Antagonistic anti-tumor necrosis factor receptor superfamily antibodies

Abstract

The present invention relates to antagonistic TNFR2 polypeptides, such as antibodies and antigen-binding fragments thereof, and the use of these polypeptides to inhibit the proliferation of regulatory T cells (T-regs) and / or expand T effector cell populations or function. For example, antibodies of the invention include antagonistic TNFR2 antibodies and antigen-binding fragments thereof, and can be used to suppress the T-reg-mediated deactivation of tumor reactive T-lymphocytes, as well as to treat a wide variety of cancers and infectious diseases. 20 23 25 83 20 30 O ct 2 02 3 A B S T R A C T 2 0 2 3 2 5 8 3 2 0 3 0 O c t 2 0 2 3

Description

5 Field of the Invention The invention relates to polypeptides, such as antibodies and antigen-binding fragments thereof, capable of antagonizing tumor necrosis factor receptor 2. The polypeptides of the invention can be used to modulate the activity of regulatory T cells, as well as to upregulate the activity of T effector cells and to 10 directly modulate surface oncogenes, such as in the field of immunotherapy for the treatment of cell proliferation disorders and infectious diseases. Background of the Invention The use of naturally-occurring and genetically engineered T-lymphocytes is a prominent 15 paradigm for ameliorating various human pathologies. For instance, while traditional therapeutic platforms for the treatment of cancer include surgical removal of tumor mass, radiation therapy, and administration of chemotherapeutics (Shewach, Chern. Rev., 109:2859-2861,2009), the last decade has witnessed a resurgence in the application of adoptive immunotherapy to cancer treatment regimens. With the advent of chimeric antigen receptor (CAR-T) therapy, new methods have emerged for the infusion of autologous 20 and allogeneic tumor-reactive T cells to patients (June, J. Clin. Invest., 117:1466-1476, 2007). CAR-T therapies harness the resources of the adaptive immune response in order to promote cancer cell cytotoxicity and eradicate tumor material. A common motif in adoptive immunotherapy is the use of T cells that exhibit the ability to selectively potentiate cytotoxicity in cells that display distinct tumor antigens. Examples of this technique include the administration of tumor-infiltrating lymphocytes (Dudley et al., J. 25 Immunother., 26:332-342, 2003), as well as autologous or allogeneic T cells that have been genetically re-engineered so as to exhibit reactivity with a tumor-specific antigen (Yee et al., PNAS., 99:1616816173, 2002). Despite the promise of T-lymphocyte-based cancer immunotherapy, the development of this therapeutic platform has been hindered by the natural propensity of the immune system to suppress 30 immune attacks mounted on self cells. Cancer cells, like all nucleated human cells, express class I major histocompatability complex (MHC) proteins that distinguish these cells from foreign cells. In order to prevent cell fratricide, regulatory T cells (T-reg cells) have evolved that suppress the activity of T cells that exhibit reactivity against “self” MHC antigens. T-reg cells represent a heterogeneous class of T cells that can be distinguished based on their unique surface protein presentation. The most well-understood 35 populations of T-reg cells include CD4+, CD25+, FoxP3+ T-reg cells and CD17+ T-reg cells. The precise mechanisms by which these cells mediate suppression of autoreactive T cells is the subject of ongoing investigations, though it has been shown that certain classes of T reg cells inhibit production of the proliferation-inducing cytokine IL-2 in target T cells and may additionally sequester IL-2 from autoreactive cells by virtue of the affinity of CD25 (a subdomain of the IL-2 receptor) for IL-2 (Josefowicz et al., Ann. £023258330   30 Get 2023 Rev. Immun., 30:531-564, 2012). Although T-reg cells play an important role in maintaining peripheral tolerance, the same biochemical features that underlie the ability of these cells to modulate autoreactive T cell activity also serve to undermine adoptive immunotherapy and the natural immune response by suppressing the activity of tumor-reactive T lymphocytes. The development of chemical modulators of T-reg cell activity has been the subject of many pharmacological investigations, as access to an agent capable of inhibiting T-reg-mediated T cell suppression could vastly improve the scope and efficacy of adoptive cancer immunotherapy, as well as improve the ability of the immune system to eradicate pathogenic organisms that give rise to infectious diseases. Tumor necrosis factor receptor (TNFR) subtypes 1 and 2 have been identified on the T-reg cell surface as signal transduction molecules that dictate cell fate. The activation of TNFR1, for instance, potentiates the caspase signaling cascade and terminates in T-reg apoptosis, while activation of TNFR2 induces signaling through the mitogen-activated protein kinase (MAPK) signaling pathway, which orchestrates signaling through TRAF2 / 3 and the NFKB-mediated transcription of genes that promote escape from apoptosis and cell proliferation. Due to its role in directing cell survival and growth, TNFR2 represents an attractive target for preventing immune detection of turnor-reactive T lymphocytes. As such, there is currently a need for therapies that can prevent T-reg cell survival and proliferation for use in treatments targeting cell proliferation disorders, such as cancer, and a wide array of infectious diseases. 20                                    Summary of the Invention The invention provides antagonistic tumor necrosis factor receptor 2 (TNFR2) polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof. Antagonistic TNFR2 polypeptides of the invention specifically bind epitopes within human TNFR2 that contain one or more residues of the KCRPG sequence (SEQ ID NO: 19) or equivalent epitopes in TNFR2 of non-human 25 primates (e.g., bison or cattle, as described herein) and do not specifically bind residues of the KCSPG motif (SEQ ID NO: 12) within human TNFR2 or equivalent epitopes in TNFR2 of non-human primates. The polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) described herein can be used for the treatment of a variety of pathologies, including cancers and infectious diseases. 30           Antagonistic TNFR2 polypeptides of the invention are described as such since they exhibit the ability to inhibit the proliferation of, and / or promote the death of, T-reg cells. Antagonists TNFR2 polypeptides of the invention may inhibit the proliferation of, and / or promote the death of, TNFR2- and oncogene-expressing cancer cells. Antagonistic TNFR2 polypeptides of the invention can permit the reciprocal expansion of T effector cells, such as cytotoxic CD8+ T cells. This may occur, for instance, 35 due to the attenuation of T-reg cell proliferation and activity. Antagonistic TNFR2 polypeptides of the invention may directly expand T effector cells, such as CD8+ cytotoxic T cells. Therefore, the designation of TNFR2 polypeptides of the invention as antagonists refers to their capacity to attenuate the proliferation and activity of T-reg and TNFR2-expressing cancer ceils and, for clarity, does not indicate antagonism of the T effector cell response. WO 2017 / 197331                                                 PCT / US2017 / 032513 Disclosed herein are polypeptides, such as single-chain polypeptides, antibodies, or antigen 2023258320   30 Oct 2023 binding fragments thereof, capable of specifically binding human TNFR2 that contain a complementarity determining region-heavy chain 1 (CDR-H1) and a CDR-H2 derived from a TNFR2 antibody and a CDR-H3 having the amino acid sequence JZ1JZ2Z4JZ3JZ5(J)2Z5Z2Z5, JZ1JZ2Z4Z3Z5(J)2Z5Z2Z5(J)2, 5 JRJDGJSJY(J)2FDJ (SEQ ID NO: 278), JRJDGSY(J)2FD(J)3 (SEQ ID NO: 279), QZ1VZ2Z4YZ3SZ5WYZ5Z2Z5 (SEQ ID NO: 265), or AZ1 DZ2Z4Z3Z5SPZ5Z2Z5WG (SEQ ID NO: 266), wherein each J is independently a naturally occurring amino acid; each Z1 is independently a naturally occurring amino acid containing a cationic side-chain at 10 physiological pH, such as lysine, arginine, and histidine; each Z2 is independently a naturally occurring amino acid containing an anionic side-chain at physiological pH, such as aspartic acid and glutamic acid; each Z3 is independently a naturally occurring amino acid containing a polar, uncharged sidechain at physiological pH, such as serine, threonine, asparagine, and glutamine; 15           each Z4 is independently a glycine or alanine; and each Z5 is independently a naturally occurring amino acid containing a hydrophobic side-chain, such as alanine, valine, leucine, isoleucine, proline, and methionine, tryptophan, phenylalanine, and tyrosine. As used herein in the context of a polypeptide formula, numeric characters in subscript notation designate the quantity of the preceding amino acid present in the formula, and numeric characters in 20 superscript notation designate the type of the preceding amino acid present in the formula. In a first aspect, the invention features a polypeptide, such as a single-chain polypeptide, antibody, or antigen-binding fragment thereof capable of specifically binding human tumor necrosis factor receptor 2 (TNFR2), wherein the single-chain polypeptide, antibody, or antigen-binding fragment thereof contains a CDR-H3 having the amino acid sequence of: 25          (a) JRJDGSY(J)2FD(J)3(SEQ ID NO: 279); (b) AZ1DZ2Z4Z3Z5SPZ5Z2Z5WG (SEQ ID NO: 266); or (c) ARDDGSYSPFDYWG (SEQ ID NO: 259) or an amino acid sequence having up to two amino acid substitutions relative to said sequence; wherein each J is independently a naturally occurring amino acid; 30           each Z1 is independently a naturally occurring amino acid comprising a cationic side-chain at physiological pH, such as lysine, arginine, and histidine; each Z2 is independently a naturally occurring amino acid comprising an anionic side-chain at physiological pH, such as aspartic acid and glutamic acid; each Z3 is independently a naturally occurring amino acid comprising a polar, uncharged side- 35 chain at physiological pH, such as serine, threonine, asparagine, and glutamine; each Z4 is independently a glycine or alanine; and each Z5 is independently a naturally occurring amino acid comprising a hydrophobic side-chain, such as alanine, valine, leucine, isoleucine, proline, and methionine, tryptophan, phenylalanine, and tyrosine. £023258330   30 Get 2023 In some embodiments, in the presence of the above CDR-H3, the antibody or antigen-binding fragment thereof is capable of specifically binding a peptide comprising the amino acid sequence of LRKCRPGFGVA (SEQ ID NO: 285) or VVCKPCAPGTFSN (SEQ ID NO: 286). In some embodiments, in the presence of the above CDR-H3, the antibody or antigen-binding fragment thereof is capable of specifically binding an epitope within amino acids 142-149 (KCRPGFGV) or amino acids 161-169 (CKPCAPGTF) of SEQ ID NO: 7. In some embodiments, the CDR-H3 has the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259). In some embodiments, the CDR-H3 has the amino acid sequence ARDDGSYSPFDYFG (SEQ ID NO: 284). In some embodiments, the single-chain polypeptide, antibody, or antigen-binding fragment thereof contains one or more, or all, of the following regions (CDRs): (a) a CDR-H1 having the amino acid sequence GJTF(J)2YJ (SEQ ID NO: 277); (b) a CDR-H2 having the amino acid sequence (J)sGSJ; (c) a CDR-L1 having the amino acid sequence (J)sY; (d) a CDR-L2 having the amino acid sequence (J)2S; and (e) a CDR-L3 having the amino acid sequence (J)3Y(J)4T. wherein each J is independently a naturally occurring amino acid. In some embodiments, the single-chain polypeptide, antibody, or antigen-binding fragment thereof contains one or more, or all, of the following regions (CDRs): 20          (a) a CDR-H1 having the amino acid sequence Z4YZ3Z5TDZ5X; (b) a CDR-H2 having the amino acid sequence VDPEYZ4Z3T (SEQ ID NO: 264); (c) a CDR-L1 having the amino acid sequence QNINKZ5 (SEQ ID NO: 268); (d) a CDR-L2 having the amino acid sequence TYZ3 or YTZ3; and (e) a CDR-L3 having the amino acid sequence CLQZ5VNLXZ3(SEQ ID NO: 271); 25           wherein each Z1 is independently an amino acid comprising a cationic side-chain at physiological pH; each Z2 is independently an amino acid comprising an anionic side-chain at physiological pH; each Z3 is independently an amino acid comprising a polar, uncharged side-chain at physiological pH; 30           each Z4 is independently a glycine or alanine; each Z5 is independently an amino acid comprising a hydrophobic side-chain; and each X is independently leucine or isoleucine. In some embodiments, the single-chain polypeptide, antibody, or antigen-binding fragment thereof contains one or more, or ali, of the following regions (CDRs): 35          (a) a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257) or an amino acid sequence having up to two amino acid substitutions relative to the sequence; (b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258) or an amino acid sequence having up to two amino acid substitutions relative to the sequence; 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 (c) a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260) or an amino acid sequence having up to two amino acid substitutions relative to the sequence; (d) a CDR-L2 having the amino acid sequence TYS or YTS; and (e) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261) or an amino acid 5 sequence having up to two amino acid substitutions relative to the sequence; wherein each X is independently leucine or isoleucine. In some embodiments, the single-chain polypeptide, antibody, or antigen-binding fragment thereof contains one or more, or all, of the following regions (CDRs): (a) a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257); 10          (b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258); (c) a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260); (d) a CDR-L2 having the amino acid sequence TYS or YTS; and (e) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261). wherein each X is independently leucine or isoleucine. 15          In some embodiments, the CDR-H1 has the amino acid sequence GYTFTDYL (SEQ ID NO: 274). In some embodiments, the CDR-H1 has the amino acid sequence GYTFTDYI (SEQ ID NO: 275). In some embodiments, the CDR-L2 has the amino acid sequence TYS. In some embodiments, the CDR-L2 has the amino acid sequence YTS. In some embodiments, the CDR-L3 has the amino acid sequence CLQYVNLLT (SEQ ID NO: 20   272). In some embodiments, the CDR-L3 has the amino acid sequence CLQYVNLIT (SEQ ID NO: 273). In some embodiments, the CDR-H1 has the amino acid sequence GYTFTDYL (SEQ ID NO: 274) and the CDR-L3 has the amino acid sequence CLQYVNLIT (SEQ ID NO: 273). In some embodiments, the single-chain polypeptide, antibody, or antigen-binding fragment thereof contains a framework region that contains the amino acid sequence LLIR (SEQ ID NO: 262) 25 bound to the N-terminus of the CDR-L2. In some embodiments, the single-chain polypeptide, antibody, or antigen-binding fragment thereof contains a framework region that contains the amino acid sequence TLE bound to the C-terminus of the CDR-L2. 30 In some embodiments, the single-chain polypeptide, antibody, or antigen-binding fragment does not include one or more of the following CDRs: (a) a CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 23); (b) a CDR H2 having the amino acid sequence SSGGSY (SEQ ID NO: 24); and (c) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID NO: 26); (d) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO: 27); (e) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO: 28); (f) a CDR-L1 containing the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO: 29); (g) a CDR-L2 containing the amino acid sequence LASNLES (SEQ ID NO: 30); and (h) a CDR-L3 containing the amino acid sequence QHSRELPRT (SEQ ID NO: 31). £023258330   30 Get 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 In some embodiments, the antibody or antigen-binding fragment thereof contains a non-native constant region, such as a human constant region, lacks all or a portion of an Fc domain, lacks all or a portion of a native Fc domain, or lacks an Fc domain altogether. In another aspect, the invention provides constructs containing a first polypeptide domain and a second polypeptide domain, each of which contains a single-chain polypeptide of the invention. The first and second polypeptide domains may be the same. In some embodiments, the first and second polypeptide domains may be different. The first and second polypeptide domains may be bound by a linker, such as a linker containing an amide bond or a disulfide bridge. The constructs may lack a murine Fc domain. Polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention may specifically bind to a peptide containing the amino acid sequence of any one of SEQ ID NOs: 11, 19, 20, and 34-117 with a Kd of less than about 100 nM and do not bind peptides containing amino acids 56-60 (KCSPG) of SEQ ID NO: 7. Similarly, polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the invention may specifically bind a peptide containing one or more of amino acids 142-146 of SEQ ID NO: 7 (KCRPG) and do not bind peptides containing amino acids 56-60 of SEQ ID NO: 7 (KCSPG). Polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention may inhibit TNFR2 signaling. In some embodiments, the single-chain polypeptide, antibody, or antigen-binding fragment thereof reduces or inhibits the expression of one or 20 more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and CIAP2 / BIRC3. In some embodiments, the single-chain polypeptide, antibody, or antigen-binding fragment thereof inhibits NFkB activation. For instance, antagonistic TNFR2 single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the invention may reduce or inhibit the expression or post-translational modification (e.g., phosphorylation) of one or more of CHUK, NFKBIE, NFKBIA, 25 MAP3K11, TRAF2, TRAF3, relB, or clAP2 / BIRC3, e.g., 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%, or 100% relative to the expression or post-translational modification (e.g., phosphorylation) of one or more of these molecules isolated from a sample not treated with an antagonistic TNFR2 single-chain polypeptide, antibody, or antigen-binding fragment thereof of the invention. Exemplary assays that can be used to 30 determine expression level and phosphorylation state are known in the art and include Western blot assays to determine protein content and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments to determine mRNA content. In preferred embodiments, anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) are dominant TNFR2 antagonists and are thus capable of inhibiting TNFR2 activation even in the presence of a TNFR2 agonist 35 (such as TNFa) or a growth-promoting agent, such as IL-2. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs of the invention) may exhibit one or more, or all, of the following properties: 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 a. Suppression of T-reg cell proliferation, for instance, by binding and inactivating TNFR2 on the T-reg cell surface; b. Suppression of MDSC proliferation, for instance, by binding and inactivating TNFR2 on the MDSC surface; 5 c. Promotion of the expansion of T effector cells, such as CD8+ T cells; and / or d. Suppression of the proliferation of TNFR2-expressing cancer cells, such as T cell lymphoma cells (e.g., Hodgkin’s lymphoma cells and cutaneous non-Hodgkin’s lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, and renal cell carcinoma cells. For example, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or 10 antigen-binding fragments thereof of the invention may reduce the total quantity of T-reg or cancer cells in a patient (such as a human patient) or within a sample (e.g., a sample isolated from a patient, such as a human patient undergoing treatment for cancer or an infectious disease as described herein, relative to a patient or sample not treated with the antagonist). In some embodiments, the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, 15 antibody, or antigen-binding fragment thereof) reduces expression of TNFR2, e.g., by a T-reg cell or a cancer cell (such as a T cell lymphoma cell (e.g., Hodgkin’s or cutaneous non-Hodgkin’s lymphoma cell), ovarian cancer cell, colon cancer cell, multiple myeloma cell, or renal cell carcinoma cell), and / or the secretion of soluble TNFR2 by these cells. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen- 20 binding fragments thereof) of the invention may inhibit the proliferation or reduce the total quantity 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 as described herein). Polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention may be capable of reducing or inhibiting the proliferation of T-reg cells and / or 25 cancer cells that express TNFR2. For instance, the cancer cells may be selected from the group consisting of T cell lymphoma cells (e.g., Hodgkin’s and cutaneous non-Hodgkin’s lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, and renal cell carcinoma cells. Binding of TNFR2 on the cancer cell may inhibit or reduce proliferation of the cancer cell or may promote the apoptosis of the cancer cell. 30           Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention may bind TNFR2 on the surface of a myeloid-derived suppressor cell (MDSC; e.g., a ceil that expresses ail or a subset of proteins and small molecules selected from the group consisting of B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1, CD2, CD31 (PECAM-1), CD43, CD44, complement component C5a R1, F4 / 80 (EMR1), Fey RIH (CD16), Fey RII (CD32), Fey RI IA (CD32a), Fey 35 RHB (CD32b), Fey RIIB / C (CD32b / c), Fey RHC (CD32c), Fey RHIA (CD16A), Fey RI 11B (CD16b), galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1 RI, IL-4Ra, IL-6Ra, integrin a4 (CD49d), integrin aL (CD11a), integrin aM (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). Particularly, MDSCs do not express proteins selected from the group £023258330   30 Get 2023 consisting of B7-2 (CD86), B7-H4, CD11c, CD14, CD21, CD23 (FceRII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6). Binding of TNFR2 on the MDSC may inhibit or reduce proliferation of the MDSC or may promote the apoptosis of the MDSC. Polypeptides, such as singlechain polypeptides, antibodies, and antigen-binding fragments thereof of the invention do not require TNFa to reduce or inhibit the proliferation of T-reg cells, cancer cells (e.g., TNFR2-expressing cancer cells), and / or MDSCs. In some embodiments, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, reduce or inhibit the proliferation of T-reg cells with a greater potency in a patient suffering from cancer relative to a subject that does not have cancer. In some embodiments, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, reduce or inhibit the proliferation of T-reg cells with a greater potency in the microenvironment of a tumor relative to a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or in a subject without cancer. For example, in some embodiments, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, reduce or inhibit the proliferation of T-reg cells with a potency that is greater in the microenvironment of a tumor than in a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or in a subject without cancer. For instance, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may exhibit an IC50 for inhibiting the proliferation of T-reg cells in a tumor microenvironment that is less than the IC50 of the polypeptides for inhibiting the proliferation of T-reg cells in a site that is free of cancer cells by, 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, 15fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more. The polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of T-reg cells with a potency that is greater in the microenvironment of a tumor containing T cell lymphoma cells (e.g., Hodgkin’s or cutaneous nonHodgkin’s lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells than in a site that is free of such cancer cells, such as a site distal from a tumor in a patient suffering from one or more of the foregoing cancers or in a subject without cancer. In some embodiments, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, reduce or inhibit the proliferation of MDSCs with a greater potency in a patient suffering from cancer relative to a subject that does not have cancer. In some embodiments, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, reduce or inhibit the proliferation of MDSCs with a greater potency in the microenvironment of a tumor relative to a site that is free of cancer cells, such as a site distal from a turner in a patient suffering from cancer or a in a subject without cancer. For example, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention may bind TNFR2 on the surface of a MDSC present within the microenvironment of a tumor, and may inhibit or reduce proliferation of the MDSC or may 8 2023258320   30 Oct 2023 promote the apoptosis of the MDSC with a potency that is greater in the microenvironment of a tumor than at a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or in a subject without cancer. For instance, the polypeptides of the invention, such as singlechain polypeptides, antibodies, and antigen-binding fragments thereof, may exhibit an IC50 for inhibiting the proliferation of MDSCs in a tumor microenvironment that is less than the IC50 of the polypeptides for inhibiting the proliferation of MDSCs in a site that is free of cancer cells by, 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, 8fold, 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. The polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of MDSCs or may promote the apoptosis of MDSCs with a potency that is greater in the microenvironment of a tumor containing T cell lymphoma cells (e.g., Hodgkin’s 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 that is free of such cancer cells, such as a site distal from a tumor in a patient suffering from one or more of the foregoing cancers or in a subject without cancer. In some embodiments, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, expand T effector cells, such as CD8+ cytotoxic T cells, with a greater potency in a patient suffering from cancer relative to a subject that does not have cancer. In some embodiments, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, expand T effector cells, such as CD8+ cytotoxic T cells, with a greater potency in the microenvironment of a turnor relative to a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or in a subject without cancer. For instance, in some embodiments, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, directly expand T effector cells, such as CD8+ cytotoxic T cells, with a potency that is greater in the microenvironment of a tumor than in a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or in a subject without cancer. For instance, the polypeptides of the invention may have an ECso for expanding T effector cells in a cancer patient that is less than the ECso of the polypeptides for expanding T effector cells in a subject without cancer by, 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. The polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may directly expand T effector cells, such as GD8+ cytotoxic T cells, with a potency that is greater in the microenvironment of a tumor containing T cell lymphoma cells (e.g., Hodgkin's 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 that is free of such cancer cells, such as a site distal from a tumor in a patient suffering from one or more of the foregoing cancers or in 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 £023258330   30 Get 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells. In another aspect, the invention features a method of identifying a TNFR2 antagonist antibody or antigen-binding fragment thereof by: (a) exposing a heterogeneous mixture of antibodies or fragments thereof to a peptide having the amino acid sequence of ID NO: 285 or 286, or an amino acid sequence having up to two amino acid substitutions relative to said sequences; and (b) retaining antibodies or fragments thereof that specifically bind the peptide and removing antibodies or fragments thereof that do not specifically bind the peptide, thereby producing an enriched antibody mixture containing at least one the TNFR2 antagonist antibody or antigen-binding fragment thereof. In some embodiments, the method includes determining the amino acid sequence of one or more of the antibodies or antigen-binding fragments thereof in the enriched antibody mixture. In some embodiments, the peptide is bound to a surface. The antibody or antigen-binding fragment thereof may be expressed on the surface of a viral particle or a cell, such as the surface of a phage, bacterial cell, or yeast cell. The antibody or antigen-binding fragment thereof may be expressed as one or more polypeptide chains non-covalently bound to ribosomes or covalently bound to mRNA or cDNA. In some embodiments, the peptide is conjugated to a detectable label, such as a detectable label selected from the group consisting of a fluorescent molecule (e.g., green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, hoescht, 4',6-diamidino-2-phenylindole (DAPI), propidiurn iodide, fluorescein, coumarin, rhodamine, tetramethylrhoadmine, and cyanine), an epitope tag (e.g., maltose-binding protein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag, biotin, and streptavidin), and a radiolabel. In some embodiments, steps (a) and (b) of the method are sequentially repeated one or more times. In another aspect, the invention features a method of producing a TNFR.2 antagonist antibody or antigen-binding fragment thereof by immunizing a non-human mammal with a peptide containing the sequence of SEQ ID NO: 285 or 286, or an amino acid sequence having up to two amino acid substitutions relative to said sequences, and collecting serum containing the TNFR2 antagonist antibody or antigen-binding fragment thereof. The non-human mammal may be selected from the group consisting of a rabbit, mouse, rat, goat, guinea pig, hamster, horse, and sheep. In another aspect, the invention features an antibody or antigen-binding fragment thereof that is produced by the method of any of the above embodiments. Antibodies or antigen-binding fragments thereof of the invention may be full-length antibodies or antibody fragments, such as 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 prfmatized antibody or antigen-binding fragment thereof, a bispefific antibody or antigen-binding fragment thereof, a multi-specific 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 10 2023258320   30 Oct 2023 nanobody, an antibody-like protein scaffold, a domain antibody, a 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 bound to one another, e.g., by an amide bond, a thioether bond, a carbon-carbon bond, or a disulfide bridge, or by a linker, such as a linker described herein. In 5 some embodiments, the antibody or antigen-binding fragment thereof has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE. The antibody or antigen-binding fragment thereof may be conjugated, for example, to a therapeutic agent, such as a cytotoxic agent described herein. The invention features a polynucleotide encoding a single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof of the invention, as well as a vector containing such a polynucleotide. 10 The vector may be an expression vector, such as a eukaryotic expression vector, or a viral vector, such as an adenovirus (Ad, such as serotype 5, 26, 35, or 48 adenovirus), retrovirus (such as a y-retrovirus or a lentivirus), poxvirus, adeno-associated virus, baculovirus, herpes simplex virus, or a vaccinia virus (such as a modified vaccinia Ankara (MVA). The invention also features host cells, such as prokaryotic and eukaryotic (e.g., mammalian) cells containing a vector of the invention. 15            In another aspect, the invention features a pharmaceutical composition containing an antibody or antigen-binding fragment thereof, single-chain polypeptide, construct, polynucleotide, vector, or host cell of the invention (e.g., a TNFR2 antagonist antibody or antigen-binding fragment thereof) and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may contain, for example, an additional therapeutic agent, such as an immunotherapy agent. In some embodiments, the 20 immunotherapy agent is an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-L2 agent, a TNF-a cross-linking agent, a TRAIL cross-linking agent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, an anti-4-1 BB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAILR1 agent, an anti-TRAILR2 agent, or an anti-TWEAKR agent. For example, the immunotherapy agent may be an anti-CTLA-4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or 25 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-a 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-4-1 BB antibody or antigen-binding 30 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, or an anti-TWEAKR antibody or antigen-binding fragment thereof. The immijnotherapy agent may be capable of specifically binding one or more of the immunological targets described in Table 1 of Mahoney et al., Cancer Immunotherapy, 14:561-584 35    (2015), the disclosure of which is incorporated herein by reference in its entirety. For example, the immunotherapy agent may be an agent, such as an antibody or antigen-binding fragment thereof, that specifically binds one or more of OX40L, TL1A, CD40L, LIGHT, BTLA, LAG3, TIM3, Singlees, ICOS, B7-H3, B7-H4, VISTA, TMIGD2, BTNL2, CD48, KIR, LIR, LIR antibody, ILT, NKG2D, NKG2A, MICA, MICB, CD244, CSF1 R, IDO, TGFp, CD39, CD73, CXCR4, CXCL12, SIRPA, CD47, VEGF, or neuropilin. In £023258330   30 Get 2023 particular, the pharmaceutical composition contains a TNFR2 antagonist antibody or antigen-binding fragment thereof and an anti-PD-1 or anti-PDL1 antibody. In some embodiments, the immunotherapy agent is Targretin, Interferon-alpha, clobestasol, Peg Interferon (e.g., PEGASYS®), prednisone, Romidepsin, Bexarotene, methotrexate, Triamcinolone cream, anti-chemokines, Vorinostat, gabapentin, antibodies to lymphoid cell surface receptors and / or lymphokines, antibodies to surface cancer proteins, and / or small molecular therapies like Vorinostat. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, such as a chemotherapeutic agent described herein. The antibody or antigen-binding fragment thereof, singlechain polypeptide, construct, polynucleotide, vector, or host cell of the invention (e.g., a TNFR2 antagonist antibody or antigen-binding fragment thereof) may be formulated for co-administration with a chemotherapeutic agent, for instance, by admixing 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 conjugated directly to the antibody or antigen-binding fragment thereof, single-chain polypeptide, construct, polynucleotide, vector, or host cell, for instance, using bond-forming techniques described herein or known in the art. The invention also features a method of producing a polypeptide (e.g., single-chain polypeptide, construct, antibody, or antigen-binding fragment) of the invention by expressing a polynucleotide 20 encoding the single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof in a host cell and recovering the single-chain polypeptide, antibody, or antigen-binding fragment thereof from host cell medium. The invention additionally features a method of inhibiting an immune response mediated by a regulatory T cell, as well as a method of treating a cell proliferation disorder in a human, by administering 25 a single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof, polynucleotide, vector, host cell, or pharmaceutical composition (e.g., a pharmaceutical composition containing a TNFR2 antagonist antibody or antigen-binding fragment thereof and, optionally, an immunotherapy agent, such as an anti-PD-1 or anti-PDL1 antibody) of the invention to the human in need of treatment. Additionally, the invention features a composition containing a single-chain polypeptide, construct, antibody, or 30 antigen-binding fragment thereof, polynucleotide, vector, or host cell of the invention for inhibiting an immune response mediated by a regulatory T cell, as well as for treating a cell proliferation disorder in a human. In some embodiments, the cell proliferation disorder may be a cancer, such as leukemia, lymphoma» liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, 35 breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, or throat cancer. In particular cases, the ceil proliferation disorder may be a cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia 2023258320   30 Oct 2023 (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, 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 tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wilms tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom macroglobulinemia. The invention also features methods of treating T cell lymphoma (e.g., Hodgkin’s or cutaneous non-Hodgkin’s lymphoma cells), ovarian cancer, colon cancer, multiple myeloma, or renal cell carcinoma by administration of an antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof), a polynucleotide, vector, or host cell of the invention to a patient (e.g., a mammalian patient, such as a human patient). For instance, the invention provides a method of treating ovarian cancer by administration of an antagonistic TNFR2 antibody or antigen-binding fragment thereof of the invention to a patient (e.g., a mammalian patient, such as a human patient). The invention additionally features a composition containing a single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof, polynucleotide, vector, or host ceil of the invention for treating Hodgkin’s or cutaneous non-Hodgkin’s lymphoma, T cell lymphoma, ovarian cancer, colon cancer, multiple myeloma, or renal cell carcinoma in a patient (e.g., a human patient). The invention also features a method of treating an infectious disease in a patient (e.g., a human patient) by administering a single-chain polypeptide, construct, antibody, or antigen-binding fragment thereof, polynucleotide, vector, or host cell of the invention to the human in need of treatment, as well as a composition containing a single-chain polypeptide, construct, antibody, or antigen-binding fragment 13 £023258330   30 Get 2023 thereof, polynucleotide, vector, or host cell of the invention for treating an infectious disease in a patient (e.g., a human patient). In some embodiments, the infectious disease is caused by a virus, a bacterium, a fungus, or a parasite. For instance, viral infections that can be treated according to the methods of the invention include hepatitis C virus, Yellow fever virus, Kadam virus, Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill virus, Negishi virus, Meaban virus, Saumarez Reef virus, Tyuleniy virus, Aroa virus, dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza virus, llheus virus, Israel turkey meningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik 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 Island virus, Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, cell fusing agent virus, Ippy virus, Lassa virus, lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal 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, Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Marburg virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O’nyong’nyong virus, and the chikungunya virus, smallpox virus, monkeypox virus, vaccinia virus, herpes simplex virus, human herpes virus, 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 (VSV), human respiratory syncytial virus (RSV), Newcastle disease virus, hendravirus, nipahvirus, 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 papilloma virus, adeno-associated virus, astrovirus, JC virus, BK virus, SV40 virus, Norwalk virus, rotavirus, human immunodeficiency virus (HIV), human T-lymphotropic virus Types I and II, and transmissible spongiform encephalopathy, such as chronic wasting disease. In some embodiments, bacterial infections that can be treated according to the methods of the invention include those caused by a bacterium belonging to a genus selected from the group consisting of Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriacece (e.g., E. coli, such as Q157:H7), Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordatella, Legionella, Pasturella, Franc!sella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus. In addition, parasitic infections that can be treated according to the methods of the invention include those caused by Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, 14 2023258320   30 Oct 2023 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 helminthic parasites include richuris trlchiura, 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, or Echinococcus granulosus. The invention also features kits, such as a kit that contains a single-chain polypeptide, construct, antibody, or antigen-binding fragment of the invention (e.g., an antagonist TNFR2 antibody), a polynucleotide of the invention, a vector of the invention, or a host cell of the invention. In some cases, kits of the invention may contain instructions for transfecting a vector of the invention into a host cell of the invention. Optionally, kits may contain instructions for (and optionally, a reagent that can be used for) expressing a single-chain polypeptide, antibody, or antigen-binding fragment of the invention in a host cell of the invention. A kit of the invention may also contain instructions for administering a single-chain polypeptide, construct, antibody or antigen-binding fragment of the invention, a polynucleotide of the invention, a vector of the invention, or a host cell of the invention to a human patient. Optionally a kit may contain instructions for making or using an antibody or antigen-binding fragment of the invention, a polynucleotide of the invention, a vector of the invention, or a host cell of the invention. Definitions As used herein, the term “about” refers to a value that is no more than 10% above or below the value being described. For example, the term “about 5 nM” indicates a range of from 4.5 nM to 5.5 nM. As used herein, the term “antibody” (Ab) refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodies, including e.g., Fab', F(ab’)2, Fab, Fv, rlgG, and scFv fragments. Moreover, unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab')2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of the animal, and may have less non-specific tissue binding than an intact antibody (see Wahl et al., J. Nucl. Med. 24:316, 1983; incorporated herein by reference). 