Anti-TNFR2 antibodies and methods of use

Abstract

To provide anti-tumor necrosis factor receptor 2 (TNFR2) antibodies and related compositions, which may be used in any of a variety of therapeutic or diagnostic methods, including the treatment or diagnosis of oncological diseases, inflammatory and / or autoimmune diseases, and others.SOLUTION: Provided is an isolated antibody, or an antigen-binding fragment thereof, that binds to TNFR2, comprising: a heavy chain variable (VH) region comprising VHCDR1, VHCDR2, and VHCDR3 regions having specific sequences; and a light chain variable (VL) region comprising VLCDR1, VLCDR2, and VLCDR3 regions having specific sequences.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 901,364, filed September 17, 2019, U.S. Provisional Patent Application No. 62 / 985,509, filed March 5, 2020, U.S. Provisional Patent Application No. 63 / 047,824, filed July 2, 2020, and U.S. Provisional Patent Application No. 63 / 058,016, filed July 29, 2020, each of which is incorporated by reference in its entirety.

[0002] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence listing is named APEX-025 / 04WO_ST25.txt. This text file is approximately 265KB, was created on September 11, 2020, and has been submitted electronically via EFS-Web. [Background technology]

[0003] The present disclosure relates to anti-Tumor Necrosis Factor Receptor 2 (TNFR2) antibodies and related compositions that may be used in any of a variety of therapeutic or diagnostic methods, including neoplastic, inflammatory and / or autoimmune diseases, as well as other therapeutic or diagnostic methods.

[0004] 2. Description of Related Art TNF-α is an essential inflammatory mediator that plays a key role in both physiological and pathological conditions. It is mainly produced by macrophages and monocytes and can exist as both a 26 kDa membrane-bound trimer (mTNF-α) and a 17 kDa soluble trimer (sTNF-α). TNF-α exerts its effects through two receptors, TNFR1 (TNFRSF1A; 55 kDa) and TNFR2 (TNFRSF1B; 75 kDa), which have similar extracellular domains containing four repeated cysteine-rich motifs, but also distinct intracellular domains that activate distinct signaling pathways.

[0005] TNFR1 is a ubiquitously expressed protein that can be engaged by both mTNF-α and sTNF-α to signal cell survival / inflammation or apoptosis depending on the context. In contrast, TNFR2 expression is controlled by activation state and is primarily restricted to T cells, as well as immunosuppressive myeloid cells also known as myeloid-derived suppressor cells (MDSCs), including mononuclear and granulocytic myeloid cells. Mononuclear myeloid cells include terminally differentiated macrophages and dendritic cells (DCs), as well as monocytes that differentiate into macrophages and DCs in tissues under inflammatory conditions. Granulocytic myeloid cells include a population of terminally differentiated polymorphonuclear neutrophils, eosinophils, basophils, and mast cells. TNFR2 can only be fully engaged through mTNF-α. Unlike TNFR1, TNFR2 does not contain a death domain in its cytoplasm and instead can signal through the TRAF and NFkB pathways to control cell survival and immunosuppression. mTNF also exhibits reverse signaling in cells in which it is expressed upon receptor binding. Both TNFRs can be cleaved by the TACE enzyme and converted to soluble forms that can act to desensitize cells to TNF-α by removing the receptor from the cell or by acting as a decoy for sTNF-α.

[0006] TNFR2 plays an important role in regulating the immune system, likely through its effects on regulatory T cells (Tregs), which express high levels of TNFR2. In mice, deletion of TNFR2 exacerbates autoimmune disease and colitis by reducing Treg function. In humans, known polymorphisms in TNFRSF1B result in reduced TNFR2 expression or reduced binding to TNFα in regulatory T cells, disrupting TNFR2-mediated signaling. These polymorphisms strongly correlate with autoimmune diseases, including systemic lupus erythematosus (SLE), Crohn's disease, and ulcerative colitis. In both mice and humans, TNFR2 is expressed in highly suppressive Tregs, including those found in tumors, but is not strongly expressed in effector T cells. TNFR2 expression strongly correlates with a suppressive tumor microenvironment in a number of tumor types. It has also been shown that in ovarian cancer, TNFR2+ Tregs can impair T effector responses within the TME (Govindaraj C). Mouse models have provided further evidence for the role of TNFR2 in interfering with the immune response to cancer. Moreover, TNFR2 has been identified in more than 25 tumor types, including renal, colon, and ovarian cancers. Gain-of-function TNFR2 mutations occur in Sézary syndrome patients with a rare form of CTCL that is resistant to treatment.

[0007] Thus, there remains a need in the art for therapeutic antibodies that effectively inhibit or otherwise antagonize TNFR2 and related methods of treating cancer and inflammatory diseases. Summary of the Invention [Means for solving the problem]

[0008] The present disclosure relates to antibodies and antigen-binding fragments thereof that specifically bind to tumor necrosis factor receptor 2 (TNFR2), and methods of use thereof. One embodiment is an isolated antibody or antigen-binding fragment thereof that binds to TNFR2, including human TNFR2, comprising: a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 1-3, respectively; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 4-6, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 7 to 9, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 10 to 12, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 13 to 15, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 16 to 18, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 19 to 21, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 22 to 24, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 25 to 27, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 28 to 30, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 31 to 33, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 34 to 36, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 37 to 39, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 40 to 42, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 43-45, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 46-48, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 49-51, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 52-54, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 55 to 57, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 58 to 60, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 61-63, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 64-66, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 67-69, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 70-72, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 73-75, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 76-78, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 79-81, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 82-84, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 85 to 87, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 88 to 90, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 91-93, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 94-96, respectively; or a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 97 to 99, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 100 to 102, respectively; Alternatively, the present invention provides an isolated antibody, or antigen-binding fragment thereof, including a variant of the antibody, or antigen-binding fragment thereof, that comprises heavy and light chain variable regions identical to the heavy and light chain variable regions of (i) and (ii) except for up to 1, 2, 3, 4, 5, 6, 7, or 8 total amino acid substitutions across the CDR regions. In some embodiments, the VH region comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135. In some embodiments, the VL region comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 134, and 136.

[0009] Certain antibodies, or antigen-binding fragments thereof, a VH region set forth in SEQ ID NO: 103, and a VL region set forth in SEQ ID NO: 104; a VH region set forth in SEQ ID NO: 105, and a VL region set forth in SEQ ID NO: 106; a VH region set forth in SEQ ID NO: 107, and a VL region set forth in SEQ ID NO: 108; a VH region set forth in SEQ ID NO: 109, and a VL region set forth in SEQ ID NO: 110; a VH region set forth in SEQ ID NO:111, and a VL region set forth in SEQ ID NO:112; a VH region set forth in SEQ ID NO: 113, and a VL region set forth in SEQ ID NO: 114; a VH region set forth in SEQ ID NO: 115, and a VL region set forth in SEQ ID NO: 116; a VH region set forth in SEQ ID NO: 117, and a VL region set forth in SEQ ID NO: 118; a VH region set forth in SEQ ID NO: 119, and a VL region set forth in SEQ ID NO: 120; a VH region set forth in SEQ ID NO: 121, and a VL region set forth in SEQ ID NO: 122; a VH region set forth in SEQ ID NO: 123, and a VL region set forth in SEQ ID NO: 124; a VH region set forth in SEQ ID NO: 125, and a VL region set forth in SEQ ID NO: 126; a VH region set forth in SEQ ID NO: 127, and a VL region set forth in SEQ ID NO: 128; a VH region set forth in SEQ ID NO: 129, and a VL region set forth in SEQ ID NO: 130; a VH region set forth in SEQ ID NO: 131, and a VL region set forth in SEQ ID NO: 132; a VH region set forth in SEQ ID NO: 133, and a VL region set forth in SEQ ID NO: 134; or It comprises a VH region set forth in SEQ ID NO:135, and a VL region set forth in SEQ ID NO:136.

[0010] Some embodiments include an isolated antibody, or antigen-binding fragment thereof, that binds tumor necrosis factor receptor 2 (TNFR2), comprising a heavy chain variable (VH) region comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and a light chain variable (VL) region comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 134, and 136, respectively.

[0011] Some embodiments include an isolated antibody, or antigen-binding fragment thereof, that binds to tumor necrosis factor receptor 2 (TNFR2), comprising a heavy chain variable (VH) region comprising VHCDR1, VHCDR2, and VHCDR3 regions selected from the underlined sequences in Table R1; and a light chain variable (VL) region comprising VLCDR1, VLCDR2, and VLCDR3 regions, respectively, selected from the underlined sequences in Table R2.

[0012] Some embodiments include the isolated antibody of claim 6, or an antigen-binding fragment thereof, comprising a VH region comprising an amino acid sequence selected from Table R1, and a VL region comprising an amino acid sequence selected from Table R2, respectively.

[0013] Some embodiments include an isolated antibody, or antigen-binding fragment thereof, that binds to human tumor necrosis factor receptor 2 (TNFR2) at an epitope comprising, consisting of, or consisting essentially of one or more residues selected from R21, Y23, T27, S33, K34, T51, and S55, as defined by the mature human TNFR2 sequence (residues 23-461 of FL human TNFR2), including an epitope comprising, consisting of, or consisting essentially of one or more residues selected from REY, TAQMCCSK (SEQ ID NO:328), and TVCDS (SEQ ID NO:329). In some embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs:37-39, respectively; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs:40-42, respectively. In some embodiments, the VH region comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 115. In some embodiments, the VL region comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 116. In certain embodiments, the isolated antibody, or antigen-binding fragment thereof, comprises a VH region set forth in SEQ ID NO: 115, and a VL region set forth in SEQ ID NO:116.

[0014] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, binds to human TNFR2, eg, soluble and / or cell-expressed human TNFR2.

[0015] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, binds to at least one, two, three, four, or five human TNFR2 peptide epitopes selected from Table T1.

[0016] In some embodiments, the antibody is humanized. In some embodiments, the antibody is selected from the group consisting of a single chain antibody, an scFv, a monovalent antibody lacking a hinge region, a minibody, and a probody. In some embodiments, the antibody is a Fab or Fab' fragment. In some embodiments, the antibody is a F(ab')2 fragment. In some embodiments, the antibody is a whole antibody.

[0017] In some embodiments, the antibody comprises a human IgG constant domain. In some embodiments, the IgG constant domain comprises an IgG1 CH1 domain. In some embodiments, the IgG constant domain comprises an IgG1 Fc region, optionally a modified Fc region, optionally modified by one or more amino acid substitutions.

[0018] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, has a K of about 2 nM or less. D In some embodiments, the isolated antibody, or antigen-binding fragment thereof, binds to at least one peptide epitope of human TNFR2, e.g., Table T1, with a K of about 0.7 nM or less. D binds to human TNFR2 with a K of approximately 50 pm or less D and primary T cells, optionally T reg It binds to human TNFR2.

[0019] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, has the following characteristics: (a) inhibiting TNF-α binding to TNFR2; (b) inhibiting TNFR2 signaling; (c) activating TNFR2 signaling; (d) inhibiting TNFR2 trimerization; (e) cross-reactive binding to human TNFR2 and cynomolgus monkey TNFR2; (f) Tumor cells, T reg , and / or increase / induce cell killing / depletion of suppressive myeloid cells (optionally macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs)); (g) Macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) of tumor cells, T reg , and / or increase / induce cell killing / depletion of suppressor myeloid cells (optionally macrophages, neutrophils, and MDSC); (h) reducing immune suppression by myeloid cells (optionally macrophages, neutrophils, and MDSCs); (i) converting MDSCs and / or M2 macrophages into pro-inflammatory M1 macrophages; (j) T to effector T cells reg Convert; (k) converting a cold tumor into a hot tumor; (l) T reg reduce mediated immunosuppression; or (m) Having one or more of any one or more combinations of (a) to (k).

[0020] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, does not substantially bind to TNFR1, herpes virus entry mediator (HVEM), CD40, death receptor 6 (DR6), and / or osteoprotegerin (OPG). In some embodiments, the isolated antibody, or antigen-binding fragment thereof, is a TNFR2 antagonist. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, is a TNFR2 agonist. In some embodiments, the isolated antibody, or antigen-binding fragment thereof, is a bispecific or multispecific antibody.

[0021] Certain embodiments include an isolated polynucleotide encoding an isolated antibody, or antigen-binding fragment thereof, an expression vector comprising the isolated polynucleotide, or an isolated host cell comprising the vector, as described herein.

[0022] Also included are compositions comprising a physiologically acceptable carrier and a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof described herein.

[0023] Also included are methods of treating a patient having cancer, eg, a cancer associated with aberrant TNFR2 expression, comprising administering to the patient a composition described herein, thereby treating the cancer.

[0024] Also included are methods of treating a patient having cancer, e.g., a cancer associated with TNFR2 antagonist-mediated immunosuppression, comprising administering to the patient a composition described herein, thereby treating the cancer. In some embodiments, the antibody, or antigen-binding fragment thereof, is a TNFR2 antagonist.

[0025] Also included is a method of treating a patient with inflammatory disease and / or autoimmune disease, comprising administering to the patient a composition as described herein, thereby treating inflammation.In some embodiments, the inflammatory disease and / or autoimmune disease is associated with abnormal TNFR2 expression, for example, the antibody, or antigen-binding fragment thereof, is a TNFR2 agonist.In some embodiments, the inflammatory disease and / or autoimmune disease is associated with TNFR2 agonist-mediated immune activation. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 illustrates the proposed mechanism for the activity of anti-TNFR2 antibodies in cancer immunotherapy. [Diagram 2] FIG. 2 illustrates the antibody immunization and screening scheme described herein. [Diagram 3]Figures 3A-3D show the results of the first set of human chimeric antibody characterization. In Figures 3A-3B, human (3A) or cynomolgus monkey (3B) TNFR2-His tagged proteins were coated onto 96-well ELISA plates at 1 μg / mL and incubated overnight at 4°C. Plates were washed and blocked using 1% BSA. Antibodies were added for 1 h at room temperature (RT), washed, and detected using anti-human HRP. Assays were developed for 3-5 min using TMB substrate. In Figure 3C, FACS binding was performed on CHO cells engineered to express human TNFR2. Briefly, antibodies were incubated with 200,000 cells in MACS buffer for 1 h, followed by washing and detection using anti-human IgG BV421 for 20 min. Samples were analyzed using a Cytoflex or MACSQuant flow cytometer. In Figure 3D, a ligand blocking ELISA was performed by coating plates with 1 μg / mL TNFR-Fc overnight at 4 °C. Plates were washed, blocked, and antibodies were incubated for 1 h at RT. Antibodies were washed, and 100 ng / mL TNF-a was added and incubated for 1 h at RT. TNF-α was detected after washing with mouse anti-human TNF-α and anti-mouse IgG HRP. The assay was developed using TMB substrate for 5-7 min. [Figure 4] Figures 4A-4D show the results of a second set of human chimeric antibody characterization, including antibody binding to soluble (4A) and cell-based human TNFR2 (4C), cynomolgus monkey TNFR2 (4B), and ELISA-based TNF-α blocking (4D), performed as described above in Figures 3A-3D. [Diagram 5]Figure 5 shows TNFR signaling by human chimeric antibodies on the NFkB HEK reporter line. Promega HEK TNFR reporter cells were seeded at 50,000 cells per well in flat bottom plates. 10 μg / mL indicated antibody or 0.2 ng / mL TNF-α was added and cells were incubated at 37°C for 20 hours. Reporter activity was detected using QuantiBlue reagent at a 4:1 ratio with the supernatant for 10 minutes. Plates were read on a SpectroMax at 655 nm. The data show that 55F6 has some agonist activity, although the signal level was low, while the other antibodies have no significant activity. [Figure 6-1] 6A-6D show humanized antibody binding to soluble (6A) and cell-based human TNFR2 (6C), cynomolgus monkey TNFR2 (6B), and ELISA-based TNF-α blocking (6D). [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. [Figure 7] Figure 7 shows a cell-based TNFα blocking assay with humanized candidates. CHO cells overexpressing TNFR2 were seeded at 100,000 cells per well. Cells were incubated with the indicated antibodies for 30 min on ice. Cells were washed and 14 ng / mL biotinylated TNFα was added for 30 min. TNFα was washed and detected with SA-PE for 15 min. Cells were analyzed using a Cytoflex or MACSQuant flow cytometer. Assays were repeated twice. [Figure 8] Figure 8 shows a soluble TNFR1 binding assay. TNFR1-His tagged protein was coated onto ELISA plates at 1 μg / mL overnight at 4°C. Plates were washed, blocked and antibodies were added at the indicated concentrations for 1 hour at room temperature. Antibodies were washed and detected with anti-human IgG HRP for 1 hour. The assay was developed using TMB substrate for 10 minutes. [Figure 9]Figures 9A-9B show ADCC of TNFR2-CHO cells by reporter assay. Promega's ADCC Reporter Bioassay Core kit was used according to the manufacturer's protocol. Briefly, 25,000 TNFR2-CHO cells were added to a flat-well plate in assay buffer. Antibodies were added at the indicated concentrations. 75,000 effector cells were added per well (E:T=3:1). Plates were incubated at 37°C for 6 hours. After 6 hours, plates were equilibrated to room temperature and reporter activity was detected using Bio-Glo luciferase substrate measured after 5 minutes on a SpectroMAX plate reader. Figure 9A shows fold change relative to isotype, calculated as RLU (inducible bkgd) / RLU (isotype control bkgd). Figure 9B shows RLU rather than fold change. [Figure 10-1] Figures 10A-10F show high affinity monovalent binding of test antibodies to TNFR2 protein by Octet [humanized clones 25-71 (A; Kon=3.58E+05 1 / Ms; Koff=5.53E-04 1 / s) and 25-108 (B; Kon=3.76E+05 1 / Ms; Koff=2.33E-04 1 / s)] and human TNFR2 protein by ELISA (10C), as well as binding to cynomolgus monkey TNFR2 protein by ELISA (10D), cell-based human TNFR2 (10E), activated human Tregs (10F). [Figure 10-2] Same as above. [Figure 11-1] Figures 11A-11E show that 25-71 and 25-108 are specific for TNFR2, as shown by the lack of binding to TNFR1 (11A), herpes virus entry mediator (HVEM; 11B), CD40 (11C), death receptor 6 (DR6; 11D), and osteoprotegerin (OPG; 11E). [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 12]12A-12B show cell killing / depletion of TNFR2-expressing cells (12A, transfectants) and TNFR2-expressing tumor cells (12B, K562, human AML cell line) by 25-71 and 25-108 via antibody-dependent cellular cytotoxicity (ADCC). [Figure 13] 13A-13B show cell killing / depletion of TNFR2-expressing human Tregs by test antibodies via ADCC. [Figure 14] 14A-14B show cell killing of TNFR2-expressing tumor cells by 5-71 and 25-108 via macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). [Figure 15-1] Figures 15A-15C show that 25-71 and 25-108 can reverse myeloid-derived suppressor cell (MDSC)-mediated immune suppression from two different donors (15B and 15C). Percent suppression = T alone - [(T + MDSC) / T alone] x 100. [Figure 15-2] Same as above. [Figure 16] FIG. 16 shows the epitope site between antibody clone 25-71 and a region of full-length human TNFR2 (SEQ ID NO:327), which epitope includes residues 43, 45, 49, 55, 56, 73, and 77 of full-length TNFR2. [Figure 17] Figures 17A-17J show the interaction between antibody clone 25-71 and human TNFR2. The TNFR2 PDB structure 3ALQ colors in grey over the epitope sites, which correspond to residues 43-45 (REY); residues 49-56 (TAQMCCSK; SEQ ID NO: 328); and residues 73-77 (TVCDS; SEQ ID NO: 329) of the full-length human TNFR2 sequence. Ribbon / surface representations of the front view (A), back view (B), side view 1 (C), side view 2 (D), and top view (E); and ribbon representations of the front view (F); back view (G), side view 1 (H), side view 2 (I), and top view (J) are shown. [Figure 18-1]Figures 18A-18E show that clone 25-71 reverses Treg suppression of effector T cells. In 18A, purified Tregs express TNFR2 when added to the suppression assay. In 18B-18C, data are plotted as % proliferation of T responder cells (B) or percent Treg suppression (C). Figures 18D-18E show exemplary proliferation histograms of the 1:2 Tresp:Treg ratio condition (D) and the IgG1 control (E). [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 19-1] 19A-19C show the antitumor efficacy of clone 25-71 (48% TGI) in female nude mice injected with Colo205 cells. 19A outlines the treatment protocol, 19B shows the effect of test agents on tumor volume, and 19C shows the effect on body weight. [Figure 19-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present disclosure relates to antibodies and antigen-binding fragments thereof that specifically bind to tumor necrosis factor receptor 2 (TNFR2), particularly antibodies with specific epitope specificity and functional properties. Some embodiments encompass specific humanized antibodies and fragments thereof that are capable of binding to TNFR2, blocking TNFR2 binding with its ligand, tumor necrosis factor-α (TNF-α), and inhibiting the induced downstream cell signaling and biological effects. In certain embodiments, the anti-TNFR2 antibodies, or antigen-binding fragments thereof, are TNFR2 antagonists or inhibitors. In some cases, TNFR2 antagonists enhance immune responses, for example, by blocking the immunosuppressive effects of TNFR2 in the tumor microenvironment. The TNFR2 antagonist antibodies described herein are useful, for example, in the treatment and prevention of cancers, including TNFR2-expressing cancers.

