Antagonistic Anti-tumor necrosis factor receptor superfamily polypeptides

Abstract

To provide antagonistic TNFR2 polypeptides.SOLUTION: Provided is an antibody or antigen-binding fragment thereof that specifically binds human tumor necrosis factor receptor 2 (TNFR2) at an epitope within cysteine-rich domain (CRD) 3 (CRD3) and / or CRD4 and does not specifically bind TNFR2 at an epitope defined by one or more amino acids within CRD1, the antibody or antigen-binding fragment thereof: (a) comprising a human lgG2 hinge region that lacks a cysteine residue at positions 232 and / or 233 of the amino acid sequence of the lgG2 hinge region; and / or (b) comprising antigen-binding sites separated from one another by a distance of at least about 133 Å.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, and is incorporated herein by reference in its entirety. The ASCII copy (created on August 20, 2019) is titled 00786-0083WO2_Sequence_Listing_08.20.19_ST25 and is 193,232 bytes in size. Technical Field The present invention relates to "antagonistic anti-tumor necrosis factor receptor superfamily polypeptides." [Background technology]

[0002] The use of natural and genetically engineered T lymphocytes is a prominent paradigm for ameliorating various human pathologies. For example, while traditional therapeutic platforms for treating cancer include surgical removal of tumor masses, radiation therapy, and chemotherapy (Shewach, Chem. Rev., 109:2859-2861, 2009), the past decade has seen a resurgence in the application of adoptive immunotherapy to cancer treatment regimens. With the advent of chimeric antigen receptor (CAR-T) therapy, a new method for infusing autologous and allogeneic tumor-reactive T cells into patients has emerged (June, J. Clin. Invest., 117:1466-1476, 2007). CAR-T therapy harnesses the resources of the adaptive immune response to enhance cancer cytotoxicity and eradicate tumor material. A common feature of adoptive immunotherapy is the use of T cells that exhibit the ability to selectively enhance cytotoxicity against cells presenting distinct tumor antigens. Examples of this technique include the administration of tumor-infiltrating lymphocytes (Dudley et al., J. Immunother., 26:332-342, 2003) as well as the administration of autologous or allogeneic T cells genetically engineered to react with tumor-specific antigens (Yee et al., PNAS., 99:16168-16173, 2002).

[0003] Despite the promise of T lymphocyte-based cancer immunotherapy, development of this therapeutic platform has been hindered by the immune system's natural tendency to suppress immune attacks initiated against self-cells. Cancer cells express class I major histocompatibility complex (MHC) proteins, which distinguish these self cells from foreign cells. To prevent cellular silencing, regulatory T cells (T-reg cells) have evolved to suppress the activity of T cells reactive to "self" MHC antigens. T-reg cells represent a distinct class of T cells that can be distinguished based on their unique surface protein presentation. The best-understood T-reg cell populations include CD4+, CD25+, FoxP3+, and CD17+ T-reg cells. Although the exact mechanism by which these cells suppress autoreactive T cells is the subject of ongoing research, it has been shown that a specific class of T-reg cells can inhibit the production of the proliferation-inducing cytokine IL-2 in target T cells and, furthermore, 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. Immun., 30:531-564, 2012).

[0004] Although T-reg cells play a crucial role in maintaining peripheral immune tolerance, the same biochemical features that underlie their ability to regulate autoreactive T cells also allow them to fundamentally disrupt adoptive immunotherapy and innate immune responses by suppressing the activity of tumor-reactive T lymphocytes. The development of chemical modulators of T-reg cell activity has been the subject of considerable pharmacological research, as access to drugs capable of inhibiting T-reg-mediated T cell suppression could significantly improve the scope and efficacy of adoptive cancer immunotherapy, as well as enhance the immune system's ability to eradicate pathogenic microorganisms that cause infectious diseases.

[0005] There is a need for improved therapeutic agents for treating cell proliferative disorders, such as cancer, and a wide variety of infectious diseases. Summary of the Invention

[0006] Described herein are antagonistic tumor necrosis factor receptor superfamily polypeptides, including single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs. For example, antagonistic tumor necrosis factor receptor 2 (TNFR2)-binding polypeptides, including single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs, are featured. 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). Antagonistic TNFR2 polypeptides described herein include antagonistic TNFR2 polypeptides that bind to one or more epitopes in CRD3 of TNFR2 and / or one or more epitopes in CRD4 of TNFR2, such as antagonistic TNFR2 polypeptides that bind to TNFR2 only within one or more epitopes in CRD3 and / or one or more epitopes in CRD4, without binding to TNFR2 within CRD1 and / or CRD2.

[0007] The 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, when these molecules are in the form of the IgG2 isotype, exhibit clearly superior TNFR2 antagonist properties compared to other antibody isotypes. The 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"), the binding sites being spatially separated from each other by about 133 Å or more, because it has been discovered herein that such polypeptides unexpectedly exhibit superior TNFR2 antagonist activity compared to polypeptides that specifically bind to TNFR2 at one or more of the above-mentioned epitopes but contain TNFR2 binding sites (e.g., antigen-binding sites) that are separated from each other by less than about 133 Å, such as IgG1 antibodies and antigen-binding fragments thereof, which contain antigen-binding sites that are separated from each other by about 117 Å, and IgG3 antibodies and antigen-binding fragments thereof, which contain antigen-binding sites that are separated from each other by 125 Å.

[0008] Also featured are anti-TNFR2 polypeptides that take a single disulfide-bonded isoform and pharmaceutical compositions containing them. For example, pharmaceutical compositions of the present disclosure include those containing antagonistic TNFR2-binding polypeptides 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 are present in a single disulfide-bonded isoform. Antagonistic TNFR2-binding polypeptides that take the human IgG2-A isoform exhibit substantially superior TNFR2 antagonist activity compared to TNFR2-binding polypeptides that take other human IgG2 isoforms, such as IgG2-B, IgG2-A / B1, and IgG2-A / B2. Therefore, TNFR2 polypeptides in single disulfide-bonded isoforms may be formulated as pharmaceutical compositions and administered using the therapeutic methods described herein to promote potent TNFR2 antagonism.

[0009] Antagonistic 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 (MDSCs) and / or the ability to promote the death of regulatory T cells (T-reg cells) and / or myeloid-derived suppressor cells (MDSCs). Antagonistic TNFR2 polypeptides may be used to inhibit the proliferation and / or promote the death of TNFR2-expressing cancer cells and oncogene-expressing cancer cells. Additionally or alternatively, antagonistic TNFR2 polypeptides may be administered to promote the reciprocal proliferation of T effector cells, such as cytotoxic CD8+ T cells. This reciprocal proliferation can occur, for example, by attenuating the proliferation and activity of T-reg cells or by directly promoting the proliferation of T effector cells, such as cytotoxic CD8+ T cells. Thus, reference to a TNFR2 polypeptide as an antagonist refers to the ability of the TNFR2 polypeptide to attenuate the proliferation and activity of T-reg cells, MDSCs, and / or TNFR2-expressing cancer cells, and does not specifically refer to antagonism of T effector cell responses. The polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) may be used to treat a variety of conditions, including cancer and infectious diseases.

[0010] In one aspect, the disclosure features polypeptides, such as single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, 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, wherein the polypeptide is (a) containing 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) contain antigen-binding sites separated from each other by a distance of at least about 133 Å;

[0011] Exemplary antagonistic TNFR2 polypeptides (e.g., antibodies and antigen-binding fragments thereof) of the present disclosure that exhibit the above-described properties are set forth below in Table 1. Table 1 provides a description of various antagonistic TNFR2 antibodies and antigen-binding fragments thereof defined by their heavy chain and light chain amino acid sequences. Antagonistic TNFR2 antibodies and antigen-binding fragments thereof of the present disclosure include antagonistic TNFR2 antibodies and antigen-binding fragments thereof having heavy and / or light chains shown in Table 1, as well as antibodies and antigen-binding fragments thereof containing heavy 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 and / or light chains shown in Table 1. Complementarity determining regions are shown in bold.

[0012] Table 1. Exemplary antagonistic TNFR2 antibodies of the present disclosure JPEG2025118591000002.jpg248161JPEG2025118591000003.jpg241163JPEG2025118591000004.jpg247166 JPEG2025118591000005.jpg223151JPEG2025118591000006.jpg223151JPEG2025118591000007.jpg223151 JPEG2025118591000008.jpg223151JPEG2025118591000009.jpg223151JPEG2025118591000010.jpg223151 JPEG2025118591000011.jpg223151JPEG2025118591000012.jpg223151JPEG2025118591000013.jpg223151

[0013] For example, in some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 302.

[0014] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 303.

[0015] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 304.

[0016] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 305.

[0017] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having an amino acid sequence 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 antigen-binding fragment thereof contains a heavy chain having the amino acid sequence of SEQ ID NO: 306.

[0018] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having the amino acid sequence of SEQ ID NO: 297.

[0019] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having the amino acid sequence of SEQ ID NO: 298.

[0020] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having the amino acid sequence of SEQ ID NO: 299.

[0021] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having the amino acid sequence of SEQ ID NO: 300.

[0022] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having an amino acid sequence 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 antigen-binding fragment thereof contains a light chain having the amino acid sequence of SEQ ID NO: 301.

[0023] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 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: 297.

[0029] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 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: 298.

[0030] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 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: 299.

[0031] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 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: 301.

[0033] In some embodiments, the disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 disclosure features an antagonistic TNFR2 antibody or antigen-binding fragment thereof containing a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence 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 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 antigen-binding fragment thereof comprises 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 the 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, antibody, antigen-binding fragment thereof, or construct thereof) may contain a human IgG2 hinge region having an amino acid other than cysteine, such as a serine residue, at positions 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 and / or a C233S amino acid substitution.

[0050] In some embodiments, 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 a serine residue at one or both of positions 232 and 233 of the IgG2 hinge amino acid sequence. For example, when an 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 an 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 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, such as 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). In some embodiments, the antigen binding sites are separated from one another by a distance of at least about 134 Å (e.g., a distance of about 134 Å to about 160 Å, such as 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). In some embodiments, the antigen binding sites are separated from one another by a distance of at least about 139 Å (e.g., a distance of about 139 Å to about 160 Å, such as, for example, 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). In some embodiments, the antigen binding sites are separated from one another by a distance of at least about 150 Å (e.g., a distance of about 150 Å to about 160 Å, such as 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).

[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 one another by a distance of about 133 Å to about 150 Å, e.g., 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 one another by a distance of about 133 Å to about 145 Å, e.g., 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 bonds are separated from one another by a distance of about 133 Å to about 139 Å, e.g., about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, or 139 Å. In some embodiments, the antigen bonds are separated from one another by a distance of about 134 Å to about 139 Å, e.g., about 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, or 139 Å.

[0053] The polypeptide (e.g., single-chain polypeptide, antibody, antigen-binding fragment thereof, or 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 naturally occurring amino acid. In some embodiments, the polypeptide (e.g., single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) (a) CDR-H2 having the amino acid sequence (J)3GSJ or (J)5GSJ; (b) 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) CDR-L1 having the amino acid sequence (J)Y or (J)Y; (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)Y(J)T or (J)Y(J)T; Further comprising: Each J is independently a naturally occurring amino acid.

[0054] A polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may comprise the amino acid sequence Z 4 FZ 3 Z 5 SSZ 5 or Z 4 YZ 3 Z 5 TDZ 5 and optionally containing a CDR-H1 having X, Each Z 3 are independently amino acids containing a polar side chain that is uncharged at physiological pH, Each Z 4 are independently glycine or alanine, Each Z 5 are independently amino acids 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) (a) Amino acid sequence SSGZ 4 Z 3 Y (SEQ ID NO: 263) or VDPEYZ 4 Z 3 CDR-H2 having T (SEQ ID NO: 264), (b) 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 (SEQ ID NO: 266), (c) Amino acid sequence SASSSVYYMZ 5 (SEQ ID NO: 267) or QNINKZ 5 CDR-L1 having (SEQ ID NO: 268), (d) Amino acid sequence STSNLAZ 3 (SEQ ID NO: 269), TYZ 3 , or YTZ 3 and / or, (e) Amino acid sequence QQRRNZ 5 PYZ 3 (SEQ ID NO: 270) or CLQZ 5 VNLXZ 3 CDR-L3 having (SEQ ID NO: 271), Further comprising: Each Z 1 are independently amino acids containing a cationic side chain at physiological pH, Each Z 2 are independently amino acids containing an anionic side chain at physiological pH, Each Z 3 are independently amino acids containing a polar side chain that is uncharged at physiological pH, Each Z 4 are independently glycine or alanine, Each Z 5 are independently amino acids containing a hydrophobic side chain; Each X is independently leucine or isoleucine.

[0056] The polypeptide (e.g., single chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) may contain a CDR-H1 having the amino acid sequence 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., single chain polypeptide, antibody, antigen-binding fragment thereof, or construct thereof) comprises: (a) a CDR-H2 having the amino acid sequence 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 the amino acid sequence 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) CDR-L1 having the amino acid sequence 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 the amino acid sequence 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 the amino acid sequence 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; Further includes:

[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) 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) can have, for example, the following CDRs: (a) CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257); (b) 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) CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO: 27), and (c) CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO: 28), The antibody may contain a light chain having one or more of: In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs: (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 light chain having one or more of the following CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261); 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., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may be (a) CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 23); (b) 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); and (d) CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID NO: 26), (e) CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO: 27), and (f) CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO: 28), and optionally further comprising three light chain CDRs comprising:

[0062] In some embodiments, the polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) (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) 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 comprising three heavy chain CDRs comprising: (d) 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) 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), and further comprising three light chain CDRs comprising: Each X is independently leucine or isoleucine.

[0063] In some embodiments, a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) comprises a framework region having the amino acid sequence LLIR (SEQ ID NO: 262) attached to the N-terminus of CDR-L2, and / or a framework region having the amino acid sequence TLE attached 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 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 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 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 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 (e.g., K of about 10 pM to about 99 nM) D For example, 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. D), specifically binds to a peptide having any one of the amino acid sequences of SEQ ID NOs: 11, 19, 20, and 34 to 117, but does not specifically bind to a peptide containing amino acids 56 to 60 (KCSPG) of SEQ ID NO: 7. Polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof) may be present at concentrations of, for example, 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, 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, 300 pM, 305 pM, 310 pM, 315 pM, 320 pM, 325 pM, 330 pM, 335 pM, 340 pM, 345 pM, 35 0pM, 155pM, 160pM, 165pM, 170pM, 175pM, 180pM, 185pM, 190pM, 195pM, 200pM, 205pM, 210pM, 215pM, 220pM, 225pM, 230pM, 235pM, 240pM , 245pM, 250pM, 255pM, 260pM, 265pM, 270pM, 275pM, 280pM, 285pM, 290pM, 295pM, 300pM, 305pM, 310pM, 315pM, 320pM, 325pM, 330pM, 335 pM, 340pM, 345pM, 350pM, 355pM, 360pM, 365pM, 370pM, 375pM, 380pM, 385pM, 390pM, 395pM, 400pM, 405pM, 410pM, 415pM, 420pM, 425pM, 430pM, 435pM, 440pM, 445pM, 450pM, 455pM, 460pM, 465pM, 470pM, 475pM, 480pM, 485pM, 490pM, 495pM, 500pM, 505pM, 510pM, 515pM, 520p M, 525pM, 530pM, 535pM, 540pM, 545pM, 550pM, 555pM, 560pM, 565pM, 570pM, 575pM, 580pM, 585pM, 590pM, 595pM, 600pM, 605pM, 610pM, 61 5pM, 620pM, 625pM, 630pM, 635pM, 640pM, 645pM, 650pM, 655pM, 660pM, 665pM, 670pM, 675pM, 680pM, 685pM, 690pM, 695pM, 700pM, 705pM,710pM, 715pM, 720pM, 725pM, 730pM, 735pM, 740pM, 745pM, 750pM, 755pM , 760pM, 765pM, 770pM, 775pM, 780pM, 785pM, 790pM, 795pM, 800pM, 805p M, 810pM, 815pM, 820pM, 825pM, 830pM, 835pM, 840pM, 845pM, 850pM, 855 pM, 860pM, 865pM, 870pM, 875pM, 880pM, 885pM, 890pM, 895pM, 900pM, 90 5pM, 910pM, 915pM, 920pM, 925pM, 930pM, 935pM, 940pM, 945pM, 950pM, 9 55pM, 960pM, 965pM, 970pM, 975pM, 980pM, 985pM, 990pM, 995pM, 1nM, 5n M, 10nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM, 96nM, 97nM, 98nM, or 99nM, K; D and can bind to a peptide having any one of the amino acid sequences of SEQ ID NOs: 11, 19, 20, and 34 to 117.

