in mammalian subjects (e.g., humans). In addition, the antagonist TNFR2 antibodies and antigen-binding fragments thereof described herein may also be used to treat a wide variety of infectious diseases, such as Mycobacterium tuberculosis, etc., in humans or agricultural livestock (e.g., bovine mammals, pigs, cows, horses, sheep, goats, cats, dogs, rabbits, hamsters, guinea pigs, or other non-human mammals).

[0210] Direct action against TNFR2+ cancer cells The antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof, etc., can bind to TNFR2 on the surface of cancer cells, such as TNFR2+ tumor cells, etc., and inactivate TNFR2. For example, the antagonist TNFR2 antibodies and antigen-binding fragments thereof described herein can bind, inter alia, to TNFR2 on the surface of T cell lymphoma cells (e.g., Hodgkin lymphoma cells or cutaneous non-Hodgkin lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells. The ability of the antagonist TNFR2 antibodies and antigen-binding fragments thereof described herein to directly bind to TNFR2 on cancer cells provides another pathway by which these molecules can attenuate the survival rate and proliferation of cancer cells. For example, the antagonist TNFR2 polypeptides described herein, such as antagonist TNFR2 single-chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs, etc., can directly bind to TNFR2 on the surface of cancer cells (e.g., cutaneous T cell lymphoma cells, ovarian cancer cells, colon cancer cells, or multiple myeloma cells, such as ovarian cancer cells, etc.) to inhibit the ability of proliferating cells and / or to promote apoptosis of cells.

[0211] The TNFR2 antagonist polypeptide is independent of other TNFR2 binding factors in terms of activity. Importantly, the antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof, can bind to TNFR2 and inhibit TNFR2-mediated signal transduction without the need for endogenous TNFR2 binding factors such as TNFα. The antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, do not require TNFα to attenuate the proliferation of T-reg and / or cancer cells. Without being limited by mechanism, the antagonistic TNFR2 antibodies or antigen-binding fragments thereof described herein may exhibit this property due to the ability of these antibodies or antigen-binding fragments thereof to bind to TNFR2 at specific epitopes, where binding stabilizes the antiparallel dimer structure of this receptor. In this structural form, NFκB signal transduction cannot be enhanced. The antagonistic TNFR2 polypeptides described herein can prevent TNFR2 agonists from restoring cell proliferation by maintaining TNFR2 in an inactive structural state and / or can result in direct killing (e.g., by apoptosis) of TNFR2+ cells such as T-reg cells, MDSC, or TNFR2+ cancer cells.

[0212] For example, the antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, their antigen-binding fragments, and constructs thereof, can bind to TNFR2 on the surface of TNFR2+ cells, such as T-reg cells, cancer cells, or myeloid-derived suppressor cells (MDSC), and inhibit the proliferation of such cells in the presence or absence of TNFα. For example, the antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, and their antigen-binding fragments, can inhibit the proliferation of such cells by, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, compared to such cells not treated with the TNFR2 antagonist polypeptide. The antagonist TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or its antigen-binding fragment) may exhibit an IC 50 value that does not vary significantly depending on the presence or absence of TNFα (e.g., in the same cell proliferation assay in the absence of TNFα, the IC 50 value of the antagonist TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or its antigen-binding fragment) compared to the IC 50value). Examples of cell death assays that can be used to measure the antagonist activity of a TNFR2 antibody are described, for example, in Example 2 below. Similarly, antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., when evaluated by measuring the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3, can inhibit TNFR2 signaling by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, compared to such cells not treated with a TNFR2 antagonist polypeptide. An antagonist TNFR2 polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) can exhibit an IC 50 value that does not vary significantly depending on the presence or absence of TNFα (e.g., in the same gene expression assay in the absence of TNFα, the IC 50 value of an antagonist TNFR2 polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) changes by less than 50%, less than 45%, less than 40%, less than 35%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% compared to the IC 50 value in the presence of TNFα).

[0213] Direct killing of T-reg cells, MDSC, and TNFR2+ cancer cells The antagonist TNFR2 polypeptides disclosed herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can, for example, not only inhibit the proliferation of T-reg cells, TNFR2+ cancer cells, and / or MDSCs in a sample (e.g., within a patient, e.g., within a human patient), but can also induce the death of T-reg cells, TNFR2+ cancer cells, and / or MDSCs. The antagonist TNFR2 polypeptides described herein can, for example, reduce the total amount of T-reg cells, cancer cells (e.g., in particular, cutaneous T cell lymphoma cells, ovarian cancer cells, colon cancer cells, renal cell carcinoma cells, or multiple myeloma cells, etc.), and / or MDSCs in a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infectious disease as described herein) treated with an antagonist TNFR2 antibody or an antigen-binding fragment thereof, compared to a sample not treated with an antagonist TNFR2 antibody or an antigen-binding fragment thereof, by, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more.

[0214] The ability of the antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) described herein to attenuate the proliferation of T-reg, MDSC, and / or cancer cells may be due in part to the ability of these polypeptides to reduce the amount of soluble TNFR2 in a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infection as described herein). In the absence of this beneficial effect, soluble TNFR2 can be secreted, for example, by T-reg cells and can interfere with the ability of TNFR2 antagonists localized on the surface of T-reg cells, TNFR2+ cancer cells, or MDSC by binding to TNFR2 antagonists in the extracellular environment and pulling them away. The antagonistic TNFR2 antibodies or antigen-binding fragments thereof described herein can, by suppressing the secretion of TNFR2, make T-reg cells, TNFR2+ cancer cells, and / or MDSC more susceptible to the effects of therapeutic molecules, such as antagonistic TNFR2 antibodies or antigen-binding fragments thereof, and / or other anti-cancer agents, such as anti-cancer agents described herein or well-known in the art that can be used in combination with the compositions and methods described herein.

[0215] Active (CD25 Hi and CD45RA Low ) Selective regulation of T-reg cells The antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) described herein can inhibit the proliferation of T-reg cells or reduce the total amount of T-reg cells in a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infection as described herein) and can act selectively on T-reg cells in an actively dividing state. The antagonistic TNFR2 antibodies or antigen-binding fragments thereof described herein, for example, CD25 Med and CD45RA Hi Compared to resting T-reg cells expressing Hi and CD45RA Lowcan selectively target activated T-reg cells that express it. For example, the antagonistic TNFR2 antibody or antigen-binding fragment thereof described herein binds to CD25 Hi and CD45RA Low and inhibits the proliferation of T-reg cells that express it compared to T-reg cells that do not express the CD25 Hi protein and CD45RA Low protein, such as T-reg cells that do not express the CD25 Med protein and CD45RA Hi protein, by, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more.