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 fragments of a full-length antibody. The antibody fragments can be a Fab, F(ab’)2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed of the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the Vl, Vh, Cl, and 15 £023258330   30 Get 2023 Ch1 domains; (ii) a F(ab')2fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the Vh and Ch1 domains; (iv) a Fv fragment consisting of the Vl and Vh domains of a single arm of an antibody, (v) a dAb including Vh and Vl domains; (vi) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a Vh domain; (vii) a dAb which consists of a Vh or a Vl domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, Vl and Vh, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the Vl and Vh regions pair to form monovalent molecules (known as 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). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art. As used herein, the terms “anti-tumor necrosis factor receptor 2 antibody,” “TNFR2 antibody,” “anti-TNFR2 antibody portion,” and / or “anti-TNFR2 antibody fragment” and the like include any protein or peptide-containing molecule that includes 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 a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, that is capable of specifically binding to TNFR2. For instance, two or more portions of an immunoglobulin molecule may be covalently bound to one another, e.g., via an amide bond, a thioether bond, a carbon-carbon bond, a disulfide bridge, or by a linker, such as a linker described herein or known in the art. TNFR2 antibodies also include antibody-like protein scaffolds, such as the tenth fibronectin type III domain (10Fn3), which contains BC, DE, and FG structural loops similar in structure and solvent accessibility to antibody CDRs. The tertiary structure of the 10Fn3 domain resembles that of the variable region of the IgG heavy chain, and one of skill in the art can graft, e.g., the CDRs of a TNFR2 monoclonal antibody onto the fibronectin scaffold by replacing residues of the BC, DE, and FG loops of 10Fn3 with residues from the CDR-H1, CDR-H2, or CDR-H3 regions of a TNFR2 monoclonal antibody. As used herein, the terms “antagonist TNFR2 antibody” and “antagonistic TNFR2 antibody” refer to TNFR2 antibodies that are capable of inhibiting or reducing activation of TNFR2 and / or attenuating one or more signal transduction pathways mediated by TNFR2. For example, antagonistic TNFR2 antibodies can inhibit or reduce the growth and proliferation of regulatory T cells. Antagonistic TNFR2 antibodies may inhibit or reduce TNFR2 activation by blocking TNFR2 from binding TNFa. In this way, antagonistic TNFR2 antibodies may block the trimerization of TNFR2 that would otherwise be induced by interacting with TNFa, thus resulting in suppression of TNFR2 activity. As used herein, the term “bispeeific antibodies” refers to monoclonal, often human or humanized antibodies that have binding specificities for at least two different antigens. In the invention, one of the 16 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 binding specificities can be directed towards TNFR2, the other can be for any other antigen, e.g., for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc. As used herein, the phrase "chemotherapeutic agent" refers to any chemical agent with therapeutic usefulness in the treatment of cancer, such as a cancer described herein. Chemotherapeutic agents encompass both chemical and biological agents. These agents can function to inhibit a cellular activity upon which a cancer cell depends for continued survival. Categories of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones, hormone analogs, and antineoplastic drugs. Exemplary chemotherapeutic agents suitable for use in conjunction with the compositions and methods described herein include, without limitation, those set forth in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal medicine, 14th edition; Perry et al., Chemotherapeutic, Chapter 17 in Abeloff, Clinical Oncology 2nd ed., 2000; Baltzer L. and Berkery R. (eds): Oncology Pocket Guide to Chemotherapeutic, 2nd ed. St. Luois, mosby-Year Book, 1995; Fischer D. S., Knobf M. F., Durivage H.J. (eds): The Cancer Chemotherapeutic Handbook, 4th ed. St. Luois, Mosby-Year Handbook, the disclosures of each of which are incorporated herein by reference as they pertain to chemotherapeutic agents. As used herein, the term “chimeric” antibody refers to an antibody having variable domain sequences (e.g., CDR sequences) derived from an immunoglobulin of one source organism, such as rat or mouse, and constant regions derived from an immunoglobulin of a different organism (e.g., a human, another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, member of the bovidae family (such as cattle, bison, buffalo, elk, and yaks, among others), cow, sheep, horse, or bison, among others). Methods for producing chimeric antibodies are 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. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397; incorporated herein by reference. As used herein, the term “complementarity determining region” (CDR) refers to a hypervariable region found both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). As is appreciated in the art, the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. The invention includes antibodies comprising modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a p-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the p-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, 17 2023258320   30 Get 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 Bethesda, Md. 1987; incorporated herein by reference). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al, unless otherwise indicated. As used herein, the terms “conservative mutation,” “conservative substitution,” or “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in table 1 below. Table 1. Representative physicochemical properties of naturally-occurring amino acids Amino Acid 3 Letter Code 1 Letter Code Sidechain Polarity Electrostatic character at physiological pH (7.4) Steric Volume1 Alanine Ala A nonpolar neutral small Arginine Arg R polar cationic large Asparagine Asn N polar neutral intermediate Aspartic acid Asp D polar anionic intermediate Cysteine Cys C nonpolar neutral intermediate Glutamic acid Giu E polar anionic intermediate Glutamine Gin Q polar neutral intermediate Glycine Gly G nonpolar neutral Both neutral and small Histidine His H polar cationic forms in equilibrium at pH 7.4 large Isoleucine lie I nonpolar neutral large Leucine Leu L nonpolar neutral large Lysine Lys K polar cationic large Methionine Met M nonpolar neutral large Phenylalanine Pho F nonpolar neutral large Proline Pro P nonpolar neutral intermediate Serine Ser S polar neutral small Threonine Thr T polar neutral intermediate Tryptophan Trp W nonpolar neutral bulky Tyrosine Tyr Y polar neutral large Valine Vai V nonpolar neutral intermediate tbased on volume in A3: 50-100 is small, 100-150 is intermediate, 150-200 is large, and >200 is bulky From this table it is appreciated that the conservative amino acid families include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (Hi) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the 15 same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg). As used herein, the term “conjugate” refers to a compound formed by the chemical bonding of a reactive functional group of one molecule with an appropriately reactive functional group of another molecule. 2023258320   30 Oct 2023 As used herein in the context of a TNFR2 antagonist, the term “construct” refers to a fusion protein containing a first polypeptide domain bound to a second polypeptide domain. The polypeptide domains may each independently be antagonistic TNFR2 single chain polypeptides, for instance, as described herein. The first polypeptide domain may be covalently bound to the second polypeptide domain, for instance, by way of a linker, such as a peptide linker or a disulfide bridge, among others. Exemplary linkers that may be used to join the polypeptide domains of an antagonistic TNFR2 construct include, without limitation, those that are described in Leriche et al., Bioorg. Med. Chern., 20:571-582 (2012), the disclosure of which is incorporated herein by reference in its entirety. As used herein, the term “derivatized antibodies” refers to antibodies that are modified by a chemical reaction so as to cleave residues or add chemical moieties not native to an isolated antibody. Derivatized antibodies can be obtained by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by addition of known chemical protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein. Any of a variety of chemical modifications can be carried out by known techniques, including, without limitation, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. using established procedures. Additionally, the derivative can contain one or more non-natural amino acids, e.g., using amber suppression technology (see, e.g., US Patent No. 6,964,859; incorporated herein by reference). As used herein, the term “diabodies” refers to bivalent antibodies comprising two polypeptide chains, in which each polypeptide chain includes Vh and Vl domains joined by a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of VH and VL domains on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain so as to form a homodimeric structure. Accordingly, the term “triabodies” refers to trivalent antibodies comprising three peptide chains, each of which contains one VH domain and one VL domain joined by a linker that is exceedingly short (e.g., a linker composed of 1 -2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain. In order to fold into their native structure, peptides configured in this way typically trimerize so as to position the VH and VL domains of neighboring peptide chains spatially proximal to one another to permit proper folding (see Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993; incorporated herein by reference). As used herein, a “dominant antagonist” of TNFR2 is an antagonist (e.g., an antagonistic polypeptide, such as a single-chain polypeptide, antibody, or antigen-binding fragment thereof) that is capable of inhibiting TNFR2 activation even in the presence of a TNFR2 agonist, such as TNFa, or IL-2. For example, a TNFR2 antagonist is a dominant antagonist if the ICso of the antagonist increases by less than 200% (e.g., less than 200%, 100%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less) in the presence of a TNFR2 agonist (e.g., TNFa) or IL-2 relative to the ICso of the antagonist as measured in the same assay in the absence of a TNFR2 agonist, such as TNFa, or IL-2. Inhibition of TNFR2 activation can be assessed, for instance, by measuring the inhibition of proliferation of TNFR2+ cells, such as T-reg cells, cancer cells that express TNFR2, or myeloid-derived suppressor cells, as well as by measuring the inhibition of NFkB signaling (eg., by monitoring the reduction in expression of one or 19 £023258330   30 Get 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and clAP2 / BIRC3 in a conventional gene expression assay). Cell proliferation assays and gene expression assays that can be used to monitor TNFR2 activation are described herein, for instance, in Examples 9 and 12, respectively. As used herein, a "dual variable domain immunoglobulin” (“DVD-lg”) refers to an antibody that combines the target-binding variable domains of two monoclonal antibodies via linkers to create a tetravalent, dual-targeting single agent. (Gu et al., Meth. Enzymol., 502:25-41,2012; incorporated by reference herein). Suitable linkers for use in the light chains of the DVDs of the invention include those identified on Table 2.1 on page 30 of Gu et al.: the short K chain linkers ADAAP (SEQ ID NO: 118) (murine) and TVAAP (SEQ ID NO: 119) (human); the long k chain linkers ADAAPTVSIFP (SEQ ID NO: 120) (murine) and TVAAPSVFIFPP (SEQ ID NO: 121) (human); the short A chain linker QPKAAP (SEQ ID NO: 122) (human); the long A chain linker QPKAAPSVTLFPP (SEQ ID NO: 123) (human); the GS-short linker GGSGG (SEQ ID NO: 124), the GS-medium linker GGSGGGGSG (SEQ ID NO: 125), and the GS-long linker GGSGGGGSGGGGS (SEQ ID NO: 126) (all GS linkers are murine and human). Suitable linkers for use in the heavy chains of the DVDs include those identified on Table 2.1 on page 30 of Gu & Ghayur, 2012, Methods in Enzymology 502:25-41, incorporated by reference herein: the short linkers AKTTAP (SEQ ID NO: 127) (murine) and ASTKGP (SEQ ID NO: 128) (human); the long linkers AKTTAPSVYPLAP (SEQ ID NO: 129) (murine) and ASTKGPSVFPLAP (SEQ ID NO: 130) (human); the GS-short linker GGGGSG (SEQ ID NO: 131), the GS-medium linker GGGGSGGGGS (SEQ ID NO: 132), and the GS-long linker GGGGSGGGGSGGGG (SEQ ID NO: 133) (all GS linkers are murine and human). As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). As used herein, the term “exogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted there from. As used herein, the term “framework region” or “FW region” includes amino acid residues that are adjacent to the CDRs. FW region residues may be present in, for example, 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. As used herein, the term “fusion protein” refers to a protein that is joined via a covalent bond to another molecule. A fusion protein can be chemically synthesized by, e.g., an amide-bond forming reaction between the N-terminus of one protein to the C-terminus of another protein. Alternatively, a fusion protein containing one protein covalently bound to another protein can be expressed recombinantly in a cell (e.g., a eukaryotic cell or prokaryotic cell) by expression of a polynucleotide encoding the fusion protein, for example, from a vector or the genome of the ceil. A fusion protein may contain one protein that is covalently bound to a 20 2023258320   30 Oct 2023 linker, which in turn is covalently bound to another molecule. Examples of linkers that can be used for the formation of a fusion protein include peptide-containing linkers, such as those that contain naturally occurring or non-naturally occurring amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally-occurring proteins and are thus more resistant to 5 degradation by endogenous proteases. Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of 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. Chern., 20:571-582, 2012). 10           As used herein, the term “heterospecific antibodies” refers to monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. Traditionally, the recombinant production of heterospecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537, 1983). Similar procedures are disclosed, e.g., in WO 93 / 08829, U.S. Pat. Nos. 15    6,210,668; 6,193,967; 6,132,992; 6,106,833; 6,060,285; 6,037,453; 6,010,902; 5,989,530; 5,959,084; 5,959,083; 5,932,448; 5,833,985; 5,821,333; 5,807,706; 5,643,759, 5,601,819; 5,582,996, 5,496,549, 4,676,980, WO 91 / 00360, WO 92 / 00373, EP 03089, Traunecker et al., EMBOJ. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986); incorporated herein by reference. Heterospecific antibodies can include Fc mutations that enforce correct chain association in multi-specific antibodies, as 20 described by Klein et al, mAbs 4(6):653-663, 2012; incorporated herein by reference. As used herein, the term “human antibody” refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, Cl, Ch domains (e.g., Ch1 , Ch2, Ch3), hinge, (Vl, Vh)) is substantially non-immunogenic in humans, with only minor sequence changes or variations. A human antibody can be produced in a human cell (e.g., by recombinant expression), or by a non-human animal 25 or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. Further, when a human antibody is a singlechain antibody, it can include a linker peptide that is not found in native human antibodies. For example, an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such 30 linker peptides are considered to be of human origin. Human antibodies can be made by a variety of methods 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 WO 1998 / 46645; WO 1998 / 50433; WO 1998 / 24893; WO 1998 / 16654; WO 1996 / 34096; WO 1996 / 33735; and WO 1991 / 10741; incorporated herein by reference. Human antibodies can also be produced using 35 transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT publications WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 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; incorporated by reference herein. £023258330   30 Get 2023 As used herein, the term “humanized” antibodies refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other target-binding subdomains of antibodies) which contain minimal sequences derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. All or substantially all of the FR regions may also be those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art. See, e.g., Riechmann et al., Nature 332:323-7, 1988; U.S. Patent Nos: 5,530,101; 5,585,089: 5,693,761; 5,693,762; and 6,180,370 to Queen et al; EP239400; PCT publication WO 91 / 09967; U.S. Patent No. 5,225,539; EP592106; and EP519596; incorporated herein by reference. As used herein, the term “hydrophobic side-chain” refers to an amino acid side-chain that exhibits low solubility in water relative due to, e.g., the steric or electronic properties of the chemical moieties present within the side-chain. Examples of amino acids containing hydrophobic side-chains include those 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. As used herein, the term “immunotherapy agent” refers to a compound, such as an antibody, 20 antigen-binding fragment thereof, single-chain polypeptide, or construct as described herein, that specifically binds an immune checkpoint receptor or ligand and exerts an antagonistic effect on the receptor or ligand, thereby reducing or inhibiting the signal transduction of the receptor or ligand that would otherwise lead to a downregulation of the immune response. Immunotherapy agents include compounds, such as antibodies, antigen-binding fragments, single-chain polypeptides, and constructs 25 capable of specifically binding receptors expressed on the surfaces of hematopoietic cells, such as lymphocytes (e.g., T cells), and suppressing the signaling induced by the receptor or ligand that would otherwise lead to tolerance towards an endogenous (“self”) antigen, such as a tumor-associated antigen. Immunotherapy agents may reduce the signaling induced by the receptor or ligand by, for example, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 30    99.9%, or 100% relative to the signaling induced by the receptor or ligand exhibited in the absence of the immunotherapy agent. Exemplary assays that can be used to measure the extent of receptor or ligand signaling include, for example, enzyme-linked immunosorbant assay (ELISA) techniques to measure protein expression alterations that are associated with a particular signal transduction pathway, as well as polymerase chain reaction (PCR)-based techniques, such as quantitative PGR, reverse-transcription 35 PCR, and real-time PCR experiments useful for determining changes in gene expression associated with a particular signal transduction pathway, among others. Exemplary methods that can be used to determine whether an agent is an “immunotherapy agent” include the assays described in Mahoney et al., Cancer Immunotherapy, 14:561 -584 (2015), the disclosure of which is incorporated herein by reference in its entirety. Examples of immunotherapy agents include, e.g., antibodies or antigen-binding fragments 2023258320   30 Oct 2023 thereof that specifically bind one or more of OX40L, TL1 A, 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, TGFp, CD39, CD73, CXCR4, CXCL12, SIRPA, CD47, VEGF, and neuropilin. Additional example of immunotherapy agents include Targretin, Interferon-alpha, clobestasol, 5 Peg Interferon (e.g., PEGASYS®), prednisone, Romidepsin, Bexarotene, methotrexate, Triamcinolone cream, anti-chemokines, Vorinostat, gabapentin, antibodies to lymphoid cell surface receptors and / or lymphokines, antibodies to surface cancer proteins, and / or small molecular therapies like Vorinostat. 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. 10             As used herein, the term “multi-specific antibodies” refers to antibodies that exhibit affinity for more than one target antigen. Multi-specific antibodies can have structures similar to full immunoglobulin molecules and include Fc regions, for example IgG Fc regions. Such structures can include, but not limited to, IgG-Fv, lgG-(scFv)2, DVD-lg, (scFv)2-(scFv)2-Fc and (scFv)2-Fc-(scFv)2. In case of lgG-(scFv)2, the scFv can be attached to either the N-terminal or the C- terminal end of either the heavy chain or the 15 light chain. Exemplary multi-specific molecules that include Fc regions and into which anti-TNFR2 antibodies or antigen-binding fragments thereof can be incorporated have been reviewed 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, 20 antibody fragments can be components of multi-specific molecules without Fc regions, based on fragments of IgG or DVD or scFv. Exemplary multi-specific molecules that lack Fc regions and into which antibodies or antibody fragments can be incorporated include scFv dimers (diabodies), trimers (triabodies) and tetramers (tetrabodies), Fab dimers (conjugates by adhesive polypeptide or protein domains) and Fab trimers (chemically conjugated), are described by Hudson and Souriau, 2003, Nature 25 Medicine 9:129-134: incorporated herein by reference. As used herein, the term “myeloid-derived suppressor cell” or “MDSC” refers to a cell of the immune system that modulates the activity of a variety of 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, and express all or a subset of proteins and small 30 molecules selected from the group consisting of B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1 d, CD1 d1, CD2, CD31 (PECAM-1), CD43, CD44, complement component C5a R1, F4 / 80 (EMR1), Fey RIH (CD16), Fey RH (CD32), Fey RIIA (CD32a), Fey RUB (CD32b), Fey RIIB / C (CD32b / c), Fey RIIC (CD32c), Fey RIIIA (CD16A), Fey RIIIB (CD16b), galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1 RI, IL-4Ra, IL-6Ra, integrin a4 (CD49d), integrin aL (CD11a), integrin aM (CD11b), M-CSFR, MGL1 (CD301a), MGL1 / 2 35    (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). Particularly, MDSCs do not express proteins selected from the group consisting of B7-2 (CD86), B7-H4, CD11 c, CD14, CD21, CD23 (FceRII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6). £023258330   30 Get 2023 As used herein, the terms “neutral TNFR2 polypeptide” and “phenotype-neutral TNFR2 polypeptide” refer to a polypeptide (such as a single-chain polypeptide, an antibody, or an antibody fragment) that binds TNFR2 and does not exert an antagonistic or an agonistic effect on TNFR2 activation. For instance, a TNFR2 polypeptide is a neutral TNFR2 polypeptide if the polypeptide binds TNFR2 and neither potentiates nor suppresses TNFR2 activation, for instance, as assessed 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 NFkB target genes, such as CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and / or clAP2 / BIRC3. Exemplary assays for measuring cell proliferation and gene expression are described, e.g., in Examples 9 and 12, respectively. As used herein, the term “non-native constant region” refers to an antibody constant region that is derived from a source that is different from the antibody variable region or that is a human-generated synthetic polypeptide having an amino sequence that is different from the native antibody constant region sequence. For instance, an antibody containing a non-native constant region may have a variable region derived from a non-human source (e.g., a 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 bovidae family (such as cattle, bison, buffalo, elk, and yaks, among others), cow, sheep, horse, or bison, among others). As used herein, the term “percent (%) sequence identity” refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits from 50% to 100% sequence identity across the full length of the candidate sequence or a selected portion of contiguous 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 is occupied by the same amino acid residue as the corresponding position in the reference sequence, then the molecules are identical at that position. As used herein, the term “primatized antibody” refers to an antibody comprising framework regions from primate-derived antibodies and other regions, such as CDRs and / or constant regions, from antibodies of a non-primate source. Methods for producing primatized antibodies are 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 instance, a primatized antibody or antigen-binding fragment thereof of the invention can be produced by 24 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 inserting the CDRs of a non-primate antibody or antigen-binding fragment thereof into an antibody or antigen-binding fragment thereof that contains one or more framework regions of a primate. As used herein, the term “operatively linked” in the context of a polynucleotide fragment is intended to mean that the two polynucleotide fragments are joined such that the amino acid sequences encoded by the two polynucleotide fragments remain in-frame. As used herein, the term “pharmacokinetic profile” refers to the absorption, distribution, metabolism, and clearance of a drug over time following administration of the drug to a patient. As used herein, a “recessive antagonist” of TNFR2 is an antagonist (e.g., an antagonistic polypeptide, such as a single-chain polypeptide, antibody, or antigen-binding fragment thereof) that inhibits TNFR2 activation to a significantly lesser extent in the presence of a TNFR2 agonist, such as TNFa, or IL-2 relative to the extent of inhibition of the same antagonist as measured in the absence of a TNFR2 agonist, such as TNFa, or IL-2. For example, a TNFR2 antagonist is a recessive antagonist if the IC50 of the antagonist increases by, e.g., 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more in the presence of a TNFR2 agonist (e.g., TNFa) or IL-2 relative to the IC50 of the antagonist as measured in the same assay the absence of a TNFR2 agonist, such as TNFa, or IL-2. Inhibition of TNFR2 activation can be assessed, for instance, by measuring the inhibition of proliferation of TNFR2+ cells, such as T-reg cells, cancer cells that express TNFR2, or myeloid-derived suppressor cells, as well as by measuring the inhibition of NFkB signaling (e.g., by monitoring the reduction in expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and clAP2 / BIRC3 in a conventional gene expression assay). Cell proliferation assays and gene expression assays that can be used to monitor TNFR2 activation are described herein, for instance, in Examples 9 and 12, respectively. 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 the 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); incorporated herein by reference. As used herein, the term “scFv” refers to a single-chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain. scFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and / or CDR-L3) and the variable region of an antibody heavy chain (VH) (e.g., CDR-H1, CDR-H2, and / or CDR-H3) separated by a linker. The linker that joins the VL and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), in order to enhance 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., a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites). scFv molecules are 25 £023258330   30 Get 2023 known in the art and are described, e.g., in US 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 a scFv molecule can be derived from one or more antibody molecules. It will also be understood by one of ordinary skill in the art that the variable regions of the scFv molecules of the invention can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived. For example, in one embodiment, nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and / or framework residues). Alternatively or in addition, mutations are made to CDR amino acid residues to optimize antigen binding using art recognized techniques. scFv fragments are described, for example, in WO 2011 / 084714; incorporated herein by reference. As used herein, the phrase “specifically binds” refers to a binding reaction which is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological molecules that is recognized, e.g., by an antibody or antigen-binding fragment thereof, with particularity. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a Kd of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a Kd of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a Kd of greater than 100 nM (e.g., greater than 500 nm, 1 pM, 100 pM, 500 pM, or 1 mM) for that particular antigen or epitope thereof. 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. 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), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. As used herein, the terms “subject” and “patient” refer to an organism that receives treatment for a particular disease or condition as described herein (such as cancer or an infectious disease). Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovidae family (such as cattle, bison, buffalo, elk, and yaks, among others), cows, sheep, horses, and bison, among others, receiving treatment for diseases or conditions, for example, cell proliferation disorders, such as cancer or infectious diseases. As used herein, the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection and the like. As used herein, the terms “treat” or “treatment” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of a cell proliferation disorder, such as cancer, or an infectious disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, 26 2023258320   30 Oct 2023 stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. As used herein, the term “tumor microenvironment" refers to cancer cells that form a turmor and the population of non-cancer cells, molecules, and / or blood vessels within the tumor or that border or surround the cancer cells. As used herein, the terms “tumor necrosis factor receptor superfamily,” “TNFR superfamily,” or “TNFRS” refer 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 cellular signaling as a consequence of binding to one or more ligands in the TNF superfamily. The TNFR superfamily can be divided into two subgroups: receptors containing the intracellular death domain and those lacking this domain. The death domain is an 80 amino acid motif that propagates apoptotic signal transduction cascades following receptor activation. Exemplary TNFR super family members that contain the intracellular death domain include TNFR1, while TNFR2 represents a TNFR super family protein that does not contain this domain. Members of the TNFR superfamily include TNFR1, TNFR2, RANK, CD30, CD40, Lymphotoxin beta receptor (LT-pR), 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), Osteoprotegrin, 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. As used herein the term “variable region CDR” includes amino acids in a CDR or complementarity determining region as identified using sequence or structure based methods. As used herein, the term “CDR” or “complementarity determining region” refers to the noncontiguous antigenbinding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chern. 