[0028] Some embodiments relate to the use of anti-TNFR2 antibodies, or antigen-binding fragments thereof, for the diagnosis, evaluation, and treatment of diseases and disorders associated with TNFR2 activity or its aberrant expression. The subject antibodies are used in the treatment or prevention of cancer, among other diseases.

[0029] The practice of the present disclosure will utilize, unless specifically indicated to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the skill of the art, many of which are described below for purposes of illustration. Such techniques are fully explained in the literature. See, for example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et al., Short Protocols in Molecular Biology, 3 rd ed.,Wiley & Sons,1995;Sambrook and Russell,Molecular Cloning:A Laboratory Manual(3rd Edition,2001);Maniatis et al.Molecular Cloning:A Laboratory See, for example, The Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0030] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0031] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by up to 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0032] "Antagonist" refers to an agent (e.g., an antibody) that interferes with or otherwise reduces the physiological action of another agent or molecule. In some cases, an antagonist specifically binds to the other agent or molecule. Full and partial antagonists are included.

[0033] "Agonist" refers to an agent (e.g., an antibody) that increases or enhances the physiological effect of another agent or molecule. In some cases, an agonist specifically binds to the other agent or molecule. Full and partial agonists are included.

[0034] Throughout this specification, unless the context requires otherwise, it will be understood that the word "comprise", or variations such as "comprises" or "comprising", imply the inclusion of a specified element or integer or group of elements or integers, but not the exclusion of other elements or integers or groups of elements or integers.

[0035] "Consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that other elements may not be present. "Consisting essentially of" means including any elements recited after the phrase and limited to other elements that do not interfere with or participate in the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but other elements may or may not be present depending on whether they substantially affect the activity or action of the recited elements.

[0036] Each embodiment herein is applied mutatis mutandis to all other embodiments unless expressly stated otherwise.

[0037] The terms "modulating" and "altering" include "increasing," "enhancing," or "stimulating," as well as "decreasing," "reducing," or "inhibiting," typically in a statistically significant or physiologically significant amount or degree compared to a control. An "increased," "stimulated," or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or more (e.g., 500, 1000 times) (including all integers and ranges between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) that is the amount produced by no composition (e.g., no agent present) or a control composition. A "decreased," "reduced," or "inhibited" amount is typically a "statistically significant" amount and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease (including all integers and ranges therebetween) of the amount produced by no composition (e.g., no agent present) or a control composition. Examples of comparisons and "statistically significant" amounts are described herein.

[0038] "Substantially" or "essentially" means nearly all or the entirety of a given quantity, e.g., 95%, 96%, 97%, 98%, 99% or more.

[0039] "Statistically significant" means that the result is unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include p-value, which is the frequency or probability that the observed phenomenon occurs if the null hypothesis is true. If the p-value obtained is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is defined as a p-value of 0.05 or less.

[0040] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures are well known in the art and may generally be performed according to conventional methods described in various general and more specific references cited and discussed throughout this specification. Unless specific definitions are provided, the nomenclature and laboratory procedures and techniques utilized in connection with molecular biology, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological synthesis, microbiological synthesis, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0041] In certain embodiments, the antibody, or antigen-binding fragment thereof, comprises a complementarity determining region V H CDR1, V H CDR2, and V H The heavy chain variable region (V H ) sequence, and complementarity determining region V L CDR1, V L CDR2, and V L The light chain variable region (V L ) sequence. H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, and V L The CDR3 sequences are provided in Table H1 below. [Table H1-1] [Table H1-2] [Table H1-3]

[0042] Thus, in certain embodiments, the antibody or antigen-binding fragment thereof binds to TNFR2 and Complementarity determining region V selected from Table H1 H CDR1, V H CDR2, and V H The heavy chain variable region (V H ) sequence; and Complementarity determining region V selected from Table H1 L CDR1, V L CDR2, and V L The light chain variable region (V L ) sequence, This includes at least one TNFR2 polypeptide or variant thereof that specifically binds to an epitope (eg, selected from Table T1). In certain embodiments, the CDR sequences are as follows: a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 1-3, respectively; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 4-6, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 7 to 9, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 10 to 12, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 13 to 15, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 16 to 18, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 19-21, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 22-24, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 25 to 27, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 28 to 30, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 31 to 33, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 34 to 36, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 37 to 39, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 40 to 42, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 43-45, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 46-48, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 49-51, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 52-54, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 55 to 57, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 58 to 60, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 61-63, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 64-66, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 67-69, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 70-72, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 73-75, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 76-78, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 79-81, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 82-84, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 85 to 87, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 88 to 90, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 91-93, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 94-96, respectively; or A VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 97 to 99, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 100 to 102, respectively. Variants thereof, including affinity matured variants that bind to TNFR2, e.g., CDR regions, e.g., one or more V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, and / or V L Also included are variants having 1, 2, 3, 4, 5, 6, 7, or 8 total alterations across the CDR3 sequence. Exemplary "alterations" include amino acid substitutions, additions, and deletions.

[0043] In certain embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (V H ) sequence, and the light chain variable region (V L ) sequence. H and V L Sequences are provided in Table H2 below, with an exemplary rabbit V H The sequences are provided in Table R1 below. H CDR1, V H CDR2, and V H CDR3 regions are underlined), an exemplary rabbit V L The sequences are provided in Table R2 below. L CDR1, V L CDR2, and V L The CDR3 region is underlined). [Table H2-1] [Table H2-2] [Table H2-3] [Table R1-1] [Table R1-2] [Table R1-3] [Table R1-4] [Table R1-5]

Table R1-6

Table R1-7

Table R2-1

Table R2-2

Table R2-3

Table R2-4

Table R2-5

Table R2-6

Table R2-7

[0044] Thus, in certain embodiments, the antibody, or antigen-binding fragment thereof, binds TNFR2 and comprises a VH and a corresponding VL region selected from Table H2. In certain embodiments, the VH region comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135. In some embodiments, the VL region comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 134, and 136. In certain embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH region set forth in SEQ ID NO: 103, and a VL region set forth in SEQ ID NO: 104; a VH region set forth in SEQ ID NO: 105, and a VL region set forth in SEQ ID NO: 106; a VH region set forth in SEQ ID NO: 107, and a VL region set forth in SEQ ID NO: 108; a VH region set forth in SEQ ID NO: 109, and a VL region set forth in SEQ ID NO: 110; a VH region set forth in SEQ ID NO:111, and a VL region set forth in SEQ ID NO:112; a VH region set forth in SEQ ID NO: 113, and a VL region set forth in SEQ ID NO: 114; a VH region set forth in SEQ ID NO: 115, and a VL region set forth in SEQ ID NO: 116; a VH region set forth in SEQ ID NO: 117, and a VL region set forth in SEQ ID NO: 118; a VH region set forth in SEQ ID NO: 119, and a VL region set forth in SEQ ID NO: 120; a VH region set forth in SEQ ID NO: 121, and a VL region set forth in SEQ ID NO: 122; a VH region set forth in SEQ ID NO: 123, and a VL region set forth in SEQ ID NO: 124; a VH region set forth in SEQ ID NO: 125, and a VL region set forth in SEQ ID NO: 126; a VH region set forth in SEQ ID NO: 127, and a VL region set forth in SEQ ID NO: 128; a VH region set forth in SEQ ID NO: 129, and a VL region set forth in SEQ ID NO: 130; a VH region set forth in SEQ ID NO: 131, and a VL region set forth in SEQ ID NO: 132; a VH region set forth in SEQ ID NO: 133, and a VL region set forth in SEQ ID NO: 134; or It comprises a VH region set forth in SEQ ID NO:135, and a VL region set forth in SEQ ID NO:136.

[0045] In some embodiments, the antibody, or antigen-binding fragment thereof, binds tumor necrosis factor receptor 2 (TNFR2) and comprises a heavy chain variable (VH) region comprising VHCDR1, VHCDR2, and VHCDR3 regions selected from the underlined sequences in Table R1; and a corresponding (by clone name) light chain variable (VL) region comprising VLCDR1, VLCDR2, and VLCDR3 regions selected from the underlined sequences in Table R2. In some embodiments, the VH region comprises an amino acid sequence selected from Table R1, and the VL region comprises a corresponding (by clone name) amino acid sequence selected from Table R2.

[0046] In some embodiments, as described above, the antibody or its antigen-binding fragment binds to tumor necrosis factor receptor 2 (TNFR2), for example, soluble or cell-expressed TNFR2. In certain embodiments, the TNFR2 is human TNFR2, or a peptide epitope thereof. Exemplary peptide epitopes of human TNFR2 are provided in Table T1 below. [Table T1]

[0047] In certain embodiments, the antibody, or antigen-binding fragment thereof, specifically binds to human TNFR2, e.g., at least one human TNFR2 peptide epitope selected from Table T1, e.g., at least one, two, three, four, or five peptide epitopes selected from Table T1. In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds to human TNFR2 at a peptide epitope comprising, consisting of, or consisting essentially of one or more residues selected from R21, Y23, T27, S33, K34, T51, and S55, as defined by the mature human TNFR2 sequence (residues 23-461 of FL human TNFR2). In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds to human TNFR2 at a peptide epitope comprising, consisting of, or consisting essentially of one or more residues selected from REY, TAQMCCSK (SEQ ID NO: 328), and TVCDS (SEQ ID NO: 329). In certain ... about 2 nM or less. D , or a K of about 0.7 nM or less D In some embodiments, the antibody, or antigen-binding fragment thereof, binds to cynomolgus monkey TNFR2, e.g., it binds cross-reactively to human TNFR2 and cynomolgus monkey TNFR2.

[0048] In some embodiments, the antibody, or antigen-binding fragment thereof, is a TNFR2 antagonist. For example, in some embodiments, the antibody, or antigen-binding fragment thereof, inhibits or otherwise reduces TNF-α binding to TNFR2. In some embodiments, the antibody, or antigen-binding fragment thereof, inhibits or otherwise reduces TNFR2 multimerization or trimerization. In some embodiments, the antibody, or antigen-binding fragment thereof, inhibits or otherwise reduces TNFR2-mediated activation of T regulatory cells (Tregs), for example, systemically or in the tumor microenvironment. In certain embodiments, the antibody, or antigen-binding fragment thereof, binds to TNFR2, is a TNFR2 antagonist, and does not substantially bind to tumor necrosis factor receptor 1 (TNFR1), e.g., human TNFR1. In some embodiments, the antibody, or antigen-binding fragment thereof, does not substantially bind to herpes virus entry mediator (HVEM), CD40, death receptor 6 (DR6), and / or osteoprotegerin (OPG).

[0049] In some embodiments, for example, in vitro or in vivo, an anti-TNFR2 antibody, or antigen-binding fragment thereof, inhibits the proliferation and proliferation of tumor cells (e.g., TNFR2-expressing tumor cells) by antibody-dependent cellular cytotoxicity (ADCC). reg , and / or increase cell killing / depletion of myeloid-derived suppressor cells (MDSCs), e.g., by about or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000%, as compared to a control or reference. In some embodiments, the anti-TNFR2 antibody, or antigen-binding fragment thereof, is capable of targeting tumor cells (e.g., TNFR2-expressing tumor cells) by macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), T reg, and / or increase cell killing / depletion of MDSCs by, for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more, as compared to a control or reference. In some embodiments, the anti-TNFR2 antibody, or antigen-binding fragment thereof, reduces MDSC-mediated immune suppression by about or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000%, e.g., compared to a control or reference. In some embodiments, the anti-TNFR2 antibody, or antigen-binding fragment thereof, converts MDSCs and / or M2 macrophages to pro-inflammatory M1 macrophages, and / or T cells, e.g., by increasing said conversion by about or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to a control or reference. reginto effector T cells. In some embodiments, the anti-TNFR2 antibody, or antigen-binding fragment thereof, converts a "cold" tumor into a "hot" tumor. A "cold" tumor is one that is not recognized or does not elicit a strong response by the immune system. For example, the microenvironment of a cold tumor typically has low or insignificant levels of CD4+ or CD8+ T cells, and instead often has relatively high levels of myeloid-derived suppressor cells and / or Tregs that secrete immunosuppressive cytokines to prevent migration of CD4+ or CD8+ T cells into the tumor microenvironment. In contrast, a "hot" tumor is one that has high levels of CD4+ or CD8+ T cells, thereby resulting in an inflamed tumor microenvironment. In certain embodiments, the anti-TNFR2 antibody, or antigen-binding fragment thereof, has any one or more combinations of the aforementioned characteristics.

[0050] For illustrative purposes only, binding interactions between an antibody, or antigen-binding fragment thereof, and a TNFR2 polypeptide can be detected and quantified using a variety of routine methods, including Biacore assays (e.g., using appropriately tagged soluble reagents bound to a sensor chip), FACS analysis using cells expressing the TNFR2 polypeptide on their cell surface (either natural or recombinant), immunoassays, fluorescent staining assays, ELISA assays, and microcalorimetric approaches such as ITC (Isothermal Titration Calorimetry).

[0051] As is well known in the art, an antibody is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one epitope recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal or monoclonal antibodies, as well as fragments thereof (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chain (scFv), synthetic variants thereof, naturally occurring variants, fusion proteins with antigen-binding fragments of the required specificity, including antibody portions, humanized antibodies, chimeric antibodies, and any other modified configurations of immunoglobulin molecules that contain an antigen-binding site or fragment (epitope recognition site) of the required specificity. "Bispecific antibodies", multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) are also specific forms of antibodies contemplated herein. Minibodies comprising scFvs linked to CH3 domains are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). For example, Ward,ESet al.,Nature 341,544-546 (1989);Bird et al.,Science,242,423-426,1988;Huston et al.,PNAS USA,85,5879-5883,1988);PCT / US92 / 09965;WO94 / 13804;P.Holliger et al. al.,Proc.Natl.Acad.Sci.USA 90 6444-6448,1993;Y.Reiter et al.,Nature Biotech,14,1239-1245,1996;S.Hu See, et al., Cancer Res., 56, 3055-3061, 1996.

[0052] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and / or light chain that binds to an antigen of interest, particularly TNFR2. In this regard, the antigen-binding fragment of an antibody described herein can contain one, two, three, four, five, or all six CDRs of the VH and VL sequences described herein from an antibody that binds to TNFR2.

[0053] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent such as an antibody and which can be used in an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes.

[0054] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural features, and / or specific charge characteristics. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. An antibody is said to specifically bind an antigen when it has an equilibrium dissociation constant of ≦10 -7 or 10 -8 M. In some embodiments, the equilibrium dissociation constant is ≦10 -9 M or ≦10 -10 It can be M.

[0055] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein each comprise a set of heavy and light chain CDRs interposed between a set of heavy and light chain framework regions (FRs), which provide support for the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of the heavy or light chain V region. Proceeding from the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2", and "CDR3", respectively. Thus, an antigen-binding site comprises six CDRs, including the CDRs set from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit". Crystallography of numerous antigen-antibody complexes has shown that the amino acid residues of the CDRs make extensive contacts with the bound antigen, with the most extensive antigen contacts being with the heavy chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0056] As used herein, the term "FR set" refers to four adjacent amino acid sequences that frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen, but are primarily responsible for folding the V region into the antigen binding site, specifically the FR residues immediately adjacent to the CDRs. Within the FR, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form an antigen binding surface. It is generally recognized that, regardless of the exact CDR amino acid sequence, there are conserved structural regions of the FR that affect the shape of the folded CDR loop into a certain "canonical" structure. In addition, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction of antibody heavy and light chains.

[0057] The structure and location of immunoglobulin variable domains may be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updated versions thereof, currently available on the Internet (immuno.bme.nwu.edu).

[0058] "Monoclonal antibody" refers to a homogenous antibody population, which consists of amino acids (naturally occurring and non-naturally occurring) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed to a single epitope. The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies, as well as fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv), variants thereof, fusion proteins containing antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) of the required specificity and ability to bind to the epitope. It is not intended to be limited with respect to the source of the antibody or the method of making the antibody (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments, etc., described herein under the definition of "antibody".

[0059] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer with an intact antigen binding site. The enzyme pepsin can cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which contains both antigen binding sites. Fv fragments for use according to certain embodiments of the present disclosure may be produced by preferential proteolytic cleavage of IgM, and rarely IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments are non-covalently linked V fragments containing an antigen binding site that retain most of the antigen recognition and binding ability of the native antibody molecule. H ::V L Heterodimers are included. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich See, e.g., et al. (1980) Biochem 19:4091-4096.

[0060] In certain embodiments, single chain Fv or scFv antibodies are contemplated, such as kappabodies (Ill et al., Prot. Eng. 10:949-57 (1997); minibodies (Martin et al., EMBO J 13:5305-9 (1994); diabodies (Holliger et al., PNAS 90:6444-8 (1993); or janusins ​​(Traunecker et al., EMBO J 10:3655-59 (1991) and Traunecker et al., Int. J. Cancer 1999). Suppl. 7:51-52 (1992) can be prepared using standard molecular biology techniques following the teachings of the present application for the selection of antibodies with desired specificity. In some embodiments, bispecific or chimeric antibodies can be produced that include the ligands of the present disclosure. For example, chimeric antibodies can include CDRs and framework regions from different antibodies, while bispecific antibodies can be produced that specifically bind to TNFR2 through one binding domain and to a second molecule through a second binding domain. These antibodies can be produced through recombinant molecular biology techniques or can be physically conjugated together.

[0061] Single-chain Fv (scFv) polypeptides are composed of V H and V L A covalently linked V expressed from a gene fusion containing a gene encoding H ::V L It is a heterodimer. See Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Numerous methods have been described to find chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to that of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Patent No. 4,946,778 to Ladner et al.

[0062] Certain embodiments include "probodies," or antibodies whose binding sites are masked or otherwise inactive until activated by proteolytic cleavage in target or disease tissue. Some of these and related embodiments include one or more masking moieties that sterically hinder the antigen binding site of the antibody and are fused to the antibody via one or more proteolytically cleavable linkers (see, e.g., Polu and Lowman, Expert Opin. Biol. Ther. 14:1049-1053, 2014).

[0063] In certain embodiments, the TNFR2 binding antibody described herein is in the form of a bispecific antibody.A bispecific antibody is a multimer of polypeptides, each polypeptide comprises a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site, the antigen-binding site being formed by the association of the first domain of one polypeptide in the multimer with the second domain of another polypeptide in the multimer (WO 94 / 13804).

[0064] A dAb fragment of an antibody consists of the VH domain (Ward, ES et al., Nature 341, 544-546 (1989)).

[0065] If bispecific or multispecific antibodies are to be used, these may be conventional bispecific antibodies, which may be produced in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), e.g., prepared chemically or from hybrid hybridomas, or any of the bispecific antibody fragments mentioned above. Bispecific antibodies and scFvs can be constructed without an Fc region, using only variable domains, thereby potentially reducing the effects of anti-idiotypic reaction.

[0066] Bispecific antibodies, as opposed to whole bispecific antibodies, may be particularly useful because they can be easily constructed and expressed in E. coli. Bispecific antibodies (and many other polypeptides such as antibody fragments) of appropriate binding specificity can be easily selected from libraries using phage display (WO 94 / 13804). If one arm of the bispecific antibody is to be kept constant, for example with specificity directed against antigen X, then a library can be created in which the other arm is varied and antibodies of appropriate specificity can be selected. Whole bispecific antibodies may be produced by knobs-into-hole engineering (JBB Rodgeway et al., Protein Eng., 9, 616-621, 1996).

[0067] In certain embodiments, the antibodies described herein may be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US Publication No. 20090226421). This unique antibody technology creates a stable, small antibody format with a predicted longer therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and therefore do not interact with the immune system. Fully human IgG4 antibodies may be modified by removing the hinge region of the antibody to obtain half-molecule fragments with distinct stability characteristics compared to the corresponding intact IgG4 (GenMab, Utrecht). Halving the IgG4 molecule leaves only one region on the UniBody® that can bind to the cognate antigen (e.g., disease target), and thus the UniBody® binds monovalently to only one site on the target cell. For certain cancer cell surface antigens, this monovalent binding may not stimulate cancer cells to grow as seen using bivalent antibodies with the same antigen specificity, and therefore UniBody® technology may provide a treatment option for some types of cancer that may be resistant to treatment with traditional antibodies. The small size of UniBody® may be of great benefit when treating some forms of cancer, allowing for better distribution of the molecule across larger solid tumors, potentially increasing efficacy.

[0068] In certain embodiments, the antibodies of the present disclosure may take the form of Nanobodies®. Nanobodies® are encoded by a single gene and are efficiently produced in almost all prokaryotic as well as eukaryotic hosts, such as E. coli (see, e.g., U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma) and yeasts (e.g., Saccharomyces, Kluyveromyces, Hansenula or Pichia (see, e.g., U.S. Pat. No. 6,838,254). The production process is scalable and multi-kilograms of Nanobodies® have been produced. Nanobodies may be formulated as a ready-to-use solution with a long shelf life. The Nanoclone® method (see, e.g., WO 06 / 079372) is a unique method for generating Nanobodies against desired targets, based on automated high-throughput selection of B cells.