[0216] Regulation of T-reg cells, MDSCs, and T effector cells in the tumor microenvironment The antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, can inhibit the proliferation of T-reg cells at higher titers in patients with cancer compared to subjects without cancer. The antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, can inhibit the proliferation of T-reg cells at higher titers in the tumor microenvironment compared to sites that do not contain cancer cells, such as sites distant from the tumor, in patients with cancer or in subjects without cancer. For example, this effect can be measured using the cell death assays described herein. For example, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, are the IC of the polypeptide for suppressing or inhibiting the proliferation of T-reg cells at sites that do not contain cancer cells 50Thus, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more-fold lower, an IC for suppressing or inhibiting the proliferation of T-reg cells in the tumor microenvironment 50 can be shown. Examples of cell death assays that can be used to measure the antagonistic action of an anti-TNFR2 polypeptide are described, for example, in Example 2 below. The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., are more potent than in a site that does not contain such cancer cells, such as a site far from the tumor, in a patient suffering from one or more of the above cancers or in a subject without cancer, in the tumor microenvironment containing TNFR2+ cancer cells (such as Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T-cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic cancer cells or lymphoma cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.), and can inhibit the proliferation of T-reg cells or promote the apoptosis of T-reg cells.

[0217] Additionally or alternatively, the polypeptides described herein, such as single-chain polypeptides, antibodies, their antigen-binding fragments, and constructs thereof, etc., can inhibit the proliferation of MDSCs at a higher titer in patients suffering from cancer compared to subjects without cancer. The polypeptides described herein, such as single-chain polypeptides, antibodies, their antigen-binding fragments, and constructs thereof, etc., can inhibit the proliferation of MDSCs at a higher titer in the tumor microenvironment compared to sites without cancer cells, such as sites far from the tumor, etc., in patients suffering from cancer or in subjects without cancer. For example, this effect may be measured using the cell death assay described herein. For example, the polypeptides described herein, such as single-chain polypeptides, antibodies, their antigen-binding fragments, and constructs thereof, etc., are IC for suppressing or inhibiting the proliferation of MDSCs at a site without cancer cells 50 50, such as 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more-fold lower for suppressing or inhibiting the proliferation of MDSCs in the tumor microenvironment 50It can be shown. For examples of cell death assays that can be used to measure the antagonistic effect of an anti-TNFR2 polypeptide, see, for example, Example 2 below. The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., in the microenvironment of tumors containing TNFR2+ cancer cells (such as Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic cancer cells or lymphoma cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.), in patients suffering from one or more of the above cancers or in sites not containing such cancer cells in a subject without cancer, such as sites far from the tumor, etc., can inhibit the proliferation of MDSC, or promote the apoptosis of MDSC, at a higher titer.

[0218] Additionally or alternatively, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., can proliferate T effector cells, such as CD8+ cytotoxic T cells, etc., at a higher titer in patients with cancer compared to subjects without cancer. In some embodiments, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, etc., can proliferate T effector cells, such as CD8+ cytotoxic T cells, etc., at a higher titer in the microenvironment of tumors compared to sites not containing cancer cells in patients with cancer or in a subject without cancer, such as sites far from the tumor, etc. For example, this effect can be measured using the cell proliferation assays described herein. For example, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., are the EC of the polypeptide for proliferating T effector cells in a site not containing cancer cells. 50Thus, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more-fold lower, an EC for proliferating T effector cells in the tumor microenvironment 50 may have. Examples of cell proliferation assays that can be used to measure the effect of an anti-TNFR2 polypeptide on T effector cells are described, for example, in Example 2 below. The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, etc., are present in the tumor microenvironment containing TNFR2+ cancer cells (e.g., Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic cancer cells or lymphoma cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.) at a higher titer than in a site that does not contain such cancer cells, such as a site far from the tumor, etc., in a patient suffering from one or more of the above cancers or in a subject without cancer, and can directly proliferate T effector cells, such as CD8+ cytotoxic T cells, etc. T effector cells (e.g., CD8+ cytotoxic T cells) specifically react with an antigen present on, for example, one or more types of cancer cells, such as among the other cancer cells described herein, Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic cancer cells or lymphoma cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.

[0219] Activity of the antigen-binding fragment of a full-length TNFR2 antagonist antibody The antagonist TNFR2 antibodies described herein can inhibit the proliferation of, for example, T-reg, cancer cells, and / or MDSC, or promote the proliferation of T effector cells, at titers similar to those exhibited by antigen-binding fragments of such antibodies. For example, even if the Fc region of the antagonist TNFR2 antibody described herein is removed, the ability of a molecule to attenuate the proliferation of T-reg cells, MDSC, and / or cancer cells, or reduce the total amount of T-reg cells, MDSC, and / or cancer cells, in a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infection as described herein) may not change. The antagonist TNFR2 antibodies and antigen-binding fragments thereof described herein can function via a pathway different from antibody-dependent cell-mediated cytotoxicity (ADCC), which requires the Fc region to recruit effector proteins to induce cell death. In addition, the antagonist TNFR2 antibody or antigen-binding fragment thereof can be in various forms, such as single-chain polypeptides (e.g., single-chain polypeptides containing one or more CDRs covalently linked to each other by, for example, amide bonds, thioether bonds, carbon-carbon bonds, or disulfide bridges), monoclonal antibodies or antigen-binding fragments thereof, polyclonal antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, primatized antibodies or antigen-binding fragments thereof, bispecific antibodies or antigen-binding fragments thereof, multispecific antibodies or antigen-binding fragments thereof, dual variable immunoglobulin domains, monovalent antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, single-chain Fv molecules (scFv), diabodies, triabodies, nanobodies, antibody-like protein scaffolds, domain antibodies, Fv fragments, Fab fragments, F(ab’)2 molecules, and tandem scFv (taFv), etc., and can exhibit therapeutic activity.

[0220] Specific binding properties of antagonist TNFR2 polypeptides The specific binding to human TNFR2 of the polypeptides described herein, such as single-chain polypeptides, antibodies, or antibody fragments, can be measured using any of a variety of established methods. Affinity can be quantitatively represented using various measurements, including the concentration of antibody required to achieve 50% inhibition of the TNFα-TNFR2 interaction in vitro (IC 50 ), and the equilibrium dissociation constant (K D ) of the antibody-TNFR2 complex dissociation. The equilibrium constant, K D , describing the interaction of TNFR2 with an antibody described herein, is the chemical equilibrium constant for the reaction in which the TNFR2-antibody complex dissociates into TNFR2 and antibody molecules separated in a solvent where they do not interact with each other.

[0221] Examples of the polypeptides described herein (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) include those with a K DExamples include polypeptides that specifically bind to TNFR2 at a value. In some embodiments, the polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) have a K of less than 1 nM (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620 pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM). D Specifically bind to TNFR2 at a value.