252:6609-6616, 1977 and Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242, 1991; by Chothia et al., (J. Mol. Biol. 196:901 -917, 1987), and by MacCallum et al., (J. Mol. Biol. 262:732-745, 1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. In certain embodiments, the term “CDR” is a CDR as defined by Kabat based on sequence comparisons. As used herein, the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, a RNA vector, virus or other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994 / 11026; incorporated herein by reference. Expression vectors of the invention contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and / or the integration 27 £023258330   30 Get 2023 of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of antibodies and antibody fragments of the invention include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors of the invention may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin. As used herein, the term “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. References to “VL” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a native antibody has at the amino terminus a variable domain (VH) followed by a number of constant domains. Each light chain of a native antibody has a variable domain at the amino terminus (VL) and a constant domain at the carboxy terminus. Brief Description of the Figures Figures 1A and 1B show the DNA and amino acid sequences of the heavy and light chains of the antagonistic TNFR2 antibody TNFRAB1. Figure 1A shows the DNA sequence that encodes the heavy chain of TNFRAB1 (top) and amino acid sequence of the heavy chain (bottom). The amino acid sequences of the three complementarity-determining regions (CDRs) are shown in bold. Figure 1B shows the DNA sequence that encodes the light chain of TNFRAB1 (top) and amino acid sequence of the light chain (bottom). The amino acid sequences of the three CDRs are shown in bold. Figures 2A and 2B show the amino acid sequence of human TNFR2 (SEQ ID NO: 7). Notably, human TNFR2 is numbered herein starting with an N-terminal methionine at position 1 and concluding with a C-terminal serine at position 461 (SEQ ID NO: 7). All references to amino acid positions within TNFR2 are made in the context of the TNFR2 numbering scheme shown in Figures 2A and 2B. (Figure 2A) Shaded residues KCRPGFGV (SEQ ID NO: 20) define an epitope that is specifically bound by the antagonistic TNFR2 antibody TNFRAB1. Significantly, the ability of TNFRAB1 to selectively bind residues within this region without binding underlined residues KCSPG (SEQ ID NO: 12) promotes antagonism of TNFR2 signaling. The poor (or lack of) affinity of the antibodies of the invention for residues within the region of, or near, the underlined residues is consistent with the antagonistic activity of these antibodies, 28 2023258320   30 Oct 2023 as binding to epitopes containing the underlined residues has been correlated with attenuation of the inhibitory activity among TNFR2 antibodies. TNFRAB1 additionally binds an epitope that includes shaded residues CKPCAPGTF (SEQ ID NO: 21). Though these residues are not consecutive in primary sequence with the KCRPG motif, they are likely spatially proximal in the three dimensional tertiary structure of TNFR2 and may be appropriately positioned for interaction with an antagonistic TNFR2 antibody of the invention (see Figure 4). (Figure 2B) Shaded residues CAPLRKRCR (SEQ ID NO: 11) define an epitope that is specifically bound by the antagonistic TNFR2 antibody TNFRAB2. TNFRAB2 may additionally bind one or more regions that include the residues DSTYTQL (SEQ ID NO: 8), PECLSCGS (SEQ ID NO: 9), and RICTCRPG (SEQ ID NO: 10), which may be part of a discontinuous epitope. Though these residues are not consecutive in primary sequence, they are likely spatially proximal in the three dimensional tertiary structure of TNFR2 and may be appropriately positioned for interaction with an antagonistic TNFR2 antibody of the invention. Figures 3A and 3B are tables showing the raw data obtained from enzyme-linked immunosorbant assay (ELISA) experiments that were conducted to determine the affinity of TNFRAB1 and TNFRAB2 for various continuous and discontinuous epitopes within TFNR2 (see Example 1). Raw luminescence values are shown in the fourth column of the tables (right). The peptide sequences shown represent those that contain a portion of the conformational epitope within TNFR2 that interacts with TNFRAB1 (Figure 3A) or TNFRAB2 (Figure 3B). Amino acid residues with the single-digit code “2” designate cysteine residues that were chemically protected at the thiol position with an acetamidomethyl (ACM) moiety during peptide synthesis. These residues are not reactive with bromomethyl-containing electrophiles and were therefore not cross-linked during the cyclization and bicyclization phases of peptide synthesis. The third column in the tables indicates the general structure of the peptide scaffold. “CYS.S” indicates a 27-residue peptide in which positions 1-11 and 17-27 of the peptide represent 11-residue peptides derived from TNFR2 that contain cysteine residues that form disulfide bridges in the native protein based on information available for UniProt entry P20333. The sequence Gly-Gly-Ser-Gly-Gly was incorporated into positions 12-16 of peptides of this group. Native Cys residues that do not form disulfide bridges were protected with acetamidomethyl (ACM) protecting groups and are designated with the single-digit code “2”. Figure 4 is a schematic illustrating the conformational epitopes within TNFR2 that may interact with antagonist TNFR2 antibodies, such as TNFRAB1 and TNFRAB2, as well as residues that do not interact with antagonist TNFR2 antibodies. The KCSPG motif is shown in the expansion at the top left of the figure; the KCRPG motif is shown in the expansion at the right of the figure. Exterior surface of the protein designates the van der Waals surface of TNFR2. Figure 4 is a rendering of a monomer of TNFR2 isolated from the X-ray crystal structure of TNFR2 (PDB ID: 3ALQ, Mukai, et al., Sci. Signal., 3:ra83, 2010). Figure 5 is a graph showing that TNFRAB1 suppresses the growth of cultured T-reg cells in vitro. Values represent the fraction of viable T-reg cells relative to untreated control cells that remained in culture after exposure of the cells to a particular condition. Bars shown on the far left represent T-reg cells treated with either IL-2 at 200 U / ml (control), TNFa (20 ng / ml), TNFR2 agonist (2.5 pg / ml), antagonistic 29 £023258330   30 Get 2023 TNFR2 antibody TNFRAB1 (2.5 pg / ml), or antagonistic TNFR2 antibody TNFRAB2 (2.5 pg / ml). Bars shown second from the left demonstrate the dose-dependent variation in T-reg viability upon treatment of cells with TNFRAB1. Bars shown second from the right show the ability of TNFRAB1 to inhibit the growthpromoting activity of TNFa. T-reg cells were treated with a constant concentration of TNFa (20 ng / ml) and varying concentrations of TNFRAB1 (from 0.0008 to 25 pg / ml). TNFRAB1 was capable of suppressing TNFa-induced proliferation in a concentration-dependent manner, indicating that TNFRAB1 antagonizes TNFR2. Bars shown on the far right demonstrate the effect of TNFa on the growth of T-reg cells. Incubation of T-reg cells with 20 ng / ml TNFa resulted in approximately 130% proliferation relative to untreated cells. Figure 6A is a graph showing the ability of TNFa to induce aT-reg cell proliferation in a dosedependent manner. Figure 6B is a graph showing the ability of IL-2 to induce aT-reg cell proliferation in a dosedependent manner. Incubation of freshly isolated human CD4+ cells for up to 48 hours with TNFa and IL-2 (200 U / ml) induces T-reg cell expansion in a dose-dependent manner and the presence of IL-2 is important for promoting T-reg expansion. Figure 6C is a graph showing the effect of TNFa and dominant antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2 on T-reg cell proliferation. Values on the x-axis represent the percent change in the quantity of T-reg cells relative to a sample treated with IL-2. Figures 6D and 6E are graphs showing the results of duplicate experiments conducted in order to determine the effect of TNFRAB2 on T-reg cell proliferation. Figures 6F and 6G are graphs showing the results of duplicate experiments conducted in order to determine the effect of TNFRAB1 on T-reg cell proliferation. Figure 7A is a graph showing the effect of TNFa on the proliferation of CD4+ T-reg cells. Values on the x-axis indicate the percent change in the quantity of T-reg cells upon treatment with TNFa relative to treatment with IL-2. Figure 7B is a graph showing the ability of TNFRAB1 to dominantly inhibit T-reg cell proliferation in the presence and absence of TNFa. Figure 7C is a graph showing the ability of TNFRAB2 to inhibit T-reg cell proliferation in the presence and absence of TNFa. Figures 7D and 7E are graphs showing the effect of TNFR2 dominant antagonists on proliferation of T-reg cells in the presence and absence of TNFa. Figure 8A is a graph showing the ability of TNFRAB1 and TNFRAB2 to inhibit the proliferation of T-reg cells generally. Figure 8B is a graph showing the effect of TNFRAB1 and TNFRAB2 on T-reg cell proliferation relative to the effect induced by treatment with IL-2. Figure 8C is a graph showing the effect of TNFRAB1 and TNFRAB2 on total T-reg quantity. Figure 8D is a graph showing the effect of TNFRAB1 and TNFRAB2 on total T-reg quantity relative to the effect induced by treatment with IL-2. Figure 8E is a graph demonstrating the effect of TNFRAB1 and TNFRAB2 on activated T-reg 30 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 cells (aT-reg cells) that express CD25 in a high-affinity state (CD25Hi) and CD45RA in a low-affinity state (CD45RALow). Figure 8F is a graph showing the effect of TNFRAB1 and TNFRAB2 on a population of T-reg cells that express CD45ROHi and CD25Hi. Figure 8G is a graph showing the effect of TNFRAB2 on CD25HLexpressing CD4+ T cells. Figure 8H is a graph demonstrating the effect of TNFRAB2 on CD25H'-expressing T-reg cells. These data demonstrate that, while the proliferation of both activated (aT-reg) and resting (rT-reg) cells is inhibited upon treatment with TNFRAB1 or TNFRAB2, antagonistic TNFR2 antibodies preferentially inhibit the proliferation of aT-reg cells. Figure 9A is a series of 2-dimensional flow cytometry plots demonstrating the effect of agents that direct the growth of T-reg cells (e.g., TNFa, IL-2, and TNFR2 agonists) as well as a TNFR2 antagonist, TNFRAB2, on the proliferation of T-reg cells of various phenotypes. Treatment with a TNFR2 antagonist antibody preferentially inhibits the proliferation of CD25Hi-expressing T-reg cells. The proportion of CD4+, CD25H'+ cells expressing TNFR2 after up to 48 hours of incubation with either IL-2 (200 U / ml) alone, with TNFa (20 ng / ml), or with TNFR2 antagonist antibodies (12.5 pg / ml) is shown. Figure 9B is a graph showing the effect of TNFRAB1 on the secretion of TNFR2, shown in units of pg / ml. Figure 9C is a series of 1-dimensional flow cytometry plots showing the effect of TNFRAB2 on CD8+ T cell count as measured by carboxyfluorescein (CFSE) labeling. Figure 10A is a graph showing the ability of full-length TNFRAB1 (IgG) as well as F(ab')2 fragment of TNFRAB1 to inhibit the proliferation of T-reg cells. Figure 10B is a graph showing the ability of full-length TNFRAB2 (IgG), as well as a F(ab’)2 fragment of TNFRAB2, to inhibit the proliferation of T-reg cells. These data demonstrate that specific binding of the Fab regions of these antagonistic TNFR2 antibodies to TNFR2 is likely responsible for modulating T-reg cell growth, rather than non-specific binding of the Fc regions of these antibodies. Incubation of freshly isolated CD4+ cells for up to 48 hrs with IL-2 (200 U / ml) plus either the full antibody or F(ab’)2 fragment of TNFRAB1 or TNFRAB2 produces similar dose-dependent inhibition of T-reg cells in the presence or absence of TNFa (20 ng / ml). Figure 10C is a graph showing the results of a dose-response assay in which T-reg cells were treated with TNFRAB1 in the presence of anti-IgG antibodies. Figure 10D is a graph showing the results of a dose-response assay in which T-reg cells were treated with TNFRAB2 in the presence of anti-IgG antibodies. The dose-dependent suppression of T-reg cell growth induced by TNFR2 antagonist antibodies was unaffected by the presence of anti-IgG molecules, indicating that non-specific cross-linking mediated by TNFRAB1 or TNFRAB2 is not responsible for the inhibitory effect of these antibodies on T-reg cell proliferation. Co-incubation of crosslinking antibody (2.5 pg / ml) with TNFR2 antagonist antibodies (0.02 - 25 pg / ml) does not affect the ability of the antibodies to inhibit T-reg proliferation in a dose-dependent manner. Data are presented as a single representative example. Figure 11A is an image of a polyacrylamide gel showing the results of SDS-PAGE analysis 31 £023258330   30 Get 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 conducted following the expression of TNFRAB1. Figure 11B is an image of a polyacrylamide gel showing the results of SDS-PAGE analysis conducted following the expression of TNFRAB2. Analysis of reduced and non-reduced TNFR2 antagonist antibodies (2.5 pg) is shown. Figure 11C is an image of a polyacrylamide gel showing the results of a SDS-PAGE analysis conducted following the expression of F(ab’)2 fragments of TNFRAB1. Figure 11D is an image of a polyacrylamide gel showing the results of a SDS-PAGE analysis conducted following the expression of F(ab’)2 fragments of TNFRAB2. Analysis of TNFR2 antagonist antibodies before and after digestion in F(ab’)2 fragment preparation is shown. Figure 12A is a graph showing the effect of TNFR2 antagonist antibodies on the expression of TNFa and lymphotoxin. Figure 12B is a graph showing the effect of TNFR2 antagonist antibodies on the expression of FoxP3 and CD25. Figure 12C is a graph showing the effect of TNFR2 antagonist antibodies on the expression of genes that promote NFkB activation: conserved helix-loop-helix ubiquitous kinase (CHUK), nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor epsilon (NFKBIE), nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha (NFKBIA), mitogen-activated protein kinase 11 (MAP3K11), TNFa receptor-associated factor 2 (TRAF2), TNFa receptor-associated factor 3 (TRAF3), transcription factor relB, and baculoviral IAP repeat containing 3 protein (clAP2 / BIRC3). Real-time PCR analysis was used to detect RNA isolated from fresh CD4+ cells after incubation with IL-2 (50 U / ml) in combination with either TNFa (20 ng / ml) or the TNFR2 antagonist (2.5 pg / ml) for 3 hours. Figure 12D is a graph showing the ability of TNFRAB1 to suppress NFkB activation as measured using a cell-based ELISA assay. Figure 12E is a graph showing the ability of TNFRAB2 to suppress NFkB activation as measured using a cell-based ELISA assay. Phosphorylated RelA / NFKB p65 was used as a marker of NFkB activity. Treatment of T-reg cells with antagonistic TNFR2 antibodies resulted in attenuated NFkB activation relative to treatment with TNFa. Using a cell-based ELISA, the phosphorylation RelA / NFKB p65 was measured after 10 minute incubation of fresh CD4+ cells with IL-2 (200 U / ml) and various concentrations of TNFa (0.2 - 20 ng / ml) or TNFR2 antagonist antibodies (0.02 - 25 pg / ml). Phosphorylation of RelA / NFKB p65 is induced by TNFa and inhibited by the TNFR2 antagonist mAbs in a dose-dependent manner. Figure 13A is a table showing the kinetic and thermodynamic parameters of the binding of TNFRAB1 and TNFRAB2 to TNFR2. Association rate constants are shown in units of M^s-1, dissociation rate constants are shown in units of s-1, and equilibrium constants are shown in units of M. Figure 13B is a table showing the affinity of TNFRAB1 and TNFRAB2 for various linear peptide sequences within human TNFR.2. Relative affinity is indicated as a series of “+” symbols, such that higher quantities of this symbol represent elevated affinity values. Raw data derived from ELISA binding experiments are provided in the “Reading" column. Figure 13C is a table showing the effect of TNFa on the affinity of TNFRAB1 and TNFRAB2 for 32 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 various linear peptide sequences within human TNFR2. Relative affinity is indicated as a series of “+” symbols, such that higher quantities of this symbol represent elevated affinity values. Raw data derived from ELISA binding experiments are provided in the “Reading" column. Figures 14A and 14B are graphs showing the results of duplicate experiments conducted in order to determine the effect of a recessive-TNFR2 antagonist antibody that mildly inhibits TNFR2 activity (recessive-antagonist TNFR2 antibody A) on the proliferation of T-reg cells. Figures 14C and 14D are graphs showing the results of duplicate experiments conducted in order to determine the effect of a second recessive-TNFR2 antagonist that mildly inhibits TNFR2 activity (recessive-antagonist TNFR2 antibody B) on the proliferation of T-reg cells. These recessive-antagonist antibodies were raised against the exterior region of TNFR2 in order to prevent TNFR2 trimerization that leads to NFkB activation. Figure 15A is a structural model showing a three-dimensional structure of the anti-parallel dimer and parallel dimer of human TNFR2. Figure 15B is a structural model showing the three-dimensional structure of the TNFa-TNFR2 complex. Shaded residues represent amino acids within human TNFR2 that are bound by TNFRAB1. Proteins are portrayed in ribbon form beneath a Van der Waals surface. The data described herein clearly shows that for the full antibody or the F(ab’)2 fragment thereof to bind to TNFR2 in an anti-parallel conformation, the TNFR2 receptor would bind the antibody or fragment thereof at the indicated amino acid motifs. The binding sites of the parallel dimer are too close to one another and would thus preclude antibody binding. Moreover, the trimeric TNFa-TNFR2 complex masks the epitopes bound by antagonistic TNFR2 antibodies, as these residues are located within the interior of the trimeric structure. Figure 15C is an image showing the known and published trimeric structural model of TNFR2 as a trimer with contained trimer TNF. Also shown in this structure are shaded residues that represent amino acids within human TNFR2 that are bound by the recessive-antagonist TNFR2 antibodies A and B. Figure 16A is a graph showing the effect of TNFRAB1 on the proliferation of T-reg cells isolated from a patient presenting with ovarian cancer (grey shade) and of T-reg cells isolated from a subject not presenting with ovarian cancer (black shade). Figure 16B is a graph showing the effect of TNFRAB2 on the proliferation of T-reg cells isolated from a patient presenting with ovarian cancer (grey shade) and of T-reg cells isolated from a subject not presenting with ovarian cancer (black shade). Figure 17A is a graph showing the effect of TNFRAB1 on the proliferation of T-reg cells isolated from a subject not presenting with ovarian cancer. Figure 17B is a graph showing the effect of TNFRAB2 on the proliferation of T-reg cells isolated from a subject not presenting with ovarian cancer. Figure 17C is a graph showing the effect of TNFRAB1 on the proliferation of S-reg cells isolated from a subject presenting with ovarian cancer. Figure 17D is a graph showing the effect of TNFRAB2 on the proliferation of K-reg cells isolated from a subject presenting with ovarian cancer. Figure 17E is a graph showing the effect of TNFRAB1 on the proliferation of T-reg cells isolated 33 £023258330   30 Get 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 from a subject not presenting with ovarian cancer (duplicate data set relative to Figure 17A). Figure 17F is a graph showing the effect of TNFRAB2 on the proliferation of T-reg cells isolated from a subject not presenting with ovarian cancer (duplicate data set relative to Figure 17B). Figure 17G is a graph showing the effect of TNFRAB1 on the proliferation of T-reg cells isolated from a subject presenting with ovarian cancer (duplicate data set relative to Figure 17C). Figure 17H is a graph showing the effect of TNFRAB2 on the proliferation of T-reg cells isolated from a subject presenting with ovarian cancer (duplicate data set relative to Figure 17D). Figure 18A is a table showing the fluorescence values obtained from an ELISA-based binding assay to determine the TNFR2 binding affinity of twelve murine antibodies. The twelve antibodies were arrayed from columns 1 through 12 of a 96-well microtiter plate and were serially diluted from rows A through F of the plate. Figure 18B is a table showing the fluorescence values obtained from an ELISA-based binding assay to determine the affinity of twelve murine antibodies for target peptide 1 having the amino acid sequence LRKCRPGFGVA (SEQ ID NO: 285) bound to bovine serum albumin (BSA) at the N-terminus. The twelve antibodies were arrayed from columns 1 through 12 of a 96-well microtiter plate and were serially diluted from rows A through F of the plate. Figure 18C is a table showing the fluorescence values obtained from an ELISA-based binding assay to determine the binding affinity of twelve murine antibodies for target peptide 2 having the amino acid sequence VVCKPCAPGTFSN (SEQ ID NO: 286) bound to BSA at the C-terminus. The twelve antibodies were arrayed from columns 1 through 12 of a 96-well microtiter plate and were serially diluted from rows A through F of the plate. Figure 19 is a table showing the fluorescence values obtained from an ELISA-based binding assay to determine the binding affinity of twelve murine antibodies for an unrelated TNFR2 epitope. The twelve antibodies were arrayed from columns 1 through 12 of a 96-well microtiter plate were serially diluted from rows A through F of the plate Figure 20 is a table showing the fluorescence values obtained from an ELISA-based binding assay to determine the affinity of BSA conjugates of target peptide 1 (LRKCRPGFGVA, SEQ ID NO: 285) and target peptide 2 (VVCKPCAPGTFSN, SEQ ID NO: 286) for TNFR2A3. Figure 21A is a graph showing the percent change in a population of cultured T-reg cells following incubation with IL-2, TNFa, a TNFR2 agonist, and varying concentrations of the antagonistic TNFR2 antibody TNFRAB2. Numerical values shown along the X-axis are in units of pg / ml. Figure 21B is a graph showing the percent change in a population of cultured T-reg cells following incubation with IL-2, TNFa, a TNFR2 agonist, and varying concentrations of the antagonistic TNFR2 antibody TNFRAB1. Numerical values shown along the X-axis are in units of pg / ml. Figure 21C is a graph showing the percent change in a population of cultured T effector cells following incubation with varying concentrations of the antagonistic TNFR2 antibody TNFRAB1 and TNFRAB2. Numerical values shown along the X-axis are in units of pg / ml. Figure 21D is a graph showing the percent change in a population of cultured OVCAR3 cells, a TNFR2-expressing ovarian cell cancer line, following incubation with varying concentrations of the 34 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 antagonistic TNFR2 antibody TNFRAB1. Numerical values shown along the X-axis are in units of pg / ml. Figure 22A is a graph showing the percent change in a population of T-reg cells in a sample isolated from a patient suffering from ovarian cancer or a human subject without cancer following incubation with varying concentrations of the antagonistic TNFR2 antibody TNFRAB1. Numerical values shown along the X-axis are in units of pg / ml. Figure 22B is a graph showing the percent change in a population T effector cells in a sample isolated from a patient suffering from ovarian cancer or a human subject without cancer following incubation with varying concentrations of the antagonistic TNFR2 antibody TNFRAB1. Numerical values shown along the X-axis are in units of pg / ml. Figure 23A is a graph showing the percent change in a population of T effector cells in a sample isolated from a patient suffering from cutaneous T cell lymphoma and that is undergoing treatment with an immunotherapy agent or a human subject without cancer following incubation with varying concentrations of the antagonistic TNFR2 antibody TNFRAB1. Numerical values shown along the X-axis are in units of pg / ml. Figure 23B is a graph showing the percent change in a population T-reg cells in a sample isolated from a patient suffering from cutaneous T cell lymphoma and that is undergoing treatment with an immunotherapy agent or a human subject without cancer following incubation with varying concentrations of the antagonistic TNFR2 antibody TNFRAB1. Numerical values shown along the X-axis are in units of pg / ml. Figure 23C is a graph showing the percent change in a population TNFR2+ CD26- cells in a sample isolated from a patient suffering from cutaneous T cell lymphoma and that is undergoing treatment with an immunotherapy agent or a human subject without cancer following incubation with varying concentrations of the antagonistic TNFR2 antibody TNFRAB1. Numerical values shown along the X-axis are in units of pg / ml. Detailed Description Antagonistic TNFR2 polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments inhibit the activation of TNFR2 on TNFR2-expressing cells by binding this receptor (e.g., on the exterior surface of a T-reg cell, a cancer cell that expresses TNFR2, or a myeloid-derived suppressor cell (MDSC) and thus prevent the protein from recruiting its cognate ligand, TNFa. TNFa potentiates TNFR2 signaling by nucleating a trimer of TNFR2 proteins. It is this trimerization event that brings individual TNFR2 proteins into close proximity and initiates signaling via the MAPK / NFkB / TRAF2 / 3 pathway, which ultimately leads to cell growth and escape from apoptosis. TNFR2 antibodies can antagonize this interaction by binding the receptor and preventing TNFa from triggering this structural change. For instance, one mechanism by which this may occur is through the formation of an anti-parallel TNFR2 dimer, which is an inactive structural form of the receptor. The invention is based in part on the discovery of epitopes within TNFR2 that promote receptor antagonism, as well as on the finding that the binding specificity of an antagonistic TNFR2 antibody or antigen-binding fragment thereof is dictated primarily by the CDR-H3 sequence of the antibody or 35 £023258330   30 Get 2023 fragment thereof. It has been discovered that binding of distinct residues within TNFR2 promote receptor antagonism, such as residues containing the KCRPG motif (SEQ ID NO: 19) within TNFR2, as well as downstream amino acids (for instance, the LRKCRPGFGVA (SEQ ID NO: 285) and VVCKPCAPGTFSN (SEQ ID NO: 286) epitopes). Additionally, it has been found that replacement of the CDR-H3 sequence of a neutral anti-TNFR2 antibody (i.e., an antibody that is neither antagonistic nor agonistic in function) with the CDR-H3 of an antagonistic TNFR2 antibody converts the phenotype-neutral antibody to an antagonistic TNFR2 antibody, such as a dominant antagonistic TNFR2 antibody. Collectively, these discoveries enable the production of TNFR2-binding polypeptides (e.g., single-chain polypeptides containing a CDR-H3 region optionally bound to one or more additional CDRs, antibodies, and antigenbinding fragments thereof), such as dominant antagonist polypeptides that bind TNFR2 and suppress receptor activation. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs described herein) may exhibit one or more, or all, of the following properties: a. Suppression of T-reg cell proliferation, for instance, by binding and inactivating TNFR2 on the T* reg cell surface; b. Suppression of MDSC proliferation, for instance, by binding and inactivating TNFR2 on the MDSC surface; c. Promotion of the expansion of T effector cells, such as CD8+ T cells; and / or d. Suppression of the proliferation of TNFR2-expressing cancer ceils, such as T cell lymphoma cells (e.g., Hodgkin’s or cutaneous non-Hodgkin’s lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, and renal cell carcinoma cells. In some embodiments, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs described herein) exert one or more, or all, of the above characteristics with a greater potency in the microenvironment of a tumor than in a site that is free of cancer cells. For instance, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs described herein) may exert one or more, or all, of properties (a), (b), and (c) preferentially in a patient (such as a mammalian patient, e.g., a human) suffering from cancer relative to a subject (such as a mammalian subject, e.g., a human) that does not have cancer. The sections that follow provide a description of exemplary characteristics of antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs of the invention, and their use in therapeutic methods. Antagonistic TNFR2 polypeptides Effects on TNFR2 / MAPK / TRAF2 / 3 signal transduction cascades Anti-TFNR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention are capable of interacting with and inhibiting the activity of TNFR2. Thus, the anti-TNFR2 polypeptides of the invention can selectively antagonize the TNFa-TNFR2 36 2023258320   30 Oct 2023 interaction rather than promote TNFR2 signaling. This is particularly important for therapeutic applications, such as cancer immunotherapy, as TNFR2 activation upon association with TNFa leads to propagation of the MARK and TRAF2 / 3 signal cascade and activation of NFKB-mediated transcription of genes involved in T-reg cell growth and escape from apoptosis (Faustman, et al., Nat. Rev. Drug Disc., 5    9:482-493, 2010). The TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen binding fragments thereof) of the invention may bind TNFR2 with high affinity and may sterically sequester the receptor from TNFa rather than allow TNFa binding to TNFR2 initiate TNFR2 signaling, e.g., by binding TNFR2 in an anti-parallel dimer conformation in which TNFa binding sites are sterically inaccessible. This, in turn, prevents TNFa from nucleating an activated trimer of TNFR2, which triggers 10 TNFR2 signal transduction. The antibodies of the invention can therefore be used to suppress T-reg cell growth and proliferation, thereby allowing, for example, the proliferation of T effector cells that can mount an immune response against, e.g., a cancer cell or foreign pathogen. Thus, antagonistic TNFR2 polypeptides described herein can be administered to a mammalian subject, such as a human patient with a cell proliferation disorder or an infectious disease, in order to enhance the effectiveness of an 15 immune response (e.g., an immune response against cancer cells or pathogenic organisms) in the patient. Effects on T-reg cell proliferation Antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigen- 20 binding fragments thereof of the invention can be used to attenuate the activity (e.g., proliferation) of T-reg cells that typically accompanies T cell-mediated cytotoxicity against self cells, such as the attack of a tumor cell by a T-lymphocyte. Antagonistic TNFR2 antibodies can be administered to a mammalian subject, such as a human (e.g., by any of a variety of routes of administration described herein) in order to prolong the duration of an adaptive immune response, such as a response against a cancer cell or a 25 pathogenic organism. In this way, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the invention may synergize with existing techniques to enhance T-lymphocyte-based therapy for cancer and for infectious diseases. For instance, TNFR2 antagonists of the invention may be administered to suppress T-reg cell activity, thereby enhancing the cytotoxic effect of tumor reactive T cells. TNFR2 antagonists may also synergize with existing strategies 30 to promote tumor-reactive T cell survival, such as lymphodepletion and growth factor therapy, and in turn prolong the duration of anti-tumor reactivity in vivo. Antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigenbinding fragments thereof can also be used to treat a broad array of infectious diseases in a mammalian subject (e.g., a human), as inhibition of T-reg proliferation promotes the activity of CD8+ T-lymphocytes 35 capable of mounting an attack on pathogenic organisms. Additionally, antagonistic TNFR2 antibodies and antigen-binding fragments thereof of the invention can be used to treat a wide variety of infectious diseases, such as Mycobacterium tuberculosis, in a human or an agricultural farm animal (e.g., a bovine mammal, pig, cow, horse, sheep, goat, cat, dog, rabbit, hamster, guinea pig, or other non-human mammal). £023258330   30 Get 2023 Direct effects on TNFR2+ cancer cells Antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigenbinding fragments thereof of the invention may bind and inactivate TNFR2 on the surface of a cancer cell, such as a TNFR2+ tumor cell. For instance, antagonistic TNFR2 antibodies and antigen-binding fragments thereof described herein may bind TNFR2 on the surface a T cell lymphoma cell (e.g., a Hodgkin’s or cutaneous non-Hodgkin’s lymphoma cell), ovarian cancer cell, colon cancer cell, multiple myeloma cell, or renal cell carcinoma cell, among others. The ability of antagonistic TNFR2 antibodies and antigen-binding fragments thereof of the invention to bind TNFR2 directly on a cancer cell provides another pathway by which these molecules may attenuate cancer cell survival and proliferation. For instance, an antagonistic TNFR2 antibody or antigen-binding fragment thereof of the invention, such as an antibody or antigen-binding fragment thereof that contains the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3, may bind TNFR2 directly on the surface of a cancer cell (e.g., a cutaneous T cell lymphoma cell, ovarian cancer cell, colon cancer cell, or multiple myeloma cell, such as an ovarian cancer cell) in order to suppress the ability of the cell to proliferate and / or to promote apoptosis of the cell. TNFR2 antagonist polypeptides are not reliant on additional TNFR2-binding agents for activity Significantly, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the invention are capable of binding TNFR2 and suppressing 20 TNFR2-mediated signalling without the need for an endogenous TNFR2-binding agent, such as TNFa. Antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention do not require TNFa to attenuate T-reg and / or cancer cell proliferation. Without being limited by mechanism, antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may exhibit this property due to the ability of these antibodies or antigen-binding 25 fragments thereof to bind TNFR2 and stabilize the anti-parallel dimer conformation of this receptor. This structural configuration is not capable of potentiating NFkB signaling. By maintaining TNFR2 in an inactive structural state, antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may prevent TNFR2 agonists from restoring cell growth. For instance, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, 30 and antigen-binding fragments thereof of the invention may bind TNFR2 on the surface of a TNFR2+ cell, such as a T-reg cell, cancer cell, or myeloid-derived suppressor cell (MDSC) and inhibit the proliferation of such cells in the presence or absence of TNFa. For example, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention may inhibit the proliferation of such cells by, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 35   25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, relative to such cells that are not treated with the TNFR2 antagonist polypeptide. The antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-biding fragment thereof) may exhibit an ICso value in such a cell proliferation assay that is largely unchanged by the presence or absence of TNFa (e.g., an ICso value in the presence of TNFa that is changed by less than 50%, 45%, 38 2023258320   30 Oct 2023 40%, 35%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% relative to the ICso value of the antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment thereof) in the same cell proliferation assay in the absence of TNFa). Examples of cell death assays that can be used to measure the antagonistic effects of TNFR2 antibodies are described herein, e.g., in Example 9, below. Similarly, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention may inhibit TNFR2 signaling 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 clAP2 / BIRC3 by, e.g., 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, relative to such cells that are not treated with the TNFR2 antagonist polypeptide. The antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-biding fragment thereof) may exhibit an ICso value in such a gene expression assay that is largely unchanged by the presence or absence of TNFa (e.g., an ICso value in the presence of TNFa that is changed by less than 50%, 45%, 40%, 35%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% relative to the ICso value of the antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment thereof) in the same gene expression assay in the absence of TNFa). Examples of gene expression assays that can be used to measure the antagonistic effects of TNFR2 antibodies are described herein, e.g., in Example 12, below. Direct killing of T-reg cells, TNFR2+ cancer cells, and MDSCs Antagonistic TNFR2 polypeptides disclosed herein, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof, can not only reduce T-reg cell, TNFR2+ cancer cell, and / or MDSC proliferation, but can also induce the death of T-reg cells, TNFR2+ cancer cells, and / or MDSCs within a sample (e.g., within a patient, such as a human patient). Antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may be capable of reducing the total quantity 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 an 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 as described herein) by, e.g., 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, relative to a sample not treated with an antagonist TNFR2 antibody or antigen-binding fragment thereof. The ability of antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention to attenuate T-reg, MDSC, and / or cancer cell growth may be due in part to the ability of these antibodies or antigen-binding fragments to diminish the quantity of soluble TNFR2 within a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infectious disease as described herein). In the absence of this beneficial activity, soluble TNFR2 can be secreted by, e.g., T-reg cells, and could otherwise interfere with the ability of TNFR2 antagonists to localize to TNFR2 at the surface of a T-reg cell, TNFR2+ cancer ceil, or MDSC by binding 39 £023258330   30 Get 2023 and sequestering such antagonists in the extracellular environment. By reducing TNFR2 secretion, antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may render T-reg cells, TNFR2+ cancer cells, and / or MDSCs increasingly susceptible to therapeutic molecules, such as an antagonistic TNFR2 antibody or antigen-binding fragment thereof, and / or additional anti-cancer agents described herein or known in the art that may be used in conjunction with the compositions and methods of the invention. Selective modulation of active (CD25Hi and CD45RALow) T-reg cells Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigenbinding fragments) of the invention may be capable of inhibiting the proliferation or reducing the total quantity of T-reg cells in a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infectious disease as described herein) and may act selectively on T-reg cells in an actively-dividing state. Antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may selectively target active T-reg cells that express CD25Hi and CD45RALow, e.g., over resting T-reg cells that express CD25Med and CD45RAHi. For instance, antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may be capable of reducing the proliferation of T-reg cells expressing CD25Hi and CD45RALow by, e.g., 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 relative to T-reg cells that do not express the CD25Hi and CD45RALow proteins, such as T-reg cells that express CD25Med and CD45RAHi proteins. Modulation of T-reg cells, MDSCs, and T effector cells in the tumor microenvironment Antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of T-reg cells with a greater potency in a patient suffering from cancer relative to a subject that does not have cancer. The antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of T-reg cells with a greater potency in the microenvironment of a tumor relative to a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or in a subject without cancer. This effect may be determined using, for example, a cell death assay as described herein. For instance, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may exhibit an ICso for reducing or inhibiting the proliferation of T-reg cells in the microenvironment of a tumor that is less than the IC50 of the polypeptides for reducing or inhibiting the proliferation of T-reg cells in a site that is free of cancer cells by, 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, 30fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more. Examples of cell death assays that can be used to measure the antagonistic effects of anti-TNFR2 polypeptides are described herein, e.g., in Example 9, below. The polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments {hereof, may reduce or inhibit the proliferation of T-reg cells 40 2023258320   30 Oct 2023 with a potency that is greater in the microenvironment of a tumor containing T cell lymphoma cells (e.g., Hodgkin’s 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 that is free of such cancer cells, such as a site distal from a tumor in a patient suffering from one or more of the foregoing cancers or a in a subject without cancer. Additionally or alternatively, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of MDSCs with a greater potency in a patient suffering from cancer relative to a subject that does not have cancer. The polypeptides described herein, such as single-chain polypeptides, antibodies, and antigenbinding fragments thereof, may reduce or inhibit the proliferation of MDSCs with a greater potency in the microenvironment of a tumor relative to a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or in a subject without cancer. This effect may be determined using, for example, a cell death assay described herein. For instance, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may have an IC50 for reducing or inhibiting the proliferation of MDSCs in the microenvironment of a tumor that is less than the IC50 of the polypeptides for reducing or inhibiting the proliferation of MDSCs in a site that is free of cancer cells by, 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, 35fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more. Examples of cell death assays that can be used to measure the antagonistic effects of anti-TNFR2 polypeptides are described herein, e.g., in Example 9, below. The polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of MDSCs or may promote the apoptosis of MDSCs with a potency that is greater in the microenvironment of a tumor containing 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 than in a site that is free of such cancer cells, such as a site distal from a tumor in a patient suffering from one or more of the foregoing cancers or in a subject without cancer. Additionally or alternatively, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may expand T effector cells, such as CD8+ cytotoxic T cells, with a greater potency in a patient suffering from cancer relative to a subject that does not have cancer. In some embodiments, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, expand T effector cells, such as CD8+ cytotoxic T cells, with a greater potency in the microenvironment of a tumor relative to a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or a in a subject without cancer. This effect may be determined using, for example, a cell proliferation assay described herein. For instance, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may have an EC50 for the expansion of T effector cells in the microenvironment of a tumor that is less than the EC50 of the polypeptides for expanding T effector cells in a site that is free of cancer cells by, for example, 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 41 £023258330   30 Get 2023 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. Examples of cell proliferation assays that can be used to measure the effects of anti-TNFR2 polypeptides on T effector cells are described herein, e.g., in Example 18, below. The polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may directly expand T effector cells, such as CD8+ cytotoxic T cells, with a potency that is greater in the microenvironment of a tumor containing \ T cell lymphoma ceils (e.g., Hodgkin’s 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 that is free of such cancer cells, such as a site distal from a tumor in a patient suffering from one or more of the foregoing cancers or in a subject without cancer. The T effector cells (e.g., CD8+ cytotoxic T cells) may, for example, 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, among cells of other cancers described herein. Activity of antigen-binding fragments of full-length TNFR2 antagonist antibodies Antagonistic TNFR2 antibodies of the invention may inhibit, e.g., T-reg, cancer cell, and / or MDSC growth, or promote T effector cell growth, with a similar potency as that exhibited by antigen-binding fragments of such antibodies. For instance, removal of the Fc region of an antagonistic TNFR2 antibody 20 of the invention may not alter the ability of the molecule to attenuate the proliferation or reduce the total quantity 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). Antagonistic TNFR2 antibodies and antigen-binding fragments thereof of the invention may function by a pathway distinct from antibody-dependent cellular cytotoxicity (ADCC), in which a Fc region is required to recruit 25 effector proteins in order to induce cell death. Additionally, antagonistic TNFR2 antibodies or antigenbinding fragments thereof may not be susceptible to a loss of inhibitory capacity in the presence of crosslinking agents. Antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may therefore exhibit therapeutic activity in a variety of isotypes, such as IgG, IgA, IgM, IgD, or IgE, or in a variety of forms, such as a single-chain polypeptide (e.g., a single-chain polypeptide one or more CDRs 30 covalently bound to one another, for instance, 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 multi-specific antibody or antigen-binding fragment thereof, a dual-variable 35 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, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, and a tandem scFv (taFv). 