[0069] In certain embodiments, the anti-TNFR2 antibody or antigen-binding fragment thereof disclosed herein is humanized. This refers to a chimeric molecule, generally prepared using recombinant techniques, based on the structure and / or sequence of a human immunoglobulin, with an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule. The antigen-binding site may comprise either a complete variable domain fused onto a constant domain, or only the CDRs grafted into the appropriate framework regions of the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but leaves open the possibility of an immune response to the foreign variable region (LoBuglio, AF et al., (1989) Proc Natl Acad Sci USA 86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327). Exemplary methods of anti-TNFR2 antibody humanization disclosed herein include those described in U.S. Pat. No. 7,462,697. Exemplary humanized antibodies according to certain embodiments include the humanized sequences provided in Table H1 and Table H2.

[0070] Another approach focuses not only on providing constant regions of human origin, but also on modifying the variable regions to reshape them as closely as possible to the human form. It is known that the variable regions of both heavy and light chains contain three complementarity determining regions (CDRs) that change in response to an epitope of interest and determine the binding ability, and are flanked by four framework regions (FRs) that are relatively conserved in a given species and putatively provide a scaffold for the CDRs. When a non-human antibody is prepared for a specific epitope, the variable region can be "reshaped" or "humanized" by grafting the CDRs, which are derived from the non-human antibody, onto the FRs present in the human antibody to be modified. The application of this approach to various antibodies has been reported by Sato, K., et al., (1993) Cancer Res 53:851-856; Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al., (1999) Nature 332:323-327; al.,(1988) Science 239:1534-1536;Kettleborough,CA,et al.,(1991)Protein Engineering 4:773-3783;Maeda,H.,et al.,(1991)Human Antibodies Hybridoma 2:124-134;Gorman,SD,et al.,(1991)Proc Natl Acad Sci USA 88:4181-4185;Tempest,PR,et al.,(1991)Bio / Technology 9:266-271;Co,MS,et al.,(1991)Proc Natl Acad Sci USA 88:2869-2873;Carter,P.,et al.,(1992)Proc Natl Acad Sci USA 89:4285-4289; and Co, MS et al., (1992) J Immunol 148:1149-1154. In some embodiments, a humanized antibody preserves all CDR sequences (e.g., a humanized rabbit antibody that contains all six CDRs from a rabbit antibody). In some embodiments, a humanized antibody has one or more CDRs (one, two, three, four, five, six) that are modified with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0071] In certain embodiments, the antibody of the present disclosure may be a chimeric antibody. In this regard, a chimeric antibody consists of an antigen-binding fragment of an anti-TNFR2 antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In some embodiments, the heterologous Fc domain may be derived from a different Ig class than the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may consist of CH2 and CH3 domains from one or more of the different Ig classes. As described above with respect to humanized antibodies, the anti-TNFR2 antigen-binding fragment of a humanized antibody may include only one or more of the CDRs of the antibodies described herein (e.g., one, two, three, four, five, or six CDRs of the antibodies described herein) or may include the entire variable domain (VL, VH, or both).

[0072] In certain embodiments, the TNFR2 binding antibody comprises one or more of the CDRs of the antibodies described herein. In this regard, it has been shown in some cases that transfer of only the VHCDR3 of an antibody can be performed while still retaining the desired specific binding (Barbas et al., PNAS (1995) 92: 2529-2533). See also McLane et al., PNAS (1995) 92: 5214-5218, Barbas et al., J. Am. Chem. Soc. (1994) 116: 2161-2162.

[0073] Marks et al. (Bio / Technology, 1992, 10:779-783) describe a method for generating a repertoire of antibody variable domains using a consensus primer directed to or adjacent to the 5' end of the variable domain span in conjunction with a consensus primer for the third framework region of the human VH gene, resulting in a repertoire of VH variable domains lacking CDR3. Marks et al. further describe how this repertoire is combined with the CDR3 of a particular antibody. Using similar techniques, the CDR3-derived sequences of the presently described antibodies may be shuffled with a repertoire of VH or VL domains lacking CDR3, and the shuffled complete VH or VL domains may be combined with the cognate VL or VH domain to provide an antibody or antigen-binding fragment thereof that binds to TNFR2. The repertoire may then be displayed in a suitable host system, such as the phage display system of WO 92 / 01047, so that suitable antibodies or antigen-binding fragments thereof may be selected. The repertoire is at least about 10 4 may consist of individual members of, for example, about 10 6 ~10 8 or 10 10 The number of members may range from several orders of magnitude upwards, such as more than one member. Similar mixed or combinatorial techniques have also been disclosed by Stemmer (Nature, 1994, 370:389-391), who describes the technique in relation to β-lactamase genes, but observes that the approach can be used for the generation of antibodies.

[0074] A further alternative is to use random mutagenesis of one or more selected VH and / or VL genes to generate new VH or VL regions carrying one or more CDR-derived sequences as described herein, generating mutations throughout the variable domain. Such a technique is described by Gram et al (1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who uses error-prone PCR. Another method that can be used is to direct mutagenesis to the CDR regions of the VH or VL genes. Such a technique is disclosed by Barbas et al (1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al (1996, J. Mol. Biol. 263:551-567).

[0075] In certain embodiments, a particular VH and / or VL of an antibody described herein may be used to screen a library of complementary variable domains to identify an antibody with desirable properties, such as increased affinity for TNFR2. Such methods are described, for example, in Portolano et al., J. Immunol. (1993) 150:880-887; Clarkson et al., Nature (1991) 352:624-628.

[0076] Other methods may also be used to mix and match CDRs to identify antibodies with desired binding activity, such as binding to TNFR2. For example, Klimka et al., British Journal of Cancer (2000) 83:252-260, describe a screening process using mouse VL and human VH libraries with CDR3 and FR4 retained from mouse VH. After obtaining antibodies, the VHs were screened against a human VL library to obtain antibodies that bound to the antigen. Beiboer et al., J. Mol. Biol. (2000) 296:833-849, describe a screening process using an entire mouse heavy chain and a human light chain library. After obtaining antibodies, one VL was combined with a human VH library that retained mouse CDR3. Antibodies capable of binding the antigen were obtained. Rader et al., PNAS (1998) 95:8910-8915, describe a process similar to that of Beiboer et al., supra.

[0077] These just described techniques are as such known in the art, however, one of ordinary skill in the art, using routine methods in the art, can use such techniques to obtain antibodies or antigen-binding fragments thereof according to some embodiments described herein.

[0078] Also disclosed herein is a method for obtaining an antibody antigen-binding domain specific for the TNFR2 antigen, the method comprising providing a VH domain that is an amino acid sequence variant of the VH domain, for the purpose of adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence of the VH domain described herein, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH / VL combination or combinations to identify a particular binding member or antibody antigen-binding domain specific for TNFR2, optionally having one or more desired properties. The VL domain may have an amino acid sequence substantially as described herein. Similar methods may be employed in which one or more sequence variants of the VL domain disclosed herein are combined with one or more VH domains.

[0079] An epitope that "specifically binds" or "preferentially binds" (used interchangeably herein) to an antibody or polypeptide is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, faster, longer, and / or with higher affinity with a particular cell or substance than it does with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or for longer than it binds to other substances. For example, an antibody that specifically or preferentially binds to a TNFR2 epitope is an antibody that binds to one TNFR2 epitope with higher affinity, avidity, more readily, and / or for longer than it binds to other TNFR2 epitopes or non-TNFR2 epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding. Generally, but not necessarily, reference to binding implies preferential binding.

[0080] Immunological binding generally refers to non-covalent interactions of the type that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin molecule is specific, for example, as a result of, for example, by way of illustration and not limitation, electrostatic, ionic, hydrophilic and / or hydrophobic attractions or forces, steric forces, hydrogen bonding, van der Waals forces, and other interactions. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ), and K dA smaller K represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on-rate constant" (K on ) and the "off rate constant" (K off ) can be determined by calculation of the concentration and the actual rates of association and dissociation. off / K on The ratio of the affinity and dissociation constant K d See generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

[0081] The term "immunologically active" with respect to an epitope that is immunologically active or "remains immunologically active" refers to the ability of an anti-TNFR2 antibody to bind to the epitope under different conditions, for example, after the epitope has been subjected to reducing and denaturing conditions.

[0082] The antibody or antigen-binding fragment thereof according to some embodiments may (i) specifically bind to the antigen, and (ii) compete for binding to TNFR2 with any of the antibodies described herein that contain the VH and / or VL domains disclosed herein, or contain the VH CDR3 disclosed herein, or any variant thereof. Competition between antibodies can be easily assayed in vitro, for example, using ELISA and / or by tagging one antibody with a specific reporter molecule that can be detected in the presence of other untagged antibodies, allowing the identification of specific antibodies that bind to the same or overlapping epitopes. Thus, provided herein are specific antibodies or antigen-binding fragments thereof that contain a human antibody antigen-binding site that competes with the antibodies described herein that bind to TNFR2.

[0083] In this regard, as used herein, the terms "compete," "inhibit binding," and "block binding" (e.g., with reference to inhibiting / blocking binding of a ligand (e.g., TNF-α) and / or counter-receptor to TNFR2, or with reference to inhibiting / blocking binding of an anti-TNFR2 antibody to TNFR2) are used interchangeably and encompass both partial and complete inhibition / blocking. Inhibition / blocking of a ligand and / or counter-receptor to TNFR2 preferably reduces or alters the normal level or type of cell signaling that occurs when a ligand and / or counter-receptor binds to TNFR2 without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in binding of a ligand and / or counter-receptor to TNFR2 when contacted with an anti-TNFR2 antibody disclosed herein, e.g., blocking of a ligand (e.g., TNF-α) and / or counter-receptor to TNFR2 by at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, as compared to a ligand not contacted with the anti-TNFR2 antibody.

[0084] The constant regions of immunoglobulins are less diverse in sequence than the variable regions and are responsible for binding a large number of natural proteins to initiate important biochemical events. In humans, there are five different classes of antibodies, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The distinguishing characteristic between these antibody classes is their constant regions, although subtle differences may exist in the V regions.

[0085] The Fc region of an antibody interacts with a number of Fc receptors and ligands, conferring a series of important functions, called effector functions. In one embodiment, an anti-TNFR2 antibody comprises an Fc region. For IgG, the Fc region comprises Ig domains CH2 and CH3, and an N-terminal hinge that connects to CH2. An important family of Fc receptors in the IgG class is the Fc gamma receptor (FcγR). These receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans, this protein family includes FcγRI (CD64), which contains the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which contains the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which contains the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65). These receptors typically have an extracellular domain that mediates binding to Fc, a transmembrane region, and an intracellular domain that may mediate some signaling event within the cell. These receptors are expressed on a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells. Formation of the Fc / FcγR complex recruits these effector cells to the site of bound antigen, which typically results in intracellular signaling events and important subsequent immune responses, such as release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack.

[0086] The ability to mediate cytotoxic and phagocytic effector functions is a mechanism by which antibodies may destroy target cells. The cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibodies on target cells and subsequently cause lysis of the target cells is called antibody-dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). The cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibodies on target cells and subsequently cause phagocytosis of the target cells is called antibody-dependent cell-mediated phagocytosis (ADCP). All FcγRs bind to the same region on Fc, N-terminal to the Cg2 (CH2) domain and the preceding hinge. This interaction has been well characterized structurally (Sondermann et al., 2001, J Mol Biol 309:737-749), and several structures of human Fc bound to the extracellular domain of human FcγRIIIb have been solved (pdb accession code 1E4K) (Sondermann et al., 2000, Nature 406:267-273.) (pdb accession codes 1IIS and 1IIX) (Radaev et al., 2001, J Biol Chem 276:16469-16477).

[0087] Different IgG subclasses have different affinities for FcγR with IgG1 and IgG3 typically binding to the receptor substantially better than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65). All FcγRs bind to the same region on the IgG Fc, yet with different affinities, with the higher affinity binder FcγRI binding to IgG1 at 10 -8 M -1 K dwhereas the low affinity receptors FcγRII and FcγRIII generally have 10 -6 and 10 -5 The extracellular domains of FcγRIIIa and FcγRIIIb are 96%, but FcγRIIIb does not have an intracellular signaling domain. Furthermore, FcγRI, FcγRIIa / c, and FcγRIIIa are positive regulators of immune complex-induced activation, characterized by having an intracellular domain with an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb has an immunoreceptor tyrosine-based inhibitory motif (ITIM) and is therefore inhibitory. Thus, the former are referred to as activating receptors, and FcγRIIb as inhibitory receptors. The receptors also differ in their expression patterns and levels on different immune cells. Yet another level of complexity is the presence of numerous FcγR polymorphisms in the human proteome. A particularly relevant polymorphism with clinical significance is the V158 / F158 FcγRIIIa. Human IgG1 binds with higher affinity to the V158 allotype than to the FV158 allotype. This difference in affinity, and perhaps its effect on ADCC and / or ADCP, has been shown to be a significant determinant of the efficacy of the anti-CD20 antibody rituximab (Rituxan®, a registered trademark of IDEC Pharmaceuticals Corporation). Patients with the V158 allotype respond favorably to rituximab treatment, whereas patients with the lower affinity F158 allotype respond poorly (Cartron et al., 2002, Blood 99:754-758). Approximately 10-20% of humans are V158 / V158 homozygous, 45% are V158 / F158 heterozygous, and 35-45% of humans are F158 / F158 homozygous (Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758). Thus, 80-90% of humans are poor responders, i.e., they have at least one allele of F158 FcγRIIIa.

[0088] Fc region also participates in the activation of complement cascade. In classical complement pathway, C1 binds with its C1q subunit to the Fc fragment of IgG or IgM that is complexed with antigen. In certain embodiments, the modification to Fc region includes a modification that changes (either enhances or decreases) the ability of the TNFR2-specific antibody described herein to activate the complement system (see, for example, U.S. Patent No. 7,740,847). To evaluate complement activation, a complement-dependent cytotoxicity (CDC) assay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol.Methods, 202:163 (1996)).

[0089] Thus, in certain embodiments, the present disclosure provides anti-TNFR2 antibodies having modified Fc regions with altered functional properties, such as reduced or enhanced CDC, ADCC, or ADCP activity, or enhanced binding affinity for a specific FcγR, or increased serum half-life. Other modified Fc regions contemplated herein are described, for example, in issued U.S. Patent Nos. 7,317,091; 7,657,380; 7,662,925; 6,538,124; 6,528,624; 7,297,775; 7,364,731; U.S. Publication Nos. 2009092599; 20080131435; 20080138344; and WO 2006 / 105338; 2004 / 063351; 2006 / 088494; 2007 / 024249.

[0090] Thus, in certain embodiments, antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences. In certain embodiments, the fusion comprises at least part of the hinge, C H 2, and C Hand an Ig heavy chain constant domain, which comprises three regions. In some cases, a first heavy chain constant region (C) containing the site necessary for light chain binding, present in at least one of the fusions, is H 1) is preferred. The DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host cell. This allows for greater flexibility to adjust the mutual proportions of the three polypeptide fragments in embodiments when an unequal ratio of the three polypeptide chains used in the construct results in the optimal yield of the desired bispecific antibody. However, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when expression of at least two polypeptide chains in equal ratio results in high yields or when the ratio has no significant effect on the yield of the desired chain combination.

[0091] The antibody (and its antigen-binding fragment and variant) of the present disclosure may also be modified to include epitope tags or labels, for example, for use in purification or diagnostic applications.There are many linking groups known in the art for making antibody conjugates, including those disclosed in U.S. Patent No. 5,208,020 or European Patent No. 0 425 235 B1, and Chari et al., Cancer Research 52:127-131 (1992).Linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups as disclosed in the patents identified above, with disulfide groups and thioether groups being preferred.

[0092] In some embodiments, the TNFR2 specific antibody described herein may be conjugated or operatively linked to another drug or therapeutic compound, referred to herein as conjugate.The drug or therapeutic compound may be a polypeptide drug, a polynucleotide drug, a cytotoxic drug, a chemotherapeutic drug, a cytokine, an antiangiogenic drug, a tyrosine kinase inhibitor, a toxin, a radioisotope, or other therapeutically active agent.Chemotherapeutic drugs, cytokines, antiangiogenic drugs, tyrosine kinase inhibitors, and other therapeutic agents are described herein, and all of these aforementioned therapeutic agents can be used as antibody conjugates.Such conjugates can be used to target drugs or compounds to, for example, sites of action, such as tumors or tumor microenvironments characterized by the expression of TNFR2.

[0093] In some embodiments, the antibody is conjugated or operably linked to a toxin, including, but not limited to, enzymatically active toxins of bacterial, fungal, plant or animal origin, including small molecule toxins, polypeptides, nucleic acids, and fragments and / or variants thereof. Small molecule toxins include, but are not limited to, saporin (Kuroda K, et al., The Prostate 70:1286-1294(2010); Lip, WL. et al., 2007 Molecular Pharmaceutics 4:241-251; Quadros EV., et al., 2010 Mol Cancer Ther;9(11);3033-40; Polito L., et al. 2009 British Journal of Haematology,147,710-718), calicheamicin, maytansine (US Pat. No. 5,208,020), trichothenes, and CC1065. Toxins include, but are not limited to, RNase, gelonin, enediyne, ricin, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin (PE40), Shigella toxin, Clostridium perfringens toxin, and pokeweed antiviral protein.

[0094] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Later, certain microorganisms were also found to produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309, and 4,371,533. Immunoconjugates containing maytansinoids and their therapeutic uses have been disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent No. 0425235B1. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described an immunoconjugate containing the maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic to cultured colon cancer cells and exhibited antitumor activity in in vivo tumor growth assays.

[0095] Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly decreasing the biological activity of either the antibody or the maytansinoid molecule. An average of 3-4 conjugated maytansinoid molecules per antibody molecule have shown effectiveness in enhancing cytotoxicity of target cells without adversely affecting the function or solubility of the antibody, but even one molecule of toxin / antibody is expected to enhance cytotoxicity over naked antibodies. Maytansinoids are well known in the art and may be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No. 5,208,020 and other patent and non-patent publications referred to herein above. Preferred maytansinoids are maytansinol and maytansinol analogs modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.

[0096] Another conjugate of interest comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics can produce double-stranded DNA breaks at sub-picomolar concentrations. Structural analogues of calicheamicin can also be used (Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928) (U.S. Patent No. 5,714,586; U.S. Patent No. 5,712,374; U.S. Patent No. 5,264,586; U.S. Patent No. 5,773,001). Dolastatin 10 analogs such as auristatin E (AE) and monomethylauristatin E (MMAE) may find use as conjugates of the antibodies of the present disclosure, or variants thereof (Doronina et al., 2003, Nat Biotechnol 21(7):778-84; Francisco et al., 2003 Blood 102(4):1458-65). Useful enzymatically active toxins include, but are not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and trichothecin. See, e.g., PCT Publication No. 93 / 21232. The present disclosure further contemplates embodiments in which a conjugate or fusion is formed between a TNFR2-specific antibody described herein and a compound having nucleolytic activity, e.g., a DNA endonuclease such as a ribonuclease or deoxyribonuclease (DNase).

[0097] In some embodiments, the antibodies disclosed herein may be conjugated or operably linked to a radioisotope to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include: 90 Y, 123 I, 125 I, 131 I, 186 Re, 188 Re, 211 At, and 212 These include, but are not limited to, Bi.

[0098] The antibodies described herein may, in certain embodiments, be conjugated to a therapeutic moiety, such as a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent, or a radioactive agent (e.g., an alpha-emitter, a gamma-emitter, etc.). A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxel / paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. One exemplary cytotoxin is saporin (available from Advanced Targeting Systems, San Diego, Calif.). Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepaclorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and antimitotic agents (e.g., vincristine and vinblastine).

[0099] Additionally, TNFR2-specific antibodies (including functional fragments thereof provided herein, such as antigen-binding fragments) may, in certain embodiments, be conjugated to a therapeutic moiety, such as a radioactive substance or a macrocyclic chelator useful for conjugating radioactive metal ions. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which may be attached to the antibody via a linker molecule. Such linker molecules are generally known in the art and are described in Denardo et al., 1998, Clin. Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zimmerman et al. al., 1999, Nucl. Med. Biol. 26:943-50.

