Anti-tumor necrosis factor receptor 2 (TNFR2) antibodies and methods of use

Abstract

The instant disclosure is directed to anti-tumor necrosis factor receptor 2 (TNFR2 or TNFRSF1B) antibodies or antigen-binding fragments thereof, and uses thereof. Also contemplated are compositions as well as methods of use and treatment for the disclosed antibodies.

Description

ANTI TUMOR NECROSIS FACTOR RECEPTOR 2 (TNFR2) ANTIBODIES AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 776,885, filed March 24, 2025; U.S. Provisional Application No. 63 / 717,187, filed November 6, 2024; U.S. Provisional Application No. 63 / 658.844, filed June 11, 2024; and U.S. Provisional Application No. 63 / 652,668, filed May 28, 2024, all of which are herein incorporated by reference in their entireties.BACKGROUND

[0002] Tumor necrosis factor receptor 2 (TNFR2 or TNFRSF1B) has emerged as a pivotal regulator of immunosuppressive functions and lineage stability in regulatory T cells (Tregs). TNFR2 is a cell surface receptor that is constitutively and predominantly expressed by highly suppressive Tregs. TNFR2 binds to tumor necrosis factor-alpha (TNF ), a key pleiotropic cytokine involved in proinflammatory and / or immune regulatory response by signaling through tumor necrosis factor receptor 1 (TNFR1) and TNFR2 (Y ang, et al., Front Immunol, (2018) 9:784. doi: 10.3389 / fimmu.2018.00784). TNFa plays a critical role in upregulation or downregulation of Treg activity (Yang, ibid) . This binding activates several downstream signaling pathways, curbing pro-inflammatory responses (Moatti and Cohen, Front. Cell Dev. Biol.. (2021) 9:725473. doi: 10.3389 / fcell.2021.725473). TNFR2-mediated activation of the canonical and alternative NFKB path ays and RelA, a canonical NFKB member, is critical for development and maintenance of fully suppressive effector Treg and tissue Treg cells (Vasanthakumar. et al., Cell Reports (2017) 20:2906-2920), especially in non-lymphoid organs.

[0003] Deficiency of TNFR2 expression has been linked to various autoimmune diseases. It has been shown that TNFR2-deficient mice developed exacerbated experimental autoimmune encephalomyelitis (EAE) associated with reduced numbers of Treg cells with altered molecular signature and impaired function both ex vivo and in vivo (Tsakiri, et al., Eur. J. Immunol. (2012) 42(2):403-412). Polymorphisms in TNFR2 have also been identified in some patients with familial rheumatoid arthritis, Crohn’s disease, ankylosing spondylitis, ulcerative colitis, and immune-related conditions such as graft versus host disease associatedwith scleroderma risk (Faustman and Davis, Front Immunol (2013) 4:article 478). TNFR2 agonists selectively destroy autoreactive T cells but not healthy T cells in human blood samples from type I diabetes patients, as well as in patients with multiple sclerosis, Graves disease, and Sjogren’s syndrome (see, Ban et al., PNAS (2008) 105(36): 13644-13649). TNFR2 agonists for use as therapeutic agents can potentially activate and expand immune suppressive Tregs, thus providing a disease-modifying effect, such as an anti-inflammatory effect, immune resolution, and / or tissue repair, by potentiating effector regulatory T cells in autoimmune diseases. In addition to acting upon Treg cells, TNFR2 signaling can drive immunosuppressive function through myeloid-derived suppressor cells and mesenchymal stem cells (Yang, et al., Immunotargets Ther. (2021) 10: 103-122) and regulatory B cells (Ticha et al., Eur J Immunol (2021) 51(5): 1195-1205). Further. TNFR2 signaling drives a direct protective effect for tissue cells such as neuronal cells and oligodendrocytes (Dong, et al., PNAS (2016) 113(43): 12304-12309)).BRIEF SUMMARY

[0004] In a first aspect, an anti-tumor necrosis factor receptor 2 (TNFR2) antibody or antigen-binding fragment thereof which exhibits agonistic activity towards TNFR2 is provided herein. The antibody or antigen-binding fragment thereof may be defined as described further herein. In embodiments, the anti-TNFR2 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain selected from (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain comprising the amino acid sequence of SEQ ID NO: 12; (ii) a heavy chain having at least 80%, 85%, 90%, or 95% identify to the amino acid sequence of SEQ ID NO: 6 and a light chain having at least 80%. 85%. 90%. or 95% identify to the amino acid sequence of SEQ ID NO: 12. wherein the amino acid at position 228 of SEQ ID NO: 6 is proline; (iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 24; and (iv) a heavy chain having at least 80%, 85%, 90%, or 95% identify to the amino acid sequence of SEQ ID NO: 18 and a light chain having at least 80%, 85%, 90%, or 95% identify to the ammo acid sequence of SEQ ID NO: 24, wherein the amino acids at positions 234 and 235 of SEQ ID NO: 18 are both alanine and the amino acid at position 329 of SEQ ID NO: 18 is glycine. In further embodiments, the anti -tumor necrosis factor receptor 2 (TNFR2) antibody or antigen-binding fragment thereof comprises aheavy chain and a light chain variable region (VL) selected from (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; (ii) a heavy chain having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 7, wherein the amino acid at position 228 of SEQ ID NO: 6 is proline; (iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19; and (iv) a heavy chain having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 19, wherein the amino acids positions 234 and 235 of SEQ ID NO: 18 are both alanine and the amino acid at position 329 of SEQ ID NO: 18 is glycine. In additional embodiments, the anti-TNFR2 antibody or antigen-binding fragment thereof comprises a set of three heavy chain variable region complementarity determining regions (CDRs), VHCDR1, VHCDR2, and VHCDR3, that are comprised within a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13. and a set of three light chain variable region complementarity determining regions, VLCDR1, VLCDR2 and VLCDR3, comprised within a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 19. In some embodiments, the anti- TNFR2 antibody or antigen-binding fragment thereof comprises a set of three heavy chain variable region complementarity determining regions (CDRs) on a heavy chain, VHCDR1 , VHCDR2, and VHCDR3, and a set of three light chain variable region complementarity determining regions on a light chain, VLCDR1, VLCDR2 and VLCDR3, wherein the VHCDR1, VHCDR2, VHCDR3, VLCDR1. VLCDR2 and VLCDR3, collectively, are selected from (i) the VHCDR1 of SEQ ID NO: 2, the VHCDR2 of SEQ ID NO: 3, the VHCDR3 of SEQ ID NO: 4, the VLCDR1 of SEQ ID NO: 8, the VLCDR2 of SEQ ID NO: 9 and the VLCDR3 of SEQ ID NO: 10; (ii) the VHCDR1 of SEQ ID NO: 14, the VHCDR2 of SEQ ID NO: 15, the VHCDR3 of SEQ ID NO: 16, the VLCDRl of SEQ ID NO: 20. the VLCDR2 of SEQ ID NO: 21 and the VLCDR3 of SEQ ID NO: 22; (hi) the VHCDR1 of SEQ ID NO: 2 comprising 0-2 amino acid substitutions, the VHCDR2 of SEQ ID NO: 3 comprising 0-2 amino acid substitutions, the VHCDR3 of SEQ ID NO: 4 comprising 0-2 amino acid substitutions, the VLCDR1 of SEQ ID NO: 8 comprising 0-2 amino acidsubstitutions, the VLCDR2 of SEQ ID NO: 9 comprising 0-2 amino acid substitutions, and the VLCDR3 of SEQ ID NO: 10 comprising 0-2 amino acid substitutions; and (iv) the VHCDR1 of SEQ ID NO: 14 comprising 0-2 amino acid substitutions, the VHCDR2 of SEQ ID NO: 15 comprising 0-2 amino acid substitutions, the VHCDR3 of SEQ ID NO: 16 comprising 0-2 amino acid substitutions, the VLCDR1 of SEQ ID NO: 20 comprising 0-2 amino acid substitutions, the VLCDR2 of SEQ ID NO: 21 comprising 0-2 amino acid substitutions, and the VLCDR3 of SEQ ID NO: 22 comprising 0-2 amino acid substitutions.

[0005] The anti-TNFR2 antibody or antigen-binding fragment thereof may comprise a human IgGl CHl-hinge-CH2-CH3 region (e.g. IgGl subclass) and which may include one, two, three, four or more mutations. In some particular embodiments, the mutation(s) may be selected from one or more of (i) a proline at position 329 of the human IgGl Fc region substituted with another amino acid, optionally wherein the proline at position 329 is substituted with a glycine (P329G); (ii) a leucine at position 234 of the human IgGl Fc region is substituted with another amino acid, optionally wherein the leucine at position 234 is substituted with an alanine (L234A); and / or (iii) a leucine at position 235 of the human IgGl Fc region is substituted with another amino acid, optionally wherein the leucine at position 235 is substituted with an alanine (L235A). In some particular embodiments, the amino acid substitutions are L234A and L235A of the human IgGl Fc region. In some other embodiments, the amino acid substitutions are P329G and / or L234A and L235A of the human IgGl Fc region. In yet further embodiments, the amino acid substitution is the proline at position 329 of the human IgGl Fc region substituted with another amino acid.

[0006] In other embodiments, the anti-TNFR2 antibody or antigen-binding fragment thereof may comprise a human IgG4 CHl-hinge-CH2-CH3 region (e.g. IgG4 subclass) and which may include one. two, three, four or more mutations. In some embodiments, the serine at position 228 of the human IgG4 constant heavy region is substituted with another amino acid. In a particular embodiment, the serine at position 228 (in the hinge region) is substituted with proline (S228P).

[0007] The anti-TNFR2 antibody or antigen-binding fragment thereof may comprise either of a human kappa constant light chain or a human lambda constant light chain.

[0008] In embodiments, the anti-TNFR2 antibody or antigen-binding fragment thereof as described herein are capable of selectively promoting the proliferation of a population of T regulator ' (Treg) cells. In some embodiments, the anti-TNFR2 antibody or antigen-bindingfragment thereof as described herein activates TNFR2 -mediated nuclear factor kappa-B (NFKB) signaling. In embodiments, the NFKB signaling activation is characterized by an increase of alkaline phosphatase secretion level from an engineered reporter cell line. In further embodiments, the alkaline phosphatase secretion level induced and / or increased by said anti-TNFR2 antibody or antigen-binding fragment thereof in the reporter cell line is at least about 1.1-fold, 1.2-fold, 1.3-fold. 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold. 2.5-fold. 2.75-fold, 3-fold. 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold.4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75- fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold, 8-fold, 8.25-fold, 8.5-fold, 8.75-fold, 9-fold, 9.25- fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11 -fold, 11.25-fold, 11.5- fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold. 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75- fold, 16-fold, 16.25-fold, 16.5-fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18- fold, 18.25-fold, 18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold. 20.5-fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than an isotype control; and about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold. 1.1 -fold to 2.5- fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1 -fold to 20-fold, 1.1-fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22- fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold. 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7- fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22 -fold, 11 -fold to 14-fold, 11-fold to 18-fold, 11-fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14- fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22 -fold, 17-fold to 19-fold, 17-fold to 21- fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21-fold to 22-fold higher than an isotype control.

[0009] In further embodiments, the NFKB signaling activation is characterized as an increase of RelA phosphorylation (pRelA) in primary' human Tregs which may optionally be definedas CD3+CD4+CD25+FOXP3+. In some embodiments, the RelA phosphorylation induced and / or increased by the antibody has a highest activity’ of at least about 1.1 -fold, 1.2-fold, 1.3- fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75- fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25- fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold,7.75-fold, 8-fold. 8.25-fold, 8.5-fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold. 10-fold, 10.25-fold. 10.5-fold, 10.75-fold, 11-fold. 11.25-fold. 11.5-fold, 11.75-fold, 12-fold. 12.25- fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5- fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold. 18.25-fold, 18.5-fold,18.75-fold. 19-fold, 19.25-fold, 19.5-fold. 19.75-fold. 20-fold, 20.25-fold, 20.5-fold, 20.75- fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than an isotype control; and about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1 -fold to 2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1 -fold to 15-fold, 1.1 -fold to 20-fold, 1.1 -fold to 22-fold, 2-fold to 2.5-fold, 2- fold to 5-fold, 2-fold to 7.5-fold. 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold, 4- fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold. 4-fold to 22-fold, 5-fold to 8-fold. 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10- fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14- fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18- fold, 8-fold to 22-fold. 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22- fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to18-fold, 11-fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14- fold to 22-fold. 15-fold to 18-fold. 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22- fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22 -fold higher than an isotype control.

[0010] In some embodiments where the anti-TNFR2 antibody or antigen-binding fragment thereof is of the IgG4 subclass and has a S228P mutation, the anti-TNFR2 antibody or antigen-binding fragment thereof has an enhanced ability to activate NFKB signaling in a larger fraction of Tregs as compared to IgGl Fc-based variants. In some embodiments the enhanced ability of NFKB signaling activation is characterized by an increase of RelAphosphorylation (pRelA) in primary human Tregs, and wherein the Tregs which are optionally defined as CD3+CD4+CD25+FOXP3+. In further embodiments, the RelA phosphorylation (pRelA) in primary human Tregs is induced and / or increased by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2- fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold,4.5-fold, 4.75-fold. 5-fold, 5.25-fold, 5.5-fold. 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75- fold, 7-fold, 7.25-fold. 7.5-fold, 7.75-fold. 8-fold. 8.25-fold, 8.5-fold. 8.75-fold, 9-fold, 9.25- fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11 -fold, 11.25-fold, 11.5- fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75- fold, 16-fold. 16.25-fold, 16.5-fold, 16.75-fold, 17-fold. 17.25-fold. 17.5-fold, 17.75-fold, 18- fold, 18.25-fold, 18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than IgGl Fc-based variants; and about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to2.5-fold, 1.1 -fold to 5 -fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1- fold to 22-fold. 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold. 2-fold to 10-fold. 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22- fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold. 8-fold to 11-fold. 8- fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22 -fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11-fold to 18-fold, 11-fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12- fold to 22-fold. 13-fold to 14-fold. 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22- fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21-fold to 22-fold higher than IgGl Fc-based variants.

[0011] In some embodiments where the anti-TNFR2 antibody or antigen-binding fragment thereof (i) comprises an IgGl Fc region having a P329G mutation, and (ii) has an enhanced ability to activate NFKB signaling of Tregs the enhanced ability of NFKB signaling activation is characterized by an increase of RelA phosphorylation (pRelA) in primary human Tregs.These Tregs may optionally be defined as CD3+CD4+CD25+FOXP3+. In some of these embodiments, the RelA phosphorylation (pRelA) in primary human Tregs induced and / or increased by said anti-TNFR2 antibody or antigen-binding fragment thereof comprising an IgGl Fc region having a P329G mutation is at least: a) about 1.1 -fold, 1.2-fold, 1.3-fold, 1.4- fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3- fold, 3.25-fold. 3.5-fold, 3.75-fold, 4-fold. 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold,5.5-fold, 5.75-fold. 6-fold. 6.25-fold, 6.5-fold. 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75- fold, 8-fold, 8.25-fold, 8.5-fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25- fold, 12.5-fold, 12.75-fold. 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold,14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5- fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold, 20.75- fold, 21-fold, 21.25-fold. 21.5-fold, 21.75-fold, or 22-fold, as compared to a control without the substitution; and b) about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1 -fold to 5 -fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold. 1.1 -fold to 20-fold. 1.1-fold to 22- fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5- fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22 -fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6- fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold. 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 1 -fold, 7-fold to 22-fold, 8-fold to 11 -fold, 8-fold to15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19- fold, 9-fold to 22-fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22 -fold, 11-fold to 14-fold, 11-fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold. 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14- fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold,16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21- fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold higher, as compared to a control without the substitution.

[0012] In additional embodiments where the anti-TNFR2 antibody or antigen-binding fragment thereof comprises an IgG4 constant heavy region having a S228P mutation, theantibody or fragment thereof has an enhanced ability to activate NFKB signaling as compared to IgGl Fc-based variants. The enhanced ability to activate NFKB signaling may be characterized by an increase of alkaline phosphatase secretion level from an engineered reporter cell line. In some embodiments, the antibody or fragment thereof preferentially binds to Tregs over non-Treg cells and cytotoxic T cells. In some embodiments, the Tregs are optionally defined as CD3+CD4+CD25+FoxP3+. In further embodiments, a maximum response resulting from the anti-TNFR2 antibody or antigen-binding fragment thereof binding to CD3+CD4+CD25+FoxP3+ Tregs cells is at least: about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, 525%, 550%, 575%. 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%. 925%, 950%, 975%, or 1000% higher than the maximum response resulting from the binding to non-Treg cells or to cytotoxic T cells; and about 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%. 55% to 100%, 58% to 100%, 60% to 100%. 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, 100% to 400%, 110% to 400%, 120% to 400%, 130% to 400%, 140% to 400%. 150% to 400%, 160% to 400%, 170% to 400%. 180% to 400%, 190% to 400%, 100% to 500%, 110% to 500%, 120% to 500%, 130% to 500%, 140% to 500%, 150% to 500%, 160% to 500%, 170% to 500%, 180% to 500%, 190% to 500%, 100% to 600%, 110% to 600%, 120% to 600%, 130% to 600%, 140% to 600%, 150% to 600%, 160% to 600%, 170% to 600%, 180% to 600%, 190% to 600%, 100% to 700%, 110% to 700%, 120% to 700%, 130% to 700%, 140% to 700%, 150% to 700%, 160% to 700%, 170% to 700%, 180% to 700%, 190% to 700%, 100% to 800%, 110% to 800%, 120% to 800%, 130% to 800%, 140% to 800%, 150% to 800%, 160% to 800%, 170% to 800%, 180% to 800%, 190% to 800%, 100% to 900%, 110% to 900%, 120% to 900%, 130% to 900%, 140% to 900%, 150% to 900%. 160% to 900%, 170% to 900%, 180% to 900%. 190% to 900% 110% to 1000%, 120% to 1000%, 130% to 1000%, 140% to 1000%, 150% to 1000%, 160% to 1000%, 170% to 1000%, 180% to 1000%, 190% to 1000%, 200% to 1000%, 250% to 1000%, 300% to 1000%, 350% to 1000%, 400% to 1000%. 450% to 1000%, 500% to1000%, 600% to 1000%, 700% to 1000%, 800% to 1000%. 900% to 1000%, or 950% to 1000% higher than the maximum response resulting from the binding to non-Treg cells or to cytotoxic T cells.

[0013] In some further embodiments, the anti-TNFR2 antibody or antigen-binding fragment described herein is characterized by a preferential binding to Tregs over non-Treg cells. In preferred embodiments, the Tregs comprise CD4+ Tregs, CD4+CD25+FoxP3+ Tregs, FoxP3+ CD4+ Tregs, CD25+FoxP3+ Tregs, or a combination of any one or more thereof. In some further embodiments, where the Tregs are FoxP3+, the FoxP3 -expressing cells comprise a subpopulation of FoxP3 high-expressing cells and a subpopulation of FoxP3 low- expressing cells and the anti-TNFR2 antibody or antigen-binding fragment thereof exhibits a higher level of binding to the FoxP3 high-expressing cells than to the FoxP3 low-expressing cells. In some of these embodiments, the non-Treg cells comprise conventional CD4+ T cells (CD4+ Tconv), conventional CD8+ T cells (CD8+ T conv), NK cells, monocytes and / or neutrophils, or a combination of any one or more of thereof. In some embodiments, the preferential binding is measured by a percent of binding positive cells, wherein the percent of binding positive cells in Tregs is at least: about 1.1-fold, 1.2-fold. 1.3-fold. 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold,3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75- fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold, 8-fold, 8.25- fold, 8.5-fold, 8.75-fold, 9-fold, 9.25-fold. 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 1 1-fold, 11.25-fold, 11.5-fold, 1 1.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75- fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15- fold, 15.25-fold, 15.5-fold. 15.75-fold, 16-fold, 16.25-fold, 16.5-fold, 16.75-fold, 17-fold, 17.25-fold. 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold, 18.75-fold, 19-fold, 19.25- fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold, 20.75-fold, 21-fold, 21.25-fold,21.5-fold, 21.75-fold, or 22-fold higher than the percent of binding positive cells to non-Treg cells, or about or 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5- fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold. 1.1 -fold to 20-fold, 1.1 -fold to 22-fold, 2-fold to2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold. 2-fold to 10-fold. 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8- fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold. 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22 -fold, 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold. 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11-fold to 14-fold, 11-fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13- fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18- fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21-fold to 22-fold higher than the percent of binding positive cells to non-Treg cells. In yet further embodiments, the preferential binding is measured by a total median fluorescence intensity, wherein the total median fluorescence intensity of Tregs is at least: a) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2- fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75- fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold. 8-fold, 8.25-fold, 8.5-fold. 8.75-fold, 9-fold, 9.25- fold, 9.5-fold, 9.75-fold. 10-fold, 10.25-fold, 10.5-fold. 10.75-fold. 11-fold, 11.25-fold, 11.5- fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75- fold, 16-fold. 16.25-fold, 16.5-fold, 16.75-fold, 17-fold. 17.25-fold. 17.5-fold, 17.75-fold, 18- fold, 18.25-fold, 18.5-fold. 18.75-fold, 19-fold, 19.25-fold, 19.5-fold. 19.75-fold, 20-fold, 20.25-fold, 20.5-fold, 20.75-fold, 21 -fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than the total median fluorescence intensity of non-Treg cells; and b) about or 1.1 -fold to 1.2- fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold. 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14- fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold. 7- fold to 22-fold. 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold. 8-fold to 22 -fold. 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22 -fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22- fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold higher than the total median fluorescence intensity of non-Treg cells.

[0014] In some particular embodiments, the anti-TNFR2 antibody or antigen-binding fragment thereof is characterized by specific or preferential binding to TNFR2 over a tumor necrosis factor 1 (TNFR1) protein.

[0015] In some embodiments, the specific binding is measured by a signal-to-noise ratio (SNR) value indicating an interaction between the anti-TNFR2 antibody or antigen-binding fragment thereof and a binding protein, wherein the SNR value indicating the interaction of the anti-TNFR2 antibody or antigen-binding fragment thereof with a TNFR2 receptor is at least: a) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25- fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold. 5.5-fold, 5.75-fold, 6-fold. 6.25-fold, 6.5-fold,6.75-fold, 7-fold. 7.25-fold, 7.5-fold, 7.75-fold. 8-fold, 8.25-fold. 8.5-fold, 8.75-fold. 9-fold. 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11 -fold, 11.25 -fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5- fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold. 15.25-fold, 15.5-fold,15.75-fold. 16-fold, 16.25-fold, 16.5-fold. 16.75-fold. 17-fold, 17.25-fold, 17.5-fold. 17.75- fold, 18-fold, 18.25-fold, 18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20- fold, 20.25-fold, 20.5-fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than the SNR value indicating the interaction with TNFR1; and b) about or 1.1 -fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1- fold to 15-fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2- fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4- fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold. 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14- fold, 6-fold to 18-fold. 6-fold to 22-fold. 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18- fold, 7-fold to 22-fold, 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22- fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14- fold, 10-fold to 18-fold, 10-fold to 22-fold, 11-fold to 14-fold, 11-fold to 18-fold, 11-fold to22-fold, 12-fold to 15-fold, 12-fold to 18-fold. 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13 -fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15- fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20- fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold higher than the SNR value indicating the interaction with TNFR1.

[0016] In some embodiments, the TNFR2 receptor is TNFR isoform 1. In other embodiments, the TNFR2 receptor is TNFR isoform 2.

[0017] In further embodiments, the agonistic activity toward TNFR2 is characterized by an increase in expression of Treg functional markers associated with immunosuppressive ability, wherein the functional markers are selected from the group consisting of CD25, FoxP3, TIGIT, and Ki67, and combinations of any one or more of the foregoing. In some embodiments, the increase of Treg functional marker expression induced and / or increased by said anti-TNFR2 antibody or antigen-binding fragment thereof is at least: a) about 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold. 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold. 2-fold, 2.25-fold,2.5-fold, 2.75-fold. 3-fold. 3.25-fold, 3.5-fold. 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75- fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25- fold, 7.5-fold, 7.75-fold, 8-fold, 8.25-fold, 8.5-fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75- fold, 12-fold. 12.25-fold, 12.5-fold, 12.75-fold, 13-fold. 13.25-fold. 13.5-fold, 13.75-fold, 14- fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25- fold, 18.5-fold, 18.75-fold. 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold,20.5-fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher as compared to an isotype control; and b) about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1- fold to 2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2- fold to 12.5-fold. 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4- fold to 22-fold. 5-fold to 8-fold, 5-fold to 12-fold. 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 11- fold, 8-fold to 15-fold, 8-fold to 18-fold. 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold. 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22- fold, 11-fold to 14-fold, 11-fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15- fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20- fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21-fold to 22-fold higher as compared to an isotype control.

[0018] In embodiments where the anti-TNFR2 antibody or antigen-binding fragment thereof comprises an IgG4 constant heavy region having a S228P mutation, the antibody or fragment thereof has an enhanced ability- to induce and / or increase expression of Treg functional markers, as compared to IgGl Fc-based variants. In a particular embodiment, the IgGl Fc- based variant is Antibody 1. In further embodiments, the expression of Treg functional markers induced and / or increased by the anti-TNFR2 antibody or antigen-binding fragment thereof comprising an IgG4 constant region having a S228P mutation are at least: a) aboutI.1-fold, 1.2-fold, 1.3-fold, 1.4-fold. 1.5-fold. 1.6-fold, 1.7-fold, 1.8-fold. 1.9-fold. 2-fold,2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5- fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7- fold, 7.25-fold. 7.5-fold, 7.75-fold, 8-fold, 8.25-fold, 8.5-fold, 8.75-fold, 9-fold, 9.25-fold,9.5-fold, 9.75-fold. 10-fold, 10.25-fold, 10.5-fold. 10.75-fold. 11-fold, 11.25-fold, 11.5-fold.I I.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75- fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16- fold, 16.25-fold, 16.5-fold. 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold. 18-fold,18.25-fold. 18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25- fold, 20.5-fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than IgGl Fc-based variants; and b) about or 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1- fold to 22-fold. 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold. 2-fold to 12.5-fold, 2-fold to 20-fold. 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold. 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22- fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 11-fold. 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22 -fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22 -fold. 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11-fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12- fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22- fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold. 18-fold to 22-fold. 19-fold to 20-fold. 19-fold to 22-fold, 20-fold to 22-fold, or 21-fold to 22-fold higher than IgGl Fc-based variants.

[0019] In a second aspect, a composition comprising the anti-TNFR2 antibody or antigenbinding fragment thereof and a pharmaceutically acceptable carrier is provided herein.