[0067] The polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may be (a) amino acids 142 to 146 (KCRPG) of SEQ ID NO: 7; (b) amino acids 142 to 149 of SEQ ID NO: 7 (KCRPGFGV); (c) amino acids 137 to 144 of SEQ ID NO: 7 (CAPLRKCR); (d) amino acids 150 to 190 of SEQ ID NO: 7 (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI); (e) amino acids 161 to 169 of SEQ ID NO: 7 (CKPCAPGTF); (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 to 184 (SSTDICRPHQI) of SEQ ID NO: 7; (h) amino acids 126 to 140 of SEQ ID NO: 7 (CALSKQEGCRLCAPL), and / or (i) amino acids 156 to 165 (TSDVVCKPCA) of SEQ ID NO: 7; It can specifically bind to TNFR2 at an epitope within the

[0068] In some embodiments, a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) specifically binds to TNFR2 at two or more of the above epitopes (e.g., at 2, 3, 4, 5, 6, 7, 8, 9, 10, or more epitopes within the above 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 D For example, a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) specifically binds to TNFR2 with a K of about 1 pM to about 10 nM. D, for example, 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, among other values. pM, 215pM, 220pM, 225pM, 230pM, 235pM, 240pM, 245pM, 250pM, 255pM, 260pM , 265pM, 270pM, 275pM, 280pM, 285pM, 290pM, 295pM, 300pM, 305pM, 310pM, 3 15pM, 320pM, 325pM, 330pM, 335pM, 340pM, 345pM, 350pM, 355pM, 360pM, 365 pM, 370pM, 375pM, 380pM, 385pM, 390pM, 395pM, 400pM, 405pM, 410pM, 415pM, 420pM, 425pM, 430pM, 435pM, 440pM, 445pM, 450pM, 455pM, 460pM, 465pM, 47 0pM, 475pM, 480pM, 485pM, 490pM, 495pM, 500pM, 505pM, 510pM, 515pM, 520p M, 525pM, 530pM, 535pM, 540pM, 545pM, 550pM, 555pM, 560pM, 565pM, 570pM, 575pM, 580pM, 585pM, 590pM, 595pM, 600pM, 605pM, 610pM, 615pM, 620pM, 625 pM, 630pM, 635pM, 640pM, 645pM, 650pM, 655pM, 660pM, 665pM, 670pM, 675pM , 680pM, 685pM, 690pM, 695pM, 700pM, 705pM, 710pM, 715pM, 720pM, 725pM, 7 30pM, 735pM, 740pM, 745pM, 750pM, 755pM, 760pM, 765pM, 770pM, 775pM, 780 pM, 785pM, 790pM, 795pM, 800pM, 805pM, 810pM, 815pM, 820pM, 825pM, 830pM,K such as 835pM, 840pM, 845pM, 850pM, 855pM, 860pM, 865pM, 870pM, 875pM, 880pM, 885pM, 890pM, 895pM, 900pM, 905pM, 910pM, 915pM, 920pM, 925pM, 930pM, 935pM, 940pM, 945pM, 950pM, 955pM, 960pM, 965pM, 970pM, 975pM, 980pM, 985pM, 990pM, 995pM, 1nM, 5nM, or 10nM; D In some embodiments, the polypeptide (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) can specifically bind to TNFR2 with a K of about 621 pM. D 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 with a K of about 44 pM. D It specifically binds to TNFR2.

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

[0071] The polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) specifically binds to TNFR2 and binds, e.g., about 10 -3 s -1 The following K off , for example, about 10-6 s -1 ~about 10 -3 s -1 K such as 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 K off) In some embodiments, the antibody-antigen complex may form an antibody-antigen complex that dissociates at about 2.2 x 10 -4 s -1 K off dissociates at

[0072] The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can be used to inhibit, for example, TNFR2-expressing cells, such as T-reg cells (e.g., CD25 HiIn some embodiments, the single chain polypeptide, antibody, or antigen-binding fragment thereof may inhibit TNFR2 signaling in cells such as TNFR2-expressing T-reg cells, myeloid-derived suppressor cells (MDSCs), and / or TNFR2+ cancer cells. In some embodiments, the single chain polypeptide, antibody, or antigen-binding fragment thereof reduces or inhibits expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3, as assessed, for example, by observing a decrease in expression of one or more of the above genes or by using other methods known in the art for assessing gene activation. For example, the antagonistic TNFR2 single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof can inhibit the expression or post-translational modification (e.g., phosphorylation) of one or more of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, or cIAP2 / BIRC3, by inhibiting 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 a manner similar to that described herein. The expression or post-translational modification (e.g., phosphorylation) of one or more of these proteins may be inhibited 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% compared to that of one or more of these proteins isolated from a sample not treated with the construct. 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 to measure protein content and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments to measure mRNA content.In preferred embodiments, the anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) are dominant TNFR2 antagonists, such that they are able to inhibit TNFR2 activation even in the presence of a TNFR2 agonist (e.g., TNFα or Bacillus Calmette-Guerin (BCG)) 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) inhibiting the proliferation of T-reg cells and / or directly killing T-reg cells, for example, by binding to and inactivating TNFR2 on the surface of T-reg cells (e.g., thereby reducing the amount of T-reg cells in a population of cells 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 population of cells not exposed to the polypeptide); (b) inhibiting proliferation of MDSCs and / or directly killing MDSCs, for example, by binding to and inactivating TNFR2 on the surface of MDSCs (e.g., thereby reducing the amount of MDSCs in a population of cells 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 population of cells not exposed to the polypeptide); (c) promoting the proliferation of T effector cells, e.g., CD8+ T cells (e.g., by increasing the amount of CD8+ effector T cells in a population of cells 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, 5.2x, 5.3x, 5.4x, 5.5x, 5.6x, 5.7x, 5.8x, 5.9x, 6x, 6.1x, 6.2x, 6.3x, 6.4x, 6.5x, 6.6x, 6.7x, 6.8x, 6.9x, 7x, 7.1x, 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8x, 8.1x, 8. 2x, 8.3x, 8.4x, 8.5x, 8.6x, 8.7x, 8.8x, 8.9x, 9x, 9.1x, 9.2x, 9.3x, 9.4x, 9.5x, 9.6x, 9.7x, 9.8x, 9.9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, or more increase), and / or (d) inhibiting the proliferation of and / or directly killing TNFR2-expressing cancer cells (e.g., Hodgkin's lymphoma cells, cutaneous non-Hodgkin's 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 or lymphatic cancer cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.); wound (e.g., thereby reducing the amount of TNFR2-expressing cancer cells in a population of cells 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 population of cells not exposed to the polypeptide); may represent one or more or all of:

[0074] For example, the antagonistic TNFR2 polypeptides described herein, such as single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may be used to reduce the total amount of T-reg cells or cancer cells in a patient (e.g., a human patient) 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), compared to a patient not treated with the polypeptide or a sample not treated with the polypeptide, respectively.

[0075] In some embodiments, the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, an antibody, or an antigen-binding fragment thereof) reduces the expression of TNFR2 by T-reg cells or cancer cells (e.g., TNFR2+ cancer cells, such as Hodgkin's lymphoma cells, cutaneous non-Hodgkin's 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 or lymphatic cancer cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, or upper gastrointestinal cancer cells), and / or reduces the secretion of soluble TNFR2 by one or more of the above cells.

[0076] The antagonistic TNFR2 polypeptides described herein (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) may be used to inhibit or suppress the proliferation of T-reg cells or reduce the total amount 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).

[0077] The antagonistic TNFR2 polypeptides described herein (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) can be used to inhibit TNFR2-expressing T-reg cells (e.g., CD25 Hi The compound may inhibit or suppress the proliferation of TNFR2-expressing activated T-reg cells and / or cancer cells, and / or directly kill TNFR2-expressing T-reg cells and / or cancer cells. For example, the cancer cells may be selected from the group consisting of Hodgkin's lymphoma cells, cutaneous non-Hodgkin's 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 or lymphatic cancer 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, the compound may bind to TNFR2 on the cancer cells to inhibit or suppress the proliferation of the cancer cells and / or directly kill the cancer cells, for example, by promoting apoptosis of the cancer cells.

[0078] The antagonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein inhibit MDSCs (e.g., B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1, CD2, CD31 (PECAM-1), CD43, CD44, complement components 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 a protein 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). By binding to TNFR2 on MDSCs, the polypeptides may inhibit or suppress MDSC proliferation and / or directly kill MDSCs, for example, by promoting MDSC apoptosis. The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, 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 and / or directly kill T-reg cells with greater potency 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 and / or directly kill T-reg cells with greater potency in the tumor microenvironment compared to sites not containing cancer cells, such as sites distant from the tumor, in patients with cancer.

[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 with higher potency in the tumor microenvironment than in sites not containing cancer cells, e.g., sites distant from the tumor, in patients with cancer, or compared to subjects not having cancer. For example, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, inhibit the proliferation of T-reg cells in sites not containing cancer cells, and / or directly kill T-reg cells with higher potency in the tumor microenvironment than in sites not containing cancer cells, e.g., sites distant from the tumor, in patients with cancer, or compared to subjects not having cancer. 50 IC50 for inhibiting T-reg cell proliferation in the tumor microenvironment 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 fold lower 50The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may inhibit the proliferation of T-reg cells with greater potency in the microenvironment of a tumor containing T-cell lymphoma cells (e.g., Hodgkin's lymphoma cells or cutaneous non-Hodgkin's lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells than in a site not containing such cancer cells, such as a site distant from the tumor, in a patient suffering from one or more of the above cancers, or compared to a subject without cancer.

[0081] In some embodiments, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, inhibit or suppress the proliferation of MDSCs and / or directly kill MDSCs with greater potency 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 or suppress the proliferation of MDSCs and / or directly kill MDSCs with greater potency 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 compared to subjects without cancer.

[0082] For example, the antagonistic TNFR2 polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) can bind to TNFR2 on the surface of MDSCs present in the tumor microenvironment and can inhibit or suppress MDSC proliferation or promote MDSC apoptosis with higher potency in the tumor microenvironment than in sites not containing cancer cells in patients with cancer, e.g., sites distant from the tumor, or compared to subjects not having cancer. For example, the polypeptides described herein, e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can be used to inhibit MDSC proliferation in sites not containing cancer cells. 50IC for inhibiting proliferation of MDSCs in the tumor microenvironment 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 fold lower 50 The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may inhibit MDSC proliferation or promote MDSC apoptosis with greater potency in the microenvironment of a tumor containing T-cell lymphoma cells (e.g., Hodgkin's lymphoma cells or cutaneous non-Hodgkin's lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells than in sites not containing such cancer cells, such as sites distant from the tumor, in patients with one or more of the above cancers, or compared to subjects without cancer.

[0083] In some embodiments, the polypeptides described herein, such as single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, expand T effector cells, such as CD8+ cytotoxic T cells, at higher titers 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, expand T effector cells, such as CD8+ cytotoxic T cells, at higher titers in the tumor microenvironment in patients with cancer compared to sites that do not contain cancer cells, such as sites distant from the tumor, or compared to subjects 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, directly expand T effector cells, such as CD8+ cytotoxic T cells, at higher titers in the tumor microenvironment than in sites that do not contain cancer cells, such as sites distant from the tumor, in patients with cancer, or compared to subjects without cancer. For example, the polypeptides described herein can be used to expand T effector cells in subjects without cancer, by increasing the EC of the polypeptide. 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 than EC2 for expanding T effector cells in cancer patients. 50 The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may directly expand T effector cells, such as CD8+ cytotoxic T cells, at higher titers in the microenvironment of a tumor containing T-cell lymphoma cells (e.g., Hodgkin's lymphoma cells or cutaneous non-Hodgkin's lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells, than in a site not containing such cancer cells, such as a site distant from the tumor, in a patient with one or more of the above cancers or a subject without cancer. In some embodiments, the T effector cells (e.g., CD8+ cytotoxic T cells) specifically react with an antigen present on one or more cancer cells, such as Hodgkin's lymphoma cells, cutaneous non-Hodgkin's 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 antigen-binding fragment thereof. Additionally or alternatively, the polypeptide may be an antibody or antigen-binding fragment thereof selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or 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, a F(ab')2 molecule, and a tandem scFv (taFv). In some embodiments, the antibody or 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 those described herein. In some embodiments, the antibody or 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] The antagonistic TNFR2 antibody of any of the above aspects may be a bispecific antibody, such as 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 PD-1, PD-L1, or CTLA-4, among others, as described herein. The arm of a bispecific antibody that specifically binds to TNFR2 may, for example, specifically bind to an epitope of human TNFR2 defined by one or more amino acids in CRD3 and / or an epitope defined by one or more amino acids in CRD4. In some embodiments, the arm of a bispecific antibody that specifically binds to TNFR2 is (a) amino acids 142 to 146 (KCRPG) of SEQ ID NO: 7; (b) amino acids 142 to 149 of SEQ ID NO: 7 (KCRPGFGV); (c) amino acids 137 to 144 of SEQ ID NO: 7 (CAPLRKCR); (d) amino acids 150 to 190 of SEQ ID NO: 7 (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI); (e) amino acids 161 to 169 of SEQ ID NO: 7 (CKPCAPGTF); (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 to 184 (SSTDICRPHQI) of SEQ ID NO: 7; (h) amino acids 126 to 140 of SEQ ID NO: 7 (CALSKQEGCRLCAPL), and (i) amino acids 156 to 165 (TSDVVCKPCA) of SEQ ID NO: 7; The present invention specifically binds to an epitope of human TNFR2 selected from the group consisting of:

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

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

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

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

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

[0093] A fourth aspect features a vector encoding the polynucleotide of the third aspect. The vector may be an expression vector, such as a eukaryotic expression vector. In some embodiments, the vector is a viral vector, such as an adenovirus (e.g., an adenovirus of serotypes 1-57, such as an adenovirus of serotypes 2, 5, 11, 12, 24, 26, 34, 35, 40, 48, 49, 50, 52, or Pan9), a retrovirus (e.g., a gamma-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).

[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 can be a prokaryotic or eukaryotic cell, such as a mammalian cell (e.g., a Chinese hamster ovary (CHO) cell). The host cell can 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 antagonistic effects on TNFR2 activity upon binding. The polypeptide may be, for example, an antibody or antigen-binding fragment of the first aspect or any of its embodiments. Additionally or alternatively, the antibody or antigen-binding fragment may be an antibody or antigen-binding fragment thereof that specifically binds to human TNFR2 at an epitope within CRD3 and / or CRD4 but does not bind to TNFR2 at an epitope defined by one or more amino acids within CRD1, and at least 10% of the antibodies or antigen-binding fragments thereof in the pharmaceutical composition are present in a single disulfide-linked isoform, such as an IgG2-A disulfide-linked isoform or an IgG2-B disulfide-linked isoform. In some embodiments, the pharmaceutical composition contains between about 10% and about 99.999% of the antibody or antigen-binding fragment thereof, for example, between about 11% and about 99.9%, between about 12% and about 99.9%, between about 13% and about 99.9%, between about 14% and about 99.9%, between about 15% and about 99%, between about 16% and about 99.9%, between about 17% and about 99.9%, between about 18% and about 99.9%, between about 19% and about 99.9%, between about 20% and about 99.9%, between about 21% and about 99.9%, between about 22% and about 99.9%, between about 23% and about 99.9%, between about 24% and about 99.9%, between about 25% and about 99.9%, between about 26% and about 99.9%, between about 27% and about 99.9%, between about 28% and about 99.9%, % to approximately 99.9%, approximately 29% to approximately 99.9%, approximately 30% to approximately 99.9%, approximately 31% to approximately 99.9%, approximately 32% to approximately 99.9%, approximately 33% to approximately 99.9%, approximately 34% to approximately 99.9%, approximately 35% to approximately 99.9%, approximately 36% to approximately 99.9%, approximately 37% to approximately 99.9%, approximately 38% to approximately 99.9%, approximately 39% to approximately 99.9%, Approximately 40% to approximately 99.9%, approximately 41% to approximately 99.9%, approximately 42% to approximately 99.9%, approximately 43% to approximately 99.9%, approximately 44% to approximately 99.9%, approximately 45% to approximately 99.9%, approximately 46% to approximately 99.9%, approximately 47% to approximately 99.9%, approximately 48% to approximately 99.9%, approximately 49% to approximately 99.9%, approximately 50% to approximately 99.9%, approximately 51% to approximately 99.9%, approximately 52% to approximately 99.9%, approximately 53% to approximately 99.9%, approximately 54% to approximately 99.9%, approximately 55% to approximately 99.9%, approximately 56% to approximately 99.9%, approximately 57% to approximately 99.9%, approximately 58% to approximately 99.9%, approximately 59% to approximately 99.9%, approximately 60% to approximately 99.9%, approximately 61% to approximately 99.9%, approximately 62% to approximately 99.9%, approximately 63% to approximately 99.9%, approximately 64% to approximately 99.9%, approximately 65% to approximately 99.9%, approximately 66% to approximately 99.9%, approximately 67% to approximately 99.9%, approximately 68% to approximately 99.9%, approximately 69% to approximately 99.9%, approximately 70% to approximately 99.9%, approximately 71% to approximately 99.9%, approximately 72% to approximately 99.9%, approximately 73% to approximately 99.9%, approximately 74% to approximately 99.9%, approximately 75% to approximately 99.9%, approximately 76% to approximately 99.9%, approximately 77 % to approximately 99.9%, approximately 78% to approximately 99.9%, approximately 79% to approximately 99.9%, approximately 80% to approximately 99.9%, approximately 81% to approximately 99.9%, approximately 82% to approximately 99.9%, approximately 83% to approximately 99.9%, approximately 84% to approximately 99.9%, approximately 85% to approximately 99.9%, approximately 86% to approximately 99.9%, approximately 87% to approximately 99.9%, approximately 88% to approximately 99.9%, approximately 89% to approximately 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 in a single disulfide bond isoform.