[0100] In some embodiments, the antibody may be conjugated to a "receptor" (e.g., streptavidin) for use in pre-tumor targeting, and the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide). In some embodiments, the antibody is conjugated or operably linked to an enzyme for use in antibody-dependent enzyme-mediated prodrug therapy (ADEPT). ADEPT may be used by conjugating or linking the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see PCT Publication No. 81 / 01145) into an active anti-cancer drug. See, e.g., PCT Publication No. 88 / 07378 and U.S. Patent No. 4,975,278. The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way as to convert it into its more active, cytotoxic form.Enzymes that are useful in the methods of these and related embodiments include alkaline phosphatases, which are useful for converting phosphate-containing prodrugs to free drugs; arylsulfatases, which are useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminases, which are useful for converting the non-toxic 5-fluorocytosine to the anti-cancer drug 5-fluorouracil; proteolytic enzymes, such as Serratia protease, thermolysin, subtilisin, carboxypeptidases, and cathepsins (e.g., cathepsins B and L), which are useful for converting peptide-containing prodrugs to free drugs; These include, but are not limited to, D-alanyl carboxypeptidase, which is useful for converting prodrugs containing a substituent; carbohydrate cleavage enzymes, such as beta-galactosidase and neuraminidase, which are useful for converting glycosylated prodrugs to free drugs; beta-lactamase, which is useful for converting drugs derivatized with beta-lactams to free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, which are useful for converting drugs derivatized with phenoxyacetyl or phenylacetyl groups, respectively, at their amine nitrogens to free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," may be used to convert prodrugs to free active drugs (see, for example, Massey, 1987, Nature 328:457-458). Conjugates of antibodies and abzymes can be prepared for delivery of the abzymes to tumor cell populations.

[0101] Immunoconjugates may be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Particular coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737

[1978] ) and N-succinimidyl-4-(2-pyridylthio)pentoate (SPP) to provide disulfide bonds. The linker may be a "cleavable linker" that facilitates the release of one or more cleavable elements. For example, acid-labile linkers may be used (Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0102] Other modifications of the antibodies (and polypeptides) of the disclosure are also contemplated herein. For example, the antibodies may be linked to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibodies may also be encapsulated, for example, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, by coacervation techniques, or in microcapsules prepared by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethialate) microcapsules, respectively). Such techniques are described in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0103] As used herein, "carrier" includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers are often aqueous pH buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or polysorbate 20 (TWEEN®), polyethylene glycols (PEG), and poloxamers (PLURONICS®), non-ionic surfactants.

[0104] The desired functional properties of anti-TNFR2 antibodies may be assessed using a variety of methods known to those of skill in the art using in vitro or in vivo models, affinity / binding assays (e.g., surface plasmon resonance, competitive inhibition assays); cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays, cancer cell and / or tumor growth inhibition. Other assays may test the ability of the antibodies described herein to block normal TNFR2-mediated responses. The antibodies described herein may also be tested for in vitro and in vivo efficacy. Such assays may be performed using well-established protocols known to those of skill in the art (see, for example, Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); or may be performed using commercially available kits.

[0105] The present disclosure further provides, in certain embodiments, an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof described herein, such as a nucleic acid encoding a CDR or a VH or VL domain described herein. Nucleic acids include DNA and RNA. These and related embodiments may include a polynucleotide encoding an antibody that binds to TNFR2 described herein. As used herein, the term "isolated polynucleotide" refers to a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, and by its origin, an isolated polynucleotide is (1) not associated with all or part of a polynucleotide found in nature, (2) linked to a polynucleotide to which it is not naturally linked, or (3) not occurring in nature as part of a larger sequence.

[0106] The term "operably linked" means that the elements to which the term is applied are in a relationship that allows them to carry out their inherent functions under appropriate conditions. For example, a transcription control sequence "operably linked" to a protein coding sequence is ligated thereto such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0107] As used herein, the term "control sequence" refers to polynucleotide sequences that can affect the expression, processing or subcellular localization of coding sequences to which they are ligated or operably linked. The nature of such control sequences may depend on the host organism. In certain embodiments, a prokaryotic transcription control sequence may include a promoter, a ribosomal binding site, and a transcription termination sequence. In other particular embodiments, a eukaryotic transcription control sequence may include a promoter that includes one or more recognition sites for transcription factors, transcription enhancer sequences, transcription termination sequences, and polyadenylation sequences. In certain embodiments, a "control sequence" may include a leader sequence and / or a fusion partner sequence.

[0108] The term "polynucleotide" as referred to herein means a single-stranded or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides comprising a polynucleotide can be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, and phosphoroamidate. The term "polynucleotide" includes single-stranded and double-stranded forms of DNA.

[0109] The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups, etc. The term "oligonucleotide linkages" includes oligonucleotide linkages such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoraniladates, phosphoroamidates, and the like. See, e.g., LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), Oxford University Press, Oxford England; U.S. Patent No. 5,151,510 to Stec et al.; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are incorporated herein by reference for any purpose. The oligonucleotide may contain a detectable label that allows for detection of the oligonucleotide or its hybridization.

[0110] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. The term "expression vector" refers to a vector suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control the expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.

[0111] As will be understood by those of skill in the art, polynucleotides may include genomic, extragenomic and plasmid coding sequences, as well as smaller engineered gene segments that express, or can be adapted to express, proteins, polypeptides, peptides, etc. Such segments may be naturally isolated or synthetically modified by those of skill in the art.

[0112] As will also be appreciated by those skilled in the art, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules, which contain introns and correspond one-to-one to DNA molecules, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may or may not be present within a polynucleotide according to the present disclosure, and the polynucleotide may or may not be linked to other molecules and / or supporting materials. Polynucleotides may contain naturally occurring sequences or may contain sequences that code for variants or derivatives of such sequences.

[0113] Thus, in accordance with these and related embodiments, the present disclosure also provides polynucleotides encoding the anti-TNFR2 antibodies described herein. In certain embodiments, polynucleotides are provided that include part or all of the polynucleotide sequences encoding the antibodies described herein, as well as the complementary strands of such polynucleotides.

[0114] In other related embodiments, polynucleotide variants may have substantial identity with the polynucleotide sequences encoding the anti-TNFR2 antibodies described herein.For example, the polynucleotide may be a polynucleotide that comprises at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, compared to a reference polynucleotide sequence, such as the sequence encoding the antibody described herein, using the methods described herein (e.g., BLAST analysis using standard parameters, as described below).Those skilled in the art will recognize that these values ​​can be appropriately adjusted to take into account codon degeneracy, amino acid similarity, reading frame positioning, etc., to determine the corresponding identity of the protein encoded by two nucleotide sequences.

[0115] Typically, polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions, such that the binding affinity of antibodies encoded by the variant polynucleotides is preferably not substantially reduced compared to antibodies encoded by the polynucleotide sequences specifically described herein.

[0116] In certain embodiments, the polynucleotide fragments may comprise or consist essentially of contiguous stretches of various lengths of sequences identical to, or complementary to, the sequences encoding the antibodies described herein. For example, polynucleotides encoding an antibody, or antigen-binding fragment thereof, disclosed herein, comprising or consisting essentially of a sequence of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, or 1000 or more contiguous nucleotides of length, as well as all intervals therebetween, are provided. It will be readily understood that a "length between" in this context means any length between the recited values, such as 200-500; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc., including all integers, such as 500-1,000. The polynucleotide sequences described herein may be extended at one or both ends by additional nucleotides not found in the native sequence. This additional sequence may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides at either end of the polynucleotide encoding the antibody described herein, or at both ends of the polynucleotide encoding the antibody described herein.

[0117] In some embodiments, polynucleotides capable of hybridizing to the polynucleotides encoding the antibodies or antigen-binding fragments thereof provided herein, or to the fragments thereof, or to the complementary sequences thereof, under moderate to high stringency conditions are provided. Hybridization techniques are well known in the art of molecular biology. For illustrative purposes, moderately stringent conditions suitable for testing the hybridization of the polynucleotides provided herein with other polynucleotides include pre-washing in a solution of 5xSSC, 0.5%SDS, 1.0mM EDTA (pH 8.0); hybridization at 50°C to 60°C, 5xSSC, overnight; followed by two washes at 65°C for 20 minutes with 2x, 0.5x and 0.2xSSC, each containing 0.1% SDS. Those skilled in the art will understand that the stringency of hybridization can be easily manipulated, such as by changing the salt content of the hybridization solution and / or the temperature at which hybridization is performed. For example, in some embodiments, suitable highly stringent hybridization conditions include those described above, except that the temperature of hybridization is increased, for example, to 60°C-65°C or 65°C-70°C.

[0118] In certain embodiments, the polynucleotides described above, such as polynucleotide variants, fragments and hybridizing sequences, code for antibodies that bind to TNFR2 or antigen-binding fragments thereof.In certain embodiments, such polynucleotides code for antibodies or antigen-binding fragments or CDRs that bind to TNFR2 at least about 50%, at least about 70%, and in certain embodiments at least about 90% as well as the antibody sequences specifically described herein.In further embodiments, such polynucleotides code for antibodies or antigen-binding fragments or CDRs that bind to TNFR2 with higher affinity than the antibodies described herein, for example, quantitatively bind at least about 105%, 106%, 107%, 108%, 109%, or 110%, as well as the antibody sequences specifically described herein.

[0119] As described elsewhere herein, the determination of the three-dimensional structure of a representative polypeptide (e.g., a variant TNFR2-specific antibody described herein, e.g., an antibody protein having an antigen-binding fragment provided herein) can be made through routine methodology such that one or more amino acid substitutions, additions, deletions, or insertions using selected natural or unnatural amino acids can be virtually modeled for the purpose of determining whether the derived structural variant retains the space-filling properties of the species disclosed herein. For example, various computer programs are known to those skilled in the art for determining suitable amino acid substitutions (or suitable polynucleotides encoding amino acid sequences) within an antibody such that affinity is maintained or better affinity is achieved.

[0120] The polynucleotides described herein, or fragments thereof, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably, regardless of the length of the coding sequence itself. Thus, nucleic acid fragments of almost any length may be utilized, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative polynucleotide segments having lengths, total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs, etc., including all lengths in between, are contemplated as being useful.

[0121] When comparing polynucleotide sequences, two sequences are said to be "identical" if the sequences of nucleotides in the two sequences are the same when aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, usually 30 to about 75, 40 to about 50 consecutive positions, and a sequence can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned.

[0122] Optimal alignment of sequences for comparison may be performed using the Megalign™ program of the Lasergene® suite of bioinformatics software (DNASTAR, Inc., Madison, WI) using default parameters. This program is based on the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990); Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, CABIOS 5: 151-153 (1989); Myers, EW and Muller W., CABIOS 4: 11-17 (1988); Robinson, ED, Comb. Theor 11:105(1971);Santou,N.Nes,M.,Mol.Biol.Evol.4:406-425(1987);Sneath,PHAand We embody several alignment schemes described in Sokal, R. R., Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif. (1973); Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad., Sci. USA 80:726-730 (1983).

[0123] Alternatively, optimal alignment of sequences for comparison can be performed by the local identity algorithm of Smith and Waterman, Add. APL. Math 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), using these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 This may be done by computerized implementations of the algorithms described herein (GAP, BESTFIT, BLAST, FASTA, and TFASTA at Science Dr., Madison, WI), or by inspection.

[0124] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, respectively, described in Altschul et al. et al., Nucl. Acids Res. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol. 215:403-410 (1990). BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine the percent sequence identity between two or more polynucleotides. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, the cumulative score can be calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. The extension of word hits in each direction is stopped when the cumulative alignment score falls by a quantity X from its maximum achieved value; when the cumulative score falls below zero due to the accumulation of alignments that negatively score one or more residues; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignment, (B) 50, expectation (E) 10, M=5, N=-4, and a comparison of both strands.

[0125] In certain embodiments, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, where the portion of the polynucleotide sequence in the comparison window may contain 20 percent or less, typically 5-15 percent, or 10-12 percent additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) for the optimal alignment of the two sequences. The percentage is calculated by determining the number of identical nucleobases that occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and dividing the result by 100 to obtain the percentage of sequence identity.

[0126] It will be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that code for the antibodies described herein. Some of these polynucleotides have minimal sequence identity to the nucleotide sequence of the natural or original polynucleotide sequence that codes for the antibody that binds to TNFR2. Nevertheless, polynucleotides that vary due to differences in codon usage are expressly contemplated by this disclosure. In certain embodiments, sequences that are codon-optimized for mammalian expression are specifically contemplated.

[0127] In some embodiments, mutagenesis approaches such as site-directed mutagenesis may be utilized for the preparation of variants and / or derivatives of the antibodies described herein. By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. These techniques provide a direct approach to preparing and testing sequence variants that incorporate one or more of the above considerations, for example, by introducing one or more nucleotide sequence changes into the polynucleotide.

[0128] Site-directed mutagenesis allows for the production of mutants through the use of specific oligonucleotide sequences that code for the DNA sequence of the desired mutation, and a sufficient number of flanking nucleotides to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex at both ends of the deletion junction across. Mutations may be utilized in a selected polynucleotide sequence to improve, alter, reduce, modify, or otherwise change the properties of the polynucleotide itself, and / or to change the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[0129] In certain embodiments, the inventors contemplate mutagenesis of the polynucleotide sequence encoding the antibody or antigen-binding fragment thereof disclosed herein to alter one or more properties of the encoded polypeptide, such as the binding affinity of the antibody or antigen-binding fragment thereof, or the function of a particular Fc region, or the affinity of the Fc region for a particular FcγR. The technique of site-directed mutagenesis is well known in the art and is widely used to generate variants of both polypeptides and polynucleotides. For example, site-directed mutagenesis is often used to alter specific portions of DNA molecules. In such embodiments, primers may be utilized that contain about 5 to about 10 residues on either side of the junction of the altered sequence, typically about 14 to about 25 nucleotides in length.

[0130] As will be understood by those skilled in the art, site-directed mutagenesis techniques often utilize phage vectors that exist in both single-stranded and double-stranded forms.Typical vectors useful for site-directed mutagenesis include vectors such as M13 phage.These phages are readily commercially available, and their use is generally well known to those skilled in the art.Double-stranded plasmids are also routinely used in site-directed mutagenesis, which does not involve the step of transferring the gene of interest from the plasmid to the phage.

[0131] Generally, site-directed mutagenesis according to the present invention is carried out by first obtaining a single-stranded vector or melting the two strands of a double-stranded vector that contains within its sequence a DNA sequence encoding the desired peptide. An oligonucleotide primer carrying the desired mutated sequence is generally prepared synthetically. This primer is then annealed with the single-stranded vector and subjected to a DNA polymerizing enzyme, such as, for example, E. coli polymerase I Klenow fragment, to complete the synthesis of the strand carrying the mutation. Thus, a heteroduplex is formed, with one strand encoding the original unmutated sequence and the second strand carrying the desired mutation. This heteroduplex vector is then used to transform an appropriate cell, such as an E. coli cell, and clones are selected that contain a recombinant vector carrying the mutated sequence arrangement.

[0132] The preparation of sequence variants of selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting, as there are other methods by which sequence variants of peptides and the DNA sequences encoding them can be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents such as hydroxylamine to obtain sequence variants. Specific details regarding these methods and protocols can be found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each of which is incorporated herein by reference for that purpose.

[0133] As used herein, the term "oligonucleotide-directed mutagenesis technique" refers to a template-dependent process and vector-mediated amplification, which results in an increase in the concentration of a particular nucleic acid molecule, or an increase in the concentration of a detectable signal, such as amplification, compared to its initial concentration. As used herein, the term "oligonucleotide-directed mutagenesis technique" is intended to refer to a process involving template-dependent extension of a primer molecule. The term template-dependent process refers to nucleic acid synthesis of an RNA or DNA molecule, where the sequence of the newly synthesized strand of nucleic acid is determined by the well-known rules of complementary base pairing (see, for example, Watson, 1987). Typically, vector-mediated methodologies involve the introduction of a nucleic acid fragment into a DNA or RNA vector, clonal amplification of the vector, and recovery of the amplified nucleic acid fragment. An example of such a methodology is provided by U.S. Pat. No. 4,237,224, the entire contents of which are specifically incorporated herein by reference.

[0134] Another approach for the production of polypeptide variants may utilize recursive sequence recombination, as described in U.S. Patent No. 5,837,458. In this approach, repeated cycles of recombination and screening or selection are performed to "evolve" individual polynucleotide variants, e.g., with increased binding affinity. Certain embodiments also provide constructs in the form of plasmids, vectors, transcription or expression cassettes that include at least one polynucleotide described herein.

[0135] In many embodiments, the nucleic acid encoding the subject monoclonal antibody is directly introduced into the host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded antibody. The antibodies of the present disclosure are prepared using standard techniques well known to those skilled in the art in combination with the polypeptide and nucleic acid sequences provided herein. The polypeptide sequence may be used to determine the appropriate nucleic acid sequence that encodes the particular antibody disclosed thereby. The nucleic acid sequence may be optimized to reflect the particular codon "preference" for various expression systems according to standard methods well known to those skilled in the art.

[0136] According to certain related embodiments, there are provided recombinant host cells comprising one or more constructs described herein, nucleic acids encoding any antibody, CDR, VH or VL domain, or antigen-binding fragment thereof, and methods of producing the encoded product, which methods include expression from the nucleic acid encoding it. Expression may be suitably achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. After production by expression, the antibody or antigen-binding fragment thereof may be isolated and / or purified using any suitable technique and then used as desired.

[0137] The antibody or antigen-binding fragment thereof encoding nucleic acid molecules and vectors provided herein may be isolated and / or purified, for example, from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acids, free or substantially free of nucleic acids or genes of origin other than the sequence encoding a polypeptide having a desired function. Nucleic acids may include DNA or RNA, and may be fully or partially synthetic. Reference to a nucleotide sequence described herein encompasses DNA molecules having the particular sequence, and, unless the context requires otherwise, encompasses RNA molecules having the particular sequence in which U is replaced by T.

[0138] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for the expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common and preferred bacterial host is E. coli.

[0139] The expression of antibodies and antigen-binding fragments in prokaryotic cells such as E. coli is well established in the art.See, for example, Pluckthun (Bio / Technology 9:545-551, 1991).Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for producing antibodies or antigen-binding fragments thereof, see recent reviews, for example, Ref, ME (1993) Curr. Opinion Biotech. 4:573-576; Trill JJ et al. (1995) Curr. Opinion Biotech 6:553-560.

[0140] Suitable vectors can be chosen or constructed, containing appropriate control sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral, e.g., phage, or phagemid, as appropriate. For further details, see, for example, Molecular Cloning: a Laboratory See, Molecular Biology, Second Edition, Ausubel et al., eds., John Wiley & Sons, 1992, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for the manipulation of nucleic acids, for example in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in the current protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, or subsequent updates.

[0141] The term "host cell" is used to refer to a cell into which a nucleic acid sequence encoding one or more of the antibodies described herein has been introduced or is capable of being introduced, and which further expresses or is capable of expressing a selected gene of interest, such as a gene encoding an antibody described herein. The term includes the progeny of a parent cell, regardless of whether the progeny is identical in morphology or genetic make-up to the original parent, so long as the selected gene is present. Thus, methods involving introducing such nucleic acids into a host cell are also contemplated. The introduction may utilize any available technique. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection and retroviruses or other viruses, such as vaccinia, or for insect cells, baculovirus. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation and transfection using bacteriophage. Following introduction, expression from the nucleic acid may be caused or permitted, for example, by culturing the host cells under conditions for gene expression. In some embodiments, the nucleic acid is integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

[0142] The disclosure also provides methods that include, in certain embodiments, using the constructs described above in an expression system to express a particular polypeptide, such as the TNFR2-specific antibody described herein. The term "transduction" is used to refer to the transfer of genes from one bacterium to another, usually by a phage. "Transduction" also refers to the acquisition and transfer of eukaryotic cellular sequences by retroviruses. The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell is "transduced" when the exogenous DNA is introduced into the cell membrane. Numerous transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories; Davis et al., 1986, BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier; and Chu et al. See, et al., 1981, Gene 13: 197. Using such techniques, one or more exogenous DNA moieties can be introduced into a suitable host cell.

[0143] As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell; a cell is transformed when it has been modified to contain new DNA. For example, a cell is transformed and it has been genetically modified from its natural state. After transfection or transduction, the transforming DNA may recombine with that of the cell by being physically integrated into the cell's chromosome, or may be maintained transiently as an episomal element without being replicated, or may be replicated independently as a plasmid. A cell is considered stably transformed when the DNA is replicated with the division of the cell. The terms "naturally occurring" or "natural" when used in reference to biological materials such as nucleic acid molecules, polypeptides, host cells, etc., refer to materials that are found in nature but have not been manipulated by humans. Similarly, as used herein, "non-naturally occurring" or "non-natural" refers to materials that are not found in nature or that have been structurally modified or synthesized by humans.

[0144] The terms "polypeptide", "protein" and "peptide" and "glycoprotein" are used interchangeably and refer to a polymer of amino acids, not limited to any particular length. The terms do not exclude modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of a signal sequence. The terms "polypeptide" or "protein" refer to one or more chains of amino acids, each chain comprising amino acids covalently linked by peptide bonds, and the polypeptide or protein may comprise multiple chains non-covalently and / or covalently linked together by peptide bonds, having the sequence of a native protein, i.e., a protein produced by a cell occurring in nature and a particular non-recombinant cell, or a genetically engineered or recombinant cell, and include molecules having the amino acid sequence of a native protein or molecules having deletions, additions, and / or substitutions from one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass the antibodies that bind to TNFR2 of the present disclosure, or sequences having deletions, additions, and / or substitutions from one or more amino acids of an anti-TNFR2 antibody. Thus, a "polypeptide" or "protein" may contain one (referred to as a "monomer") or multiple (referred to as a "multimer") amino acid chains.