[0020] In a third aspect, use of the anti-TNFR2 antibody or antigen-binding fragment thereof as described herein is provided for the treatment of an autoimmune disease. In some embodiments, the autoimmune disease is an autoimmune disease of the skin, lungs, gastrointestinal tract, central nervous system, endocrine system, or kidneys. In further embodiments, the autoimmune disease is selected from the group consisting of acute febrile mucocutaneous lymph node syndrome, amyloidosis, asthma such as allergic asthma, autoimmune hepatitis, celiac disease, chronic obstructive pulmonary disease, Crohn’s disease, dermatitis, diabetes mellitus ty pe 1, glomerulonephritis including rapidly progressive glomerulonephritis, graft versus host disease, immunoglobulin a vasculitis, an inflammatory bowel disease, interstitial cystitis, lupus erythematosus, lupus nephritis, membranous nephropathy, multiple sclerosis including relapsing remitting multiple sclerosis, myocarditis, neuromyelitis optica, optic neuritis, pancreatitis, pemphigus, psoriasis, rheumatoid arthritis, scleroderma, Sjogren’s syndrome, ulcerative colitis, uveitis, vasculitis, vitiligo including vitiligo-associated multiple autoimmune disease susceptibility’ 1. In additional embodiments, the autoimmune disease is selected from multiple sclerosis, vitiligo, and ulcerative colitis. In embodiments, use of the anti-TNFR2 antibody or antigen-binding fragment thereof is capable of (or effective to) inducing and / or enhancing the immunosuppressive ability of a population of Treg cells, (e.g. levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by at least: a) about 0.00001%. 0.00002%. 0.00003%. 0.00004%. 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%, 70.0%, 80.0%, 90.0%, or 100% as compared to a control or untreated subject; and b) about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%, 0.00005% to 0.009%, 0.000055% to 0.01%, 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%. 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%.0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%, 0.002% to 2%, 0.003% to 3%, 0.004% to 4%, 0.005% to 5%, 0.006% to 6%, 0.007% to 7%, 0.008% to 8%. 0.009% to 9%. 0.01% to 10%. 0.02% to 20%. 0.03% to 30%. 0.04% to 40%. 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to 100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to100%, 28% to 100%, 30% to 100%. 33% to 100%, 35% to 100%, 38% to 100%. 40% to100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to100%, 88% to 100%, 90% to 100%. 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subject. In further embodiments, use of the anti-TNFR2 antibody or antigen-binding fragment thereof is capable of inducing and / or enhancing the proliferation of a population of Treg cells, (e.g., levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by at least: a) about 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%. 4.0%, 5.0%, 6.0%. 7.0%, 8.0%, 9.0%. 10.0%. 20.0%. 30.0%. 40.0%.50.0%, 60.0%, 70.0%, 80.0%, 90.0%, or 100%, as compared to a control or untreated subject; and b) about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to0.008%, 0.00005% to 0.009%, 0.000055% to 0.01%, 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%, 0.002% to 2%, 0.003% to 3%, 0.004% to 4%, 0.005% to 5%, 0.006% to 6%, 0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%. 0.03% to 30%. 0.04% to 40%. 0.05% to 50%. 0.06% to 60%. 0.07% to 70%. 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to 100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, l% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%. 25% to 100%, 28% to 100%, 30% to 100%. 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subject. In some further embodiments, use of the anti-TNFR2 antibody or antigen-binding fragment thereof is effective in suppressing, reducing, and / or decreasing antigen-induced skin inflammation, wherein the skin inflammation is suppressed and / or reduced and / or decreased by a) about 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001 %, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%, 70.0%, 80.0%, 90.0%, or 100%, as compared to a control or untreated subject; and b) about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%. 0.000045% to 0.008%, 0.00005% to 0.009%, 0.000055% to 0.01%. 0.00006% to 0.02%. 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%. 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%,0.002% to 2%, 0.003% to 3%, 0.004% to 4%, 0.005% to 5%, 0.006% to 6%, 0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to 100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%. 33% to 100%, 35% to 100%, 38% to 100%. 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%. 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subject.

[0021] In some embodiments, use of the anti-TNFR2 antibody or antigen-binding fragment thereof is effective in increasing and / or enhancing survival rate of subjects with GvHD, wherein the survival rate is increased and / or enhanced by at least: a) about 10%, 20%, 30%, 40%. 50%. 60%. 70%. 80%. 90%. 100%, 125%, 150%. 175%, 200%, 225%. 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, 525%, 550%, 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000%, as compared to a control or untreated subject; and b) 10% to 100%. 13% to 100%, 15% to 100%, 18% to 100%. 20% to 100%, 23% to 100%, 25% to 100%. 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, 100% to 400%, 110% to 400%, 120% to 400%, 130% to 400%, 140% to 400%, 150% to 400%, 160% to 400%, 170% to 400%, 180% to 400%, 190% to 400%, 100% to 500%, 110% to 500%, 120% to 500%, 130% to 500%, 140% to 500%, 150% to 500%, 160% to 500%, 170% to 500%, 180% to 500%, 190% to 500%. 100% to 600%, 110% to 600%, 120% to 600%. 130% to 600%. 140% to 600%, 150% to 600%, 160% to 600%, 170% to 600%, 180% to 600%, 190% to 600%, 100% to 700%, 110% to 700%, 120% to 700%, 130% to 700%, 140% to 700%, 150% to 700%, 160% to 700%, 170% to 700%, 180% to 700%, 190% to 700%, 100% to 800%, 110%to 800%, 120% to 800%, 130% to 800%, 140% to 800%, 150% to 800%, 160% to 800%, 170% to 800%, 180% to 800%, 190% to 800%, 100% to 900%, 110% to 900%, 120% to 900%, 130% to 900%, 140% to 900%, 150% to 900%, 160% to 900%, 170% to 900%, 180% to 900%, 190% to 900% 110% to 1000%, 120% to 1000%, 130% to 1000%, 140% to 1000%, 150% to 1000%, 160% to 1000%, 170% to 1000%, 180% to 1000%, 190% to 1000%, 200% to 1000%, 250% to 1000%, 300% to 1000%, 350% to 1000%, 400% to 1000%, 450% to 1000%, 500% to 1000%, 600% to 1000%. 700% to 1000%. 800% to 1000%. 900% to 1000%, or 950% to 1000%, as compared to a control or untreated subject.

[0022] In a fourth aspect, a method of treating an autoimmune disease in a subject by administering an anti-TNFR2 antibody or antigen-binding fragment thereof as described herein is provided herein. In some embodiments, the autoimmune disease for treatment is an autoimmune disease of the skin, lungs, gastrointestinal tract, central nervous system, endocrine system, or kidneys. In some further embodiments, the autoimmune disease for treatment is selected from the group consisting of acute febrile mucocutaneous lymph node syndrome, amyloidosis, asthma such as allergic asthma, autoimmune hepatitis, celiac disease, chronic obstructive pulmonary disease, Crohn’s disease, dermatitis, diabetes mellitus type 1. glomerulonephritis including rapidly progressive glomerulonephritis, graft versus host disease, immunoglobulin a vasculitis, an inflammatory bowel disease, interstitial cystitis, lupus erythematosus, lupus nephritis, membranous nephropathy, multiple sclerosis including relapsing remitting multiple sclerosis, myocarditis, neuromyelitis optica, optic neuritis, pancreatitis, pemphigus, psoriasis, rheumatoid arthritis, scleroderma, Sjogren’s syndrome, ulcerative colitis, uveitis, vasculitis, and vitiligo including vitiligo-associated multiple autoimmune disease susceptibility 1. In some particular embodiments, the autoimmune disease for treatment is selected from multiple sclerosis, vitiligo, and ulcerative colitis. In further embodiments, the anti-TNFR2 antibody or antigen-binding fragment thereof is capable of inducing and / or enhancing the immunosuppressive ability of a population of Treg cells such as the levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells by at least: a) about 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%. 0.0001%. 0.0002%, 0.0003%, 0.0004%. 0.0005%, 0.0006%, 0.0007%, 0.0008%. 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%,9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%. 70.0%, 80.0%, 90.0%, or 100%. as compared to a control or untreated subject; and b) about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%, 0.00005% to 0.009%, 0.000055% to 0.01%, 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%. 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%. 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%, 0.002% to 2%, 0.003% to 3%, 0.004% to 4%, 0.005% to 5%, 0.006% to 6%, 0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%. 0.06% to 60%. 0.07% to 70%. 0.08% to 80%. 0.09% to 90%, 0.1% to 100%. 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to 100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%. 48% to 100%, 50% to 100%, 53% to 100%. 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subject. In additional embodiments, the anti-TNFR2 antibody or antigen-binding fragment thereof is capable of inducing and / or enhancing the proliferation of a population of Treg cells, (e.g., levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by at least: a) about 0.00001, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%. 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%. 70.0%. 80.0%. 90.0%. or 100%. as compared to a control or untreated subject; and b) about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%, 0.00005% to 0.009%, 0.000055% to 0.01%, 0.00006% to 0.02%, 0.000065% to 0.03%,0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%. 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%, 0.002% to 2%, 0.003% to 3%, 0.004% to 4%, 0.005% to 5%, 0.006% to 6%, 0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%. 0.09% to 90%. 0. 1% to 100%. 0.2% to 100%. 0.3% to 100%. 0.4% to 100%. 0.5% to 100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, l% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%. 40% to 100%, 43% to 100%, 45% to 100%. 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subject.

[0023] In some embodiments, the anti-TNFR2 antibody or antigen-binding fragment thereof is effective in suppressing, reducing, and / or decreasing antigen-induced skin inflammation, wherein the skin inflammation is suppressed and / or reduced and / or decreased by at least: a) about 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%. 0.0002%, 0.0003%, 0.0004%, 0.0005%. 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%, 70.0%, 80.0%, 90.0%, or 100%, as compared to a control or untreated subject; and b) 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%, 0.00005% to 0.009%, 0.000055% to 0.01%, 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%. 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%.0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%, 0.002% to 2%, 0.003% to 3%, 0.004% to 4%, 0.005% to 5%, 0.006% to 6%, 0.007% to 7%,0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to 100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%. 48% to 100%, 50% to 100%, 53% to 100%. 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subject.

[0024] In some further embodiments, administering the anti-TNFR2 antibody or antigenbinding fragment thereof is effective in increasing survival rate of subjects with GvHD, wherein the survival rate is increased and / or enhanced by at least: a) about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%. 375%, 400%, 425%. 450%, 475%, 500%. 525%, 550%. 575%. 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000%, as compared to a control or untreated subject; and b) about 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%. 33% to 100%, 35% to 100%, 38% to 100%. 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, 100% to 400%, 110% to 400%, 120% to 400%, 130% to 400%, 140% to 400%, 150% to 400%, 160% to 400%, 170% to 400%, 180% to 400%, 190% to 400%, 100% to 500%, 110% to 500%, 120% to 500%, 130% to 500%, 140% to 500%, 150% to 500%, 160% to 500%, 170% to 500%, 180% to 500%, 190% to 500%, 100% to 600%, 110% to 600%, 120% to 600%, 130% to 600%. 140% to 600%, 150% to 600%, 160% to 600%. 170% to 600%, 180% to 600%, 190% to 600%, 100% to 700%, 110% to 700%, 120% to 700%, 130% to 700%, 140% to 700%, 150% to 700%, 160% to 700%, 170% to 700%, 180% to 700%, 190% to 700%, 100% to 800%, 110% to 800%, 120% to 800%, 130% to 800%, 140% to 800%, 150% to 800%,160% to 800%, 170% to 800%, 180% to 800%, 190% to 800%, 100% to 900%, 110% to 900%, 120% to 900%, 130% to 900%, 140% to 900%, 150% to 900%, 160% to 900%, 170% to 900%, 180% to 900%, 190% to 900% 110% to 1000%, 120% to 1000%, 130% to 1000%, 140% to 1000%, 150% to 1000%, 160% to 1000%, 170% to 1000%, 180% to 1000%, 190% to 1000%, 200% to 1000%, 250% to 1000%, 300% to 1000%, 350% to 1000%, 400% to 1000%, 450% to 1000%, 500% to 1000%, 600% to 1000%. 700% to 1000%. 800% to 1000%, 900% to 1000%, or 950% to 1000%, as compared to a control or untreated subject.

[0025] In a fifth aspect, a nucleic acid composition comprising a) a first nucleic acid encoding a heavy chain variable region comprising a VHCDR1, VHCDR2, VHCDR3 and optionally a heavy chain constant region, of an anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 2-40; and b) a second nucleic acid encoding a light chain variable region comprising a VLCDRL VLCDR2 and VLCDR3, of an anti- TNFR2 antibody or antigen-binding fragment is provided.

[0026] In a sixth aspect, an expression vector composition comprising: a) a first expression vector comprising a first nucleic acid described herein; and b) a second expression vector comprising said second nucleic acid described herein is provided.

[0027] In a seventh aspect, an expression vector composition comprising an expression vector comprising a first nucleic acid and a second nucleic acid described herein is provided.

[0028] In an eighth aspect a host cell comprising a nucleic acid composition described herein is provided.

[0029] In a ninth aspect, a host cell comprising an expression vector composition described herein is provided.

[0030] In a tenth aspect, a method of making an anti TNFR2 antibody or antigen-binding fragment thereof comprises: a) culturing a host cell under conditions wherein the antibody or antigen-binding fragment thereof is expressed; and b) recovering the antibody or antigenbinding fragment thereof is provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figures 1A, IB, 1C, ID, IE, and IF are graphs showing Antibody 1 dose response binding curves of the %+ binding (percentage binding to cells that are positive for expression of a particular marker) and total median fluorescence intensity7(tMFI) binding data from one donor as detailed in Example 4. FIGs. 1 A and IB show the total Treg %+ binding (FIG. 1 A)and tMFI binding (FIG. IB) of Antibody 1 (•) or an isotype control (■). FIGs. 1C and ID show the CD25-high Treg %+binding (FIG. 1C) and tMFI binding (FIG. ID) of Antibody 1 (•) or an isotype control (■). FIGs. IE and IF show the CD25-low Treg %+ binding (FIG. IE) and tMFI binding (FIG. IF) of Antibody 1 (•) or an isotype control (■).

[0032] Figures 2A, 2B,2C and 2D are graphs showing Antibody 1 dose response binding curves in non-Treg T cell populations as detailed in Example 4. FIGs. 2A and 2B show the CD4+ non-Treg %+ binding (FIG. 2A) and tMFI binding (FIG. 2B) of Antibody 1 (•) or an isotype control (■). FIGs. 2C and 2D show the CD8+ T cells %+ binding (FIG. 2C) and tMFI binding (FIG. 2D) of Antibody 1 (•) or an isotype control (■).

[0033] Figures 3A, 3B, 3C, 3D, 3E, and 3F are graphs showing the dose response curves of the average %+ pRelA from two donors for TNFR2-binding antibody variants. Antibodies 1- 4, as detailed in Example 5. FIGs. 3A and 3B show the total Treg %+pRelA for PBMCs from donor 1 (FIG. 3 A) and donor 2 (FIG. 3B) treated with Antibody 1 (■), Antibody 2 (A), Antibody 3 (▼), Antibody 4 (♦), or an isotype control (o). FIGs. 3C and 3D show the CD25 / FoxP3high Treg %+pRelA for PBMCs from donor 1 (FIG. 3C) and donor 2 (FIG. 3D) treated with Antibody 1 (■). Antibody 2 ( A). Antibody 3 ( ▼), Antibody 4 (♦). or an isotype control (o). FIGs. 3E and 3F show the CD25 / FoxP31ow Treg %+pRelA for PBMCs from donor 1 (FIG. 3E) and donor 2 (FIG. 3F) treated with Antibody 1 (■), Antibody 2 (A), Antibody 3 (▼), Antibody 4 (♦), or an isotype control (o).

[0034] Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H are graphs showing dose-response pRel A signaling curves of the %+ data from two donors for Antibodies 1 -4 as detailed in Example 5. FIGs. 4A-4B shows the CD4+ non-Treg %pRelA for PBMCs from donor 1 (top) and donor 2 (bottom) treated with Antibody 1 (■). Antibody 2 (A), Antibody 3 (▼), Antibody 4 (♦), or an isotype control (o). FIGs. 4C-4D shows the CD8+ T cells %pRelA for PBMCs from donor 1 (top) and donor 2 (bottom) treated with Antibody 1 (■), Antibody 2 (A), Antibody 3 (▼), Antibody 4 (♦), or an isotype control (o). FIGs. 4E-4F shows the CD14+ monocyte %pRelA for PBMCs from donor 1 (top) and donor 2 (bottom) treated with Antibody 1 (■), Antibody 2 (A), Antibody 3 (▼), Antibody 4 (♦), or an isotype control (o). FIGs. 4G-4H show the CD56+ NK cell %pRelA for PBMCs from donor 1 (top) and donor 2 (bottom) treated with Antibody 1(B), Antibody 2 (A), Antibody 3 (▼), Antibody 4 (♦), or an isotype control (o).

[0035] Figure 5 is a graph of dose response curves in an HEK-Blue™ TNFa reporter cell assay of the average OD655 data for TNFR2 binding antibody variants, Antibody 1 and Antibody 2 as well as for the Fab labeled as Antibody 5 as detailed in Example 6. FIG. 5 shows the average ODess data for Antibody 1 (lower A), Antibody 2 (top A), and Antibody 5 (-).

[0036] Figures 6A, 6B, and 6C are graphs of the average dose response of expression of CD25 (FIG. 6A). and FoxP3 (FIG. 6B) in Treg cells from one donor after 5-days culture of human PBMCs with vehicle (far left ■ only), isotype control (♦), or Antibody 1 (•), Antibody 2 ( ■), Antibody 3 (A), and Antibody 4 (▼) variants as detailed in Example 7. Expression of CD25 and FoxP3 are reported as each marker's positive population (+MFI) within the total Treg population in Fig. 6A for CD25 and in Fig. 6B for FoxP3. The fraction of CD25 / FoxP3 high Tregs (FIG. 6C) or frequency is reported as the % CD25 / FoxP3 high fraction of total Treg cells. Antibody 1, Antibody 4, and Antibody 2 showed induction of CD25 and FoxP3 expression in Treg cells while induction by Antibody 3 was less in comparison.

[0037] Figures 7A and 7B are graphs of the average dose response induction of TIGIT in Tregs in human PBMC by Fc variants. The graphs show the expression level of TIGIT (FIG. 7 A) in Tregs (posMFI TIGIT), and the frequency of the total Tregs expressing TIGIT (%+ TIGIT) (FIG. 7B) on human PBMC Treg cells from one donor after 5-days of treatment with vehicle (far left ■ only), isotype control (♦), Antibody 1 (•), Antibody 2 (■). Antibody 3 (A), or Antibody 4 (▼) Fc variants as detailed in Example 7. Expression is posMFI reported as the positive MFI (MFI of TIGIT-expressing Tregs), and the frequency is the fraction of the total Tregs expressing TIGIT (%+TIGIT). Treatment with Antibody 1, Antibody 4 and Antibody 2 showed similar induction of TIGIT on Treg cells while induction by Antibody 3 was less in comparison.

[0038] Figure 8 is a graph of the average dose response induction of Ki67 by Fc variants in human PBMC Treg cells from one donor after 5-days of treatment. The graph shows the percentage of the total Tregs expressing Ki67 (Treg%+Ki67) in human PBMC Treg cells from one donor after 5-days of treatment with vehicle (far left ■ only), isotype control (♦), Antibody 1 (•), Antibody 2 (■). Antibody 3 ( A), or Antibody 4 (▼) Fc vanants as detailed in Example 7. Administration of Antibody 2 showed the most potent induction of Ki67 on Treg cells, followed by Antibody 1 and Antibody 4, while induction by Antibody 3 was less in comparison.

[0039] Figures 9A, 9B, 9C and 9D are graphs of the average dose response induction of CD25 and Ki67 by Fc variants in human PBMC non-Treg T cells from one donor after 5- days of treatment. FIGS. 9A-9B show the expression of CD25 (CD25 positive cells MFI (posMFI CD25)) of human PBMC CD4+ non-Treg (FIG. 9A) or CD8+ T cells (FIG. 9B) cells. FIGS. 9C-9D show the fraction of cells expressing Ki67 (%Ki67) of human PBMC CD4+ non-Treg (FIG. 9C) or CD8+ T cells (FIG. 9D) cells from one donor after 5-days of dose-response treatment with vehicle (far left ■ only), isotype control (♦), or Antibody I (•), Antibody 2 (■), Antibody 3 (A), and Antibody 4 (▼) variants as detailed in Example 7.

[0040] Figures 10A, 10B and 10C are graphs showing the effect of Antibody 1 (A), an isotype control (•), or dexamethasone (▼) as compared to no KLH challenge (o) on inflammatory response in a KLH-induced DTH model in hTNFR2 knock-in mice as described further in Example 8. FIG. 10A is a graph of the mean ear thickness (mm ±SEM) measured at 6, 7, 8, 9, and 10 days. FIG. 10B shows the area under the curve (AUC ±SEM) for days 6-10 for Antibody 1 (bar 3), an isoty pe control (bar 2), dexamethasone (bar 4), and mice with no KLH challenge (bar 1). FIG. 10C shows the ear thickness (mm ±SEM) at the peak inflammation (on day 9) for Antibody 1 (A), an isotype control (•), dexamethasone (▼), and mice with no KLH challenge (o).

[0041] FIG. 11 is a graph of the relative body weight changes as the % change in body weight (mean ±SEM) in mice on days 6, 7, 8, 9, and 10 in response to KLH challenge and treatment with Antibody 1 (A), an isotype control (•), dexamethasone (▼), and mice with no KLH challenge (o) as detailed in Example 8.

[0042] Figures 12A, 12B, 12C and 12D are graphs of spleen (FIG. 12A) and lymph node (FIG. 12C) weights and organ weight normalized Treg counts (spleen in FIG. 12B and lymph node in FIG. 12D) in hTNFR2 knock-in mice (Groups 1-4) on Day 10 in response to KLH challenge and treatment with Antibody 1 (A), an isotype control (•), dexamethasone (▼), and mice with no KLH challenge (o) as detailed in Example 8.

[0043] Figure 13 is a graph of KLH challenged ear epidermal thickness as measured on Day 10 in hTNFR2 knock-in mice after treatment with Antibody 1 (A), an isolype control (•), dexamethasone (▼ ). and mice with no KLH challenge (naive) (o) as detailed in Example 8.

[0044] Figures 14A and 14B are graphs showing efficacy in a hPBMC xenotransplantation GvHD model after treatment in vivo with Antibody 2 (FIG. 14A) and treatment in vitro with hPBMC pretreatment (FIG. 14B) as detailed in Example 8. FIG. 14A is a graph of thepercent survival for 0 to 75 days after hPBMC transfer after treatment in vivo with a vehicle (no hPBMC transfer) (1), vehicle (twice weekly (BIW)) (4), Antibody 2 (BIW) (5), or 60 mg / kg of 10-C1-BBQ (thrice weekly) (6). FIG. 14B is a graph of the percent survival for 0 to 75 days after hPBMC transfer after in vitro pre-treatment with a vehicle (no hPBMC transfer) (1), vehicle pretreated hPBMC (2), or Antibody 2 pretreated hPBMC (3).

[0045] Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, 151, 15J, 15K, 15L, 15M and 15N are graphs showing the ability of anti-TNFR2 antibodies to induce TNFR2 -mediated NFKB signaling in a HEK-Blue™ TNFa reporter cell assay as described further in Example 3. The graphs show the optical density at 655 nm (ODess) for antibodies 1-12, Antibody 1, and an isotype control. The antibodies were added at a dose of 6.67 nM, 66.7 nM, or 667 nM.

[0046] Figure 16 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 2.

[0047] Figure 17 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 1.

[0048] Figure 18 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 17.

[0049] Figure 19 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 13.

[0050] Figure 20 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 8.

[0051] Figure 21 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 10.

[0052] Figure 22 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl. vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 12.

[0053] Figure 23 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl. vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 11.

[0054] Figure 24 depicts the variable heavy7region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl. vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 7.

[0055] Figure 25 depicts the variable heavy7region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 16.

[0056] Figure 26 depicts the variable heavy7region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 6.

[0057] Figure 27 depicts the variable heavy' region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 14.

[0058] Figure 28 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 15.

[0059] Figure 29 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 4.

[0060] Figure 30 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl. vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 3.

[0061] Figure 31 depicts the variable heavy region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl. vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 5.

[0062] Figure 32 depicts the variable heavy7region, heavy chain constant region, full length heavy chain, variable light region, light chain constant region, and full-length light chain as well as the vhCDRl. vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 amino acid sequences of Antibody 9.

[0063] Figures 33A-K are graphs of dose-dependent cell binding curves showing the fraction of binding positive cells (% Binding) after IV dosing of Antibody 1 at a dose of 2 mg / kg (■), 6.6 mg / kg (A), or 20 mg / kg (▼) for certain T cell subsets as compared to a vehicle (•) as measured at 3 hours, 24 hours, and 96 hours after dosing in hTNFR2 knock-in mice as detailed in Example 10. FIGs. 33 A-33K show the % binding (mean ± SD) for total T cells (CD4+CD25+FoxP3+ Treg cells (FIG. 33 A), CD4+CD25-FoxP3+ cells (FIG. 33B), non-Treg CD4+ T cells (FIG. 33C), Ly6G+ neutrophils (FIG. 33D), CD25+FoxP3high Treg cells (FIG. 33E), CD25-FoxP3high Treg cells (FIG. 33F). CD8+ T cells (FIG. 33 G), Ly6C+ monocytes (FIG. 33 H), CD25+FoxP31ow Treg cells (FIG. 331), CD25-FoxP31ow Treg cells (FIG. 33J), and NKp46+ NK cells (FIG. 33K).

[0064] Figures 34A-34E are graphs showing Antibody 1 dose-dependent binding curves of the tMFI binding data after IV dosing of Antibody 1 at a dose of 2 mg / kg (■), 6.6 mg / kg (A), or 20 mg / kg (▼) for certain T cell subsets as compared to a vehicle (•) as measured at 3 hours, 24 hours, and 96 hours in hTNFR2 knock-in mice as detailed in Example 10. FIGs. 34A-34E show the Antibody 1 tMFI for total Treg cells (FIG. 34A), CD4+ non-Treg cells(FIG. 34B), CD25+FoxP3high Treg cells (FIG. 34C), CD8+ T cells (FIG. 34D), and CD25+FoxP31ow Treg cells (FIG. 34E).

[0065] Figure 35 is a graph comparing the mean specific binding (background non-specific signal subtracted) between Total CD4+CD25+FoxP3+ Treg, Total CD4+CD25-FoxP3+ Treg, CD4+CD25+FoxP31ow Treg, CD4+CD25+FoxP3high Treg, CD4+CD25-FoxP31ow Treg, CD4+ Tconv (non-Treg), CD8+ Tconv (CD8+ T cells), natural killer (NK) cells, monocytes, and neutrophil immune cell types as percent binding positivity (% specific binding) at peak binding (3 hours timepoint) for Antibody 1 in the highest dose group (20 mg / kg) in hTNFR2 knock-in mice as detailed in Example 10.

[0066] Figure 36 is a graph of the mean plasma concentration of Antibody 1 after IV dosing in hTNFR2 knock-in mice as detailed in Example 10. The graph shows the plasma concentration of Antibody 1 (pg / mL) (mean ±SD, n=4) at 3, 24 and 96 hours after administration of Antibody 1 at a dose of 2 mg / mL (•), 6.6 mg / mL (■) or 20 mg / mL (A).

[0067] Figures 37A-37J are graphs showing percent binding and tMFI binding data after IV administration of Antibody 1 or Antibody 2 at a dose of 20 mg / kg in vivo in mice expressing knock-in of human TNFR2 receptor as detailed in Example 11. FIGs. 37A and 37B show the total Treg %+ binding (FIG. 37A) and tMFI binding (FIG. 37B) for Antibody 1 (A), Antibody 2 (▼), or a vehicle (•). FIG. 37C and 37D show7the CD25+FoxP3high Treg %+binding (FIG. 37C) and tMFI binding (FIG. 37D) for Antibody 1 (A), Antibody 2 (▼), or a vehicle (•). FIGs. 37E and 37F show the CD25+FoxP31ow Treg %binding (FIG. 37E) and tMFI binding (FIG. 37F) for Antibody 1 (A), Antibody 2 (▼), or a vehicle (•). FIGs. 37G and 37H show the non-Treg CD4+ T cell %binding (FIG. 37G) and tMFI binding (FIG. 37H) for Antibody 1 (A), Antibody 2 (▼), or a vehicle (•). FIGs. 371 and 37J show the CD8+ T cell %binding (FIG. 371) and tMFI binding (FIG. 37J) for Antibody 1 (A), Antibody 2 (▼), or a vehicle (•).

[0068] Figures 38A and 38B are graphs showing specific binding of Antibody 1 (left bar) or Antibody 2 (right bar) for total Treg cells, FoxP3high Tregs, FoxP31ow Tregs, CD4+ Tconv (non-Treg) cells, or CD8+ T cells 24 hours (FIG. 38A) or 96 hours (FIG. 38B) after IV dosing in vivo at a dose of 20 mg / kg in mice expressing knock-in of human TNFR2 receptor as detailed in Example 11.

[0069] Figures 39A and 39B are graphs showing the results of a library screen assay of Antibody 2 against HEK293 cells expressing 6105 individual full-length human plasmamembrane proteins, secreted and cell surface-tethered human secreted proteins, as well as a further 400 human heterodimers as detailed in Example 12. FIG. 39A is a graph showing the binding of Antibody 2 for 19 of the proteins as the median SNR. FIG. 39B is a graph show ing the binding of Antibody 2 to TNFR2 isoform 1 (transmembrane) or TNFR2 isoform 2 (tethered soluble) as the median SNR.