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 Specific binding properties of antagonistic TNFR2 polypeptides The specific binding of a polypeptide, such as a single-chain polypeptide, antibody, or antibody fragment of the invention, to human TNFR2 can be determined by any of a variety of established methods. The affinity can be represented quantitatively by various measurements, including the concentration of antibody needed to achieve half-maximal inhibition of the TNFa-TNFR2 interaction in vitro (IC50) and the equilibrium constant (Kd) of the antibody-TNFR2 complex dissociation. The equilibrium constant, Kd, that describes the interaction of TNFR2 with an antibody of the invention is the chemical equilibrium constant for the dissociation reaction of a TNFR2-antibody complex into solvent-separated TNFR2 and antibody molecules that do not interact with one another. Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention include those that specifically bind to TNFR2 with a Kd value 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). In some embodiments, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention are those that specifically bind to TNFR2 with a Kd value of less than 1 nM (e.g., (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620 pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM). Polypeptides of the invention can also be characterized by a variety of in vitro binding assays. Examples of experiments that can be used to determine the Kd or IC50 of an anti-TNFR2 polypeptide include, e.g., 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 typically require minimal concentrations of antibodies. A common signal that is analyzed in a typical ELISA assay is luminescence, which is typically the result of the activity of a peroxidase conjugated to a secondary antibody that specifically binds a primary antibody (e.g., a TNFR2 antibody of the invention). Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention are capable of binding TNFR2 and epitopes derived thereof, such as epitopes containing one or more of residues 142-146 of SEQ ID NO: 7 within human TNFR2 (KCRPG, as shown in Figures 2A and 2B), as well as isolated peptides derived from TNFR2 that structurally pre-organize various residues in a manner that may simulate the conformation of these amino acids in the native protein. For instance, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may bind peptides containing the amino acid sequence of any one of SEQ ID NOs: 11,19,20, 34-117, 285, or 286, or a peptide containing between about 10 and about 30 continuous or discontinuous amino acids between positions 80 and 130 of SEQ ID NO: 7. In a direct ELISA £023258330   30 Get 2023 experiment, this binding can be quantified, e.g., by analyzing the luminescence that occurs upon incubation of an HRP substrate (e.g., 2,2’-azino-di-3- ethylbenzthiazoline sulfonate) with an antigenantibody complex bound to a HRP-conjugated secondary antibody. For instance, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may induce a luminescence response of about 400 absorbance units or more when incubated with surface-immobilized antigen and a HRP-conjugated secondary antibody in the presence of an HRP substrate (see, e.g., Example 3). In some embodiments, the luminescence observed can be from about 400 to about 900 absorbance units (e.g., 400-900 absorbance units, 500-800 absorbance units, or 600-700 absorbance units). In some embodiments, the luminescence observed can be from about 600 to about 900 absorbance units (e.g., 600-900 absorbance units or 700-800 absorbance units). Kinetic properties of antagonistic TNFR2 polypeptides In addition to the thermodynamic parameters of a TNFR2-polypeptide interaction, it is also possible to quantitatively characterize the kinetic association and dissociation of a polypeptide of the invention with TNFR2. This can be done, e.g., by monitoring the rate of antibody-antigen complex formation according to established procedures. For example, one can use surface plasmon resonance (SPR) to determine the rate constants for the formation (kon) and dissociation (kott) of an antibody-TNFR2 complex. These data also enable calculation of the equilibrium constant of (Kd) of antibody-TNFR2 complex dissociation, since the equilibrium constant of this unimolecular dissociation can be expressed 20 as the ratio of the kOft to kon values. SPR is a technique that is particularly advantageous for determining kinetic and thermodynamic parameters of receptor-antibody interactions since the experiment does not require that one component be modified by attachment of a chemical label. Rather, the receptor is typically immobilized on a solid metallic surface which is treated in pulses with solutions of increasing concentrations of antibody. Antibody-receptor binding induces distortion in the angle of reflection of 25 incident light at the metallic surface, and this change in refractive index over time as antibody is introduced to the system can be fit to established regression models in order to calculate the association and dissociation rate constants of an antibody-receptor interaction. Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may exhibit high kon and low kott values upon interaction with TNFR2, consistent with high-30 affinity receptor binding. For example, antibodies of the invention may exhibit kon values in the presence of TNFR2 of greater than 104 M s’: (e.g., 1.0 x 104 M4S‘1, 1.5 x 104 M :s , 2.0 x 104 M-'s-1, 2.5 x 104 Ms 1,3.0 x 104 M 's-1,3.5 x 104 M-1s1,4.0 x 104       4.5 x 104 M1s-1,5.0 x 104 M-1s-\ 5.5 x 104 M 's1,6.0 x 104 M-1sT 6.5 x 104 M-1s-1,7.0x 104 M1s-1, 7.5 x 104 M's-1,8.0 x 104 M 's1,8.5 x 104 M-1s1,9.0 x 104 M V, 9.5 x 104 M-1s1,1.0 x 105 M-'s1, 1.5 x 105 M-1s1, 2.0 x 105 M1S'1,2.5 x 105 M-1sT3.0 x 105 M's1, 3.5 35 x 105 M-1s-1, 4.0 x 105 M 1s-1, 4.5 x 105 M-1s1,5.0 x 105 M-1S’1,5.5 x 105 M 's1, 6.0 x 105 M-1S'1, 6.5 x 105 Ms', 7.0 x 105 M^S'1, 7.5 x 105 M's', 8.0 x 105 M-1S'1, 8.5 x 105 M 1S'1, 9.0 x 105 M^S'1, 9.5 x 105 M-'s'1, or 1.0 x 106 Ms'). Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may exhibit low kott values when bound to TNFR2, as these polypeptides are capable of interacting with distinct TNFR2 epitopes with a high affinity. Residues within these epitopes 2023258320   30 Oct 2023 form strong intermolecular contacts with TFNR2, which serves to slow the dissociation of the antibody-TNFR2 complex. This high receptor affinity is manifested in low kOff values. For instance, antibodies of the invention may exhibit kotf values of less than 10-3 s when complexed to TNFR2 (e.g., 1.0x10-3 s1,9.5 x 10’4 S, 9.0 x 10’4 s, 8.5 x 10’4 s-1,8.0 x 10 4 s', 7.5 x 10 4 s’,7.0 x 10 4 s’,6.5x1 O’4 s',6.0x1 O’4 s', 5.5 x 10'4 S'1,5.0 x 10'4 S'1,4.5 x 10"4 s’,4.0 x 104 s’,3.5 x 10'4 S’1,3.0 x 10'4 S’1, 2.5 x 10"4 s,2.0 x 10’4 s-1, 1.5 x 104 S'1,1.0 x 104 S'1, 9.5 x 10 5 s1,9.0 x 10 5 S’1, 8.5 X TO 5 s1,8.0 x 10’5 s4, 7.5 x 10 5 s1,7.0 x 10 5 S, 6.5 x 10’5 s-1, 6.0 x 10 5 s-1, 5.5 x 10 5 s', 5.0 x 10 5 s',4.5 x 10’5 s',4.0 x 10 s s', 3.5 x 10’5 S, 3.0 x 103 s-1,2.5 x 105 s-1,2.0 x 10 5 s ’, 1.5 x 10 5 s1, or 1.0 x 10 5 s1). Epitopes within TNFR2 bound by antagonistic TNFR2 polypeptides Among the difficulties in developing anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) that are capable of antagonizing TNFR2 has been the elucidation of epitopes within TNFR2 that participate in antagonistic complex formation rather than epitopes that promote signal transduction. The present invention is based in part on the discovery of epitopes within TNFR2 that, when bound, promote receptor antagonism. Particularly important epitopes that bind antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention and promote receptor antagonism are those that contain one or more residues of the KCRPG motif (SEQ ID NO: 19), located at positions 142-146 of SEQ ID NO: 7 within human TNFR2. One or more of these residues reside within larger epitopes (e.g., residues 142-149 of SEQ ID NO: 7, shown in Figure 2A, and residues 137-144 of SEQ ID NO: 7, shown in Figure 2B) that may interact with antagonistic TNFR2 antibodies of the invention. The knowledge of those residues that selectively bind antagonistic TNFR2 antibodies can be used to identify and design a wide array of antagonistic TNFR2 antibodies and antigen-binding fragments thereof using library screening techniques, e.g., those described herein or known in the art. For instance, peptides containing one or more the residues within the KCRPG sequence (e.g., the LRKCRPGFGVA motif (SEQ ID NO: 285) within human TNFR2) can be used to screen and select for antibodies and antibody-like scaffolds that bind these epitopes with high affinity and selectivity. Importantly, antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention are capable of selectively binding an epitope of TNFR2 that contains one or more of the residues of the KCRPG motif (SEQ ID NO: 19) and distinctly do not exhibit specific binding to an epitope containing residues 56-60 of SEQ ID NO: 7 within human TNFR2 (KCSPG, SEQ ID NO: 12). Polypeptides that exhibit the ability to bind an epitope containing one or more residues of amino acids 142-146 of SEQ ID NO: 7 within human TNFR2 and an epitope containing residues 56-60 of SEQ ID NO: 7 within human TNFR2 have been shown to lack inhibitory (antagonistic) activity. As such, the ability of a TNFR2 polypeptide to discriminate among these epitopes and specifically interact with an epitope including one or more of residues 142-146 of SEQ ID NO: 7 within human TNFR2 and to not engage in specific binding with an epitope composed of residues 56-60 of SEQ ID NO: 7 within human TNFR2 characterizes antibodies of the invention that antagonize TNFR2 signaling. £023258330   30 Get 2023 In addition to one or more residues of amino acids 142-146 of SEQ ID NO: 7, antagonistic TNFR2 polypeptides, such a dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may also bind one or more residues of a larger epitope that includes at least five continuous or discontinuous residues from positions 130-149 of SEQ ID NO: 7 within human TNFR2 (KQEGCRLCAPLRKCRPGFGV, SEQ ID NO: 17), or an epitope that exhibits at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. For example, antagonistic TNFR2 antibodies of the invention may specifically bind an epitope containing residues 142-149 of SEQ ID NO: 7 within human TNFR2 (KCRPGFGV, SEQ ID NO: 20), or an epitope that exhibits at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence (so long as one or more residues of the KCRPG sequence is present in the epitope). Additionally or alternatively, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may specifically bind an epitope including residues 137-144 of SEQ ID NO: 7 within human TNFR2 (CAPLRKCR, SEQ ID NO: 11), or an epitope that exhibits at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence (so long as one or more residues of the KCRPG sequence is present in the epitope). In addition to the KCRPG motif (SEQ ID NO: 19), it has been discovered that another important epitope present within human TNFR2 that promotes receptor antagonism contains residues 159-171 of SEQ ID NO: 7 (VVCKPCAPGTFSN, SEQ ID NO: 286). Antagonistic TNFR2 polypeptides (e.g., singlechain polypeptides, antibodies, and antigen-binding fragments) of the invention may therefore bind an epitope downstream of the KCRPG sequence (SEQ ID NO: 19) that contains, for instance, at least five continuous or discontinuous residues from positions 150-190 of SEQ ID NO: 7 within human TNFR2 (ARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI, SEQ ID NO: 22), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. For example, in some embodiments, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention) may specifically bind an epitope that includes residues from positions 161 -169 of SEQ ID NO: 7 within human TNFR2 (CKPCAPGTF, SEQ ID NO: 21), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention may specifically bind an epitope that includes residues from positions 140-150 of SEQ ID NO: 7 within human TNFR2 (LRKCRPGFGVA, SEQ ID NO: 285), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. Additionally or alternatively, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention may specifically bind an epitope that includes residues from positions 159-171 of SEQ ID NO: 7 within human 46 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 TNFR2 (VVCKPCAPGTFSN, SEQ ID NO: 286), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. In addition to the above-described epitopes, antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may also specifically bind an epitope within human TNFR2 that includes at least five continuous or discontinuous residues from positions 75-128 of SEQ ID NO: 7 within human TNFR2 (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL, SEQ ID NO: 13), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. Anti-TNFR2 polypeptides of the invention may also specifically bind an epitope within human TNFR2 that includes at least five continuous or discontinuous residues from positions 75-91 of SEQ ID NO: 7 within human TNFR2 (CDSCEDSTYTQLWNWVP, SEQ ID NO: 14), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. In some embodiments, anti-TNFR2 polypeptides of the invention may specifically bind an epitope that includes residues at positions 80-86 of SEQ ID NO: 7 within human TNFR2 (DSTYTQL, SEQ ID NO: 8), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. Antagonistic TNFR2 polypeptides of the invention also may specifically bind an epitope that includes at least five continuous or discontinuous residues from positions 86-103 of SEQ ID NO: 7 within human TNFR2 (LWNWVPECLSCGSRCSSD, SEQ ID NO: 15), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. In particular cases, antibodies of the invention may specifically bind an epitope that includes residues from positions 91 -98 of SEQ ID NO: 7 within human TNFR2 (PECLSCGS, SEQ ID NO: 9), as well as an epitope that exhibits at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. The polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may also specifically bind an epitope that include at least five continuous or discontinuous residues from positions 111 -128 of SEQ ID NO: 7 within human TNFR2 (TREQNRICTCRPGWYCAL, SEQ ID NO: 16), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. In some embodiments, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may specifically bind an epitope that includes residues from positions 116-123 of SEQ ID NO: 7 within human TNFR2 (RICTCRPG, SEQ ID NO: 10), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. For £023258330   30 Get 2023 example, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigenbinding fragments) of the invention may specifically bind an epitope containing residues 116-123 of SEQ ID NO: 7 within human TNFR2 (RICTCRPG, SEQ ID NO: 10) and residues 137-144 of SEQ ID NO: 7 within human TNFR2 (CAPLRKCR, SEQ ID NO: 11). One exemplary procedure that can be used to predict the inhibitory activity of a TNFR2 polypeptide of the invention is to compare the affinity of the antibody or antibody fragment for a peptide containing the KCRPG motif (e.g., a linear peptide having the sequence LRKCRPGFGVA (SEQ ID NO: 285) or VVCKPCAPGTFSN (SEQ ID NO: 286) to the affinity of the same antibody or antibody fragment for a peptide containing the KCSPG sequence (e.g., a linear peptide having the sequence QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18). For instance, antagonistic TNFR2 antibody TNFRAB1 specifically binds the peptide fragment defined by residues 130-149 of SEQ ID NO: 7 within human TNFR2 (KQEGCRLCAPLRKCRPGFGV, SEQ ID NO: 17) with a 40-fold greater affinity than the peptide fragment defined by residues 48-67 of SEQ ID NO: 7 within human TNFR2 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18). Antagonistic TNFR2 antibodies and antigen-binding fragments of the invention bind an epitope containing one or more residues of the KCRPG sequence (SEQ ID NO: 19), e.g., with an affinity that is at least 10-fold greater than the affinity of the same antibody or antigen-binding fragment for a peptide that contains the KCSPG sequence of human TNFR2 (SEQ ID NO: 12). For example, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may bind an epitope containing one or more residues of the 20 KCRPG sequence (SEQ ID NO: 19) of human TNFR2 with an affinity that is 10-fold, 20-fold, 30-fold, 40fold, 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 than the affinity of the same antibody or antigen-binding fragment for a peptide that contains the KCSPG sequence (SEQ ID NO: 12) of human TNFR2. Antibodies or antibody fragments that bind epitopes containing one or more residues of 25 the KCRPG sequence (amino acids 142-146 of SEQ ID NO: 7 within human TNFR2) and epitopes containing the KCSPG motif (amino acids 56-60 of SEQ ID NO: 7 within human TNFR2) with similar affinity (e.g., less than a 10-fold difference in affinity) are not considered antagonistic TNFR2 antibodies of the invention. 30 Antagonistic TNFR2 polypeptides that bind TNFR2 from non-human animals In addition to binding epitopes within human TFNR2 that contain the KCRPG motif, antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention also include those that specifically bind epitopes containing the equivalent motif within TNFR2 derived from non-human animals, 35 such as the KCGPG motif (SEQ ID NO: 287) within TNFR2 of non-human mammals, e.g., in TNFR2 obtained from a cow, bison, mouse, or rat, among others. The location of sequences equivalent to the human KCRPG motif in TNFR2 derived from exemplary non-human mammals is shown in Table 2, below: Table 2. Location of sequences equivalent to KCRPG in TNFR2 from non-human mammals Source of TNFR2 Sequence equivalent to KCRPG Amino acid positions of equivalent sequence within TNFR2 SEQ ID NO. of full-length TNFR2 sequence Genbank Accession No. of full-length TNFR2 sequence Human KCRPG 142-146 7 P20333.3 Cattle KCGPG 142-146 280 AAI05223 Bison KCGPG 142-146 281 XP 010848145 Mouse KCGPG 144-148 282 AAA39752.1 Rat KCGPG 144-148 283 Q80WY6 2023258320   30 Oct 2023 Epitopes within TNFR2 derived from the non-human mammals discussed above that may be bound by antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., 5 single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention are illustrated in the sequence alignment below. This sequence alignment shows partial sequences of TNFR2 derived from human, cattle, bison, mouse, and rat, as well as epitopes (highlighted in grey) equivalent to the human KCRPG motif. 10 15 UI o 20 25 2023258320   30 Oct 2023 Alignment of partial TNFP.2 sequences derived from human and select non-human mammals Human :  1    MAP VAWAAL AvGLE LWAAAHALPAQVAF TP TAPE PGGTCRL —REYYDQTAQMC C SKCSPGQHAKVE CTK.TS DTVCDS C Cattle: 1    MAPiAFWAALAVGLQFWAAGRAVPAQAVFTPYIPEPGSSCRQ—qeyynqkiqmccskcpbgyrvqslcnmtlijticasc Bison:  1    MAPTAFWAALAVGLQFWAAGRAVPAQAVF.TPYIPEPGSSCRQ—QEYYNHKIQMCCSKCPPGYRVQSLCNXTL.IJZI'CASC Mouse:  1    MAPAALWVAEVFELQLWATGHWPAQVVLIPYKPEPGYECQIS—QEYYDRKAQMCCAKCPPGQYVKHFCNKTSDTVCADC Rati   i   MAPAALWVALVVELQLmTGHTWAKWLTPYKPEPGNQCQIS-QEYYDKKAQMCCAKCPPGQYAKHFCNKTSBTVCADC Human: 79 EDSl'Yl'QLwNwVPECLSCCSRCSSDQVEl'QACl'REQNR_Cl'CRPGWYCALSKQEC CRLCAPLRKCRPCt GVARPGThTS Cattle : 79 ESSfrrQLWNLVTACk SCNSRCSSDQVE'iQACl"iKQNR_Cl'CKPGWYCtLGRQEC CRLCVALRKCGPGt CVAKPGTA1"! Bison : 79 ESSlY'iQLwNLVlACb SCNSRCSSDQVE1QACHKQNR-C1CKPGWYC1LCRQEC CRLCVALRKCGPGh CVAKPClAl'l Mouse : 80 EASMYTQVWNQh RiXLSCSSSCitXQVE-RAClKQQNRVCACEACRYCALKlHSGSCRQCMRLSKCGPGl GVASSRAPNG Rat: 80 AACMt 'JQVWNHLH'_ CLSCSSSCSBL)QVE'_HNCYXKQNRVCACNADSYCALKLHSCNCRQCMKLSKCGPGb CVARSR1SNG Human : 158 DWCKPCAPG'TFSNSTSSTPICRPHQICNVVAIPGNASMBAVCIX——SISP’JRSMAPGAVHLPQPVSTRSQHIQPTP Cattle: 158 NVICAPCGE’G'TFSDSTCYTpiCKPHRNCSSVAIPGi'ASTDAVCl--——SVLPTRKVARG------PAZTRSQHMEPTL Bison: 158 NVICAPCGPGTFSDTTSYTDTCKPHRNCSSVAIPGTASTDAVCT— —SVLPTRKVARG— ------PATTRSQHMEPTL Mouse : 16 0 NVLCKACAPGXFSBTTSSTBVCRPHRICSXLAIPGNACTBAVCAPESPTLSAZPR------—TLYVSQPEPTRSQPLDQEP Rati 160 WXCSACAPGTF.SBTTS&TDVCRPHRXC.SItAIPGNASTDAVQASESPTPSAVPR-------“XETYVSQPEPTRSQPMDQEP Human :  232  EPSXAPSXSFLLPMGPSPPA----EGSTGBFALPVGLI’VGVTALGLLIlCWNCVIMXQVKKKPLCLQREAKVPHLPABK Cattle: 2 26 GPSXAPSXEFLLPKVPSPPSSPVEQPNTGNIMPIXLTVGVTALGLLLXVVVNCVIMXQKKKKPFCLQGBAKVPHLPANK Bison:  226 GPSXAPSXEFLLPKVPSPPSSPVEQPNAGNXSLPIELl'VGVXALGL.LtlVWNCVIMXQKKKKPFCLQGBAKVPHLPANK Mouse :  23 4 GPSQXPS—■-ILTSLGSTPlX-TEQSXKGGTSLPXGLXXGYTSLGLLMLGLVNCiILVQRKKKPSCLQRBAKVPHVPBEK Rat; 23 4 GPSQXPH---IPVSLGSTEX— IEPSX'XGGISLPIGLIVGlTXLGL'LMLGLANCFIXVQRKKKPSCLQRETMVPHLPBBK Human:  308  ARGTQGPEQQHLLITAPSSSSSSLESSASALBRRAPXRNQPQAPGVE-A.SGAGEARASTGSSBSSPGGHGXQVNVXCIVN Cattle: 306 AQGAPGPEQQHLLZTAPSSSSSSLESSXSSXBKRAPXRSQLQSPGVEKASXSGEAQTQCSSSEASSGGHGXQVNVXCIVN Bison:  306  AQGAPGPEQQHLLTTAPSSSSSSLESSXSSTBKRAP'IRSQLQSPGVE-ANXSGEAQTGCSSSEASSGGHGTQVJWXCIVN 30 Mouse:  309  SQDAVGLEQQHLLTZAPSSSSSSLESSASAGDRRAPPGGHPQARVMAEAQGFQEARASSRISDSSHGSHGTHVNVTCIVN Rati 309 SQDAIGLEQQHLLTTAPSSSSSSLESSA.SAGDRRAPPGGHPQARVTAEAQGSQEACAGSRSSDSSHGSHGTHVNVTC1VN 78 7 8 78 79 79 157 157 157 159 159 231 225 225 233 233 307 305 305 308 308 386 385 38 4 388 388 WO 2017 / 197331                                                 PCT / US2017 / 032513 2023258320   30 Oct 2023 The antagonistic TNFR2 antibody TNFRAB1 Antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigenbinding fragments thereof of the invention may contain the CDR-H3 sequence of TNFRAB1, also referred to herein as TNFR2 antagonist 1, which is a murine antibody that antagonizes the TNFRa-TNFR2 5 interaction. For instance, the CDR-H3 of TNFRAB1 and variants thereof (e.g., variants that exhibit conservative amino acid substitutions relative to this CDR-H3 sequence) can be used to make an antagonistic TNFR2 antibody or antigen-binding fragment thereof of the invention, for instance, using antibody humanization methods described herein or known in the art. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen- 10 binding fragments) of the invention may exhibit binding properties that are the same as or similar to those of TNFRAB1. These properties are as follows: In the presence of TNFR2, TNFRAB1 exhibits a high kon value of 4.98 x 106 M'1s-1, as well as a low kotf of 2.21 x 10-4 s-1 and a Kd of about 44.4 pM in complex with TNFR2. The KCRPGFGV motif (SEQ ID NO: 20), and specifically, the KCRPG sequence (SEQ ID NO: 19), has been identified as a particularly important component of the functional epitope that establishes 15 intermolecular contacts with TNFRAB1 as determined by epitope mapping analysis (Figures 2 and 3). The interaction of these residues with anti-TNFR2 antibodies of the invention selectively promotes antagonistic activity. Significantly, a TNFR2 epitope including amino acid residues 56-60 of SEQ ID NO: 7 within human TNFR2 (KCSPG, SEQ ID NO: 12) is distinctly not a part of the conformational epitope that is specifically bound by TNFRAB1 or antagonistic TNFR2 antibodies or antibody fragments of the 20 invention, as specific binding to both of these epitopes has been shown to lead to a loss of, or significant reduction in, antagonistic activity. As such, TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) that specifically bind both of these epitopes (KCSPG and an epitope containing at least the KCR sequence, and more specifically, the KCRPG sequence of human TNFR2) are not considered antagonistic TNFR2 antibodies of the invention. 25           In addition to binding an epitope contained within the sequence KCRPGFGV (SEQ ID NO: 20), TNFRAB1 also binds to a downstream epitope contained within a sequence defined by positions 161 -169 of SEQ ID NO: 7 within human TNFR2 (CKPCAPGTF, SEQ ID NO: 21). TNFR2 antibodies and antibody fragments of the invention may also bind this epitope or a larger region within TNFR2 containing this epitope (e.g., a sequence that includes at least five continuous or discontinuous residues from positions 30   150-190 of SEQ ID NO: 7 within human TNFR2 (ARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI, SEQ ID NO: 22). TNFRAB1 contains two heavy chains, as well as two light chains, as shown in Figures 1A and 1B. The heavy chains of TNFRAB1 contain the following amino acid sequence (CDRs are indicated in bold): 35 EVQLQESGGGLVKPGGSLKLSCAASGFTFSSYVMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRF TISRDNAKNTLYLQMSSLRSEDTAMYYCARQRVDGYSSYWYFDVWGAGTAVTVSS (SEQ ID NO: 2) The sequence of the TNFRAB1 light chain is as follows (CDRs are indicated in bold): £023258330   30 Get 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 DIVLTQSPAIMSASPGEKVTITCSASSSVYYMYWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSY SLTISRMEAEDAATYYCQQRRNYPYTFGGGTKLEIKRA (SEQ ID NO: 4) The antagonistic TNFR2 antibody TNFRAB2 An antagonistic TNFR2 antibody or antibody fragment of the invention may contain, for instance, the CDR-H3 sequence of TNFRAB2, also referred to herein as TNFR2 antagonist 2, an antibody that selectively binds and inhibits TNFR2 by virtue of specifically binding various epitopes within this receptor. For instance, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigenbinding fragments) of the invention may exhibit binding properties that are the same as or similar to those of TNFRAB2. These properties are as follows: In the presence of TNFR2, TNFRAB2 exhibits a high kon value of 3.6099 x 105 M-1s-1, as well as a low kotf of 2.24 x 10"4 S’1 and a Kd of about 621 pM in complex with TNFR2. An epitope containing residues 137-144 of SEQ ID NO: 7 within human TNFR2 (CAPLRKCR, SEQ ID NO: 11) has been identified as a particularly important component of the functional epitope that establishes intermolecular contacts with TNFRAB2 as determined by epitope mapping analysis (see, e.g., Example 1 and Figures 2B and 3B). Included in the invention are TNFR2 antibodies and antibody fragments that specifically bind this epitope. In addition to binding an epitope containing residues CAPLRKCR (SEQ ID NO: 11), TNFRAB2 also binds to epitopes that include one or more residues within positions 80-86 of SEQ ID NO: 7 within human TNFR2 (DSTYTQL, SEQ ID NO: 8), positions 91-98 of SEQ ID NO: 7 within human TNFR2 (PECLSCGS, SEQ ID NO: 9), as well as positions 116-123 of SEQ ID NO: 7 within human TNFR2 (RICTCRPG, SEQ ID NO: 10). TNFR2 antibodies and antibody fragments of the invention may also bind one or more of these epitopes. Antibodies and antibody fragments of the invention can be designed and identified using the knowledge of the epitopes specifically bound by TNFRAB2. For instance, one can use any of a variety of in vitro peptide display techniques or combinatorial antibody library screens as described herein or known in the art in order to screen for antibodies capable of binding these epitopes with high affinity and selectivity. The heavy chain and light chain CDRs of TNFRAB2 are shown below: TNFRAB2 CDR-H1: GYTFTDY(LZI) (SEQ ID NO: 257) 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 or YTS TNFRAB2 CDR-L3: CLQYVNL(L / I)T (SEQ ID NO: 261) As shown above, the CDR-H1 sequence of TNFRAB2 may contain either a leucine or isoleucine residue at the eighth position of this region. Similarly, the TNFRAB2 CDR-L2 may include a TYS or YTS tripeptide, and the TNFRAB2 CDR-L3 may contain either a leucine or isoleucine residue at the eighth 52 2023258320   30 Oct 2023 position of this region. Notably, the CDR-L2 of TNFRAB2 is flanked by the N-terminal framework residues LLIR (SEQ ID NO: 262) and the C-terminal framework residues TLE. Accordingly, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention include those that contain one or more of the above CDRs of TNFRAB2, as well as N-terminal LLIR (SEQ ID NO: 262) and C-terminal TLE residues that flank the CDR-L2 sequence of the antagonistic TNFR2 antibody or antigen-binding fragment thereof. The antagonistic TNFR2 antibody TNFR2A3 A representative antagonist TNFR2 antibody of the invention is TNFR2A3, a murine antibody that was discovered by immunization of a mouse with human TNFR2 and subsequent CDR mutagenesis. A precursor antibody to TNFR2A3 was identified from murine immunization experiments as a TNFR2-binding antibody that was neither antagonistic nor agonistic of TNFR2. Using recombinant gene expression techniques, TNFR2A3 was produced by replacing the CDR-H3 sequence of the precursor murine antibody with the CDR-H3 sequence ARDDGSYSPFDYFG (SEQ ID NO: 284). Upon insertion of this CDR-H3 sequence into the precursor scaffold, the resulting TNFR2A3 antibody was capable of exhibiting an antagonistic effect on the TNFR2 target. Additionally, the TNFR2A3 antibody was found to bind epitopes within human TNFR2 that are consistent with those bound by dominant antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2. As described in Example 15, TNFR2A3 binds to two distinct epitopes within human TNFR2. The first epitope spans residues 140-150 of human TNFR2 (LRKCRPGFGVA, SEQ ID NO: 285) and contains the KCRPG motif (SEQ ID NO: 19). The second epitope is a downstream sequence that contains residues 159-171 of human TNFR2 (VVCKPCAPGTFSN, SEQ ID NO: 286). For instance, dominant antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may exhibit binding properties that are the same as or similar to those of TNFR2A3. Importantly, the binding of TNFR2A3 to these epitopes within TNFR2 is sitespecific, as the TNFR2A3 antibody does not bind a human TNFR2-derived peptide containing an unrelated, distal amino acid sequence (see, e.g., Example 15). Collectively, these findings demonstrate two significant features of the CDR-H3 sequence with respect to TNFR2 antagonism: (i) that the CDR-H3 sequence of an antagonistic TNFR2 antibody largely dictates the antigen-binding properties of the system, and (ii) that the CDR-H3 motif is a modular domain that can be substituted into anti-TNFR2 antibodies that do not exhibit antagonistic activity in order to impart such antibodies with TNFR2 dominant antagonistic features. Molecular determinants of TNFR2 affinity and antagonism Notably, there are distinct sequence similarities between the CDR-H3 regions of the antagonistic TNFR2 antibodies TNFRAB1, TNFRAB2, and TNFR2A3. An analysis of the residues common to the CDR-H3 sequences of these antibodies provides insight into the molecular features of antibodies that bind TNFR2 and exhibit an antagonistic effect, such as a dominant antagonistic effect. Epitope mapping analysis has shown that both TNFRAB1, TNFRAB2, and TNFR2A3 bind epitopes within TNFR2 that contain residues 142--146 of SEQ ID NO: 7 and do not bind epitopes containing residues 56-60 of SEQ ID 53 20232583^0   30 Get 2023 NO: 7. The structural similarities between corresponding CDR-H3 regions provide a basis for predicting residue substitutions that may preserve or enhance TNFR2 affinity and antagonism (e.g., dominant antagonism). Inspection of the CDR-H3 sequences of these antibodies demonstrates that several residues and physicochemical characteristics are conserved throughout this region, while other positions within this CDR can be varied significantly without loss of affinity and antagonistic function. The CDR-H3 sequences of TNFRAB1, TNFRAB2, and TNFR2A3 are shown below: QRVDGYSSYWYFDV (TNFRAB1 CDR-H3, SEQ ID NO: 25) ARDDG-S-YSPFDYWG (TNFRAB2 CDR-H3, SEQ ID NO: 259) ARDDG-S-YSPFDYFG (TNFR2A3 CDR-H3, SEQ ID NO: 284) -R-DG-S-Y—FD---- (Consensus sequence) Inspection of the CDR-H3 sequences of TNFRAB1, TNFRAB2, and TNFR2A3 reveals conserved arginine, aspartic acid, glycine, serine, tyrosine, and phenylalanine residues throughout this CDR. Notably, residues of varying steric and electrostatic properties are tolerated in the remaining positions. For instance, the first position of the CDR-H3 sequence tolerates amino acid residues of contrasting size and hydrogen bond-forming tendencies, as the first position of CDR-H3 in TNFRAB1 features a polar glutamine residue containing a carboxamide side-chain with hydrogen bond donor and acceptor moieties, while an alanine residue bearing an unfunctionalized methyl side-chain is found at the corresponding position in TNFRAB2 and TNFR2A3. Additionally, the third position in the above CDR-H3 sequences features a hydrophobic valine in TNFRAB1 and an anionic aspartic acid moiety in the corresponding position of TNFRAB2. Similarly, positions ten and eleven of the CDR-H3 of TNFRAB1 contain aromatic systems, while the corresponding residues in TNFRAB2 and TNFR2A3 contain polar and cyclic aliphatic substituents. Collectively, the shared structural features of the above CDR-H3 sequences provide insight into those residues that are important for selectively binding residues within TNFR2 that promote receptor antagonism (e.g., dominant receptor antagonism), such as residues 140-150 of human TNFR2 (LRKCRPGFGVA, SEQ ID NO: 285) and residues 159-171 of human TNFR2 (VVCKPCAPGTFSN, SEQ ID NO: 286) and demonstrate that other amino acids can be varied while retaining affinity and dominant antagonistic activity. For instance, antagonistic TNFR2 polypeptides, such a dominant antagonistic TNFR2 polypeptides (e.g., ^'ngle-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention may contain a CDR-H3 represented by the formula JZ1 JZ2Z4JZ3JZ5(J)2Z5Z2Z5 or JZ1JZ2Z4Z3Z5(J)2Z5Z2Z5(J)2, wherein each J is independently a naturally occurring amino acid, each Z1 is independently a naturally occurring amino acid containing a cationic side-chain at physiological pH, each Z2 is independently a naturally occurring amino acid containing an anionic side-chain at physiological pH, each Z3 is independently a naturally occurring amino acid containing a polar, uncharged side-chain at physiological pH, each Z4 is independently a glycine or alanine, and each Z§ is independently a naturally occurring amino acid containing a hydrophobic side-chain. Similarly, antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention may contain a CDR-H3 represented by the formula JRJDGJSJY(J)2FDJ (SEQ ID NO: 278), 54 2023258320   30 Oct 2023 JRJDGSY(J)2FD(J)3(SEQ ID NO: 279), QZ1 VZ2Z4YZ3SZ5WYZ5Z2Z5 (SEQ ID NO: 265), or AZ1DZ2Z4Z3Z5SPZ5Z2Z5WG (SEQ ID NO: 266). For instance, the CDR-H3 may be derived from TNFRAB1 and have the amino acid sequence QRVDGYSSYWYFDV (SEQ ID NO: 25). The CDR-H3 may be derived from TNFRAB2 and have the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 5   259). In some embodiments, the CDR-H3 is derived from TNFR2A3 and has the amino acid sequence ARDDGSYSPFDYFG (SEQ ID NO: 284). In addition to the above CDR-H3 sequences, other CDR sequences can be include in antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention. Additional CDR 10 sequences that promote dominant TNFR2 antagonism can be determined, for instance, by alignment of the CDR sequences of TNFRAB1 and TNFRAB2. For example, the CDR-H1 sequences of TNFRAB1 and TNFRAB2 are shown below: GFTFSSY (TNFRAB1 CDR-H1, SEQ ID NO: 23) 15                GYTFTDY(L / I) (TNFRAB2 CDR-H1, SEQ ID NO: 257) g-tf—y-     (Consensus sequence) Alignment of the sequences reveals a shared GXTFXXY motif, wherein “X" designates any amino acid. These CDR-H1 sequences feature a conserved glycine residue at the first position and conserved 20 threonine, phenylalanine, and tyrosine residues at the third, fourth, and seventh positions, respectively. Inspection of these sequences demonstrates that the CDR-H1 region is tolerant of substitutions at the remaining positions. Side-chains of varying polarity are tolerated at the second position, for example, as both phenylalanine, containing an unsubstituted and hydrophobic phenyl moiety, and tyrosine, containing a protic hydroxyl substituent, are found in this position in the CDR-H1 region of TNFRAB1 and TNFRAB2, 25 respectively. Additionally, while the fifth and sixth positions are occupied by polar serine residues in the CDR-H1 of TNFRAB1, these positions feature a threonine, containing an additional hydrophobic methyl substituent, and aspartic acid, which is anionic at physiological pH, in TNFRAB2. This diversity demonstrates that these positions can be substituted with amino acids of diverse electrostatic properties without loss of TNFR2 affinity and antagonism. 