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

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

[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 comprise the construct of the second aspect or any embodiment thereof, the polynucleotide of the third aspect or any embodiment thereof, the vector of the fourth aspect or any embodiment thereof, and / or the host cell of the fifth aspect or any embodiment thereof. The pharmaceutical composition may further comprise 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 present in the pharmaceutical composition in an amount of about 0.001 mg / ml to about 100 mg / ml, such as about 0.01 mg / ml to about 10 mg / ml.

[0101] The pharmaceutical composition may further comprise another therapeutic agent, such as an immunotherapeutic agent. 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, or an anti-B7 agent. The immunotherapeutic agent is selected from the group consisting of an 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. 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 antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-PD-L2 antibody or antigen-binding fragment thereof, a TNF-α cross-linking antibody or antigen-binding fragment thereof, a TRAIL cross-linking antibody or antigen-binding fragment thereof, an anti-CD27 antibody or antigen-binding fragment thereof, an anti-CD30 antibody or antigen-binding fragment thereof, an anti-CD40 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, an anti-GITR antibody or antigen-binding fragment thereof, an anti-OX40 antibody or antigen-binding fragment thereof, an anti-TRAILR1 antibody or antigen-binding fragment thereof, an anti-TRAILR2 antibody or antigen-binding fragment thereof, an anti-TWEAK antibody or antigen-binding fragment thereof, an anti-TWEAKR antibody or antigen-binding fragment thereof, anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, anti-BRAF antibody or antigen-binding fragment thereof, anti-MEK antibody or antigen-binding fragment thereof, anti-CD33 antibody or antigen-binding fragment thereof, anti-CD20 antibody or antigen-binding fragment thereof, anti-HLA-DR antibody or antigen-binding fragment thereof, anti-HLA class I antibody or antigen-binding fragment thereof, anti-CD52 antibody or antigen-binding fragment thereof, anti-A33 antibody or antigen-binding fragment thereof, anti-GD3 antibody or antigen-binding fragment thereof, anti-PSMA antibody or antigen-binding fragment thereof, anti-Ceacan1 antibody or antigen-binding fragment thereof, anti-Galedin9 antibody or antigen-binding fragment thereof, anti-HVEM antibody or antigen-binding fragment thereof, anti-VISTA antibody or antigen-binding fragment thereof, anti-B7H4 antibody or antigen-binding fragment thereof, anti-HHLA2 antibody or antigen-binding fragment thereof, anti-CD155 antibody or antigen-binding fragment thereof, anti-CD80 antibody or antigen-binding fragment thereof, anti-BTLA antibody or antigen-binding fragment thereof, anti-CD160 antibody or antigen-binding fragment thereof, anti-CD28 antibody or antigen-binding fragment thereof, anti-CD226 antibody or antigen-binding fragment thereof, anti-CEACAM1 antibody or antigen-binding fragment thereof, anti-TIM3 antibody or antigen-binding fragment thereof, anti-TIGIT antibody or antigen-binding fragment thereof, anti-CD96 antibody or antigen-binding fragment thereof, anti-CD70 antibody or antigen-binding fragment thereof, anti-CD27 antibody or antigen-binding fragment thereof, anti-LIGHT antibody or antigen-binding fragment thereof, anti-CD137 antibody or antigen-binding fragment thereof, anti-DR4 antibody or antigen-binding fragment thereof, anti-CR5 antibody or antigen-binding fragment thereof The immunotherapeutic agent may be selected from the group consisting of an anti-TNFRS antibody or antigen-binding fragment thereof, an anti-TNFR1 antibody or antigen-binding fragment thereof, an anti-FAS antibody or antigen-binding fragment thereof, an anti-CD95 antibody or antigen-binding fragment thereof, an anti-TRAIL antibody or antigen-binding fragment thereof, an anti-DR6 antibody or antigen-binding fragment thereof, an anti-EDAR antibody or antigen-binding fragment thereof, an anti-NGFR antibody or antigen-binding fragment thereof, an anti-OPG antibody or antigen-binding fragment thereof, an anti-RANKL antibody or antigen-binding fragment thereof, an anti-LTβ receptor antibody or antigen-binding fragment thereof, an anti-BCMA antibody or antigen-binding fragment thereof, an anti-TACI antibody or antigen-binding fragment thereof, an anti-BAFFR antibody or antigen-binding fragment thereof, an anti-EDAR2 antibody or antigen-binding fragment thereof, an anti-TROY antibody or antigen-binding fragment thereof, and an anti-RELT antibody or antigen-binding fragment thereof. For example, the immunotherapeutic agent may be an anti-CTLA-4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, or an anti-PD-L1 antibody or antigen-binding fragment thereof.

[0103] In some embodiments, the pharmaceutical composition contains an anti-CTLA-4 antibody or antigen-binding fragment thereof, such as ipilimumab or tremelimumab. Additionally or alternatively, the pharmaceutical composition may contain an anti-PD-1 antibody or 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 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, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD3 D42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, C D67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, C D92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD108, CD109, CD110, CD111, CD112, CD113, CD 114, CD115, CD116, CD117, CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD131, CD132, CD133, CD134, C D135, 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,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, CD23 3, CD234, CD235, CD236, CD237, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD250, CD251, CD25 2, CD253, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD2 71, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD287, CD288, CD289, CD2 and antibodies or antigen-binding fragments thereof that bind to one or more of CD90, 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.

[0105] 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 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 and inhibits the activity of one or more or all of CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5. 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 and inhibits the activity of one or more or all of CCL3, CCL4, CCL8, and CCL22.

[0106] The immunotherapeutic agent may be capable of specifically binding to one or more of the immunological targets listed 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 agent, such as an antibody or antigen-binding fragment thereof, that specifically binds 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 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, clobestasol, pegylated interferon (e.g., PEGASYS®), prednisone, romidepsin, bexarotene, methotrexate, trimcinolone cream, anti-chemokines, vorinostat, gabapentin, antibodies against lymphoid cell surface receptors and / or lymphokines, antibodies against surface oncoproteins, and / or small molecule therapeutic agents 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 arm specifically binds to an immune checkpoint protein, such as PD-1, PD-L1, or CTLA-4, among others, as described herein. The arm of the bispecific antibody that specifically binds to TNFR2 may, for example, specifically bind to an epitope on human TNFR2 defined by one or more amino acids in CRD3 and / or an epitope defined by one or more amino acids in CRD4, such as an epitope on human TNFR2 described above and herein, resulting in an antagonistic (predominantly antagonistic) phenotype.

[0109] In some embodiments, a bispecific antibody contains one arm that specifically binds to TNFR2, such as an epitope of human TNFR2 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 may specifically bind to the same epitope(s) on PD-1 as nivolumab, pembrolizumab, avelumab, durvalumab, or atezolizumab. For example, the arm of the bispecific antibody that specifically binds to PD-1 may competitively inhibit the binding of PD-1 to nivolumab, pembrolizumab, avelumab, durvalumab, and / or atezolizumab, as assessed, for example, using a competitive binding assay described herein or one well known in the art, such as a competitive ELISA.

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

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

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

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

[0114] In some embodiments, the other therapeutic agent in the pharmaceutical composition is a chemotherapeutic agent, such as a chemotherapeutic agent described herein. The antibody or antigen-binding fragment thereof, single-chain polypeptide, construct, polynucleotide, vector, or host cell described herein (e.g., a TNFR2 antagonist antibody or antigen-binding fragment thereof) 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, the additional 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, the other 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 a polypeptide (e.g., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) of the first aspect, and / or a construct of the second aspect or any embodiment thereof. The method can 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 mediated by T-reg cells in a mammal (e.g., a human) by administering to the mammal 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 embodiment thereof, a construct of the second aspect or any embodiment thereof, a polynucleotide of the third aspect or any embodiment thereof, a vector of the fourth aspect or any embodiment thereof, a host cell of the fifth aspect or any embodiment thereof, and / or a pharmaceutical composition of the sixth aspect or any embodiment thereof.

[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 (e.g., a single chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) of the first aspect or any embodiment thereof, a construct of the second aspect or any embodiment thereof, a polynucleotide of the third aspect or any embodiment thereof, a vector of the fourth aspect or any embodiment thereof, a host cell of the fifth aspect or any embodiment thereof, and / or a pharmaceutical composition of the sixth aspect or any embodiment thereof.

[0120] The cell proliferation disorder may be, for example, cancer, such as 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. In some embodiments, the cancer is selected from the group consisting of Hodgkin's lymphoma, cutaneous non-Hodgkin's lymphoma, T-cell lymphoma, ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, skin cancer, lung cancer, liver cancer, endometrial cancer, cancer of the hematopoietic or lymphatic system, cancer of the central nervous system, breast cancer, pancreatic cancer, stomach cancer, esophageal cancer, and cancer of the upper gastrointestinal tract. 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 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 / rod tumor, basal cell carcinoma, cholangiocarcinoma, extrahepatic carcinoma, Ewing's sarcoma family, osteosarcoma, and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasm, colon cancer, extrahepatic cholangiocarcinoma, ductal carcinoma in situ ( DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, 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, childhood brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal carcinoma, pancreatic 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 carcinoma), multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low-grade ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma , pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, kidney cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, rhabdomyosarcoma, Sezary 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 cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom's macroglobulinemia.

[0122] A tenth aspect features a method for treating an infectious disease in a mammal (e.g., a human) by administering to the mammal 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 embodiment thereof, a construct of the second aspect or any embodiment thereof, a polynucleotide of the third aspect or any embodiment thereof, a vector of the fourth aspect or any embodiment thereof, a host cell of the fifth aspect or any embodiment thereof, and / or a pharmaceutical composition of the sixth aspect or any embodiment thereof. 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 selected from the group consisting of Hepatitis C virus, Yellow Fever virus, Kadam virus, Kyasanur Forest disease virus, Rangat 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, Tyureni virus, Aroa virus, Dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Ustu virus, West Nile virus, Yaounde virus, Kocobera virus, Bagaza virus, Ileus virus, Israeli turkey meningoencephalitis virus, Untaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge virus, Hill virus, Jugra virus, Saboya virus, Sepic virus, Uganda S virus, Wesselsbron virus, Yellow fever virus, Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey virus Island virus, Dakar bat virus, Montana myotis bat leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, fusogenic agent virus, Ippy virus, Lassa virus, Lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexar virus, Guanarito virus, Junin 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, Hantan virus, Sin Nombre virus, Dugbe virus, Bunyambera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virusCrimean-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, smallpox virus, monkeypox virus, vaccinia virus, herpes simplex virus, human herpesvirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus, Kaposi's sarcoma-associated herpesvirus (KSHV), influenza virus, severe acute respiratory syndrome (SARS) virus, rabies virus, and vesicular stomatitis virus The virus is caused by a virus selected from the group consisting of: human respiratory syncytial 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), and human T-lymphotropic virus types I and II.

[0124] In some embodiments, the infection is caused by a bacterium 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, Pasteurella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus.

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

[0126] In some embodiments, the infection 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 to the human an immunotherapeutic agent, such as 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, or an anti-B7 agent. The antibody may be selected from the group consisting of an 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 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, an anti-CTLA-4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-PD-L2 antibody or antigen-binding fragment thereof, a TNF-α cross-linking antibody or antigen-binding fragment thereof, a TRAIL cross-linking antibody or antigen-binding fragment thereof, an anti-CD27 antibody or antigen-binding fragment thereof, an anti-CD30 antibody or antigen-binding fragment thereof, an anti-CD40 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, an anti-GITR antibody or antigen-binding fragment thereof, an anti-OX40 antibody or antigen-binding fragment thereof, an anti-TRAILR1 antibody or antigen-binding fragment thereof, an anti-TRAILR2 antibody or antigen-binding fragment thereof, an anti-TWEAK ... R antibody or antigen-binding fragment thereof, anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, anti-BRAF antibody or antigen-binding fragment thereof, anti-MEK antibody or antigen-binding fragment thereof, anti-CD33 antibody or antigen-binding fragment thereof, anti-CD20 antibody or antigen-binding fragment thereof, anti-HLA-DR antibody or antigen-binding fragment thereof, anti-HLA class I antibody or antigen-binding fragment thereof, anti-CD52 antibody or antigen-binding fragment thereof, anti-A33 antibody or antigen-binding fragment thereof, anti-GD3 antibody or antigen-binding fragment thereof, anti-PSMA antibody or antigen-binding fragment thereof, anti-Ceacan1 antibody or antigen-binding fragment thereof, anti-Galedin9 antibody or antigen-binding fragment thereof, anti-HVEM antibody or antigen-binding fragment thereof, anti-VISTA antibody or antigen-binding fragment thereof, anti-B7H4 antibody or antigen-binding fragment thereof, anti-HHLA2 antibody or antigen-binding fragment thereof, anti-CD155 antibody or antigen-binding fragment thereof, anti-CD80 antibody or antigen-binding fragment thereof, anti-BTLA antibody or antigen-binding fragment thereof, anti-CD160 antibody or antigen-binding fragment thereof, anti-CD28 antibody or antigen-binding fragment thereof, anti-CD226 antibody or antigen-binding fragment thereof, anti-CEACAM1 antibody or antigen-binding fragment thereof, anti-TIM3 antibody or antigen-binding fragment thereof, anti-TIGIT antibody or antigen-binding fragment thereof, anti-CD96 antibody or antigen-binding fragment thereof, anti-CD70 antibody or antigen-binding fragment thereof, anti-CD27 antibody or antigen-binding fragment thereof, anti-LIGHT antibody or antigen-binding fragment thereof, anti-CD137 antibody or antigen-binding fragment thereof, anti-DR4 antibody or antigen-binding fragment thereof, anti-CR5 antibody or antigen-binding fragment thereof the immunotherapeutic agent may be selected from the group consisting of an anti-TNFRS antibody or antigen-binding fragment thereof, an anti-TNFR1 antibody or antigen-binding fragment thereof, an anti-FAS antibody or antigen-binding fragment thereof, an anti-CD95 antibody or antigen-binding fragment thereof, an anti-TRAIL antibody or antigen-binding fragment thereof, an anti-DR6 antibody or antigen-binding fragment thereof, an anti-EDAR antibody or antigen-binding fragment thereof, an anti-NGFR antibody or antigen-binding fragment thereof, an anti-OPG antibody or antigen-binding fragment thereof, an anti-RANKL antibody or antigen-binding fragment thereof, an anti-LTβ receptor antibody or antigen-binding fragment thereof, an anti-BCMA antibody or antigen-binding fragment thereof, an anti-TACI antibody or antigen-binding fragment thereof, an anti-BAFFR antibody or antigen-binding fragment thereof, an anti-EDAR2 antibody or antigen-binding fragment thereof, an anti-TROY antibody or antigen-binding fragment thereof, and an anti-RELT antibody or antigen-binding fragment thereof. In some embodiments, the immunotherapeutic agent administered to a human is an anti-CTLA-4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, or an anti-PD-L1 antibody or antigen-binding fragment thereof.

[0129] In some embodiments of the eighth, ninth, and / or tenth aspects, the method includes administering to a mammal (e.g., a human) an anti-CTLA-4 antibody or antigen-binding fragment thereof, such as ipilimumab or tremelimumab. Additionally or alternatively, the method may include administering to the human an anti-PD-1 antibody or 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 includes administering to a subject an anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, such as, for example, 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, CD1 11, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD131, CD 132, CD133, CD134, CD135, CD136, CD137, CD138, CD139, CD140, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD149, CD150, CD151, CD152, C D153, CD154, CD155, CD156, CD157, CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167, CD168, CD169, CD170, CD171, CD172, CD173,CD174, CD175, CD176, CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD187, CD188, CD189, CD190, CD191, CD192, CD19 3, CD194, CD195, CD196, CD197, CD198, CD199, CD200, CD201, CD202, CD203, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CD211, CD212, CD 213, 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, CD2 72, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD287, CD288, CD289, CD290, CD291, CD and administering to a mammal (e.g., a human) an antibody or antigen-binding fragment thereof that binds to one or more of 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.

[0131] In some embodiments of the eighth, ninth, and / or tenth aspects, the method includes 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 and inhibits the activity of one or more or all of CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5. 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 and inhibits the activity of one or more or all of CCL3, CCL4, CCL8, and CCL22.

[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 capable of specifically binding to one or more of the immunological targets set forth 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 can be an agent such as an antibody or antigen-binding fragment thereof that specifically binds 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 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 targretin, interferon-α, clobestasol, pegylated interferon (e.g., PEGASYS®), prednisone, romidepsin, bexarotene, methotrexate, trimcinolone cream, anti-chemokines, vorinostat, gabapentin, antibodies against lymphoid cell surface receptors and / or lymphokines, antibodies against surface oncoproteins, and small molecule therapeutics such as vorinostat.

[0134] In some embodiments, the method includes administering to a mammal (e.g., a human) a CAR-T drug, a chemotherapeutic agent, a small molecule anti-cancer drug, or a cancer vaccine, such as a CAR-T drug, a chemotherapeutic agent, a small molecule anti-cancer drug, or a cancer vaccine 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, 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 about 0.01 mg / kg to about 10 mg / kg.

[0136] An 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 embodiment thereof, a construct of the second aspect or any embodiment thereof, a polynucleotide of the third aspect or any embodiment thereof, a vector of the fourth aspect or any embodiment thereof, a host cell of the fifth aspect or any embodiment thereof, and / or a pharmaceutical composition of the sixth aspect or any embodiment thereof.