[0145] As referred to herein, the term "isolated protein" means that the subject protein is (1) free of at least some other proteins with which it is typically found in nature, (2) essentially free of other proteins from the same source, e.g., from the same species, (3) expressed by cells from a different species, (4) separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) the "isolated protein" is not associated (by covalent or non-covalent interactions) with portions of proteins with which it is naturally associated, (6) operably linked (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (7) not occurring in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, may be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, preventive, research, or otherwise).

[0146] The term "polypeptide fragment" refers to a polypeptide, which may be monomeric or multimeric, having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a naturally occurring or recombinantly produced polypeptide. In certain embodiments, a polypeptide fragment may comprise an amino acid chain at least 5 to about 500 amino acids in length. It will be appreciated that in certain embodiments, the fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Particularly useful polypeptide fragments include functional domains, including antigen binding domains or fragments of antibodies. In the case of anti-TNFR2 antibodies, useful fragments include, but are not limited to, the CDR regions, in particular the CDR3 regions of the heavy or light chains, the variable regions of the heavy or light chains, portions of the antibody chains or just the variable regions thereof comprising two CDRs.

[0147] Polypeptides may contain signal (or leader) sequences at the N-terminus of proteins, which direct the transport of proteins co-translationally or post-translationally. Any polypeptide amino acid sequence provided herein that contains a signal peptide is also contemplated for any use described herein without such a signal or leader peptide. As will be recognized by those skilled in the art, signal peptides are usually cleaved during processing and are not included in active antibody proteins. Polypeptides may also be fused in frame to or conjugated to linkers or other sequences for ease of synthesis, purification or identification of polypeptides (e.g., poly-His), or to enhance binding of polypeptides to solid support.

[0148] Peptide linker / spacer sequences may also be utilized to separate multiple polypeptide components by a distance sufficient to ensure that each polypeptide will fold into its secondary and / or tertiary structure, if desired. Such peptide linker sequences can be incorporated into the fusion polypeptide using standard techniques well known in the art.

[0149] A particular peptide spacer sequence may be selected based, for example, on (1) its ability to adopt a flexible extended conformation, (2) its ability to adopt secondary structure that can interact with functional epitopes on the first and second polypeptides, and / or (3) its lack of hydrophobic or charged residues that can react with polypeptide functional epitopes.

[0150] In one illustrative embodiment, the peptide spacer sequence contains, for example, Gly, Asn, and Ser residues. Other near-neutral amino acids, such as Thr and Ala, may also be included in the spacer sequence.

[0151] Other amino acid sequences which may be usefully employed as spacers include those described in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.

[0152] Other illustrative spacers may include, for example, Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO: 324) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070) and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp (SEQ ID NO: 325) (Bird et al., 1988, Science 242:423-426).

[0153] In some embodiments, a spacer sequence is not required when the first and second polypeptides have a non-essential N-terminal amino acid region that can be used to separate the functional domains and prevent steric interference. The two coding sequences can be fused directly without a spacer or by using a flexible polylinker consisting of, for example, the pentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 326) repeated 1-3 times. Such spacers have been used in the construction of single chain antibodies (scFv) by inserting them between VH and VL (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5979-5883).

[0154] The peptide spacer is, in certain embodiments, designed to allow for correct interactions between the two beta sheets that form the variable region of a single chain antibody.

[0155] In certain embodiments, the peptide spacer is 1-5 amino acids, 5-10 amino acids, 5-25 amino acids, 5-50 amino acids, 10-25 amino acids, 10-50 amino acids, 10-100 amino acids, or any intervening range. In some embodiments, the peptide spacer comprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids in length.

[0156] Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. For example, amino acid sequence variants of the antibody can be prepared by introducing appropriate nucleotide changes into the polynucleotide encoding the antibody or its chain, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions and / or substitutions into, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final antibody, provided that the final construct has the desired characteristics (e.g., high affinity binding to TNFR2). Amino acid changes can also alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites. Any of the variations and modifications described above for the polypeptides of the present disclosure can be included in the antibodies of the present disclosure.

[0157] The present disclosure provides the variant of the antibody disclosed herein.In certain embodiments, such variant antibody or its antigen-binding fragment or CDR binds to TNFR2 at least about 50%, at least about 70%, in certain embodiments at least about 90%, and binds to the antibody sequence specifically described herein.In further embodiments, such variant antibody or its antigen-binding fragment or CDR binds to TNFR2 with higher affinity than the antibody described herein, for example, quantitatively binds at least about 105%, 106%, 107%, 108%, 109% or 110%, and binds to the antibody sequence specifically described herein.

[0158] Determination of the three-dimensional structure of a representative polypeptide (e.g., a variant TNFR2-specific antibody described herein, e.g., an antibody protein having an antigen-binding fragment provided herein) can be made through routine methodologies such that one or more amino acid substitutions, additions, deletions or insertions with selected natural or unnatural amino acids can be virtually modeled for purposes of determining whether the induced structural variant retains the space-filling properties of the species disclosed herein. For example, Donate et al.,1994 Prot.Sci.3:2378;Bradley et al.,Science 309:1868-1871(2005);Schueler-Furman et al.,Science 310:638(2005);Dietz et al.,Proc.Nat.Acad.Sci.USA 103:1244 (2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature 450:259 (2007); Raman et al. Science 327:1014-1018 (2010). Some additional non-limiting examples of computer algorithms that can be used for these and related embodiments, such as for the rational design of the TNFR2-specific antibody antigen-binding domains provided herein, include VMD, a molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3-D drawing techniques and built-in scripts (see the website for the Theoretical and Computational Biophysics Group, University of Illinois at Urbana-Champagne at ks.uiuc.edu / Research / vmd / ).Many other computer programs that allow the determination of atomic diameters from space-filling models of energy-minimized conformations (van der Waals ranges): GRID, which determines regions of high affinity for different chemical groups and thus seeks to enhance binding; Monte Carlo search, which calculates mathematical alignments; and CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and AMBER (Weiner et al. (1981) J. Comput. Chem. 106:765), which evaluate and analyze force field calculations, are known in the art and available to the skilled artisan (Eisenfield et al. (1991) Am. J. Physiol. 261:C376-386; Lybrand (1991) J. Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam). (See also, Pedersen (1985) Environ. Health Perspect. 61:185-190; Kini et al. (1991) J. Biomol. Struct. Dyn. 9:475-488). A variety of suitable calculation computer programs are also commercially available, such as from Schroudinger (Munich, Germany).

[0159] In some embodiments, anti-TNFR2 antibodies and humanized versions thereof are derived from rabbit monoclonal antibodies, and are particularly produced using APXiMAB™ technology. These antibodies are advantageous because they require minimal sequence modification, which promotes the retention of functional properties after humanization using mutation-guided gene (MLG) humanization technology (see, for example, U.S. Pat. No. 7,462,697). Thus, illustrative methods for producing anti-TNFR2 antibodies of the present disclosure include, for example, the APXiMAB™ rabbit monoclonal antibody technology described in U.S. Pat. Nos. 5,675,063 and 7,429,487. In this regard, in certain embodiments, anti-TNFR2 antibodies of the present disclosure are produced in rabbits. In certain embodiments, rabbit spleen cells or rabbit-derived immortalized B lymphocytes capable of fusing with peripheral B lymphocytes are used to generate hybrid cells that produce antibodies. The immortalized B lymphocytes do not detectably express endogenous immunoglobulin heavy chains and, in certain embodiments, may contain an altered immunoglobulin heavy chain encoding gene.

[0160] Compositions and Methods of Use The present disclosure provides compositions comprising TNFR2-specific antibodies, or antigen-binding fragments thereof, and the administration of such compositions in a variety of therapeutic settings, including the treatment of cancer, inflammatory and autoimmune diseases, and other diseases.

[0161] Administration of the TNFR2-specific antibodies described herein, either in pure form or in a suitable pharmaceutical composition, can be via any of the accepted modes of administration of drugs to achieve similar utility. Pharmaceutical compositions can be prepared by combining the antibody or antibody-containing compositions with suitable physiologically acceptable carriers, diluents or excipients, and can be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition, other pharmacologic active ingredients (including other anticancer agents described elsewhere herein) and / or suitable excipients such as salts, buffers and stabilizers may be present in the composition, but are not required. Administration can be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, subcutaneous or topical. The preferred mode of administration depends on the nature of the condition to be treated or prevented. An amount that reduces, inhibits, prevents, or delays the progression and / or metastasis of cancer after administration is considered effective.

[0162] In certain embodiments, the amount administered is sufficient to cause tumor regression, as indicated by a statistically significant reduction in the amount of viable tumor, e.g., at least a 50% reduction in tumor mass, or by an altered (e.g., statistically significantly reduced) scan dimension.

[0163] The exact dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known test protocols or by testing the composition in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosage may also vary according to the severity of the condition to be alleviated. Pharmaceutical compositions are generally formulated and administered to produce a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered once or divided into a number of smaller doses to be administered at time intervals. The particular dosage regimen for any particular subject may be adjusted over time according to individual needs.

[0164] The TNFR2-specific antibody-containing composition may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, etc. The composition may also be administered in combination with an antibiotic.

[0165] Thus, typical routes of administration of these and related pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, intravitreal, and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. Pharmaceutical compositions according to certain embodiments are formulated so that the active ingredient contained therein is bioavailable upon administration of the composition to a patient. The composition administered to a subject or patient may take the form of one or more dosage units, for example, a tablet may be a single dosage unit, and a container of the TNFR2-specific antibody described herein in aerosol form may hold multiple dosage units. Actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of an antibody of the present disclosure for treatment of the disease or condition of the subject in accordance with the teachings herein.

[0166] The pharmaceutical composition may be in solid or liquid form. In one embodiment, the carrier is particulate, and thus the composition is, for example, in tablet or powder form. The carrier may be liquid, and the composition is, for example, oral oil, injectable liquid, or aerosol, which is useful for, for example, inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, and semi-solid, semi-liquid, suspension, and gel forms are included in the forms considered as either solid or liquid herein.

[0167] As a solid composition for oral administration, the pharmaceutical composition may be formulated into powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrin; disintegrating agents such as alginic acid, sodium alginate, primogel, corn starch; lubricants such as magnesium stearate or sterotex; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate or orange flavor; and coloring agents. When the pharmaceutical composition is in the form of a capsule, for example a gelatin capsule, it may contain, in addition to the above-mentioned types of materials, liquid carriers such as polyethylene glycol or oil.

[0168] The pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or delivery by injection, as two examples. When intended for oral administration, certain compositions contain, in addition to the compound, one or more of a sweetener, a preservative, a dye / colorant and a flavor enhancer. In a composition intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer and an isotonicity agent may be included.

[0169] Liquid pharmaceutical compositions, whether in solution, suspension or other similar form, may contain one or more of the following adjuvants: water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, sterile diluents such as fixed oils, polyethylene glycol, glycerin, propylene glycol or other solvents that can act as solvents or suspending media, such as synthetic mono- or diglycerides; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting osmolarity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Saline is an exemplary adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0170] Liquid pharmaceutical compositions intended for either parenteral or oral administration should contain an amount of TNFR2-specific antibody disclosed herein such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% antibody in the composition. When intended for oral administration, this amount may vary to be 0.1 to about 70% by weight of the composition. Certain oral pharmaceutical compositions contain about 4% to about 75% antibody. In certain embodiments, pharmaceutical compositions and preparations are prepared such that a parenteral dosage unit contains 0.01 to 10% antibody by weight, prior to dilution.

[0171] The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base may, for example, comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. A thickening agent may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device. The pharmaceutical composition may be intended for rectal administration, for example, in the form of a suppository that dissolves in the rectum and releases the drug. A composition for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

[0172] Pharmaceutical compositions may contain various substances that modify the physical form of a solid or liquid dosage unit. For example, the composition may contain substances that form a coating shell around the active ingredient. The substances that form the coating shell are typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. Pharmaceutical compositions in solid or liquid form may contain an agent that binds to the antibody and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include other monoclonal or polyclonal antibodies, one or more proteins, or liposomes. Pharmaceutical compositions may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to indicate a variety of systems, from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas, or by a suitable pump system that dispenses the active ingredient. Aerosols may be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Delivery of the aerosol includes the necessary containers, activators, valves, subcontainers, etc., which can together form a kit. One of ordinary skill in the art can determine a preferred aerosol without undue experimentation.

[0173] Pharmaceutical compositions may be prepared by methodologies well known in the pharmaceutical field. For example, pharmaceutical compositions intended to be administered by injection can be prepared by combining a composition comprising the TNFR2-specific antibody described herein and, optionally, one or more of salts, buffers and / or stabilizers with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with antibody compositions to facilitate the dissolution or homogeneous suspension of the antibody in an aqueous delivery system.

[0174] The compositions may be administered in therapeutically effective amounts, which vary depending on a variety of factors, including the activity of the particular compound (e.g., TNFR2-specific antibody) utilized; the metabolic stability and length of action of the compound; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject being treated. Generally, a therapeutically effective daily dose is about 0.001 mg / kg (i.e., 0.07 mg) to about 100 mg / kg (i.e., 7.0 g) (for a 70 kg mammal), preferably, a therapeutically effective dose is about 0.01 mg / kg (i.e., 0.7 mg) to about 50 mg / kg (i.e., 3.5 g) (for a 70 kg mammal), and more preferably, a therapeutically effective dose is about 1 mg / kg (i.e., 70 mg) to about 25 mg / kg (i.e., 1.75 g) (for a 70 kg mammal).

[0175] The composition comprising the TNFR2-specific antibody of the present disclosure may be administered simultaneously with, before, or after the administration of one or more other therapeutic agents. Such combination therapy may include administration of a single pharmaceutical dosage formulation containing the antibody and one or more additional active agents, as well as administration of the composition comprising the antibody of the present disclosure and each active agent in its own separate pharmaceutical dosage formulation. For example, the antibody and other active agents described herein may be administered to the patient together in a single oral dosage composition, such as a tablet or capsule, or each agent may be administered in a separate oral dosage formulation. Similarly, the antibody and other active agents described herein may be administered to the patient together in a single parenteral dosage composition, such as in a saline solution or other physiologically acceptable solution, or each agent may be administered in a separate parenteral dosage formulation. When separate dosage formulations are used, it is understood that the composition comprising the antibody and one or more additional active agents may be administered essentially simultaneously, i.e., simultaneously, or separately at different times, i.e., sequentially and in any order, and combination therapy includes all of these regimens.

[0176] Thus, in certain embodiments, administration of the anti-TNFR2 antibody composition of the present disclosure in combination with one or more other therapeutic agents is also contemplated. Such therapeutic agents may be accepted in the art as standard treatment for certain disease conditions described herein, such as rheumatoid arthritis, inflammation, or cancer. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMD, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, or other effective drugs and adjuvants.

[0177] In certain embodiments, the anti-TNFR2 antibody disclosed herein is administered in combination with one or more cancer immunotherapeutic agents. In certain cases, the immunotherapeutic agent modulates the immune response of the subject, for example, to increase or maintain a cancer-related or cancer-specific immune response, thereby resulting in increased immune cell inhibition or reduced cancer cells. Exemplary immunotherapeutic agents include polypeptides, such as antibodies and their antigen-binding fragments, ligands, and small peptides, and mixtures thereof. Immunotherapeutic agents also include small molecules, cells (e.g., immune cells such as T cells), various cancer vaccines, gene therapy or other polynucleotide-based agents, including viral agents such as oncolytic viruses, and others known in the art. Thus, in certain embodiments, the cancer immunotherapeutic agent is selected from one or more of immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-based immunotherapeutic agents.

[0178] In certain embodiments, the cancer immunotherapeutic agent is an immune checkpoint modulator. Specific examples include "antagonists" of one or more inhibitory immune checkpoint molecules, and "agonists" of one or more stimulatory immune checkpoint molecules. In general, immune checkpoint molecules are elements of the immune system that either turn up signals (costimulatory molecules) or turn down signals, and targeting them has therapeutic potential in cancer, since cancer cells can interfere with the natural function of immune checkpoint molecules (see, for example, Sharma and Allison, Science. 348: 56-61, 2015; Topalian et al., Cancer Cell. 27: 450-461, 2015; Pardoll, Nature Reviews Cancer. 12: 252-264, 2012). In some embodiments, immune checkpoint modulators (e.g., antagonists, agonists) "bind" or "specifically bind" to one or more immune checkpoint molecules described herein.

[0179] In some embodiments, the immune checkpoint modulator is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules. Exemplary inhibitory immune checkpoint molecules include programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), programmed death 1 (PD-1), V-domain Ig inhibitor of T-cell activation (VISTA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), B- and T-lymphocyte attenuator (BTLA), CD160, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and signal regulatory protein alpha (SIRPα).

[0180] In certain embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, the targeting of which has been shown to restore immune function in the tumor environment (see, e.g., Phillips et al., Int Immunol. 27:39-46, 2015). PD-1 belongs to the immunoglobulin superfamily and is a cell surface receptor expressed on T cells and pro-B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 functions as an inhibitory immune checkpoint molecule, for example, reducing or preventing the activation of T cells, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is achieved at least in part through a dual mechanism that promotes apoptosis in antigen-specific T cells in lymph nodes while also reducing apoptosis in regulatory T cells (suppressor T cells). Some examples of PD-1 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-1 and reduce one or more of its immunosuppressive activities, such as its downstream signaling or its interaction with PD-L1. Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pidilizumab, and antigen-binding fragments thereof (see, e.g., U.S. Pat. Nos. 8,008,449; 8,993,731; 9,073,994; 9,084,776; 9,102,727; 9,102,728; 9,181,342; 9,217,034; 9,387,247; 9,492,539; 9,492,540; and U.S. Application Nos. 2012 / 0039906; 2015 / 0203579).

[0181] In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As mentioned above, PD-L1 is one of the natural ligands of the PD-1 receptor. Common examples of PD-L1 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L1 and reduce one or more of its immunosuppressive activities, such as its binding to the PD-1 receptor. Specific examples of PD-L1 antagonists include antibodies atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), as well as antigen-binding fragments thereof (see, for example, U.S. Patent Nos. 9,102,725; 9,393,301; 9,402,899; and 9,439,962).

[0182] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As mentioned above, PD-L2 is one of the natural ligands of the PD-1 receptor. Common examples of PD-L2 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L2 and reduce one or more of its immunosuppressive activities, such as its binding to the PD-1 receptor.

[0183] In certain embodiments, the agent is a VISTA antagonist or inhibitor. VISTA is approximately 50 kDa in size and belongs to the immunoglobulin superfamily (has one IgV domain) and the B7 family. It is mainly expressed in leukocytes, and its transcription is partially controlled by p53. There is evidence that VISTA can act as both a ligand and a receptor on T cells to inhibit T cell effector function and maintain peripheral tolerance. VISTA is produced at high levels in tumor-infiltrating lymphocytes, such as myeloid-derived suppressor cells and regulatory T cells, and its blockade with antibodies results in delayed tumor growth in mouse models of melanoma and squamous cell carcinoma. Exemplary anti-VISTA antagonist antibodies include, for example, those described in WO 2018 / 237287, which is incorporated by reference in its entirety.

[0184] In some embodiments, the agent is a CTLA-4 antagonist or inhibitor. CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an inhibitory immune checkpoint molecule by transmitting an inhibitory signal to T cells, for example, when it binds to CD80 or CD86 on the surface of an antigen-presenting cell. Common examples of CTLA-4 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4. Specific examples include the antibodies ipilimumab and tremelimumab, and antigen-binding fragments thereof. It is believed that at least some of the activity of ipilimumab is mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) killing of suppressor Tregs that express CTLA-4.

[0185] In some embodiments, the agent is an IDO antagonist or inhibitor, or a TDO antagonist or inhibitor. IDO and TDO are tryptophan catabolic enzymes with immune inhibitory properties. For example, IDO is known to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. Common examples of IDO and TDO antagonists or inhibitors include those that specifically bind to IDO or TDO (see, e.g., Platten et al., Frontier Research, 2003, 144:131-132, 2003). Immunol. 5:673, 2014), including antibodies or antigen-binding fragments or small molecules that reduce or inhibit one or more immunosuppressive activities. Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norherman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g., Sheridan, Nature Biotechnology. 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, e.g., Pilotte et al., PNAS USA. 109:2497-2502, 2012).

[0186] In some embodiments, the agent is a TIM-3 antagonist or inhibitor. T cell immunoglobulin domain and mucin domain 3 (TIM-3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function by triggering cell death upon interaction with its ligand, galectin-9. TIM-3 is involved in the suppressive tumor microenvironment, and its overexpression is associated with poor prognosis in various cancers (see, for example, Li et al., Acta Oncol. 54:1706-13, 2015). Common examples of TIM-3 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3 and reduce or inhibit one or more of its immunosuppressive activities.