30          Sequence analysis of the CDR-H2 regions of TNFRAB1 and TNFRAB2 similarly reveals a set of conserved amino acids at various positions throughout these regions: 35 SSG—GSY (TNFRAB1 CDR-H2, SEQ ID NO: 24) VDPEYGST (TNFRAB2 CDR-H2, SEQ ID NO: 258) -----GS- (Consensus sequence) Analysis of this sequence alignment demonstrates that the CDR-H2 sequences exhibit a conserved GS motif at the C-terminal end of the CDR-H2 region, with side-chains of variable molecular 2023258330   30 Get 2023 size, polarity, and electrostatic charge tolerated at the remaining positions. A similar analysis reveals molecular features common to the CDR-L sequences of TNFRAB1 and TNFRAB2. For instance, the CDR-L1 sequences of TNFRAB1 and TNFRAB2 are shown below: SASSSVYYMY (TNFRAB1 CDR-L1, SEQ ID NO: 26) Q-N— INK-Y (TNFRAB2 CDR-L1, SEQ ID NO: 260) y (Consensus residue) Inspection of these sequences reveals that a hydroxyl-containing tyrosine residue is featured at the final position of CDR-L1, while residues of varying physicochemical properties are tolerated at the remaining positions. Similarly, analysis of the CDR-L2 regions of TNFRAB1 and TNFRAB2 reveals a conserved amino acid at the final position in both regions: STSNLAS (TNFRAB1 CDR-L2, SEQ ID NO: 27) TY----S or (TNFRAB2 CDR-L2) 15                 YT----S s (Consensus residue) Analysis of the above sequence alignment demonstrates that serine residues are featured at the third position of these CDR-L2 sequences, while substitutions are widely tolerated at the remaining 20 residues. Similarly, the CDR-L3 sequences of TNRAB1 and TNFRAB2 are as follows: Q-QRRNYPY------T (TNFRAB1 CDR-L3, SEQ ID NO: 28) CLQ---YVNL(L / I)T (TNFRAB2 CDR-L3, SEQ ID NO: 261) ------y--------t (Consensus sequence) 25 Analysis of the CDR-L3 sequences of TNFRAB1 and TNFRAB2 reveals a preference for tyrosine and threonine residues at distinct positions within these regions, while amino acids of a wide range of physicochemical characteristics are tolerated at other positions, including residues with cationic sidechains (Arg), conformationally restricted side-chains (Pro), and side-chains of varying polarity (e.g., Gin, 30 Asn, Leu, and Vai). Collectively, the shared structural features of the above CDR-H and CDR-L sequences provide insight into those residues that are important for selectively binding one or more residues of the KCRPG epitope of TNFR2 (positions 142-146 of SEQ ID NO: 7, shown in SEQ ID NO: 19) in an anti-parallel dimer configuration and demonstrate that certain amino acids can be varied while retaining affinity and dominant antagonistic activity. 35           Antagonistic TNFR2 polypeptides of the invention, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may therefore have heavy chain and light chain CDRs that contain the above consensus sequences. For instance, TNFR2 antagonists of the invention may have a CDR-H1 having the amino acid sequence 2023258320   30 Oct 2023 Z4JZ3Z5(J)2Z5J; a CDR-H2 having the amino acid sequence (J)sZ4Z3J; a CDR-L1 having the amino acid sequence (J)sZ5; a CDR-L2 having the amino acid sequence (J)2Z3; and / or a CDR-L3 having the amino acid sequence (J)aZ5(J)4Z3; wherein each J is independently a naturally occurring amino acid; each Z' is independently a naturally occurring amino acid containing a cationic side-chain at physiological pH; each Z2 is independently a naturally occurring amino acid containing an anionic side-chain at physiological pH; each Z3 is independently a naturally occurring amino acid containing a polar, uncharged side-chain at physiological pH; each Z4 is independently a glycine or alanine; and each Z5 is independently a naturally occurring amino acid containing a hydrophobic side-chain. In some embodiments, antagonistic TNFR2 polypeptides of the invention, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 having the amino acid sequence GJTF(J)2YJ (SEQ ID NO: 277); a CDR-H2 having the amino acid sequence (J)sGSJ; a CDR-L1 having the amino acid sequence (J)sY; a CDR-L2 having the amino acid sequence (J)2S; and / or a CDR-L3 having the amino acid sequence (J)3Y(J)4T; wherein each J is independently a naturally occurring amino acid. Antagonistic TNFR2 polypeptides of the invention, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 having the amino acid sequence Z4YZ3Z5TDZ5X; a CDR-H2 having the amino acid sequence VDPEYZ4Z3T (SEQ ID NO: 264); a CDR-L1 having the amino acid sequence QNINKZ5(SEQ ID NO: 268); a CDR-L2 having the amino acid sequence TYZ3 or YTZ3; and / or a CDR-L3 having the amino acid sequence CLQZ5VNLXZ3(SEQ ID NO: 271); wherein each Z1 is independently an amino acid containing a cationic side-chain at physiological pH; each Z2 is independently an amino acid containing an anionic side-chain at physiological pH; each Z3 is independently an amino acid containing a polar, uncharged side-chain at physiological pH; each Z4 is independently a glycine or alanine; each Z5 is independently an amino acid containing a hydrophobic side-chain; and each X is independently leucine or isoleucine. In some embodiments, antagonistic TNFR2 polypeptides of the invention, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257), or an amino acid sequence having up to two amino acid substitutions relative to this sequence; a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258), or an amino acid sequence having up to two amino acid substitutions relative to this sequence; a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260), or an amino acid sequence having up to two amino acid substitutions relative to this sequence; a CDR-L2 having the amino acid sequence TYS or YTS; and / or a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261), or an amino acid sequence having up to two amino acid substitutions relative to this sequence. For example, in some embodiments, antagonistic TNFR2 polypeptides of the invention, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigenbinding fragments thereof) may have a CDR-H1 having the amino acid sequence GYTFTDYL (SEQ ID NO: 274), or an amino acid sequence having up to two amino acid substitutions relative to this sequence; and a CDR-L3 having the amino acid sequence CLQYVNLIT (SEQ ID NO: 273), or an amino acid 57 £023258330   30 Get 2023 sequence having up to two amino acid substitutions relative to this sequence. The present invention is based in part on the discovery that this particular combination of CDR-H1 and CDR-L3 regions promote the selective killing of activated T-reg cells and potentiate augmented T effector cell proliferation. As described herein, these phenotypes are beneficial for the treatment of cancers and infectious diseases, as the ability to deplete activated T-reg cell populations in a patient suffering from such pathologies can lessen the attenuation of cytotoxic CD8+ T cells, thereby enabling effector cells to mount an immune response against cancerous and infectious cells (see, e.g., Example 17). Humanized, primatized, and chimeric antibodies Antibodies of the invention include human, humanized, primatized, and chimeric antibodies that contain the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3, or a CDR-H3 that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences that contain conservative mutations relative to these CDR-H3 sequences. Antibodies of the invention also include human, humanized, primatized, and chimeric antibodies that contain the CDR-H3 of TNFRAB1, TNFAB2, or TNFR2A3, or a CDR-H3 that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences that contain conservative mutations relative to these CDR-H3 sequences. For instance, antibodies of the invention also include human, humanized, primatized, and chimeric antibodies that contain a CDR-H3 that is identical to the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 except for conservative amino acid substitutions. In some embodiments, a humanized, primatized, or chimeric antibody may contain the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3, or a CDR that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences that contain conservative mutations relative to these CDR-H3 sequences. For example, antagonistic TNFR2 antibodies of the invention can be generated by incorporating any of the above CDR-H3 sequences into the framework regions (e.g., FW1, FW2, FW3, and FW4) of a human antibody. Exemplary framework regions that can be used for the development of a humanized anti-TNFR2 antibody containing one or more of the above CDRs include, without limitation, those described in US Patent No. 7,732,578, US Patent No. 8,093,068, and WO 2003 / 105782; incorporated herein by reference. One strategy that can be used to design humanized antibodies of the invention is to align the sequences of the heavy chain variable region and light chain variable region of an antagonistic TNFR2 antibody, such as TNFRAB1, TNFRAB2, or TNFR2A3, with the heavy chain variable region and light chain variable region of a consensus human antibody. Consensus human antibody heavy chain and light chain sequences are known in the art (see e.g., the “VBASE” human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242, 1991; Tomlinson et al., J. Mol. Biol. 227:77698, 1992; and Cox et al, Eur. J. Immunol. 24:827-836, 1994; incorporated herein by reference). In this way, the variable domain framework residues and CDRs can be identified by sequence alignment (see Kabat, supra). One can substitute, for example, the CDR-H3 of the consensus human antibody with the 58 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 CDR-H3 of an antagonistic TNFR2 antibody, such as a CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3, in order to produce a humanized TNFR2 antagonist antibody. Exemplary variable domains of a consensus human antibody include the heavy chain variable domain: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGSDTYYADSVKGR FTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS (SEQ ID NO: 32) and the light chain variable domain: DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 33) identified in US Patent No. 6,054,297; incorporated herein by reference (CDRs are shown in bold were determined according to the method of Chothia, et al., J. Mol. Biol, 196:901-917, 1987). These amino acid substitutions can be made, for example, by recombinant expression of polynucleotides encoding the heavy and light chains of a humanized antibody in a host cell using methods known in the art or described herein. Similarly, this strategy can also be used to produce primatized anti-TNFR2 antibodies, as one can substitute, for example, the CDR-H3 of a primate antibody consensus sequence with, for example, the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3. Consensus primate antibody sequences 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). In some embodiments, it may be desirable to import particular framework residues in addition to CDR sequences from a TNFR2 antibody, such as TNFRAB1, TNFRAB2, or TNFR2A3 into the heavy and / or light chain variable domains of a human antibody. For instance, US Patent No. 6,054,297 identifies several instances when it may be advantageous to retain certain framework residues from a particular antibody heavy chain or light chain variable region in the resulting humanized antibody. In some embodiments, framework residues may engage in non-covalent interactions with the antigen and thus contribute to the affinity of the antibody for the target antigen. In other cases, individual framework residues may modulate the conformation of a CDR, and thus indirectly influence the interaction of the antibody with the antigen. Alternatively, certain framework residues may form the interface between VH and VL domains, and may therefore contribute to the global antibody structure. In other cases, framework residues may constitute functional glycosylation sites (e.g., Asn-X-Ser / Thr) which may dictate antibody structure and antigen affinity upon attachment to carbohydrate moieties. In cases such as those described above, it may be beneficial to retain certain framework residues of a TNFR2 antagonist antibody (e.g., TNFRAB1, TNFRAB2, or TNFR2A3) in the antagonistic antibodies and antigen-binding fragments thereof of the invention, such as humanized antibodies, as various framework residues may promote high epitope affinity and improved biochemical activity of the antibody or antigen-binding fragment thereof. Antibodies of the invention also include antibody fragments, Fab domains, F(ab’) molecules, 59 £023258330   30 Get 2023 F(ab’)2 molecules, single-chain variable fragments (scFvs), tandem scFv fragments, diabodies, triabodies, dual variable domain immunoglobulins, multi-specific antibodies, bispecific antibodies, and heterospecific antibodies that contain the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 (e.g., QRVDGYSSYWYFDV (SEQ ID NO: 25), ARDDGSYSPFDYWG (SEQ ID NO: 259), or ARDDGSYSPFDYFG (SEQ ID NO: 284)) or a CDR-H3 that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences. Antibodies and antigen-binding fragments thereof of the invention include those that also contain CDR-H3 sequences having between one and three amino acid substitutions (e.g., conservative or nonconservative substitutions) relative to the CDR-H3 sequences of TNFRAB1, TNFRAB2, or TNFR2A3. These molecules can be expressed recombinantly, e.g., by incorporating polynucleotides encoding these proteins into expression vectors for transfection in a eukaryotic or prokaryotic cell using techniques described herein or known in the art, or synthesized chemically, e.g., by solid phase peptide synthesis methods described herein or known in the art. Polypeptides of the invention additionally include antibody-like scaffolds that contain, for example, the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3, or a CDR-H3 sequence that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences that contain between one and three amino acid substitutions (e.g., conservative or nonconservative substitutions) relative to the CDR-H3 sequences of TNFRAB1, TNFRAB2, or TFNR2A3. Examples of antibody-like scaffolds include proteins that contain a tenth fibronectin type III domain (10Fn3), which contains BC, DE, and FG structural loops analogous to canonical antibodies. It has been shown that the tertiary structure of the 10Fn3 domain resembles that of the variable region of the IgG heavy chain, and one of skill in the art can graft, e.g., the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3, or sequences having at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences containing conserved amino acid substitutions relative to these CDR-H3 sequences onto the fibronectin scaffold by replacing residues of the BC, DE, and FG loops of 10Fn3 with residues of the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3. This can be achieved by recombinant expression of a modified 10Fn3 domain in a prokaryotic or eukaryotic cell (e.g., using the vectors and techniques described herein). Examples of using the 10Fn3 domain as an antibody-like scaffold for the grafting of CDRs from antibodies onto the BC, DE, and FG structural loops are reported in WO 2000 / 034784, WO 2009 / 142773, WO 2012 / 088006, and U.S. Patent No. 8,278,419; incorporated herein by reference. Antagonistic TNFR2 single-chain polypeptides TNFR2 antagonists of the invention may be in the form of a single-chain polypeptide, such as a single-chain polypeptide that contains a CDR-H3 region described herein (e.g., the CDR-H3 region of TNFRAB1, TNFRAB2, or TNFR2A3), optionally in combination with a CDR derived from a phenotypeneutral TNFR2 antibody (i.e., an antibody that is neither antagonistic nor agonistic of TNFR2 activation). Single-chain polypeptides may be in the form of an antibody fragment, e.g., an antibody fragment described herein or known in the art, such as a scFv fragment. Single chain polypeptides may alternatively contain one or more CDRs described herein covalently bound to one another using 60 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 conventional bond-forming techniques known in the art, for instance, by an amide bond, a thioether bond, a carbon-carbon bond, or by a linker, such as a peptide linker or a multi-valent electrophile (e.g., a bis(bromomethyl) arene derivative, such as a bis(bromomethyl)benzene or bis(bromomethyl)pyridine) described herein or known in the art. For instance, antagonistic TNFR2 single-chain polypeptides of the invention may have a CDR-H3 region that contains one of the above-described consensus sequences that promote selective binding to TNFR2 epitopes, such as the KCRPG motif (SEQ ID NO: 19), and induce TNFR2 antagonism. For instance, antagonistic TNFR2 single-chain polypeptides of the invention may have a CDR-H3 having the amino acid sequence 32^32224323^5(3)22522 / 5 or 321322242325(3)2252225(3)2, wherein each 3 is independently a naturally occurring amino acid; each 21 is independently a naturally occurring amino acid containing a cationic side-chain at physiological pH; each 22 is independently a naturally occurring amino acid containing an anionic side-chain at physiological pH; each 23 is independently a naturally occurring amino acid containing a polar, uncharged side-chain at physiological pH; each 24 is independently a glycine or alanine; and each 25 is independently a naturally occurring amino acid containing a hydrophobic side-chain. In some embodiments, antagonistic TNFR2 single-chain polypeptides of the invention may have a CDR-H3 having the amino acid sequence 3R3DG3S3Y(3)2FD3 (SEQ ID NO: 278) or 3R3DGSY(3)2FD(3)3 (SEQ ID NO: 279), wherein each 3 is independently a naturally occurring amino acid. Antagonistic TNFR2 single-chain polypeptides of the invention may have a CDR-H3 having the amino acid sequence QZ1VZ2Z4YZ3SZ5WYZ5Z2Z5 (SEQ ID NO: 265) or A21D222W5SPZ52225WG (SEQ ID NO: 266), wherein each 2 is independently an amino acid containing a cationic side-chain at physiological pH; each 22 is independently an amino acid containing an anionic side-chain at physiological pH; each 23 is independently an amino acid containing a polar, uncharged side-chain at physiological pH; each 24 is independently a glycine or alanine; each 25 is independently an amino acid containing a hydrophobic side-chain; and each X is independently leucine or isoleucine. In some embodiments, antagonistic TNFR2 single-chain polypeptides of the invention may have a CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ ID NO: 25), ARDDGSYSPFDYWG (SEQ ID NO: 259), or an amino acid sequence having up to two amino acid substitutions relative to these sequences (e.g., one or two amino acid substitutions, such as conservative amino acid substitutions) Single-chain polypeptides can be produced by a variety of recombinant and synthetic techniques, such as by recombinant gene expression or solid-phase peptide synthesis procedures described herein or known in the art. For instance, one of skill in the art can design polynucleotides encoding, e.g., two or more CDRs operably linked to one another in frame so as to produce a continuous, single-chain peptide containing these CDRs. Optionally, the CDRs may be separated by a spacer, such as by a framework region (e.g., a framework sequence described herein or a framework region of a germline consensus sequence of a human antibody) or a flexible linker, such as a poly-glycine or glycine / serine linker described herein or known in the art. When produced by chemical synthesis methods, native chemical ligation can optionally be used as a strategy for the synthesis of long peptides (e.g., greater than 50 61 £023258330   30 Get 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 amino acids). Native chemical ligation protocols are known in the art and have been described, e.g., by Dawson et al. (Science, 266:776-779, 1994); incorporated herein by reference. A detailed description of techniques for the production of single-chain polypeptides, full-length antibodies, and antibody fragments is provided in the sections that follow. Nucleic acids and expression systems Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention can be prepared by any of a variety of established techniques. For instance, an antagonistic TNFR2 antibody or antigen-binding fragment thereof of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, optionally, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel et al., eds., Greene Publishing Associates, 1989), and in U.S. Patent No. 4,816,397; incorporated herein by reference. Vectors for expression of antagonistic TNFR2 polypeptides Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding antibody light and heavy chains or antibody fragments of the invention include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, 62 2023258320   30 Oct 2023 Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (U.S. Patent. No. 5,801,030); Incorporated herein by reference. Genome editing techniques In addition to viral vectors, a variety of additional methods have been developed for the incorporation of genes, e.g., those encoding antibody light and heavy chains, single-chain polypeptides, single-chain variable fragments (scFvs), tandem scFvs, Fab domains, F(ab’)s domains, diabodies, and triabodies, among others, into the genomes of target cells for polypeptide expression. One such method that can be used for incorporating polynucleotides encoding anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) into prokaryotic or eukaryotic cells includes transposons. Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by excision sites at the 5’ and 3’ positions. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon. This activity is mediated by the sitespecific recognition of transposon excision sites by the transposase. In some embodiments, these excision sites may be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the genome of a prokaryotic or eukaryotic cell by transposase-catalyzed cleavage of similar excision sites that exist within nuclear genome of the cell. This allows the gene encoding an anti-TNFR2 antibody or fragment or domain thereof to be inserted into the cleaved nuclear DNA at the excision sites, and subsequent ligation of the phosphodiester bonds that join the gene of interest to the DNA of the prokaryotic or eukaryotic cell genome completes the incorporation process. In some embodiments, the transposon may be a retrotransposon, such that the gene encoding the antibody is first transcribed to an RNA product and then reverse-transcribed to DNA before incorporation in the prokaryotic or eukaryotic cell genome. Exemplary transposon systems include the piggybac transposon (described in detail in WO 2010 / 085699) and the sleeping beauty transposon (described in detail in US20050112764); incorporated herein by reference. Another useful method for the integration of nucleic acid molecules encoding anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) into the genome of a prokaryotic or eukaryotic cell is the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system, which is a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against infection by viruses. The CRISPR / Cas system consists of palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host ceil to create a guide RNA, which can subsequently 63 £023258330   30 Get 2023 anneal to a target sequence and localize the Cas9 nuclease to this site. In this manner, highly sitespecific cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings cas9 within close proximity of the target DNA molecule is governed by RNA:DNA hybridization. As a result, one can theoretically design a CRISPR / Cas system to cleave any target DNA molecule of interest. This technique has been exploited in order to edit eukaryotic genomes (Hwang et al., Nat. Biotech., 31:227-229, 2013) and can be used as an efficient means of site-specifically editing eukaryotic or prokaryotic genomes in order to cleave DNA prior to the incorporation of a polynucleotide encoding an anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention. The use of CRISPR / Cas to modulate gene expression has been described in US Patent No. 8,697,359, which is incorporated herein by reference. Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a polynucleotide encoding a TNFR2 antibody or antibody fragment of the invention include the use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes. Zinc finger nucleases and TALENs for use in genome editing applications are described in Urnov et al. (Nat. Rev. Genet., 11:636-646, 2010); and in Joung et al., (Nat. Rev. Mol. Cell. Bio. 14:49-55, 2013); incorporated herein by reference. Additional genome editing techniques that can be used to incorporate polynucleotides encoding antibodies of the invention into the genome of a prokaryotic or eukaryotic cell 20 include the use of ARCUS™ meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA. The use of these enzymes for the incorporation of polynucleotides encoding antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention into the genome of a prokaryotic or eukaryotic cell is particularly advantageous in view of the structure-activity relationships that have been established for such enzymes. 25 Single-chain meganucleases can thus be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations. These single-chain nucleases have been described extensively, e.g., in U.S. Patent Nos. 8,021,867 and 8,445,251; incorporated herein by reference. 30 Polynucleotide sequence elements To express antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention, polynucleotides encoding partial or full-length light and heavy chains, e.g., polynucleotides that encode a CDR-H3 region as described herein, can be inserted into expression vectors such that the genes are operatively linked to transcriptional and 35 translational control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Polynucleotides encoding the light chain gene and the heavy chain of a TNFR2 antibody can be inserted into separate vectors, or, optionally, both polynucleotides can be incorporated into the same expression vector using established techniques described herein or known in the art. 2023258320   30 Oct 2023 In addition to polynucleotides encoding the heavy and light chains of an antibody (or a polynucleotide encoding a single-chain polypeptide or an antibody fragment, such as a scFv molecule), the recombinant expression vectors of the invention may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed or the level of expression of protein desired. For instance, suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Patent No. 5, 168,062, U.S. Patent No. 4,510,245, and U.S. Patent No. 4,968,615. In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patents Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR’ host cells with methotrexate selection / amplification) and the neo gene (for G418 selection). In order to express the light and heavy chains of a TNFR2 antibody or a TNFR2 antibody fragment, the expression vector(s) containing polynucleotides encoding the heavy and light chains can be transfected into a host cell by standard techniques. Polynucleotides encoding modified antagonistic TNFR2 polypeptides Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention may contain the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3, but feature differences in the sequence of one or more of the remaining CDRs relative to the corresponding sequence in TNFRAB1, TNFRAB2, or TNFR2A3. Similarly, polypeptides of the invention may contain the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 but may feature differences in one or more framework regions. For instance, one or more framework regions of TNFRAB1, TNFRAB2, or TNFR2A3 may be substituted with the framework region of a human antibody. Exemplary framework regions include, for example, human framework regions described in US 7,829,086, and primate framework regions as described in EP 1945668; incorporated herein by reference. To generate nucleic acids encoding such TNFR2 antibodies, DNA fragments encoding, e.g., at least one, or both, of the light chain variable regions and the heavy chain variable regions can be produced by chemical synthesis (e.g., by soiid phase polynucleotide synthesis techniques), in vitro gene amplification (e.g., by polymerase chain reaction techniques), or by replication of the polynucleotide in a host organism. For instance, 65 £023258330   30 Get 2023 nucleic acids encoding anti-TNFR2 antibodies of the invention may be obtained by amplification and modification of germline DNA or cDNA encoding light and heavy chain variable sequences so as to incorporate the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 into the framework residues of a consensus antibody. In some embodiments, a humanized antagonistic TNFR2 antibody may include the CDR-H3 of TFNRAB1, TNFRAB2, or TNFR2A3, or a variant thereof that has at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences that contain between one and three amino acid substitutions (e.g., conservative or nonconservative substitutions) relative to the CDR-H3 sequences of TNFRAB1, TNFRAB2, or TNFR2A3. This can be achieved, for example, by performing site-directed mutagenesis of germline DNA or cDNA and amplifying the resulting polynucleotides using the polymerase chain reaction (PCR) according to established procedures. Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see, e.g., the “VBASE” human germline sequence database; see also Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242, 1991; Tomlinson et al., J. Mol. Biol. 227:776-798, 1992; and Cox et al., Eur. J. Immunol. 24:827- 836, 1994; incorporated herein by reference). Chimeric nucleic acid constructs encoding human heavy and light chain variable regions containing the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 can be produced, e.g., using established cloning techniques known in the art. Additionally, a polynucleotide encoding a heavy chain variable region containing the CDR-H3 of TNFRAB1, TNFRAB2, or TFNR2A3 can be synthesized and used as a template for mutagenesis to 20 generate a variant as described herein using routine mutagenesis techniques. Alternatively, a DNA fragment encoding the variant can be directly synthesized (e.g., by established solid phase nucleic acid chemical synthesis procedures). Once DNA fragments encoding VH segments containing the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 are obtained, these DNA fragments can be further manipulated by standard recombinant DNA 25 techniques, e.g., to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The isolated DNA encoding the Vh region of an anti-TNFR2 antibody of the invention can be 30 converted to a full-length heavy chain gene (as well as a Fab heavy chain gene), e.g., by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant region domains (CH1, CH2, CH3, and, optionally, CH4). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242, 1991) and DNA 35 fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG 1, lgG2, lgG3, lgG4, IgA, IgE, IgM or IgD constant region, and in certain embodiments is an lgG1 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively ffnked to another DNA molecule encoding only the heavy chain CH1 domain. 2023258320   30 Oct 2023 Isolated DNA encoding the VL region of an anti-TNFR2 antibody can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat et al., Sequences of Proteins of 5 Immunological Interest, Fifth Edition (U.S. Department of Health and Human Services, NIH Publication No. 91 -3242, 1991)) and DNA fragments encompassing these regions can be obtained, e.g., by amplification in a prokaryotic or eukaryotic cell of a polynucleotide encoding these regions, by PCR amplification, or by chemical polynucleotide synthesis. The light chain constant region can be a kappa (k) or lambda (A) constant region, but in certain embodiments is a kappa constant region. To create a scFv 10 gene, the VH and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., a polynucleotide encoding a flexible, hydrophilic amino acid sequence, such as the amino acid sequence (Gly4Ser)3, such that the Vh and Vl sequences can be expressed as a contiguous single-chain protein, with the Vl and Vh regions joined by the linker (see e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988: McCafferty et al., 15 Nature 348:552-554, 1990). Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to TNFR2. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies can be produced in which one heavy contains a CDR-H3 sequence 20 derived from TNFRAB1, TNFRAB2, or TNFR2A3, and the other heavy chain and / or the light chains are specific for an antigen other than TNFR2. Such antibodies can be generated, e.g., by crosslinking a heavy chain containing the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3 and a light chain of an anti-TNFR2 antibody, such as an anti-TNFR2 antibody that is neither agonistic nor antagonistic, to a heavy chain and light chain of a second antibody by standard chemical crosslinking methods (e.g., by 25 disulfide bond formation). Bifunctional antibodies can also be made by expressing a nucleic acid molecule engineered to encode a bifunctional antibody in a prokaryotic or eukaryotic cell. Dual specific antibodies, i.e., antibodies that bind TNFR2 and a different antigen using the same binding site, can be produced by mutating amino acid residues in the light chain and / or heavy chain CDRs. In some embodiments, dual specific antibodies that bind two antigens, such as TNFR2 and a 30 second cell-surface receptor, can be produced by mutating amino acid residues in the periphery of the antigen binding site (Bostrom et al., Science 323: 1610-1614, 2009). Dual functional antibodies can be made by expressing a polynucleotide engineered to encode a dual specific antibody. Modified antagonistic TNFR2 antibodies and antibody fragments of the invention can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd 35 ed., 1984 The Pierce Chemical Co., Rockford, 111; incorporated herein by reference). Variant antibodies can also be generated using a cell-free synthetic platform (see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals); incorporated herein by reference). £023258330   30 Get 2023 Host cells for expression of antagonistic TNFR2 polypeptides It is possible to express the polypeptides (e.g., single-chain polypeptides, antibodies, or antigenbinding fragments thereof) of the invention in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) is performed in eukaryotic cells, e.g., mammalian host cells, for optimal secretion of a properly folded and immunologically active antibody. Exemplary mammalian host cells for expressing the recombinant antibodies or antigen-binding fragments thereof of the invention include Chinese Hamster Ovary (CHO cells) (including DHFR CHO cells, described in Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA 77:4216-4220), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982, Mol. Biol. 159:601 -621), NSO myeloma cells, COS cells, 293 cells, and SP2 / 0 cells. Additional cell types that may be useful for the expression of antibodies and fragments thereof include bacterial cells, such as BL-21(DE3) E. co / / cells, which can be transformed with vectors containing foreign DNA according to established protocols. Additional eukaryotic cells that may be useful for expression of antibodies include yeast cells, such as auxotrophic strains of S. cerevisiae, which can be transformed and selectively grown in incomplete media according to established procedures known in the art. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. 20            Polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. The invention also includes methods in which the above procedure is varied according to established protocols known in the art. For example, it can be desirable to transfect a host cell with DNA encoding 25 either the light chain or the heavy chain (but not both) of an anti-TNFR2 antibody of the invention in order to produce an antigen-binding fragment of the antibody. Once an anti-TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment) thereof of the invention has been produced by recombinant expression, it can be purified by any method known in the art, such as a method useful for purification of an immunoglobulin molecule, for example, by 30 chromatography (e.g., ion exchange, affinity, particularly by affinity for TNFR2 after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the anti TNFR2 polypeptides of the invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification or to produce therapeutic conjugates (see “Antagonistic TNFR2 35 polypeptide conjugates,” below). Once isolated, an anti-TNFR2 single-chain polypeptide, antibody, or antigen-binding fragments thereof can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology (Work and Burdon, eds.a Elsevier, 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 1980); incorporated herein by reference), or by gel filtration chromatography, such as on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden). Platforms for generating and affinity-maturing antagonistic anti-TNFR2 polypeptides Mapping epitopes of TNFR2 that promote receptor antagonism Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigenbinding fragments thereof) of the invention can be produced by screening libraries of polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) for functional molecules that are capable of binding epitopes within TNFR2 that selectively promote receptor antagonism rather than receptor activation. Such epitopes can be modeled by screening antibodies or antigen-binding fragments thereof against a series of linear or cyclic peptides containing residues that correspond to a desired epitope within TNFR2. As an example, peptides containing individual fragments isolated from TNFR2 that promote receptor antagonism can be synthesized by peptide synthesis techniques described herein or known in the art. These peptides can be immobilized on a solid surface and screened for molecules that bind antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof), such as TNFRAB1, TNFRAB2, or TNFR2A3, e.g., using an ELISA-based screening platform using established procedures. Using this assay, peptides that specifically bind TNFRAB1, TNFRAB2, or TNFR2A3 with high affinity therefore contain residues within epitopes of TNFR2 that preferentially bind these antibodies. Peptides identified in this manner (e.g., peptides having the sequence of any one of SEQ ID NOs: 11, 19, 20, 34-117, 285, and 286, or a peptide containing between about 10 and about 30 continuous or discontinuous amino acids between positions 80 and 130 of SEQ ID NO: 7) can be used to screen libraries of antibodies and antigen-binding fragments thereof in order to identify anti-TNFR2 antibodies of the invention. Moreover, since these peptides act as surrogates for epitopes within TNFR2 that promote receptor antagonism, antibodies generated using this screening technique may bind the corresponding epitopes in TNFR2 and are expected to be antagonistic of receptor activity. Screening of libraries for antagonistic TNFR2 polypeptides Methods for high throughput screening of polypeptide (e.g., single-chain polypeptide, antibody, or antibody fragment) libraries for molecules capable of binding epitopes within TNFR2 (e.g., peptides having the sequence of SEQ ID NO: 285 or 286) include, without limitation, display techniques including phage display, bacterial display, yeast display, mammalian display, ribosome display, mRNA display, and cDNA display. The use of phage display to isolate ligands that bind biologically relevant molecules has been reviewed, e.g., in Felici etal. (Biotechnol. Annual Rev. 1:149-183, 1995), Katz (Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997), and Hoogenboom et al. (Immunotechnology 4:1-20, 1998). Several randomized combinatorial peptide libraries have been constructed to select for polypeptides that bind different targets, e.g., cell surface receptors or DNA (reviewed by Kay (Perspect. Drug Discovery Des. 2, 251 -268, 1995), Kay et al., (Moi. Divers. 1:139-140, 1996)). Proteins and multimeric proteins have 69 £023258330   30 Get 2023 been successfully phage-displayed as functional molecules (see EP 0349578A, EP 4527839A, EP 0589877A; Chiswell and McCafferty (Trends Biotechnol. 