[0222] The polypeptides described herein can also be characterized using various in vitro binding assays. The K of the anti-TNFR2 polypeptide D Or IC 50Examples of experiments that can be used to measure include, for example, among others, surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, and ELISA-based assays. ELISA is a representative example of a particularly useful method for analyzing antibody activity because such assays generally require extremely low concentrations of antibodies. A common signal analyzed using a standard ELISA assay is fluorescence, which is generally the result of the activity of peroxidase conjugated to a secondary antibody that specifically binds to a primary antibody (e.g., the TNFR2 antibody described herein). The polypeptides described herein (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) can bind to TNFR2 and epitopes within it, such as epitopes containing one or more contiguous or non-contiguous residues within CRD3 and / or CRD4 of human TNFR2. The antagonistic polypeptides described herein can further bind to isolated peptides derived from TNFR2 in which various residues are pre-structured to mimic the structure of the above epitopes within the native protein. For example, the polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) can bind to a peptide containing any one of the amino acid sequences of SEQ ID NOs: 11, 19, 20, and 34-117, or a peptide having a maximum of 5 amino acid substitutions compared to any one of the amino acid sequences of SEQ ID NOs: 11, 19, 20, and 34-117 (e.g., a peptide having a maximum of 5 conservative amino acid substitutions compared to any one of the amino acid sequences of SEQ ID NOs: 11, 19, 20, and 34-117, etc.), and / or a peptide having an amino acid sequence that is at least 85% identical (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the amino acid sequences of SEQ ID NOs: 11, 19, 20, and 34-117. In a direct ELISA experiment, this binding can be quantified by analyzing the fluorescence generated during the incubation of an HRP substrate (e.g., 2,2'-azino-di-3-ethylbenzothiazoline sulfonate) with an antigen-antibody complex bound to a secondary antibody conjugated with HRP.

[0223] Kinetic properties of antagonist TNFR2 polypeptides In addition to the thermodynamic parameters of the TNFR2-polypeptide interaction, it is also possible to quantitatively characterize the kinetic binding and dissociation of the polypeptides described herein with TNFR2. This can be accomplished, for example, by monitoring the rate of formation of polypeptide-antigen (e.g., antibody-antigen) complexes according to established techniques. For example, surface plasmon resonance (SPR) can be used to measure the rate constants for the formation (k on ) and dissociation (K off ) of antibody-TNFR2 complexes. Since the equilibrium constant for this unimolecular dissociation can be expressed as the ratio of the K off value to the k on value, these data also allow for the calculation of the equilibrium constant (K D ) for antibody-TNFR2 complex dissociation. SPR is a particularly advantageous technique for measuring the kinetic and thermodynamic parameters of receptor-antibody interactions since its experiments do not require modification of one of the components by the attachment of chemical labels. Conversely, the receptor is typically immobilized on a solid metal surface, and the surface is treated with pulses of solutions of increasing antibody concentration. Since antibody-receptor binding induces a distortion in the angle of reflection of incident light at the metal surface, this change in refractive index over time upon introduction of the antibody into the system may be fit to an established regression model to calculate the association and dissociation rate constants for the antibody-receptor interaction.

[0224] The polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) may exhibit high k on values and low K off values when interacting with TNFR2, which is consistent with high-affinity receptor binding. For example, the polypeptides described herein may have a rate of association greater than 10 4 M -1 s -1 sup (e.g., 1.0×10 4 M -1 s -1, 1.5×10 4 M -1 s -1 , 2.0×10 4 M -1 s -1 , 2.5×10 4 M -1 s -1 , 3.0×10 4 M -1 s -1 , 3.5×10 4 M -1 s -1 , 4.0×10 4 M -1 s -1 , 4.5×10 4 M -1 s -1 , 5.0×10 4 M -1 s -1 , 5.5×10 4 M -1 s -1 , 6.0×10 4 M -1 s -1 , 6.5×10 4 M -1 s -1 , 7.0×10 4 M -1 s -1 , 7.5×10 4 M -1 s -1 , 8.0×10 4 M -1 s -1 , 8.5×10 4 M -1 s -1 , 9.0×10 4 M -1 s -1 , 9.5×10 4 M -1 s -1 , 1.0×10 5 M -1 s -1 , 1.5×10 5 M -1 s -1 , 2.0×10 5 M -1 s -1 , 2.5×10 5 M -1 s-1 , 3.0×10 5 M -1 s -1 , 3.5×10 5 M -1 s -1 , 4.0×10 5 M -1 s -1 , 4.5×10 5 M -1 s -1 , 5.0×10 5 M -1 s -1 , 5.5×10 5 M -1 s -1 , 6.0×10 5 M -1 s -1 , 6.5×10 5 M -1 s -1 , 7.0×10 5 M -1 s -1 , 7.5×10 5 M -1 s -1 , 8.0×10 5 M -1 s -1 , 8.5×10 5 M -1 s -1 , 9.0×10 5 M -1 s -1 , 9.5×10 5 M -1 s -1 , or 1.0×10 6 M -1 s -1 )'s k on value can be shown. The polypeptides described in this specification (e.g., single-chain polypeptides, antibodies, antigen-binding fragments, and their constructs) can show low K off values when binding to TNFR2 because these polypeptides can interact with different TNFR2 epitopes with high affinity. The residues within these epitopes can form strong intermolecular contacts with TNFR2, which may slow down the dissociation of the antibody-TNFR2 complex. This high receptor affinity results in low K offIt can be manifested by a value. For example, when the polypeptide described in this specification forms a complex with TNFR2, it is less than 10 -3 s -1 (for example, 1.0×10 -3 s -1 , 9.5×10 -4 s -1 , 9.0×10 -4 s -1 , 8.5×10 -4 s -1 , 8.0×10 -4 s -1 , 7.5×10 -4 s -1 , 7.0×10 -4 s -1 , 6.5×10 -4 s -1 , 6.0×10 -4 s -1 , 5.5×10 -4 s -1 , 5.0×10 -4 s -1 , 4.5×10 -4 s -1 , 4.0×10 -4 s -1 , 3.5×10 -4 s -1 , 3.0×10 -4 s -1 , 2.5×10 -4 s -1 , 2.0×10 -4 s -1 , 1.5×10 -4 s -1 , 1.0×10 -4 s -1 , 9.5×10 -5 s -1 , 9.0×10 -5 s -1 , 8.5×10 -5 s -1 , 8.0×10 -5 s -1 , 7.5×10 -5 s -1 , 7.0×10 -5 s -1 , 6.5×10 -5 s -1 , 6.0×10 -5 s -1, 5.5×10 -5 s -1 , 5.0×10 -5 s -1 , 4.5×10 -5 s -1 , 4.0×10 -5 s -1 , 3.5×10 -5 s -1 , 3.0×10 -5 s -1 , 2.5×10 -5 s -1 , 2.0×10 -5 s -1 , 1.5×10 -5 s -1 , or 1.0×10 -5 s -1 ) of K off values can be shown.