[0187] In some embodiments, the agent is a LAG-3 antagonist or inhibitor.Lymphocyte activation gene 3 (LAG-3) is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells.It negatively regulates T cell proliferation, activation, and homeostasis in a manner similar to CTLA-4 and PD-1 (see, for example, Workman and Vignali.European Journal of Immun.33:970-9,2003; and Workman et al.,Journal of Immun.172:5450-5,2004), and has been reported to play a role in Treg suppressive function (see, for example, Huang et al.,Immunity.21:503-13,2004).LAG3 also maintains CD8+T cells in a tolerogenic state, and in combination with PD-1, maintains CD8 T cell exhaustion. General examples of LAG-3 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to LAG-3 and inhibit one or more of its immunosuppressive activities. Specific examples include the antibody BMS-986016, and antigen-binding fragments thereof.

[0188] In some embodiments, the agent is a BTLA antagonist or inhibitor. B and T lymphocyte attenuator (BTLA; CD272) expression is induced during T cell activation and inhibits T cells through interaction with tumor necrosis family receptor (TNF-R) and B7 cell surface receptor family. BTLA is a ligand for tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). BTLA-HVEM complex negatively regulates T cell immune response, for example, by inhibiting the function of human CD8+ cancer-specific T cells (see, for example, Derre et al., J Clin Invest 120:157-67, 2009). Common examples of BTLA antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA-4 and reduce one or more of its immunosuppressive activities.

[0189] In some embodiments, the agent is an HVEM antagonist or inhibitor, e.g., an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD 160. Common examples of HVEM antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to HVEM and optionally reduce HVEM / BTLA and / or HVEM / CD 160 interactions, thereby reducing one or more of the immunosuppressive activities of HVEM.

[0190] In some embodiments, the agent is a CD160 antagonist or inhibitor, e.g., an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. Common examples of CD160 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CD160 and optionally reduce the CD160 / HVEM interaction, thereby reducing or inhibiting one or more of the immunosuppressive activities.

[0191] In some embodiments, the agent is a TIGIT antagonist or inhibitor. T cell Ig and ITIM domain (TIGIT) is a co-inhibitory receptor found on the surface of various lymphoid cells, suppressing anti-tumor immunity, for example, via Treg (Kurtulus et al., J Clin Invest.125:4053-4062,2015). Common examples of TIGIT antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIGIT and reduce one or more of its immunosuppressive activities (see, for example, Johnston et al., Cancer Cell.26:923-37,2014).

[0192] In certain embodiments, the agent is a SIRPα antagonist or inhibitor. SIRPα is a regulatory membrane glycoprotein expressed primarily by myeloid cells that interacts with the widely expressed transmembrane protein CD47 to negatively regulate the effector functions of innate immune cells, such as host cell phagocytosis. Certain cancer cells, for example, by overexpressing CD47, activate the inhibitory SIRPα-CD47 signaling pathway, thereby inhibiting macrophage-mediated phagocytosis. SIRPα inhibitors have been shown to reduce cancer growth and metastasis, both alone and in synergy with other cancer treatments (see, e.g., Yanagita, JCI Insight. 2017 Jan 12; 2(1): e89140). Common examples of SIRPα antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to SIRPα and interfere with SIRPα-CD47 signaling (see Id.).

[0193] In certain embodiments, the immune checkpoint modulator is an agonist of one or more stimulatory immune checkpoint molecules. Exemplary stimulatory immune checkpoint molecules include CD40, OX40, glucocorticoid-inducible TNFR family related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0194] In some embodiments, the agent is a CD40 agonist. CD40 is expressed on antigen-presenting cells (APCs) and some malignant tumors. Its ligand is CD40L (CD154). On APCs, ligation leads to upregulation of costimulatory molecules, potentially bypassing the need for T cell help in antitumor immune responses. CD40 agonist therapy plays a key role in APC maturation and their migration from tumors to lymph nodes, leading to elevated antigen presentation and increased T cell activation. Anti-CD40 agonist antibodies generate substantial responses and sustained anticancer immunity in animal models, an effect that is at least partially mediated by cytotoxic T cells (see, for example, Johnson et al. Clin Cancer Res. 21:1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Res. 19:1035-43, 2013). General examples of CD40 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD40 and increase one or more of its immunostimulatory activities. Specific examples include CP-870,893, dacetuzumab, Chi Lob 7 / 4, ADC-1013, CD40L, rhCD40L, and antigen-binding fragments thereof. Specific examples of CD40 agonists include, but are not limited to, APX005 (see, e.g., U.S. Publication No. 2012 / 0301488) and APX005M (see, e.g., U.S. Publication No. 2014 / 0120103).

[0195] In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotes the expansion of effector and memory T cells and inhibits the differentiation and activity of T regulatory cells (see, for example, Croft et al., Immunol Rev.229:173-91,2009). Its ligand is OX40L (CD252). OX40 signaling plays an important role in initiating anti-tumor immune responses in lymph nodes and maintaining anti-tumor immune responses in tumor microenvironment, as it affects both T cell activation and survival. Common examples of OX40 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40 and increase one or more of its immunostimulatory activities. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (humanized OX40 agonist), MEDI6469 (murine OX40 agonist), and MEDI6383 (OX40 agonist), and antigen-binding fragments thereof.

[0196] In some embodiments, the agent is a GITR agonist. Glucocorticoid-induced TNFR family related gene (GITR) increases T cell expansion, inhibits the suppressive activity of Treg, and prolongs the survival of T effector cells. GITR agonists have been shown to promote anti-tumor responses through the loss of Treg lineage stability (see, for example, Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These diverse mechanisms indicate that GITR plays an important role in initiating immune responses in lymph nodes and maintaining immune responses in tumor tissues. Its ligand is GITRL. Common examples of GITR agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to GITR and increase one or more of its immunostimulatory activities. Specific examples include INCAGN01876, DTA-1, MEDI1873, and antigen-binding fragments thereof.

[0197] In some embodiments, the agent is a CD137 agonist. CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T cells, such as CD8+ T cells, from activation-induced cell death. Common examples of CD137 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137 and increase one or more of its immunostimulatory activities. Specific examples include the antibody utomirumab, which includes CD137 (or 4-1BB) ligands (see, e.g., Shao and Schwarz, J Leukoc Biol. 89:21-9, 2011) and antigen-binding fragments thereof.

[0198] In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific expansion of naive T cells and is involved in the long-term maintenance of T cell memory and T cell immunity. Its ligand is CD70. Targeting human CD27 with agonistic antibodies stimulates T cell activation and antitumor immunity (see, for example, Thomas et al., Oncoimmunology. 2014;3:e27255.doi:10.4161 / onci.27255; and He et al., J Immunol. 191:4174-83, 2013). Common examples of CD27 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD27 and increase one or more of its immunostimulatory activities. Specific examples include antibodies warilumab and CDX-1127 (1F5), which include CD70 and antigen-binding fragments thereof.

[0199] In some embodiments, the agent is a CD28 agonist. CD28 is constitutively expressed on CD4+ T cells, some CD8+ T cells. Its ligands include CD80 and CD86, and its stimulation increases T cell expansion. Common examples of CD28 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD28 and increase one or more of its immunostimulatory activities. Specific examples include CD80, CD86, antibody TAB08, and antigen-binding fragments thereof.

[0200] In some embodiments, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares a ligand with TIGIT, and CD226 engagement, as opposed to TIGIT, enhances T cell activation (see, for example, Kurtulus et al., J Clin Invest. 125:4053-4062, 2015; Bottino et al., J Exp Med. 1984:557-567, 2003; and Tahara-Hanaoka et al., Int Immunol. 16:533-538, 2004). Common examples of CD226 agonists include antibodies or antigen-binding fragments or small molecules or ligands (e.g., CD112, CD155) that specifically bind to CD226 and increase one or more of its immunostimulatory activities.

[0201] In some embodiments, the agent is an HVEM agonist. Herpes virus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF receptor superfamily. HVEM is found on various cells, including T cells, APCs, and other immune cells. Unlike other receptors, HVEM is expressed at high levels on resting T cells and is downregulated upon activation. HVEM signaling has been shown to play an important role in the early stages of T cell activation and in the expansion of tumor-specific lymphocyte populations in lymph nodes. Common examples of HVEM agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM and increase one or more of its immunostimulatory activities.

[0202] In certain embodiments, the anti-TNFR2 antibody disclosed herein is administered in combination with one or more bispecific or multispecific antibodies. For example, certain bispecific or multispecific antibodies can (i) bind to and inhibit one or more inhibitory immune checkpoint molecules, and (ii) bind to and stimulate one or more stimulatory immune checkpoint molecules. In certain embodiments, the bispecific or multispecific antibodies (i) bind to and inhibit one or more of PD-L1, PD-L2, PD-1, CTLA-4, IDO, TDO, TIM-3, LAG-3, BTLA, CD160, and / or TIGIT, and (ii) bind to and stimulate one or more of CD40, OX40 glucocorticoid-inducible TNFR family related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and / or herpes virus entry mediator (HVEM).

[0203] In some embodiments, the anti-TNFR2 antibodies disclosed herein are administered in combination with one or more cancer vaccines.In certain embodiments, the cancer vaccine is selected from one or more of Oncophage, human papillomavirus HPV vaccine, optionally Gardasil or Cervarix, Hepatitis B vaccine, optionally Engerix-B, Recombivax HB, or Twinrix, and Sipuleucel-T (Provenge), or a combination of human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33 , CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin phospho-αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SL and a cancer antigen selected from one or more of the following: AMF7), EGP40 pan-carcinoma antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death 1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis Y antigen, GD2 (disialoganglioside expressed on tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

[0204] In some embodiments, the anti-TNFR2 antibodies disclosed herein are administered in combination with one or more oncolytic viruses. In some embodiments, the oncolytic viruses are selected from one or more of Talimogene Laherparepvec (T-VEC), Coxsackievirus A21 (CAVATAK™), Oncoline (H101), Peraleorep (REOLYSIN®), Seneca Valley Virus (NTX-010), Seneca Virus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0205] In certain embodiments, the cancer immunotherapeutic agent is a cytokine. Exemplary cytokines include interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0206] In certain embodiments, the cancer immunotherapeutic agent is a cell-based immunotherapy, such as a T cell-based adoptive immunotherapy. In some embodiments, the cell-based immunotherapy comprises cancer antigen-specific T cells, optionally ex vivo derived T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-mediated T cells, and T cell receptor (TCR)-mediated T cells, tumor infiltrating lymphocytes (TIL), and peptide-induced T cells. In certain embodiments, the CAR-modified T cells are targeted against CD-19 (see, e.g., Maude et al., Blood. 125:4017-4023, 2015).

[0207] In some embodiments, the anti-TNFR2 antibodies disclosed herein are used as part of an adoptive immunotherapy, e.g., an autoimmune therapy. Accordingly, certain embodiments include a method of treating cancer in a patient in need thereof, the method comprising:

[0208] (a) incubating the ex vivo derived immune cells with an anti-TNFR2 antibody, or antigen-binding fragment thereof, as described herein; and

[0209] (b) Administering autoimmune cells to the patient.

[0210] In some cases, the ex vivo derived immune cells are autologous cells obtained from the patient to be treated. In some embodiments, the autologous immune cells include lymphocytes, natural killer (NK) cells, macrophages, and / or dendritic cells (DCs). In some embodiments, the lymphocytes include T cells, optionally cytotoxic T lymphocytes (CTLs). For a description of adoptive T cell and NK cell immunotherapy, see, e.g., June, J Clin. Invest.117:1466-1476,2007;Rosenberg and See Restifo, Science. 348:62-68, 2015; Cooley et al., Biol. of Blood and Marrow Transplant. 13:33-42, 2007; and Li and Sun, Chin J Cancer Res. 30:173-196, 2018. In some embodiments, the T cells comprise cancer antigen-specific T cells directed against at least one "cancer antigen" described herein. In certain embodiments, the anti-TNFR2 antibody, or antigen-binding fragment thereof, enhances the efficacy of adoptively transferred immune cells.

[0211] In certain embodiments, the anti-TNFR2 antibodies disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodepa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenetriphosphaolamide, and trimethylolmelamine; chlorambucil, chlornaphazine. , nitrogen mustards such as chlorophosphatamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosuureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, and caliche mycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin s, peplomycin, potofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex , zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU;Androgens such as calcineurin, dromostanol propionate, epithiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopida mol; nitracrine; pentostatin; phenameth; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (Taxol®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (Taxotere®, Rhne-Poulenc Rorer, Antony, France);Chlorambucil;Gemcitabine;6-Thioguanine;Mercaptopurine;Methotrexate;Platinum analogues such as cisplatin and carboplatin;Vinblastine;Platinum;Etoposide (VP-16);Ifosfamide;Mitomycin C;Mitoxantrone;Vincristine;Vinorelbine;Navelbine;Novantrone;Teniposide;Daunomycin;Aminopterin;Xeloda;Ibandronate;CPT-11;Topoisomerase inhibitors RFS2000;Difluoromethylmycin (DMFO);Retinoic acid derivatives such as Targretin™ (Bexarotene), Panretin™ (Alitretinoin);ONTAK™ (Denileukin diftitox);Esperamicin;Capecitabine;Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onapristone, and toremifene (falcetone); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and also pharmaceutically acceptable salts, acids, or derivatives of any of the above.;

[0212] Various other therapeutic agents may be used together with the anti-TNFR2 antibody described herein.In some embodiments, the antibody is administered together with an anti-inflammatory agent.Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolate.

[0213] Exemplary NSAIDs are selected from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib), and sialic acid adducts. Exemplary analgesics are selected from the group consisting of acetaminophen, oxycodone, tramadol of proporoxifene hydrochloride. Exemplary glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0214] In certain embodiments, the antibodies described herein are administered in conjunction with a cytokine. As used herein, "cytokine" refers to a collective term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and classical polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and beta; Mullerian inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factor, such as NGF-beta; platelet growth factor; transforming factor, such as TGF-alpha and TGF-beta. Examples of cytokines include transforming growth factor (TGF); insulin-like growth factor I and II; erythropoietin (EPO); osteoinductive factor; interferons such as interferon-alpha, beta, and gamma; colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; interleukins (ILs) such as IL-15, tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or recombinant cell culture, and biologically active equivalents of the native sequence cytokines.

[0215] In some embodiments, the compositions comprising the TNFR2-specific antibodies described herein are administered to individuals suffering from diseases described herein, including, but not limited to, cancer, inflammatory diseases, and autoimmune diseases. Cancers include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colon, gastrointestinal, prostate, bladder, renal, ovarian, cervical, breast, lung, nasopharyngeal carcinoma, and malignant melanoma, among others. Thus, certain embodiments include a method of treating a patient with cancer, comprising administering to the patient a composition described herein, thereby treating the cancer. In some embodiments, the cancer is associated with abnormal TNFR2 expression and / or TNFR2 antagonist-mediated immune suppression. In certain embodiments, the antibody for use in treating cancer is a TNFR2 antagonist.

[0216] In some embodiments, the inflammatory disease or autoimmune disease is associated with abnormal TNFR2 expression. In some embodiments, the inflammatory disease or autoimmune disease is associated with TNFR2 agonist-mediated immune activation. Exemplary autoimmune diseases include arthritis (including rheumatoid arthritis, reactive arthritis), systemic lupus erythematosus (SLE), psoriasis and inflammatory bowel disease (IBD), encephalomyelitis, uveitis, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Addison's disease, celiac disease, chronic fatigue syndrome, autoimmune hepatitis, autoimmune alopecia, ankylosing spondylitis, ulcerative colitis, Crohn's disease, fibromyalgia, pemphigus vulgaris, Sjogren's syndrome, Kawasaki disease, hyperthyroidism / Graves' disease, hypothyroidism / Hashimoto's disease, endometriosis, scleroderma, pernicious anemia, Goodpasture's disease, and others. syndrome, Guillain-Barre syndrome, Wegener's disease, glomerulonephritis, aplastic anemia (including multiple transfused aplastic anemia patients), paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Evans syndrome, Factor VIII inhibitor syndrome, systemic vasculitis, dermatomyositis, polymyositis and rheumatic fever, autoimmune lymphoproliferative syndrome (ALPS), autoimmune bullous pemphigoid, Parkinson's disease, sarcoidosis, vitiligo, primary biliary cirrhosis, and autoimmune myocarditis.

[0217] Exemplary inflammatory diseases include, but are not limited to, Crohn's disease, colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematosus, nephritis, Parkinson's disease, ulcerative colitis, multiple sclerosis (MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma, and various cardiovascular diseases such as atherosclerosis and vasculitis.In certain embodiments, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, diabetes, gout, cryopyrin-associated periodic syndrome, and chronic obstructive pulmonary disorder.In certain embodiments, the antibody for use in treating inflammatory disease or autoimmune disease is a TNFR2 agonist.

[0218] For example, certain embodiments provide a method of treating an inflammatory disease or reducing the severity of an inflammatory disease by administering to a patient in need thereof a therapeutically effective amount of a composition disclosed herein comprising an agonist anti-TNFR2 antibody. Some embodiments provide a method of treating graft-versus-host disease or reducing the severity of graft-versus-host disease by administering to a patient in need thereof a therapeutically effective amount of a composition disclosed herein comprising an agonist anti-TNFR2 antibody. Some embodiments provide a method of treating graft rejection or reducing the severity of graft rejection or preventing graft rejection by administering to a patient in need thereof a therapeutically effective amount of a composition disclosed herein comprising an agonist anti-TNFR2 antibody.

[0219] Certain embodiments provide methods of treating, reducing the severity of, or preventing an infectious disease by administering to a patient in need thereof a therapeutically effective amount of a composition disclosed herein comprising an agonist anti-TNFR2 antibody, including, but not limited to, a viral infection, a bacterial infection, a fungal infection, optionally a yeast infection, and a protozoal infection.

[0220] For in vivo use for the treatment of human disease, the antibodies described herein are generally incorporated into pharmaceutical compositions prior to administration. The pharmaceutical compositions include one or more of the antibodies described herein in combination with physiologically acceptable carriers or excipients described elsewhere herein. To prepare pharmaceutical compositions, an effective amount of one or more compounds is mixed with any pharmaceutical carrier or excipient known to those skilled in the art to be suitable for a particular mode of administration. Pharmaceutical carriers may be liquid, semi-liquid, or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application may contain, for example, a sterile diluent (e.g., water), saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents (e.g., benzyl alcohol and methylparabens); antioxidants (e.g., ascorbic acid and sodium sulfite) and chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); buffers (e.g., acetates, citrates, and phosphates). If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening agents and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0221] Compositions comprising the TNFR2-specific antibodies described herein may be prepared with carriers that protect the antibodies against rapid elimination from the body, such as time-release formulations or coatings.Such carriers include, but are not limited to, controlled release formulations, such as implants and microencapsulated delivery systems, and biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and others known to those skilled in the art.

[0222] Therapeutic methods using antibodies that bind to TNFR2 are provided herein. In one embodiment, the antibody of the present disclosure is administered to patients with diseases involving inappropriate expression of TNFR2, which in the context of the present disclosure means to include diseases and disorders characterized by abnormal TNFR2 expression or activity, for example, due to the amount of protein present, or the presence of mutant proteins, or both being altered (e.g., statistically significant increase or decrease). The excess can be due to any cause, including, but not limited to, overexpression at the molecular level of TNFR2, prolonged or accumulated appearance at the site of action, or increased (e.g., in a statistically significant manner) activity, compared to what is normally detectable. Such excess of TNFR2 can be measured compared to the normal expression, appearance, or activity of TNFR2 signaling events, and such measurement can play an important role in the development and / or clinical testing of the antibodies described herein.

[0223] In particular, the antibody is useful for treating various cancers, including cancers associated with TNFR2 expression or overexpression.For example, certain embodiments provide a method for treating cancer, including but not limited to non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colonic, gastrointestinal, prostate, bladder, renal, ovarian, cervical, breast, lung, nasopharyngeal carcinoma, and malignant melanoma, by administering a therapeutically effective amount of the TNFR2-specific antibody disclosed herein to cancer patients.The amount that inhibits, prevents, or delays the progression and / or metastasis of cancer in a statistically significant manner (i.e., compared to a suitable control known to those skilled in the art) after administration is considered to be effective.

[0224] Some embodiments provide a method of reducing or preventing metastasis of cancer, including, but not limited to, non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colonic, gastrointestinal, prostate, bladder, renal, ovarian, cervical, breast, lung, nasopharyngeal carcinoma, and malignant melanoma, by administering to a cancer patient a therapeutically effective amount of a TNFR2-specific antibody disclosed herein (e.g., an amount that inhibits, prevents, or delays metastasis of the cancer in a statistically significant manner following administration, i.e., compared to a suitable control known to one of skill in the art).

[0225] Some embodiments provide a method for preventing cancer, including but not limited to non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colonic, gastrointestinal, prostate, bladder, renal, ovarian, cervical, breast, lung, nasopharyngeal carcinoma, and malignant melanoma, by administering to a cancer patient a therapeutically effective amount of a TNFR2-specific antibody disclosed herein.