10, 80-84 1992)). In addition, functional antibody fragments (e.g. Fab, single-chain Fv [scFv]) have been expressed (McCafferty et al. (Nature 348: 552554, 1990), Barbas et al. (Proc. Natl. Acad Sci. USA 88:7978-7982, 1991), Clackson et al. (Nature 352:624-628, 1991)). These references are hereby incorporated by reference in their entirety. (i) Phage display techniques As an example, phage display techniques can be used in order to screen libraries of polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) for functional molecules capable of binding cyclic or polycyclic peptides containing epitopes within TNFR2 that promote receptor antagonism (e.g., peptides having the sequence of SEQ ID NO: 285 or 286). For instance, libraries of polynucleotides encoding single-chain antibody fragments, such as scFv fragments, that contain randomized hypervariable regions can be obtained using established procedures (e.g., solid phase polynucleotide synthesis or error-prone PCR techniques, see McCullum et al. (Meth. Mol. Biol., 634:103-109, 2010); incorporated herein by reference). These randomized polynucleotides can subsequently be incorporated into a viral genome such that the randomized antibody chains encoded by these genes are expressed on the surface of filamentous phage, e.g., by a covalent bond between the antibody chain and a coat protein (e.g., pill coat protein on the surface of M13 phage). This provides a physical connection between the genotype and phenotype of the antibody chain. In this way, libraries of phage that display diverse antibody chains containing random mutations in hypervariable regions can be screened for the ability of the exterior antibody chains to bind TNFR2 epitopes (e.g., peptides having the sequence of SEQ ID NO: 285 or 286) that are immobilized to a surface using established procedures. For instance, such peptides can be physically bound to the surface of a microtiter plate by forming a covalent bond between the peptide and an epitope tag (e.g., biotin) and incubating the peptide in wells of a microtiter plate that have been previously coated with a complementary tag (e.g., avidin) that binds the tag attached to the peptide with high affinity. Suitable epitope tags include, without limitation, maltose-binding protein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag, biotin, streptavidin. Peptides containing the epitopes presented by these molecules are capable of being immobilized on surfaces containing such complementary molecules as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively. In this way, phage can be incubated with a surface containing an immobilized TNFR2-derived peptide for a time suitable to allow binding of the antibody to the constrained peptide and in the presence of an appropriate buffer system (e.g., one that contains physiological salt concentration, ionic strength, and is maintained at physiological pH by a buffering agent). The surface can then be washed (e.g., with phosphate buffer containing 0.1% Tween-20) so as to remove phage that do not present antibody chains that interact with the TNFR2-derived peptides with an affinity greater than a particular threshold value. The affinity of the polypeptides that remain after this initial panning (i.e., screening) step can be modulated by adjusting the conditions of the washing step (e.g., by including mildly acidic or basic 70 2023258320   30 Oct 2023 components, or by including other TNFR2-derived peptides at a low concentration in order to compete with immobilized peptides for antigen-binding sites). In this way, the population of phage that remains bound to the surfaces of the microtiter plate following the washing step is enriched for phage that bind TNFR2-derived peptide epitopes that promote receptor antagonism. The remaining phage can then be amplified by eluting the phage from the surface containing these peptides (e.g., by altering the ambient pH, ionic strength, or temperature) so as to diminish protein-protein interaction strength. The isolated phage can then be amplified, e.g., by infecting bacterial cells, and the resulting phage can optionally be subjected to panning by additional iterations of screening so as to further enrich the population of phage for those harboring higher-affinity anti-TNFR2 polypeptides. Following these panning stages, phage that display high-affinity antibodies or antigen-binding fragments thereof can subsequently be isolated and the genomes of these phage can be sequenced in order to identify the polynucleotide and polypeptide sequences of the encoded antibodies. Phage display techniques such as this can be used to generate, e.g., antibody chains, such as scFv fragments, tandem scFv fragments, and other antigen-binding fragments of the invention that can be used as antagonists of TNFR2. Exemplary phage display protocols for the identification of antibody chains and antigen-binding fragments thereof that bind a particular antigen with high affinity are well-established and are described, e.g., in US Patent No. 7,846,892, WO 1997 / 002342, US Patent No. 8,846,867, and WO 2007 / 132917; incorporated herein by reference. Similar phage display techniques can be used to generate antibody-like scaffolds (e.g., 10Fn3 domains) of the invention that bind epitopes within TNFR2 that promote receptor antagonism (e.g., epitopes presented by peptides with the sequence of SEQ ID NO: 285 or 286). Exemplary phage display protocols for the identification of antibody-like scaffold proteins are described, e.g., in WO 2009 / 086116; incorporated herein by reference). (ii) Cell-based display techniques Other in vitro display techniques that exploit the linkage between genotype and phenotype of a solvent-exposed polypeptide include yeast and bacterial display. Yeast display techniques are established in the art and are often advantageous in that high quantities of antibodies (often up to 30,000) can be presented on the surface of an individual yeast cell (see, e.g., Boder et al. (Nat Biotechno. 15:553, 1997); incorporated herein by reference). The larger size of yeast cells over filamentous phage enables an additional screening strategy, as one can use flow cytometry to both analyze and sort libraries of yeast. For instance, established procedures can be used to generate libraries of bacterial cells or yeast cells that express polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) containing randomized hypervariable regions (see, e.g., see US Patent No. 7,749,501 and US 2013 / 0085072; the teachings of each which are incorporated herein by reference). For instance, large libraries of yeast cells that express polynucleotides encoding naive scFv fragments can be made using established procedures (de Bruin et al., Nat Biotechnol 17:397, 1999; incorporated herein by reference). Yeast cells expressing these polynucleotides can then be incubated with two different fluorescent molecules during the panning steps: one dye that binds conserved residues within the antibody and thus reflects the amount of antibody displayed, and another dye that fluoresces at a different 71 £023258330   30 Get 2023 wavelength and binds the antigen and thus indicates the amount of antigen bound. For instance, one of skill in the art can use a TNFR2-derived peptide containing the sequence of SEQ ID NO: 285 or 286 that has been conjugated to an epitope tag (e.g., biotin), optionally at the N- or C-terminus of the peptide or at a residue that is not expected to interfere with antibody-antigen binding. This enables a fluorescent dye labeled with a complementary tag (e.g., avidin) to localize to the antibody-antigen complex. This results in great flexibility and immediate feedback on the progress of a selection. In contrast to phage display, by normalizing to antibody display levels, antibodies with higher affinities, rather than greater expression levels can easily be selected. In fact, it is possible to distinguish and sort antibodies whose affinities differ by only two-fold (VanAntwerp and Wittrup (Biotechnol Prog 16:31,2000)). (Hi) Nucleotide display techniques Display techniques that utilize in vitro translation of randomized polynucleotide libraries also provide a powerful approach to generating anti-TNFR2 antibodies of the invention. For instance, randomized DNA libraries encoding polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) that contain mutations within designated hypervariable regions can be obtained, e.g., using established PCR-based mutagenesis techniques as described herein. The polynucleotides of these libraries may contain transcription regulating sequences, such as promoters and transcription terminating sequences, and may additionally encode sequences that increase the rate of translation of the resulting mRNA construct (e.g., IRES sequences, 5’ and 3’ UTRs, a poly-adenylation 20 tract, etc). These polynucleotide libraries can be incubated in an appropriately buffered solution containing RNA polymerase and RNA nucleoside triphosphates (NTPs) in order to enable transcription of the DNA sequences to competent mRNA molecules, which can subsequently be translated by large and small ribosomal subunits, aminoacyl tRNA molecules, and translation initiation and elongation factors present in solution (e.g., using the PURExpress® In Vitro Protein Synthesis Kit, New England Biolabs®). 25 Designed mRNA modifications can enable the antibody product to remain covalently bound to the mRNA template by a chemical bond to puromycin (e.g., see Keefe (Curr. Protoc. Mol. Biol., Chapter 24, Unit 24.5, 2001); incorporated herein by reference). This genotype-phenotype linkage can thus be used to select for antibodies that bind a TNFR2-derived peptide (e.g., a peptide that has the sequence SEQ ID NO: 285 or 286) by incubating mRNA:antibody fusion constructs with a peptide immobilized to a surface 30 and panning in a fashion similar to phage display techniques (see, e.g., WO 2006 / 072773; incorporated herein by reference). Optionally, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be generated using a similar technique, except the antibody product may be bound non-covalently to the ribosome-mRNA complex rather than covalently via a 35 puromycin linker. This platform, known as ribosome display, has been described, e.g., in US Patent No. 7,074,557; incorporated herein by reference. Alternatively, antibodies can be generated using cDNA display, a technique analogous to mRNA display with the exception that cDNA, rather than mRNA, is covalently bound to an antibody product via a puromycin linker. cDNA display techniques offer the advantage of being able to perform panning steps under increasingly stringent conditions, e.g., under 2023258320   30 Oct 2023 conditions in which the salt concentration, ionic strength, pH, and / or temperature of the environment is adjusted in order to screen for antibodies with particularly high affinity for TNFR2-derived peptides. This is due to the higher natural stability of double-stranded cDNA over single-stranded mRNA. cDNA display screening techniques are described, e.g., in Ueno et al. (Methods Mol. Biol., 805:113-135, 2012); incorporated herein by reference. In addition to generating anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention, in vitro display techniques (e.g., those described herein and those known in the art) also provide methods for improving the affinity of an anti-TNFR2 polypeptide of the invention. For instance, rather than screening libraries of antibodies and fragments thereof containing completely randomized hypervariable regions, one can screen narrower libraries of antibodies and antigen-binding fragments thereof that feature targeted mutations at specific sites within hypervariable regions. This can be accomplished, e.g., by assembling libraries of polynucleotides encoding antibodies or antigen-binding fragments thereof that encode random mutations only at particular sites within hypervariable regions. These polynucleotides can then be expressed in, e.g., filamentous phage, bacterial cells, yeast cells, mammalian cells, or in vitro using, e.g., ribosome display, mRNA display, or cDNA display techniques in order to screen for antibodies or antigen-binding fragments thereof that specifically bind TNFR2 epitopes (e.g., peptides containing the sequence of SEQ ID NO: 285 or 286) with improved binding affinity. Yeast display, for instance, is well-suited for affinity maturation, and has been used previously to improve the affinity of a single-chain antibody to a Kd of 48 fM (Boder et al. (Proc Natl Acad Sci USA 97:10701,2000)). Additional in vitro techniques that can be used for the generation and affinity maturation of antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention include the screening of combinatorial libraries of antibodies or antigen-binding fragments thereof for functional molecules capable of specifically binding TNFR2-derived peptides (e.g., a peptide having the amino acid sequence of SEQ ID NO: 285 or 286). Combinatorial antibody libraries can be obtained, e.g., by expression of polynucleotides encoding randomized hypervariable regions of an antibody or antigen-binding fragment thereof in a eukaryotic or prokaryotic cell. This can be achieved, e.g., using gene expression techniques described herein or known in the art. Heterogeneous mixtures of antibodies can be purified, e.g., by Protein A or Protein G selection, sizing column chromatography), centrifugation, differential solubility, and / or by any other standard technique for the purification of proteins. Libraries of combinatorial libraries thus obtained can be screened, e.g., by incubating a heterogeneous mixture of these antibodies with a peptide derived from TNFR2 that has been immobilized to a surface (e.g., a peptide having the amino acid sequence of SEQ ID NO: 285 or 286 immobilized to the surface of a solid-phase resin or a well of a microtiter plate) for a period of time sufficient to allow antibody-antigen binding. Non-binding antibodies or fragments thereof can be removed by washing the surface with an appropriate buffer (e.g., a solution buffered at physiological pH (approximately 7.4) and containing physiological salt concentrations and ionic strength, and optionally containing a detergent, such as TWEEN-20). Antibodies that remain bound can subsequently be detected, e.g., using an ELISA-based detection protocol (see, e.g., US Patent No. 4,661,445; £023258330   30 Get 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 incorporated herein by reference). Additional techniques for screening combinatorial libraries of polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) for those that specifically bind TNFR2-derived peptides (e.g., a peptide containing the amino acid sequence of SEQ ID NO: 285 or 286) include the screening of one-bead-one-compound libraries of antibody fragments. Antibody fragments can be chemically synthesized on a solid bead (e.g., using established split-and-pool solid phase peptide synthesis protocols) composed of a hydrophilic, water-swellable material such that each bead displays a single antibody fragment. Heterogeneous bead mixtures can then be incubated with a TNFR2-derived peptide that is optionally labeled with a detectable moiety (e.g., a fluorescent dye) or that is conjugated to an epitope tag (e.g., biotin, avidin, FLAG tag, HA tag) that can later be detected by treatment with a complementary tag (e.g., avidin, biotin, anti-FLAG antibody, anti-HA antibody, respectively). Beads containing antibody fragments that specifically bind a TNFR2-derived peptide (e.g., a peptide containing the amino acid sequence of SEQ ID NO: 285 or 286) can be identified by analyzing the fluorescent properties of the beads following incubation with a fluorescently-labeled antigen or complementary tag (e.g., by confocal fluorescent microscopy or by fluorescence-activated bead sorting; see, e.g., Muller et al. (J. Biol. Chern., 16500-16505, 1996); incorporated herein by reference). Beads containing antibody fragments that specifically bind TNFR2-derived peptides can thus be separated from those that do not contain high-affinity antibody fragments. The sequence of an antibody fragment that specifically binds a TNFR2-derived peptide can be determined by techniques known in the art, including, e.g., Edman degradation, tandem mass spectrometry, matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), and 2D gel electrophoresis, among others (see, e.g., WO 2004 / 062553; incorporated herein by reference). Negative screens of polypeptides In addition to the above-described methods for screening for a single-chain polypeptide, antibody, or antibody fragment that specifically binds to an epitope derived from human TNFR2 that promotes receptor antagonism, one can additionally perform negative screens in order to eliminate antibodies or antibody fragments that may also bind an epitope that contains the KCSPG sequence. For instance, mixtures of antibodies or antibody fragments isolated as a result of any of the above-described screening techniques can be screened for antibodies or antibody fragments that also specifically bind to a peptide derived from human TNFR2 that contains the KCSPG motif, such as a peptide containing residues 48-67 of SEQ ID NO: 7 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18). This can be accomplished using any of the above-described methods or variations thereof, e.g., such that the antibodies or antibody fragments being screened are those that were previously identified as being capable of specifically binding a peptide containing one or more residues of the KCRPG sequence (e.g., at least the KCR sequence). Exemplary techniques useful for a negative screen include those described above or known in the art, such as phage display, yeast display, bacterial display, ribosome display, mRNA display, cDNA display, or surface-based combinatorial library screens (e.g., in an ELISA format). This screening technique represents a useful strategy for identifying an antagonistic TNFR2 antibody or antibody fragment* as antibodies or antibody 74 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 fragments capable of binding TNFR2 epitopes containing the KCSPG sequence and one or more residues of the KCRPG sequence have been shown to lack, or to have significantly reduced, antagonistic activity. 5 Immunization of a non-human mammal Another strategy that can be used to produce antagonistic TNFR2 antibodies and antigen-binding fragments thereof of the invention includes immunizing a non-human mammal. Examples of non-human mammals that can be immunized in order to produce antagonistic TNFR2 antibodies and fragments thereof of the invention include rabbits, mice, rats, goats, guinea pigs, hamsters, horses, and sheep, as 10 well as non-human primates. For instance, established procedures for immunizing primates are known in the art (see, e.g., WO 1986 / 6004782; incorporated herein by reference). Immunization represents a robust method of producing monoclonal antibodies by exploiting the antigen specificity of B lymphocytes. For example, monoclonal antibodies can be prepared by the Kohler-Millstein procedure (described, e.g., in EP 0110716: incorporated herein by reference), wherein spleen cells from a non-human animal (e.g., a 15 primate) immunized with a peptide that presents a TNFR2-derived antigen that promotes receptor antagonism (e.g., a peptide containing the amino acid sequence of SEQ ID NO: 285 or 286). A clonally-expanded B lymphocyte produced by immunization can be isolated from the serum of the animal and subsequently fused with a myeloma cell in order to form a hybridoma. Hybridomas are particularly useful agents for antibody production, as these immortalized cells can provide a lasting supply of an antigen- 20 specific antibody. Antibodies from such hybridomas can subsequently be isolated using techniques known in the art, e.g., by purifying the antibodies from the cell culture medium by affinity chromatography, using reagents such as Protein A or Protein G. Antagonistic TNFR2 polypeptide conjugates 25            Prior to administration of antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention to a mammalian subject (e.g., a human), it may be desirable to conjugate the antibody or fragment thereof to a second molecule, e g.. to modulate the activity of the antibody in vivo. Antagonistic TNFR2 antibodies and fragments thereof can be conjugated to other molecules at either the N-terminus or C-terminus of a light or heavy chain of the antibody using any one 30 of a variety of established conjugation strategies that are well-known in the art. Examples of pairs of reactive functional groups that can be used to covalently tether an antagonistic TNFR2 antibody or fragment thereof to another molecule include, without limitation, thiol pairs, carboxylic acids and amino groups, ketones and amino groups, aldehydes and amino groups, thiols and alpha,beta-unsaturated moieties (such as maleimides or dehydroalanine), thiols and alpha-halo amides, carboxylic acids and hydrazides, aldehydes and 35 hydrazides, and ketones and hydrazides. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) can be covalently appended directly to another molecule by chemical conjugation as described. Alternatively, fusion proteins containing antagonistic TNFR2 antibodies and fragments thereof can be expressed recombinantiy from a cell (e.g., a eukaryotic ceil or prokaryotic cell). This can be accomplished, £023258330   30 Get 2023 for example, by incorporating a polynucleotide encoding the fusion protein into the nuclear genome of a cell (e.g., using techniques described herein or known in the art). Optionally, antibodies and fragments thereof of the invention can be joined to a second molecule by forming a covalent bond between the antibody and a linker. This linker can then be subsequently conjugated to another molecule, or the linker can be conjugated to another molecule prior to ligation to the anti-TNFR2 antibody or fragment thereof. Examples of linkers that can be used for the formation of a conjugate include polypeptide linkers, such as those that contain naturally occurring or non-naturally occurring amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally-occurring proteins and are thus more resistant to degradation by endogenous proteases. Fusion proteins containing polypeptide linkers can be made using chemical synthesis techniques, such as those described herein, or through recombinant expression of a polynucleotide encoding the fusion protein in a cell (e.g., a prokaryotic or eukaryotic cell). Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of 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. Chern., 20:571-582, 2012). Drug-polypeptide conjugates An antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, and antigen-binding fragment thereof) of the invention can additionally be conjugated to, admixed with, or administered separately from a therapeutic agent, such as a cytotoxic molecule. Conjugates of the invention may be applicable to the treatment or prevention of a disease associated with aberrant cell proliferation, such as a cancer described herein. Exemplary cytotoxic agents that can be conjugated to, admixed with, or administered separately from an antagonistic TNFR2 polypeptide include, without limitation, antineoplastic agents such as: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; adriamycin; aldesleukin; altretamine; ambomycin; a. metantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; camptothecin; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; combretestatin a-4; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daca (n- [2- (dimethyl-amino) ethyl] acridine-4-carboxamide); dactinomycin; daunorubicin hydrochloride; daunomycin; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; dolasatins; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; ellipticine; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; ethiodized oil i 131; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; 5-fdump; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; gold au 198; homocamptothecin; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interferon alfa-2a; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta-i a; interferon gamma-76 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 i b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; 5 mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peploycinsulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; rhizoxin; rhizoxin d; riboprine; rogletimide; safingol; safingol hydrochloride; 10 semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; strontium chloride sr 89; sulofenur; talisomycin; taxane; taxoid; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; thymitaq; tiazofurin; tirapazamine; tomudex; top53; topotecan hydrochloride; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate 15 glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine; vinblastine sulfate; vincristine; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride; 2-chlorodeoxyadenosine; 2' deoxyformycin; 9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid; 2chloro-2'-arabino-fluoro-2'-deoxyadenosine; 2-chloro-2'- 20 deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlor ethamine); cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-Nnitrosourea (MNU); N, N'-Bis (2-chloroethyl)-N-nitrosourea (BCNU); N- (2-chloroethyl)-N' cyclohexyl-N-nitrosourea (CCNU); N- (2-chloroethyl)-N'- (trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU); N- (2-chloroethyl)-N'- (diethyl) ethylphosphonatc-N-nitrosourea (fotemustine); streptozotocin; diacarbazine (DTIC); 25 mitozolomide; temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; cisplatin; carboplatin; ormaplatin; oxaliplatin;C1-973; DWA 2114R; JM216; JM335; Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine; 6-mercaptopurine; 6-thioguanine; hypoxanthine; teniposide 9-amino camptothecin; topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin; darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol; 14-hydroxy-retro-retinol; all-trans retinoic acid; 30 N- (4- hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-rnethyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); or 2-chlorodeoxyadenosine (2-Cda). Other therapeutic compounds that can be conjugated to, admixed with, or administered separately from an antagonistic TNFR2 single-chain polypeptide, antibody, or antigen-binding fragment thereof of the invention in order to treat, prevent, or study the progression of a disease associated with aberrant cell 35 proliferation include, but are not limited to, cytotoxic agents such as 20-pi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense £023258330   30 Get 2023 oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bleomycin A2; bleomycin B2; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives (e.g., 10-hydroxy-camptothecin); canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A ; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816 ; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; 2'deoxycoformycin (DCF); deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9- ; dioxamycin; diphenyl spiromustine; discodermolide; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epothilones (A, R = H; B, R - Me); epithilones; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide; etoposide 4'-phosphate (etopofos); exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyarnine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maytansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; rnerbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; ifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mithracin; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factorsaporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A + myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelfn; nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral 2023258320   30 Oct 2023 WO 2017 / 197331                                                 PCT / US2017 / 032513 endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; podophyllotoxin; porfimer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B 1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single-chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trirnetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. Labeled anti-TNFR2 polypeptides In some embodiments, antagonistic TNFR2 single-chain polypeptides, antibodies, or antigen-binding fragments thereof may be conjugated to another molecule (e.g., an epitope tag) for the purpose of purification or detection. Examples of such molecules that are useful in protein purification include those that present structural epitopes capable of being recognized by a second molecule. This is a common strategy that is employed in protein purification by affinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding the immobilized compound. Examples of epitope tag molecules that can be conjugated to antagonistic TNFR2 antibodies or fragments thereof for the purposes of molecular recognition include, without 79 £023258330   30 Get 2023 limitation, maltose-binding protein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag, biotin, streptavidin. Conjugates containing the epitopes presented by these molecules are capable of being recognized by such complementary molecules as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively. For example, one can purify an antagonistic TNFR2 antibody or fragment thereof of the invention that has been conjugated to an epitope tag from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc) by treating the mixture with a solid phase resin containing an complementary molecule that can selectively recognize and bind the epitope tag of the antagonistic anti-TNFR2 antibody or fragment thereof. Examples of solid phase resins include agarose beads, which are compatible with purifications in aqueous solution. An antagonistic TNFR2 single-chain polypeptide, antibody, or antigen-binding fragment thereof of the invention can also be covalently appended to a fluorescent molecule, e.g., to detect the antibody or antigen-binding fragment thereof by fluorimetry and / or by direct visualization using fluorescence microscopy. Exemplary fluorescent molecules that can be conjugated to antibodies of the invention include green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, hoescht, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhoadmine, and cyanine. Additional examples of fluorescent molecules suitable for conjugation to antibodies of the invention are well-known in the art and have been described in detail in, e.g., U.S. Patent Nos. 7,417,131 and 7,413,874, each of which is incorporated by reference herein. Antagonistic TNFR2 polypeptides containing a fluorescent molecule are particularly useful for monitoring the cell-surface localization properties of antibodies and fragments thereof of the invention. For instance, one can expose cultured mammalian cells (e.g., T-reg cells) to antagonistic TNFR2 antibodies or fragments thereof of the invention that have been covalently conjugated to a fluorescent molecule and subsequently analyze these cells using conventional fluorescent microscopy techniques known in the art. Confocal fluorescent microscopy is a particularly powerful method for determining cellsurface localization of antagonistic anti-TNFR2 antibodies or fragments thereof, as individual planes of a cell can be analyzed in order to distinguish antibodies or fragments thereof that have been internalized into a cell’s interior, e.g., by receptor-mediated endocytosis, from those that are bound to the external face of the cell membrane. Additionally, cells can be treated with antagonistic TNFR2 antibodies conjugated to a fluorescent molecule that emits visible light of a particular wavelength (e.g., fluorescein, which fluoresces at about 535 nm) and an additional fluorescent molecule that is known to localize to a particular site on the T-reg cell surface and that fluoresces at a different wavelength (e.g., a molecule that localizes to CD25 and that fluoresces at about 599 nm). The resulting emission patterns can be visualized by confocal fluorescence microscopy and the images from these two wavelengths can be merged in order to reveal information regarding the location of the antagonistic TNFR2 antibody or antigen-binding fragment thereof on the T-reg cell surface with respect to other receptors. Bioluminescent proteins can also be incorporated into a fusion protein for the purposes of detection and visualization of an antagonistic anti-TNFR2 polypeptide, such as a single-chain 80 2023258320   30 Oct 2023 polypeptide, antibody, or fragment thereof. Bioluminescent proteins, such as Luciferase and aequorin, emit light as part of a chemical reaction with a substrate (e.g., luciferin and coelenterazine). Exemplary bioluminescent proteins suitable for use as a diagnostic sequence and methods for their use are described in, e.g., U.S. Patent Nos. 5,292,658, 5,670,356, 6,171,809, and 7,183,092, each of which is herein incorporated by reference. Antagonistic TNFR2 antibodies or fragments thereof labeled with bioluminescent proteins are a useful tool for the detection of antibodies of the invention following an in vitro assay. For instance, the presence of an antagonistic TNFR2 antibody that has been conjugated to a bioluminescent protein can be detected among a complex mixture of additional proteins by separating the components of the mixture using gel electrophoresis methods known in the art (e.g., native gel analysis) and subsequently transferring the separated proteins to a membrane in order to perform a Western blot. Detection of the antagonistic TNFR2 antibody among the mixture of other proteins can be achieved by treating the membrane with an appropriate Luciferase substrate and subsequently visualizing the mixture of proteins on film using established protocols. The polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can also be conjugated to a molecule comprising a radioactive nucleus, such that an antibody or fragment thereof of the invention can be detected by analyzing the radioactive emission pattern of the nucleus. Alternatively, an antagonistic TNFR2 antibody or fragment thereof can be modified directly by incorporating a radioactive nucleus within the antibody during the preparation of the protein. Radioactive isotopes of methionine (35S), nitrogen (15N), or carbon (13C) can be incorporated into antibodies or fragments thereof of the invention by, e.g., culturing bacteria in media that has been supplemented with nutrients containing these isotopes. Optionally, tyrosine derivatives containing a radioactive halogen can be incorporated into an antagonistic TNFR2 antibody or fragment thereof by, e.g., culturing bacterial cells in media supplemented with radiolabeled tyrosine. It has been shown that tyrosine functionalized with a radioactive halogen at the C2 position of the phenol system are rapidly incorporated into elongating polypeptide chains using the endogenous translation enzymes in vivo (U.S. Patent No. 4,925,651; incorporated herein by reference). The halogens include fluorine, chlorine, bromine, iodine, and astatine. Additionally, antagonistic TNFR2 antibodies or fragments thereof can be modified following isolation and purification from cell culture by functionalizing antibodies or fragments thereof of the invention with a radioactive isotope. The halogens represent a class of isotopes that can be readily incorporated into a purified protein by aromatic substitution at tyrosine or tryptophan, e.g., via reaction of one or more of these residues with an electrophilic halogen species. Examples of radioactive halogen isotopes include 18F, 75Br, 77Br, '22l, 123| 124| 125| 129| 1311 Qr211At. Another alternative strategy for the incorporation of a radioactive isotope is the covalent attachment of a chelating group to the antagonistic anti-TNFR2 polypeptide, such as a single-chain polypeptide, antibody, or fragment thereof. Chelating groups can be covalently appended to an antagonistic TNFR.2 antibody or fragment thereof by attachment to a reactive functional group, such as a thiol, amino group, alcohol, or carboxylic acid. The chelating groups can then be modified to contain any of a variety of metallic radioisotopes, including, without limitation, such radioactive nuclides as 125l, 67Ga, 111 In, "Tc, 169Yb, 186Re, 123l, 124|, 125ij 1311, 99mTc, 111 |n> 64Cu, 67Cu, 1B6Re> 188Re, 177|_Uj 90Y, 77As> 72As, 86^, 89Zr, 211 At> 212Bi) 213Bi, or 225Ac 81 £023258330   30 Get 2023 In some embodiments, it may be desirable to covalently conjugate the polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof) of the invention with a chelating group capable of binding a metal ion from heavy elements or rare earth ions, such as Gd3+, Fe3+, Mn3+, or Cr2+. Conjugates containing chelating groups that are coordinated to such paramagnetic metals are useful as in MRI imaging applications. Paramagnetic metals include, but are not limited to, chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), and ytterbium (III). In this way, antagonistic TNFR2 antibodies can be detected by MRI spectroscopy. For instance, one can administer antagonistic TNFR2 antibodies or fragments thereof conjugated to chelating groups bound to paramagnetic ions to a mammalian subject (e.g., a human patient) in order to monitor the distribution of the antibody following administration. This can be achieved by administration of the antibody to a patient by any of the administration routes described herein, such as intravenously, and subsequently analyzing the location of the administered antibody by recording an MRI of the patient according to established protocols. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) can additionally be conjugated to other molecules for the purpose of improving the solubility and stability of the protein in aqueous solution. Examples of such molecules include PEG, PSA, bovine serum albumin (BSA), and human serum albumin (HSA), among others. For instance, one can conjugate an antagonistic TNFR2 antibody or fragment thereof to carbohydrate moieties in order to evade detection of the antibody or fragment thereof by the immune system of the patient receiving treatment. This process of hyperglycosylation reduces the immunogenicity of therapeutic proteins by sterically inhibiting the interaction of the protein with B cell receptors in circulation. Alternatively, antagonistic TNFR2 antibodies or fragments thereof can be conjugated to molecules that prevent clearance from human serum and improve the pharmacokinetic profile of antibodies of the invention. Exemplary molecules that can be conjugated to or inserted within anti-TNFR2 antibodies or fragments thereof of the invention so as to attenuate clearance and improve the pharmacokinetic profile of these antibodies and fragments include salvage receptor binding epitopes. These epitopes are found within the Fc region of an IgG immunoglobulin and have been shown to bind Fc receptors and prolong antibody half-life in human serum. The insertion of salvage receptor binding epitopes into anti-TNFR2 antibodies or fragments thereof can be achieved, e.g., as described in US Patent No. 5,739,277; incorporated herein by reference. Modified antagonistic TFNR2 polypeptides In addition to conjugation to other therapeutic agents and labels for identification or visualization, anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can also be modified so as to improve their pharmacokinetic profile, biophysical stability, or inhibitory capacity. For instance, any cysteine residue not involved in maintaining the proper conformation of the anti-TNFR2 antibody or fragment thereof may be substituted with an isosteric or isolectronic amino acid (e.g., serine) in order to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cystine bond(s) may ...