[0225] Epitopes within TNFR2 to which an antagonistic TNFR2 polypeptide binds One of the problems in the development of anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) that can antagonize TNFR2 is the elucidation of epitopes within TNFR2 that are involved in the formation of antagonistic complexes rather than epitopes that promote signal transduction. The present disclosure is in part that upon binding, promotes receptor antagonist action and has the following advantageous biological activities, (a) Inhibition of the proliferation of T-reg cells and / or direct killing of T-reg cells, for example, by binding to TNFR2 on the surface of T-reg cells and inactivating TNFR2, (b) Inhibition of the proliferation of MDSCs and / or direct killing of MDSCs, for example, by binding to TNFR2 on the surface of MDSCs and inactivating TNFR2, (c) Promotion of the proliferation of T effector cells, such as CD8+ T cells, etc., and / or, (d) Inhibition of the proliferation of TNFR2-expressing cancer cells (such as Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T-cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, skin cancer cells, lung cancer cells, liver cancer cells, endometrial cancer cells, hematopoietic cancer cells or lymphoma cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.) and / or direct killing of TNFR2-expressing cancer cells, is based on the discovery of epitopes within TNFR2 that have the ability to promote one or more or all of the following.

[0226] Antagonistic TNFR2 polypeptides, such as the dominant antagonistic TNFR2 polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof), etc., are the following epitopes on human TNFR2, (a) Amino acids 142 - 146 (KCRPG) of SEQ ID NO: 7, (b) Amino acids 142 - 149 (KCRPGFGV) of SEQ ID NO: 7, (c) Amino acids 137 - 144 (CAPLRKCR) of SEQ ID NO: 7, (d) Amino acids 150 - 190 (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI) of SEQ ID NO: 7, (e) Amino acids 161 - 169 (CKPCAPGTF) of SEQ ID NO: 7, (f) Amino acids 75 - 128 (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL) of SEQ ID NO: 7 (optionally, the epitope is within amino acids 80 - 86 (DSTYTQL), 91 - 98 (PECLSCGS), or 116 - 123 (RICTCRPG) of SEQ ID NO: 7), (g) Amino acids 174 - 184 (SSTDICRPHQI) of SEQ ID NO: 7, (h) Amino acids 126 - 140 (CALSKQEGCRLCAPL) of SEQ ID NO: 7, (i) Amino acids 156 - 165 (TSDVVCKPCA) of SEQ ID NO: 7, (j) An equivalent epitope within TNFR2 of a non-human mammal, such as the non-human mammals described herein, an epitope showing at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to any of the above epitopes, and / or an epitope containing one or more conservative amino acid substitutions compared to these epitopes, can specifically bind to one or more of them.

[0227] In some embodiments, an antagonistic TNFR2 polypeptide, such as the dominant antagonistic TNFR2 polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof), does not bind to one or more or all of residues 142-146 (KCRPG, SEQ ID NO: 19) of SEQ ID NO: 7 within human TNFR2. In addition, the antagonistic TNFR2 polypeptides described herein do not demonstrably specifically bind to an epitope containing residues 56-60 (KCSPG, SEQ ID NO: 12) of SEQ ID NO: 7 within human TNFR2. A polypeptide showing the ability to bind to one or more of the above epitopes within human TNFR2 and to an epitope containing residues 56-60 of SEQ ID NO: 7 within human TNFR2 lacks inhibitory (antagonistic) activity. Therefore, the ability of a TNFR2 polypeptide to identify these epitopes and specifically interact with one or more of the above epitopes and not specifically bind by specific binding to an epitope composed of residues 56-60 of SEQ ID NO: 7 within human TNFR2 characterizes the polypeptides described herein that neutralize TNFR2 signaling.

[0228] One exemplary approach that can be used to predict the inhibitory activity of the TNFR2 polypeptides described herein is to measure the affinity of an antibody or antibody fragment for a peptide containing the KCRPG motif (SEQ ID NO: 19), such as a linear or cyclic peptide containing this motif. The peptide may be pre-structured based on one or more structural constraints (e.g., main chain or side chain-side chain cyclization) so as to mimic the three-dimensional arrangement of the KCRPG motif (SEQ ID NO: 19). For example, the antagonistic TNFR2 polypeptides described herein can specifically bind to such peptides with a higher affinity compared to the affinity of the antagonistic TNFR2 polypeptide for the peptide fragment defined by residues 48-67 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18) of SEQ ID NO: 7 within human TNFR2. For example, the antagonistic TNFR2 polypeptides described herein can bind to peptides containing the KCRPG motif (SEQ ID NO: 19) with an affinity that is 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, or more than 1000-fold higher compared to the affinity of the antagonistic polypeptide for a peptide having the amino acid sequence of SEQ ID NO: 18.

[0229] Antagonistic TNFR2 polypeptide that binds to TNFR2 derived from non-human animals In addition to binding to the epitopes detailed above within human TNFR2, the antagonistic TNFR2 polypeptides described herein, such as, for example, dominant antagonistic TNFR2 polypeptides, also include antagonistic TNFR2 polypeptides that specifically bind to epitopes containing one or more equivalent motifs within TNFR2 from non-human animals. Regarding the positions of epitopes equivalent to those within human TNFR2 that give rise to an antagonistic phenotype upon binding, they are described, for example, in WO2016 / 187068 and WO2017 / 197331 (the entire disclosures of which are incorporated herein by reference). Exemplary non-human animal TNFR2 proteins to which the antagonistic polypeptides of the present disclosure can bind include, but are not limited to, TNFR2 proteins derived from cows, bison, and other agricultural animals described herein.

[0230] Antagonistic TNFR2 antibody TNFRAB1 Exemplary antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may include one or more or all of the CDRs of TNFRAB1, which is a mouse antibody that neutralizes the TNFRα-TNFR2 interaction. For example, antibody humanization methods described herein or well-known in the art can be used, for example, using CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 of TNFRAB1, and variants of these CDRs (e.g., variants showing conservative amino acid substitutions compared to these CDR sequences), to produce an antagonistic TNFR2 antibody or an antigen-binding fragment thereof.

[0231] The antagonist TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the present disclosure may exhibit binding properties that are identical to or similar to those of TNFRAB1. These properties are as follows. In the presence of TNFR2, TNFRAB1 exhibits a high k 6 M -1 s -1 value of 4.98×10 on and a low K -4 s -1 value of 2.21×10 off and a K D of approximately 44.4 pM. The KCRPGFGV motif (SEQ ID NO: 20), and particularly the KCRPG sequence (SEQ ID NO: 19), have been identified as particularly important components of the functional epitope that establishes intermolecular contact with TNFRAB1 when confirmed by epitope mapping analysis. The interaction of these residues with the anti-TNFR2 antibodies of the present disclosure selectively promotes antagonist action. Importantly, the TNFR2 epitope containing amino acid residues 56-60 (KCSPG, SEQ ID NO: 12) of SEQ ID NO: 7 within human TNFR2 has clearly been found to result in the loss or significant reduction of antagonist action when specific binding to both of these epitopes occurs, and thus is not part of the structural epitope to which TNFRAB1 or the antagonist TNFR2 antibodies or antibody fragments of the present disclosure specifically bind.