[0070] Figures 40A, 40B, 40C, 40D, and 40E are graphs showing the dose response curves of the average %+ pRelA from donor 3 for TNFR2 binding antibody variants Antibodies 1-2 as detailed in Example 5. FIG. 40 A shows the CD25 / FoxP3high Treg %pRelA for PBMCs from donor 3 for Antibody 1 (•), Antibody 2 (■); FIG. 40B shows the CD4+ non-Treg %pRelA for PBMCs from donor 3 for Antibody 1 (•). Antibody 2 (■); FIG. 40C shows the CD8+ T cells %pRelA for PBMCs from donor 3 for Antibody 1 (•), Antibody 2 (■); FIG. 40D shows the CD56+ NK. cell %pRelA for PBMCs from donor 3 for Antibody 1 (•), Antibody 2 (■); FIG. 40E shows the CD 14+ monocyte %pRelA for PBMCs from donor 3 for Antibody 1 (•), Antibody 2 (■).

[0071] Figures 41A and 41B are graphs showing gene set enrichment analysis (GSEA) of Antibody 2 induced transcriptome in primary human Tregs as detailed in Example 13.DETAILED DESCRIPTION

[0072] Before describing one or more embodiments of the present disclosure in detail, it is to be understood that this disclosure is not limited to the antibodies, antibody fragments, their features, methods of preparation or use, and the like, as such may vary.

[0073] It must be noted that, as used in this specification and the intended claims, the singular forms “a,” “an,’' and “the"’ include singular and plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” includes a single antibody as well as two or more of the same or different antibodies, a composition comprising two or more of the same or different antibodies, and the like. Accordingly, the terms “a”, “one or more”, and “at least one” are used interchangeably herein unless context clearly indicates otherwise.

[0074] An “antibody” as used herein is intended to encompass a polypeptide that binds to an antigen and comprises at least an antigen-binding site (the variable domains of each of a heavy chain and a light chain). Antibody structures are well known to those of skill in the art. The terms “antibody” and “immunoglobulin” may be used interchangeably herein. As usedherein, “antibody” also encompasses an “antigen-binding fragment” (also used interchangeably with “antigen-binding portion” of an antibody, “antibody fragment”, and “antibody derivative”) of the antibody unless otherwise apparent by context. That is, for the purposes described herein, an antibody has a minimum functional requirement that it binds to a TNFR2 antigen.

[0075] An “isolated” antibody (or fragment, variant, or mutant thereof) is intended to encompass an antibody that is separated / purified from other proteins and antibodies without limitation as to a particular purification level or method. A recombinantly prepared antibody is also considered “isolated”. Suitable separation techniques are known in the art and include, without limitation, ammonium sulfate precipitation, affinity purification, and chromatography. Exemplary methods of purification are described in Example 1 and Example 2.

[0076] “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions as described herein and that causes no significant adverse toxicological effects to the patient. Pharmaceutically-acceptable excipients are well- known in the art; exemplary excipients are described in the Handbook of Pharmaceutical Excipients, 9thEdition, Eds. Rowe, R.C., Sheskey, P.J., and Quinn, M.E., Pharmaceutical Press.

[0077] “Pharmacologically effective amount,” “physiologically effective amount,” and “therapeutically effective amount” are used interchangeably herein to mean the amount of a TNFR2 antibody that is needed to provide a desired level of the antibody in the bloodstream or in the target tissue and / or the amount needed to provide a desired physiological effect as described further below. The precise amount will depend upon numerous factors, e.g., the particular antibody, the components and physical characteristics of the therapeutic composition, intended patient population, individual patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein.

[0078] The term, “about” or “approximately” as used herein means in reasonable vicinity of a stated numerical value or range of values, for example, within plus or minus five percent of a given quantity.

[0079] "Substantially" or "essentially" means nearly totally or completely, for instance, 95% or greater of a given quantity', unless stated to the contrary.

[0080] “Optional” or “optionally” means that the subsequently described circumstance may but need not necessarily occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0081] The term “substantially homologous” means that a particular subject sequence, for example, a variant or mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For purposes of the present invention, sequences having greater than 80 percent (more preferably greater than 85 percent, still more preferably greater than 90 percent, with greater than 95 percent being most preferred) homology, equivalent biological activity (although not necessarily equivalent strength of biological activity), and equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncation of the mature sequence should be disregarded.

[0082] The term “patient” or “subj ect” as used herein refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of an active agent (e.g.. an anti-TNFR2 agonist antibody), and includes both humans and animals.

[0083] As used herein, “sequence identity” is determined by comparing the sequence of a reference amino acid sequence or DNA sequence to that portion of another amino acid sequence or DNA sequence so aligned so as to maximize overlap between the two sequences while minimizing sequence gaps, wherein any overhanging sequences between the two sequences are ignored. With respect to any sequence identity described herein, sequences having at least 80%, more preferably at least 85%, yet more preferably at least 90%, still yet more preferably at least 95% sequence identity, with sequence identities of at least 96%, at least 97%, at least 98%, and at least 99% being most preferred. Various software programs for determining similarity between sequences are available, including, for example, BLAST, Ident and Sim, ClustalW, MMseqs2, and the like.

[0084] Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is He or I; Methionine is Met or M; Valine is Vai or V; Serine is Ser or S: Proline is Pro or P; Threonine is Thr or T: Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.OVERVIEW

[0085] The instant disclosure is directed to, among other things, novel and unique anti- TNFR2 antibodies, and compositions, kits, methods, combinations and uses thereof. Such antibodies, compositions and / or methods will ideally possess one or more of several advantageous and unpredictable features, such as, for example, (i) preferential and selective activation of the NFKB pathway in Tregs, (ii) preferential binding to Treg cells (e.g. cells expressing one or more of CD3+, CD4+, CD25+, and FoxP3+ cell surface markers) over conventional T cells and / or cytotoxic T cells; (iii) promotion of Treg proliferation; and (iv) selective stimulation of TNFR2 receptor activity and / or upregulation of FoxP3 on Tregs. among other features. Such anti-TNFR2 antibodies can find use, for example, in the treatment of inflammatory conditions and / or autoimmune diseases.

[0086] Provided herein are agonistic TNFR2-antibodies having an antigen-binding domain that specifically binds to TNFR2. Such antibodies can comprise, for example: (i) a heavy chain having from about 80-100% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO: 18; and / or (ii) a heavy chain variable region (VH or vh) having from about 80-100% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13. and / or (iii) a light chain variable region (VL or vl) having from about 80-100% sequence identity to the ammo acid sequence of SEQ ID NO:7 or SEQ ID NO: 19. In one or more embodiments, the antibody comprises heavy chain CDRs, vhCDRl, vhCDR2, and vhCDR3, comprising the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and light chain CDRs. vlCDRl. vlCDR2, and vlCDR3, comprising the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NOTO, respectively. In yet another embodiment, the anti-TNFR2 antibody comprises heavy chain CDRs, vhCDRl, vhCDR2, and vhCDR3, comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, and light chain CDRs, vlCDRl, vlCDR2, and vlCDR3, comprising the amino acid sequences of SEQ ID NO:20, SEQ ID NO:21. and SEQ ID NO:22, respectively. These and other structural and functional characteristics and uses of the subject antibodies are described in detail in the sections which follow.ANTI-TNFR2 ANTIBODIES

[0087] Selective upregulation of TNFR2 in Tregs is facilitated through the NFKB (canonical and non-canonical), MAPK, and PI3K-Akt signaling pathways (Li el al., J Transl Med. (2024) 22:812). Agonistic TNFR2 antibodies or antigen-binding fragments thereof as described herein potentiate the activation of TNFR2 by binding this receptor (e.g, on the exterior surface of a Treg cell) and inducing TNFR2-mediated signaling pathways as referenced above such as the canonical and / or non-canonical (alternative) NFKB pathway. Without being limited by theory, the anti-TNFR2 antibodies described herein activate TNFR2 -mediated NFKB signaling to provide an increase in RelA phosphorylation (pRelA) in Tregs (e.g. cells expressing one or more of CD3+CD4+CD25+FoxP3+ cell markers). Increasing RelA phosphorylation in Treg cells can, for example, reduce inflammation and the occurrence or severity of inflammatory’ conditions by enhancing the suppressive function of Tregs. Activation of the NFKB pathway by the anti-TNFR2 antibodies provided herein can be monitored or determined by methods as known in the art. One exemplary’ method is by monitoring any increase in the alkaline phosphatase (ALP) secretion level and activity' in Tregs as described further in the examples below. As agonistic antibodies, the TNFR2 antibodies described herein preferentially and selectively activate the NFKB pathway in Tregs, but not to a significantly greater extent than in other immune cells such as conventional T cells (e.g. non-Treg cells) and cytotoxic T cells. This finding is unexpected considering that TNFR2 expression is not Treg selective and other T cells, monocytes and NK cell express TNFR2. Additionally, the agonistic TNFR2 antibodies as described herein also preferentially (and advantageously) bind to immunosuppressive Tregs over conventional T cells (e.g. non-Treg cells) and cytotoxic T cells.

[0088] Without being limited as to theory7, use of TNFR2 agonism (e.g., the anti- TNFR2 / agonistic TNFR2 antibodies described herein) as a treatment modality is based on one or more mechanisms of action including, but not limited to suppression of inflammation, promotion of immune resolution, and / or tissue protection and / or repair. In general, Treg cells are a subset of CD4+ T cells that are involved in maintaining immune homeostasis by inhibiting the pro-inflammatory activities of other cells, including CD4+ and CD8+ conventional T cells. Tregs may be identified as expressing one or more of CD3+, CD4+, CD25+, and / or FoxP3+ surface markers. In a preferred embodiment, the Tregs are identified as (at least) CD4+CD25+FoxP3+ T cells. Accordingly, the agonistic TNFR2 antibodies orantigen-binding fragments thereof provided herein can agonize the TNFR2 receptor and activate the TNFR2 pathway. In some preferred embodiments, an agonistic TNFR2 antibody comprises the structural features as provided in Figures 16 and 17 (e.g., including anti- TNFR2 antibodies including those with full length heavy chain sequences, light chain sequences, variable heavy and light chain sequences, Fc sequences and / or CDRs as shown in Figures 16 and / or 17, or having the CDRs of an antibody comprising the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 7 or the VH of SEQ ID NO: 13 and the VL of SEQ ID NO: 19) as well as antibodies that compete for binding with the antibodies enumerated in Figures 16 and / or 17.

[0089] As discussed below-, the term "antibody'’ is used generally . Antibodies that find use herein can take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments and mimetics, described below. In general, the term ‘‘antibody” includes any polypeptide that includes at least one antigen binding domain. Antibodies may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, as described herein, with monoclonal antibodies finding particular use in many of the embodiments described herein. In some embodiments, the antibodies described herein bind specifically or substantially specifically to TNFR2. The terms “monoclonal antibodies” and “monoclonal antibody composition”, as used herein, refer to a population of antibody molecules that contain only one species of an antigen-binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody molecules that contain multiple species of antigen-binding sites capable of interacting w ith a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.

[0090] As used herein, an “agonist antibody” of TNFR2, “TNFR2 agonist antibody”, or “anti-TNFR2 antibody” refers to an antibody that can essentially mimic the activity of a native / physiological ligand or ligands. The agonist activity7can occur when the antibody binds the TNF receptor 2 in a manner that mimics the binding of a physiological ligand, thereby resulting in antibody-mediated agonism. In some embodiments, an agonist antibody described herein mimics the activity of a native ligand. In some embodiments, an agonist antibody described herein replaces the activity7of the native ligand completely. In some embodiments, an agonist antibody described herein specifically activates the TNFR2receptor. In some embodiments, an agonist antibody described herein replaces the activity of the native ligand and specifically activates the TNFR2 receptor (e.g. TNFR2-mediated signaling). In some embodiments, “specific binding” of the TNFR2 antibody or antigenbinding fragment thereof to TNFR2 occurs when the equilibrium dissociation constant (Kd) is no greater than and / or within a range from about 0.01 nM-2000 nM. In further embodiments, the TNFR2 antibody or antigen-binding fragment thereof specifically binds to TNFR2 with an affinity characterized by having an equilibrium dissociation constant of no greater than about 2000 nM, about 1000 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, or about 0. 1 nM. Various methods are known in the art and may be used to measure the dissociation rate constant (koff) and the association rate constant (kon) and calculate the Kd. One exemplary method for determining the Kd is surface plasmon resonance (SPR). Agonism of the antibodies can be determined by any method as known in the art. Exemplar}' methods of assessing agonism by canonical NFKB pathway activation can be determined, for example, by a reporter assay or pRelA assay as well as by using downstream events such as Treg markers as described in the examples.

[0091] Traditional full length antibody structural units typically comprise a tetramer. Each tetramer is ty pically composed of two identical pairs of polypeptide chains, each pair having one “light” chain (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The antibodies described herein are generally directed to the IgG class, which includes several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4. Thus, by “isotype” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. While the exemplary antibodies herein designated “Antibody 2” and “Antibody 1” are based on IgG4 and IgGl heavy chain constant regions, respectively, as shown in Figure 16 and Figure 17, the anti-TNFR2 antibodies described herein include those having IgGl. IgG2, IgG3 and IgG4 sequences, or combinations thereof. For example, as is known in the art. different IgG isotypes have different effector functions which may or may not be desirable. Accordingly, the TNFR2 antibodies described herein can also possess domains in which the IgGl constant domain is replaced by an IgG2, IgG3 or IgG4 constant domain, with IgG2 and IgG4 finding particular use in a number of situations, for example, forease of manufacture or when reduced effector function is desired, the latter being desired in some situations.

[0092] In general. Antibody 1 is a human TNFR2 binding antibody of the human IgGl isotype and containing LALA-PG mutations, described further below, in the Fc region to eliminate Fc receptor binding of the constant region without diminishment of and / or compromising agonist activity as compared to a TNFR2 binding antibody of the human IgGl isotype and containing LALA mutations with the same variable region. Antibody 1 is an agonist of human TNFR2, preferentially targeting Tregs for the enhancement of their immunosuppressive function.

[0093] In general, Antibody 2 is a human TNFR2 binding antibody of the human IgG4 isotype and containing a S228P mutation in the hinge region to eliminate Fab arm exchange (FAE), among other things. Incorporation of the IgG4 subclass in this antibody improved TNFR2 agonism activity. In particular, Antibody 2 IgG4 (S228P) exhibited higher activity than antibodies having the same variable region variants on an IgGl_LALA or an IgGl subclass. As detailed in Example 5, Antibody 2 including the IgG4 subclass showed improved agonism activity over antibodies including an IgGl, IgGI LALA, or IgGI LALAPG structural subclass features as indicated by an increase in phosphorylation of RelA (higher Emax values). As detailed in Example 6, Antibody 2 consistently possessed higher Emax values for induction of Fc-receptor independent NFKB pathway signaling in HEK-Blue™ TNFa reporter cells as compared to antibodies including IgGl, IgGl LALA, or IgGI LALAPG structural subclass features.

[0094] The amino-terminal portion of each chain includes a variable (V or v) region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, generally referred to in the art and herein as the ”Fv domain” or Tv region”. In the variable region, three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a “CDR”), in which the variation in the amino acid sequence is most significant. “Variable” refers to the fact that certain segments of the variable region differ extensively in sequence among antibodies. Variability within the variable region is not evenly distributed. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability' called “hypervariable regions”.

[0095] As used herein, the term “complementarity determining region’' or “CDR” refers to the six hypervariable region(s) of a heavy or light chain variable region. Proceeding from the N-terminus, each of a heavy or light chain polypeptide has three CDRs denoted as “CDR1,” “CDR2,” and “CDR3”. Proceeding from the N-terminus, the heavy chain variable region CDRs are denoted as vhCDRl, vhCDR2, and vhCDR3. Proceeding from the N-terminus, the light chain variable region CDRs are denoted as vlCDRl, vlCDR2, and vlCDR3. Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with a bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the CDR regions are primarily responsible for the specificity of an antigen-binding site. In one embodiment, an antigen-binding site includes six CDRs, comprising the CDRs from each of a heavy and a light chain variable region denoted as vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2, and vlCDR3. These CDRs are short, non-contiguous sequences that form the antigen-binding domain of the antibody in its three-dimensional configuration provided, at least in part, by the framework region described below.

[0096] As used herein, the term “framework region” or “FR” refers to the four flanking amino acid sequences (FR residues) w hich frame the CDRs of a heavy or light chain variable region. Some FR residues may contact bound antigen, however, FR residues are primarily responsible for folding the variable region into the antigen-binding site. In some embodiments, the FR residues responsible for folding the variable regions comprise residues directly adjacent to the CDRs. Within the FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all variable region sequences contain an internal disulfide loop of around 90 amino acid residues. When a variable region folds into an antigen binding site, the CDRs are displayed as projecting loop motifs that form an antigen binding surface. It is generally recognized that there are conserved structural regions of FRs that influence the folded shape of the CDR loops into certain “canonical” structures regardless of the precise CDR amino acid sequence. Furthermore, certain FR residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.

[0097] Each VH and VL is composed of three hypervariable regions (“complementary determining regions,” “CDRs”) and four FRs, arranged from amino-terminus to carboxyterminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0098] Accordingly, provided herein are variable heavy domains, variable light domains, heavy constant domains, light constant domains, hinge regions and Fc domains of anti- TNFR2 agonist antibodies. By “variable region’’ as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the VK or V , and / or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively. Accordingly, the variable heavy domain comprises vhFRl-vhCDRl-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4. and the variable light domain comprises vlFRl-vlCDRl-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. By “heavy constant region” is meant the CHl-hinge-CH2-CH3 portion of an antibody. By “Fc” or “Fc region” or “Fc domain” as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain and, in some cases, part of the hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, the Fc domain comprises immunoglobulin domains Cy2 and Cy3 (Cy2 and Cy3) and the lower hinge region between Cyl (Cyl) and Cy2 (Cy2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. In some embodiments, as is more fully described below, amino acid modifications are made to the Fc region, for example to alter binding to one or more FcyR receptors or to the FcRn receptor.

[0099] A number of antibody numbering systems are routinely used in the art for identification and / or comparison of antibody variable regions (including CDRs). Commonly used numbering schemes / sy stems known and routinely used in the art include Kabat. Chothia, and IMGT®. It will be appreciated that the CDRs are defined by the different schemes / systems and a skilled artisan could readily convert CDR positions from one system to another.

[0100] Wu and Kabat (Wu and Kabat, “An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity”, Journal of Experimental Medicine (1970) 132(2):8; Kabat et al. , “Sequence of proteins of immunological interest”, Bethesda: National Institutes of Health,' 323 (1983)) pioneered the alignment of antibody peptide sequences, and their contributions inthis area were several-fold. Through study of sequence similarities between variable domains, they identified correspondent residues that to a greater or lesser extent were homologous across all antibodies in all vertebrate species, inasmuch as they adopted similar three- dimensional structure, played similar functional roles, interacted similarly with neighboring residues, and existed in similar chemical environments. Secondly, they devised a peptide sequence numbering system in which homologous immunoglobulin residues were assigned the same position number. Accordingly, one skilled in the art can unambiguously assign to any variable domain sequence using what is now commonly called Kabat numbering without reliance on any experimental data beyond the sequence itself. Thirdly, Kabat and Wu calculated variability for each Kabat-numbered sequence position, which is the finding of few or many possible amino acids when variable domain sequences are aligned. They identified three contiguous regions of high variability embedded within four less variable contiguous regions. Kabat and Wu formally demarcated residues constituting these variable tracts and designated these “complementarity' determining regions” (CDRs), referring to chemical complementarity between antibody and antigen. A role in three-dimensional folding of the variable domain, but not in antigen recognition, was ascribed to the remaining less-variable regions, which are now termed “framework regions”. Fourthly, Kabat and Wu established a public database of antibody peptide and nucleic acid sequences, which continues to be maintained and is well known to those skilled in the art.

[0101] Chothia and coworkers (Cyrus Chothia and Arthur M. Lesk. Journal of Molecular Biology (1987) 196(4): 901 -917) subsequently found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2 and L3 or HI. H2 and H3, where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs.

[0102] More recent studies have shown that virtually all antibody binding residues fall within regions of structural consensus. (Kunik, V., et al., PloS Computational Biology (2012) 8(2):el002388). In some embodiments, these regions are referred to as antibody binding regions. It was shown that these regions can be identified from the antibody sequence as well. “Paratome”, an automated tool for the identification of antigen binding regions of an antibody involving implementation of a structural approach for the identification of structuralconsensus in antibodies, may be used for this purpose. (Ofran, Y. et al, J. Immunol. (2008) 757:6230-6235). While residues identified by Paratome cover virtually all the antibodybinding sites, the CDRs (as identified by the commonly used CDR identification tools) miss significant portions of them. Antibody binding residues identified by Paratome but not identified by any of the common CDR identification methods are referred to as Paratomeunique residues. Similarly, antibody binding residues that are identified by any of the common CDR identification methods but are not identified by Paratome are referred to as CDR-unique residues. Paratome-unique residues make crucial energetic contribution to antibody-antigen interactions, while CDRs-unique residues have a rather minor contribution. These results allow for better identification of antigen binding sites.

[0103] IMGT® is the international ImMunoGeneTics information system®, (See, Lefranc et al.. Nucleic Acids Res. (2015) 43(D1):D413-D422. doi.org / 10.1093 / nar / gkul056; Lefranc et al., Dev Comp Immunol. (2003) 27(l):55-77). IMGT® is a unique numbering system for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains (Lefranc et al. , Dev Comp Immunol. (2003) 27: 55-77). IMGT® presents a uniform numbering system for these IG and TcR variable domain sequences, based on aligning 5 or more IG and TcR variable region sequences, taking into account and combining the Kabat definition of FRs and CDRs, structural data, and Chothia’s characterization of the hypervariable loops. The IMGT® system may routinely be used to analyze the variable domains and / or to identify a variable region (including the CDRs). The IMGT® is considered well known in the art as a universal numbering scheme for antibodies.

[0104] In describing variant amino acid positions present in the VH and VL domains, in some embodiments any common CDR definition may be used to describe the CDR regions, for example but not limited to the approaches used by IMGT®. Kabat, Chothia and Paratome. One of ordinary skill in the art can routinely determine the scheme / system used for CDR sequence numbering (e.g., Kabat, Chothia, or IMGT®).

[0105] CDRs may be defined as set forth herein. A number of programs are available to determine the CDRs of a given antibody including, without limitation, abYsis. AlphaFold, IgFold. NovoPro, Rosetta Antibody, or any of the Molecular Operating Environment (MOE) programs as appropriate and available from the Chemical Computing Group. It will be appreciated that different systems and / or schemes use different numbering conventions and CDR definitions. It will be appreciated that different numbering schemes may utilizedifferent definitions of CDR lengths, which may be readily determined by the skilled person using routine methods and tools as known in the art. Moreover, it will be appreciated that, having the heavy chain variable and light chain variable amino acid sequences in hand, a skilled artisan can readily determine the CDRs comprised within such variable domains.

[0106] An antibody may exist in various forms or have various domains including, without limitation, a complementarity determining region (CDR), a variable region (Fv). a VH domain, a VL domain, a single chain variable region (scFv), and a Fab fragment. A person of ordinary skill in the art will appreciate that a scFv is a fusion polypeptide comprising the variable heavy chain (VH) and variable light chain (VL) regions of an immunoglobulin, connected by a short linker peptide. The linker may have, for example, 10 to about 25 amino acids.

[0107] By ‘'Fab” or “Fab region” as used herein is meant the polypeptide that comprises the VH, CHI, VL, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody. As will be appreciated by those in the art, these generally are made up of two chains.

[0108] The carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat el al. collected numerous primary- sequences of the variable regions of heavy- chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof (see, SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., incorporated herein by reference in its entirety).

[0109] In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By “immunoglobulin (Ig) domain” as used herein is meant a region of an immunoglobulin having a distinct tertiary- structure. Of interest are the heavychain domains including the constant heavy (CH) domains and the hinge domains. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, “CH” domains in the context of IgG are as follows: “CHI” refers to positions 1 18-220 according to the EU index as in Kabat, “CH2” refers to positions 237-340 according to the EU index as in Kabat, and “CH3” refers to positions 341-447 according to the EU index as in Kabat.

[0110] Thus, “Fc variant” or “variant Fc” as used herein is meant to embody a protein comprising at least one amino acid modification in the antibody Fc domain. The Fc variants described herein are defined according to the amino acid modifications comprised therein. Thus, for example, P329G is an Fc variant with the substitution of proline at position 329 with glycine relative to the parent Fc polypeptide, wherein the numbering is according to the EU index. The identity of the wild type amino acid may be unspecified, in which case the aforementioned variant is referred to as P329G. For all positions discussed herein, numbering is according to the EU index. The EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody (Edelman, et al. Proc Natl Acad Sci USA (1969) 63:78-85; hereby incorporated by reference in its entirety). The modification can be an addition, deletion, or substitution. Substitutions can include naturally occurring amino acids and non-naturally occurring amino acids. Variants may comprise non-natural amino acids.

[0111] A “variant Fc region” comprises an amino acid sequence which differs from that of a “native” or “wildtype” sequence Fc region by virtue of at least one “amino acid modification” as herein defined. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will in some embodiments possess at least about 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, in some embodiments possess at least about 80% or at least about 85% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, and in some embodiments at least about 90%, at least about homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, and in some embodiments at least about 95% or about 99% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide. In embodiments, the Fc region of the antibodies described herein have at least about 80% to about 99% homology7with a native Fc region. In embodiments, the Fc region of the antibodies described herein have at least about 80% to about 95%. at least about 80% to about 90%, at least about 80% to about 85%. at least about 85% to about 99%, at least about 85% to about 95%, or at least about 85% to about 90%, homology7with a native Fc region. Further contemplated are antibodies having at least about 80% homology7with an Fc region of antibodies disclosed herein such as Antibody 1and Antibody 2. In some embodiments, contemplated herein are antibodies having at least about 80%. at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology with the Fc region of the antibodies disclosed herein. In embodiments, the antibodies have at least about 80% to about 99% homology with the Fc region of the antibodies disclosed herein. Further embodiments include antibodies having at least about 80% to about 95%. at least about 80% to about 90%, at least about 80% to about 85%, at least about 85% to about 99%, at least about 85% to about 95%, or at least about 85% to about 90% homologj' to the antibodies described herein. However, it will be appreciated that where the antibody has a defined variation (e.g. LALA or LALAPG), the variation is retained.

[0112] Throughout the present specification, the Chothia numbering system, IMTG® numbering system, or the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) (e.g., Kabat et al., supra (1991); Chothia, C. et al., Nature (1989) 342:877-883; Chothia, C. and Lesk, A.M., J. Mol. Biol. (1987) 196:901-917). EU numbering as in Kabat is generally used for constant domains and / or the Fc domains.

[0113] Included within the definition of “antibody” is an “antigen-binding portion” of an antibody (also used interchangeably with “antigen-binding fragment”, “antibody fragment” and “antibody derivative”). That is. for the purposes described herein, an antibody has a minimum functional requirement that it bind to a TNFR2 antigen. As will be appreciated by those in the art, there are a large number of antigen fragments and derivatives that retain the abi 1 i ty to bind an antigen and yet have alternative structures, including, but not limited to, (i) the Fab fragment consisting of VL, VH, CL and CHI domains, (ii) the Fd fragment consisting of the VH and CHI domains, (iii) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., Science (1988) 242:423-426, Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883, entirely incorporated by reference), (iv) “diabodies” or “triabodies”, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al. , Methods Enzymol. (2000) 326:461-479; PCT Publication No. WO 94 / 13804 to Cambridge Antibody Technology' Limited and Medical Research Council; Holliger et al., Proc. Natl. Acad. Sci. USA (1993) 90:6444-6448, all entirely incorporated byreference), (v) “domain antibodies” or “dAb” (sometimes referred to as an “immunoglobulin single variable domain”, including single antibody variable domains from other species such as rodent (for example, as disclosed in PCT Publication No. WO 00 / 29004 to Peptor Ltd.), nurse shark and Camelid V-HH dAbs, (vi) SMIPs (small molecule immunopharmaceuticals), camel bodies, nanobodies and IgNAR.

[0114] Still further, an antibody or antigen-binding portion thereof (antigen-binding fragment, antibody fragment, antibody portion) may be part of larger immunoadhesion molecules (sometimes also referred to as “fusion proteins”), formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules. Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0115] The CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of antibodies. “Epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.