Claims

1. An antibody or antigen-binding fragment thereof capable of specificaily binding human tumor necrosis factor receptor 2 (TNFR2), wherein the antibody or antigen-binding fragment thereof comprises a complementarity determining region-heavy chain 3 (CDR-H3) having the amino acid sequence of:(a) JRJDGSY(J)2FD(J)3(SEQ ID NO: 279);(b) AZ1DZ2Z4Z3Z5SPZ5Z2Z5WG (SEQ ID NO: 266); or(c) ARDDGSYSPFDYWG (SEQ ID NO: 259) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;wherein each J is independently a naturally occurring amino acid;each Z1 is independently a naturally occurring amino acid comprising a cationic side-chain at physiological pH;each Z2 is independently a naturally occurring amino acid comprising an anionic side-chain at physiological pH;each Z3 is independently a naturally occurring amino acid comprising a polar, uncharged sidechain at physiological pH;each Z4 is independently a glycine or alanine; andeach Z5 is independently a naturally occurring amino acid comprising a hydrophobic side-chain.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein in the presence of said CDR-H3, the antibody or antigen-binding fragment thereof is capable of specifically binding a peptide comprising the amino acid sequence of LRKCRPGFGVA (SEQ ID NO: 285) or VVCKPCAPGTFSN (SEQ ID NO: 286).

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein in the presence of said CDR-H3, the antibody or antigen-binding fragment thereof is capable of specifically binding an epitope within amino acids 142-149 (KCRPGFGV) or amino acids 161-169 (CKPCAPGTF) of SEQ ID NO: 7.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 -3, wherein said CDR-H3 has the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259).

5. The antibody or antigen-binding fragment thereof of any one of claims 1 -3, wherein said CDR-H3 has the amino acid sequence ARDDGSYSPFDYFG (SEQ ID NO: 284).

6. The antibody or antigen-binding fragment thereof of any one of claims 1 -5, wherein said antibody or antigen-binding fragment further comprises one or more, or all, of the following regions (CDRs):(a) a CDR-H1 having the amino acid sequence GJTF(J)2YJ (SEQ ID NO: 277);(b) a CDR-H2 having the amino acid sequence (J)sGSJ;(c) a CDR-L1 having the amino acid sequence (J)sY;2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 032513(d) a CDR-L2 having the amino acid sequence (J)zS; and(e) a CDR-L3 having the amino acid sequence (J)3Y(J)4T;wherein each J is independently a naturally occurring amino acid.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 -5, wherein said antibody or antigen-binding fragment further comprises one or more, or all, of the following regions (CDRs):(a) a CDR-H1 having the amino acid sequence Z4YZ3Z5TDZ5X;(b) a CDR-H2 having the amino acid sequence VDPEYZ4Z3T (SEQ ID NO: 264);(c) a CDR-L1 having the amino acid sequence QNINKZ5 (SEQ ID NO: 268);(d) a CDR-L2 having the amino acid sequence TYZ3 or YTZ3; and(e) a CDR-L3 having the amino acid sequence CLQZ5VNLXZ3 (SEQ ID NO: 271);wherein each Z1 is independently an amino acid comprising a cationic side-chain at physiological pH;each Z2 is independently an amino acid comprising an anionic side-chain at physiological pH;each Z3 is independently an amino acid comprising a polar, uncharged side-chain at physiological pH;each Z4 is independently a glycine or alanine;each Z5 is independently an amino acid comprising a hydrophobic side-chain; andeach X is independently leucine or isoleucine.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 -5, wherein said antibody or antigen-binding fragment further comprises one or more, or all, of the following regions (CDRs):(a) a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;(b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;(c) a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;(d) a CDR-L2 having the amino acid sequence TYS or YTS; and(e) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;wherein each X is independently leucine or isoleucine.

9. The antibody or antigen-binding fragment thereof of any one of claims 6-8, wherein said antibody or antigen-binding fragment comprises one or more, or all, of the following regions (CDRs):(a) a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257);(b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258);(c) a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260);2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 032513(d) a CDR-L2 having the amino acid sequence TYS or YTS; and(e) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261);wherein each X is independently leucine or isoieucine.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein said CDR-H1 has the amino acid sequence GYTFTDYL (SEQ ID NO: 274).

11. The antibody or antigen-binding fragment thereof of claim 9, wherein said CDR-H1 has ihe amino acid sequence GYTFTDYI (SEQ ID NO: 275).

12. The antibody or antigen-binding fragment thereof of any one of claims 9-11, wherein said CDR-L2 has the amino acid sequence TYS.

13. The antibody or antigen-binding fragment thereof of any one of claims 9-11, wherein said CDR-L2 has the amino acid sequence YTS.

14. The antibody or antigen-binding fragment thereof of any one of claims 9-13, wherein said CDR-L3 has the amino acid sequence CLQYVNLLT (SEQ ID NO: 272).

15. The antibody or antigen-binding fragment thereof of any one of claims 9-13, wherein said CDR-L3 has the amino acid sequence CLQYVNLIT (SEQ ID NO: 273).

16. The antibody or antigen-binding fragment thereof of any one of ciaims 9-15, wherein said antibody or antigen-binding fragment thereof comprises a framework region comprising the amino acid sequence LLIR (SEQ ID NO: 262) bound to the N-terminus of said CDR-L2.

17. The antibody or antigen-binding fragment thereof of any one of claims 9-16, wherein said antibody or antigen-binding fragment thereof comprises a framework region comprising the amino acid sequence TLE bound to the C-terminus of said CDR-L2.

18. The antibody or antigen-binding fragment thereof of any one of claims 1 -17, wherein said antibody or antigen-binding fragment does not comprise one or more of the following regions (CDRs):(a) a CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 23);(b) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO: 24);(c) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID NO: 26);(d) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO: 27);(e) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO: 28);(f) a CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO: 29);(g) a CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO: 30); and(h) a CDR-L3 comprising the amino acid sequence QHSRELPRT (SEQ ID NO: 31).

19. The antibody or antigen-binding fragment thereof of any one of claims 1 -18, wherein said antibody or antigen-binding fragment thereof comprises a non-native constant region.2023258320   30 Oct 202320. The antibody or antigen-binding fragment thereof of claim 19, wherein said non-native constant region is a human constant region.

21. The antibody or antigen-binding fragment thereof of any one of claims 1 -18, wherein said antibody or antigen-binding fragment thereof lacks all or a portion of an Fc domain, lacks all or a portion of a native Fc domain, or lacks an Fc domain altogether.

22. The antibody or antigen-binding fragment thereof of any one of claims 1 -21, wherein said antibody or antigen-binding fragment thereof specifically binds to a peptide comprising the ammo acid sequence of any one of SEQ ID NOs: 11,19, 20, and 34-117 with a Kd of less than about 100 nM and does not bind a peptide comprising amino acids 56-60 (KCSPG) of SEQ ID NO: 7.

23. The antibody or antigen-binding fragment thereof of any one of claims 1 -22, wherein said antibody or antigen-binding fragment thereof specifically binds a peptide comprising one or more of amino acids 142-146 of SEQ ID NO: 7 (KCRPG) and does not bind a peptide comprising amino acids 56-60 of SEQ ID NO: 7 (KCSPG).

24. The antibody or antigen-binding fragment thereof of any one of claims 1 -23, wherein said antibody or antigen-binding fragment thereof inhibits TNFR2 signaling.

25. The antibody or antigen-binding fragment thereof of any one of claims 1 -24, wherein said antibody or antigen-binding fragment thereof inhibits the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and clAP2 / BIRC3.

26. The antibody or antigen-binding fragment thereof of any one of claims 1 -25, wherein said antibody or antigen-binding fragment thereof inhibits NFkB activation.

27. The antibody or antigen-binding fragment thereof of any one of claims 1 -26, wherein said antibody or antigen-binding fragment thereof is capable of reducing or inhibiting the proliferation of T-reg ceils.

28. The antibody or antigen-binding fragment thereof of any one of claims 1 -27, wherein said antibody or antigen-binding fragment thereof is capable of reducing or inhibiting the proliferation of cancer cells that express TNFR2.

29. The antibody or antigen-binding fragment thereof of claim 28, wherein said cancer cells are selected from the group consisting of T cell lymphoma cells, such as Hodgkin’s or cutaneous nonHodgkin’s lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, and renal cell carcinoma cells.

30. The antibody or antigen-binding fragment thereof of any one of claims 1 -29, wherein said antibody or antigen-binding fragment thereof is capable of reducing or inhibiting the proliferation of myeloid-derived suppressor cells.2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 03251331. The antibody or antigen-binding fragment thereof of any one of claims 1 -30, wherein said antibody or antigen-binding fragment thereof does not require TNFa to reduce or inhibit the proliferation of T-reg cells.

32. The antibody or antigen-binding fragment thereof of any one of claims 1 -31, wherein said antibody or antigen-binding fragment thereof promotes proliferation of T effector cells.

33. A method of identifying a TNFR2 antagonist antibody or antigen-binding fragment thereof comprising:(a) exposing a heterogeneous mixture of antibodies or fragments thereof to a peptide having the amino acid sequence of ID NO: 285 or 286, or an amino acid sequence having up to two amino acid substitutions relative te said sequences; and(b) retaining antibodies or fragments thereof that specifically bind said peptide and removing antibodies or fragments thereof that do not specifically bind said peptide, thereby producing an enriched antibody mixture comprising at least one said TNFR2 antagonist antibody or antigen-binding fragment thereof.

34. The method of claim 33, wherein said method comprises determining the amino acid sequence of one or more of the antibodies or antigen-binding fragments thereof in said enriched antibody mixture.

35. The method of claim 33 or 34, wherein said peptide is bound to a surface.

36. The method of any one of claims 33-35, wherein said antibody or antigen-binding fragment thereof is expressed on the surface of a phage, bacterial cell, or yeast cell.

37. The method of any one of claims 33-35, wherein said antibody or antigen-binding fragment thereof is expressed as one or more polypeptide chains non-covalently bound to ribosomes or covalently bound to mRNA or cDNA.

38. The method of any one of claims 33-37, wherein said peptide is conjugated to a detectable label.

39. The method of claim 38, wherein said detectable label is selected from the group consisting of a fluorescent molecule, an epitope tag, and a radiolabel.

40. The method of claim 39, wherein said fluorescent molecule is selected from the group consisting of green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, hoescht, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhoadmine, and cyanine.2023258320   30 Oct 202341, The method of claim 39, wherein said epitope tag is selected from the group consisting of a maltose-binding protein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag, biotin, and streptavidin.

42. The method of any one of claims 33-41, wherein steps (a) and (b) are sequentially repeated one or more times.

43. A method of producing a TNFR2 antagonist antibody or antigen-binding fragment thereof comprising immunizing a non-human mammal with a peptide comprising the sequence of SEQ ID NO: 285 or 286, or an amino acid sequence having up to two amino acid substitutions relative to said sequences, and collecting serum comprising said TNFR2 antagonist antibody or antigen-binding fragment thereof.

44. The method of claim 43, wherein said non-human mammal is selected from the group consisting of a rabbit, mouse, rat, goat, guinea pig, hamster, horse, and sheep.

45. An antibody or antigen-binding fragment thereof that is produced by the method of any one of claims 33-44.

46. The antibody or antigen-binding fragment thereof of any one of claims 1 -32 and 45, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of 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 multi-specific 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, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, and a tandem scFv (taFv).

47. The antibody or antigen-binding fragment thereof of claim 46, wherein said antibody or antigen-binding fragment thereof is a F(ab’)2 molecule.

48. The antibody or antigen-binding fragment thereof of any one of claims 1 -32, 45, and 46, wherein said antibody or antigen-binding fragment thereof has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.

49. The antibody or antigen-binding fragment thereof of any one of claims 1-32 and 45-48, wherein said antibody is conjugated to a therapeutic agent.

50. The antibody or antigen-binding fragment thereof of claim 49, wherein said therapeutic agent is a cytotoxic agent.2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 03251351. A single-chain polypeptide capable of specifically binding human TNFR2, wherein the singlechain polypeptide comprises a CDR-H1, a CDR-H2, and a CDR-H3, wherein the CDR-H3 comprises an amino acid sequence of:(a) JRJDGSY(J)2FD(J)3(SEQ ID NO: 279);(b) AZ'DZ2Z4Z3Z5SPZ5Z2Z5WG (SEQ ID NO: 266); or(c) ARDDGSYSPFDYWG (SEQ ID NO: 259) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;wherein each J is independently a naturally occurring amino acid;each Z1 is independently a naturally occurring amino acid comprising a cationic side-chain at physiological pH;each Z2 is independently a naturally occurring amino acid comprising an anionic side-chain at physiological pH;each Z3 is independently a naturally occurring amino acid comprising a polar, uncharged sidechain at physiological pH;each Z4 is independently a glycine or alanine; andeach Z5 is independently a naturally occurring amino acid comprising a hydrophobic side-chain.

52. The single-chain polypeptide of claim 51, wherein said CDR-H1 and CDR-H2 are from an anti-TNFR2 antibody capable of specifically binding an epitope within amino acids 70-180 of SEQ ID NO: 7.

53. The single-chain polypeptide of claim 52, wherein said CDR-H1 and CDR-H2 are from an anti-TNFR2 antibody capable of specifically binding an epitope within amino acids 142-149 of SEQ ID NO: 7 (KCRPGFGV).