[0232] In addition to binding to the epitope contained within the KCRPGFGV array (array number: 20), TNFRAB1 also binds to a downstream epitope contained within the sequence defined by positions 161 - 169 of SEQ ID NO: 7 in human TNFR2 (CKPCAPGTF, SEQ ID NO: 21). The TNFR2 antibodies and antibody fragments of the present disclosure also bind to this epitope, or a larger region within TNFR2 containing this epitope (e.g., a sequence containing at least 5 contiguous or non - contiguous residues derived from positions 150 - 190 of SEQ ID NO: 7 in human TNFR2 (ARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI, SEQ ID NO: 22)). TNFRAB1 contains two light chains in addition to two heavy chains. The heavy chain of TNFRAB1 contains the following amino acid sequence (CDRs are shown in bold). TIFF0007713880000015.tif14168

[0233] The sequence of the TNFRAB1 light chain is as follows (CDRs are shown in bold). TIFF0007713880000016.tif20169

[0234] The heavy - chain CDRs and light - chain CDRs of TNFRAB2 are shown below. TNFRAB1 CDR - H1: GFTFSSY (SEQ ID NO: 23) TNFRAB1 CDR - H2: SSGGSY (SEQ ID NO: 24) TNFRAB1 CDR - H3: QRVDGYSSYWYFDV (SEQ ID NO: 25) TNFRAB1 CDR - L1: SASSSVYYMY (SEQ ID NO: 26) TNFRAB1 CDR - L2: STSNLAS (SEQ ID NO: 26) TNFRAB1 CDR - L3: QQRRNYPYT (SEQ ID NO: 28)

[0235] The antagonist TNFR2 antibody TNFRAB2 The antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may include one or more or all of the CDRs of TNFRAB2, which is another antibody that neutralizes the TNFRα-TNFR2 interaction. For example, antibody humanization methods described herein or well-known in the art may be used, such as CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 of TNFRAB2, and variants of these CDRs (e.g., variants showing conservative amino acid substitutions compared to these CDR sequences), to produce an antagonist TNFR2 antibody or an antigen-binding fragment thereof.

[0236] For example, the antagonist TNFR2 polypeptides (e.g., single-chain polypeptide...

Claims

1. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to human tumor necrosis factor receptor 2 (TNFR2) and inhibits the human TNFR2, wherein at least 50% of the antibody or the antigen-binding fragment thereof in the pharmaceutical composition is present as a single disulfide bond IgG2-A isoform, each of the antibody or the antigen-binding fragment thereof, (a) specifically binds to TNFR2 at an epitope within cysteine-rich domain (CRD) 3 (CRD3) and / or CRD4 and does not specifically bind to TNFR2 at an epitope defined by one or more amino acids within CRD1, wherein each of the antibody or the antigen-binding fragment thereof, (i) comprises a human IgG2 hinge region lacking the cysteine residues at positions 232 and / or 233 of the amino acid sequence of the human IgG2 hinge region; and (ii) comprises antigen-binding sites separated from each other by a distance of at least about 133 Å; and, (b) (i) a heavy chain having an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 302 to 306, the heavy chain comprising the following complementarity-determining regions (CDRs): CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257), CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258), and CDR-H3 having the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259), wherein X is leucine or isoleucine, the heavy chain; and (ii) a light chain having an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 297 to 301, the light chain comprising the following CDRs: CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260), CDR-L2 having the amino acid sequence YTS or TYS, and CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261), wherein X is leucine or isoleucine, the light chain comprising, the pharmaceutical composition.

2. (a) whether each heavy chain of the antibody or the antigen-binding fragment thereof has an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 302 to 306, and / or (b) each light chain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 297 to 301 The pharmaceutical composition according to claim 1.

3. (a) each heavy chain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 303, and each light chain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 297 to 301; or (b) each heavy chain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 304, and each light chain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 297 to 301; or (c) each heavy chain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 305, and each light chain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 297 to 301; or (d) each heavy chain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 306, and each light chain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 297 to 301; or (e) each IgG2 hinge region of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 291; and / or (f) (i) each heavy chain variable domain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 2, and / or (ii) each light chain variable domain of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 4 The pharmaceutical composition according to claim 1.

4. Each heavy chain variable domain of the antibody or its antigen-binding fragment has the amino acid sequence of SEQ ID NO: 2, and each light chain variable domain of the antibody or its antigen-binding fragment has the amino acid sequence of SEQ ID NO:

4. The pharmaceutical composition according to claim 3.

5. The human IgG2 hinge region is (a) Amino acids other than cysteine at positions 232 and 233 of the amino acid sequence of the IgG2 hinge region; (b) Serine residues at positions 232 and 233 of the amino acid sequence of the IgG2 hinge region; and / or (c) An amino acid deletion in one or both of cysteine residues 232 and 233 The pharmaceutical composition according to any one of claims 1 to 4.

6. (a) Are the antigen-binding sites separated from each other by a distance of about 133 Å to about 150 Å; and / or (b) Each of the antibody or its antigen-binding fragment has the amino acid sequence: (i) LLIR (SEQ ID NO: 262) bound to the N-terminus of the CDR-L2, or (ii) TLE bound to the C-terminus of the CDR-L2 And includes a framework region having The pharmaceutical composition according to any one of claims 1 to 5.

7. (a) Does the CDR-H1 have the amino acid sequence GYTFTDYL (SEQ ID NO: 274) or GYTFTDYI (SEQ ID NO: 275); and / or (b) Does the CDR-L3 have the amino acid sequence CLQYVNLLT (SEQ ID NO: 272) or CLQYVNLIT (SEQ ID NO: 273), The pharmaceutical composition according to any one of claims 1 to 6.

8. (a) Each of the antibody or its antigen-binding fragment specifically binds to a peptide having any one of the amino acid sequences of SEQ ID NOs: 11, 19, 20, and 34 to 117 with a K of less than about 100 nM, and does not bind to a peptide containing amino acids 56 to 60 (KCSPG) of SEQ ID NO: 7; or D ​ (b) Each of the antibody or its antigen-binding fragment (i) Amino acids 142-146 (KCRPG) of SEQ ID NO: 7, (ii) Amino acids 142-149 (KCRPGFGV) of SEQ ID NO: 7, (iii) Amino acids 137-144 (CAPLRKCR) of SEQ ID NO: 7, (iv) Amino acids 150-190 (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI) of SEQ ID NO: 7, (v) Amino acids 161-169 (CKPCAPGTF) of SEQ ID NO: 7, (vi) Amino acids 75-128 (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL) of SEQ ID NO: 7, (vii) Amino acids 174-184 (SSTDICRPHQI) of SEQ ID NO: 7, (viii) Amino acids 126 to 140 (CALSKQEGCRLCAPL) of SEQ ID NO: 7, and / or (ix) Amino acids 156 to 165 (TSDVVCKPCA) of SEQ ID NO: 7, which specifically binds to the TNFR2 at an internal epitope of The pharmaceutical composition according to any one of claims 1 to 7.