[0137] In some embodiments, the kit contains instructions for transfecting the 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 contains instructions for administering the agent to a mammal (e.g., a human), such as a human patient, suffering from a cell proliferative disorder and / or an infectious disease described herein. In some embodiments, the kit contains instructions for making or using the agent.

[0138] definition As used herein, the term "about" refers to a value that is 10% or less than the stated value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0139] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or immunologically reacts with a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including, but not limited to, chimeric, humanized, primatized, heteroconjugate (e.g., bispecific, trispecific, and tetraspecific) antibodies, diabodies, triabodies, and tetrabodies, and antigen-binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments. Furthermore, unless otherwise specified, the term "monoclonal antibody" (mAb) is intended to include both intact molecules capable of specifically binding to a target protein and antibody fragments (e.g., Fab fragments and F(ab')2 fragments) capable of specifically binding to a target protein. Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody and, compared to an intact antibody, are cleared more rapidly from the circulation of an animal and may exhibit lower non-specific tissue binding (see Wahl et al., J. Nucl. Med. 24:316, 1983, incorporated herein by reference).

[0140] The term "antigen-binding fragment," as used herein, refers to 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 a full-length antibody. An antibody fragment may be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) V L Domain, V H Domain, C L Domain, and C H(ii) a Fab fragment, which is a monovalent fragment consisting of one domain; (iii) a V(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; H Domain and C H Fd fragment consisting of one domain, (iv) V of a single arm of an antibody L Domain and V H Fv fragment consisting of domains, (v)V H Domain and V L (vi) V domain-containing dAbs H (vii) V domain-based dAb fragments (Ward et al., Nature 341:544-546, 1989). H Domain or V L These include, but are not limited to, dAbs composed of two domains, (viii) isolated complementarity determining regions (CDRs), and (ix) combinations of two or more isolated CDRs optionally linked by a synthetic linker. Additionally, the two domains of an Fv fragment, V, L and V H Even if V is encoded by separate genes, recombinant methods can be used to L Area and V H A pair of domains combines them into a single protein chain that forms a monovalent molecule (known as a single-chain Fv (scFv), see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). L and V H By using a linker that can generate V L and V Hcan be linked to a target antibody. These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as are intact antibodies. Antigen-binding fragments may be produced 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 portion," and / or "anti-TNFR2 antibody fragment," etc., include any protein- or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity-determining region (CDR) of a heavy or light chain or ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, or any portion thereof, capable of specifically binding to TNFR2. For example, two or more portions of an immunoglobulin molecule may be covalently linked to each other, for example, by an amide bond, a thioether bond, a carbon-carbon bond, a disulfide bridge, or a linker, such as those described herein or known in the art. TNFR2 antibodies also include antibody-like protein scaffolds, such as the tenth fibronectin type III domain ( ) containing the BC, DE, and FG structural loops whose structure and solvent accessibility are similar to antibody CDRs. 10 Fn3) and others. 10 The tertiary structure of the Fn3 domain is similar to the tertiary structure of the variable region of an IgG heavy chain, and those skilled in the art will recognize, for example, 10 The CDRs of a TNFR2 monoclonal antibody can be grafted onto a fibronectin scaffold by replacing residues in the BC, DE, and FG loops of Fn3 with residues from the CDR-H1, CDR-H2, or CDR-H3 regions of the TNFR2 monoclonal antibody.

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

[0143] As used herein, the term "bispecific antibody" refers to an antibody (e.g., a monoclonal antibody, often a human or humanized antibody) that has binding specificities for at least two different antigens. For example, one of the binding specificities may be for 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, virus-encoded envelope protein, bacterial-derived protein, or bacterial surface protein.

[0144] As used herein, the phrase "chemotherapeutic agent" refers to any chemical agent that has therapeutic utility in the treatment of cancer, such as those cancers described herein. Chemotherapeutic agents include both chemical and biological agents. These agents may function to inhibit cellular activities that cancer cells depend on 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 those described in 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 DS, Knobf MF, Durivage HJ (eds): The Cancer Chemotherapeutic Handbook, 4 th chemotherapeutic agents include, but are not limited to, those described in the Mosby-Year Handbook, ed. St. Luois, Mosby-Year Handbook (the disclosures of each of which, where relevant, are incorporated herein by reference).

[0145] As used herein, the term "chimeric" antibody refers to an antibody having variable domain sequences (e.g., CDR sequences) derived from the immunoglobulin of one source organism, such as a rat or mouse, and constant regions derived from the immunoglobulin of a different organism (e.g., a human, another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, a member of the bovine family (e.g., cow, bison, water buffalo, elk, and yak, among others), cow, sheep, horse, or bison, among others). Methods for producing 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 (incorporated herein by reference).

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

[0147] As used herein, the terms "conservative mutation," "conservative substitution," or "conservative amino acid substitution" refer to the replacement 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 bulk, as summarized in Table 2 below for each of the 20 naturally occurring amino acids.

[0148] JPEG2025118591000014.jpg127167JPEG2025118591000015.jpg90159

[0149] From this table, it can be seen 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. Thus, a conservative variation or substitution is one in which one amino acid is substituted with a member of the same amino acid family (e.g., Ser for Thr, or Lys for Arg).

[0150] As used herein, amino acid substitutions may be represented using the convention: (AA1)(N)(AA2), where "AA1" represents the amino acid normally present at a particular site in the amino acid sequence, "N" represents the residue number in the amino acid sequence where the substitution occurred, and "AA2" represents the amino acid present in the amino acid sequence after the substitution. For example, the designation "C232S" in reference to an antibody hinge region, such as an IgG2 antibody hinge region, refers to the substitution of a serine residue for the native cysteine residue at amino acid residue 232 of the designated hinge amino acid sequence. Similarly, the designation "C233S" in reference to an antibody hinge region, such as an IgG2 antibody hinge region, refers to the substitution of a serine residue for the native cysteine 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 with a suitable reactive functional group of another molecule.

[0152] As used herein with respect to TNFR2 antagonists, the term "construct" refers to a fusion protein containing a first polypeptide domain linked to a second polypeptide domain. Each polypeptide domain may be, independently, 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 a peptide linker or a disulfide bridge, among others. Exemplary linkers that can be used to link the polypeptide domains of an antagonistic TNFR2 construct include, but are not limited to, the linkers described in Leriche et al., Bioorg.Med.Chem., 20:571-582 (2012) (the entire disclosure of which is incorporated herein by reference).

[0153] As used herein, the term "derivatized antibody" refers to an antibody that has been modified by chemical reaction to cleave residues or add non-naturally occurring chemical moieties to the isolated antibody. Derivatized antibodies can be obtained by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by addition of well-known chemical protecting / blocking groups, proteolytic cleavage, or conjugation to a cellular ligand or other protein. Any of a variety of chemical modifications can be performed using well-known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like, using established procedures. Additionally, derivatives can include one or more non-natural amino acids, for example, using amber suppression techniques (see, e.g., U.S. Pat. No. 6,964,859, incorporated herein by reference).

[0154] As used herein, the term "diabody" refers to a VH domain and a VL domain connected by a linker that is too short to allow intramolecular binding of the VH and VL domains on the same peptide chain (e.g., a linker consisting of five amino acids). H Domain and V L "Triabody" refers to a bivalent antibody comprising two polypeptide chains, each containing a complementary domain. This structure forces each domain to pair with a complementary domain on another polypeptide chain to form a homodimeric structure. Thus, the term "triabody" refers to a trivalent antibody comprising three peptide chains, each of which contains one VH domain and one VL domain connected by a linker (e.g., a linker consisting of one to two amino acids) that is too short to allow intramolecular binding of the VH and VL domains within the same peptide chain. Peptides constructed in this manner typically trimerize to fold into their native structure, with the VH and VL domains of adjacent peptide chains positioned in close spatial proximity to one another to allow proper folding (see Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993, incorporated herein by reference).

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

[0156] As used herein, a "dominant antagonist" of TNFR2 is an antagonist (e.g., an antagonistic polypeptide, e.g., 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, 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 IC of antagonist in the presence of TNFR2 agonist (e.g., TNFα) or IL-2 compared to 50 is increased by 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 than 0.5). Inhibition of TNFR2 activation can be assessed, for example, by measuring inhibition of NFκB signaling (e.g., by monitoring decreased expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2 / BIRC3 using conventional gene expression assays), in addition to measuring inhibition of proliferation of TNFR2+ cells, such as T-reg cells, TNFR2-expressing cancer cells, 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 those identified in Table 2.1 on page 30 of Gu et al.: short K chain linkers ADAAP (SEQ ID NO: 118) (mouse) and TVAAP (SEQ ID NO: 119) (human); long K chain linkers 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 GGSGGGSGGGGS (SEQ ID NO: 126) (all GS linkers are mouse and human). Suitable linkers for use in the heavy chain of the DVD include those identified in Table 2.1 on page 30 of Gu & Ghayur, 2012, Methods in Enzymology 502:25-41 (incorporated herein by reference): short linkers AKTTAP (SEQ ID NO: 127) (mouse) and ASTKGP (SEQ ID NO: 128) (human); long linkers 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 in a particular organism (e.g., a human) or in 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" refers to a portion of an antigen recognized and bound by a polypeptide, such as an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct described herein. In the context of a protein antigen (e.g., TNFR2, e.g., human TNFR2 as set forth in SEQ ID NO:7, or TNFR2 from a non-human mammal, e.g., a non-human mammal, such as those described herein), an epitope may be a continuous epitope, which is a single, uninterrupted segment of one or more amino acids covalently linked to each other by peptide bonds, with all constituent amino acids bound to the polypeptide (e.g., an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct thereof). An exemplary assay for measuring the binding of an antagonistic TNFR2 polypeptide to specific amino acids within an antigen is described in Example 1 below. A continuous epitope may consist of, for example, 1, 5, 10, 15, 20, or more amino acids within an antigen, such as a TNFR2 protein described herein (e.g., human TNFR2 as set forth in SEQ ID NO:7). For example, a continuous epitope may consist 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 within an antigen. Examples of continuous epitopes on TNFR2 to which the antagonistic polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) bind include one or more consecutive residues or all of the residues of the SSTDICRPHQI motif (SEQ ID NO: 288), one or more consecutive residues or all of the residues of the CALSKQEGCRLCAPL motif (SEQ ID NO: 289), and one or more consecutive residues or all of the residues of the TSDVVCKPCA motif (SEQ ID NO: 290), and corresponding regions on TNFR2 proteins of non-human mammals (e.g., bison, cow, and other non-human mammals described herein).In some embodiments, an epitope may be a discontinuous epitope containing two or more segments of amino acids separated from each other by one or more intervening amino acid residues in the amino acid sequence of the antigen. A discontinuous epitope may be composed of, for example, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) segments containing amino acids from one or more of the amino acid residues of 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) in human TNFR2, and corresponding regions on the TNFR2 protein of non-human mammals (e.g., bison, cow, and other non-human mammals described herein). Despite this separation by intervening amino acids, the segments comprising a discontinuous epitope may be spatially adjacent to each other, for example, in the three-dimensional structure of the antigen. Exemplary discontinuous epitopes on TNFR2 to which the antagonistic polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) 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). Further examples of discontinuous epitopes on TNFR2 to which the antagonistic polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) bind include 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 "exogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that does not naturally occur in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell). Exogenous materials include foreign substances introduced into an organism from an outside source or culture materials extracted from the foreign substance.

[0161] As used herein, the term "framework region" or "FW region" includes amino acid residues adjacent to the CDRs. FW region residues may be present, for example, within human antibodies, rodent-derived antibodies (e.g., murine 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, among others.

[0162] As used herein, the term "fusion protein" refers to a protein covalently linked to another molecule. Fusion proteins 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 one protein covalently linked to another protein can be recombinantly expressed in a cell (e.g., a eukaryotic or prokaryotic cell) by expression of a polynucleotide encoding the fusion protein from a vector or the cell's genome. A fusion protein can also contain one protein covalently linked to a linker (which in turn is covalently linked to another molecule). Examples of linkers that can be used to form fusion proteins include peptide-containing linkers, such as peptide-containing linkers containing natural or unnatural amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker because these residues are not present in natural proteins and are therefore more resistant to degradation by endogenous proteases. Linkers can be prepared using a variety of strategies that are well known in the art and depend on the reactive components of the linker; 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" refers to a monoclonal antibody, preferably a human or humanized antibody, that has binding specificities for at least two different antigens. Traditionally, recombinant production of heterospecific antibodies has been based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537, 1983). Similar techniques are described, for example, in WO 93 / 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, and 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 association 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" refers to the domain of an antibody or antigen-binding fragment thereof (e.g., an IgG2 antibody or antigen-binding fragment thereof) 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 located approximately in the center of each heavy chain, linking the CH1 domain to the CH2 and CH3 domains. The hinge region of an antibody or antigen-binding fragment thereof may provide chemical bonding between the molecular chains of the antibody or antigen-binding fragment thereof. For example, in monoclonal antibodies, cysteine residues in the hinge region form interchain disulfide bonds, thereby providing defined covalent bonding between the heavy chains. 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 numbering system of 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. For example, using the numbering scheme of Kabat et al., the wild-type human IgG2 hinge region set forth in SEQ ID NO:292 would be 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 this disclosure, variant IgG2 hinge regions, such as the variant set forth in SEQ ID NO:291 (ERKCCVECPPCP), will be numbered according to the convention of Kabat et al., unless expressly stated to the contrary.

[0165] As used herein, the term "human antibody" refers to a human antibody that has only minor sequence changes or mutations, but is composed of proteins (e.g., CDRs, frameworks, C L , C H Domain (e.g., C H 1. C H 2. C H 3), Hinge, (V L , V H)) is an antibody in which substantially all of its components 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 and / or light chain) genes. Furthermore, when a human antibody is a single-chain antibody, the human antibody may contain a linker peptide not present in naturally occurring human antibodies. For example, an Fv may contain a linker peptide, such as two to about eight glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region. Such a linker peptide is considered to be of human origin. Human antibodies can be produced using a variety of methods well known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent 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 which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT Publications WO98 / 24893, WO92 / 01047, WO96 / 34096, WO96 / 33735, U.S. Patent 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 refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other target-binding subdomains of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Generally, humanized antibodies comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to the CDR regions 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 comprise at least a portion of an immunoglobulin constant region (Fc), typically that 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; Queenetal U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370, EP 239400, PCT Publication WO 91 / 09967, U.S. Patent No. 5,225,539, EP 592106, and EP 519596 (incorporated herein by reference).

[0167] As used herein, the term "hydrophobic side chain" refers to an amino acid side chain that exhibits relatively low solubility in water, for example, due to the steric or electronic properties of the compound moiety present in the side chain. Examples of amino acids containing hydrophobic side chains include amino acids containing unsaturated aliphatic hydrocarbons, such as alanine, valine, leucine, isoleucine, proline, and methionine, as well as amino acids containing aromatic ring systems that are electrostatically neutral 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 thereof, 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 signal transduction of the receptor or ligand that would otherwise result in 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 receptor- or ligand-induced signal transduction that would otherwise cause immune tolerance to endogenous ("self") antigens, such as tumor-associated antigens. Immunotherapeutic agents may reduce receptor- or ligand-induced signaling 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 receptor- or ligand-induced signaling in the absence of the immunotherapeutic agent. Exemplary assays that can be used to measure the degree of receptor or ligand signaling include, for example, enzyme-linked immunosorbent assay (ELISA) techniques for measuring changes in protein expression involving specific signaling pathways, and polymerase chain reaction (PCR)-based techniques useful for measuring changes in gene expression involving specific signaling pathways, such as quantitative PCR experiments, reverse transcription PCR experiments, and real-time PCR experiments. Exemplary methods that can be used to determine whether an agent is an "immunotherapeutic agent" include the assay 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. Further examples of immunotherapeutic agents include targretin, interferon-α, clobestasol, pegylated interferon (e.g., PEGASYS®), prednisone, romidepsin, bexarotene, methotrexate, trimcinolone cream, anti-chemokines, vorinostat, gabapentin, antibodies against lymphoid cell surface receptors and / or lymphokines, antibodies against surface oncoproteins, 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, as well as 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" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0170] As used herein, the term "multispecific antibody" refers to an antibody that exhibits affinity for two or more target antigens. Multispecific antibodies may have a structure similar to that of a complete immunoglobulin molecule and may include an Fc region, for example, an IgG Fc region. 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 C-terminus of either the heavy or light chain. Exemplary multispecific molecules that include an Fc region and can incorporate anti-TNFR2 antibodies or antigen-binding fragments 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 contain an Fc region, based on IgG or DVD or scFv fragments. Exemplary multispecific molecules that lack an Fc region and can incorporate antibodies or antibody fragments include scFv dimers (diabodies), trimers (triabodies), and tetramers (tetrabodies), Fab dimers (conjugated with an adhesive polypeptide or protein domain), and Fab trimers (chemically conjugated), as described in Hudson and Souriau, 2003, Nature Medicine 9:129-134, incorporated herein by reference.

[0171] As used herein, the term "myeloid-derived suppressor cells" or "MDSCs" refers to cells of the immune system that regulate the activity of various effector cells and antigen-presenting cells, such as T cells, NK cells, dendritic cells, and macrophages, among others. Myeloid-derived suppressor cells are distinguished by their gene expression profile, which includes B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1, CD2, CD31 (PECAM-1), CD43, CD44, complement components 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). In particular, 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).