54. The single-chain polypeptide of claim 52, wherein said CDR-H1 and CDR-H2 are from an anti-TNFR2 antibody capable of specifically binding an epitope within amino acids 137-144 of SEQ ID NO: 7 (CAPLRKCR).

55. The single-chain polypeptide of claim 52, wherein said CDR-H1 and CDR-H2 are from an anti-TNFR2 antibody capable of specifically binding an epitope within amino acids 161 -169 of SEQ ID NO: 7 (CKPCAPGTF).

56. The single-chain polypeptide of claim 52, wherein said CDR-H1 and CDR-H2 are from an anti-TNFR2 antibody capable of specifically binding an epitope within amino acids 80-86 (DSTYTQL), 9198 (PECLSCGS), and 116-123 (RICTCRPG) of SEQ ID NO: 7.

57. The single-chain polypeptide of any one of claims 51 -56, wherein in the presence of said CDR-H3, the antibody or antigen-binding fragment thereof is capable of specifically binding a peptide comprising the amino acid sequence of LRKCRPGFGVA (SEQ ID NO: 285) or VVCKPCAPGTFSN (SEQ ID NO: 286).2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 03251358. The single-chain polypeptide of any one of claims 51 -57, wherein said CDR-H3 has the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259).

59. The single-chain polypeptide of any one of claims 51 -57, wherein said CDR-H3 has the amino acid sequence ARDDGSYSPFDYFG (SEQ ID NO: 284).

60. The single-chain polypeptide of any one of claims 51 -59, wherein said CDR-H1 has the amino acid sequence:(a) GJTF(J)2YJ (SEQ ID NO: 277);(b) Z4YZ3Z5TDZ5X; or(c) GYTFTDYX (SEQ ID NO: 257) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;wherein each J is independently a naturally occurring amino acid;each Z1 is independently a naturally occurring amino acid comprising a cationic side-chain at physiological pH;each Z2 is independently a naturally occurring amino acid comprising an anionic side-chain at physiological pH;each Z3 is independently a naturally occurring amino acid comprising a polar, uncharged sidechain at physiological pH;each Z4 is independently a glycine or alanine; andeach Z5 is independently a naturally occurring amino acid comprising a hydrophobic side-chain; andeach X is independently leucine or isoleucine.

61. The single-chain polypeptide of claim 60, wherein said CDR-H1 has the amino acid sequence GYTFTDYX (SEQ ID NO: 257).

62. The single-chain polypeptide of claim 61, wherein said CDR-H1 has the amino acid sequence GYTFTDYL (SEQ ID NO: 274).

63. The single-chain polypeptide of claim 61, wherein said CDR-H1 has the amino acid sequence GYTFTDYI (SEQ ID NO: 275).

64. The single-chain polypeptide of any one of claims 51 -63, wherein said CDR-H2 has the amino acid sequence:(a) (J)5GSJ;(b) VDPEYZ4Z3T (SEQ ID NO: 264); or(c) VDPEYGST (SEQ ID NO: 258) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;wherein each J is independently a naturally occurring amino acid;2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 032513each Z1 is independently a naturally occurring amino acid comprising a cationic side-chain at physiological pH;each Z2 is independently a naturally occurring amino acid comprising an anionic side-chain at physiological pH;each Z3 is independently a naturally occurring amino acid comprising a polar, uncharged sidechain at physiological pH;each Z4 is independently a glycine or alanine; andeach Z5 is independently a naturally occurring amino acid comprising a hydrophobic side-chain.

65. The single-chain polypeptide of claim 64, wherein said CDR-H2 has the amino acid sequence VDPEYGST (SEQ ID NO: 258).

66. The single-chain polypeptide of any one of claims 51 -65, wherein said single-chain polypeptide further comprises one or more, or ail, of the following CDRs:(a) a CDR-L1 having the amino acid sequence (J)sY;(b) a CDR-L2 having the amino acid sequence (J)2S; and(c) a CDR-L3 having the amino acid sequence (J)3Y(J)4T.wherein each J is independently a naturally occurring amino acid.

67. The single-chain polypeptide of any one of claims 51 -65, wherein said single-chain polypeptide further comprises one or more, or all, of the following CDRs:(a) a CDR-L1 having the amino acid sequence QNINKZ5 (SEQ ID NO: 268);(b) a CDR-L2 having the amino acid sequence TYZ3 or YTZ3; and(c) a CDR-L3 having the amino acid sequence CLQZ5VNLXZ3 (SEQ ID NO: 271);wherein each Z1 is independently an amino acid comprising a cationic side-chain at physiological pH;each Z2 is independently an amino acid comprising an anionic side-chain at physiological pH;each Z3 is independently an amino acid comprising a polar, uncharged side-chain at physiological pH;each Z4 is independently a glycine or alanine;each Z5 is independently an amino acid comprising a hydrophobic side-chain; andeach X is independently leucine or isoleucine.

68. The single-chain polypeptide of any one of claims 51 -65, wherein said single-chain polypeptide further comprises one or more, or all, of the following CDRs:(a) a CDR-L1 having the amino acid sequence QNINKY [SEQ ID NO: 260) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;(b) a CDR-L2 having the amino acid sequence TYS or YTS; and(c) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 032513wherein each X is independently leucine or isoleucine.

69. The single-chain polypeptide of any one of claims 66-68, wherein said CDR-L2 has the amino acid sequence TYS.

70. The single-chain polypeptide of any one of claims 66-68, wherein said CDR-L2 has the amino acid sequence YTS.

71. The single-chain polypeptide of any one of claims 66-70, wherein said CDR-L3 has the amino acid sequence CLQYVNLLT (SEQ ID NO: 272).

72. The single-chain polypeptide of any one of claims 66-70, wherein said CDR-L3 has the amino acid sequence CLQYVNLIT (SEQ ID NO: 273).

73. The single-chain polypeptide of any one of claims 66-72, wherein said single-chain polypeptide comprises a framework region comprising the amino acid sequence LLIR (SEQ ID NO: 262) bound to the N-terminus of said CDR-L2.

74. The single-chain polypeptide of any one of claims 66-72, wherein said single-chain polypeptide comprises a framework region comprising the amino acid sequence TLE bound to the C-terminus of said CDR-L2.

75. The single-chain polypeptide of any one of claims 51 -74, wherein said single-chain polypeptide does not comprise one or more of the following CDRs:(a) a CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 23);(b) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO: 24); and(c) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID NO: 26);(d) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO: 27);(e) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO: 28);(f) a CDR-L1 having the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO: 29);(g) a CDR-L2 having the amino acid sequence LASNLES (SEQ ID NO: 30); and(h) a CDR-L3 having the amino acid sequence QHSRELPRT (SEQ ID NO: 31).

76. A construct comprising a first polypeptide domain and a second polypeptide domain, wherein said first polypeptide domain and said second polypeptide domain each independently comprise a singlechain polypeptide of any one of claims 51 -75.

77. The construct of claim 76, wherein said first polypeptide domain and said second polypeptide domain are bound by a covalent linker.

78. The construct of claim 77, wherein said covalent linker comprises an amide bond.

79. The construct of claim 77, wherein said covalent linker comprises a disulfide bond.2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 03251380. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-32.

81. A polynucleotide encoding the single-chain polypeptide of any one of claims 51 -75.

82. A polynucleotide encoding the construct of any one of claims 76-79.

83. A vector comprising the polynucleotide of any one of claims 80-82.

84. The vector of claim 83, wherein said vector is an expression vector.

85. The vector of claim 84, wherein said expression vector is a eukaryotic expression vector.

86. The vector of claim 83, wherein said vector is a viral vector.

87. The vector of claim 86, wherein said viral vector is selected from the group consisting ofadenovirus (Ad), retrovirus, poxvirus, adeno-associated virus, baculovirus, herpes simplex virus, and a vaccinia virus.

88. The vector of claim 87, wherein said adenovirus is a serotype 5, 26, 35, or 48 adenovirus.

89. The vector of claim 87, wherein said retrovirus is a y-retrovirus or a lentivirus.

90. The vector of claim 87, wherein said vaccinia virus is a modified vaccinia Ankara (MVA).

91. An isolated host cell comprising the vector of any one of claims 83-90.

92. The host cell of claim 91, wherein said host cell is a prokaryotic cell.

93. The host cell of claim 91, wherein said host cell is a eukaryotic cell.

94. The host cell of claim 93, wherein said eukaryotic cell is a mammalian cell.

95. The host cell of claim 94, wherein said mammalian cell is a CHO cell.

96. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 -32 and 45-50, the single-chain polypeptide of any one of claims 51 -75, or the construct of any one of claims 76-79 and a pharmaceutically acceptable carrier or excipient.

97. The pharmaceutical composition of claim 96, wherein said pharmaceutical composition further comprises an additional therapeutic agent.

98. The pharmaceutical composition of claim 97, wherein said additional therapeutic agent is an immunotherapy agent.

99. The pharmaceutical composition of claim 98, wherein said immunotherapy agent is selected from the group consisting of an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-1422023258320   30 Oct 2023L2 agent, a TNF-a cross-linking agent, a TRAIL cross-linking agent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, an anti-4-1 BB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAILR1 agent, an anti-TRAILR2 agent, an anti-TWEAKR agent, Targretin, Interferon-alpha, clobestasol, Peg Interferon, prednisone, Romidepsin, Bexarotene, methotrexate, Triamcinolone cream, anti-chemokines, Vorinostat, and / or gabapentin.

100. A method of producing the antibody or antigen-binding fragment thereof of any one of claims 1-32, said method comprising expressing a polynucleotide encoding said antibody or antigenbinding fragment thereof in a host cell and recovering the antibody or antigen-binding fragment thereof from host cell medium.

101. A method of producing the single-chain polypeptide of any one of claims 51 -75, said method comprising expressing a polynucleotide encoding said single-chain polypeptide in a host cell and recovering the single-chain polypeptide from host cell medium.

102. A method of producing the construct of any one of claims 76-79, said method comprising expressing a polynucleotide encoding said construct in a host cell and recovering the construct from host cell medium.

103. A method of inhibiting an immune response mediated by a regulatory T cell in a human, said method comprising administering to the human the antibody or antigen-binding fragment thereof of any one of claims 1 -32 and 45-50, the single-chain polypeptide of any of claims 51 -75, the construct of any one of claims 76-79, the polynucleotide of any one of claims 80-82, the vector of any one of claims 83-90, the host cell of any one of claims 91 and 93-95, or the pharmaceutical composition of any one of claims 96-99.

104. A method of treating a cell proliferation disorder in a human, said method comprising administering to the human the antibody or antigen-binding fragment thereof of any one of claims 1-32 and 45-50, the single-chain polypeptide of any of claims 51-75, the construct of any one of claims 76-79, the polynucleotide of any one of claims 80-82, the vector of any one of claims 83-90, the host cell of any one of claims 91 and 93-95, or the pharmaceutical composition of any one of claims 96-99.

105. The method of claim 104, wherein said cell proliferation disorder is a cancer selected from the group consisting of leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and throat cancer.

106. The method of claim 104, wherein said cell proliferation disorder is a cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical2023258320   30 Oct 2023teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, 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 tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wilms tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom macroglobulinemia.

107. A method of treating an infectious disease in a human, said method comprising administering to the human the antibody or antigen-binding fragment thereof of any one of claims 1 -32 and 45-50, the single-chain polypeptide of any of claims 51 -75, the construct of any one of claims 76-79, the polynucleotide of any one of claims 80-82, the vector of any one of claims 83-90, the host cell of any one of claims 91 and 93-95, or the pharmaceutical composition of any one of claims 96-99.

108. The method of claim 107, wherein said infectious disease is caused by one or more agents selected from the group consisting of a virus, a bacterium, a fungus, or a parasite.

109. The method of claim 108, wherein said infectious disease is caused by a virus selected from the group consisting of hepatitis C virus, Yellow fever virus, Kadam virus, Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill virus, Negishi virus, Meaban virus, Saumarez Reef virus, Tyuleniy virus, Aroa virus, dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza virus, llheus virus, Israel2023258320   30 Oct 2023turkey meningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik 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 Island virus, Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, cell fusing agent virus, Ippy virus, Lassa virus, lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal 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, Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Marburg virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O’nyong’nyong virus, and the chikungunya virus, smallpox virus, monkeypox virus, vaccinia virus, herpes simplex virus, human herpes virus, 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 (VSV), human respiratory syncytial virus (RSV), Newcastle disease virus, hendravirus, nipahvirus, 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 papilloma virus, 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.

110. The method of claim 108, wherein said infectious disease is caused by a bacterium belonging to a genus selected from the group consisting of Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriacece, Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordatella, Legionella, Pasturella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus.

111. The method of claim 108, wherein said infectious disease is caused by a fungus selected from the group consisting of Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, and Absidia.

112. The method of claim 108, wherein said infectious disease is caused by a parasite selected from the group consisting of Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crust, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. Exemplary helminthic parasites include richuris trichiura, Ascaris2023258320   30 Oct 2023lumbricoides, 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.

113. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-32 and 45-50, the single-chain polypeptide of any of claims 51-75, the construct of any one of claims 76-79, the polynucleotide of any one of claims 80-82, the vector of any one of claims 83-90, the host cell of any one of claims 91 and 93-95, or the pharmaceutical composition of any one of claims 9699 for inhibiting an immune response mediated by a regulatory T cell in a human.

114. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-32 and 45-50, the single-chain polypeptide of any of claims 51-75, the construct of any one of claims 76-79, the polynucleotide of any one of claims 80-82, the vector of any one of claims 83-90, the host cell of any one of claims 91 and 93-95, or the pharmaceutical composition of any one of claims 9699 for treating a cell proliferation disorder in a human.

115. The composition of claim 114, wherein said cell proliferation disorder is a cancer selected frpm the group consisting of leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and throat cancer.

116. The composition of claim 114, wherein said cell proliferation disorder is a cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneurobiastoma, extracranial germ cell tumor, extragonadai germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wiims tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma / piasma cell2023258320   30 Oct 2023neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom macroglobulinemia.

117. A composition the antibody or antigen-binding fragment thereof of any one of claims 1 -32 and 45-50, the single-chain polypeptide of any of claims 51-75, the construct of any one of claims 76-79, the polynucleotide of any one of claims 80-82, the vector of any one of claims 83-90, the host cell of any one of claims 91 and 93-95, or the pharmaceutical composition of any one of claims 96-99 for treating an infectious disease in a human.

118. The composition of claim 117, wherein said infectious disease is caused by one or more agents selected from the group consisting of a virus, a bacterium, a fungus, or a parasite.

119. The composition of claim 118, wherein said infectious disease is caused by a virus selected from the group consisting of hepatitis C virus, Yellow fever virus, Kadam virus, Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill virus, Negishi virus, Meaban virus, Saumarez Reef virus, Tyuleniy virus, Area virus, dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza virus, llheus virus, Israel turkey meningoencephaio-myelitis virus, Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik 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, Bukaiasa bat virus, Carey island virus, Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, cell fusing agent virus, Ippy virus, Lassa virus, lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal 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, Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Marburg virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O’nyong’nyong virus, and the chikungunya virus, smallpox virus,2023258320   30 Oct 2023monkeypox virus, vaccinia virus, herpes simplex virus, human herpes virus, 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 (VSV), human respiratory syncytial virus (RSV), Newcastle disease virus, hendravirus, nipahvirus, 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 papilloma virus, 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.

120. The composition of claim 118, wherein said infectious disease is caused by a bacterium belonging to a genus selected from the group consisting of Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriacece, Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordatella, Legionella, Pasturella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus.

121. The composition of claim 118, wherein said infectious disease is caused by a fungus selected from the group consisting of Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, and Absidia.

122. The composition of claim 118, wherein said infectious disease is caused by a parasite selected from the group consisting of Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meieagridis. Exemplary helminthic 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.

123. A kit comprising an agent selected from the group consisting of the antibody or antigenbinding fragment thereof of any one of claims 1 -32 and 45-50, the single-chain polypeptide of any of claims 51 -75, the construct of any one of claims 76-79, the polynucleotide of any one of claims 80-82, the vector of any one of claims 83-90, the host cell of any one of claims 91 and 93-95, and the pharmaceutical composition of any one of claims 96-99.

124. The kit of claim 123, wherein said kit comprises the antibody or antigen-binding fragment thereof any one of claims 1 -32 and 45-50.2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 032513125. The kit of claim 123, wherein said kit comprises the single-chain polypeptide of any of claims 51-75.

126. The kit of claim 123, wherein said kit comprises the construct of any of claims 76-79.

127. The kit of claim 123, wherein said kit comprises the polynucleotide of any one of claims80-82.

128. The kit of claim 123, wherein said kit comprises the vector of any one of claims 83-90.

129. The kit of claim 128, wherein said kit further comprises instructions for transfecting saidvector into a host cell.

130. The kit of claim 129, wherein said kit further comprises instructions for expressing said antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct in said host cell.

131. The kit of claim 123, wherein said kit comprises the host ceil of any one of claims 91 -95.

132. The kit of claim 123, wherein said kit comprises the pharmaceutical composition of anyone of claims 96-99.

133. The kit of claim 123, wherein said kit further comprises a reagent that can be used to express the antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct in said host cell.

134. The kit of claim 123, further comprising instructions for administering said agent to a human patient.

135. The kit of claim 123, further comprising instructions for making or using said agent.

136. The antibody or antigen-binding fragment thereof of claim 1, wherein said CDR-H3 has the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259).

137. The antibody or antigen-binding fragment thereof of claim 1, wherein said CDR-H3 has the amino acid sequence ARDDGSYSPFDYFG (SEQ ID NO: 284).

138. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment further comprises one or more, or all, of the following regions (CDRs):(a) a CDR-H1 having the amino acid sequence GJTF(J)2YJ (SEQ ID NO: 277);(b) a CDR-H2 having the amino acid sequence (J)sGSJ;(c) a CDR-L1 having the amino acid sequence (J)sY;(d) a CDR-L2 having the amino acid sequence (J)2S; and(e) a CDR-L3 having the amino acid sequence (J)sY(J)4T;wherein each J is independently a naturally occurring amino acid.2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 032513139. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment further comprises one or more, or all, of the following regions (CDRs):(a) a CDR-H1 having the amino acid sequence Z4YZ3Z5TDZ5X;(b) a CDR-H2 having the amino acid sequence VDPEYZ4Z3T (SEQ ID NO: 264);(c) a CDR-L1 having the amino acid sequence QNINKZ5 (SEQ ID NO: 268);(d) a CDR-L2 having the amino acid sequence TYZ3 or YTZ3; and(e) a CDR-L3 having the amino acid sequence CLQZ5VNLXZ3(SEQ ID NO: 271);wherein each Z1 is independently an amino acid comprising a cationic side-chain at physiological pH;each Z2 is independently an amino acid comprising an anionic side-chain at physiological pH;each Z3 is independently an amino acid comprising a polar, uncharged side-chain at physiological pH;each Z4 is independently a glycine or alanine;each Z5 is independently an amino acid comprising a hydrophobic side-chain; andeach X is independently leucine or isoleucine.

140. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment further comprises one or more, or all, of the following regions (CDRs):(a) a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;(b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;(c) a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;(d) a CDR-L2 having the amino acid sequence TYS or YTS; and(e) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;wherein each X is independently leucine or isoleucine.

141. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment comprises one or more, or all, of the following regions (CDRs):(a) a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257);(b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258);(c) a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260);(d) a CDR-L2 having the amino acid sequence TYS or YTS; and(e) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261);wherein each X is independently leucine or isoleucine.

142. The antibody or antigen-binding fragment thereof of claim 141, wherein said CDR-H1 has the amino acid sequence GYTFTDYL (SEQ ID NO: 274).2023258320   30 Oct 2023143. The antibody or antigen-binding fragment thereof of claim 141, wherein said CDR-H1 has the amino acid sequence GYTFTDYI (SEQ ID NO: 275).

144. The antibody or antigen-binding fragment thereof of claim 141, wherein said CDR-L2 has the amino acid sequence TYS.

145. The antibody or antigen-binding fragment thereof of claim 141, wherein said CDR-Lf! has the amino acid sequence YTS.

146. The antibody or antigen-binding fragment thereof of claim 141, wherein said CDR-L3 has the amino acid sequence CLQYVNLLT (SEQ ID NO: 272).

147. The antibody or antigen-binding fragment thereof of claim 141, wherein said CDR-L3 has the amino acid sequence CLQYVNLIT (SEQ ID NO: 273).

148. The antibody or antigen-binding fragment thereof of claim 141, wherein said antibody or antigen-binding fragment thereof comprises a framework region comprising the amino acid sequence LLIR (SEQ ID NO: 262) bound to the N-terminus of said CDR-L2.

149. The antibody or antigen-binding fragment thereof of claim 141, wherein said antibody or antigen-binding fragment thereof comprises a framework region comprising the amino acid sequence TLE bound to the C-terminus of said CDR-L2.

150. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment does not comprise one or more of the following regions (CDRs):(a) a CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 23);(b) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO: 24);(c) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID NO: 26);(d) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO: 27);(e) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO: 28);(f) a CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO: 29);(g) a CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO: 30); and(h) a CDR-L3 comprising the amino acid sequence QHSRELPRT (SEQ ID NO: 31).

151. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof comprises a non-native constant region.

152. The antibody or antigen-binding fragment thereof of claim 151, wherein said non-native constant region is a human constant region.

153. The antibody or antigen-binding fragment thereof of claim 1 wherein said antibody or antigen-binding fragment thereof lacks all or a portion of an Fc domain, lacks all or a portion of a native Fc domain, or lacks an Fc domain altogether.2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 032513154. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof specifically binds to a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 11, 19, 20, and 34-117 with a Kd of less than about 100 nM and does not bind a peptide comprising amino acids 56-60 (KCSPG) of SEQ ID NO: 7.

155. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof specifically binds a peptide comprising one or more of amino acids 142146 of SEQ ID NO: 7 (KCRPG) and does not bind a peptide comprising amino acids 56-60 of SEQ ID NO: 7 (KCSPG).

156. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof inhibits TNFR2 signaling.

157. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof inhibits the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA. MAP3K11, TRAF2, TRAF3, relB, and clAP2 / BIRC3.

158. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof inhibits NFkB activation.

159. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof is capable of reducing or inhibiting the proliferation of T-reg cells.

160. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof is capable of reducing or inhibiting the proliferation of cancer cells that express TNFR2.

161. The antibody or antigen-binding fragment thereof of claim 160, wherein said cancer cells are selected from the group consisting of \ T cell lymphoma cells, such as Hodgkin’s or cutaneous nonHodgkin’s lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, and renal cell carcinoma cells.

162. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof is capable of reducing or inhibiting the proliferation of myeloid-derived suppressor cells.

163. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof does not require TNFa to reduce or inhibit the proliferation of T-reg cells.

164. The antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment thereof is capable of directly expanding T effector cells.2023258320   30 Oct 2023165. The single-chain polypeptide of any one of claims 51 -56, wherein in the presence of said CDR-H3, the antibody or antigen-binding fragment thereof is capable of specifically binding a peptide comprising the amino acid sequence of LRKCRPGFGVA (SEQ ID NO: 285) or VVCKPCAPGTFSN (SEQ IDNO:286).

166. The single-chain polypeptide of claim 51, wherein said CDR-H3 has the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259).

167. The single-chain polypeptide of claim 51, wherein said CDR-H3 has the amino acid sequence ARDDGSYSPFDYFG (SEQ ID NO: 284).

168. The single-chain polypeptide of claim 51, wherein said CDR-H1 has the amino acid sequence:(a) GJTF(J)2YJ (SEQ ID NO: 277);(b) Z4YZ3Z5TDZ3X; or(c) GYTFTDYX (SEQ ID NO: 257) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;wherein each J is independently a naturally occurring amino acid;each Z1 is independently a naturally occurring amino acid comprising a cationic side-chain at physiological pH;each Z2 is independently a naturally occurring amino acid comprising an anionic side-chain at physiological pH;each Z3 is independently a naturally occurring amino acid comprising a polar, uncharged sidechain at physiological pH;each Z4 is independently a glycine or alanine; andeach Z5 is independently a naturally occurring amino acid comprising a hydrophobic side-chain; andeach X is independently leucine or isoleucine.

169. The single-chain polypeptide of claim 168, wherein said CDR-H1 has the amino acid sequence GYTFTDYX (SEQ ID NO: 257).

170. The single-chain polypeptide of claim 169, wherein said CDR-H1 has the amino acid sequence GYTFTDYL (SEQ ID NO: 274).

171. The single-chain polypeptide of claim 169, wherein said CDR-H1 has the amino acid sequence GYTFTDYI (SEQ ID NO: 275).

172. The single-chain polypeptide of claim 51, wherein said CDR-H2 has the amino acid sequence:(a) (J)5GSJ;(b) VDPEYZ4Z3T (SEQ ID NO: 264); or2023258320   30 Oct 2023WO 2017 / 197331                                                 PCT / US2017 / 032513(c) VDPEYGST (SEQ ID NO: 258) or an amino acid sequence having up to two amino acid substitutions relative to said sequence ;wherein each J is independently a naturally occurring amino acid;each Z' is independently a naturally occurring amino acid comprising a cationic side-chain at physiological pH;each Z2 is independently a naturally occurring amino acid comprising an anionic side-chain at physiological pH;each Z3 is independently a naturally occurring amino acid comprising a polar, uncharged sidechain at physiological pH;each Z4 is independently a glycine or alanine; andeach Z5 is independently a naturally occurring amino acid comprising a hydrophobic side-chain.

173. The single-chain polypeptide of claim 172, wherein said CDR-H2 has the amino acid sequence VDPEYGST (SEQ ID NO: 258).

174. The single-chain polypeptide of ciaim 51, wherein said single-chain polypeptide further comprises one or more, or all, of the following DDRs:(a) a CDR-L1 having the amino acid sequence (J)sY;(b) a CDR-L2 having the amino acid sequence (J)2S; and(c) a CDR-L3 having the amino acid sequence (J)3Y(J)4T.wherein each J is independently a naturally occurring amino acid.

175. The single-chain polypeptide of claim 51, wherein said single-chain polypeptide further comprises one or more, or all, of the following DDRs:(a) a CDR-L1 having the amino acid sequence QNINKZ5 (SEQ ID NO: 268);(b) a CDR-L2 having the amino acid sequence TYZ3 or YTZ3; and(c) a CDR-L3 having the amino acid sequence GLQZ5VNLXZ3(SEQ ID NO: 271);wherein each Z1 is independently an amino acid comprising a cationic side-chain at physiological pH;each Z2 is independently an amino acid comprising an anionic side-chain at physiological pH;each Z3 is independently an amino acid comprising a polar, uncharged side-chain at physiological pH;each Z4 is independently a glycine or alanine;each Z5 is independently an amino acid comprising a hydrophobic side-chain; andeach X is independently leucine or isoleucine.

176. The single-chain polypeptide of claim 51, wherein said single-chain polypeptide further comprises one or more, or all, of the following DDRs:(a) a DDR-L1 having the amino acid sequence QNINKY [SEQ ID NO: 260) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;2023258320   30 Oct 2023(b) a CDR-L2 having the amino acid sequence TYS or YTS; and(c) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261) or an amino acid sequence having up to two amino acid substitutions relative to said sequence;wherein each X is independently leucine or isoleucine.

177. The single-chain polypeptide of claim 174, wherein said CDR-L2 has the amino acid sequence TYS.

178. The single-chain polypeptide of claim 174, wherein said CDR-L2 has the amino acid sequence YTS.

179. The single-chain polypeptide of claim 174, wherein said CDR-L3 has the amino acid sequence CLQYVNLLT (SEQ ID NO: 272).

180. The single-chain polypeptide of claim 174, wherein said CDR-L3 has the amino acid sequence CLQYVNLIT (SEQ ID NO: 273).

181. The single-chain polypeptide of claim 174, wherein said single-chain polypeptide comprises a framework region comprising the amino acid sequence LLIR (SEQ ID NO: 262) bound to the N-terminus of said CDR-L2.

182. The single-chain polypeptide of claim 174, wherein said single-chain polypeptide comprises a framework region comprising the amino acid sequence TLE bound to the C-terminus of said CDR-L2.

183. The single-chain polypeptide of claim 51, wherein said singie-chain polypeptide does not comprise one or more of the following CDRs:(a) a CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 23);(b) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO: 24); and(c) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID NO: 26);(d) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO: 27);(e) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID NO: 28);(f) a CDR-L1 having the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO: 29);(g) a CDR-L2 having the amino acid sequence LASNLES (SEQ ID NO: 30); and(h) a CDR-L3 having the amino acid sequence QHSRELPRT (SEQ ID NO: 31).

184. A method of inhibiting an immune response mediated by a regulatory T cell in a human, said method comprising administering to the human the antibody or antigen-binding fragment thereof of claim 1.

185. A method of treating a cell proliferation disorder in a human, said method comprising administering to the human the antibody or antigen-binding fragment thereof of claim 1.2023258320   30 Oct 2023186. The method of claim 185, wherein said cell proliferation disorder is a cancer selected from the group consisting of leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and throat cancer.

187. The method of claim 185, wherein said cell proliferation disorder is a cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, 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 tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wilms tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom macroglobulinemia.

188. A method of treating an infectious disease in a human, said method comprising administering to the human the antibody or antigen-binding fragment thereof of claim 1.

189. The method of claim 188, wherein said infectious disease is caused by one or more agents selected from the group consisting of a virus, a bacterium, a fungus, or a parasite.2023258320   30 Oct 2023190. The method of claim 189, wherein said infectious disease is caused by a virus selected from the group consisting of hepatitis C virus, Yellow fever virus, Kadam virus, Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill virus, Negishi virus, Meaban virus, Saumarez Reef virus, Tyuleniy virus, Aroa virus, dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza virus, llheus virus, Israel turkey meningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik 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 Island virus, Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, cell fusing agent virus, Ippy virus, Lassa virus, lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal 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, Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Marburg virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O’nyong’nyong virus, and the chikungunya virus, smallpox virus, monkeypox virus, vaccinia virus, herpes simplex virus, human herpes virus, 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 (VSV), human respiratory syncytial virus (RSV), Newcastle disease virus, hendravirus, nipahvirus, 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 papilloma virus, 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.

191. The method of claim 189, wherein said infectious disease is caused by a bacterium belonging to a genus selected from the group consisting of Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriacece, Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordatella, Legionella, Pasturella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, and Staphylococcus.2023258320   30 Oct 2023192. The method of claim 189, wherein said infectious disease is caused by a fungus selected from the group consisting of Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, and Absidia.

193. The method of claim 189, wherein said infectious disease is caused by a parasite selected from the group consisting of Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crust, Leishmania mexicana, Leishmania braziiiensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. Exemplary helminthic 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.

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