9. The pharmaceutical composition according to claim 8, wherein the epitope within amino acids 75 to 128 of SEQ ID NO: 7 is within amino acids 80 to 86 (DSTYTQL), 91 to 98 (PECLSCGS), or 116 to 123 (RICTCRPG) of SEQ ID NO:

7.

10. Each of the antibody or its antigen-binding fragment (a) inhibits TNFR2 signaling; (b) reduces the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3 in cells expressing TNFR2; (c) reduces NFκB activation in cells expressing TNFR2; (d) K of about 10 nM or less D specifically binds to the TNFR2 at; (e) at least about 10 4 M -1 s -1 of k on that specifically binds to said TNFR2 to form an antibody-antigen complex; (f) specifically binds to the TNFR2 to form an antibody-antigen complex, and the complex dissociates at about 10 -3 s -1 of the following k off or dissociates; (g) reduces or inhibits the proliferation of the population of T-reg cells and / or induces the proliferation of the population of CD8+ effector T cells; (h) reduces or inhibits the proliferation of the population of cancer cells expressing TNFR2; or (i) reduces or inhibits the proliferation of the population of myeloid-derived suppressor cells, The pharmaceutical composition according to any one of claims 1 to 9.

11. (a) whether the cell is a regulatory T cell (T-reg cell) or a cancer cell; (b) each of the antibody or its antigen-binding fragment (i) K of about 1 nM or less D , (ii) K of about 621 pM D or (iii) K of approximately 44 pM D specifically binds to the TNFR2 at (c) each of the antibody or its antigen-binding fragment (i) Approximately 4.9×10 6 M -1 s -1 of k on or (ii) approximately 3.6×10 5 M -1 s -1 of k on specifically binds to the TNFR2 to form an antibody-antigen complex at (d) each of the antibody or its antigen-binding fragment (i) about 10 -3 s -1 or less of the following k off , (ii) about 3.0×10 -5 s -1 of the following k off or (iii) Approximately 2.2×10 -4 s -1 of k off dissociates from the TNFR2 at (e) whether the T-reg cells express CD25 Hi ; or (f) each of the antibody or its antigen-binding fragment reduces or inhibits the proliferation of the population of T-reg cells in the presence of TNFα, and / or (g) the cancer cells are selected from the group consisting of Hodgkin lymphoma cells, cutaneous non-Hodgkin lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, and renal cell carcinoma cells, The pharmaceutical composition according to claim 10.

12. (a) Each of the antibody or its antigen-binding fragment is selected from the group consisting of a monoclonal antibody or its antigen-binding fragment, a polyclonal antibody or its antigen-binding fragment, a human antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a primatized antibody or its antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, a multispecific antibody or its antigen-binding fragment, a dual variable immunoglobulin domain, a monovalent antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a single-chain Fv molecule (scFv), a diabody, a triabody, an antibody-like protein scaffold, a Fab fragment, F(ab') 2 molecule, and tandem scFv (taFv); or (b) each of the antibody or its antigen-binding fragment is a human IgG2 isotype antibody or its antigen-binding fragment, and / or (c) each of the antibodies is conjugated to a therapeutic agent, The pharmaceutical composition according to any one of claims 1 to 11.

13. (a) each of the antibody or its antigen-binding fragment is a human antibody, humanized antibody, or chimeric antibody, or its antigen-binding fragment; and / or (b) the therapeutic agent is a cytotoxic agent, The pharmaceutical composition according to claim 12.

14. The pharmaceutical composition according to any one of claims 1 to 13, further comprising a pharmaceutically acceptable carrier or excipient.

15. (a) is the single disulfide bond isoform IgG2-A; and / or (b) the pharmaceutical composition further comprises another therapeutic agent, The pharmaceutical composition according to claim 14.

16. (a) each of the antibody or its antigen-binding fragment is present in the pharmaceutical composition in an amount of about 0.001 mg / ml to about 100 mg / ml; and / or (b) the another therapeutic agent is an immunotherapeutic agent, The pharmaceutical composition according to claim 14 or 15.