[0172] As used herein, the terms "neutral TNFR2 polypeptide" and "phenotypically neutral TNFR2 polypeptide" refer to 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, a TNFR2 polypeptide is a neutral TNFR2 polypeptide if it binds to TNFR2 and does not enhance or inhibit TNFR2 activation, as assessed, for example, by measuring the proliferation of TNFR2-expressing cells (e.g., T-reg cells, TNFR2+ cancer cells, and / or MDSCs) and / or by measuring the expression of one or more NFκB target genes, such as CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and / or cIAP2 / BIRC3.

[0173] As used herein, the term "non-native constant region" refers to an antibody constant region derived from a source other than the antibody variable region, or an antibody constant region that is a human-made synthetic polypeptide having an amino acid sequence different from the sequence of a native antibody constant region. For example, an antibody containing a non-native 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 a constant region derived from another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, member of the bovine family (e.g., cow, bison, water buffalo, elk, and yak, among others), cow, sheep, horse, or bison, among others.

[0174] As used herein, the term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after aligning the sequences, optionally introducing gaps to achieve the maximum percent sequence identity (e.g., gaps may be introduced in one or both of the candidate and reference sequences to optimize alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment for purposes of measuring percent sequence identity can be performed in a variety of ways well known to those of skill in the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared. For example, alignment of a reference sequence to a candidate sequence for comparison may show that the candidate sequence exhibits 50% to 100% sequence identity over the entire length of the candidate sequence or over a selected portion of the consecutive amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. When a position in the candidate sequence contains the same amino acid residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0175] As used herein, the term "primatized antibody" refers to an antibody that comprises the framework region of a primate-derived antibody and other regions, such as CDRs and / or constant regions, of an antibody of non-primate origin. Methods for producing primatized antibodies are well known in the art. See, e.g., U.S. Patent Nos. 5,658,570, 5,681,722, and 5,693,780 (incorporated herein by reference). For example, the primatized antibodies or antigen-binding fragments 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 containing one or more primate framework regions.

[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, MDSCs, or TNFR2+ cancer cells), refers to mitosis and cytokinesis to produce a plurality of cells. Cell proliferation can be demonstrated, 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 one or more hours, one or more days, or one or more weeks. Those skilled in the art can monitor cell proliferation using a variety of well-known techniques, such as visual 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 reduced compared to the rate of proliferation of a control population of cells, such as a population of TNFR2+ cells not contacted with an antagonistic TNFR2 polypeptide. A decrease in the rate of proliferation can be manifested, for example, by a decrease in the amount of cells of interest in a sample over a period of time, e.g., a 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 decrease in the amount of cells of interest in a sample over a period of time. Additionally or alternatively, inhibition of cell proliferation may be evidenced by a result showing, for example, a %, 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 decrease in the rate at which the cells of interest (e.g., TNFR2+ cells contacted with an antagonistic TNFR2 polypeptide described herein) divide compared to the rate at which control cells (e.g., TNFR2+ cells not contacted with an antagonistic TNFR2 polypeptide) divide.

[0177] As used herein, the term "operably linked" in reference 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 remain in frame.

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

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

[0180] As used herein, the term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory 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" refers to a single-chain Fv antibody in which the variable domains of the antibody heavy and light chains are linked to form a single molecular chain. An scFv fragment contains a single polypeptide chain comprising the variable region 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 connecting the VL and VH regions of the scFv fragment may be a peptide linker composed of proteinogenic amino acids. Alternative linkers may be used to increase the resistance of the scFv fragment to proteolysis (e.g., linkers containing D-amino acids), to increase the solubility of the scFv fragment (e.g., hydrophilic linkers, such as polyethylene glycol-containing linkers, or polypeptides containing repeating 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 reduce 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 scFv molecules 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 to vary in amino acid sequence from the antibody molecule from which the variable regions are derived.For example, in one embodiment, nucleotide or amino acid substitutions resulting in conservative substitutions or variations in amino acid residues may be made (e.g., in CDR and / or framework residues). Alternatively or additionally, CDR amino acid residues may be mutated to optimize antigen binding using art-recognized techniques. scFv fragments are described, for example, in WO2011 / 084714 (incorporated herein by reference).

[0182] As used herein, the phrase "specifically binds" refers to a binding reaction that determines the presence of an antigen within a heterogeneous population of proteins and other biomolecules that are specifically recognized, for example, by an antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof that specifically binds to an antigen has a K of less than 100 nM. D For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen may have a K of up to 100 nM (e.g., 1 pM to 100 nM). D An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will bind to an antigen with a K of 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 epitope. D A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. For a description of immunoassay formats and conditions that can be used to measure specific immune responses, 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" refer to an organism receiving treatment for a particular disease or condition (e.g., cancer or an infectious disease, etc.) described herein. Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovine family (e.g., cows, bison, water buffalo, elk, and yaks, among others), cows, sheep, horses, and bison, that are being treated for a disease or condition, e.g., a cell proliferative disorder such as cancer, or an infectious disease, etc.

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

[0185] As used herein, the terms "treat" or "treatment" refer to therapeutic procedures aimed at preventing or slowing (alleviating) an undesirable physiological change or disorder, such as a cell proliferative disorder such as cancer or the progression of an infectious disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, lessening of disease severity, stable (i.e., not worsening) disease, delayed or slowed disease progression, improvement or palliation of the pathology, and remission (whether partial or complete). Subjects and patients in need of treatment include those already with the condition or disorder, as well as those prone to having the condition or disorder, or those in whom the condition or disorder is to be prevented.

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

[0187] As used herein, the term "tumor necrosis factor receptor superfamily," "TNFR superfamily," or "TNFRS" refers to a group of type I transmembrane proteins with a carboxy-terminal intracellular domain and an amino-terminal extracellular domain characterized by 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 divided into two subgroups: receptors that contain an intracellular death domain and receptors that lack this domain. The death domain is an 80-amino acid motif that propagates the apoptotic signaling cascade following receptor activation. Exemplary TNFR superfamily members that contain an intracellular death domain include TNFR1, while TNFR2 represents a TNFR superfamily protein that does not contain this domain. Members of the TNFR superfamily include TNFR1, TNFR2, RANK, CD30, CD40, lymphotoxin beta 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," and the like are used interchangeably to 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 increased 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, TNFR2 signaling is considered to be "inhibited" if, upon contacting the cells with an agent, such as a TNFR2 antagonist polypeptide described herein, 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 reduced in TNFR2+ cells compared to TNFR2+ cells that have not been contacted with the agent (e.g., a TNFR2 antagonist polypeptide). For example, TNFR2 signaling is considered to be "inhibited" if 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 antagonistic TNFR2 polypeptide is reduced 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%, or 100% compared to the expression or post-translational modification (e.g., phosphorylation) of one or more of these genes in TNFR2+ cells not contacted with an antagonistic TNFR2 polypeptide.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 to measure protein content and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments to measure mRNA content.

[0189] As used herein, the term "variable region CDR" includes amino acids within the CDRs, or complementarity determining regions, identified using sequence- or structure-based methods. As used herein, the term "CDR" or "complementarity determining region" refers to the noncontiguous antigen-binding sites present within the variable regions of both heavy and light chain polypeptides. These particular 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, USDapartment of Health and Human Services, NIH Publication No. 91-3242, 1991; Chothia et al., (J. Mol. Biol. 196:901-917, 1987), and MacCallum et al., (J. Mol. Biol. 262:732-745, 1996), and the definitions include overlapping or subsets of amino acid residues when compared with each other. The term "CDR" may refer to the CDR defined by Kabat, for example, based on sequence comparisons.

[0190] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors, e.g., plasmids, RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various 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 WO 1994 / 11026 (incorporated herein by reference). The expression vectors described herein contain, in addition to the polynucleotide sequences, other sequence elements used, for example, to express proteins and / or 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 regulatory sequences, such as promoter and enhancer regions that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that increase the translation rate of these genes or that improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, an internal ribosome entry site (IRES), and a polyadenylation signal site for inducing efficient transcription of the gene carried on the expression vector. The expression vectors described herein may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0191] As used herein, the term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including a heavy chain of an Fv, scFv, or Fab. Reference to "VL" refers to the variable region of an immunoglobulin light chain, including a light chain of an Fv, scFv, dsFv, or Fab. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with identical structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Natural antibodies and immunoglobulins are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a natural antibody has a variable domain (VH) at its amino terminus followed by a number of constant domains. Each light chain of a natural antibody has a variable domain (VL) at its amino terminus and a constant domain at its carboxy terminus. [Brief explanation of the drawings]

[0192] [Figure 1] The amino acid sequence of human TNFR2 (SEQ ID NO:7) is shown. Human TNFR2 is numbered herein starting with methionine at position 1 at the N-terminus and ending with serine at position 461 at the C-terminus (SEQ ID NO:7). All references to amino acid positions within TNFR2 are made relative to the TNFR2 numbering scheme shown in Figure 1. In addition to the residues shown in bold and underlined font (KCRPG, SEQ ID NO:19), binding of other epitopes present within cysteine-rich domain 3 (CRD3) and CRD4 of human TNFR2 (residues 121-162 and 162-202, respectively) and within equivalent regions within TNFR2 of non-humans, e.g., non-human mammals, promotes antagonism of TNFR2 signaling. In addition to the italicized residues (KCSPG, SEQ ID NO: 12), binding of other epitopes present within the CRD1 of human TNFR2 (residues 48-76 of SEQ ID NO: 7) and within equivalent regions within TNFR2 of non-humans, e.g., non-human mammals, inhibits TNFR2 antagonist activity. [Figure 2]Figure 1 shows a graph comparing the effect of the murine monoclonal TNFR2 antagonist antibody TNFRAB2 on regulatory T (T-reg) cell viability in vitro (left) with the effect of a human chimeric version of TNFRAB2 on T-reg cell viability under the same assay conditions (right). TNFRAB2 is a murine IgG2 antibody, while the human chimeric TNFR2 antagonist antibody tested in this example exhibits a human IgG1 isotype. Values along the x-axis represent antibody concentration in μg / ml. Values along the y-axis represent the percentage of T-reg cells present in an in vitro cell sample after incubation of the sample with the indicated concentrations of TNFR2 antibody. [Figure 3] Figure 1 is a graph comparing the effect of the murine monoclonal TNFR2 antagonist antibody TNFRAB2 on the abundance of effector T cells in an in vitro sample (left) with the effect of a human chimeric form of TNFRAB2 on the abundance of effector T cells in an in vitro sample under the same assay conditions (right). The human chimeric TNFR2 antagonist antibody is the same as that described in Figure 2. Values along the x-axis represent antibody concentration in μg / ml. Values along the y-axis represent the percentage of T effector cells contained in the in vitro cell sample after incubation of the sample with the indicated concentrations of TNFR2 antibody. [Figure 4] Figure 1 is a graph comparing the effect of the murine monoclonal TNFR2 antagonist antibody TNFRAB2 on the amount of TNFR2+SW480 colon cancer cells in an in vitro sample (left) with the effect of a human chimeric form of TNFRAB2 on the amount of TNFR2+SW480 colon cancer cells in an in vitro sample under the same assay conditions (right). The human chimeric TNFR2 antagonist antibody is the same as that described in Figure 2. Values along the x-axis represent antibody concentration in μg / ml. Values along the y-axis represent the amount of TNFR2+SW480 colon cancer cells in the in vitro cell sample after incubation of the sample with the indicated concentrations of TNFR2 antibody. [Figure 5]Figure 1 is a graph comparing the effect of the murine monoclonal TNFR2 antagonist antibody TNFRAB2 on the abundance of effector T cells in an in vitro sample (left) with the effect of a human chimeric form of TNFRAB2 on the abundance of effector T cells in an in vitro sample under the same assay conditions (right). The human chimeric TNFR2 antagonist antibody is the same as that described in Figure 2. Values along the x-axis represent antibody concentration in μg / ml. Values along the y-axis represent the percentage of T effector cells contained in the in vitro cell sample after incubation of the sample with the indicated concentrations of TNFR2 antibody. [Figure 6] Figure 1 is a graph comparing the effect of the murine monoclonal TNFR2 antagonist antibody TNFRAB2 on T-reg cell viability in vitro (left) with the effect of a human chimeric version of TNFRAB2 on T-reg cell viability under the same assay conditions (right). The human chimeric TNFR2 antagonist antibody is the same as that described in Figure 2. Values along the x-axis represent antibody concentration in μg / ml. Values along the y-axis represent the percentage of T-reg cells present in an in vitro cell sample after incubation of the sample with the indicated concentrations of TNFR2 antibody. [Figure 7] Figure 1 is a graph comparing the effect of the murine monoclonal TNFR2 antagonist antibody TNFRAB2 on the amount of TNFR2+SW480 colon cancer cells in an in vitro sample (left) with the effect of a human chimeric form of TNFRAB2 on the amount of TNFR2+SW480 colon cancer cells in an in vitro sample under the same assay conditions (right). The human chimeric TNFR2 antagonist antibody is the same as that described in Figure 2. Values along the x-axis represent antibody concentration in μg / ml. Values along the y-axis represent the amount of TNFR2+SW480 colon cancer cells in the in vitro cell sample after incubation of the sample with the indicated concentrations of TNFR2 antibody. [Figure 8]Figure 1 shows a graph comparing the effect of the murine monoclonal TNFR2 antagonist antibody TNFRAB2 on the abundance of effector T cells in an in vitro sample (left) with the effect of a human chimeric form of TNFRAB2 on the abundance of effector T cells in an in vitro sample under the same assay conditions (right). The human chimeric TNFR2 antagonist antibody tested in this example exhibits a human IgG2 isotype and has a human IgG2 hinge region characterized by C232S and C233S amino acid substitutions. Values along the x-axis represent antibody concentration in μg / ml. Values along the y-axis represent the percentage of T effector cells contained in an in vitro cell sample after incubation of the sample with the indicated concentrations of TNFR2 antibody. [Figure 9] This graph compares the effect of the murine monoclonal TNFR2 antagonist antibody TNFRAB2 on T-reg cell viability in vitro (left) with the effect of a human chimeric version of TNFRAB2 on T-reg cell viability under the same assay conditions (right). The human chimeric TNFR2 antagonist antibody tested in this example exhibits a human IgG2 isotype and has a human IgG2 hinge region characterized by C232S and C233S amino acid substitutions. Values along the x-axis represent antibody concentration in μg / ml. Values along the y-axis represent the percentage of T-reg cells present in an in vitro cell sample after incubation of the sample with the indicated concentrations of TNFR2 antibody. [Figure 10]This graph compares the effect of the murine monoclonal TNFR2 antagonist antibody TNFRAB2 on the amount of TNFR2+SW480 colon cancer cells in an in vitro sample (left) with the effect of a human chimeric form of TNFRAB2 on the amount of TNFR2+SW480 colon cancer cells in an in vitro sample under the same assay conditions (right). The human chimeric TNFR2 antagonist antibody tested in this example exhibits a human IgG2 isotype and has a human IgG2 hinge region characterized by C232S and C233S amino acid substitutions. Values along the x-axis represent antibody concentration in μg / ml. Values along the y-axis represent the amount of TNFR2+SW480 colon cancer cells present in the in vitro cell sample after incubation of the sample with the indicated concentrations of TNFR2 antibody. [Figure 11] 1 is a graph showing the TNFR2+ cancer cell killing properties of chimeric variants of monoclonal antibody TNFRAB2. The human chimeric TNFR2 antagonist antibody tested in this example exhibits a human IgG2 isotype and has a human IgG2 hinge region characterized by C232S and C233S amino acid substitutions. Values along the x-axis represent the number of days after treatment of TNFR2+ SW480 tumor cells with the chimeric TNFRAB2 variant antibody. Values along the y-axis represent SW480 tumor volume in cubic millimeters after treatment with the TNFRAB2 variant antibody. The tumor volume values observed after treatment with the TNFRAB2 variant antibody (squares) are compared to those observed after treatment with a solvent control (circles). [Figure 12]1 is an image showing the results of a polyacrylamide gel electrophoresis separation of a chimeric TNFRAB2 variant antibody containing, in addition to a human IgG2 constant domain, a wild-type human IgG2 hinge region and the variable domain of the murine TNFRAB2 monoclonal antibody described herein. The gel electrophoresis separation shown in this figure was performed under non-reducing conditions. This separation revealed four unique bands, corresponding to the IgG2-A, IgG2-B, IgG2-A / B1, and IgG2-A / B2 disulfide-linked isoforms of the human IgG2 isotype. These bands are highlighted with white boxes for clarity. [Figure 13] Figures A-D are a series of schematic diagrams comparing the arrangement of disulfide bonds present within each of the IgG2-A (A), IgG2-B (B), IgG2-A / B1 (C), and IgG2-A / B2 (D) isoforms of human IgG2 isotype antibodies. Thin lines represent disulfide bonds connecting various portions of each antibody heavy or light chain (represented by shaded rectangles). Heavy chains are represented by the longer, outermost rectangles of each antibody. Within each heavy chain, black shading represents the constant region, and lighter shading represents the variable region. Light chains are represented by the shorter, innermost rectangles of each antibody. Within each light chain, darker shading represents the constant region, and lighter shading represents the variable region. DETAILED DESCRIPTION OF THE INVENTION

[0193] The antagonistic 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, TNFR2-expressing cancer cells, or myeloid-derived suppressor cells (MDSCs)) and preventing the receptor from adopting a three-dimensional structure suitable for binding to its cognate ligand, TNFα. TNFα enhances TNFR2 signaling by aggregating trimers of TNFR2 proteins. This trimerization event brings individual TNFR2 proteins into close proximity, triggering TNFR2 signaling via the MAPK / NFκB / TRAF2 / 3 pathway, ultimately leading to cell proliferation and avoidance of apoptosis. The antagonistic 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 cancellation may occur is through the formation of antiparallel TNFR2 dimers, which are the inactive structural form of the receptor.