[0226] Some embodiments provide methods of treating, inhibiting the progression of, or preventing non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colonic, gastrointestinal, prostate, bladder, renal, ovarian, cervical, breast, lung, nasopharyngeal carcinoma, or malignant melanoma by administering a therapeutically effective amount of a TNFR2-specific antibody disclosed herein to a patient suffering from one or more of these diseases.

[0227] In some embodiments, an anti-TNFR2 antibody can be used to determine the structure of the bound antigen, e.g., a conformational epitope, which can then be used to develop compounds that have or mimic this structure, e.g., through chemical modeling and SAR methods.

[0228] Some embodiments relate in part to diagnostic applications for detecting the presence of cells or tissues expressing TNFR2.Thus, the present disclosure provides a method for detecting TNFR2 in a sample, such as detecting cells or tissues expressing TNFR2.Such methods can be applied in various known detection formats, including but not limited to immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), whole mount in situ hybridization (WISH), fluorescent DNA in situ hybridization (FISH), flow cytometry, enzyme immunoassay (EIA) and enzyme-linked immunoassay (ELISA).

[0229] ISH is a type of hybridization that uses labeled complementary DNA or RNA strands (i.e., primary binders) to localize specific DNA or RNA sequences in parts or sections of cells or tissues (in situ), or in whole tissues (whole mount ISH) if the tissue is small enough. Those skilled in the art will understand that this is distinct from immunohistochemistry, which uses antibodies as primary binders to localize proteins in tissue sections. DNA ISH can be used with genomic DNA to determine the structure of chromosomes. Fluorescent DNA ISH (FISH) can be used, for example, in medical diagnostics to evaluate the integrity of chromosomes. RNA ISH (hybridization immunohistochemistry) is used to measure and localize mRNA and other transcripts in tissue sections or whole mounts.

[0230] In various embodiments, the antibody described herein is conjugated to a detectable label that can be directly or indirectly detected. In this regard, antibody "conjugate" refers to an anti-TNFR2 antibody that is covalently bound to a detectable label. In the present disclosure, DNA probes, RNA probes, monoclonal antibodies, antigen-binding fragments thereof, and antibody derivatives thereof, such as single-chain variable fragment antibodies or epitope-tagged antibodies, may be covalently bound to a detectable label. In "direct detection", only one detectable antibody, i.e., the primary detectable antibody, is used. Thus, direct detection means that the antibody conjugated to a detectable label can be detected by itself without the need to add a second antibody (secondary antibody).

[0231] A "detectable label" is a molecule or substance that can produce a detectable (e.g., visual, electronic or other) signal that indicates the presence and / or concentration of the label in a sample. When conjugated to an antibody, a detectable label can be used to locate and / or quantify the target to which a particular antibody is directed. Thus, the presence and / or concentration of a target in a sample can be detected by detecting the signal generated by the detectable label. A detectable label can be detected directly or indirectly, and several different detectable labels conjugated to different specific antibodies can be combined to detect one or more targets.

[0232] Examples of detectable labels that can be directly detected include fluorescent dyes and radioactive substances and metal particles. In contrast, indirect detection requires the application of one or more additional antibodies, i.e., a secondary antibody after the application of a primary antibody. Thus, detection is performed by detecting the binding of a secondary antibody or binder to a primary detectable antibody. Examples of primary detectable binders or antibodies that require the addition of a secondary binder or antibody include enzyme-detectable binders and hapten-detectable binders or antibodies.

[0233] In some embodiments, the detectable label is conjugated to a nucleic acid polymer that comprises a first binding agent (e.g., in an ISH, WISH, or FISH process). In certain embodiments, the detectable label is conjugated to an antibody that comprises a first binding agent (e.g., in an IHC process).

[0234] Examples of detectable labels that can be conjugated to antibodies used in the methods of the present disclosure include fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, bioluminescent labels, polymers, polymeric particles, metal particles, haptens, and dyes.

[0235] Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamidohexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optionally substituted coumarins including AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeton Red, green fluorescent protein (GFP) and analogs thereof, and conjugates of R-phycoerythrin or allophycoerythrin, inorganic fluorescent labels such as particles based on semiconductor materials, such as coated CdSe nanocrystals.

[0236] Examples of polymeric particle labels include microparticles or latex particles of polystyrene, PMMA or silica that can be embedded with fluorescent dyes, or polymeric micelles or capsules containing dyes, enzymes or substrates.

[0237] Examples of metal particle labels include gold particles and coated gold particles that can be converted by silver staining. Examples of haptens include DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin. Examples of enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosamimidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO). Examples of commonly used substrates for horseradish peroxidase are 3,3'-diaminobenzidine (DAB), diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), Hankel-Yates reagent (HYR), indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), alpha-naphtholpyrroline (alpha-Naphtholpyrroline (NaP)), and alpha-naphtholpyrroline (NAP). .-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolylphospho-chelate (BCIP), nitro blue tetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT), tetranitro blue tetrazolium (TNBT), 5-bromo-4-chloro-3-indoxy-l-beta-D-galactoside / ferro-ferricyanide (BCIG / FF).

[0238] Examples of commonly used substrates for alkaline phosphatase include naphthol-AS-B1-phosphate / Fast Red TR (NABP / FR), naphthol-AS-MX-phosphate / Fast Red TR (NAMP / FR), naphthol-AS-B1-phosphate / -Fast Red TR (NABP / FR), naphthol-AS-MX-phosphate / Fast Red TR (NAMP / FR), naphthol-AS-B1-phosphate / Neufustin (NABP / NF), bromochloroindolyl phosphate / nitro blue tetrazolium (BCIP / NBT), 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside (BCIG).

[0239] Examples of luminescent labels include luminol, isoluminol, acridinium esters, 1,2-dioxetanes, and pyridopyridazines. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels include radioisotopes of iodide, cobalt, selenium, tritium, carbon, sulfur, and phosphorous.

[0240] The detectable label may be linked to the antibody described herein or other molecules that specifically bind to the biological marker of interest, such as antibodies, nucleic acid probes, or polymers. In addition, those skilled in the art will understand that the detectable label may also be conjugated to a second, and / or third, and / or fourth, and / or fifth binding agent or antibody, etc. In addition, those skilled in the art will understand that each binding agent or antibody used to characterize the biological marker of interest may act as a signal amplification step. The biological marker may be visually detected, for example, using a light microscope, a fluorescent microscope, an electron microscope, and the detectable substance is, for example, a dye, a colloidal gold particle, a luminescent reagent. The visually detectable substance bound to the biological marker may also be detected using a spectrophotometer. If the detectable substance is a radioisotope, detection may be visual by autoradiography or non-visual using a scintillation counter. For example, Larsson, 1988, Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.); Methods in Molecular Biology, vol. 80 1998, John D. Pound (ed.) (Humana Press, Totowa, NJ.

[0241] The present disclosure further provides kits for detecting TNFR2 or cells or tissues expressing TNFR2 in a sample, the kits containing at least one antibody, polypeptide, polynucleotide, vector or host cell described herein. In certain embodiments, the kits may include buffers, enzymes, labels, substrates, beads or other surfaces to which the antibodies of the present disclosure bind, and the like, as well as instructions for use. EXAMPLES

[0242] Example 1 Immunotherapy and Primary Screening To prepare antibodies for screening, four New Zealand White rabbits were immunized and then boosted with either human 293-TNFR2 overexpressing cells or human TNFR2-Fc fusion protein. All rabbits had serum titers specific for human TNFR2, which were used to generate hybridomas using APXiMAB™ technology and antibody by B cell culture method (RevMAb). During primary screening, 460 antibodies were identified that bound to CHO-TNFR2 overexpressing cells. The screening process to identify potential lead candidates is shown in Figure 2.

[0243] Hybridoma and B cell culture supernatants were then used to screen for antibodies that could block TNF-α binding to soluble TNFR2-His (Sino Biological; 10417-H08H) in an ELISA-based receptor-ligand binding assay. Of the 460 antibodies identified, 173 showed inhibition of TNF-α binding to TNFR2 by more than 75% of maximum binding.

[0244] 110 antibodies were selected to proceed to the human IgG chimera stage. Of the 110 antibodies that progressed from B cell cloning, 28 clones failed to amplify. The heavy and light chains of the remaining 82 clones were amplified and directly cloned into a human IgG1 backbone. The 82 chimeric antibodies expressed and supernatants that tested positive for binding to cell-based TNFR2 were sequenced. All 10 antibodies identified from hybridomas that passed the initial screening funnel were chimerized, sequenced, and progressed further.

[0245] Using sequence analysis (uniqueness and limited potential liabilities such as glycosylation, deamination, and CDR3 length), 25 antibodies were selected from the B cell cultures for further study. All 10 hybridoma clones were carried forward independently of sequence analysis. Two sets of hybridoma-derived clones had identical sequences as shown in the summary table (Table E1) with asterisks or hashtags (8G11.5=37D1.4 and 28B7.3=37H4.1). To express recombinant chimeric antibodies, heavy (H) and light (L) chain plasmids were co-transfected into 293 cells and supernatants were harvested after 7-10 days. Antibodies were purified through a protein A column and dialyzed against PBS.

[0246] In Vitro Characterization of Human Chimeric Antibodies Chimeric antibodies were screened for binding to soluble and cell-expressed human TNFR2, soluble cynomolgus and mouse TNFR2, and ELISA-based TNF-α blockade. The data sets in Figures 3A-3D show the first set of antibodies, and the data sets in Figures 4A-4D show the second set of antibodies. Antibodies were also screened in peptide binding ELISAs for preliminary epitope binding using peptides from the PLAD or CRD1 (aa17-54; TCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCD), CRD2 (aa58-93; DSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQN), CRD3 (aa106-133; LSKQEGCRLCAPLRKCRPGFGVARPGTE and aa114-133; LCAPLRKCRPGFGVARPGTE) and CRD4 (peptide 5; aa146-174; and TFSNTTSSTDICRPHQICNVVAIPGNAS) domains (see data summary in Table E1 below). [Table E1-1] [Table E1-2]

[0247] Fifteen of the 35 antibodies were selected for humanization based on binding affinity, cynomolgus cross-reactivity, blocking potency, and ability to capture broad epitope bins (highlighted in bold in Table E1). Of the 15 humanized clones, three clones did not block TNF-α binding by ELISA, but were hypothesized to block TNF-α on the cell surface by binding to peptide 5 and blocking TNFR2 trimerization. Clones with identical sequences that did not bind strongly to cynomolgus or human TNFR2 or block TNF-α binding were deselected.

[0248] The antibodies were humanized using a proprietary Mutation Lineage Guidance (MLG) technology on a human IgG1 framework. One antibody out of 15 lost binding to the antigen in ELISA and could not be recovered. The other 14 antibodies were screened for soluble human TNFR1 and TNFR2 binding, cell-based TNFR2 binding, cynomolgus monkey TNFR2 binding, and TNF-α blockade by both ELISA and FACS (see Figures 5-8). Antibodies that bound peptide 5 and did not block TNF-α at the chimeric stage did not block TNF-α binding by cell-based ELISA and were therefore deselected. Antibodies that retained strong, cell-based TNFR2 binding were tested for ADCC of TNFR2-overexpressing CHO cell lines using the Promega ADCC Jurkat NFAT reporter kit (Figures 9A-9B). Characteristics of the humanized candidates are shown in Table E2 below. Five lead candidates (highlighted in bold) met the desired criteria. [Table E2-1] [Table E2-2]

[0249] Selection of Lead Candidates and Back-Up ADCC assays on overexpressing cell lines were completed along with TNFR family member specificity, cytokine release assays, and developability analysis to aid in the selection of two lead candidates to proceed to CLD. Table E3 below shows the percent humanization analysis. [Table E3]

[0250] Example 2 Binding and activity characteristics of clones h600-25-71 and h600-25-108 Humanized clones h600-25-71 and h600-25-108 were tested for binding and activity characteristics. To test binding to TNFR family members, target proteins were coated on ELISA plates at 1 μg / mL overnight at 4° C. Plates were washed, blocked, and antibodies were added at the indicated concentrations for 1 hour at room temperature. Positive control antibodies for each protein were used at approximately 1 μg / mL. Antibodies were washed and detected with anti-human IgG HRP for 1 hour. Assays were developed using TMB substrate for 10 minutes. For cell binding, human CD4+ T cells purified from buffy coats were cultured with anti-CD3 / CD28 in flat-bottom plates for 24 hours. Cells were harvested and stained for viability, CD4, CD25, and FOXP3. Anti-human IgG APC was used to detect test antibody staining. Binding titration of test antibodies was assessed on CD4+CD25hiFOXP3+ regulatory T cells and plotted as percent binding.

[0251] The results in Figures 10A-10F show that 25-71 and 25-108 inhibited the expression of human TNFR2 protein (10C), cynomolgus monkey TNFR2 protein (10D), cell-expressed human TNFR2 (10E), and activated human T reg(10F), all of which bind to TNFR2 with high affinity. The results in Figures 11A-11E show that 25-71 and 25-108 are specific for TNFR2 and do not bind to TNFR1, HVEM, CD40, DR6, or OPG. The IgG isotype control is indicated by a triangle and the positive control is indicated by an asterisk.

[0252] To test the effect of the antibodies on TNFR2-expressing cells via antibody-dependent cellular cytotoxicity (ADCC), an ADCC assay was performed as described above. As shown in Figures 12A-12B, 25-71 and 25-108 induced ADCC against TNFR2-expressing cells, including tumor cells (12B).

[0253] T reg PBMCs were isolated from the leukocyte reduction chamber to test the effect of antibodies against 2 × 10 5 PBMCs were stimulated with 0.1 μg / mL anti-CD3 (clone: ​​OKT3) and treated with anti-TNFR2 antibody for 16-24 hours at 37°C in a humidified 5% CO2 incubator in a 96-well flat-bottom plate. Cells were cultured in complete RPMI (10% heat-inactivated FBS, 1x penicillin-streptomycin, 1 mM sodium pyruvate, 1x MEM non-essential amino acids, 50 mM β-mercaptoethanol). Cells were harvested and CD4 Tregs were identified as CD4+CD25hi FoxP3+ by flow cytometry. As shown in Figures 13A-13B, 25-71 and 25-108 were isolated from human PBMCs and treated with anti-TNFR2 antibody for 16-24 hours at 37°C in a humidified 5% CO2 incubator ... reg Both graphs show the average of 5 donors, each condition performed in duplicate. % Depletion = 100 x (no depletion) * -Experimental) / (No depletion), no depletion refers to PBMC stimulated with anti-CD3 only.

[0254] To test the effect of antibodies on macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) of TNFR2-expressing tumor cells (see FIG. 14A), macrophages were generated by culturing human CD14+ monocytes with 10 μg / mL M-CSF in a 5% CO2, 37°C incubator for 6 days. The resulting macrophages were labeled with CellTrace CFSE and Violet, respectively, along with TNFR2-overexpressing CHO cells (targets). Target cells were incubated with anti-TNFR2 antibodies or isotype controls for 30 minutes on ice. The labeled cells were co-cultured for 3 hours in RPMI with low human IgG serum at a ratio of 100,000 macrophages to 50,000 target cells. The cells were analyzed using a Cytoflex flow cytometer. As shown in FIG. 14B, 25-71 and 25-108 induced macrophage-mediated ADCP of TNFR2+ cells. Percent phagocytosis was calculated as follows: ((% double positive cells) / (total % of target cells)×100).

[0255] To test the effect of antibodies on myeloid-derived suppressor cell (MDSC) suppression of CD8 T cells, MDSC were generated by co-culture of human PBMC with 786-O tumor cell line for 7 days. CD33+MDSC were isolated by positive selection using microbeads (Miltenyi). Autologous CD8 T cells were labeled with cell tracing violet and co-cultured with MDSC at a ratio of 4:1 with CD3 / CD28 stimulation for 3 days. After 3 days, cells were labeled with viability dye and CD8 T cell proliferation was measured using violet dye dilution. See experimental summary in Figure 15A.

[0256] As shown in Figures 15B-15C, 25-71 and 25-108 significantly reduced MDSC suppression of CD8 T cells. Percent suppression was calculated as follows: [T cells alone-(T+MDSC)] / T alone x 100. Data are plotted as mean ± SEM. IgG1 isotype is plotted using black bars, 25-71 is grey bars, and 25-108 is open bars. Statistics are presented by ANOVA as well as* = P < 0.01 and ** = P < 0.001.

[0257] To further test the effect of the antibodies on regulatory T cells, CD4+ T cells were isolated from PBMCs derived from healthy human buffy coats by negative selection using magnetic beads (Miltenyi). reg were depleted using CD25 magnetic beads according to the manufacturer's protocol (Miltenyi), and the resulting CD4+ cells (T responder cells) were cryopreserved until the assay was set up. Tregs from autologous donors were isolated according to the manufacturer's protocol, and T reg Expander beads (DynaBeads) and 100 nM rapamycin were used for 15 days to expand. The day before the assay was set up, T responder cells were thawed and left overnight. The following day, T responder cells were labeled with Cell Trace Violet and T reg were added at the indicated ratios along with 10 μg / mL 25-71 or isotype control in round-bottom plates. reg Test beads (Miltenyi) were added and the assay was incubated for 5 days. On day 5, cells were harvested, stained with a viability dye, and analyzed on a MACSQuant analyzer. Percent T cell suppression was calculated as (proliferation of stimulated T responders only - proliferation of test value) / (stimulated T responders only). Experiments were performed on at least four donors. As shown in Figures 18A-18E, clone 25-71 suppressed the T cell proliferation of effector T cells. reg Reverse inhibition.

[0258] Binding affinity (KD) and EC 50 The values ​​are summarized in Table E4 below. Table E4

[0259] To test the effect of the antibodies in mice, female nude mice were injected with Colo205 tumor cells SQ. At a tumor volume of 100 mm3, mice were treated as shown in Figure 19A. A significant antitumor effect (48% TGI) was observed with antibody 25-71 compared to the control (see Figure 19B). No changes in body weight were observed in the treatment group compared to the control group (see Figure 19C).

[0260] Example 3 Identification of the epitope site of clone 25-71 Experiments were performed to determine the epitopes of the TNFR2 / 25-71 complex with high resolution.

[0261] First, high-mass MALDI analysis was performed on samples of TNFR2 alone and clone 25-71 alone to confirm the integrity and aggregation levels. Measurements were performed using an Autoflex II MALDI ToF mass spectrometer (Bruker) equipped with an HM4 interaction module (CovalX) with a detection system designed to optimize detection up to 2 Mda with nanomolar sensitivity. TNFR2 sample powder was dissolved in distilled water to a concentration of 1 mg / ml, and 20 μl of each protein sample of TNFR2 and clone 25-71 were pipetted to prepare eight dilutions in a final volume of 10 μl. Next, 1 μl of each dilution was mixed with 1 μl of substrate consisting of sinapinic acid substrate (10 mg / ml) recrystallized in acetonitrile / water (1:1, v / v), 0.1% TFA (K200 MALDI kit). After mixing, 1 μl of each sample was spotted onto a MALDI plate (SCOUT 384). After crystallization at room temperature, the plate was introduced into the MALDI mass spectrometer and immediately analyzed in high-mass MALDI mode.

[0262] Cross-linking experiments. Cross-linking experiments allow direct analysis of non-covalent interactions by high-mass MALDI mass spectrometry. By mixing protein samples containing non-covalent interactions with a cross-linking mixture (Bich et al., Anal. Chem. 82 (1), pp 172-179, 2010), it is possible to specifically detect non-covalent complexes with high sensitivity. The resulting covalent bonds allow interacting species to survive the sample preparation process and MALDI ionization. High-mass detection systems allow characterization of interactions in the high mass range.

[0263] Each mixture (leaving 9 μl) prepared for the control experiment was cross-linked using the K200 MALDI MS analysis kit (CovalX). 9 μl (1-1 / 128) of the mixture was mixed with 1 μl of K200 stabilizer reagent (2 mg / ml) and incubated at room temperature. After the incubation time (180 min), the samples were prepared for MALDI analysis as in the control experiment. The samples were analyzed by high-mass MALDI analysis immediately after crystallization using an HM4 interaction module (CovalX) with a standard nitrogen laser, focusing on different mass ranges from 0 to 1500 kDa.

[0264] Results: High-mass MALDI mass spectrometry and chemical cross-linking analyses did not detect non-covalent aggregates of clone 25-71 or multimers of TNFR2.

[0265] Then, Autoflex II with HM4 interaction module (CovalX) The TNFR2 / 25-71 complex was characterized using a MALDI ToF mass spectrometer (Bruker). 10 μl of TNFR2 / 25-71 mixture was prepared at respective concentrations of 1.25 μM / 0.5 μM. 1 μl of the mixture was mixed with 1 μl of substrate consisting of sinapinic acid substrate (10 mg / ml) recrystallized in acetonitrile / water (1:1, v / v), 0.1% TFA (K200 MALDI kit). After mixing, 1 μl of each sample was spotted onto a MALDI plate (SCOUT 384). After crystallization at room temperature, the plate was introduced into the MALDI mass spectrometer and analyzed immediately.