17. (a) The immunotherapeutic agent is selected from the group consisting of an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-L2 agent, a TNF-α crosslinking agent, a TRAIL crosslinking agent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, an anti-4-1BB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAILR1 agent, an anti-TRAILR2 agent, an anti-TWEAK agent, an anti-TWEAKR agent, an anti-cell surface lymphocyte protein agent, an anti-BRAF agent, an anti-MEK agent, an anti-CD33 agent, an anti-CD20 agent, an anti-HLA-DR agent, an anti-HLA class I agent, an anti-CD52 agent, an anti-A33 agent, an anti-GD3 agent, an anti-PSMA agent, an anti-Ceacan1 agent, an anti-Galedin9 agent, an anti-HVEM agent, an anti-VISTA agent, an anti-B7 H4 agent, an anti-HHLA2 agent, an anti-CD155 agent, an anti-CD80 agent, an anti-BTLA agent, an anti-CD160 agent, an anti-CD28 agent, an anti-CD226 agent, an anti-CEACAM1 agent, an anti-TIM3 agent, an anti-TIGIT agent, an anti-CD96 agent, an anti-CD70 agent, an anti-CD27 agent, an anti-LIGHT agent, an anti-CD137 agent, an anti-DR4 agent, an anti-CR5 agent, an anti-TNFRS agent, an anti-TNFR1 agent, an anti-FAS agent, an anti-CD95 agent, an anti-TRAIL agent, an anti-DR6 agent, an anti-EDAR agent, an anti-NGFR agent, an anti-OPG agent, an anti-RANKL agent, an anti-LTβ receptor agent, an anti-BCMA agent, an anti-TACI agent, an anti-BAFFR agent, an anti-EDAR2 agent, an anti-TROY agent, and an anti-RELT agent; and / or (b) The immunotherapeutic agent is an anti-CTLA-4 antibody or its antigen-binding fragment, an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, an anti-PD-L2 antibody or its antigen-binding fragment, a TNF-α bridging antibody or its antigen-binding fragment, a TRAIL bridging antibody or its antigen-binding fragment, an anti-CD27 antibody or its antigen-binding fragment, an anti-CD30 antibody or its antigen-binding fragment, an anti-CD40 antibody or its antigen-binding fragment, an anti-4-1BB antibody or its antigen-binding fragment, an anti-GITR antibody or its antigen-binding fragment, an anti-OX40 antibody or its antigen-binding fragment, an anti-TRAILR1 antibody or its antigen-binding fragment, an anti-TRAILR2 antibody or its antigen-binding fragment, an anti-TWEAK antibody or its antigen-binding fragment, an anti-TWEAKR antibody or its antigen-binding fragment, an anti-cell surface lymphocyte protein antibody or its antigen-binding fragment, an anti-BRAF antibody or its antigen-binding fragment, an anti-MEK antibody or its antigen-binding fragment, an anti-CD33 antibody or its antigen-binding fragment, an anti-CD20 antibody or its antigen-binding fragment, an anti-HLA-DR antibody or its antigen-binding fragment, an anti-HLA class I antibody or its antigen-binding fragment, an anti-CD52 antibody or its antigen-binding fragment, an anti-A33 antibody or its antigen-binding fragment, an anti-GD3 antibody or its antigen-binding fragment, an anti-PSMA antibody or its antigen-binding fragment, an anti-Ceacan1 antibody or its antigen-binding fragment, an anti-Galedin9 antibody or its antigen-binding fragment, an anti-HVEM antibody or its antigen-binding fragment, an anti-VISTA antibody or its antigen-binding fragment, an anti-B7An H4 antibody or an antigen-binding fragment thereof, an anti-HHLA2 antibody or an antigen-binding fragment thereof, an anti-CD155 antibody or an antigen-binding fragment thereof, an anti-CD80 antibody or an antigen-binding fragment thereof, an anti-BTLA antibody or an antigen-binding fragment thereof, an anti-CD160 antibody or an antigen-binding fragment thereof, an anti-CD28 antibody or an antigen-binding fragment thereof, an anti-CD226 antibody or an antigen-binding fragment thereof, an anti-CEACAM1 antibody or an antigen-binding fragment thereof, an anti-TIM3 antibody or an antigen-binding fragment thereof, an anti-TIGIT antibody or an antigen-binding fragment thereof, an anti-CD96 antibody or an antigen-binding fragment thereof, an anti-CD70 antibody or an antigen-binding fragment thereof, an anti-CD27 antibody or an antigen-binding fragment thereof, an anti-LIGHT antibody or an antigen-binding fragment thereof, an anti-CD137 antibody or an antigen-binding fragment thereof, an anti-DR4 antibody or an antigen-binding fragment thereof, an anti-CR5 antibody or an antigen-binding fragment thereof, an anti-TNFRS antibody or an antigen-binding fragment thereof, an anti-TNFR1 antibody or an antigen-binding fragment thereof, an anti-FAS antibody or an antigen-binding fragment thereof, an anti-CD95 antibody or an antigen-binding fragment thereof, an anti-TRAIL antibody or an antigen-binding fragment thereof, an anti-DR6 antibody or an antigen-binding fragment thereof, an anti-EDAR antibody or an antigen-binding fragment thereof, an anti-NGFR antibody or an antigen-binding fragment thereof, an anti-OPG antibody or an antigen-binding fragment thereof, an anti-RANKL antibody or an antigen-binding fragment thereof, an anti-LTβ receptor antibody or an antigen-binding fragment thereof, an anti-BCMA antibody or an antigen-binding fragment thereof, an anti-TACI antibody or an antigen-binding fragment thereof, an anti-BAFFR antibody or an antigen-binding fragment thereof, an anti-EDAR2 antibody or an antigen-binding fragment thereof, an anti-TROY antibody or an antigen-binding fragment thereof, and an anti-RELT antibody or an antigen-binding fragment thereof, selected from the group consisting of The pharmaceutical composition according to claim 16.

18. The pharmaceutical composition according to claim 16, wherein the immunotherapeutic agent is ipilimumab, tremelimumab, nivolumab, pembrolizumab, avelumab, durvalumab, or atezolizumab.

19. The pharmaceutical composition according to any one of claims 14 to 18 for use in the treatment of a disease or disorder in a human.

20. The disease or disorder is (a) an immune response involving T-reg cells; (b) a cell proliferation disorder; or (c) an infectious disease The pharmaceutical composition according to claim 19, comprising.

21. The cell proliferation disorder is (a) a cancer selected from the group consisting of leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, heart cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and throat cancer; (b) cancer selected from the group consisting of Hodgkin lymphoma, cutaneous non-Hodgkin lymphoma, T-cell lymphoma, ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, skin cancer, lung cancer, endometrial cancer, hematopoietic or lymphatic cancer, central nervous system cancer, gastric cancer, esophageal cancer, and upper gastrointestinal cancer; or (c) acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, extrahepatic cancer, Ewing sarcoma family, osteosarcoma, and malignant fibrous histiocytoma, central nervous system fetal tumor, central nervous system embryonal tumor, craniopharyngioma, ependymoma, bronchial tumor, Burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative tumor, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, sensory neuroblastoma, extracranial embryonal tumor, extragonadal embryonal tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumor, pancreatic neuroendocrine tumor, Wilms tumor, and other childhood kidney tumors, Langerhans cell histiocytosis, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, nasal and paranasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low-grade ovarian cancer, pancreatic neuroendocrine tumor, papillomatosis, paraganglioma, paranasal and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, renal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, rhabdomyosarcoma, Sézary syndrome, small intestine cancer, soft tissue sarcoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia which is the pharmaceutical composition according to claim 19 or 20. Claim 22 wherein the infectious disease is (a) one or more factors selected from the group consisting of viruses, bacteria, fungi, and parasites; (b)Hepatitis C virus, Yellow fever virus, Kadam virus, Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, Calicivirus, Tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill virus, Negishi virus, Meaban virus, Saumarez Reef virus, Turure virus, Aroa virus, Dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israeli fowl meningitis virus, Untanaya virus, Tembusu virus, Zika virus, Banj virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus, Yellow fever virus, Eptesicus virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, Cell fusion factor virus, Ippy virus, Lassa virus, Lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus, Guanarito virus, Funin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, Chapare virus, Lujo virus, Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus,Marburg virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O'nyong'nyong virus, and Chikungunya virus, variola virus, simian pox virus, vaccinia virus, herpes simplex virus, human herpes virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus, Kaposi's sarcoma-associated herpes virus (KSHV), influenza virus, severe acute respiratory syndrome (SARS) virus, rabies virus, vesicular stomatitis virus (VSV), human respiratory syncytial virus (RSV), Newcastle disease virus, Hendra virus, Nipah virus, measles virus, rinderpest virus, canine distemper virus, Sendai virus, human parainfluenza virus, rhinovirus, mumps virus, poliovirus, human enterovirus (A, B, C, and D), hepatitis A virus, coxsackievirus, hepatitis B virus, human papillomavirus, adeno-associated virus, astrovirus, JC virus, BK virus, SV40 virus, Norwalk virus, rotavirus, human immunodeficiency virus (HIV), human T-lymphotropic virus type I and type II; a virus selected from the group consisting of; (c) bacteria belonging to a genus selected from the group consisting of Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriaceae, Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordatella, Legionella, Pasturella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus; (d) a fungus selected from the group consisting of Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, and Absidia; or (e) Parasites selected from the group consisting of Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, Histomonas meleagridis, Richuris trichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, and Dracunculus medinensis, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyes, Paragonimus westermani, Taenia solium, Taenia saginata, Hymenolepis nana, and Echinococcus granulosus; The pharmaceutical composition according to any one of claims 19 to 21, caused by

23. The pharmaceutical composition according to any one of claims 19 to 22, wherein the pharmaceutical composition is for administration to a human together with an immunotherapeutic agent.