[0194] The TNFR2 polypeptides described herein specifically bind to epitopes within TNFR2 that promote receptor antagonism and various beneficial 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 antagonistic TNFR2 polypeptides described herein specifically bind to TNFR2 at one or more epitopes within CRD3 and / or CRD4. In some embodiments, the antagonistic TNFR2 polypeptides do not bind to epitopes within CRD1 and / or CRD2. For example, polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof) of the present disclosure may contain the following residues: (a) amino acids 142 to 146 (KCRPG) of SEQ ID NO: 7; (b) amino acids 142 to 149 of SEQ ID NO: 7 (KCRPGFGV); (c) amino acids 137 to 144 of SEQ ID NO: 7 (CAPLRKCR); (d) amino acids 150 to 190 of SEQ ID NO: 7 (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI); (e) amino acids 161 to 169 of SEQ ID NO: 7 (CKPCAPGTF); (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 to 184 (SSTDICRPHQI) of SEQ ID NO: 7; (h) amino acids 126 to 140 of SEQ ID NO: 7 (CALSKQEGCRLCAPL), and / or (i) amino acids 156 to 165 (TSDVVCKPCA) of SEQ ID NO: 7; one or more internal epitopes of or an equivalent epitope within TNFR2 of a non-human mammal, such as a non-human mammal described herein. It can bind to human TNFR2 in

[0195] The present 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 an IgG2 isotype. As described in the Examples below, it has now been discovered that this class of TNFR2 polypeptides exhibits surprisingly superior abilities to inhibit TNFR2 signaling, attenuate the proliferation of T-reg cells and cancer cells, and enhance the proliferation of effector T cells compared to TNFR2-binding polypeptides of other isotypes.

[0196] Another discovery underlying the present disclosure is that antagonistic TNFR2 polypeptides that specifically bind to TNFR2 at one or more of the above epitopes but contain antigen-binding sites that are spatially separated by about 133 Å or more unexpectedly exhibit superior TNFR2 antagonist activity compared to polypeptides that contain antigen-binding sites that are separated by less than about 133 Å. Examples of such polypeptides include IgG1 antibodies and antigen-binding fragments thereof, which contain antigen-binding sites that are separated by about 117 Å, and IgG3 antibodies and antigen-binding fragments thereof, which contain antigen-binding sites that are separated by 125 Å.

[0197] The antagonistic TNFR2 polypeptide of the present disclosure can be formulated into a pharmaceutical composition. Preferably, the polypeptide contained in the pharmaceutical composition is in a single disulfide bond isoform. For example, the pharmaceutical composition of the present disclosure includes a pharmaceutical composition containing an antagonistic 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 polypeptide in the pharmaceutical composition is in a single disulfide bond isoform. Antagonistic TNFR2 polypeptides of the present disclosure may advantageously adopt the IgG2-A disulfide-bonded isoform, which has surprisingly been found to promote substantially more potent levels of TNFR2 antagonism compared to other IgG2 disulfide-bonded isoforms, such as the IgG2-B isoform, the IgG2-A / B1 isoform, and the IgG2-A / B2 isoform. These isoforms are illustrated in Figures 13A-13D. For example, polypeptides of the present disclosure may be modified to adopt predominantly the IgG2-A isoform by introducing mutations into the IgG2 hinge region that inhibit the formation of other disulfide-bonded isoforms. 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 isoforms listed above, include deletion and / or substitution of cysteine residues at positions 232 and 233 of the wild-type human IgG2 hinge amino acid sequence, as set forth in SEQ ID NO: 291. For example, to modify an IgG2 antibody or antigen-binding fragment thereof to adopt predominantly the IgG2-A isoform, conservative amino acid substitutions may be introduced at cysteine residues 232 and / or 233 of SEQ ID NO: 291. An exemplary IgG2 hinge region that is present predominantly in the IgG2-A isoform has the amino acid sequence of SEQ ID NO: 292, and contains the C232S and C233S substitutions compared to SEQ ID NO: 291.

[0198] The following biological activities: (a) inhibiting the proliferation of T-reg cells and / or directly killing T-reg cells, for example, by binding to and inactivating TNFR2 on the surface of T-reg cells; (b) inhibiting MDSC proliferation and / or directly killing MDSC, for example, by binding to and inactivating TNFR2 on the surface of MDSC; (c) promoting the proliferation of T effector cells, such as CD8+ T cells, and / or (d) inhibiting the proliferation of and / or directly killing TNFR2-expressing cancer cells (e.g., Hodgkin's lymphoma cells, cutaneous non-Hodgkin's 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 or lymphatic cancer cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.); are examples of antagonistic TNFR2 phenotypes that are exhibited to a greater extent by polypeptides of the present disclosure compared to TNFR2-binding polypeptides that (i) exhibit an isotype other than IgG2, (ii) contain antigen-binding sites that are less than 133 Å apart, and / or (iii) are predominantly present in single disulfide-bonded isoforms (e.g., IgG2-A isoforms).

[0199] The following sections provide exemplary properties of the antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, as well as a description of their use in therapeutic methods.

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

[0201] To stabilize the IgG2-A disulfide-bonded isoform, mutations may be introduced into the IgG2 hinge region to prevent or suppress the formation of disulfide bonds between cysteine residues that exist as unbound thiols in the IgG2-A isoform. An example of such a mutation is an amino acid substitution or deletion at residues C232 and C233 in the human IgG2 hinge region. By removing one or both of these residues and, optionally, replacing them with amino acids that cannot form disulfide bonds, the disulfide bonding pattern in a population of IgG2 isoforms can be biased toward 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 and C233S amino acid substitutions. Due to the similar molecular volume and polarity of cysteine and serine, the C232S and C233S substitutions are characterized by the advantageous effect of inhibiting disulfide bond formation at positions 232 and / or 233 in the IgG2 hinge region while retaining the steric and electronegativity properties of natural cysteine residues. Introducing the C232S and / or C233S substitution into a TNFR2 antibody or fragment thereof can result in a population of TNFR2 antagonist antibodies or fragments that exhibit the IgG2-A isoform. Methods for generating amino acid substitutions and deletions in antibodies or antigen-binding fragments thereof include mutagenesis techniques described herein and well known in the art.

[0202] Spacing between antigen-binding sites The antagonist TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs thereof) described herein may contain antigen-binding sites (i.e., antigen-binding arms) separated from each other by a distance of at least about 133 Å, which is the spacing found between the antigen-binding arms of human IgG2 isotype antibodies. As described in the Examples below, this spacing has been discovered to result in antibodies with optimal TNFR2 antagonist properties. TNFR2 antagonist polypeptides of the present disclosure include, for example, TNFR2 antagonist polypeptides that contain antigen-binding arms separated by a distance of about 133 Å to about 160 Å, e.g., 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., a single-chain polypeptide, an antibody, an antigen-binding fragment thereof, or a construct thereof) may contain antigen-binding sites that are separated from one another by a distance of about 133 Å to about 150 Å, e.g., 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 one another by a distance of about 133 Å to about 145 Å, e.g., 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 bonds are separated from one another by a distance of about 133 Å to about 139 Å, e.g., about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, or 139 Å. In some embodiments, the antigen bonds are separated from one another by a distance of about 134 Å to about 139 Å, e.g., 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, two, three, four, five, 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, among others. Methods for producing these antibody fragments include peptide synthesis 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, for example, 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 of which are incorporated herein by reference).

[0205] Homogeneity of the population of TNFR2 antagonist polypeptides Pharmaceutical compositions can be prepared in which the TNFR2 antagonist polypeptides described herein (e.g., antibodies, antigen-binding fragments thereof, single-chain polypeptides, or constructs thereof) are present as a single disulfide-bonded isoform. For example, at least 10% or more of the polypeptides in the pharmaceutical composition may be present as a single disulfide-bonded isoform (e.g., IgG2-A isoform). This can be achieved, for example, by making amino acid substitutions or deletions at one or both of cysteine residues 232 and 233 to prevent or suppress the formation of disulfide bonds that would give rise to IgG2 isoforms other than IgG2-A (see, e.g., Figures 13A-13D). Pharmaceutical compositions of the present disclosure include, for example, pharmaceutical compositions in which about 10% to about 99.999% of the antagonist TNFR2 polypeptides in the pharmaceutical composition are present as a single disulfide-bonded isoform, such as the IgG2-A isoform. For example, pharmaceutical compositions of the present disclosure include those containing antagonist TNFR2 polypeptides, wherein, 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 in a single disulfide-bonded isoform.

[0206] Techniques for measuring the relative amounts of various disulfide-linked isoforms in a sample of an antagonist TNFR2 polypeptide include liquid chromatography techniques well known in the art and described herein, such as those 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 TNFR2 / MAPK / TRAF2 / 3 signaling cascade The anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) described herein can interact with TNFR2 and inhibit TNFR2 activity. Therefore, the anti-TNFR2 polypeptides described herein can selectively antagonize TNFα-TNFR2 interaction rather than promoting TNFR2 signaling. This antagonism is particularly important in therapeutic applications, such as cancer immunotherapy, because TNFR2 activation upon binding to TNFα leads to the propagation of MAPK and TRAF2 / 3 signaling cascades, activation of NFκB-mediated transcription of genes involved in T-reg cell proliferation, and escape from apoptosis (Faustman, et al., Nat. Rev. Drug Disc., 9:482-493, 2010). The TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) described herein bind to TNFR2 at one or more specific epitopes that prevent the receptor from trimerizing with adjacent TNFR2 proteins. This trimerization activates intracellular signaling by TNFR2, which in turn 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 a specific epitope such that the TNFα binding site stabilizes TNFR2 in an antiparallel dimeric structure that is sterically inaccessible. This binding prevents TNFα from aggregating TNFR2 trimers, which would otherwise induce TNFR2 signaling. Therefore, the polypeptides described herein can be used to suppress the growth and proliferation of TNFR2+ cells, such as T-reg cells, MDSCs, and TNFR2+ cancer cells. For example, suppressing the proliferation of T-regs and MDSCs allows the proliferation of T effector cells, which can initiate immune responses against, for example, cancer cells or foreign pathogens.Thus, the antagonistic TNFR2 polypeptides described herein may be administered to a mammalian subject, such as a human, having a cell proliferative disorder or an infectious disease to enhance the effectiveness of the immune response in the subject (e.g., an immune response against cancer cells or pathogenic microorganisms).

[0208] Effect on T-reg cell proliferation The antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof, may be used to attenuate the activity of T-reg cells, which are typically involved in T cell-mediated cytotoxicity against autologous cells, e.g., the attack of tumor cells by T lymphocytes. This attenuation can be achieved, for example, by the ability of the antagonistic TNFR2 polypeptides described herein to inhibit the proliferation of and / or directly kill T-reg cells. Thus, antagonistic TNFR2 polypeptides may be administered (e.g., by any of the various administration routes described herein) to a mammalian subject, e.g., a human, to prolong the duration of an adaptive immune response, such as a response to cancer cells or pathogenic microorganisms. In this manner, for example, the antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof, may be synergized with existing technologies for enhancing T lymphocyte-based therapies for cancer and infectious diseases. For example, the TNFR2 antagonists described herein may be administered to suppress the activity of T-reg cells, thereby enhancing the cytotoxic effect of tumor-reactive T cells. TNFR2 antagonists may also be synergized with existing strategies for enhancing the survival of tumor-reactive T cells, such as lymphodepletion therapy and growth factor therapy, thereby extending the duration of anti-tumor reactivity in vivo.

[0209] Because inhibition of T-reg proliferation enhances the activity of CD8+ T lymphocytes, which can mount an attack against pathogenic microorganisms, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may also be used to treat a wide variety of infectious diseases in mammalian subjects (e.g., humans). In addition, the antagonistic 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, 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 effect on TNFR2+ cancer cells The antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof, can bind to TNFR2 on the surface of cancer cells, such as TNFR2+ tumor cells, and inactivate TNFR2. For example, the antagonistic TNFR2 antibodies and antigen-binding fragments thereof described herein can bind to TNFR2 on the surface of T-cell lymphoma cells (e.g., Hodgkin's lymphoma cells or cutaneous non-Hodgkin's lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells, among others. The ability of the antagonistic 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 viability and proliferation of cancer cells. For example, the antagonistic TNFR2 polypeptides described herein, such as antagonistic TNFR2 single-chain polypeptides, antibodies, antigen-binding fragments thereof, or constructs, 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, e.g., ovarian cancer cells, etc.) to inhibit the ability of the cells to proliferate and / or promote apoptosis of the cells.

[0211] TNFR2 antagonist polypeptides are independent of additional TNFR2 binding factors for 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 signaling 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 T-reg and / or cancer cell proliferation. Without being limited by mechanism, the antagonistic TNFR2 antibodies or antigen-binding fragments described herein may exhibit this property due to their ability to bind to TNFR2 at specific epitopes, which, upon binding, stabilize the antiparallel dimeric structure of the receptor. This structural form is unable to enhance NFκB signaling. The antagonistic TNFR2 polypeptides described herein can prevent TNFR2 agonists from restoring cell proliferation by maintaining TNFR2 in an inactive conformational state and / or can result in direct killing (e.g., by apoptosis) of TNFR2+ cells, such as T-reg cells, MDSCs, or TNFR2+ cancer cells.

[0212] For example, the antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, 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 (MDSCs), and inhibit the proliferation of such cells in the presence or absence of TNFα. For example, the antagonistic TNFR2 polypeptides described herein, such as single chain polypeptides, antibodies, and antigen-binding fragments thereof, may 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. Antagonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, or antigen-binding fragments thereof) may exhibit IC in such cell proliferation assays that are not significantly altered by the presence or absence of TNFα. 50 values (e.g., IC values of antagonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, or antigen-binding fragments thereof) in the same cell proliferation assay in the absence of TNFα). 50 IC in the presence of TNFα changed 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 value 50An example of a cell death assay that can be used to measure the antagonistic effects of TNFR2 antibodies is described, for example, in Example 2 below. Similarly, the antagonistic TNFR2 polypeptides described herein, such as single chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may 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 the TNFR2 antagonist polypeptide, as assessed 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. Antagonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, or antigen-binding fragments thereof) exhibit IC in such gene expression assays that are not significantly altered by the presence or absence of TNFα. 50 values (e.g., IC of an antagonistic TNFR2 polypeptide (e.g., single chain polypeptide, antibody, or antigen-binding fragment thereof) in the same gene expression assay in the absence of TNFα). 50 IC in the presence of TNFα changed 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 value 50 value).

[0213] Direct killing of T-reg cells, MDSCs, and TNFR2+ cancer cells The antagonistic TNFR2 polypeptides disclosed herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can not only inhibit the proliferation of T-reg cells, TNFR2+ cancer cells, and / or MDSCs, for example, in a sample (e.g., in a patient, e.g., in a human patient), but can also induce the death of T-reg cells, TNFR2+ cancer cells, and / or MDSCs. The antagonistic TNFR2 polypeptides described herein may be capable of reducing, for example, 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 the total amount of T-reg cells, cancer cells (such as cutaneous T-cell lymphoma cells, ovarian cancer cells, colon cancer cells, renal cell carcinoma cells, or multiple myeloma cells, among others), and / or MDSCs in a sample treated with the antagonist TNFR2 antibody or antigen-binding fragment thereof (such as a sample isolated from a human patient undergoing treatment for cancer or an infectious disease described herein) compared to a sample not treated with the antagonist TNFR2 antibody or antigen-binding fragment thereof.

[0214] The ability of the antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) described herein to attenuate proliferation of T-regs, MDSCs, 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 infectious disease as described herein). Without this beneficial effect, soluble TNFR2 may be secreted, for example, by T-reg cells, and may bind to and sequester TNFR2 antagonists from the extracellular milieu, thereby interfering with the ability of TNFR2 antagonists to localize to TNFR2 on the surface of T-reg cells, TNFR2+ cancer cells, or MDSCs. The antagonistic TNFR2 antibodies or antigen-binding fragments thereof described herein may inhibit the secretion of TNFR2, thereby making T-reg cells, TNFR2+ cancer cells, and / or MDSCs more susceptible to the effects of a therapeutic molecule, e.g., an antagonistic TNFR2 antibody or antigen-binding fragment thereof, and / or another anti-cancer agent, e.g., an anti-cancer agent described herein or known in the art that can be used in conjunction with the compositions and methods described herein.