[0116] The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide; in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide.

[0117] Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and non conformational epitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0118] An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, for example “binning”. Specific bins are described below.

[0119] Half maximal effective concentration (ECso) refers to the concentration of a compound, drug, antibody or toxicant which induces a response halfway between the baseline and maximum responses after a specified exposure time. In some embodiments, the response comprises a binding affinity. In some embodiments, the response comprises a functional response for example an agonistic response. A skilled artisan would appreciate that as used herein in certain embodiments, the EC50 measurement of an anti-TNFR2 antibody disclosed herein provides a measure of a half-maximal binding of the anti-TNFR2 antibody to the TNFR2 antigen (ECso binding). Measurement of ECso binding affinity comprises measuring the binding of an anti-TNFR2 antibody described herein to the TNFR2 antigen. The skilled artisan would appreciate that as used herein in certain embodiments, the ECso measurement of an anti-TNFR2 antibody disclosed herein provides a measure of a half-maximal effective concentration of the anti-TNFR2 antibody to induce an agonist response (ECso functional agonism). Measure of ECso functional agonism comprises measuring the effects of the anti- TNFR2 antibodies described herein on cellular signaling of TNFR2, as exemplified in the Examples provided herein. At least Example 4 provides an exemplary method of assessing binding of an antibody to lymphocyte subsets using human PBMCs. Example 5 provides an exemplary method of evaluating signaling ability of antibodies using PBMCs by determining phosphorylation of RelA (pRelA).

[0120] In some embodiments, ECso comprises the concentration of antibody required to obtain a 50% agonist response that would be observed following binding of the antibody to receptors of the cell. In certain embodiments, a measure of ECso is commonly used as a measure of a drug’s potency and may in some embodiments, reflect the binding of the antibody to the receptor. In certain embodiments, an anti-TNFR2 antibody disclosed herein comprises an agonist for the TNFR2 receptor. In certain embodiments, an anti-TNFR2 antibody disclosed herein is a low potency binder / agonist for the TNFR2 receptor.

[0121] In embodiments, the antibody is a humanized antibody. A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-humanHVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two. variable domains, in which all or substantially all of the HVRs (e.g, CDRs) correspond to those of a nonhuman antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibodyconstant region derived from a human antibody. A “humanized form” of an antibody, e.g. , a non-human antibody, refers to an antibody that has undergone humanization.

[0122] In preferred embodiments, the anti-TNFR2 antibodies described herein are recombinant. “Recombinant” as used herein, refers broadly with reference to a product, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0123] The term “recombinant antibody”, as used herein, includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g, from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0124] “Modulation of an immune response” includes, for example, the following: elimination, deletion, or sequestration of immune cells; induction or generation of immunecells that can modulate the functional capacity of other cells such as autoreactive lymphocytes, antigen presenting cells, or inflammatory cells; induction of an unresponsive state in immune cells (z.e., anergy): increasing, decreasing, or changing the activity or function of immune cells or the capacity to do so, including, but not limited to, altering the pattern of proteins expressed by these cells. Examples include altered production and / or secretion of certain classes of molecules such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors; or any combination of these modulatory events. In some embodiments, modulation includes suppression or immunosuppression of certain immune responses, e.g. , in conditions or diseases where the immune system displays an excessive or overactive response such as autoimmune diseases, allergies, bone marrow or organ transplant, and other inflammatory diseases. Suppression or immunosuppression as used herein includes reduction, inhibition and lessening of an immune response or inflammation.Specific anti-TNFR2 antibodies

[0125] It will be appreciated that the description and embodiments as described above for anti-TNFR2 antibodies applies equally to the specific anti-TNFR2 antibodies described below;

[0126] Generation of a reliable and well-engineered TNFR2 agonist antibody can open new avenues in therapeutic intervention for inflammatory and / or autoimmune diseases. It is an object to provide anti-TNFR2 antibodies for use in disease treatment. This need is met herein by providing anti-TNFR2 antibodies which, among other things, provide effective TNFR2 signaling activity to treat autoimmune diseases and disorders.

[0127] Anti-TNFR2 antibodies with agonistic activity were designed and assessed for their in vitro pharmacological properties in primary human Tregs and in vivo efficacy in a skin inflammation humanized-mouse model as well as in a graft versus host mouse model. The designed antibodies have unique monovalent Fab TNFR2 binders recognizing a “signaling epitope”. The antibodies have Treg-selective agonistic biological activities, which is a desirable property in therapeutic intervention for various autoimmune diseases.

[0128] Described herein are anti-TNFR2 antibodies. For convenience, “anti-TNFR2 antibodies” and “TNFR2 antibodies” are used interchangeably. The anti-TNFR2 antibodiesspecifically bind to human TNFR2. In embodiments, the, e.g., anti-TNFR2 antibodies including those with CDRs identical to those shown in Figures 16 and 17.

[0129] In one embodiment, each of the anti-TNFR2 antibodies comprises a set of three complementarity determining regions (CDRs) on a heavy chain (vhCDRl, vhCDR2, and vhCDR3) and a set of three CDRs on a light chain (vlCDRl, vlCDR2 and vlCDR3). Preferably the CDRs are selected from the variable heavy chain as set forth in SEQ ID NOs: 1 or 13 and the variable light chain as set forth in SEQ ID NOs: 7 or 19. In specific embodiments, the set of vhCDRl, vhCDR2, and vhCDR3 comprises a combination of the amino acid sequences as shown in Figure 16 and Figure 17, and the set of vlCDRl, vlCDR2 and vlCDR3 on a corresponding light chain comprises a combination of the amino acid sequences as shown in Figure 16 and Figure 17.

[0130] In some embodiments, the anti-TNFR2 antibody is Antibody 2. The set of vlCDRl, vlCDR2 and vlCDR3 for this antibody comprises the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 respectively (Figure 16), whereas the set of vhCDRl, vhCDR2, and vhCDR3 for the same antibody comprises the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 respectively (Figure 16). As noted above, different approaches to determining CDR sequences may provide different definitions of the CDRs. For example, the set of CDRs for Antibody 2 as determined by Chothia are vlCDRl having the sequence of RASQDVSTAVA (SEQ ID NO:205), vlCDR2 having the sequence of SASSLYS (SEQ ID NO:206), vlCDR3 having the sequence of QQPTYTPPT (SEQ ID NO:207), vhCDRl having the sequence GFNIRDT (SEQ ID NO:208), vhCDR2 having the sequence DPDNGD (SEQ ID NO:209), and vhCDR3 having the sequence DLADGYNAFDR (SEQ ID NO:210).

[0131] In some embodiments, the anti-TNFR2 antibody is Antibody 1. The set of vlCDRl, vlCDR2 and vlCDR3 for this antibody comprises the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 respectively (Figure 17), whereas the set of vhCDRl, vhCDR2, and vhCDR3 for the same antibody comprises the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively (Figure 17).

[0132] In one embodiment, each of the anti-TNFR2 antibodies presented herein comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0133] In some embodiments, the anti-TNFR2 antibody is Antibody 2. The antibody comprises the VH comprises the amino acid sequences of SEQ ID NO: 1 and the VL comprises the amino acid sequences of SEQ ID NO: 7 of Figure 16.

[0134] In some embodiments, the anti-TNFR2 antibody is Antibody 1. The antibody comprises the VH comprises the amino acid sequences of SEQ ID NO: 13 and the VL comprises the amino acid sequences of SEQ ID NO: 19 of Figure 17.

[0135] In some embodiments, an anti-TNFR2 antibody comprises a heavy chain (HC) and a light chain (LC).

[0136] In some embodiments, the anti-TNFR2 antibody is Antibody 2. The HC comprises the amino acid sequences of SEQ ID NO: 6 and LC comprises the amino acid sequences of SEQ ID NO: 12 of Figure 16.

[0137] In some embodiments, the anti-TNFR2 antibody is Antibody 1. The HC comprises the amino acid sequences of SEQ ID NO: 18 and LC comprises the amino acid sequences of SEQ ID NO: 24 of Figure 17.

[0138] In another embodiment, the anti-TNFR2 antibodies comprise heavy chain and light chain CDR sequences that are at least 80% (e.g.. at least about 85%. 90%. 95%. 96%. 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example but not limited to identity as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. A skilled artisan would appreciate that percent identity (% identity) provides a number that describes how similar the query sequence is to the target sequence (e.g., how many amino acids in each sequence are identical). The higher the percent identity is, the more significant the match.

[0139] As discussed herein, further provided are variants of the above components, including variants in the CDRs, as outlined above. In addition, variable heavy chains can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the “VH” sequences herein, and / or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes, or more, when Fc variants are used. Variable light chains are provided that can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the “VL” sequences herein, and / or contain from I, 2, 3. 4, 5, 6, 7. 8, 9, 10 amino acid changes, or more, when Fc variants are used. Similarly, heavy and light chains are provided that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the “HC” and “LC” sequences herein, and / or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9,10 amino acid changes, or more, when Fc variants are used. It will be appreciated, however, where the antibody includes a mutation or variation in the sequence, that mutation or variation is maintained. For example looking to Antibody 2, the S228P mutation in the hinge region is maintained in any variants as encompassed by the presently described antibodies.

[0140] In some embodiments, the anti-TNFR2 antibodies also comprise framework regions. The framework regions of the variable heavy and variable light chains can be humanized as is known in the art (with occasional variants generated in the CDRs as needed), and thus humanized variants of the VH and VL chains of Figures 16 and 17 can be generated.

[0141] Furthermore, the humanized variable heavy and light domains can then be fused with human constant regions, such as the constant regions of anti-TNFR2 antibodies are from IgG class of antibodies. In a specific embodiment, the anti-TNFR2 antibodies are of the IgGl, IgG2, IgG3, or IgG4 subclass. In another specific embodiment, the anti-TNFR2 antibodies are of the IgGl subclass. In some other embodiments, the anti-TNFR2 antibodies are of the IgG2 subclass. In yet some other embodiments, the anti-TNFR2 antibodies are of the IgG3 subclass. In some further embodiments, the anti-TNFR2 antibodies are of the IgG4 subclass. In some further embodiments, the anti-TNFR2 antibodies are suitable for therapeutic use, e.g., a full-length human or humanized IgGl, IgG2, IgG3, or IgG4 antibody.

[0142] In some embodiments, the anti-TNFR2 antibody is Antibody 2. The constant heavy region Antibody 2 antibody comprises the amino acid sequences of SEQ ID NO: 5 of Figure 16, which is of the IgG4 subclass. In some embodiments, the anti-TNFR2 antibody is Antibody 1. The constant heavy region Antibody 1 antibody comprises the amino acid sequences of SEQ ID NO: 17 of Figure 17, which is of the IgGl subclass.

[0143] In some embodiments, the anti-TNFR2 antibody is Antibody 3. The constant heavy region Antibody 3 antibody comprises the amino acid sequences of SEQ ID NO: 173 of Figure 30, w hich is of IgGl subclass.

[0144] In some embodiments, the anti-TNFR2 antibody is Antibody 4. The constant heavy region Antibody 4 antibody comprises the amino acid sequences of SEQ ID NO: 161 of Figure 29, which is of IgGl subclass.

[0145] In addition, also included are sequences that may have the identical CDRs but have changes in the variable domain, framework regions, or constant regions. For example, anti-TNFR2 antibodies include those with CDRs identical to those shown in Figures 16-17 but whose identity along the variable region can be lower, for example about 80%, about85%, about 88%. about 90%, about 92%, about 95% or about 98% percent identical, and in some embodiments at least about 95% or at least about 98% identical.

[0146] The percent identity between two amino acid sequences can be determined using methods known in the art such as using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. (1988) 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity betw een two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (1970) 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available commercially), using either a Blossum 62 matrix or a PAM250 matrix, and a gap w eight of 16, 14, 12, 10, 8. 6, or 4 and a length weight of 1, 2. 3, 4, 5. or 6.

[0147] Additionally or alternatively, the protein sequences described herein can further be used as a “query' sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. J. Mol. Biol. (1990) 215:403-10. BLAST protein searches can be performed with the XBLAST program. score=50, word length=3 to obtain amino acid sequences homologous to the antibody molecules according to at least some of the embodiments described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul, et al., Nucleic Acids Res. (1997) 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0148] In general, the percentage identify for comparison between anti-TNFR2 antibodies is at least about 75%, at least about 80%, at least about 85%. at least about 90%, with at least about 95%, 96%, 97%, 98% or about 99% percent identify being preferred. The percentage identify may be along the whole amino acid sequence, for example the entire heavy or light chain or along a portion of the chains. For example, included within the definition of the anti-TNFR2 antibodies described herein are those that share identify along the entire variable region (for example, where the identify is about 80%, about 85%, about 90%. about 95% or about 98% identical along the variable regions, and in some embodiments at least about 80%, about 85%, about 90%, 95% or at least about 98%), or along the entire constant region, or along just the Fc domain.

[0149] In some embodiments, the anti-TNFR2 antibodies encompasses whole antibody molecules, including monoclonal and polyclonal antibodies, such as Antibody 1, Antibody 2, Antibody 3, or Antibody 4. In some embodiments, an antibody encompasses an antibody fragment or fragments that retain binding specificity including, but not limited to, variable heavy chain (VH) fragments, variable light chain (VL) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies. In a specific embodiment, the anti-TNFR2 antibodies is Antibody 5 which comprises the Fab region having the amino acid sequence of SEQ IDs: 181-192 as shown in Figure 31. In a specific embodiment, the anti-TNFR2 antibodies is Antibody 9 which comprises the Fab region having the amino acid sequence of SEQ IDs: 193-204 as shown in Figure 32.

[0150] In one embodiment, the anti-TNFR2 antibodies disclosed herein can be incorporated as part of a bispecific antibody. As generally known in the art, a bispecific antibody is a recombinant protein that includes antigen-binding fragments of two different monoclonal antibodies and is thereby capable of binding two different antigens. In embodiments, the anti-TNFR2 antibodies disclosed herein includes an antigen-binding fragment as described herein and an antigen-binding fragment of a second and different antigen target. In some embodiments, bispecific antibodies are used for cancer immunotherapy by simultaneously targeting, for example, both CTLs (such as a CTL receptor component such as CD3) or effector natural killer (NK) cells, and a tumor antigen. Similarly, a multi specific antibody is a recombinant protein that includes antigen-binding fragments of at least two different monoclonal antibodies, such as two, three or four different monoclonal antibodies.Anti-TNFR2 variants

[0151] In some embodiments, a variant Fc region of anti-TNFR2 antibody (Fc variant) may be generated according to the methods herein disclosed. It will be appreciated that the description and embodiments with reference to anti-TNFR2 antibodies and specific anti-TNFR2 antibodies as described above apply equally to the anti-TNFR2 variants described below. The IgGl, IgG2, IgG3, or IgG4 antibodies comprise an Fc region. Generally, the Fc region of the IgGl, IgG2, IgG3, or IgG4 polypeptide will comprise a native or wildtype sequence Fc region, and preferably a human native sequence Fc region (humanFc region). However, the Fc region of the wildtype polypeptide may have one or more preexisting amino acid sequence alterations or modifications from a native sequence Fc region. For example, the C Iq or Fey binding activity of the Fc region may have been previously altered (other types of Fc region modifications are described in more detail below). In a further embodiment the parent polypeptide Fc region is “conceptual” and, while it does not physically exist, the antibody engineer may decide upon a desired variant Fc region amino acid sequence and generate a polypeptide comprising that sequence or a DNA encoding the desired variant Fc region amino acid sequence.

[0152] In an alternative embodiment, a variant Fc region of anti-TNFR2 antibody derives from a human IgGl or IgG4 Fc region. In still a further embodiment, the variants are IgGl or IgG4 antibodies.

[0153] In another specific embodiment, anti-TNFR2 antibodies are of the IgGl subclass and proline at position 329 of the human IgG Fc region is substituted with another amino acid. In a further embodiment, the proline at position 329 is substituted with glycine (P329G). wherein the residues are numbered according to the EU index of Kabat.

[0154] In another embodiment, anti-TNFR2 antibodies comprising a Pro329 Fc variant further comprise at least one addition, substitution, or deletion of an amino acid residue in the Fc region that is correlated with increased stability of the antibody.

[0155] In one embodiment, the addition, substitution, or deletion of an amino acid residue is at position 234 and / or 235 of the IgGl Fc region, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al.

[0156] In another specific embodiment, anti-TNFR2 antibodies are of the IgGl subclass and leucine at position 234 of the human IgG Fc region is substituted by another amino acid. In a further embodiment, the leucine at position 234 is substituted with alanine (L234A), wherein the residues are numbered according to the EU index of Kabat.

[0157] In another specific embodiment, anti-TNFR2 antibodies are of the IgGl subclass and leucine at position 235 of the human IgG Fc region is substituted by another amino acid. In a further embodiment, the leucine at position 235 is substituted with alanine (L235A). wherein the residues are numbered according to the EU index of Kabat.

[0158] In a specific embodiment, the anti-TNFR2 antibodies are of the IgGl subclass and comprises a triple mutation: an amino acid substitutions at positions Pro329, Leu234, and Leu235235 mutation (LALAPG).

[0159] In another specific embodiment, said anti-TNFR2 antibody is of IgGl subclass and comprises the following amino acid substitutions: L234A and L235A of the Fc region (LALA mutations). In a further specific embodiment, the anti-TNFR2 antibody is Antibody 4. Antibody 4 comprises a constant heavy region (SEQ ID NO: 161) having IgGl Fc with LALA mutation.

[0160] In another specific embodiment, anti-TNFR2 antibodies are of the IgGl subclass and comprise the following amino acid substitutions: P329G and / or L234A and L235A of the Fc region. In a further specific embodiment, the anti-TNFR2 antibody is Antibody 1. Antibody 1 comprises a constant heavy region (SEQ ID NO: 17) having IgGl Fc with each of P329G, L234A and L235A mutations.

[0161] Applicant has unexpectedly found that anti-TNFR2 antibodies of the IgG4 subclass and comprising a modified hinge region possess an enhanced ability to activate NFKB signaling in Tregs. A variant constant region (i.e. CHl-hinge-CH2-CH3) of anti- TNFR2 antibody (constant region variant) may be generated according to methods as known in the art as exemplified by the methods herein disclosed. In one particular embodiment, anti- TNFR2 antibodies are of the IgG4 subclass and comprise a serine at position 228 of the human IgG substituted with another amino acid. In a preferred embodiment, the serine at position 228 is substituted with proline (S228P), wherein the residues are numbered according to the EU index of Kabat. In a further specific embodiment, the anti-TNFR2 antibody is Antibody 2. Antibody 2 comprises an IgG4 constant heavy region (SEQ ID NO: 5) with an S228P mutation. As seen in the Examples, Antibody 2, a TNFR2 antibody of the IgG4 subclass and having a S228P substitution exhibited higher activity than antibodies having the same variable region variants of the IgGl subclass and with or without a LALA or LALA PG mutation. Antibody 2, an anti-TNFR2 antibody of the IgG4 subclass and comprising a modified hinge region, showed enhanced ability to activate NFKB signaling in Tregs as compared to IgGl Fc-based variants. As described in Example 6, Antibody 2 (IgG4 S228P) induced greater NFKB pathw ay signaling than Antibody 1 (an IgG2_LALAPG Fc- variant) as shown in a HEK-Blue™ TNFa reporter cell assay. Antibody 2 also induced greater NFKB pathway signaling than Antibody 5 (a Fab). This enhanced ability to activate signaling by the IgG4 variant antibody appears to be Fc receptor-independent as the Fc receptor is not involved in this assay system (the Tregs in this assay do not express Fc receptors). In particular and as show n in Example 6, Antibody 2, a TNFR2 antibody of theIgG4 subclass and having a S228P substitution, exhibited higher activity than the Fc-variant Antibody 1.

[0162] In certain embodiments, the modified antibodies described herein may be produced by combining a variable domain, or fragment thereof, with a constant domain / Fc domain comprising one or more of the amino acid substitutions disclosed herein. In other embodiments, modified antibodies may be produced by modifying an Fc domain-containing antibody by introducing one or more of the amino acid substitutions residues into the Fc domain.

[0163] As used herein, the abbreviations for the genetically encoded L-enantiomeric amino acids used in the disclosure methods are conventional and are as follows in Table A.Table A: Amino acid abbreviations

[0164] '’Hydrophilic Amino Acid"’ refers to an amino acid exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., J. Mol. Biol. (1984) 179:125-142. Genetically encoded hydrophilic amino acids include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gin (Q), Asp (D). Lys (K) and Arg (R).

[0165] ‘"Acidic Amino Acid" refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Glu (E) and Asp (D).

[0166] "Basic Amino Acid'’ refers to a hydrophilic amino acid having a side chain pK value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydrogen ion. Genetically encoded basic amino acids include His (H), Arg (R) and Lys (K).

[0167] “Polar Amino Acid” refers to a hydrophilic amino acid having a side chain uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Asn (N), Gin (Q), Ser (S) and Thr (T).

[0168] “Hydrophobic Amino Acid” refers to an amino acid exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity' scale of Eisenberg, J. Mol. Biol. 179: 125-142 (1984). Exemplary hydrophobic amino acids include He (I), Phe (F). Vai (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), Pro (P), and proline analogues.

[0169] “Aromatic Amino Acid” refers to a hydrophobic amino acid with a side chain having at least one aromatic or heteroaromatic ring. The aromatic or heteroaromatic ring may contain one or more substituents such as -OH. -SH, -CN, -F, -Cl, -Br. -I, -NO2. -NO. -NH2, -NHR, - NRR, -C(O)R, -C(O)OH, -C(O)OR, -C(O)NH2, -C(O)NHR, -C(O)NRR and the like where each R is independently (C1-C6) alkyl, substituted (C1-C6) alky l, (C1-C6) alkenyl, substituted (CLC6) alkenyl, (C1-C6) alkynyl, substituted (C1-C6) alkynyl, (C1-C21)) aryl, substituted (C5-C20) aryl. (C6-C26) alkaryl, substituted (C6-C26) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl or substituted 6-26 membered alkheteroaryl. Genetically encoded aromatic amino acids include Phe (F), Tyr (Y) and Trp (W).

[0170] “Nonpolar Amino Acid” refers to a hydrophobic amino acid having a side chain uncharged at physiological pH and which has bonds in which the pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (e.g., the side chain is not polar). Genetically encoded apolar amino acids include Leu (L), Vai (V), He (I), Met (M), Gly (G) and Ala (A).

[0171] “Aliphatic Amino Acid” refers to a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala (A), Vai (V), Leu (L) and He (I).

[0172] The term "non-naturally" with regard to amino acids can include any amino acid molecule not included as one of the 20 amino acids listed in Table A above as well as any modified or derivatized amino acid known to one of skill in the art. Non-naturally amino acids can include but are not limited to 0-alanine, a-amino butyric acid, y-amino butyric acid, y-(aminophenyl) butyric acid, a-amino isobutyric acid, a-amino caproic acid, 7-amino heptanoic acid. (3-aspartic acid, aminobenzoic acid, aminophenyl acetic acid, aminophenyl butyric acid, y-glutamic acid, cysteine (ACM), s-lysine. methionine sulfone, norleucine, norvaline, ornithine, d-omithine, p-nitro-phenylalanine, hydroxy proline, 1, 2,3,4, - tetrahydroisoquinoline-3-carboxylic acid, and thioproline.Anti-TNFR2 agonist activity

[0173] The anti-TNFR2 antibodies described herein are capable of interacting with and promoting signal transduction events mediated by TNFR2. Thus, these TNFR2 antibodies can selectively potentiate TNFR2-mediated Treg cell grow th. Treg cell fate stability and enhanced T reg immunosuppressive function. In particular embodiments, the anti-TNFR2 antibodies described herein are capable of selectively promoting the proliferation of a population of Treg cells, i.e. immunosuppressive Tregs. Without being limited to any particular mechanism, these phenotypes may be achieved due to the ability of an agonistic TNFR2 antibody or antigen-binding fragment thereof to induce conformational changes within TNFR2 that lead to receptor functional activation. Such spatial configuration has been shown to render TNFR2 active for TRAF 2 / 3 signal transduction, which subsequently leads to activation of NFicB-mediated transcription of genes involved in Treg cell growth and escape from apoptosis (Faustman, et al., Nat. Rev. Drug Disc. (2010) 9:482-493), the disclosure of which is incorporated herein by reference). "‘Selectively'’ as used in reference to effects on TNFR2 -mediated signaling pathways may be assessed as a mean of delta pRelA response (the highest concentration for the antibody vs. vehicle) for Tregs that is significantly higher than that for other immune cells and the mean for other immune cells is less than 20%.Methods for producing and characterizing an antibody are well-known in the art. Exemplary methods for production and purification of an antibody are provided in Example 1 . In a preferred embodiment, anti-TNFR2 antibodies and its constant domain / Fc variants are produced and screened for predefined properties using one or more cell-based assays employing TNFR2 expressing cells or in vivo assays. For such assays, the antibody istypically added exogenously such that cells can be bound, e.g., in the presence and absence of TNFa to determine the agonistic activity’. These assays are typically based on the function of the immunoglobulin; that is, the ability of the antibody to bind to TNFR2 and mediate some biochemical event, e.g., in a binding assay, receptor binding activation, the reduction / increase of cytokine expression in the presence or absence of TNF, and the like.

[0174] Such assays often involve monitoring the response of cells to the antibody, for example cell survival, cell death, change in cellular morphology, or transcriptional activation such as cellular expression of a natural gene or reporter gene. For some assays, additional cells or components in addition to the target cells may need to be added. For example, serum complement, or effector cells such as peripheral blood mononuclear cells (PBMCs), NK cells, macrophages, and the like may need to be added to some assays. Such additional cells may be from any organism, preferably humans, mice, rat, rabbit, and monkey. Exemplary methods for characterization of antibody signaling are provided at least in Examples 3 and 5- 6. Exemplary' methods for assessing antibody binding and characterization of binding to certain lymphocyte subsets are provided at least in Examples 4. 8, and 10-12

[0175] According to some embodiments, the anti-TNFR2 antibodies may be selectively targeted to a Treg by the binding of the targeting moiety7to the cell surface. For example, the anti-TNFR2 antibodies can be targeted to general Tregs by binding to general Treg surface markers such as CD3, CD4 and / or CD25. The anti-TNFR2 antibodies can also be specifically targeted to subsets of functional Tregs by binding to subset-specific markers such as FoxP3.

[0176] In some embodiments, the targeting moiety’ targets the anti-TNFR2 antibodies to one or more markers, cell surface receptors, or other molecules present on the surface of a regulatory T cell, including, for example, CD45, 9.4, CD4, CD3, CD2, CD25, CD127, CD197 (CCR7), CXCR3, CXCR4, CXCR5, CD38, CD27, CD8, CCR4, CCR5, CD137, CCR6 (CD196), CCR8, CCR10, 0X40, GITR, NRP1, CTLA4, LAG3, LRRC32, ICAM1, CD73, CD 103, CD62L, CCR2, CCR9, CDlla (via, for example, MHM24 monoclonal antibody), and / or CD18 (via, for example. chimericlB4 monoclonal antibody) In some further embodiments, the targeting moiety targets one or more general Treg markers, cell surface receptors, or other molecules present on the surface of a Treg including without limitation CTLA4 and / or ICOS. In some further embodiments, the targeting moiety’ targets one or more of the anti-TNFR2 antibodies to markers, cell surface receptors, or othermolecules present on the surface of an effector Treg including without limitation 0X40 (TNFRSF4), GITR (TNFRSF18), TIGIT, CCR8, 4-1BB (TNFRSF9), ICAM1, LRRC32, CD39 (ENTPD1), and / or LAYN In even further embodiments, the targeting moiety targets one or more of the anti-TNFR2 antibodies to markers, cell surface receptors, or other molecules including without limitation TIM3, LAG3 and / or PD1.