24. (a) Is the immunotherapeutic agent selected from the group consisting of an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-L2 agent, a TNF-α crosslinking agent, a TRAIL crosslinking agent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, an anti-4-1BB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAILR1 agent, an anti-TRAILR2 agent, an anti-TWEAK agent, an anti-TWEAKR agent, an anti-cell surface lymphocyte protein agent, an anti-BRAF agent, an anti-MEK agent, an anti-CD33 agent, an anti-CD20 agent, an anti-HLA-DR agent, an anti-HLA class I agent, an anti-CD52 agent, an anti-A33 agent, an anti-GD3 agent, an anti-PSMA agent, an anti-Ceacan1 agent, an anti-Galedin9 agent, an anti-HVEM agent, an anti-VISTA agent, an anti-B7 H4 agent, an anti-HHLA2 agent, an anti-CD155 agent, an anti-CD80 agent, an anti-BTLA agent, an anti-CD160 agent, an anti-CD28 agent, an anti-CD226 agent, an anti-CEACAM1 agent, an anti-TIM3 agent, an anti-TIGIT agent, an anti-CD96 agent, an anti-CD70 agent, an anti-CD27 agent, an anti-LIGHT agent, an anti-CD137 agent, an anti-DR4 agent, an anti-CR5 agent, an anti-TNFRS agent, an anti-TNFR1 agent, an anti-FAS agent, an anti-CD95 agent, an anti-TRAIL agent, an anti-DR6 agent, an anti-EDAR agent, an anti-NGFR agent, an anti-OPG agent, an anti-RANKL agent, an anti-LTβ receptor agent, an anti-BCMA agent, an anti-TACI agent, an anti-BAFFR agent, an anti-EDAR2 agent, an anti-TROY agent, and an anti-RELT agent; and / or (b) The immunotherapeutic agent is an anti-CTLA-4 antibody or its antigen-binding fragment, an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, an anti-PD-L2 antibody or its antigen-binding fragment, a TNF-α bridging antibody or its antigen-binding fragment, a TRAIL bridging antibody or its antigen-binding fragment, an anti-CD27 antibody or its antigen-binding fragment, an anti-CD30 antibody or its antigen-binding fragment, an anti-CD40 antibody or its antigen-binding fragment, an anti-4-1BB antibody or its antigen-binding fragment, an anti-GITR antibody or its antigen-binding fragment, an anti-OX40 antibody or its antigen-binding fragment, an anti-TRAILR1 antibody or its antigen-binding fragment, an anti-TRAILR2 antibody or its antigen-binding fragment, an anti-TWEAK antibody or its antigen-binding fragment, an anti-TWEAKR antibody or its antigen-binding fragment, an anti-cell surface lymphocyte protein antibody or its antigen-binding fragment, an anti-BRAF antibody or its antigen-binding fragment, an anti-MEK antibody or its antigen-binding fragment, an anti-CD33 antibody or its antigen-binding fragment, an anti-CD20 antibody or its antigen-binding fragment, an anti-HLA-DR antibody or its antigen-binding fragment, an anti-HLA class I antibody or its antigen-binding fragment, an anti-CD52 antibody or its antigen-binding fragment, an anti-A33 antibody or its antigen-binding fragment, an anti-GD3 antibody or its antigen-binding fragment, an anti-PSMA antibody or its antigen-binding fragment, an anti-Ceacan1 antibody or its antigen-binding fragment, an anti-Galedin9 antibody or its antigen-binding fragment, an anti-HVEM antibody or its antigen-binding fragment, an anti-VISTA antibody or its antigen-binding fragment, an anti-B7An H4 antibody or an antigen-binding fragment thereof, an anti-HHLA2 antibody or an antigen-binding fragment thereof, an anti-CD155 antibody or an antigen-binding fragment thereof, an anti-CD80 antibody or an antigen-binding fragment thereof, an anti-BTLA antibody or an antigen-binding fragment thereof, an anti-CD160 antibody or an antigen-binding fragment thereof, an anti-CD28 antibody or an antigen-binding fragment thereof, an anti-CD226 antibody or an antigen-binding fragment thereof, an anti-CEACAM1 antibody or an antigen-binding fragment thereof, an anti-TIM3 antibody or an antigen-binding fragment thereof, an anti-TIGIT antibody or an antigen-binding fragment thereof, an anti-CD96 antibody or an antigen-binding fragment thereof, an anti-CD70 antibody or an antigen-binding fragment thereof, an anti-CD27 antibody or an antigen-binding fragment thereof, an anti-LIGHT antibody or an antigen-binding fragment thereof, an anti-CD137 antibody or an antigen-binding fragment thereof, an anti-DR4 antibody or an antigen-binding fragment thereof, an anti-CR5 antibody or an antigen-binding fragment thereof, an anti-TNFRS antibody or an antigen-binding fragment thereof, an anti-TNFR1 antibody or an antigen-binding fragment thereof, an anti-FAS antibody or an antigen-binding fragment thereof, an anti-CD95 antibody or an antigen-binding fragment thereof, an anti-TRAIL antibody or an antigen-binding fragment thereof, an anti-DR6 antibody or an antigen-binding fragment thereof, an anti-EDAR antibody or an antigen-binding fragment thereof, an anti-NGFR antibody or an antigen-binding fragment thereof, an anti-OPG antibody or an antigen-binding fragment thereof, an anti-RANKL antibody or an antigen-binding fragment thereof, an anti-LTβ receptor antibody or an antigen-binding fragment thereof, an anti-BCMA antibody or an antigen-binding fragment thereof, an anti-TACI antibody or an antigen-binding fragment thereof, an anti-BAFFR antibody or an antigen-binding fragment thereof, an anti-EDAR2 antibody or an antigen-binding fragment thereof, an anti-TROY antibody or an antigen-binding fragment thereof, and an anti-RELT antibody or an antigen-binding fragment thereof, selected from the group consisting of The pharmaceutical composition according to claim 23.

25. The pharmaceutical composition according to claim 24, wherein the immunotherapeutic agent is ipilimumab, tremelimumab, nivolumab, pembrolizumab, avelumab, durvalumab, or atezolizumab.

26. The pharmaceutical composition according to any one of claims 19 to 25, wherein the antibody or antigen-binding fragment thereof that specifically binds to TNFR2 is for administration to a human in an amount of about 0.001 mg / kg to about 100 mg / kg.

27. A kit comprising the pharmaceutical composition according to any one of claims 14 to 18.

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