[0215] Activity (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 may inhibit the proliferation of 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 infectious disease described herein), and may act selectively on actively dividing T-reg cells. The antagonistic TNFR2 antibodies or antigen-binding fragments thereof described herein may be used to inhibit, for example, CD25 Med and CD45RA Hi Compared with resting T-reg cells expressing CD25 Hi and CD45RA LowFor example, the antagonistic TNFR2 antibodies or antigen-binding fragments thereof described herein may selectively target activated T-reg cells expressing CD25 Hi and CD45RA Low The proliferation of T-reg cells expressing CD25 Hi Protein and CD45RA Low T-reg cells that do not express proteins, e.g., CD25 Med Protein and CD45RA Hi Compared to T-reg cells expressing the protein, for example, the suppression may be 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 antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, can inhibit the proliferation of T-reg cells with greater potency in patients with cancer compared to subjects without cancer. The antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, can inhibit the proliferation of T-reg cells with greater potency in the tumor microenvironment compared to sites not containing cancer cells, e.g., sites distant from the tumor, in patients with cancer or in subjects without cancer. This effect can be measured, for example, 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, can inhibit the IC of the polypeptides to suppress or inhibit the proliferation of T-reg cells in sites not containing cancer cells. 50IC50 for suppressing or inhibiting T-reg cell proliferation in the tumor microenvironment 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 fold lower 50 Examples of cell death assays that can be used to measure the antagonistic effects of anti-TNFR2 polypeptides 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, may inhibit the proliferation of T-reg cells or promote the apoptosis of T-reg cells with a higher potency in tumor microenvironments containing TNFR2+ cancer cells (e.g., Hodgkin's lymphoma cells, cutaneous non-Hodgkin's 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 or lymphatic cancer cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.) than in sites not containing such cancer cells, e.g., sites distant from the tumor, in patients suffering from one or more of the above cancers or in cancer-free subjects.

[0217] Additionally or alternatively, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may inhibit MDSC proliferation with greater potency in patients with cancer compared to subjects without cancer. The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may inhibit MDSC proliferation with greater potency in the tumor microenvironment compared to sites not containing cancer cells, such as sites distant from the tumor, in patients with cancer or subjects without cancer. This effect may be measured, for example, 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, may inhibit the IC of the polypeptides to suppress or inhibit MDSC proliferation in sites not containing cancer cells. 50 IC50 for suppressing or inhibiting MDSC proliferation in the tumor microenvironment 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 fold lower 50Examples of cell death assays that can be used to measure the antagonistic effects of anti-TNFR2 polypeptides 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, may inhibit MDSC proliferation or promote MDSC apoptosis with higher potency in tumor microenvironments containing TNFR2+ cancer cells (e.g., Hodgkin's lymphoma cells, cutaneous non-Hodgkin's 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 or lymphatic cancer cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.) than in sites not containing such cancer cells, such as sites distant from the tumor, in patients suffering from one or more of the above cancers or in cancer-free subjects.

[0218] Additionally or alternatively, the polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may expand T effector cells, such as CD8+ cytotoxic T cells, at higher titers 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, expand T effector cells, such as CD8+ cytotoxic T cells, at higher titers in the tumor microenvironment compared to sites not containing cancer cells, such as sites distant from the tumor, in patients with cancer or subjects without cancer. This effect may be measured, for example, 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, may expand T effector cells at sites not containing cancer cells, for example, by measuring the EC of the polypeptide to expand T effector cells at sites not containing cancer cells. 50For 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 than EC for expanding T effector cells in the tumor microenvironment. 50 An example of a cell proliferation assay that can be used to measure the effect of an anti-TNFR2 polypeptide on T effector cells is described, for example, in Example 2 below. The polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can directly proliferate T effector cells, such as CD8+ cytotoxic T cells, at higher titers in the tumor microenvironment containing TNFR2+ cancer cells (e.g., Hodgkin's lymphoma cells, cutaneous non-Hodgkin's 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 or lymphatic cancer cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.) than at sites that do not contain such cancer cells, such as sites distant from the tumor, in patients with one or more of the above cancers or in cancer-free subjects. T effector cells (e.g., CD8+ cytotoxic T cells) specifically react with antigens present on, for example, one or more cancer cells, such as Hodgkin's lymphoma cells, cutaneous non-Hodgkin's 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 or lymphatic cancer cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, among other cancers described herein.

[0219] Activity of antigen-binding fragments of full-length TNFR2 antagonist antibodies The antagonistic TNFR2 antibodies described herein may, for example, inhibit the proliferation of T-regs, cancer cells, and / or MDSCs, or promote the proliferation of T effector cells, with potency similar to that exhibited by antigen-binding fragments of such antibodies. For example, removal of the Fc region of an antagonistic TNFR2 antibody described herein may not alter the ability of the molecule to attenuate the proliferation of T-reg cells, MDSCs, and / or cancer cells, or to reduce the total amount of T-reg cells, MDSCs, and / or cancer cells, in a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infectious disease as described herein). The antagonistic TNFR2 antibodies and antigen-binding fragments thereof described herein may function, for example, via a pathway distinct from antibody-dependent cellular cytotoxicity (ADCC), which requires the Fc region to recruit effector proteins to induce cell death. In addition, antagonistic TNFR2 antibodies or antigen-binding fragments thereof may exhibit therapeutic activity in various forms, such as, for example, a single-chain polypeptide (e.g., a single-chain polypeptide containing one or more CDRs covalently linked to one another, e.g., by an amide bond, a thioether bond, a carbon-carbon bond, or a disulfide bridge), a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multispecific antibody or antigen-binding fragment thereof, dual variable immunoglobulin domains, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or 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).

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

[0221] Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) described herein include those having a K of less than 100 nM (e.g., 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). DIn some embodiments, the polypeptides (e.g., single chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) described herein specifically bind to TNFR2 at a concentration 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, M, 830pM, 820pM, 810pM, 800pM, 790pM, 780pM, 770pM, 760pM, 750pM, 740pM, 730pM, 720pM, 710pM, 700p M, 690pM, 680pM, 670pM, 660pM, 650pM, 640pM, 630pM, 620pM, 610pM, 600pM, 590pM, 580pM, 570pM, 560p M, 550pM, 540pM, 530pM, 520pM, 510pM, 500pM, 490pM, 480pM, 470pM, 460pM, 450pM, 440pM, 430pM, 420p M, 410pM, 400pM, 390pM, 380pM, 370pM, 360pM, 350pM, 340pM, 330pM, 320pM, 310pM, 300pM, 290pM, 280p K of 200pM, 270pM, 260pM, 250pM, 240pM, 230pM, 220pM, 210pM, 200pM, 190pM, 180pM, 170pM, 160pM, 150pM, 140pM, 130pM, 120pM, 110pM, 100pM, 90pM, 80pM, 70pM, 60pM, 50pM, 40pM, 30pM, 20pM, 10pM, 5pM, or 1pM). D It specifically binds to TNFR2 at 1000kJ / mL.

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

[0223] Kinetic properties of antagonistic TNFR2 polypeptides In addition to the thermodynamic parameters of TNFR2-polypeptide interactions, it is also possible to quantitatively characterize the kinetic binding and dissociation of the polypeptides described herein with TNFR2. This can be done, 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 formation of antibody-TNFR2 complexes (k on ) and dissociation (K off The equilibrium constant for this unimolecular dissociation is K off value and k on These data also provide information on the equilibrium constant for antibody-TNFR2 complex dissociation (K D ) is a particularly advantageous technique for measuring the kinetic and thermodynamic parameters of receptor-antibody interactions because the experiment does not require modification of either component by the attachment of a chemical label. Conversely, the receptor is typically immobilized on a solid metal surface, and the surface is treated with pulses of solutions containing increasing concentrations of antibody. Because 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 as the antibody is introduced into the system can be fitted to established regression models to calculate the association and dissociation rate constants of the antibody-receptor interaction.

[0224] The polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) exhibit high kappa binding activity when interacting with TNFR2. on value and low K off For example, the polypeptides described herein may exhibit a 10 affinity value in the presence of TNFR2, which is consistent with high affinity receptor binding. 4 M -1 s -1 Super (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 The polypeptides described herein (e.g., single-chain polypeptides, antibodies, antigen-binding fragments, and constructs thereof) can exhibit low K values when binding to TNFR2 because these polypeptides can interact with different TNFR2 epitopes with high affinity. off Residues within these epitopes may form strong intermolecular contacts with TNFR2, which may delay dissociation of the antibody-TNFR2 complex. This high receptor affinity is due to the low K offFor example, the polypeptides described herein may exhibit a 10 -3 s -1 Less than (e.g., 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 )K off The value may be indicated.

[0225] Epitopes within TNFR2 to which antagonistic TNFR2 polypeptides bind One of the challenges in developing anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) that can antagonize TNFR2 is the elucidation of the epitopes within TNFR2 that are involved in the formation of antagonistic complexes, rather than epitopes that promote signal transduction. The present disclosure provides, in part, the identification of epitopes that, upon binding, promote receptor antagonism and exhibit the following beneficial biological activities: (a) inhibiting the proliferation of T-reg cells and / or directly killing T-reg cells, for example, by binding to and inactivating TNFR2 on the surface of T-reg cells; (b) inhibiting MDSC proliferation and / or directly killing MDSC, for example, by binding to and inactivating TNFR2 on the surface of MDSC; (c) promoting the proliferation of T effector cells, such as CD8+ T cells, and / or (d) inhibiting the proliferation of and / or directly killing TNFR2-expressing cancer cells (e.g., Hodgkin's lymphoma cells, cutaneous non-Hodgkin's 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 or lymphatic cancer cells, central nervous system cancer cells, breast cancer cells, pancreatic cancer cells, gastric cancer cells, esophageal cancer cells, and upper gastrointestinal cancer cells, etc.); The present invention 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), bind to the following epitopes on human TNFR2: (a) amino acids 142 to 146 (KCRPG) of SEQ ID NO: 7; (b) amino acids 142 to 149 of SEQ ID NO: 7 (KCRPGFGV); (c) amino acids 137 to 144 of SEQ ID NO: 7 (CAPLRKCR); (d) amino acids 150 to 190 of SEQ ID NO: 7 (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI); (e) amino acids 161 to 169 of SEQ ID NO: 7 (CKPCAPGTF); (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 to 184 (SSTDICRPHQI) of SEQ ID NO: 7; (h) amino acids 126 to 140 (CALSKQEGCRLCAPL) of SEQ ID NO: 7, (i) amino acids 156 to 165 (TSDVVCKPCA) of SEQ ID NO: 7, (j) equivalent epitopes within TNFR2 of a non-human mammal, such as a non-human mammal described herein, epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to any of the above epitopes, and / or epitopes that contain one or more conservative amino acid substitutions compared to these epitopes; The antibody may specifically bind to one or more of:

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

[0228] One exemplary technique 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-organized structurally based on one or more structural constraints (e.g., backbone or side chain-to-side chain cyclization) to mimic the three-dimensional configuration of the KCRPG motif (SEQ ID NO: 19). For example, the antagonistic TNFR2 polypeptides described herein may specifically bind to such a peptide with higher affinity than the affinity of the antagonistic TNFR2 polypeptide for the peptide fragment defined by residues 48-67 of SEQ ID NO: 7 in human TNFR2 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18). For example, the antagonistic TNFR2 polypeptides described herein may bind to a peptide containing a KCRPG motif (SEQ ID NO: 19) with 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 greater affinity than the affinity of the antagonistic polypeptide for a peptide having the amino acid sequence of SEQ ID NO: 18, for example.

[0229] Antagonistic TNFR2 polypeptides that bind to TNFR2 from non-human animals In addition to binding to the epitopes in human TNFR2 detailed above, the antagonistic TNFR2 polypeptides described herein, such as dominant antagonistic TNFR2 polypeptides, also include antagonistic TNFR2 polypeptides that specifically bind to epitopes containing one or more equivalent motifs in TNFR2 derived from non-human animals. The locations of epitopes equivalent to epitopes in human TNFR2 that result in an antagonistic phenotype upon binding 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 cattle, bison, and other agricultural animals described herein.

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

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

[0232] In addition to binding to the epitope contained within the sequence KCRPGFGV (SEQ ID NO: 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). TNFR2 antibodies and antibody fragments of the present disclosure may also bind to this epitope or a larger region within TNFR2 containing this epitope (e.g., a sequence comprising at least five consecutive or non-consecutive residues 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): JPEG2025118591000016.jpg14168

[0233] The sequence of the TNFRAB1 light chain is as follows (CDRs are shown in bold): JPEG2025118591000017.jpg20169

[0234] The heavy 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] Antagonistic TNFR2 antibody TNFRAB2 The antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, may comprise one or more or all of the CDRs of TNFRAB2, another antibody that antagonizes TNFRα-TNFR2 interaction. For example, using antibody humanization methods described herein or well known in the art, antagonistic TNFR2 antibodies or antigen-binding fragments thereof may be generated using, for example, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 of TNFRAB2, as well as variants of these CDRs (e.g., variants exhibiting conservative amino acid substitutions compared to these CDR sequences).

[0236] For example, antagonistic TNFR2 polypeptides (e.g., single chain polypeptides, antibodies, and antigen-binding fragments) of the present disclosure can exhibit binding properties that are identical to or similar to the binding properties of TNFRAB2. These properties are as follows: In the presence of TNFR2, TNFRAB2 binds to 3.6099×10 5 M -1 s -1 High K on value, as well as 2.24 x 10 -4 s -1 Low K off and a K of approximately 621 pM D An epitope comprising residues 137-144 of SEQ ID NO:7 (CAPLRKCR, SEQ ID NO:11) within human TNFR2 has been identified as a particularly important component of the functional epitope that establishes intermolecular contacts with TNFRAB2, as determined by epitope mapping analysis. TNFR2 antibodies and antibody fragments that specifically bind to this epitope are included in the present disclosure.

[0237] In addition to binding to the epitope containing residues CAPLRKCR (SEQ ID NO:11), TNFRAB2 also binds to epitopes comprising one or more residues within positions 80-86 of SEQ ID NO:7 (DSTYTQL, SEQ ID NO:8) in human TNFR2, positions 91-98 of SEQ ID NO:7 (PECLSCGS, SEQ ID NO:9) in human TNFR2, and positions 116-123 of SEQ ID NO:7 (RICTCRPG, SEQ ID NO:10) in human TNFR2. TNFR2 antibodies and antibody fragments of the present disclosure may also bind to one or more of these epitopes. Information about the epitopes to which TNFRAB2 specifically binds can be used to design and identify antibodies and antibody fragments of the present disclosure. For example, any of the various in vitro peptide display techniques or combinatorial antibody library screening methods described herein or known in the art can be used to screen for antibodies capable of binding to these epitopes with high affinity and selectivity.

[0238] The heavy and light chain CDRs of TNFRAB2 are shown below. TNFRAB2 CDR-H1: GYTFTDYL (SEQ ID NO: 274) TNFRAB2 CDR-H2: VDPEYGST (SEQ ID NO: 258) TNFRAB2 CDR-H3: ARDDGSYSPFDYWG (SEQ ID NO: 259) TNFRAB2 CDR-L1: QNINKY (SEQ ID NO: 260) TNFRAB2 CDR-L2:TYS TNFRAB2 CDR-L3: CLQYVNLLT (SEQ ID NO: 272)

[0239] In addition, the CDR-L2 of TNFRAB2 is flanked by N-terminal framework residues LLIR (SEQ ID NO: 262) and C-terminal framework residues TLE. Thus, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the present disclosure include antagonistic TNFR2 polypeptides that contain, in addition to one or more of the above-described CDRs of TNFRAB2, the N-terminal LLIR residues (SEQ ID NO: 262) and C-terminal TLE residues that flank the CDR-L2 sequence of an antagonistic TNFR2 antibody or antigen-binding fragment thereof.

[0240] Antagonistic TNFR2 antibody TNFRAB3 The antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can exhibit binding characteristics identical to or similar to those of TNFRAB3. TNFRAB3 is a monoclonal antibody and a dominant TNFR2 antagonist. The monoclonal antibody TNFRAB3 has the CDR-H1 amino acid sequence of GYTFTDVI (SEQ ID NO: 293). TNFRAB3 binds to an epitope within CDR3 and / or CRD4 of human TNFR2, excluding an epitope within CRD1 of human TNFR2.

[0241] As described in detail below, the antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) described herein can be produced by generating and identifying antibodies that exhibit epitope-binding properties similar to those of TNFRAB3. Exemplary techniques for generating polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof) having epitope-binding properties similar to those of TNFRAB3 include, but are not limited to, generating fully human, humanized, primatized, and chimeric antibodies incorporating one or more or all of the complementarity-determining regions (CDRs) of TNFRAB3, as well as screening for polypeptides that specifically bind to one or more or all of the epitopes on TNFR2 to which TNFRAB3 specifically binds.

[0242] Antagonistic TNFR2 antibody TNFRAB4 The antagonistic TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs thereof, can exhibit binding properties identical to or similar to those of TNFRAB4. TNFRAB4 is a monoclonal murine antibody described herein. This antibody is a dominant TNFR2 antagonist. Monoclonal antibody TNFRAB4 binds to the following amino acid residues...