[0177] In some embodiments, the anti-TNFR2 antibody activates TNFR2-mediated NFKB signaling through the canonical signaling pathway or an alternative (e.g. non- canonical) pathway. Considering further the canonical NFKB signaling pathway, TNFR2 signaling activity may be characterized by an increase of alkaline phosphatase secretion which can be measured by a suitable method as known in the art including in an engineered reporter cell line. One suitable reporter cell line is a HEK-Blue™ NFKB reporter cell line as described in Examples 3 and 6. In particular, the ability to induce TNFR2-mediated NFKB signaling may be evaluated using secretion of alkaline phosphatase from an engineered reporter cell line as compared to a control as described in Example 3. As described therein, Antibody 1, among others, showed dose responsive activity with Antibody 1 showing the highest maximum effect (Emax) in induction of TNFR2-mediated NFKB signaling. In a further assay as described in Example 6, anti-TNFR2 antibodies as described herein demonstrated activity in activating TNFR2-mediated NFKB signaling with different formats providing better response. The anti-TNFR2 antibody having the IgG4 S228P scaffold (Antibody 2) provided the highest activity (observed Emax). The anti-TNFR2 antibody having the IgGl LALA_PG scaffold (Antibody 1 ) also provided good activity. As measured in the assay, Antibody 2 provided an increase in observed Emax of at least about 30% or at least about 1.4 fold as compared to the observed Emax for Antibody 1. In some embodiments, the NFKB signaling activity induced and / or increased by said anti-TNFR2 is of at least about 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75- fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25- fold, 7.5-fold, 7.75-fold, 8-fold, 8.25-fold. 8.5-fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold. 10.5-fold, 10.75-fold, 11-fold, 11.25-fold. 11.5-fold, 11.75- fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14- fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold, 18.75-fold. 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold. In some embodiments, the NFKB signaling activity induced and / or increased by said anti-TNFR2 antibody is of at least about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1- fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold. 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5-fold. 2- fold to 20-fold. 4-fold to 6-fold, 4-fold to 10-fold. 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8- fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10- fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 11-fold, 8-fold to 15- fold, 8-fold to 18-fold. 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19- fold, 9-fold to 22-fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11-fold to 14-fold, 11-fold to 18-fold, 11-fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14- fold to 18-fold. 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold. 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21- fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold, higher than an isotype control.

[0178] In some embodiments, the ability to activate TNFR2-mediated NFKB signaling may be evaluated using measurement of RelA phosphorylation (pRelA) (e.g. as %+ pRelA) as described in Example 5. As described therein, Antibody 2 consistently showed the ability to activate NFKB signaling in a larger fraction of CD25 / FoxP3 high and CD25 / FoxP3 low cells as compared to the IgGl -based variants Antibodies 1 and 3-4 across donors and Treg subsets. As measured in the assay, Antibody 2 provided an increase in observed Emax of at least about 15-30% or at least about 1.3-1.6 fold when considering both CD25 / FoxP3 high and CD25 / FoxP3 low cells.. In some embodiments, the RelA phosphorylation induced and / or increased by said anti-TNFR2 has a highest activity of at least about 1.1-fold, 1.2-fold, 1.3- fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold. 2.25-fold, 2.5-fold, 2.75- fold, 3-fold, 3.25-fold. 3.5-fold, 3.75-fold. 4-fold. 4.25-fold, 4.5-fold. 4.75-fold, 5-fold, 5.25- fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold, 8-fold, 8.25-fold, 8.5-fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold. 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5- fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold, 20.75- fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold. In some embodiments, TNFR2 signaling activity was measured by phosphorylated RelA (pRelA) induction in primaryhuman Tregs. In some embodiments, the RelA phosphorylation is induced and / or increased by said anti-TNFR2 antibody and has ahighest activity of at least about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15- fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5- fold, 2-fold to 10-fold. 2-fold to 12.5-fold. 2-fold to 20-fold, 4-fold to 6-fold. 4-fold to 10- fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22 -fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6- fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold. 9-fold to 22 -fold. 10-fold to 14-fold. 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13- fold to 22-fold. 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22- fold, 17-fold to 19-fold, 17 -fold to 21 -fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold higher than an isotype control.

[0179] In some embodiments, the anti-TNFR2 antibody comprising an IgG4 having an S228P mutation has an enhanced and / or increased ability to activate NFKB signaling in a larger fraction of Tregs, as compared to IgGl Fc-based variants. In some embodiments, the enhanced and / or increased ability of NFKB signaling activation is characterized by an increase of RelA phosphorylation (pRelA) as measured by ECso for potency of the antibody and / or Emax for the maximal response of the antibody, and wherein the Tregs are defined as optionally CD3+CD4+CD25+FOXP3+. In some embodiments, the RelA phosphorylation (pRelA) in primary human Tregs induced and / or increased by said anti-TNFR2 antibody comprising an IgG4 having S228P mutation is at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3- fold, 3.25-fold. 3.5-fold, 3.75-fold, 4-fold. 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold,5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75- fold, 8-fold, 8.25-fold, 8.5-fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25- fold, 12.5-fold. 12.75-fold. 13-fold, 13.25-fold, 13.5-fold. 13.75-fold. 14-fold, 14.25-fold,14.5-fold, 14.75-fold, 15-fold. 15.25-fold. 15.5-fold, 15.75-fold, 16-fold. 16.25-fold. 16.5- fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold, 20.75- fold, 21-fold. 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold. In some embodiments, the RelA phosphorylation (pRelA) in primary human Tregs induced and / or increased by said anti- TNFR2 antibody comprising an IgG4 having S228P mutation is at least about 1.1-fold to 1.2- fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold. 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold. 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14- fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7- fold to 22-fold. 8-fold to 11 -fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22 -fold. 9-fold to 12-fold, 9-fold to 16-fold. 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 1 1 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13- fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22- fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold higher than IgGl Fc-based variants.

[0180] In some embodiments, the anti-TNFR2 antibody comprising an IgG4 having S228P mutation has an enhanced and / or increased ability to induce RelA phosphorylation (pRelA) in primary human Tregs about 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6- fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5- fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold, 8-fold, 8.25-fold,8.5-fold, 8.75-fold. 9-fold, 9.25-fold, 9.5-fold. 9.75-fold, 10-fold, 10.25-fold. 10.5-fold,10.75 -fold, 1 1 -fold, 11.25-fold, 11.5-fold, 1 1.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75- fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15- fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold, 18.75-fold, 19-fold, 19.25- fold, 19.5-fold. 19.75-fold. 20-fold, 20.25-fold, 20.5-fold. 20.75-fold. 21-fold, 21.25-fold,21.5-fold, 21.75-fold, or 22-fold. In some embodiments, the anti-TNFR2 antibody comprising an IgG4 having S228P mutation has an enhanced and / or increased ability to induce RelA phosphorylation (pRelA) in primary human Tregs about 1.1-fold to 1.2-fold, 1.1-fold to 1.5- fold, 1.1-fold to 2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold. 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1 -fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16- fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold. 6-fold to 14-fold, 6-fold to 18-fold, 6- fold to 22-fold. 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold. 7-fold to 22-fold. 8-fold to 11 -fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22 -fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22 -fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14- fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19- fold, 17-fold to 21 -fold, 17 -fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21-fold to 22-fold higher than IgGl Fc- based variants.

[0181] In some embodiments, the anti-TNFR2 antibody comprising an IgG4 having S228P mutation has an enhanced and / or increased ability' to activate NFKB signaling, as compared to IgGl Fc-based variants, and wherein the enhanced ability to activate NFKB signaling is characterized by an increase and / or enhancement of alkaline phosphatase secretion level which may be measured in an engineered reporter cell line. In some embodiments, the alkaline phosphatase secretion level induced and / or increased by said anti- TNFR2 antibody comprising an IgG4 having S228P mutation is at least about 1. 1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold, 2.5- fold, 2.75-fold. 3-fold, 3.25-fold, 3.5-fold. 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5- fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold,7.5-fold, 7.75-fold, 8-fold, 8.25-fold, 8.5-fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75- fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75-fold, 12- fold, 12.25-fold, 12.5-fold. 12.75-fold, 13-fold, 13.25-fold, 13.5-fold. 13.75-fold, 14-fold, 14.25-fold. 14.5-fold, 14.75-fold, 15-fold. 15.25-fold. 15.5-fold, 15.75-fold, 16-fold. 16.25- fold, 16.5-fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold,18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5- fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold. In some embodiments, the alkaline phosphatase secretion level induced and / or increased by said anti-TNFR2 antibody comprising an IgG4 having S228P mutation is at least about 1.1 -fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15- fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5- fold, 2-fold to 10-fold, 2-fold to 12.5-fold. 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10- fold, 4-fold to 16-fold. 4-fold to 22-fold. 5-fold to 8-fold. 5-fold to 12-fold. 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22 -fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6- fold to 18-fold, 6-fold to 22 -fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold. 9-fold to 22-fold. 10-fold to 14-fold. 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 1 1 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13- fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22- fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold higher than or as compared to IgGl Fc-based variants.

[0182] In some embodiments, the anti-TNFR2 antibody comprising an IgGl Fc having a LALA or a LALA_P329G mutation has an increased and / or enhanced ability to activate NFKB signaling of Tregs, and wherein the enhanced ability of NFKB signaling activation is characterized by an increase of RelA phosphorylation (pRelA) in primary human Tregs, and wherein the Tregs are defined as optionally CD3+CD4+CD25+FOXP3+.

[0183] In some embodiments, the RelA phosphorylation (pRelA) in primary human Tregs induced and / or increased by said anti-TNFR2 antibody comprising an IgGl Fc having a LALA or LALA_P329G mutation is increased and / or enhanced at least about 1.1-fold, 1.2- fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold, 2.5- fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5- fold, 5.25-fold. 5.5-fold, 5.75-fold, 6-fold. 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold,7.5-fold, 7.75-fold. 8-fold. 8.25-fold, 8.5-fold. 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75- fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11 -fold, 11.25 -fold, 11.5-fold, 11.75-fold, 12- fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25- fold, 16.5-fold. 16.75-fold. 17-fold, 17.25-fold, 17.5-fold. 17.75-fold. 18-fold, 18.25-fold,18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5- fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22 -fold higher. In some embodiments, the RelA phosphorylation (pRelA) in primary human Tregs induced and / or increased by said anti-TNFR2 antibody comprising an IgGl Fc having a LALA or LALA_P329G mutation is increased and / or enhanced at least about 1.1-fold to 1.2-fold, 1.1- fold to 1.5-fold, 1.1 -fold to 2.5-fold, 1.1 -fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15- fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5- fold, 2-fold to 10-fold, 2-fold to 12.5-fold. 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10- fold, 4-fold to 16-fold. 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold. 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22 -fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6- fold to 18-fold, 6-fold to 22 -fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold. 10-fold to 14-fold. 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13- fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22- fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold, as compared to a control without the substitution.

[0184] In some embodiments, the anti-TNFR2 antibody preferentially binds to Treg subsets over conventional T cells or non-Treg cells including CD4+ non-Treg cells and / or cytotoxic CD8+ T cells. '‘Preferentially binds’’ as used in this context refers to binding of the anti-TNFR2 antibodies described herein to immunosuppressive Tregs at least about 10%- 20% or greater over non-Treg cells such as CD4+ non-Treg cells and / or cytotoxic CD8+ T cells. In some embodiments, the anti-TNFR2 antibodies described herein preferentially bind by about 10%-1000% over non-Treg cells such as CD4+ non-Treg cells and / or cytotoxic CD8+ T cells. Cellular receptor binding may be determined by methods known to those of skill in the art. In an exemplary method, human peripheral blood mononuclear cells (PBMCs) are incubated with an anti-TNFR2 agonistic antibody and Treg surface binding is measured by flow cytometry as described further at least in Examples 4 and Example 11. In some embodiments, the Tregs for binding with anti-TNFR2 antibody are defined as CD3+CD4+CD25+CD127-dim cells. As described in Example 11, cell binding of Antibody 1 and Antibody 2 was evaluated in human TNFR2 knock-in mice where binding was characterized in conventional T cells, total CD4+ Tregs, and FoxP3 high and low expressing Treg subsets. Cell binding for both Antibody 1 and Antibody 2 was highly selective for Tregs. In some embodiments, the maximum response resulting from the anti-TNFR2 antibody binding to CD3+CD4+CD25+CD127- Tregs are at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%. 375%, 400%, 425%. 450%, 475%, 500%, 525%, 550%. 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000% higher than a maximum response resulting from binding to non-Treg cells and / or cytotoxic T cells; or 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, 100% to 400%, 110% to 400%. 120% to 400%, 130% to 400%, 140% to 400%, 150% to 400%, 160% to 400%, 170% to 400%, 180% to 400%, 190% to 400%, 100% to 500%, 110% to 500%, 120% to 500%, 130% to 500%, 140% to 500%, 150% to 500%, 160% to 500%, 170% to 500%, 180% to 500%, 190% to 500%, 100% to 600%, 110%to 600%, 120% to 600%, 130% to 600%, 140% to 600%, 150% to 600%, 160% to 600%, 170% to 600%, 180% to 600%, 190% to 600%, 100% to 700%, 110% to 700%, 120% to 700%, 130% to 700%, 140% to 700%, 150% to 700%, 160% to 700%, 170% to 700%, 180% to 700%, 190% to 700%, 100% to 800%, 110% to 800%, 120% to 800%, 130% to 800%, 140% to 800%, 150% to 800%, 160% to 800%, 170% to 800%, 180% to 800%, 190% to 800%, 100% to 900%, 110% to 900%, 120% to 900%, 130% to 900%, 140% to 900%, 150% to 900%. 160% to 900%, 170% to 900%, 180% to 900%. 190% to 900% 110% to 1000%. 120% to 1000%, 130% to 1000%, 140% to 1000%, 150% to 1000%, 160% to 1000%, 170% to 1000%, 180% to 1000%, 190% to 1000%, 200% to 1000%, 250% to 1000%, 300% to 1000%, 350% to 1000%, 400% to 1000%, 450% to 1000%. 500% to 1000%, 600% to 1000%, 700% to 1000%, 800% to 1000%, 900% to 1000%. or 950% to 1000%, higher than maximum response resulting from binding of non-Treg cells and / or cytotoxic T cells.

[0185] In some embodiments, the isolated anti-TNFR2 antibody is characterized by a preferential binding to Tregs over non-Treg cells. In some embodiments, the Tregs include but are not limited to CD4+ Tregs, CD4+CD25+FoxP3+ Tregs. FoxP3+ CD4+ Tregs, CD25+FoxP3+ Tregs, CD4+CD25-FoxP3+ Tregs, CD25-FoxP3+ Tregs. or a combination thereof.

[0186] In some embodiments, the FoxP3 expressing cells comprise a subpopulation of FoxP3 high expressing cells and a subpopulation of FoxP3 low expressing cells. In some embodiments, the anti-TNFR2 antibody exhibits a higher level of binding to the FoxP3 high expressing cells than to the FoxP3 low expressing cells. In some embodiments, the FoxP3 high expressing cells are characterized by having a top 50% FoxP3 expression level relative to a population of FoxP3 expressing Tregs. In some embodiments, the FoxP3 low expressing cells are characterized by having a bottom 50% FoxP3 expression level relative to a population of FoxP3 expressing Tregs.

[0187] In some embodiments, the non-Treg cells include but are not limited to conventional CD4+ T cells (CD4+ Tconv), conventional CD8+ T cells (CD8+ T conv), NK cells, monocytes, and / or neutrophils, and / or a combination thereof.

[0188] In some embodiments, conventional T cells include but are not limited to helper T cells, cytotoxic T cells, and memory T cells, and / or any other type of non-Treg T cells.

[0189] In some embodiments, the preferential binding to Tregs over non-Treg cells is measured by a percent of binding positive cells, wherein the percent of binding positive cells in Tregs is at least: a) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2- fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold. 5-fold, 5.25-fold, 5.5-fold. 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75- fold, 7-fold, 7.25-fold. 7.5-fold, 7.75-fold. 8-fold. 8.25-fold, 8.5-fold. 8.75-fold, 9-fold, 9.25- fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11 -fold, 11.25-fold, 11.5- fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75- fold, 16-fold. 16.25-fold, 16.5-fold, 16.75-fold, 17-fold. 17.25-fold. 17.5-fold, 17.75-fold, 18- fold, 18.25-fold, 18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, 22-fold higher than the percent of binding positive cells in non-Treg cells, or b) about or 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5-fold. 1.1-fold to 10-fold. 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold. 2-fold to 2.5- fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20- fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6- fold to 10-fold. 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold. 7-fold to 10-fold. 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 1 1-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22- fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold. 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13 -fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14- fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22- fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold higher than the percent of binding positive cells in non-Treg cells.

[0190] In some embodiments, the preferential binding to Tregs over non-Treg cells is measured by a total median fluorescence intensity, wherein the total median fluorescence intensity of Tregs is at least: a) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2- fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold. 5-fold, 5.25-fold, 5.5-fold. 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75- fold, 7-fold, 7.25-fold. 7.5-fold, 7.75-fold. 8-fold. 8.25-fold, 8.5-fold. 8.75-fold, 9-fold, 9.25- fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11 -fold, 11.25-fold, 11.5- fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75- fold, 16-fold. 16.25-fold, 16.5-fold, 16.75-fold, 17-fold. 17.25-fold. 17.5-fold, 17.75-fold, 18- fold, 18.25-fold, 18.5-fold, 18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, 22-fold higher than the total median fluorescence intensity of non-Treg cells; or b) about or 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5-fold. 1.1-fold to 10-fold. 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold. 2-fold to 2.5- fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20- fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6- fold to 10-fold. 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold. 7-fold to 10-fold. 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 1 1-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22- fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold. 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14- fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22- fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold higher than the total median fluorescence intensity of non-Treg cells.

[0191] In some embodiments, the anti-TNFR2 antibody is characterized by specific binding to a TNFR2 receptor over a protein that is not TNFR2 receptor. In someembodiments, the anti-TNFR2 antibody specifically binds to a TNFR2 receptor over a TNFR1 receptor.

[0192] In some embodiments, the specific binding is measured by a Signal-to-Noise Ratios (SNR) value indicating an interaction between the anti-TNFR2 antibody and a binding protein, wherein the SNR value indicating the interaction of the anti-TNFR2 antibody with a TNFR2 receptor is at least: a) about 1.1-fold, 1.2-fold. 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold. 1.7-fold. 1.8-fold, 1.9-fold, 2- fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75- fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold, 8-fold, 8.25-fold, 8.5-fold, 8.75-fold, 9-fold, 9.25- fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11-fold, 11.25-fold, 11.5- fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75- fold, 16-fold, 16.25-fold, 16.5-fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18- fold, 18.25-fold, 18.5-fold. 18.75-fold, 19-fold, 19.25-fold, 19.5-fold. 19.75-fold, 20-fold, 20.25-fold. 20.5-fold, 20.75-fold, 21-fold. 21.25-fold. 21.5-fold, 21.75-fold, 22-fold higher than the SNR value indicating the interaction with the protein that is not TNFR2 receptor; or b) about or 1.1 -fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5-fold, 1.1 -fold to 10-fold. 1.1 -fold to 15 -fold, 1.1 -fold to 20-fold, 1.1 -fold to 22-fold, 2-fold to 2.5- fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold. 2-fold to 12.5-fold, 2-fold to 20- fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6- fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22- fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11-fold to 14-fold, 11-fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14- fold to 22-fold. 15-fold to 18-fold. 15-fold to 20-fold. 15-fold to 22-fold. 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22- fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to22-fold, or 21 -fold to 22 -fold higher than the SNR value indicating the interaction with the protein that is not TNFR2 receptor.

[0193] In some embodiments, the TNFR2 receptor is TNFRSF1B isoform 1. In some embodiments, the TNFR2 receptor is TNFRSF1B, isoform 2. As described in Example 12, cell binding specificity to TNFRSF1B isoform 1 and TNSFRSF1B isoform 2 was evaluated in a HEK293 cell assay. Antibody 2 showed a significant specific interaction with TNFRSF IB isoform 1 but not TNFRSF1B isoform 2. Antibody 2 further showed significant, specific binding interaction to human TNFR2, but not to other TNFR superfamily receptors (TNFR1, 0X40, GITR, 4-1BB, BCMA, TRAILR1, HVEM, DDR3, DR6, TRAILR2, BAFFR, TweakR, RANK, TRADE, OCIF, and DcR3).

[0194] In some embodiments, the protein that is not a TNFR2 receptor is selected from a plurality of human plasma membrane proteins and membrane tethered secreted proteins. In some embodiments, the plurality of human plasma membrane proteins and membrane tethered secreted proteins comprise KCNJ12, TNFRSF13B, APOCI, CXCL12, and IGF1. In some embodiments, the plurality of human plasma membrane proteins and membrane tethered secreted proteins comprise a range of TNFR superfamily receptors. Such TNFR superfamily receptors include, but are not limited to TNFR1, 0X40, GITR, 4- IBB, BCMA, TRAILR1, HVEM, DDR3, DR6, TRAILR2, BAFFR, TweakR, RANK, TRADE, OCIF, and DcR3.

[0195] In some embodiments, the anti-TNFR2 antibody agonist activity is characterized by an increase of functional markers expressed on Treg associated with immunosuppressive ability , wherein the functional marker comprising CD25, FoxP3, TIGIT, and / or Ki67, and combinations thereof. In some embodiments, the increase of functional Treg markers expression induced and / or increased by said anti-TNFR2 is of at least about 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75- fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25- fold, 7.5-fold, 7.75-fold, 8-fold, 8.25-fold. 8.5-fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold. 10.5-fold, 10.75-fold, 11-fold, 11.25-fold. 11.5-fold, 11.75- fold, 12-fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14- fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold, 16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold, 18.75-fold. 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold,20.5-fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher. In some embodiments, the increase of functional Treg markers expression induced and / or increased by said anti-TNFR2 is of at least about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1- fold to 22-fold. 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold. 2-fold to 12.5-fold, 2-fold to 20-fold. 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold. 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22- fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8-fold to 11-fold. 8- fold to 15-fold. 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold. 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11-fold to 14-fold, 11-fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12- fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22- fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21 -fold to 22-fold, as compared to an isotype control. In some embodiments an increase or enhancement is an increase or enhancement in the expression percentage as compared to an isotype control.

[0196] In some embodiments, the increase or enhancement in the expression percentage induced and / or increased by said anti-TNFR2 is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, 525%, 550%. 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000%, or 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to100%, 53% to 100%, 55% to 100%. 58% to 100%, 60% to 100%, 63% to 100%. 65% to100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to100%, 98% to 100%, 100% to 400%, 110% to 400%, 120% to 400%, 130% to 400%, 140%to 400%, 150% to 400%, 160% to 400%, 170% to 400%, 180% to 400%, 190% to 400%, 100% to 500%, 110% to 500%, 120% to 500%, 130% to 500%, 140% to 500%, 150% to 500%, 160% to 500%, 170% to 500%, 180% to 500%, 190% to 500%, 100% to 600%, 110% to 600%, 120% to 600%, 130% to 600%, 140% to 600%, 150% to 600%, 160% to 600%, 170% to 600%, 180% to 600%, 190% to 600%, 100% to 700%, 110% to 700%, 120% to 700%, 130% to 700%, 140% to 700%, 150% to 700%, 160% to 700%, 170% to 700%, 180% to 700%. 190% to 700%, 100% to 800%, 110% to 800%. 120% to 800%, 130% to 800%, 140% to 800%, 150% to 800%, 160% to 800%, 170% to 800%, 180% to 800%, 190% to 800%, 100% to 900%, 110% to 900%, 120% to 900%, 130% to 900%, 140% to 900%, 150% to 900%, 160% to 900%, 170% to 900%, 180% to 900%, 190% to 900% 110% to 1000%, 120% to 1000%, 130% to 1000%, 140% to 1000%, 150% to 1000%, 160% to 1000%, 170% to 1000%, 180% to 1000%, 190% to 1000%, 200% to 1000%, 250% to 1000%, 300% to 1000%, 350% to 1000%, 400% to 1000%, 450% to 1000%, 500% to 1000%, 600% to 1000%, 700% to 1000%, 800% to 1000%. 900% to 1000%. or 950% to 1000%, higher as compared to an isotype control.

[0197] In some embodiments, the anti-TNFR2 antibody comprising an IgG4 having S228P mutation has increased and / or enhanced ability to induce and / or increase the expression of functional Treg markers, as compared to IgGl Fc-based variants. In some embodiments, the expression of Treg functional markers induced and / or increased by an anti- TNFR2 antibody comprising an IgG4 having S228P mutation are at least about 1.1 -fold. 1.2- fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.25-fold, 2.5- fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5- fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold,7.5-fold, 7.75-fold. 8-fold, 8.25-fold, 8.5-fold. 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75- fold, 10-fold, 10.25-fold, 10.5-fold, 10.75-fold, 11 -fold, 1 1.25 -fold, 11.5-fold, 11.75-fold, 12- fold, 12.25-fold, 12.5-fold, 12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold, 14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25- fold, 16.5-fold. 16.75-fold. 17-fold, 17.25-fold, 17.5-fold. 17.75-fold. 18-fold, 18.25-fold,18.5-fold, 18.75-fold, 19-fold. 19.25-fold. 19.5-fold, 19.75-fold, 20-fold. 20.25-fold. 20.5- fold, 20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold. In some embodiments, the expression of Treg functional markers induced and / or increased by an anti-TNFR2 antibody comprising an IgG4 having S228P mutation are at least about 1.1 -fold to 1.2-fold,1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5-fold, 1.1-fold to 10-fold, 1.1-fold to 15- fold, 1.1-fold to 20-fold, 1.1-fold to 22-fold. 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5- fold, 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10- fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22 -fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6- fold to 18-fold. 6-fold to 22 -fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold. 7-fold to 22-fold, 8-fold to 11-fold. 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22 -fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold, 13-fold to 18-fold, 13- fold to 22-fold. 14-fold to 16-fold. 14-fold to 18-fold. 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22- fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold. 20-fold to 22-fold, or 21 -fold to 22-fold higher than a control. In some embodiments an increase and / or enhancement is an increase and / or enhancement in the expression percentage as compared to IgGl Fc-based variants. In some embodiments, the increase and / or enhancement in the expression percentage induced and / or increased by said anti-TNFR2 comprising an IgG4 having S228P mutation is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%. 300%, 325%, 350%. 375%, 400%, 425%, 450%, 475%, 500%, 525%. 550%, 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000%, or 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, 100% to 400%, 110% to 400%, 120% to 400%, 130% to 400%, 140% to 400%. 150% to 400%, 160% to 400%, 170% to 400%. 180% to 400%. 190% to 400%, 100% to 500%, 110% to 500%, 120% to 500%, 130% to 500%, 140% to 500%, 150% to 500%, 160% to 500%, 170% to 500%, 180% to 500%, 190% to 500%, 100% to 600%, 110% to 600%, 120% to 600%, 130% to 600%, 140% to 600%, 150% to 600%, 160%to 600%, 170% to 600%, 180% to 600%, 190% to 600%, 100% to 700%, 110% to 700%, 120% to 700%, 130% to 700%, 140% to 700%, 150% to 700%, 160% to 700%, 170% to 700%, 180% to 700%, 190% to 700%, 100% to 800%, 110% to 800%, 120% to 800%, 130% to 800%, 140% to 800%, 150% to 800%, 160% to 800%, 170% to 800%, 180% to 800%, 190% to 800%, 100% to 900%, 110% to 900%, 120% to 900%, 130% to 900%, 140% to 900%, 150% to 900%, 160% to 900%, 170% to 900%, 180% to 900%, 190% to 900% 110% to 1000%, 120% to 1000%, 130% to 1000%, 140% to 1000%, 150% to 1000%, 160% to 1000%, 170% to 1000%, 180% to 1000%, 190% to 1000%, 200% to 1000%, 250% to1000%, 300% to 1000%, 350% to 1000%, 400% to 1000%, 450% to 1000%, 500% to1000%, 600% to 1000%, 700% to 1000%, 800% to 1000%. 900% to 1000%, or 950% to1000%, higher than or as compared to IgGl Fc-based variants.

[0198] In some embodiments, a preferred anti-TNFR2 antibody promotes Treg proliferation and upregulation of FoxP3 and TIGIT in a primary human PBMC culture system as a single agent without the need for T cell receptor stimulation and cytokine (e.g. IL-2) stimulation.NUCLEIC ACID COMPOSITIONS

[0199] Nucleic acid compositions encoding the anti-TNFR2 antibodies are also provided, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acid and / or expression vector compositions. As will be appreciated by those in the art, the protein sequences depicted herein can be encoded by any number of possible nucleic acid sequences, due to the degeneracy of the genetic code.