[0266] Cross-linking experiment. The mixture prepared for the control experiment (leaving 9 μl) was Cross-linking was performed using a MS analysis kit (CovalX). 9 μl of the mixture was mixed with 1 μl of K200 stabilizer reagent (2 mg / ml) and incubated at room temperature. After the incubation period (180 min), samples were prepared for MALDI analysis as in the control experiment. Samples were analyzed by high-mass MALDI analysis immediately after crystallization.

[0267] MALDI ToF MS analysis was performed using an HM4 interaction module (CovalX) with a standard nitrogen laser, focusing on different mass ranges from 0 to 1500 kDa.

[0268] Results: For the control experiment, TNFR2 and clone 25-71 were detected at MH+=37.179 kDa and MH+=147.708 kDa. After cross-linking, two additional peaks were detected at MH+=189.401 kDa and MH+=228.341 kDa. Using the complex tracker software, the control and cross-linking spectra were overlaid and two non-covalent protein complexes with MH+=186.113 kDa and MH+=224.384 kDa were detected.

[0269] For epitope determination, TNFR2 was first subjected to trypsin, chymotrypsin, Asp-N, elastase, and thermolysin proteolysis, followed by nLC-LTQ-Orbitrap MS / MS analysis. Composite peptide mapping confirmed coverage of approximately 94.57% of the TNFR2 sequence (data not shown).

[0270] To determine the epitopes of the TNFR2 / 25-71 complex at high resolution, the complex was incubated with a deuterated cross-linker and subjected to multiple enzymatic cleavage with trypsin, chymotrypsin, Asp-N, elastase, and thermolysin. After enrichment of the cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry (nLC-LTQ-Orbitrap MS) and the generated data were analyzed using XQuest and Stavrox software.

[0271] Reductive alkylation. 20 μL of TNFR2 / clone 25-71 mixture was mixed with 2 μL of DSS d0 / d12 (2 mg / mL; DMF) for an incubation time of 180 min at room temperature. After incubation, the reaction was stopped by adding 1 μL of ammonium bicarbonate (20 mM final concentration) followed by incubation at room temperature for 1 h. The solution was then dried using a speedvac before H2O 8 M urea suspension (20 μL). After mixing, 2 μl of DTT (500 mM) was added to the solution. The mixture was then incubated at 37° C. for 1 h. After incubation, 2 μl of iodoacetamide (1 M) was added followed by incubation at room temperature for 1 h in the dark. After incubation, 80 μl of proteolysis buffer was added. Trypsin buffer contains 50 mM Ambic pH 8.5, 5% acetonitrile, chymotrypsin buffer contains Tris HCl 100 mM, CaCl2 10 mM pH 7.8, ASP-N buffer contains phosphate buffer 50 MM pH 7.8, elastase buffer contains Tris HCl 50 mM pH 8.0, thermolysin buffer contains Tris It contained HCl 50 mM, CaCl2 0.5 mM pH 9.0.

[0272] For trypsin proteolysis, 100 μl of the reduced / alkylated TNFR2 / 25-71 mixture was mixed with 0.5 μl of trypsin (Promega) at a ratio of 1 / 100, and the proteolytic mixture was incubated overnight at 37° C. For chymotrypsin proteolysis, 100 μl of the reduced / alkylated TNFR2 / 25-71 mixture was mixed with 0.25 μl of chymotrypsin (Promega) at a ratio of 1 / 200, and the proteolytic mixture was incubated overnight at 25° C. For ASP-N proteolysis, 100 μl of the reduced / alkylated TNFR2 / 25-71 mixture was mixed with 0.25 μl of ASP-N (Promega) at a ratio of 1 / 200, and the proteolytic mixture was incubated overnight at 37° C. For elastase proteolysis, 100 μl of the reduced / alkylated TNFR2 / 25-71 mixture was mixed with 0.5 μl of elastase (Promega) at a ratio of 1 / 100, and the proteolytic mixture was incubated overnight at 37° C. For thermolysin proteolysis, 100 μl of the reduced / alkylated TNFR2 / 25-71 mixture was mixed with 1 μl of thermolysin (Promega) at a ratio of 1 / 50, and the proteolytic mixture was incubated overnight at 70° C. After digestion, 1% formic acid was added to the solution. Cross-linked peptides were analyzed using Xquest version 2.0 and Stavrox 3.6 software.

[0273] Results. After trypsin, chymotrypsin, ASP-N, elastase, and thermolysin proteolysis of the TNFR2 / 25-71 complex with deuterated d0d12, nLC-orbitrap MS / MS analysis detected 12 cross-linked peptides between TNFR2 and clone 25-71. Chemical cross-linking, high-mass MALDI mass spectrometry, and nLC-Orbitrap mass spectrometry were used to characterize the molecular interface between TNFR2 and antibody 25-71. The results of this analysis are illustrated in Figure 16 and Figures 17A-17J, which show that the TNFR2 / 25-71 interaction involves the following residues in full-length human TNFR2; R43, Y45, T49, S55, K56, T73, and S77, or the following residues in mature human TNFR2; R21, Y23, T27, S33, K34, T51, and S55. The present invention provides, for example, the following items. (Item 1) 1. An isolated antibody, or antigen-binding fragment thereof, that binds to tumor necrosis factor receptor 2 (TNFR2), a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 1-3, respectively; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 4-6, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 7 to 9, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 10 to 12, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 13 to 15, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 16 to 18, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 19 to 21, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 22 to 24, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 25 to 27, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 28 to 30, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 31 to 33, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 34 to 36, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 37 to 39, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 40 to 42, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 43-45, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 46-48, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 49-51, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 52-54, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 55 to 57, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 58 to 60, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 61-63, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 64-66, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 67-69, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 70-72, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 73-75, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 76-78, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 79-81, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 82-84, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 85 to 87, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 88 to 90, respectively; a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 91-93, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 94-96, respectively; or a VH region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 97 to 99, respectively; and a VL region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 100 to 102, respectively; or an isolated antibody, or antigen-binding fragment thereof, comprising a variant of said antibody, or antigen-binding fragment thereof, comprising heavy and light chain variable regions identical to said heavy and light chain variable regions of (i) and (ii), except for up to 1, 2, 3, 4, 5, 6, 7, or 8 total amino acid substitutions across said CDR regions. (Item 2) 2. The isolated antibody or antigen-binding fragment thereof of item 1, wherein the VH region comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135. (Item 3) 3. The isolated antibody or antigen-binding fragment thereof according to item 1 or 2, wherein the VL region comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 134, and 136. (Item 4) a VH region set forth in SEQ ID NO: 103, and a VL region set forth in SEQ ID NO: 104; a VH region set forth in SEQ ID NO: 105, and a VL region set forth in SEQ ID NO: 106; a VH region set forth in SEQ ID NO: 107, and a VL region set forth in SEQ ID NO: 108; a VH region set forth in SEQ ID NO: 109, and a VL region set forth in SEQ ID NO: 110; a VH region set forth in SEQ ID NO:111, and a VL region set forth in SEQ ID NO:112; a VH region set forth in SEQ ID NO: 113, and a VL region set forth in SEQ ID NO: 114; a VH region set forth in SEQ ID NO: 115, and a VL region set forth in SEQ ID NO: 116; a VH region set forth in SEQ ID NO: 117, and a VL region set forth in SEQ ID NO: 118; a VH region set forth in SEQ ID NO: 119, and a VL region set forth in SEQ ID NO: 120; a VH region set forth in SEQ ID NO: 121, and a VL region set forth in SEQ ID NO: 122; a VH region set forth in SEQ ID NO: 123, and a VL region set forth in SEQ ID NO: 124; a VH region set forth in SEQ ID NO: 125, and a VL region set forth in SEQ ID NO: 126; a VH region set forth in SEQ ID NO: 127, and a VL region set forth in SEQ ID NO: 128; a VH region set forth in SEQ ID NO: 129, and a VL region set forth in SEQ ID NO: 130; a VH region set forth in SEQ ID NO: 131, and a VL region set forth in SEQ ID NO: 132; a VH region set forth in SEQ ID NO: 133, and a VL region set forth in SEQ ID NO: 134; or 4. The isolated antibody or antigen-binding fragment thereof according to item 2 or 3, comprising a VH region as set forth in SEQ ID NO: 135 and a VL region as set forth in SEQ ID NO: 136. (Item 5) 1. An isolated antibody, or antigen-binding fragment thereof, that binds to tumor necrosis factor receptor 2 (TNFR2), comprising a heavy chain variable (VH) region comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and a light chain variable (VL) region comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 134, and 136, respectively. (Item 6) An isolated antibody, or antigen-binding fragment thereof, that binds to tumor necrosis factor receptor 2 (TNFR2), comprising: a heavy chain variable (VH) region comprising a VHCDR1, VHCDR2, and VHCDR3 region selected from the underlined sequences in Table R1; and a light chain variable (VL) region comprising a VLCDR1, VLCDR2, and VLCDR3 region selected from the underlined sequences in Table R2. (Item 7) 7. The isolated antibody or antigen-binding fragment thereof of item 6, comprising a VH region comprising an amino acid sequence selected from Table R1, and a VL region comprising an amino acid sequence selected from Table R2, (Item 8) An isolated antibody, or antigen-binding fragment thereof, that binds to human tumor necrosis factor receptor 2 (TNFR2) at an epitope comprising, consisting of, or consisting essentially of one or more residues selected from R21, Y23, T27, S33, K34, T51, and S55, as defined by the mature human TNFR2 sequence (residues 23-461 of FL human TNFR2), optionally wherein the epitope comprises, consists of, or consists essentially of one or more residues selected from REY, TAQMCCSK (SEQ ID NO: 328), and TVCDS (SEQ ID NO: 329). (Item 9) 9. The isolated antibody or antigen-binding fragment thereof according to item 8, comprising a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs: 37 to 39, respectively; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs: 40 to 42, respectively. (Item 10) 10. The isolated antibody or antigen-binding fragment thereof of item 8 or 9, wherein the VH region comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 115. (Item 11) 11. The isolated antibody or antigen-binding fragment thereof according to any one of items 8 to 10, wherein the VL region comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 116. (Item 12) 12. The isolated antibody or antigen-binding fragment thereof according to item 10 or 11, comprising a VH region as set forth in SEQ ID NO: 115 and a VL region as set forth in SEQ ID NO: 116. (Item 13) 13. The isolated antibody, or antigen-binding fragment thereof, of any one of items 1 to 12, which binds to human TNFR2, optionally soluble TNFR2 and cell-expressed human TNFR2. (Item 14) 14. The isolated antibody or antigen-binding fragment thereof of item 13, which binds to at least one, two, three, four, or five human TNFR2 peptide epitopes selected from Table T1. (Item 15) 15. The isolated antibody of any one of items 1 to 14, wherein the antibody is humanized. (Item 16) 16. The isolated antibody of any one of items 1 to 15, wherein the antibody is selected from the group consisting of a single chain antibody, an scFv, a monovalent antibody lacking a hinge region, a minibody, and a probody. (Item 17) 16. The isolated antibody of any one of items 1 to 15, wherein the antibody is a Fab or Fab' fragment. (Item 18) The antibody is an F(ab') 2 16. The isolated antibody of any one of items 1 to 15, which is a fragment. (Item 19) 16. The isolated antibody of any one of items 1 to 15, wherein the antibody is a whole antibody. (Item 20) 20. The isolated antibody of any one of items 1 to 19, comprising a human IgG constant domain. (Item 21) 21. The isolated antibody of claim 20, wherein the IgG constant domain comprises an IgG1 CH1 domain. (Item 22) 21. The isolated antibody of item 20, wherein the IgG constant domain comprises an IgG1 Fc region, optionally modified with one or more amino acid substitutions, optionally a modified Fc region. (Item 23) K below approximately 2 nM D 23. The isolated antibody or antigen-binding fragment thereof according to any one of paragraphs 1 to 22, which binds to human TNFR2, optionally to at least one peptide epitope of Table T1. (Item 24) K below approximately 0.7 nM D binds to human TNFR2 with a K of approximately 50 pm or less D and primary T cells, optionally T reg 24. The isolated antibody, or antigen-binding fragment thereof, according to any one of items 1 to 23, which binds to human TNFR2. (Item 25) The isolated antibody, or antigen-binding fragment thereof, (a) inhibiting TNF-α binding to TNFR2; (b) inhibiting TNFR2 signaling; (c) activating TNFR2 signaling; (d) inhibiting TNFR2 dimerization / trimerization; (e) cross-reactive binding to human TNFR2 and cynomolgus monkey TNFR2; (f) Tumor cells, T reg , and / or increase / induce cell killing / depletion of suppressive myeloid cells (optionally macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs)); (g) Macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) of tumor cells, T reg , and / or increase / induce cell killing / depletion of suppressor myeloid cells (optionally macrophages, neutrophils, and MDSC); (h) reducing immune suppression by myeloid cells (optionally macrophages, neutrophils, and MDSCs); (i) converting MDSCs and / or M2 macrophages into pro-inflammatory M1 macrophages; (j) T to effector T cellsreg Convert; (k) converting a cold tumor into a hot tumor; (l) T reg reduce mediated immunosuppression; or (m) The isolated antibody, or antigen-binding fragment thereof, according to any one of items 1 to 24, which is any one or more combinations of (a) to (k). (Item 26) 26. The isolated antibody of any one of paragraphs 1 to 25, or an antigen-binding fragment thereof, which does not substantially bind to TNFR1, herpes virus entry mediator (HVEM, CD40, death receptor 6 (DR6), and / or osteoprotegerin (OPG). (Item 27) 27. The isolated antibody, or antigen-binding fragment thereof, of any one of items 1 to 26, which is a TNFR2 antagonist. (Item 28) 27. The isolated antibody, or antigen-binding fragment thereof, of any one of items 1 to 26, which is a TNFR2 agonist. (Item 29) 29. The isolated antibody, or antigen-binding fragment thereof, of any one of items 1 to 28, which is a bispecific or multispecific antibody. (Item 30) 30. An isolated polynucleotide encoding the isolated antibody, or antigen-binding fragment thereof, according to any one of items 1 to 29, an expression vector comprising the isolated polynucleotide, or an isolated host cell comprising the vector. (Item 31) 30. A composition comprising a physiologically acceptable carrier and a therapeutically effective amount of the isolated antibody, or antigen-binding fragment thereof, of any one of items 1 to 29. (Item 32) 32. A method of treating a patient having cancer, optionally a cancer associated with aberrant TNFR2 expression, comprising administering to said patient the composition of claim 31, thereby treating said cancer. (Item 33) 32. A method of treating a patient having cancer, optionally a cancer associated with TNFR2 antagonist-mediated immunosuppression, comprising administering to said patient a composition according to item 31, thereby treating said cancer. (Item 34) 34. The method of claim 32 or 33, wherein the antibody, or antigen-binding fragment thereof, is a TNFR2 antagonist. (Item 35) 23. A method for treating a patient having an inflammatory and / or autoimmune disease, comprising administering to said patient a composition according to item 231, thereby treating inflammation. (Item 36) 36. The method of claim 35, wherein the disease is associated with aberrant TNFR2 expression, and optionally the antibody, or antigen-binding fragment thereof, is a TNFR2 agonist. (Item 37) The method of claim 35, wherein the disease is associated with TNFR2 agonist-mediated immune activation.

Claims

1. An isolated antibody, or antigen-binding fragment thereof, that binds to tumor necrosis factor receptor 2 (TNFR2), comprising: a heavy chain variable (VH) region comprising the VHCDR1, VHCDR2, and VHCDR3 regions set forth in SEQ ID NOs:37-39, respectively; and a light chain variable (VL) region comprising the VLCDR1, VLCDR2, and VLCDR3 regions set forth in SEQ ID NOs:40-42, respectively.

2. An isolated antibody, or antigen-binding fragment thereof, comprising:

2. The isolated antibody, or antigen-binding fragment thereof, of claim 1, wherein the VH region comprises an amino acid sequence having at least 90% identity to the sequence set forth in SEQ ID NO:

115.

3. An isolated antibody, or an antigen-binding fragment thereof, described in claim 1 or 2, wherein the VL region comprises an amino acid sequence having at least 90% identity to the sequence set forth in SEQ ID NO:

116.

4. An isolated antibody, or an antigen-binding fragment thereof, described in claim 2 or 3, comprising a VH region described in SEQ ID NO: 115 and a VL region described in SEQ ID NO:

116.

5. An isolated antibody, or antigen-binding fragment thereof, that binds to tumor necrosis factor receptor 2 (TNFR2), comprising a heavy chain variable (VH) region having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:115, and a light chain variable (VL) region having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:116,

6. An isolated antibody, or an antigen-binding fragment thereof, described in any one of claims 1 to 5, which binds to human TNFR2, soluble human TNFR2, and cell-expressed human TNFR2.

7. The isolated antibody of claim 6, or an antigen-binding fragment thereof, which binds to at least one, two, three, four, or five human TNFR2 peptide epitopes comprising one or more residues selected from R21, Y23, T27, S33, K34, T51, and S55 as defined by the mature human TNFR2 sequence (residues 23-461 of FL human TNFR2), or comprising one or more residues selected from REY, TAQMCCSK (SEQ ID NO:328), and TVCDS (SEQ ID NO:329).

8. An isolated antibody according to any one of claims 1 to 7, wherein the antibody is humanized.

9. The isolated antibody of any one of claims 1 to 8, wherein the antibody is selected from the group consisting of scFvs, monovalent antibodies lacking a hinge region, minibodies, and probodies.

10. The isolated antibody of any one of claims 1 to 8, wherein the antibody is a Fab or Fab' fragment.

11. The isolated antibody of any one of claims 1-8, wherein the antibody is an F(ab') 2 fragment.

12. An isolated antibody described in any one of claims 1 to 8, wherein the antibody is a whole antibody.

13. An isolated antibody according to any one of claims 1 to 12, comprising a human IgG constant domain.

14. The isolated antibody of claim 13, wherein the IgG constant domain comprises an IgG1 CH1 domain.

15. The isolated antibody of claim 13, wherein the IgG constant domain comprises an IgG1 Fc region, a modified Fc region, or a modified Fc region modified with one or more amino acid substitutions.

16. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1 to 15, which binds to human TNFR2 with a K D of about 2 nM or less.

17. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1 to 16, which binds to human TNFR2 with a K D of about 0.7 nM or less, or binds to human TNFR2 on primary T cells or Tregs with a K D of about 50 pm or less.

18. The isolated antibody, or antigen-binding fragment thereof, (a) inhibiting TNF-α binding to TNFR2; (b) inhibiting TNFR2 signaling; (c) activates TNFR2 signaling; (d) inhibiting TNFR2 dimerization / trimerization; (e) cross-reactively binds to human TNFR2 and cynomolgus monkey TNFR2; (f) increasing / inducing cell killing / depletion of tumor cells, Tregs, and / or suppressive myeloid cells, including macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs), via antibody-dependent cellular cytotoxicity (ADCC); (g) increasing / inducing cell killing / depletion of tumor cells, Tregs, and / or suppressive myeloid cells, including macrophages, neutrophils, and MDSCs, via macrophage-mediated antibody-dependent cellular phagocytosis (ADCP); (h) reducing immunosuppression by myeloid cells, including macrophages, neutrophils, and MDSCs; (i) converting MDSCs and / or M2 macrophages into pro-inflammatory M1 macrophages; (j) converting Tregs into effector T cells; (k) converting a cold tumor into a hot tumor; (l) reducing Treg-mediated immunosuppression; or (m) The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1 to 17, which is a combination of any one or more of (a) to (k).

19. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 18, which does not substantially bind to TNFR1, herpes virus entry mediator (HVEM, CD40, death receptor 6 (DR6), and / or osteoprotegerin (OPG).

20. An isolated antibody, or antigen-binding fragment thereof, described in any one of claims 1 to 19, which is a TNFR2 antagonist.

21. An isolated antibody, or antigen-binding fragment thereof, described in any one of claims 1 to 19, which is a TNFR2 agonist.

22. An isolated antibody, or an antigen-binding fragment thereof, described in any one of claims 1 to 21, which is a bispecific or multispecific antibody.

23. An isolated polynucleotide encoding an isolated antibody, or an antigen-binding fragment thereof, described in any one of claims 1 to 22.

24. An expression vector comprising the isolated polynucleotide described in claim 23.

25. An isolated host cell comprising the expression vector described in claim 24.

26. A composition comprising a physiologically acceptable carrier and a therapeutically effective amount of an isolated antibody, or antigen-binding fragment thereof, described in any one of claims 1 to 22.

27. The composition of claim 26 for treating a patient having cancer, including cancer associated with abnormal TNFR2 expression.

28. The composition of claim 26 for treating a patient having cancer, including cancer associated with TNFR2 antagonist-mediated immunosuppression.

29. The composition of claim 27 or 28, wherein the antibody, or antigen-binding fragment thereof, is a TNFR2 antagonist.

30. The composition of claim 26 for treating a patient having an inflammatory disease and / or an autoimmune disease.

31. The composition of claim 30, wherein the disease is associated with abnormal TNFR2 expression and the antibody, or antigen-binding fragment thereof, is a TNFR2 agonist.

32. The composition described in claim 30, wherein the disease is associated with TNFR2 agonist-mediated immune activation.

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