Claims

1. A humanized antibody or antigen-binding fragment thereof that specifically binds to human TNFR2, wherein the antibody or antigen-binding fragment thereof comprises: (a) a human IgG2 hinge region lacking the cysteine ​​residues at positions 232 and 233 of the amino acid sequence of the IgG2 hinge region numbered according to Kabat; (b) a heavy chain comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 302-306, wherein the heavy chain comprises the following CDRs: CDR-H1 having the amino acid sequence of SEQ ID NO: 274, CDR-H2 having the amino acid sequence of SEQ ID NO: 258, and CDR-H3 having the amino acid sequence of SEQ ID NO: 259; and (c) a light chain comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 297-301, wherein the light chain comprises the following CDRs: CDR-L1 having the amino acid sequence of SEQ ID NO: 260, CDR-L2 having the amino acid sequence YTS, and CDR-L3 having the amino acid sequence of SEQ ID NO: 273; The antibody or antigen-binding fragment thereof comprising:

2. An antibody or antigen-binding fragment thereof that specifically binds to human TNFR2, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 302, and a light chain comprising the amino acid sequence of SEQ ID NO: 297; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 302, and a light chain comprising the amino acid sequence of SEQ ID NO: 298; (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 302, and a light chain comprising the amino acid sequence of SEQ ID NO: 299; (d) a heavy chain comprising the amino acid sequence of SEQ ID NO: 302, and a light chain comprising the amino acid sequence of SEQ ID NO: 300; (e) a heavy chain comprising the amino acid sequence of SEQ ID NO: 302, and a light chain comprising the amino acid sequence of SEQ ID NO: 301; (f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 303, and a light chain comprising the amino acid sequence of SEQ ID NO: 297; (g) a heavy chain comprising the amino acid sequence of SEQ ID NO: 303, and a light chain comprising the amino acid sequence of SEQ ID NO: 298; (h) a heavy chain comprising the amino acid sequence of SEQ ID NO: 303, and a light chain comprising the amino acid sequence of SEQ ID NO: 299; (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 303, and a light chain comprising the amino acid sequence of SEQ ID NO: 300; (j) a heavy chain comprising the amino acid sequence of SEQ ID NO: 303, and a light chain comprising the amino acid sequence of SEQ ID NO: 301; (k) a heavy chain comprising the amino acid sequence of SEQ ID NO: 304, and a light chain comprising the amino acid sequence of SEQ ID NO: 297; (l) a heavy chain comprising the amino acid sequence of SEQ ID NO: 304, and a light chain comprising the amino acid sequence of SEQ ID NO: 298; (m) a heavy chain comprising the amino acid sequence of SEQ ID NO: 304, and a light chain comprising the amino acid sequence of SEQ ID NO: 299; (n) a heavy chain comprising the amino acid sequence of SEQ ID NO: 304, and a light chain comprising the amino acid sequence of SEQ ID NO: 300; (o) a heavy chain comprising the amino acid sequence of SEQ ID NO: 304, and a light chain comprising the amino acid sequence of SEQ ID NO: 301; (p) a heavy chain comprising the amino acid sequence of SEQ ID NO: 305, and a light chain comprising the amino acid sequence of SEQ ID NO: 297; (q) a heavy chain comprising the amino acid sequence of SEQ ID NO: 305, and a light chain comprising the amino acid sequence of SEQ ID NO: 298; (r) a heavy chain comprising the amino acid sequence of SEQ ID NO: 305, and a light chain comprising the amino acid sequence of SEQ ID NO: 299; (s) a heavy chain comprising the amino acid sequence of SEQ ID NO: 305, and a light chain comprising the amino acid sequence of SEQ ID NO: 300; (t) a heavy chain comprising the amino acid sequence of SEQ ID NO: 305, and a light chain comprising the amino acid sequence of SEQ ID NO: 301; (u) a heavy chain comprising the amino acid sequence of SEQ ID NO: 306, and a light chain comprising the amino acid sequence of SEQ ID NO: 297; (v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 306, and a light chain comprising the amino acid sequence of SEQ ID NO: 298; (w) a heavy chain comprising the amino acid sequence of SEQ ID NO: 306, and a light chain comprising the amino acid sequence of SEQ ID NO: 299; (x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 306 and a light chain comprising the amino acid sequence of SEQ ID NO: 300; or (y) a heavy chain comprising the amino acid sequence of SEQ ID NO: 306, and a light chain comprising the amino acid sequence of SEQ ID NO: 301; The antibody or antigen-binding fragment thereof comprising:

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 302 and the light chain comprises the amino acid sequence of SEQ ID NO:

297.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 302 and the light chain comprises the amino acid sequence of SEQ ID NO:

298.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 302 and the light chain comprises the amino acid sequence of SEQ ID NO:

299.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 302 and the light chain comprises the amino acid sequence of SEQ ID NO:

300.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 302 and the light chain comprises the amino acid sequence of SEQ ID NO:

301.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 303 and the light chain comprises the amino acid sequence of SEQ ID NO:

297.

9. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 303 and the light chain comprises the amino acid sequence of SEQ ID NO:

298.

10. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 303 and the light chain comprises the amino acid sequence of SEQ ID NO:

299.

11. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 303 and the light chain comprises the amino acid sequence of SEQ ID NO:

300.

12. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 303 and the light chain comprises the amino acid sequence of SEQ ID NO:

301.

13. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 304 and the light chain comprises the amino acid sequence of SEQ ID NO:

297.

14. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 304 and the light chain comprises the amino acid sequence of SEQ ID NO:

298.

15. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 304 and the light chain comprises the amino acid sequence of SEQ ID NO:

299.

16. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 304 and the light chain comprises the amino acid sequence of SEQ ID NO:

300.

17. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 304 and the light chain comprises the amino acid sequence of SEQ ID NO:

301.

18. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 305 and the light chain comprises the amino acid sequence of SEQ ID NO:

297.

19. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 305 and the light chain comprises the amino acid sequence of SEQ ID NO:

298.

20. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 305 and the light chain comprises the amino acid sequence of SEQ ID NO:

299.

21. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 305 and the light chain comprises the amino acid sequence of SEQ ID NO:

300.

22. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 305 and the light chain comprises the amino acid sequence of SEQ ID NO:

301.

23. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 306 and the light chain comprises the amino acid sequence of SEQ ID NO:

297.

24. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 306 and the light chain comprises the amino acid sequence of SEQ ID NO:

298.

25. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 306 and the light chain comprises the amino acid sequence of SEQ ID NO:

299.

26. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 306 and the light chain comprises the amino acid sequence of SEQ ID NO:

300.

27. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 306 and the light chain comprises the amino acid sequence of SEQ ID NO:

301.

28. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof has at least 90%, 91%, 92%, 93%, or 94% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 302-306, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof has at least 90%, 91%, 92%, 93%, or 94% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 297-301.

29. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 302-306, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 297-301.

30. The antibody or antigen-binding fragment thereof according to claim 1, wherein the human IgG2 hinge region of the antibody or antigen-binding fragment thereof has a serine residue at positions 232 and / or 233.

31. The antibody or antigen-binding fragment thereof according to claim 1, wherein the human IgG2 hinge region of the antibody or antigen-binding fragment thereof has serine residues at positions 232 and 233.

32. A humanized antibody or antigen-binding fragment thereof that specifically binds to human TNFR2, wherein the antibody or antigen-binding fragment thereof comprises: (a) a human IgG2 hinge region lacking the cysteine ​​residues at positions 232 and 233 of the amino acid sequence of the IgG2 hinge region numbered according to Kabat; (b) a heavy chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 302, wherein the heavy chain comprises the following CDRs: CDR-H1 having the amino acid sequence of SEQ ID NO: 274, CDR-H2 having the amino acid sequence of SEQ ID NO: 258, and CDR-H3 having the amino acid sequence of SEQ ID NO: 259; and (c) a light chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 299, wherein the light chain comprises the following CDRs: CDR-L1 having the amino acid sequence of SEQ ID NO: 260, CDR-L2 having the amino acid sequence YTS, and CDR-L3 having the amino acid sequence of SEQ ID NO:

273. The antibody or antigen-binding fragment thereof comprising:

33. The antibody or antigen-binding fragment thereof of claim 32, wherein the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof has at least 91%, 92%, 93%, or 94% sequence identity to the amino acid sequence of SEQ ID NO: 302, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof has at least 91%, 92%, 93%, or 94% sequence identity to the amino acid sequence of SEQ ID NO:

299.

34. The antibody or antigen-binding fragment thereof of claim 32, wherein the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 302, and the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:

299.

35. The antibody or antigen-binding fragment thereof of claim 32, wherein the human IgG2 hinge region of the antibody or antigen-binding fragment thereof has a serine residue at position 232 and / or 233.

36. 33. The antibody or antigen-binding fragment thereof of claim 32, wherein the human IgG2 hinge region of the antibody or antigen-binding fragment thereof has serine residues at positions 232 and 233.

37. 34. The antibody or antigen-binding fragment thereof of claim 33, wherein the human IgG2 hinge region of the antibody or antigen-binding fragment thereof has serine residues at positions 232 and 233.

38. 35. The antibody or antigen-binding fragment thereof of claim 34, wherein the human IgG2 hinge region of the antibody or antigen-binding fragment thereof has serine residues at positions 232 and 233.

39. 39. A construct comprising a first polypeptide domain and a second polypeptide domain, wherein the first polypeptide domain and the second polypeptide domain are each independently an antigen-binding fragment of any one of claims 1 to 38.

40. A pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 38, or a construct comprising a first polypeptide domain and a second polypeptide domain, wherein the first polypeptide domain and the second polypeptide domain are each independently an antigen-binding fragment according to any one of claims 1 to 38; and (ii) A pharmaceutically acceptable carrier.

41. 41. The pharmaceutical composition of claim 40 for use in the treatment of a human disease or disorder.

42. 42. The pharmaceutical composition of claim 41, (a) the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, an antibody-like protein scaffold, a Fab fragment, a F(ab') ２ molecule, and tandem scFv (taFv), (b) the antibody or antigen-binding fragment thereof is a human IgG2 isotype antibody or antigen-binding fragment thereof; and / or (c) the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent; The pharmaceutical composition.

43. 43. The pharmaceutical composition of claim 42, wherein the therapeutic agent is a cytotoxic agent.

44. The disease or disorder is (a) T-reg cell-mediated immune responses; (b) a cell proliferation disorder, or (c) Infectious disease 42. The pharmaceutical composition of claim 41, comprising:

45. 45. The pharmaceutical composition of claim 44, 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) a cancer selected from the group consisting of Hodgkin's lymphoma, cutaneous non-Hodgkin's lymphoma, T-cell lymphoma, ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, skin cancer, lung cancer, endometrial cancer, cancer of the hematopoietic or lymphatic system, cancer of the central nervous system, stomach cancer, esophageal cancer, and cancer of the upper gastrointestinal tract; 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, appendix cancer, astrocytoma, atypical teratoid / rod tumor, basal cell carcinoma, cholangiocarcinoma, extrahepatic cancer, Ewing's sarcoma family, osteosarcoma, and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasm, colon cancer, extrahepatic bile duct carcinoma, ductal carcinoma in situ (DCIS) ), endometrial cancer, ependymoma, esthesioneuroblastoma, 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, childhood brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal carcinoma, pancreatic 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 carcinoma), multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's 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 sinus and nasal cavity cancer, parathyroid carcinoma, penile cancer, pharyngeal cancer, brown cell carcinoma Cancers selected from the group consisting of: cysts, pituitary tumors, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, kidney cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, rhabdomyosarcoma, Sezary 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 cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom's macroglobulinemia The pharmaceutical composition,

46. 45. The pharmaceutical composition of claim 44, The infection is (a) one or more agents 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, Tyureni virus, Aroa virus, dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Ustu virus, West Nile virus, Yaounde virus, Kocobera virus, Bagaza virus, ileus virus, Israeli turkey meningoencephalitis virus, Untaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge virus Hill virus, Jugra virus, Saboya virus, Sepic virus, Uganda S virus, Wesselsbron virus, Yellow fever virus, Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey virus Island virus, Dakar bat virus, Montana myotis bat leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, fusion factor virus, Ippy virus, Lassa virus, lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexar virus, Guanarito virus, Junin 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, Hantan virus, Sin Nombre virus, Dugbe virus, Bunyambera 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, Chikungunya virus, Smallpox virus, Monkeypox virus, Vaccinia virus, Herpes simplex virus, Human herpesvirus, Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-zoster virus, Kaposi's sarcoma-associated herpesvirus (KSHV), Influenza virus, Severe acute respiratory syndrome (SARS) virus, Rabies virus, Vesicular stomatitis virus (VS) V), a virus selected from the group consisting of 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; (c) Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobac ter, Moraxella, Enterobacteriacece, Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, P Bacteria belonging to a genus selected from the group consisting of: Bacillus subtilis, Bacillus spp. ... (d) a fungus selected from the group consisting of Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, and Absidia; or (e) Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiensis, 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 a parasite selected from the group consisting of 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, wherein the

47. 42. The pharmaceutical composition of claim 41, wherein the pharmaceutical composition further comprises another therapeutic agent.

48. 48. The pharmaceutical composition of claim 47, wherein the additional therapeutic agent is an immunotherapeutic agent.

49. 41. The pharmaceutical composition of claim 40, wherein the pharmaceutical composition is for administration to a human in conjunction with an immunotherapeutic agent.

50. 49. The pharmaceutical composition of claim 48, (a) 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, or an anti-B7 agent. H4 agents, anti-HHLA2 agents, anti-CD155 agents, anti-CD80 agents, anti-BTLA agents, anti-CD160 agents, anti-CD28 agents, anti-CD226 agents, anti-CEACAM1 agents, anti-TIM3 agents, anti-TIGIT agents, anti-CD96 agents, anti-CD70 agents, anti-CD27 agents, anti-LIGHT agents, anti-CD137 agents, anti-DR4 agents, anti-CR5 agents, anti-TNFRS 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 antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-PD-L2 antibody or antigen-binding fragment thereof, a TNF-α cross-linking antibody or antigen-binding fragment thereof, a TRAIL cross-linking antibody or antigen-binding fragment thereof, an anti-CD27 antibody or antigen-binding fragment thereof, an anti-CD30 antibody or antigen-binding fragment thereof, an anti-CD40 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, an anti-GITR antibody or antigen-binding fragment thereof, an anti-OX40 antibody or antigen-binding fragment thereof, an anti-TRAILR1 antibody or antigen-binding fragment thereof, an anti-TRAILR2 antibody or antigen-binding fragment thereof, an anti-TWEAK antibody or antigen-binding fragment thereof, or an anti-TWEAKR antibody or antigen-binding fragment thereof, anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, anti-BRAF antibody or antigen-binding fragment thereof, anti-MEK antibody or antigen-binding fragment thereof, anti-CD33 antibody or antigen-binding fragment thereof, anti-CD20 antibody or antigen-binding fragment thereof, anti-HLA-DR antibody or antigen-binding fragment thereof, anti-HLA class I antibody or antigen-binding fragment thereof, anti-CD52 antibody or antigen-binding fragment thereof, anti-A33 antibody or antigen-binding fragment thereof, anti-GD3 antibody or antigen-binding fragment thereof, anti-PSMA antibody or antigen-binding fragment thereof, anti-Ceacan1 antibody or antigen-binding fragment thereof, anti-Galedin9 antibody or antigen-binding fragment thereof, anti-HVEM antibody or antigen-binding fragment thereof, anti-VISTA antibody or antigen-binding fragment thereof, anti-B7H4 antibody or antigen-binding fragment thereof, anti-HHLA2 antibody or antigen-binding fragment thereof, anti-CD155 antibody or antigen-binding fragment thereof, anti-CD80 antibody or antigen-binding fragment thereof, anti-BTLA antibody or antigen-binding fragment thereof, anti-CD160 antibody or antigen-binding fragment thereof, anti-CD28 antibody or antigen-binding fragment thereof, anti-CD226 antibody or antigen-binding fragment thereof, anti-CEACAM1 antibody or antigen-binding fragment thereof, anti-TIM3 antibody or antigen-binding fragment thereof, anti-TIGIT antibody or antigen-binding fragment thereof, anti-CD96 antibody or antigen-binding fragment thereof, anti-CD70 antibody or antigen-binding fragment thereof, anti-CD27 antibody or antigen-binding fragment thereof, anti-LIGHT antibody or antigen-binding fragment thereof, anti-CD137 antibody or antigen-binding fragment thereof, anti-DR4 antibody or antigen-binding fragment thereof, anti-CR5 antibody or antigen-binding fragment thereof an antibody or fragment thereof, an anti-TNFRS antibody or antigen-binding fragment thereof, an anti-TNFR1 antibody or antigen-binding fragment thereof, an anti-FAS antibody or antigen-binding fragment thereof, an anti-CD95 antibody or antigen-binding fragment thereof, an anti-TRAIL antibody or antigen-binding fragment thereof, an anti-DR6 antibody or antigen-binding fragment thereof, an anti-EDAR antibody or antigen-binding fragment thereof, an anti-NGFR antibody or antigen-binding fragment thereof, an anti-OPG antibody or antigen-binding fragment thereof, an anti-RANKL antibody or antigen-binding fragment thereof, an anti-LTβ receptor antibody or antigen-binding fragment thereof, an anti-BCMA antibody or antigen-binding fragment thereof, an anti-TACI antibody or antigen-binding fragment thereof, an anti-BAFFR antibody or antigen-binding fragment thereof, an anti-EDAR2 antibody or antigen-binding fragment thereof, an anti-TROY antibody or antigen-binding fragment thereof, and an anti-RELT antibody or antigen-binding fragment thereof; The pharmaceutical composition.

51. 51. The pharmaceutical composition of claim 50, wherein the immunotherapeutic agent is ipilimumab, tremelimumab, nivolumab, pembrolizumab, avelumab, durvalumab, or atezolizumab.

52. 41. The pharmaceutical composition of claim 40, wherein the pharmaceutical composition for administration to a human comprises an amount of the antibody or antigen-binding fragment thereof from about 0.001 mg / kg to about 100 mg / kg.

53. 41. A kit comprising the pharmaceutical composition of claim 40.

Images