[0200] The nucleic acid compositions that encode the anti-TNFR2 antibodies will depend on the format of the antibody. For traditional, tetrameric antibodies containing two heavy chains and two light chains are encoded by two different nucleic acids, one encoding the heavy chain and one encoding the light chain. These can be put into a single expression vector or two expression vectors, as is known in the art, transformed into host cells, where they are expressed to form the antibodies of the disclosure. In some embodiments, for example when scFv constructs are used, a single nucleic acid encoding the variable heavy chain-linker-variable light chain is generally used, which can be inserted into an expression vector for transformation into host cells. The nucleic acids can be put into expression vectors that contain the appropriate transcriptional and translational control sequences, including, butnot limited to, signal and secretion sequences, regulator ' sequences, promoters, origins of replication, selection genes, etc.

[0201] Preferred mammalian host cells for expressing the recombinant antibodies according to at least some embodiments of the disclosure include Chinese Hamster Ovary (CHO cells), PER.C6, HEK293 and others as known in the art.

[0202] The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified aw ay from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art.

[0203] To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see, e.g.. Bird et al.. Science 242:423-426 (1988); Huston et al. PNAS. 85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990)).ADMINISTRATION OF FORMULATIONS OF ANTI-TNFR2 ANTIBODIES

[0204] In some embodiments, the anti-TNFR2 antibodies and antigen-binding fragments thereof are comprised within a pharmaceutical composition for administration as a treatment. It will be appreciated that description of administration or treatment with a composition comprising antibodies herein applies equally to administration of the antibodies themselves. Administration of the pharmaceutical composition comprising anti-TNFR2 antibodies described herein (e.g., anti-TNFR2 antibodies including those with CDRs identical to those shown in Figures 16, and / or 17), preferably in the form of a sterile aqueous solution, may be carried out in a variety of ways. As is known in the art, protein therapeutics are often delivered by IV infusion. The antibodies may also be delivered using such methods. For example, administration may be veinous or by intravenous infusion with 0.9% sodium chloride as an infusion vehicle. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 23rd edition, Osol, A. Ed., 2021.

[0205] In embodiments, the anti-TNFR2 antibodies described herein may be administered as a monotherapy. In some embodiments, the anti-TNFR2 antibodies are administered as a monotherapy without the need for one or more of CD3 ligation, costimulation, or cytokine or mitogen support.

[0206] The dosing amounts and frequencies of administration are, in some embodiments, selected to be therapeutically or prophylactically effective. As is known in the art. adjustments for protein degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary7, and will be ascertainable with routine experimentation by those skilled in the art. In order to treat a patient, a therapeutically effective dose of the of the anti-TNFR2 antibodies as described herein may be administered. By '‘therapeutically effective dose” herein is meant a dose that produces the effects for which it is administered.

[0207] A composition comprising an anti-TNFR2 antibody or an antigen-binding fragment thereof, as disclosed herein, can be administered to a subject (e.g.. a human or an animal) alone, or in combination with a carrier, e.g.. a pharmaceutically acceptable earner. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. As would be well- known to one of ordinary skill in the art, the carrier is selected to minimize any degradation of the polypeptides disclosed herein and to minimize any adverse side effects in the subject. The pharmaceutical compositions may be prepared by methodologies w ell known in the pharmaceutical art.METHODS OF USING THE ANTI- TNFR2 ANTIBODYTherapeutic Uses

[0208] Agonistic TNFR2 antibodies and antigen-binding fragments thereof as described herein can therefore be used to promote Treg cell proliferation and can be administered to a mammalian subject, such as a human patient, with an autoimmune disease or disorder, in order to treat the disease or disorder or to attenuate the magnitude and durationof an immune response (e.g., quantity of CD8+ cytotoxic T lymphocytes produced in vivo in response to a self or non-threatening foreign antigen) in the patient.

[0209] In some embodiments, the composition comprises anti-TNFR2 antibodies having a set of three complementarity determining regions (CDRs) on a heavy chain (vhCDRl, vhCDR2, and vhCDR3) and a set of three CDRs on a light chain (vlCDRl, vlCDR2 and vlCDR3). In one embodiment, the set of vhCDRl. vhCDR2, and vhCDR3 comprises a combination of the ammo acid sequences as shown in Figure 16 and Figure 17, and the set of vlCDRl, vlCDR2 and vlCDR3 on a corresponding light chain comprises a combination of the amino acid sequences as show n in Figure 16 and Figure 17.

[0210] In some embodiments, the composition comprises the anti-TNFR2 Antibody 2. The set of vlCDRl, vlCDR2 and vlCDR3 for Antibody 2 comprises the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 respectively (Figure 16), whereas the set of vhCDRl, vhCDR2, and vhCDR3 for the same antibody comprises the amino acid sequences of SEQ ID NO:2, SEQ ID NO: 3 and SEQ ID NO:4 respectively (Figure 16).

[0211] In some embodiments, the composition comprises the anti-TNFR2 Antibody1. The set of vlCDRl, vlCDR2 and vlCDR3 for Antibody 1 comprises the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 respectively (Figure 17), whereas the set of vhCDRl, vhCDR2, and vhCDR3 for the same antibody comprises the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively (Figure 17).

[0212] In one embodiment, the composition comprises anti-TNFR2 antibodies having a heavy chain variable region (VH) and a light chain variable region (VL).

[0213] In some embodiments, the composition comprises the anti-TNFR2 Antibody2. Antibody 2 comprises the VH comprises the amino acid sequences of SEQ ID NO: 1 and the VL comprises the amino acid sequences of SEQ ID NO: 7 of Figure 16.

[0214] In some embodiments, the composition comprises the anti-TNFR2 Antibody 1. Antibody 1 comprises the VH comprises the amino acid sequences of SEQ ID NO: 13 and the VL comprises the amino acid sequences of SEQ ID NO: 19 of Figure 17.

[0215] In some embodiments, the composition comprises anti-TNFR2 antibodies having a heavy chain (HC) and a light chain (LC).

[0216] In some embodiments, the composition comprises the anti-TNFR2 Antibody 2. The HC comprises the amino acid sequences of SEQ ID NO: 6 and LC comprises the amino acid sequences of SEQ ID NO: 12 of Figure 16.

[0217] In some embodiments, the composition comprises the anti-TNFR2 Antibody 1. The HC comprises the amino acid sequences of SEQ ID NO: 18 and LC comprises the amino acid sequences of SEQ ID NO: 24 of Figure 17.

[0218] In another embodiment, the composition comprises anti-TNFR2 antibodies having heavy chain and light chain CDR sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example but are not limited to identity as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. A skilled artisan would appreciate that percent identity (% identity) provides a number that describes how similar the query sequence is to the target sequence (e.g., how many amino acids in each sequence are identical). The higher the percent identity7is, the more significant the match.

[0219] The anti-TNFR2 antibodies (e.g., anti-TNFR2 antibodies including those described in Figures 16 and / or 17) find use in treating patients, such as human subjects. The term ‘‘treatment” as used herein, refers to both therapeutic treatment and prophylactic or preventative measures, which in this example relates to treatment of an immunological disease. Those in need of treatment include those already with an immunological disease as well as those in which the cancer is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the immunological disease or may be predisposed or susceptible to the immunological disease. As used herein the term “treating” refers to preventing, delay ing the onset of, curing, reversing, attenuating, alleviating, minimizing, suppressing, reducing, decreasing, halting the deleterious effects or stabilizing of discernible symptoms of the above-described immunological diseases, disorders or conditions. It also includes managing the immunological disease as described above. By “manage” it is meant reducing the severity of the disease, reducing the frequency of episodes of the disease, reducing the duration of such episodes, reducing the severity of such episodes, slowing progression of at least one symptom, amelioration of at least one measurable physical parameter and the like. For example, immunosuppressive anti-TNFR2 immune molecules should modulate the proliferation and / or functions of regulatory7T cells and myeloid-derived suppressor cells., thereby treat an immunological disease.

[0220] The anti-TNFR2 antibodies described herein are provided in therapeutically effective dosages. A “therapeutically effective dosage” of an anti-TNFR2 immune molecule according to at least some embodiments preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in lifespan, disease remission, or a prevention or reduction of impairment or disability due to the disease affliction. For example, for the treatment of treating an autoimmune disease, a “therapeutically effective dosage” preferably provides antiinflammatory and immunomodulatory activity by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to induce antiinflammatory and immunomodulatory activity can be evaluated in an animal model system predictive of efficacy in human immunological disorders. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to induce such antiinflammatory and immunomodulatory activity in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can generate immunomodulatory effect, or otherwise ameliorate symptoms in a subject.

[0221] One of ordinary' skill in the art would be able to determine a therapeutically effective amount based on such factors as the subject’s size, the severity' of the subject’s symptoms, and the particular composition or route of administration selected.

[0222] In some embodiments, the anti-TNFR2 antibodies are useful for treating or alleviating a symptom associated with an immunological disease or disorders. In embodiments, the anti-TNFR2 antibodies are useful for treating or alleviating a symptom associated with a mucosal immunological disease including gastrointestinal and oral mucosal diseases. In particular, the anti-TNFR2 antibodies are useful for treating an autoimmune disease. In some embodiments, the autoimmune disease is an autoimmune disease of the skin (dermal), lungs, gastrointestinal tract, central nervous system, endocrine system, or kidneys. In some specific embodiments, the anti-TNFR2 antibodies are useful for treating a condition selected from acute febrile mucocutaneous lymph node syndrome, amyloidosis, asthma such as allergic asthma, autoimmune hepatitis, celiac disease, chronic obstructive pulmonary disease, Crohn’s disease, dermatitis, diabetes mellitus type 1, glomerulonephritis including rapidly progressive glomerulonephritis, graft versus host disease, immunoglobulin a vasculitis, an inflammatory' bowel disease, interstitial cystitis, lupus erythematosus, lupusnephritis, membranous nephropathy, multiple sclerosis including relapsing remiting multiple sclerosis, myocarditis, neuromyelitis optica, optic neuritis, pancreatitis, pemphigus, psoriasis, rheumatoid arthritis, scleroderma, Sjogren’s syndrome, ulcerative colitis, uveitis, vasculitis, vitiligo including vitiligo-associated multiple autoimmune disease susceptibility. In further specific embodiments, the anti-TNFR2 antibodies are useful for treating a condition selected from multiple sclerosis, vitiligo, and ulcerative colitis.

[0223] The pharmaceutical composition comprising an antibody as described herein, preferably in the form of a sterile aqueous solution, may be administered in a variety of ways, including, but not limited to, subcutaneously, intravenously, orally, intranasally, intraotically, trans dermally, mucosal, topically (e.g., gels, salves, lotions, creams, etc.), intraperitoneally, intramuscularly, intrapulmonary, vaginally, parenterally, rectally or intraocularly.

[0224] In some embodiments, the agonistic TNFR2 antibodies are capable of inducing the immunosuppressive ability of a population of Treg cells, (e.g., levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%. 0.0003%, 0.0004%, 0.0005%, 0.0006%. 0.0007%. 0.0008%, 0.0009%, 0.001%, 0.002%. 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%. 60.0%. 70.0%. 80.0%. 90.0%, or 100%. In some embodiments, the agonistic TNFR2 antibodies are capable of inducing the immunosuppressive ability of a population of Treg cells, (e.g., levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by 0.00001% to 0.001%, 0.000015% to 0.002%. 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%. 0.00004% to 0.007%, 0.000045% to 0.008%, 0.00005% to 0.009%, 0.000055% to 0.01%, 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%. 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%, 0.002% to 2%, 0.003% to 3%, 0.004% to 4%, 0.005% to 5%, 0.006% to 6%, 0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to 100%, 0.6% to100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%. 10% to 100%, 13% to 100%, 15% to 100%. 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%. 90% to 100%, 93% to 100%, 95% to 100%. 98% to 100%, as compared to a control or untreated subject.

[0225] In some embodiments, the agonistic TNFR2 antibodies are capable of inducing the proliferation of a population of Treg cells, (e.g., levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by 0.00001%, 0.00002%, 0.00003%. 0.00004%. 0.00005%.0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%. 6.0%, 7.0%, 8.0%. 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%. 60.0%. 70.0%, 80.0%, 90.0%, or 100%. In some embodiments, the agonistic TNFR2 antibodies are capable of inducing the proliferation of a population of Treg cells, (e.g., levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%, 0.00005% to 0.009%, 0.000055% to 0.01 %, 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%, 0.002% to 2%, 0.003% to 3%, 0.004% to 4%, 0.005% to 5%, 0.006% to 6%, 0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%. 0.3% to 100%. 0.4% to 100%. 0.5% to 100%. 0.6% to 100%. 0.7% to 100%. 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%. 70% to100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, as compared to a control or untreated subject.

[0226] In some embodiments, the agonistic TNFR2 antibodies show efficacy in suppressing, reducing, and / or decreasing antigen-induced skin inflammation. In some embodiments, the skin inflammation is suppressed, reduced, and / or decreased by about 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%. 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%. 0.004%, 0.005%, 0.006%. 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 20.0%, 30.0%, 40.0%, 50.0%, 60.0%, 70.0%, 80.0%, 90.0%, or 100%. In some embodiments, the skin inflammation is suppressed, reduced, and / or decreased by about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%, 0.00005% to 0.009%, 0.000055% to 0.01%, 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%. 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%, 0.002% to 2%, 0.003% to 3%, 0.004% to 4%, 0.005% to 5%, 0.006% to 6%, 0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0. 1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to 100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, l% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to100%, 35% to 100%, 38% to 100%. 40% to 100%, 43% to 100%, 45% to 100%. 48% to100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to100%, 95% to 100%, 98% to 100%, as compared to a control or untreated subject. The immunosuppressant efficacy of Antibody 1 was assessed in a KLH-induced delayed typehypersensitivity model in human TNFR2 knock-in mice as described in Example 8.Treatment with Antibody 1 significantly reduced inflammation as measured by a reduction in ear thickness. At peak inflammation 48 hours after KLH challenge, treatment with Antibody 1 provided a 42% reduction in inflammation (ear thickness) as compared to an isotype control.

[0227] In some embodiments, the agonistic TNFR2 antibodies show efficacy in increasing and / or enhancing survival rate of subjects with GvHD. Efficacy of Antibody 2 in prolonging survival was assessed in a human PBMC Xeno-GvHD model in immunocompromised mice as described in Example 9. Treatment with Antibody 2 significantly prolonged survival and reduced GvHD progression as compared to vehicle treatment. Median survival was 51 days for the group treated with Antibody 2 as compared to 44 days. In some embodiments, the survival rate is increased and / or enhanced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%. 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%. 325%, 350%, 375%. 400%, 425%, 450%. 475%, 500%. 525%. 550%, 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000%, as compared to a control or untreated subject. In some embodiments, the survival rate is increased and / or enhanced by about 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%. 20% to 100%, 23% to 100%, 25% to 100%. 28% to100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to100%, 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, 100% to 400%, 110% to 400%, 120% to 400%, 130% to 400%, 140% to 400%, 150% to 400%, 160% to 400%, 170% to 400%, 180% to 400%, 190% to 400%, 100% to 500%, 110% to 500%, 120% to 500%, 130% to 500%, 140% to 500%, 150% to 500%, 160% to 500%, 170% to 500%, 180% to 500%, 190% to 500%. 100% to 600%, 110% to 600%, 120% to 600%. 130% to 600%. 140% to 600%, 150% to 600%, 160% to 600%, 170% to 600%, 180% to 600%, 190% to 600%, 100% to 700%, 110% to 700%, 120% to 700%, 130% to 700%, 140% to 700%, 150% to 700%, 160% to 700%, 170% to 700%, 180% to 700%, 190% to 700%, 100% to 800%, 110%to 800%, 120% to 800%, 130% to 800%, 140% to 800%, 150% to 800%, 160% to 800%, 170% to 800%, 180% to 800%, 190% to 800%, 100% to 900%, 110% to 900%, 120% to 900%, 130% to 900%, 140% to 900%, 150% to 900%, 160% to 900%, 170% to 900%, 180% to 900%, 190% to 900% 110% to 1000%, 120% to 1000%, 130% to 1000%, 140% to 1000%, 150% to 1000%, 160% to 1000%, 170% to 1000%, 180% to 1000%, 190% to 1000%, 200% to 1000%, 250% to 1000%, 300% to 1000%, 350% to 1000%, 400% to 1000%, 450% to 1000%, 500% to 1000%, 600% to 1000%. 700% to 1000%. 800% to 1000%. 900% to 1000%, or 950% to 1000%, as compared to a control or untreated subject.

[0228] In another embodiment, the anti-TNFR2 antibody compositions used to treat an immune-related disorder are administered in combination with any of a variety' of known anti-inflammatory and / or immunosuppressive compounds.EXAMPLES

[0229] It is to be understood that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention(s) provided herein. Other aspects, advantages and modifications will be apparent to those skilled in the art to which this disclosure pertains.

[0230] In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g, amounts, temperatures, etc.) but some experimental error and deviation should be taken into account. Unless indicated otherwise, temperature is in degrees C (°C) and pressure is at or near atmospheric pressure at sea level. Each of the following examples is considered to be instructive to one of ordinary skill in the art for carrying out one or more of the embodiments described herein.EXAMPLE 1EXPRESSION AND PURIFICATION OF IgG ANTIBODY

[0231] Two separate approaches to IgG expression were utilized.

[0232] The first approach utilized transient expression using ExpiCHO™ cells (ThermoFisher Scientific, USA). Expression was carried out according to the manufacturer’s instructions. Briefly, ExpiCHO™ cells were maintained at >95% viability, cultured at 37°C. 120 rpm, 8% CO2to a density of 6 xlO6cells / ml. The day before the transfection, theExpiCHO™ cells were split (in half). The next day 1 pg total of heavy chain and light chain plasmids at a ratio of 1:2 were transfected into the ExpiCHO™ cells. The cells were grown for an additional 24 hours after which enhancer and feed (ExpiFectamine™ CHO Enhancer and ExpiCHO™ Feed, ThermoFisher Scientific) were added and growth conditions were changed to 30°C, 120 rpm, 5% CO2. Cells were grown an additional 7-10 days after transfection until the viability dropped to 70% and were harvested. The supernatants were first clarified through centrifugation (2000 xg. 30 min.) followed by filtration through a 0.22 pm filter. The clarified supernatant containing the IgGs was purified using a MabSelect PrismA™ column (Cytiva, Marlborough, MA, USA) followed by dialysis into a buffer appropriate for the isoelectric point (pl) of the antibody. Subsequent chromatography for polishing used Capto™ MMC resin (Cytiva, Marlborough, MA, USA), and the bound antibody was eluted using a linear gradient of NaCl.

[0233] The second approach utilized the creation of a stable cell line expressing the heavy chain (HC) and light chain (LC). Cell line development was performed using ATUM’s DNA2.0 gene design and synthesis (ATUM, Newark. CA). Using ATUM's Leap- In Transposase® and synthetic transposons, single copies of the entire synthetic transposon were integrated into multiple transcriptionally active genomic loci. Briefly, stable constructs were generated that expressed both the HC and LC. Constructs were transfected into the HDBIOP3 cell line (Horizon Discovery) and pools were selected in the absence of glutamine. Expression was carried out using a cell culture method for stable pools and clones. As per standard culturing techniques, the cultures were maintained at 37°C, 125 rpm, 5% CO2 and 70-80% relative humidity with passaging every 3-4 days to keep them in exponential phase (not exceeding 6 xlO6cells / mL). Clones were passaged at least 2 times with >95% viability before using the cells to start production runs. On Day 4 the culture temperature is reduced to 32°C until the end of the fed-batch run if the culture VCD >10 xlO6cells / mL.

[0234] Fed-batch cultures were fed on days 0, 3, 4, 7, 10, and 12, and after feeding the shaker speed was increased to 150 rpm for shake flasks. The pH was not adjusted in smaller scale production runs and harvested on Day 14 (or if viability <75%). The supernatants were first clarified through depth filtration using Sartoclear® cassettes (Sartorius AG) and a Sartogard NF 0.2 urn filter (Sartorius AG) for bioburden reduction. A 3M Emphaze™ AEX filter (3M™) was next used to reduce host cell proteins and other impurities. The clarified supernatant containing the IgGs was purified using a MabSelectPrismA™ column (Cytiva. Marlborough, MA, USA) followed by dialysis into a buffer appropriate for the isoelectric point (pl) of the antibody. Subsequent chromatography for polishing used Capto™ S ImpAct resin (Cytiva, Marlborough, MA, USA), and the bound antibody was eluted using a linear gradient of NaCl. Finally, ultrafiltration and diafiltration (UF / DF) was performed for buffer exchange into final formulation buffer.EXAMPLE 2ANALYTICAL SIZE EXCLUSION CHROMATOGRAPHY

[0235] To analyze the purity and homogeneity of the IgGs, 10 pg IgGs were loaded on an Agilent 1260 HPLC, equipped with Tosoh Biosciences G-4000 column (Tosoh Biosciences, Grove City7, OH, USA), with a flow rate of 0.5 ml / min and run time of 45 minutes. 100 mM NaPhosphate / 100 mM Na Sulfate served as mobile phase and retention was monitored at 280nM.EXAMPLE 3CHARACTERIZATION OF TNFR2-MEDIATED NFKB SIGNALING INDUCED BY A PANEL OF ANTI-TNFR2 ANTIBODIES IN HEK-BLUE™ TNFa REPORTER CELL ASSAY

[0236] A panel of anti-TNFR2 antibodies as shown in Table 1 below was screened for the ability7to induce TNFR2-mediated NFKB signaling by evaluating the secretion of alkaline phosphatase from an engineered reporter cell line as compared to an isotype control. The use of a human engineered reporter cell line provides a practical, reproducible, and scalable system to characterize agonistic potential of TNFR2 binding proteins.Table 1 : Test Articles

[0237] HEK-Blue™ TNFa cells (Invivogen) were cultured in DMEM medium (Fisher) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco Cat. No. 10082-147), Pen-strep antibiotic / antimycotic (100 U / ml) (Gibco Cat. No. 15240-062), 100 ug / ml Normocin, 1 pg / ml puromycin and 100 pg / ml Zeocin at 37°C / 5% CO2 in a humidified incubator. The HEK-Blue™ TNFa cell suspension was prepared by gently detaching the cells in pre-warmed DPBS medium for 2-3min and the cell pellet was resuspended in pre-warmed DMEM medium supplemented with 10% FBS and Pen-strep (100 U / ml) at 280,000 cells / ml. 180 pl of the cell suspension was plated into a flat bottom 96-well tissue culture plate (50,000cells per well). 20 pl (10X final concentration) of the anti-TNFR2 antibodies (diluted in PBS) were added into each well according to Table 2 to stimulate the cells for 24 hours.

[0238] The next day, QUANTI-Blue™ solution (Invivogen) was prepared by adding 1 ml of QB reagent and 1 ml of QB buffer to 98 ml of sterile water. After dispensing 180 pl of QUANTI-Blue™ solution per well into a flat-bottom 96-well plate, 20 pl of cell supernatant was added into QUANTI-Blue™ solution according to the study design as shown in Table 2 and the plate was incubated at 37°C for 2 hours before optical density at 655 nm was measured using a spectrophotometer with the results shown in FIGs. 15A-15N.Table 2: Experimental Study Design

[0239] Data was graphed using Prism® 9 (GraphPad Software, San Diego, California). The maximum observed response (Emax observed) values were taken from the rawdata, as average ODess value of replicate wells. The Emax as used herein is the maximum signal at any concentration for each test article. The observed Emaxobserved (also observed Emax) as used herein is the maximum signal observed at any concentration for each test article.

[0240] The induction of NFKB pathway signaling by a panel of anti-TNFR2 Fabs in HEK-Blue™ TNFa reporter cells with 24-hour stimulation was determined using detection of secreted alkaline phosphatase levels via colorimetric substrate. Cells were incubated with a 3- point dilution series of each test article to generate dose-response datasets as shown in Table 2. Antibody 6, Antibody 10, Antibody 1, and Antibody 13 showed dose-responsive reporter assay activity. Antibody 1 had the highest measured activity (E max observed). Emax observed IS the highest signal measured at any concentration for a given antibody.

[0241] The observed Emax values were obtained for each test article with the results shown in Table 3.Table 3: Observed E max values

[0242] In some instances, in vitro stimulation of HEK-Blue™ TNFa reporter cells with increasing doses of anti-TNFR2 Fabs resulted in dose dependent induction of TNFR2- mediated NFKB signaling as measured by increased secreted alkaline phosphatase enzyme after 24-hour incubation. A subset of "hit" anti-TNFR2 antibodies; Antibody 6, Antibody 10, Antibody 1, and Antibody 13 demonstrated dose-responsive agonistic activity7(Fig. 15). Antibody 1 had the highest activity7(E max observed).EXAMPLE 4IN VITRO ASSESSMENT OF BINDING TO LYMPHOCYTE SUBSETS

[0243] The ability of Antibody 1 to bind to lymphocyte subsets was determined using human PBMCs.

[0244] Cryopreserved human PBMCs from one healthy donor were thawed and rested for 2 hours in a 37°C / 5% CO2 incubator. 100 pl of the thawed PBMCs was aliquoted into a Protein LoBind®, deep well 96-well 2.0 ml plate. Serial dilutions of Antibody 1 were prepared in phosphate buffered saline (PBS), in a Protein LoBind®, deep well 96-well 0.5 ml plate. Human Fc block (BD Cat. No. 564220) was added to the aliquoted PBMC samples and incubated for 20 minutes on ice. The cells were then incubated for 45 minutes on ice in the presence of increasing doses of Antibody 1 ranging from 0.9 nM to 667 nM as shown in Table 4. Cells were washed 2 times with flow cytometry staining (FCS) buffer (eBioscience Cat. No. 00-4222-26) and resuspended in 100 pl of FCS buffer containing the surfacestaining fluorescent antibody cocktail for 30 minutes on ice. Samples were washed 2 times with FCS buffer and the cell pellet was resuspended in 200 ml FCS buffer. Samples were acquired using an LSRFortessa™ cell analyzer flow cytometer (Becton Dickinson).Table 4: Experimental Study Design

[0245] Flow cytometry data was analyzed using FlowJo™ software (FlowJo, LLC, Oregon). Binding of Antibody 1 to cells was measured via secondary binding of the phy coerythrin (PE) labeled goat anti-human IgG Fc antibody. PE positive population was determined based on the vehicle treatment. PE percent positivity (%+) and tMFI values were used to determine the ECso values. ECso values for compound dose responses were determined from 4-parameter non-linear regression curves using Prism® 10 (GraphPad Software, San Diego, California). Observed Emaxvalues were obtained from the raw data and were the largest response observed at any concentration of the test article.

[0246] The binding of Antibody 1 in subsets of lymphocytes in human PBMCs was determined using flow cytometry. After gating on lymphocytes, sub-populations were defined with lineage specific markers. Human Tregs were defined as CD3+CD4+CD25+CD127- and were also split into CD25high and CD251ow subsets based on CD25 MFI. T helper non-Treg cells were defined as CD3+CD4+CD25-, and Cytotoxic T cells as CD3+CD8+. Bound positive population was determined based on the vehicle treatment. The results are shown in Table 5 and Table 6.Table 5: EC50 and observed Emax values for Antibody 1 in human PBMC Treg populationsTable 6: ECso and observed Emax values for TNFR2 binding antibodies in human PBMC non- Treg lymphocyte populations

[0247] In vitro incubation of human PBMCs with increasing doses of Antibody 1 resulted in dose dependent binding of TNFR2 binding antibodies to lymphocytes. Antibody 1 demonstrated variable binding to different lymphocyte subsets (FIGs. 1A-1F & 2A-2D). Binding of Antibody 1 was highest on Treg cells compared to non-Treg lymphocytes. The maximal response was higher on CD4+ non-Treg cells (FIGs. 2A and 2B) compared to CD8+ cytotoxic T cells (FIGs. 2C and 2D). Within the Treg cell subset, Antibody 1 binding was increased on CD25high Tregs (FIGs. 1C and ID) compared to CD251ow Tregs (FIGs. IE and IF). This data confirms that Antibody 1 preferentially binds to immunosuppressive Tregs over other lymphocytes.EXAMPLE 5IN VITRO SIGNALING STUDY

[0248] The phosphorylation of RelA (pRelA) in lymphocyte subsets using human peripheral blood mononuclear cells (PBMCs) was evaluated in order to determine the abilityof TNFR2 binding antibodies to induce TNFR2-mediated NFKB signaling. The doses for different Anti-TNFR2 antibodies were shown in Table 7.Table 7: Experimental Study Design

[0249] Cryopreserved human PBMC from three healthy donors (see, FIG. 3A-3F and FIG. 4A-4H for donor 1 and donor 2; see, FIG. 39A-39E for donor 3) were thawed, plated in cell culture media and then rested for 2 hours in a 37C / 5% CO2 incubator. 100 pl of cells was aliquoted in Protein LoBind®. deep well 96- well 2.0ml plate. Serial dilutions of TNFR2 binding antibody Fc variants were prepared in phosphate buffered saline (PBS), in Protein LoBind®, deep well 96-well 0.5ml plate. The aliquoted PBMC samples were stimulated for 30 minutes at 37°C in the presence of increasing doses of TNFR2 binding antibodies ranging from 0.9 nM to 666.7 nM. PBMC were washed 2 times with flow cytometry staining (FCS) buffer and permeabilized with -20°C pre-chilled 100% methanol for 30 minutes on ice. The cells were then washed 3 times with FCS buffer, resuspended in 100 pl of FCS buffer containing human BD Fc block for 20 minutes on ice. The cells were then stained overnight at 4°C using a panel of fluorescent antibodies to distinguish various T, NK, and monocyte cell subpopulations and pRelA status. Samples were washed 3 times with FCS buffer and thecell pellet was resuspended in 200 pl FCS buffer. Samples were acquired using an LSRFortessa™ cell analyzer (Becton Dickinson).

[0250] Flow cytometry data was analyzed using FlowJo™ software (FlowJo, LLC, Oregon). The pRelA positive population was determined based on the vehicle treatment. pRelA percent positivity (%+ pRelA or %pRelA) values were used to determine the EC50 values. EC50 values for compound dose responses were determined from 4-parameter nonlinear regression curves using Prism® 6 (GraphPad Software. San Diego, California).

[0251] The induction of RelA by Antibody 1, Antibody 2, Antibody 3, and Antibody 4 in cell subpopulations following 30 minutes stimulation of human PBMC was determined using phospho-flow cytometry. Sub-populations were defined with lineage specific markers. Human Tregs were defined as CD3+CD4+CD25+FOXP3+, and further divided into high and low expressing subpopulations based on MFI of CD25 and / or FOXP3 markers. T helper cells were defined as CD3+CD4+, Cytotoxic T cells as CD3+CD8+, NK cells as CD3-CD56+, and monocytes as CD3-CD14+. pRelA positive population was determined based on the vehicle treatment.

[0252] Table 8 summarizes the EC50 values in nanomolar units, and the maximum observed (Observed Emax or Emax observed) pRelA-positive cell fraction expressed as %+ (also referred to as %positive), obtained for each Treg cell population for donor 1 and donor 2. Antibodies 1-4 demonstrated variable but overall similar potency (EC50) across compounds, donors, and Treg subsets. Antibody 2 consistently showed higher observed Emax values across donors and Treg subsets, while Antibody 3 and Antibody 4 had similar lower observed Emax values compared to Antibody 2.Table 8: pRelA induction EC50 values (nM) and observed Emax values for Antibodies 1 -4 in human PBMC Treg cells

[0253] In vitro stimulation of human PBMC with increasing doses of Antibody 1,Antibody 2, Antibody 3, or Antibody 4 resulted in dose dependent induction of pRelA in Treg cells, confirming that each of the compounds can effectively induce TNFR2-mediated downstream pRelA signaling. Antibody 2 demonstrated enhanced ability to activate NFKB signaling in a larger fraction of Tregs relative to three IgGl -based variants which all showed similar ability to induce signaling (Figs. 3A-3F). The low potency of each of the compounds enables Treg-selective signaling (Figs. 3A-3F; Figs. 4A-4H; Figs 40A-40E) which may increase therapeutic window and benefit, in patients with autoimmune disease.EXAMPLE 6CHARACTERIZATION OF TNFR2-MEDIATED NFKB SIGNALING INDUCED BY TNFR2 BINDING ANTIBODIES IN HEK-BLUE™ TNFa REPORTER ASSAY

[0254] The ability of different antibody formats to induce TNFR2 -mediated NFKB signaling was determined by evaluating the secretion of alkaline phosphatase from an engineered reporter cell line. The use of a human engineered reporter cell line provides a practical, reproducible, and scalable system to characterize agonistic potential ofTNFR2 binding proteins.

[0255] HEK-Blue™ TNFa cells were cultured in DMEM medium supplemented with 10% FBS, Pen-strep (100 U / ml), 100 ug / ml Normocin, lug / ml puromycin and 100 ug / ml Zeocin at 37°C / 5% CO2 in a humidified incubator. HEK-Blue™ TNFa cell suspension was prepared by gently detaching the cells in pre-warmed DPBS for 2-3min and cell pellet wasresuspended in pre-warmed DMEM medium supplemented with 10% FBS and Pen-strep (100 U / ml) at 280,000 cells / ml. 180 pl of cell suspension was plated into flat bottom 96-well tissue culture plate (50,000 cells per well). 20 pl (10X final concentration) of anti-TNFR2 antibodies (diluted in PBS) were added into each well to stimulate the cells for 24 hours.

[0256] The next day, QUANTI-Blue™ solution was prepared by adding 1 ml of QB reagent and 1 ml of QB buffer to 98 ml of sterile water. After dispensing 180 pl of QUANTI- Blue™ solution per well into a flat-bottom 96-well plate, 20 pl of cell supernatant was added into QUANTI-Blue™ solution and the plate was incubated at 37 °C for 3 hours before optical density at 655 nm was measured using a spectrophotometer. The doses for different antibodies are shown in Table 9.Table 9: Experimental Study Design

[0257] Curve fit parameters for compound dose responses were determined from 4- parameter non-linear regression curves using Prism® 10 (GraphPad Software, San Diego, California). Observed Emax values w ere taken from the raw data, as average ODess value of replicate wells.

[0258] The induction of NFKB pathway signaling by TNFR2 binding antibody variants in HEK-Blue™ TNFa reporter cells with 24-hour stimulation was determined using detection of secreted alkaline phosphatase levels via colorimetric substrate. Cells were incubated with a dilution series of each test article to generate dose-response datasets for curve fitting. Antibody 2 had the highest measured activity (observed Emax), followed byAntibody 1, and then Antibody 5 .

[0259] Table 10 summarizes the observed Emax values obtained for each sample.Antibody 2 had the highest observed Emax, followed by Antibody 1. Antibody 5 (monovalentFab format) showed agonistic activity, albeit at a reduced level compared to the bivalent mAb formats. ECso values could not be determined with confidence.Table 10: ECso values for TNFR2 binding antibody variants in HEK-Blue™ TNFa reporter cell assay

[0260] In vitro stimulation of HEK-Blue™ TNFa reporter cells with increasing doses of TNFR2 binding antibody variants resulted in dose dependent induction of TNFR2- mediated NFKB signaling as measured by increased secreted alkaline phosphatase enzyme after 24-hour incubation. Antibody 1 and Antibody 2 demonstrated variable activity depending on format (Fig. 5). Antibody 2 had the highest activity followed by Antibody 1, followed by Antibody 5. These data confirm that TNFR2 binding antibodies in multiple formats can effectively induce TNFR2 mediated downstream signaling, and furthermore that formatting of Antibody 2 enhances activity relative to Antibody 1.EXAMPLE 7CHARACTERIZATION OF Treg FUNCTIONAL MARKER EXPRESSION IN HUMAN PBMCS

[0261] The ability of TNFR2 binding antibody variants with differing fragment crystallizable (Fc) sequences to induce TNFR2-mediated expression of functional markers on T cell subsets in human peripheral blood mononuclear cell (PBMC) samples was determined.

[0262] Cryopreserved human PBMC from one healthy donor was thawed, washed, resuspended in 10 mL prewarmed growth media (RPMI-1640+10%FBS+2nM-glutamine+lX antibiotics / antimycotic + lx b-mercaptoethanol), counted, then pelleted and diluted to 1.2 million cells / mL. 180 pL (200,000 cells) was aliquoted into flat bottom 96 well tissue culture plates and rested for 2 hours in a 37C / 5% CO2 incubator. TNFR2 binding antibody Fc variants were prepared at lOx final concentration in phosphate buffered saline (PBS) diluent, in a Protein LoBind®, deep well 96-well 2 mL plate, and 20 pL was added to the well. The treated PBMC samples were stimulated for 5 days at 37°C / 5% CO2 in the presence of vehicle or TNFR2 binding antibody variants at 0.1, 0.39, 1.56, 6.25, 25, or 100 pg / mL.PBMC were washed 3 times with PBS then 500 pL / well LiveDead™ BV510 stain was added and incubated on ice for 15 minutes. Cells were washed twice with cold flow cytometry staining (FCS) buffer then 100 pL of Fc block was added and incubated on ice for 15 minutes. Next, 100 pL of cell surface staining antibody mix w as added and incubated on ice for 30 minutes. After washing twice with cold FCS buffer, cells were permeabilized with 200 pL of Fix / Perm solution. Samples were vortexed then incubated for 30 minutes on ice. The cells w ere then washed twice with 500 pL Perm buffer, then 100 pL of Fc block w as added and incubated on ice for 15 minutes. Next 100 pL of intracellular staining mix (made in Perm buffer) was added and mixed by gentle pipetting then incubated for 30 minutes on ice. Samples were washed twice with cold FCS buffer and the cell pellet was resuspended in 150 pL FCS buffer. Samples were acquired using an LSRFortessa™ cell analyzer (Becton Dickinson). The doses for different Anti-TNFR2 antibodies w ere shown in Table 11.Table 11 : Experimental Study Design

[0263] Flow cytometry data was analyzed using FlowJo™ software (FlowJo. LLC.Oregon). Graphing of data was done in Prism® 10 (GraphPad Software, San Diego, California).

[0264] The induction of CD25, FoxP3, TIGIT, and Ki67 by TNFR2 binding antibody variants in Treg and non-Treg T cells following 5 days stimulation of human PBMC was determined using cell surface and intracellular flow cytometry. Human Tregs were defined as CD3+CD4+CD8-CD25+FOXP3+. Human T helper-non-Treg cells were defined as CD3+CD4+CD8-CD25-FOXP3- and T cytotoxic cells were defined as CD3+CD4-CD8+. TIGIT and Ki67 positive population was determined based on fluorescence-minus-one (FMO) stained negative control samples.

[0265] In vitro stimulation of human PBMC with increasing doses of TNFR2 binding antibody variants resulted in dose dependent induction of CD25 (Fig. 6A), FoxP3 (Fig. 6B), TIGIT (Figs. 7A-7B), and Ki67 (Fig. 8) in Treg cells, confirming that the TNFR2 binding antibodies described herein can effectively induce Treg functional markers associated with immunosuppressive and proliferative ability. Overall, Antibody 2, Antibody 1, and Antibody 4 variants demonstrated similar abi 1 i t to induce Treg functional markers. Antibody 3 treatment led to lesser induction these same functional markers. Finally, the Treg-selectivity of the present TNFR2 binding antibodies is highlighted by comparing Figs. 6A-6C, Figs. 7A-7B, and Fig. 8 with Fig. 9A-9D, which shows minimal induction of CD25 and Ki67 markers non-Treg T cells.EXAMPLE 8EVALUATION OF TNFR2 BINDING ANTIBODY EFFICACY AND IMMUNEMODULATORY ACTIVITY IN KLH-INDUCED DELAYED TYPEHYPERSENSITIVITY MODEL OF HUMAN TNFR2 KNOCK-IN MICE

[0266] A pharmacological assessment of anti-inflammatory and immunomodulatory activity of Antibody 1 in vivo in a human TNFR2 (hTNFR2) knock-in mouse model of KLH induced delayed-type hypersensitivity (DTH) was performed. Delayed-type hypersensitivity evaluates cell-mediated immune responses associated with Thl reactivity7. The DTH reaction is divided into the afferent and efferent phases. During the afferent phase of this model, mice are immunized by subcutaneous injection with hapten KLH in its chemically reactive state and emulsified with an adjuvant. The efferent phase is typically initiated one week after sensitization, whereby the previously sensitized mice are challenged by KLH ear injection resulting in inflammatory7reaction in the injected ear. The anti-inflammatory efficacy was assessed as DTH response over 72-hours after KLH challenge. Additionally, the immunomodulatory effect of Antibody 1 was evaluated in draining lymph node and spleen and histological evaluation of ear tissue was also conducted.

[0267] Mice were distributed to 6 treatment groups as shown in Table 12. On Day 0, 30 B-11TNFR2 (hTNFR2 knock-in) mice were randomly divided into 4 groups (Group 1- Group 4) according to their ear thickness and body weights. Group 1 and Group 4 contained 5 mice whereas Group 3 and Group 2 were composed of 10 mice each. Group 5 and Group 6 had each 5 wild-type animals. G1 and G5 were non-KLH challenge control groups whereas Groups 2-4 and Group 6 received KLH challenge on Day 7 to induce DTH response. Table 12: Treatment groups

[0268] On Day 0, each animal received immunization injection of lOOpL emulsion (lOOgg KLH+CFA+IFA) in the neck subcutaneously. On day 7, one ear of animals in Groups 1 and 5 were injected with lOpL of PBS whereas animals in Groups 2, 3. 4 and 6 received an intradermal ear injection of 1 OpL solution of 1 Opg KLH in PBS to induce a DTH response.

[0269] Treatments were applied to DTH response groups as shown in Table 12 above: Group 2 received on Day 6 a single subcutaneous (s.c.) 30 mg / kg dose of human IgGl- LALA-PG isotype control antibody. Group 3 received on Day 6 a single subcutaneous 30 mg / kg dose of Antibody 1, Group 4 received daily 3 mg / kg oral (p.o.) treatment of dexamethasone starting on Day 7 and Group 6 received on Day 6 a single dose of phosphate buffered saline (PBS) (vehicle of antibody formulations). The study was terminated on Day 10, three days after KLH challenge.

[0270] Three types of measurements were collected in the study. Body weight and ear thickness time course measurements were collected on Days 0, 3, 6, 7, 8, 9, and 10. Spleen and retroauricular lymph node immunophenot ping was conducted from terminal Day 10 samples measuring CD4+ and CD8+ T cells, CD4+ regulatory' T cells, monocytes and neutrophils by flow cytometry. Ear tissue histological assessment was conducted by paraffin embedded tissue sections H&E staining and measurement of epidermal thickness and inflammatory' cell infiltration.

[0271] The anti-inflammatory efficacy of Antibody 1 was evaluated by measuring the increase in ear thickness post KLH challenge. KLH challenge was conducted on Day 7 of the study. Antibody 1 treatment was conducted 1 day prior on Day 6. Positive control dexamethasone was delivered daily starting on Day 7. The effect of KLH challenge and different treatments in the hTNFR2 knock-in mice (Groups 1-4) is shown in Figs. 10A-10C. Vehicle treated c57B16 wildtype mice that served as disease modeling controls in Group 5 (no KLH challenge) and Group 6 (with KLH challenge) showed no inflammation and inflammation equivalent to Group 1 and Group 2, respectively, confirming the animal model validity.

[0272] Ear thickness recorded from Day 6 to Day 10 is shown in Figure 10A. After KLH challenge significant skin inflammation was observed over the following 72-hours as measured by ear thickness. Peak inflammation was recorded on Day 9 of the study, 48h after KLH challenge. Antibody 1 treated mice showed significant reduction of ear thickness over the duration of DTH response as measured by area under curve quantification from Day 6 to 10 as did positive control dexamethasone treatment (FIG. 10B). At peak inflammation on Day 9, treatment with Antibody 1 reduced ear thickness significantly by 42% compared to isotype control treatment (FIG. 10C). Treatment with Antibody 1 show ed significant efficacy in reducing ear inflammation.

[0273] Individual animals' body weights ere measured in parallel to ear thickness measurements to assess tolerability of the treatments. Relative body weight changes from Days 6 to the end of study at Day 1 are shown in Fig. 11 . Treatment with Antibody 1 had no negative effect on bodyweight at an efficacious dose level. Expected minor bodyw eight loss was observed in the dexamethasone treatment group.

[0274] Key immune cell subsets (CD4+and CD8+T cells, CD4+Tregs. NK cells, monocytes and neutrophils) frequency and absolute counts were enumerated at the end of the study on Day 10. Spleen and retroauricular lymph node w ere collected, weighed, and processed for flow cytometry' analysis using an automatic cell counter (Rigel S2, Shanghai Ruiyu Biotechnology’ Co. Ltd.) and Attune NxT Flow Cytometer (Invitrogen).

[0275] Treatment yvith Antibody I did not significantly affect immune cell subsets frequency or absolute cell counts, including Tregs, in spleens or lymph nodes compared to isotype control group yvhen normalized to tissue weight (Figs. 12A-12D). Dexamethasone positive control treatment reduced spleen and lymph node size and lowered multiple immunecell subsets cell counts in both organs consistent with its strong immunosuppressive mechanism.

[0276] At the tested timepoint on Day 10, 72h after KLH challenge, treatment with Antibody 1 did not change the profile of maj or immune cell subsets in spleen or retroauricular lymph node compared to iso type control treatment.

[0277] KLH challenged and control ear tissue were collected at Day 10, processed and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin and graded for inflammatory cell infiltration, erosion / crust formation and epidermal hyperplasia. Epidermal thickness was measured from stained sections.

[0278] Treatment with Antibody 1 had no effect on inflammatory cell infiltration in dermis and subcutaneous compared with isotype control. Dexamethasone as a positive control treatment showed significant improvement on inflammatory cell infiltration in dermis and subcutaneous compared with isotype control group. As shown in Fig. 13, KLH challenge induced epidermal thickening was not significantly different between Antibody 1 and isotype control treated groups. Dexamethasone reduced epidermal thickness to same levels as mice with no KLH challenge.

[0279] In KLH-induced DTH model. Antibody 1 was efficacious in showing a decrease in inflammatory response (measured as reduction in ear thickness) induced by KLH challenge. The overall AUC for ear thickness increase over the 72-hours post KLH challenge was significantly lower in Antibody 1 treated animals compared to isotype control. At peak inflammation 48h after KLH challenge, Antibody 1 treatment reduced ear thickness significantly by 42%. Furthermore, at the efficacious dose level Antibody 1 treatment was very' well tolerated.

[0280] In addition to ear thickness primary’ efficacy endpoint measurements, ear histopathology and flow cytometry from spleen and draining lymph node were conducted at the end of study to evaluate ear epidermal thickness and immune cells profile, respectively. Treatment with Antibody 1 did not show' significant difference from iso t pe control treatment in these secondary endpoints at the terminal timepoint at the end of study.EXAMPLE 9EVALUATION OF TNFR2 BINDING ANTIBODY IN PROLONGING SURVIVAL IN HUMAN PBMC XENO-GVHD MODEL IN IMMUNOCOMPROMISED MICE

[0281] A pharmacological assessment of anti-infl ammatory activity of Antibody 2 in immunocompromised NCG mice that have been induced graft versus host disease (GvHD) by human peripheral blood mononuclear cells (hPBMC) intravenous injection was performed. hPBMC transfer leads to systemic inflammation and human immune cell attack on mouse tissues that causes mice to show loss of body weight and multiple clinical signs including skin inflammation and lesions. GvHD severity is scored by standardized evaluation of body weight and clinical signs and animals are eliminated from study at predetermined score values resulting in survival scoring. GvHD is used for Treg function enhancing anti-inflammatory drugs evaluation as they can delay GvHD disease progression and increase animal survival in the model by stimulating the Treg compartment of the injected hPMBC. Efficacy of Antibody 2 was assessed using disease scoring based survival measurements over the course of GvHD progression relative to vehicle treated animals.

[0282] Mice were distributed to 6 treatment groups as shown in Table 13. Group 1 served as control group that did not receive hPBMC transfer and were treated with vehicle (PBS) twice weekly. Group 2 animals were injected intravenously (IV) with 20 million hPBMC pretreated with IgG4 (S228P) isotype control (100 pg / ml) for 48-hours in culture whereas animals in Group 3 were injected IV with 20 million hPBMC pretreated 48-hours in culture with Antibody 2 (100 pg / ml). No in vivo drug treatments were conducted in Groups 2 and 3 after hPBMC transfer. Group 4 and 5 animals were IV injected with 20 million untreated hPBMC and received twice weekly subcutaneous injections of vehicle (Group 4) or Antibody 2 at 20 mg / kg (Group 5). As a positive control, Group 6 received 20 million untreated hPBMC by IV injection and were treated orally three times a week with aryl hydrocarbon receptor agonist 10-C1-BBQ which was previously validated to suppress GvHD development in this model. In vivo drug treatments for Groups 1, 4, 5 and 6 started on the day of hPBMC transfer. Body weight measurements and clinical signs monitoring were conducted three times a week throughout the study duration. The last drug treatments were conducted on Day 58. The study was terminated on Day 73.Table 13: Treatment groups

[0283] Survival criteria underlying the calculation of drug treatment efficacy were established by veterinarian-validated body condition grading (Grade 0, 1 or 2) of 5 parameters: body weight loss, activity, posture, fur texture and skin integrity as outlined in Table 14. Animals that reached Grade 2 in any parameter were removed from the study. Table 14: Body condition grading and criteria for survival determination

[0284] The efficacy of Antibody 2 was evaluated relative to a vehicle control by measuring survival rate of animals based on absolute survival and body condition grading driven termination of animals from study over time as hPBMC adoptive transfer induced GvHD progressed.

[0285] Figs. 14A and 14B show survival curves for all of the treatment groups. Efficacy assessment of in vivo drug treatments were conducted by comparing survival of Antibody 2 treatment Group 5 to vehicle treatment Group 4. Antibody 2 treatment was efficacious by significantly increasing survival rate compared to vehicle treatment. Antibody 2 treatment group median survival was 51 days whereas vehicle group median survival was 44 days showing a statistically significant difference (p=0.0022, Log-rank test). Efficacy of m vitro hPBMC pretreatments was evaluated by comparing survival rate of animals in Groups 2 and 3 that received in vitro vehicle or Antibody 2 pretreated hPBMC, respectively. Treatment Groups 2 and 3 showed median survival of 55 and 66.5 days, respectively, without statistically significant difference (p=0.2018, Log-rank test). Treatment Group 1 (no hPBMC transfer) and Group 6 (positive control drug treatment) served as control groups confirming disease model validity and hPBMC dependence of disease induction.

[0286] The immunosuppressive effect of Antibody 2 on hPBMC dependent GvHD induction was evaluated in two different treatment regimens. In the hPBMC pretreatment regimen, cells were incubated in culture 48-hours with Antibody 2 prior to adoptive transfer. In the second regimen. hPBMCs were adoptively transferred without pretreatment and Antibody 2 was subcutaneously injected twice a week after hPBMC transfer. hPBMC pretreatment with Antibody 2 in vitro did not yield a difference in the in vivo survival compared to vehicle pretreatment suggesting that brief exposure prior to adoptive transfer is insufficient to affect hPBMC GvHD induction. However, twice weekly Antibody 2 administration resulted in significant survival improvement as compared to vehicle treatmentshowing that Antibody 2 is efficacious in delaying hPBMC dependent GvHD progression in vivo.EXAMPLE 10EVALUATION OF TNFR2 ANTIBODY CELL BINDING AND PHARMOKINETICS IN HUMAN TNFR2 KNOCK-IN MICE

[0287] Cell binding of Antibody 1 (IgGI LALAPG) in immune cells was evaluated using flow cytometry -based characterization. Further, plasma pharmacokinetics (PK) of Antibody 1 was evaluated in human TNFR2 (hTNFR2) receptor expressing knock-in mice (B-hTNFR2, Biocytogen Pharmaceutical).

[0288] Mice were distributed to 4 treatment groups as shown in Table 15. Group 1 served as a vehicle control group. Group 2 animals were injected intravenously (IV) with a single 2 mg / kg dose of Antibody 1. Group 3 were injected IV with a single 6.6 mg / kg dose of Antibody 1. Group 4 were injected IV with a single 20 mg / kg dose of Antibody 1. At each timepoint (3, 24 and 96 hours after dosing) 4 animals in each group were sacrificed and the spleen from each animal was collected and analyzed by flow cytometry analysis and for blood plasma (EDTA anticoagulant) for pharmacokinetics measurement (PK) samples.Table 15: Treatment groups

[0289] Flow cytometry staining was conducted on single cell spleen suspensions with an immune cell detection antibody panel shown in Table 16. Unlabeled Antibody 1 detection was conducted with a PE-conjugated goat anti-human Fc secondary antibody. Stained samples were acquired on Fortessa™ X20 flow cytometer (Becton-Dickinson) and data analysis with FlowJo™ software (FlowJo, LLC). Antibody 1 binding was characterized in CD4+ and CD8+ conventional T cells (including, for example, non-Treg cells), total CD4+ CD25+ FoxP3 regulatory T cells, including FoxP3 high and low expressing CD4+ regulatoryT cells subsets, NK cells, monocytes and neutrophils as percent of binding positive cells frequency and total MFI was calculated as a per cell basis measurement.Table 16: Flow cytometry antibodies

[0290] Antibody 1 pharmacokinetics (PK) was also measured. Antibody 1 concentration from each plasma sample was measured with an ELISA based MSD assay utilizing human antibody binding capture and detection antibodies. Non-compartmental analysis was utilized to calculate PK parameters based on Antibody 1 plasma concentrations over time.Antibody Binding to Immune Cells in Spleen

[0291] Antibody 1 binding to T cell subsets, NK cells, monocytes and neutrophils was evaluated in vivo in mice expressing knock-in of human TNFR2 receptor (B-hTNFR2 mice. Biocytogen Pharmaceutical). Cell binding was measured by flow cytometry at 3 hours, 24 hours and 96 hours after IV dosing from spleen tissue. As shown in FIGS. 33A-33K and 34A-34E, by fraction of binding positive cells (absolute binding measured by percentage of positive signal (% Binding)) (FIGS. 33A-33K) and by total MFI (tMFI) (i.e. MFI of all recorded events in a given sample) (FIGS 34A-34E) of Antibody 1 detecting anti-human Fc secondary antibody signal, Tregs showed highest cell binding as a fraction of antibody bound cells (FIGS. 33A-33K), and also highest binding per cell basis (tMFI, FIGS. 34A-34E) for Antibody 1 among the T cell subsets. Minor binding was detected on monocytes and neutrophils. No Antibody 1 binding was detected on natural killer (NK) cells and conventional CD4+ and CD8+ T cells (Tconv). Antibody 1 binding in Treg subsets was clearly observed in the mid- and high dose groups (6.6 and 20 mg / kg). Cell binding was dosedependent with the highest levels of cell binding being observed at 3 hours post administration with sustained over time decreasing binding observed at later timepoints at 24 hours and 96 hours after treatment. Binding kinetics for Antibody 1 to monocytes and neutrophils was distinct from Tregs with the former showing lower percentage saturated binding profile with a uniform level binding throughout the time course lacking a clear peak at the 3 hour timepoint. FIG. 35 shows a comparison of specific binding (background nonspecific signal subtracted) between immune cell types as percent binding positivity to quantify binding selectivity between measured immune cell types and peak binding (at the 3 hour timepoint) and for the highest dose group (20 mg / kg). Mean values of specific cell binding (mean vehicle background values subtracted total binding mean values) at 3 hours after dosing in the 20 mg / kg dose group were calculated for all measured immune cell subsets. CD25 positive and negative subpopulations of Tregs (CD4+ FoxP3+) were split to FoxP3 high or FoxP3 low expressing populations based on FoxP3 expression level. FoxP3 high MFI expressing cells designated as “FoxP3high’‘ constituted on average approximately 50% of total Tregs with the remaining FoxP3 low MFI cells designated as ”FoxP31ow'’ when analyzed for Antibody 1 binding frequency. Treg binding was compared to conventional CD4+ and CD8+ T cells (CD4+ Tconv and CD8+ T conv), NK cell, monocytes and neutrophils.

[0292] All FoxP3+ CD4+ regulatory T cell subsets showed significantly higher fraction of cells bound to Antibody 1 than NK cells, conventional CD4+ and CD8+ T cells or neutrophils and monocytes. Within Treg subsets. Antibody 1 cell binding correlated with FoxP3 expression levels. The highest fraction of Antibody 1 bound cells and binding per cell was observed within the FoxP3 high expressing Tregs.

[0293] Antibody 1 showed significant sustained Treg cell binding in spleens of hTNFR2 expressing mice in vivo. Peak levels, on average 15-20%, binding in Tregs was observed in the earliest 3 hour timepoint after IV administration. Higher levels of binding were observed in FoxP3 high expressing cells compared to FoxP3 low expressing cells. Antibody 1 binding was highly selective to Tregs compared to other immune cell types.

[0294] Selective and dose dependent Treg binding of approximately 5% and 15% Tregs was observed at 6.6 and 20 mg / kg dose levels, respectively. Sustained albeit diminished Antibody 1 selective binding to Tregs was also observed at 24h and 96h after treatment at the highest dose level.Pharmacokinetics of Antibody 1 in hTNFR2 Knock-In Mice

[0295] In parallel with spleen collections for flow cytometry, blood was collected from each animal and K2EDTA anticoagulant plasma was isolated. Mean plasma concentrations over time at each dose level are plotted in FIG. 36. Calculated PK parameters (Cmax, Tmax, half-life, clearance rate and exposure values) are shown in Table 17. Cmax and observed exposure showed dose proportionality. On average 15-20% Treg cell binding in spleen at the 3 hour timepoint was observed at mean plasma concentration of 189 jrg / ml at the 20 mg / kg dose. Approximately 3-fold lower 5% binding activity was observed at the 6.6 mg / kg dose level at 3 hours at mean plasma concentration of 79 pg / rnl.Table 17: Summary' of non-compartmental analysis of Antibody 1 plasma concentrations over timeEXAMPLE 11EVALUATION OF TNFR2 ANTIBODY CELL BINDING IN HUMAN TNFR2 KNOCK-IN MICE

[0296] Cell binding of Antibody 1 (IgGI LALAPG) and Antibody 2 (IgG4_S228P) to regulatory CD25+ FoxP3+ CD4+ T cells and conventional CD4+ and CD8+ T cells in vivo in the blood of human TNFR2 (hTNFR2) receptor expressing knock-in mice that express TNFR2 receptor with human extracellular binding domain was evaluated using flow cytometry -based characterization.

[0297] B-hTNFR2 (human TNFR2 knock-in) mice (Biocytogen Pharmaceutical) were distributed to 3 treatment groups as shown in Table 18. Group 1 served as vehicle control group. Group 2 animals were injected intravenously7(IV) with a single 20 mg / kg doseof Antibody 1. Group 3 animals were injected IV with a single 20 mg / kg dose of Antibody 2.Blood for flow cytometry analysis was collected from 4 mice per timepoint into EDTA anticoagulant tubes 24 hours and 96 hours after dosing.Table 18: Treatment groups

[0298] Flow cytometry staining was conducted after red blood cell lysis with T cell subsets detecting antibodies. After surface markers staining, intracellular FoxP3 staining was conducted with a FoxP3 staining kit (eBiosciences). Unlabeled Antibody 1 and Antibody 2 detection was conducted with PE-conjugated goat anti -human Fc secondary' antibody (ThermoFisher Cat. No. 12-4998-82). Stained samples were acquired on a Fortessa™ X20 flow cytometer (Becton Dickinson) and data analysis with FlowJo™ software (FlowJo, LLC). Antibody 1 and Antibody 2 binding was characterized in CD4+ and CD8+ conventional T cells, total CD4+ regulatory' T cells and FoxP3 high and low expressing CD4+ regulatory T cells subsets as percent of binding positive cells and total MFI as a per cell basis measurement with the results shown in FIGS. 37A-37J.

[0299] Further, Antibody 1 and Antibody 2 binding to CD4+ CD25+ FoxP3+ Tregs and conventional (non-Treg) CD4+ and CD8+ T cells was evaluated in vivo in mice expressing knock-in of human TNFR2 receptor. Cell binding was measured by flow cytometry at 24 hours and 96 hours after IV dosing with the results shown in FIG. 38 A for 24 hours after dosing and FIG. 38B for 96 hours after dosing.

[0300] As shown in FIGS. 37A-37J by fraction of binding positive cells (absolute binding measured by percentage of positive signal; (% Binding)) and by total MFI (tMFI) of anti-human Fc secondary’ antibody signal, Tregs showed highest cell binding as a fraction cells and also highest binding per cell basis (tMFI) for both antibodies among the tested T cell subsets at both timepoints. Minor binding was detected on conventional CD4+ and CD8+ T cells. Highest binding was observed at 24 hours post administration with sustained reduced binding observed also at 96 hours after administration of the antibodies. Both antibodies showed comparable levels of cell binding in each T cell subset. Both Antibody 1 andAntibody 2 cell binding were highly Treg selective. FIGS. 38A and 38B show specific binding (background non-specific signal subtracted) comparison between T cell subsets as percent cell binding positivity. Mean values of specific cell binding (mean vehicle background values subtracted total binding mean values) at 24 hours (FIG. 38 A) and at 96 hours (FIG. 38B) after dosing in measured T cell subsets. Total CD4+ Treg cells (Total Treg) were split into two sub-populations based on FoxP3 expression level. FoxP3 high MFI expressing cells designated as ’ 'oxP3high ‘ constituted on average approximately 50% of total Tregs with the remaining FoxP3 low MFI cells designated as ”FoxP3low" when analyzed for Antibody 1 binding frequency. Treg binding was compared to conventional CD4+ and CD8+ T cells (CD4+ Tconv and CD8+ T conv). At 24 hours after administration, Tregs showed 8-9-fold higher specific binding compared to CD4+ and CD8+ conventional T cells. Within the Treg subsets, FoxP3 high expression cells showed higher binding (10.5- 11.5% specific binding) compared to FoxP3 low expressing cells (5.7-7.7% positivity). FoxP3 high expressing cells specific binding at 24 hours after administration was 10-fold higher compared to conventional CD4+ and CD8+ T cells. At 96 hours after administration Treg subsets specific binding of Antibody 1 and Antibody 2 was still on average 4-fold higher compared to CD4+ and CD8+ conventional T cells.

[0301] Both Antibody 1 and Antibody 2 showed significant sustained Treg cell binding in blood of hTNFR2 expressing mice in vivo. At 24 hours after administration close to 10% of total CD4+ Tregs showed specific binding. Higher levels of binding were observed in FoxP3 high expressing cells compared to FoxP3 low expressing cells. Antibody 1 and Antibody 2 binding were highly selective to Tregs, showing up to 10-fold higher binding compared to conventional CD4+ and CD8+ T cells at the 24 hour timepoint.EXAMPLE 12EVALUATION OF TNFR2 ANTIBODY CELL BINDING SPECIFICITY

[0302] Antibody 2 or an isotype control antibody (recombiMAb human IgG4_S228P) having respiratory syncytial virus as atarget / receptor at 20 mg / mL were screened for binding against 6105 human plasma membrane and membrane tethered secreted proteins (Retrogenix Cell Microarray) displayed on HEK cells.

[0303] 20 mg / mL of Antibody 2 was screened for binding against fixed HEK293 cells, individually over-expressing 6105 full-length human plasma membrane proteins, secreted and cell surface-tethered human secreted proteins plus a further 400 human heterodimers. The fluorescence of each protein screen was measured using appropriate scanning technologies and normalised against the secondary only control (PBS) value. The screen identified 15 library interactions. Each of the 15 library interactions was re-expressed, along with two control receptors (CD20 and EGFR) and re-tested with Antibody 2 or the control to determine which interactions, if any, were repeatable and specific to the test article. This screen was performed on both fixed and live cells. Signal-to-Noise Ratios (SNR) were normalised against concurrent secondary only control values. A significant protein interaction as used in the context of these screens was defined as an interaction with an SNR > 1. The results are provided in Table 19. A median SNR <1 was classified as no significant interaction, a median SNR > with negative control / PBS interaction was classified as a nonspecific interaction, a median SNR > was classified as a significant specific interaction. Table 19: Antibody ScreenClassification: PM = plasma membrane. TS = tethered secreted, M = evidence of membrane *data point excluded from analysis

[0304] On the fixed cell microarrays, 8 of the 15 library' interactions were bound by the test article and at least one of the negative control treatments (RecombiMAb human IgG4_S228P isotype control or PBS / secondary only had SNR > 1). These interactions were classified as non-specific. The non-specific interaction included the Fc gamma receptors (FCGR1 A, FCGR2A isoform 2, FCGR2A isoform 1, and FCGR2B), which were presumably Fc-domain mediated, and IGHG3, which was bound directly by the detection antibody. A further 5 of the 15 library’ interactions (KCNJ12, TNFRSF13B, APOCI, CXCL12, and IGF1) appeared to be specific to Antibody 2. However, the SNRs were below the level of a significant interaction (SNR <1) and were therefore classed as non-significant. After excluding the non-specific and non-significant interactions, only two interactions remained that were significant and specific to Antibody 2 (TNFRSF1B (TNFR2, isoform 1) and TNFRSF1B (TNFR2, isoform 2)). On a cell microarray treated in the absence of fixation (live cells), Antibody 2 showed a significant specific interaction with TNFRSF1B (TNFR2) isoform 1 only. As seen in FIG. 39A, Antibody 2 showed significant, specific binding interaction to human TNFR2, but not to other TNFR superfamily receptors (TNFR1, 0X40, GITR, 4-1BB, BCMA. TRAILR1. HVEM, DDR3, DR6, TRAILR2, BAFFR, TweakR, RANK, TRADE, OCIF, and DcR3).

[0305] Antibody 2 showed a significant specific interaction with TNFRSF1B (TNFR2) isoform 1 on both the fixed and live cell microarrays. Antibody 2 showed a significant specific interaction with TNFRSF1B (TNFR2) isoform 2 on fixed cell microarrays only. No other interactions were identified for Antibody 2, indicating high specificity of Antibody 2 for TNFR2. The only significant, specific binding interactions for Antibody 2 detected in the screen were to human TNFR2. As seen in Fig. 39B, Antibody 2 displayedhighest binding to the transmembrane hTNFR2 (isoform 1) with limited binding to soluble hTNFR2 (isoform 2).EXAMPLE 13GENE SET ENRICHMENT ANALYSIS (GSEA) OF ANTIBODY 2 INDUCED TRANSCRIPTOME IN PRIMARY HUMAN Tregs

[0306] Human peripheral blood mononuclear cells were incubated in vitro in cell culture in standard culture media for 5 days in the presence of Antibody 2 (0. 1 mg / ml) or vehicle (phosphate buffered saline). At the end of the 5 days incubation, cells were collected and processed for single cell RNA collection, cDNA library generation and sequencing. Cell clustering analysis based on individual cells similarity in transcriptional profiles was carried out and UMAP dimension reduction was performed to visualize cell clusters. Treg cluster was identified based on cell lineage marker FoxP3 expression. Further sub-clustering of Tregs identified two cell clusters composed of a major cell cluster containing majority of Tregs and a minor cluster with low cell count. The major cluster Tregs sequencing data was analyzed to identify the specific subset of Tregs transcriptome that was differentially expressed between Antibody 2 and vehicle stimulated cells. The major Tregs cluster expressed genes were compared to publicly available gene signatures using gene set enrichment analysis software (GSEA v4.3.3). GSEA was performed using two previously published gene signatures. The first signature describes TNFa induced transcriptome in HeLa cells that is specifically mediated by NFKB pathway activation (Tian et al., 2005). The second gene signature describes non-lymphoid tissue-resident effector Tregs signature that is induced in purified human thymus derived Tregs in vitro in seven days of cell culture in the presence of IL-2 cytokine, agonistic CD3 antibody and either TNFR2 agonist antibody MR2-1 or agonistic CD28 antibody co-stimulation (Mensink et al., 2024).

[0307] Figs. 41 A and 41 B show enrichment of specific gene signatures in Antibody 2 induces Tregs. Fig. 41 A is a GSEA plot showing enrichment of NFKB pathway activation gene signature (Tian et al., 2005) in the transcriptome of human primary Tregs stimulated with Antibody 2. The normalized enrichment score (NES) is depicted (FDR q-val < 0. 1). Fig. 41B is a GSEA plot showing enrichment of non-lymphoid tissue-resident effector Treggene signature (Mensink et al.. 2024) in the transcriptome of human primary Tregs stimulated with Antibody 2. The normalized enrichment score (NES) is depicted (FDR q-val < 0.01).

[0308] Significant enrichment of NFKB pathway activation gene signature is observed in Antibody 2 stimulated Tregs transcriptome as shown in Fig. 41 A. These data indicate that Antibody 2 induces NFKB pathw ay activation in primary7human Tregs.

[0309] Significant enrichment of non-lymphoid tissue-resident effector Tregs gene signature is observed in Antibody 2 stimulated Tregs transcriptome as shown in Fig. 41B. These data indicate that Antibody 2 mediated TNFR2 agonism in human Tregs can enhance expression of genes that promote Treg capacity7for survival, migration, immunosuppression, and tissue regeneration, cellular functions that are associated with the non-lymphoid tissueresident effector Tregs gene signature expression (Mensink et al.. 2024). Notably in this example, single agent stimulation with Antibody 2 was sufficient to induce non-lymphoid tissue-resident effector Tregs gene signature in human Tregs without the requirement of added IL-2 cytokine and T cell receptor stimulation (exemplified by CD3 agonism).

[0310] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the antibodies, compositions, systems and methods described herein and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the abovedescribed modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the present disclosure pertains.

[0311] All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriate according to the spirit and scope of the present disclosure described herein.

[0312] All references cited herein are hereby incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application w as specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0313] Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.

[0314] Embodiments of the present methods, therapeutic combinations and kits include, but are not limited to:Embodiment 1. An anti-tumor necrosis factor receptor 2 (TNFR2) antibody or antigenbinding fragment thereof which exhibits agonistic activity towards TNFR2.Embodiment 2.The anti-TNFR2 antibody or antigen-binding fragment thereof according to embodiment 1. comprising a heavy chain and a light chain selected from the group consisting of: i. a heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain comprising the amino acid sequence of SEQ ID NO: 12; ii. a heavy chain having at least 80%. 85%. 90%. or 95% identity to the amino acid sequence of SEQ ID NO: 6 and a light chain having at least 80%. 85%. 90%. or 95% identity7to the amino acid sequence of SEQ ID NO: 12, wherein the amino acid at position 228 of SEQ ID NO: 6 is proline; iii. a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 24; and iv. a heavy chain having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 18 and a light chain having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 24, wherein the amino acids at positions 234 and 235 of SEQ ID NO: 18 are both alanine and the amino acid at position 329 of SEQ ID NO: 18 is glycine.Embodiment 3. The anti -tumor necrosis factor receptor 2 (TNFR2) antibody or antigenbinding fragment thereof according to the combined or separate embodiments 1-2, comprising a heavy chain and a light chain variable region (VL) selected from the group consisting of: i. a heavy chain comprising the amino acid sequence of SEQ ID NO:6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;ii. a heavy chain having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having at least 80%. 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 7, wherein the amino acid at position 228 of SEQ ID NO: 6 is proline; iii. a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19; and iv. a heavy chain having at least 80%. 85%. 90%. or 95% identity to the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 19, wherein the amino acids positions 234 and 235 of SEQ ID NO: 18 are both alanine and the amino acid at position 329 of SEQ ID NO: 18 is glycine.Embodiment 4. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-3, wherein said antibody or antigen-binding fragment comprises a set of three heavy chain variable region complementarity determining regions (CDRs), VHCDR1, VHCDR2, and VHCDR3, comprised within a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13. and a set of three light chain variable region complementarity determining regions, VLCDR1, VLCDR2 and VLCDR3, comprised within a light chain variable region comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 19.Embodiment 5. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1 -4, wherein said antibody or antigen-binding fragment comprises a set of three heavy chain variable region complementarity determining regions (CDRs) on a heavy chain, VHCDR1, VHCDR2, and VHCDR3, and a set of three light chain variable region complementarity determining regions on a light chain, VLCDR1, VLCDR2 and VLCDR3, wherein the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3, collectively, are selected from: i. the VHCDR1 of SEQ ID NO: 2, the VHCDR2 of SEQ ID NO: 3, the VHCDR3 of SEQ ID NO: 4. the VLCDR1 of SEQ ID NO: 8, the VLCDR2 of SEQ ID NO: 9 and the VLCDR3 of SEQ ID NO: 10; ii. the VHCDR1 of SEQ ID NO: 14, the VHCDR2 of SEQ ID NO: 15, the VHCDR3 of SEQ ID NO: 16, the VLCDR1 of SEQ ID NO: 20, the VLCDR2 of SEQ ID NO: 21 and the VLCDR3 of SEQ ID NO: 22;iii. the VHCDR1 of SEQ ID NO: 2 comprising 0-2 amino acid substitutions, the VHCDR2 of SEQ ID NO: 3 comprising 0-2 amino acid substitutions, the VHCDR3 of SEQ ID NO: 4 comprising 0-2 amino acid substitutions, the VLCDR1 of SEQ ID NO: 8 comprising 0-2 amino acid substitutions, the VLCDR2 of SEQ ID NO: 9 comprising 0-2 amino acid substitutions, and the VLCDR3 of SEQ ID NO: 10 comprising 0-2 amino acid substitutions; and iv. the VHCDR1 of SEQ ID NO: 14 comprising 0-2 amino acid substitutions, the VHCDR2 of SEQ ID NO: 15 comprising 0-2 amino acid substitutions, the VHCDR3 of SEQ ID NO: 16 comprising 0-2 amino acid substitutions, the VLCDR1 of SEQ ID NO: 20 comprising 0-2 amino acid substitutions, the VLCDR2 of SEQ ID NO: 21 comprising 0-2 amino acid substitutions, and the VLCDR3 of SEQ ID NO: 22 comprising 0-2 amino acid substitutions.Embodiment 6. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-5, comprising a human IgGl CHl-hinge-CH2-CH3 region, wherein said hinge region optionally comprises one, two, three, four or more mutations; and / or wherein said antibody or antigen-binding fragment is of the IgGl subclass. Embodiment 7. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-6, wherein:(a) a proline at position 329 of the human IgGl Fc region is substituted with another amino acid, optionally wherein the proline at position 329 is substituted with a glycine (P329G); and / or(b) a leucine at position 234 of the human IgGl Fc region is substituted with another amino acid, optionally wherein the leucine at position 234 is substituted with an alanine (L234A); and / or(c) a leucine at position 235 of the human IgGl Fc region is substituted with another amino acid, optionally wherein the leucine at position 235 is substituted with an alanine (L235A).Embodiment 8. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-7. comprising the amino acid substitutions: L234A and L235A of the human IgGl Fc region.Embodiment 9. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-7, comprising the amino acid substitutions: P329G and / or L234A and L235A of the human IgGl Fc region.Embodiment 10. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-9, wherein the proline at position 329 of the human IgGl Fc region is substituted with another amino acid.Embodiment 11. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-10, comprising a human IgG4 CH1- hinge-CH2-CH3 region, wherein said hinge region optionally comprises one, two, three, four or more mutations; and / or wherein said antibody or antigen-binding fragment is of the IgG4 subclass.Embodiment 12. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-11, wherein the serine at position 228 of the human IgG4 constant heavy region is substituted with another amino acid.Embodiment 13. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-12, wherein the serine at position 228 is substituted with proline (S228P).Embodiment 14. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-13, comprising a constant light region of a human kappa light chain.Embodiment 15. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-14, comprising a constant light region of a human lambda light chain.Embodiment 16. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-15, wherein said antibody or antigen-binding fragment thereof is capable of selectively promoting the proliferation of a population of T regulatory (Treg) cells.Embodiment 17. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-16, wherein the anti-TNFR2 antibody or antigenbinding fragment thereof activates TNFR2-mediated nuclear factor kappa-B (NFKB) signaling.Embodiment 18. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-17, wherein the NFKB signaling activation is characterized by an increase of alkaline phosphatase secretion level from an engineered reporter cell line.Embodiment 19. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-18, wherein the reporter cell line is a HEK-Blue NFKB reporter cell line.Embodiment 20. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-19, wherein the alkaline phosphatase secretion level induced and / or increased by said anti-TNFR2 antibody or antigen-binding fragment thereof in the reporter cell line is at least: a) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4- fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25- fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold. 8-fold, 8.25-fold, 8.5- fold, 8.75-fold. 9-fold. 9.25-fold. 9.5-fold. 9.75-fold, 10-fold. 10.25-fold. 10.5-fold,10.75-fold, 11 -fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold,12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold,14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold,16.75-fold. 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold,18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold,20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than an isotype control; and b) about 1.1-fold to 1.2-fold. 1.1-fold to 1.5-fold, 1.1 -fold to 2.5 -fold. 1.1 -fold to 5 -fold,1.1 -fold to 10-fold, 1.1 -fold to 15-fold, 1.1 -fold to 20-fold, 1.1 -fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22- fold, 5-fold to 8-fold. 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold. 6-fold to 10-fold. 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8- fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12- fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold. 10-foldto 18-fold, 10-fold to 22-fold, 11-fold to 14-fold, 11-fold to 18-fold, 11-fold to 22- fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22 -fold, 13-fold to 14-fold. 13- fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21 -fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22- fold, 20-fold to 22 -fold, or 21 -fold to 22-fold higher than an isotype control.Embodiment 21. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1 -20, wherein the anti-TNFR2 antibody or antigenbinding fragment thereof activates TNFR2-mediated NFKB signaling, wherein the NFKB signaling activation is characterized as an increase of RelA phosphorylation (pRelA) in primary human Tregs, and wherein the Tregs are defined as optionally CD3+CD4+CD25+FOXP3+.Embodiment 22. The anti-TNFR2 antibody or antigen-binding fragment thereof according to the combined or separate embodiments 1-21, wherein the RelA phosphorylation induced and / or increased by said anti-TNFR2 has a highest activity of at least: a) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4- fold, 4.25-fold, 4.5-fold, 4.75-fold. 5-fold, 5.25-fold, 5.5-fold. 5.75-fold, 6-fold, 6.25- fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold. 7.5-fold, 7.75-fold. 8-fold. 8.25-fold, 8.5- fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold,10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold,12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold,14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold,16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold,18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold,20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than an isotype control; and b) about 1.1-fold to 1.2-fold. 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold. 1.1 -fold to 5 -fold,1.1 -fold to 10-fold, 1.1 -fold to 15-fold, 1.1 -fold to 20-fold, 1.1 -fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold. 4-fold to 22-fold, 5-fold to 8-fold. 5-fold to 12-fold, 5-fold to ...

Claims

WHAT IS CLAIMED IS:

1. An anti-tumor necrosis factor receptor 2 (TNFR2) antibody or antigen-binding fragment thereof which exhibits agonistic activity towards TNFR2.

2. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain and a light chain selected from the group consisting of: i. a heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain comprising the amino acid sequence of SEQ ID NO: 12; ii. a heavy chain having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 6 and a light chain having at least 80%. 85%. 90%. or 95% identity to the amino acid sequence of SEQ ID NO: 12, wherein the amino acid at position 228 of SEQ ID NO: 6 is proline; iii. a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 24; and iv. a heavy chain having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 18 and a light chain having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 24, wherein the amino acids at positions 234 and 235 of SEQ ID NO: 18 are both alanine and the amino acid at position 329 of SEQ ID NO: 18 is glycine.

3. The anti -tumor necrosis factor receptor 2 (TNFR2) antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain and a light chain variable region (VL) selected from the group consisting of: i. a heavy chain comprising the amino acid sequence of SEQ ID NO:6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; ii. a heavy chain having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 7, wherein the amino acid at position 228 of SEQ ID NO: 6 is proline; iii. a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19; and iv. a heavy chain having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having at least 80%,85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 19, wherein the amino acids positions 234 and 235 of SEQ ID NO: 18 are both alanine and the amino acid at position 329 of SEQ ID NO: 18 is glycine.

4. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 1, wherein said antibody or antigen-binding fragment comprises a set of three heavy chain variable region complementarity determining regions (CDRs), VHCDR1, VHCDR2, and VHCDR3, comprised within a heavy chain vanable region comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13, and a set of three light chain variable region complementarity determining regions, VLCDR1, VLCDR2 and VLCDR3, comprised within a light chain variable region comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 19.

5. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 1, wherein said antibody or antigen-binding fragment comprises a set of three heavy chain variable region complementarity determining regions (CDRs) on a heavy chain, VHCDR1, VHCDR2, and VHCDR3, and a set of three light chain variable region complementarity determining regions on a light chain, VLCDR1. VLCDR2 and VLCDR3, wherein the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3, collectively, are selected from: i. the VHCDR1 of SEQ ID NO: 2, the VHCDR2 of SEQ ID NO: 3, the VHCDR3 of SEQ ID NO:

4. the VLCDR1 of SEQ ID NO:

8. the VLCDR2 of SEQ ID NO: 9 and the VLCDR3 of SEQ ID NO: 10; ii. the VHCDR1 of SEQ ID NO: 14, the VHCDR2 of SEQ ID NO: 15, the VHCDR3 of SEQ ID NO: 16, the VLCDR1 of SEQ ID NO: 20, the VLCDR2 of SEQ ID NO: 21 and the VLCDR3 of SEQ ID NO: 22; iii. the VHCDR1 of SEQ ID NO: 2 comprising 0-2 amino acid substitutions, the VHCDR2 of SEQ ID NO: 3 comprising 0-2 amino acid substitutions, the VHCDR3 of SEQ ID NO: 4 comprising 0-2 amino acid substitutions, the VLCDR1 of SEQ ID NO: 8 comprising 0-2 amino acid substitutions, the VLCDR2 of SEQ ID NO: 9 compnsing 0-2 amino acid substitutions, and the VLCDR3 of SEQ ID NO: 10 comprising 0-2 amino acid substitutions; and iv. the VHCDR1 of SEQ ID NO: 14 comprising 0-2 amino acid substitutions, the VHCDR2 of SEQ ID NO: 15 comprising 0-2 amino acid substitutions, the VHCDR3of SEQ ID NO: 16 comprising 0-2 amino acid substitutions, the VLCDR1 of SEQ ID NO: 20 comprising 0-2 amino acid substitutions, the VLCDR2 of SEQ ID NO: 21 comprising 0-2 amino acid substitutions, and the VLCDR3 of SEQ ID NO: 22 comprising 0-2 amino acid substitutions.

6. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-5, comprising ahuman IgGl CHl-hinge-CH2-CH3 region, wherein said hinge region optionally comprises one. two, three, four or more mutations; and / or wherein said antibody or antigen-binding fragment is of the IgGl subclass.

7. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 6, wherein:(d) a proline at position 329 of the human IgGl Fc region is substituted with another amino acid, optionally wherein the proline at position 329 is substituted with a glycine (P329G); and / or(e) a leucine at position 234 of the human IgGl Fc region is substituted with another amino acid, optionally wherein the leucine at position 234 is substituted with an alanine (L234A); and / or(f) a leucine at position 235 of the human IgGl Fc region is substituted with another amino acid, optionally wherein the leucine at position 235 is substituted with an alanine (L235A).

8. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 7, comprising the amino acid substitutions: L234A and L235A of the human IgGl Fc region.

9. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 7, comprising the amino acid substitutions: P329G and / or L234A and L235A of the human IgGl Fc region.

10. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 6 to 9, wherein the proline at position 329 of the human IgGl Fc region is substituted with another amino acid.

11. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-5, comprising ahuman IgG4 CHl-hinge-CH2-CH3 region, wherein said hinge region optionally comprises one, two, three, four or more mutations; and / or wherein said antibody or antigen-binding fragment is of the IgG4 subclass.

12. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 11, wherein the serine at position 228 of the human IgG4 constant heavy region is substituted wi th another amino acid.

13. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 12, wherein the serine at position 228 is substituted with proline (S228P).

14. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-13, comprising a constant light region of a human kappa light chain.

15. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-14, wherein said antibody or antigen-binding fragment thereof is capable of selectively promoting the proliferation of a population of T regulatory (Treg) cells.

16. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-15, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof activates TNFR2-mediated nuclear factor kappa-B (NFKB) signaling.

17. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 16, wherein the NFKB signaling activation is characterized by an increase of alkaline phosphatase secretion level from an engineered reporter cell line.

18. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-17, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof activates TNFR2-mediated NFKB signaling, wherein the NFKB signaling activation is characterized as an increase of RelA phosphorylation (pRelA) in primary human Tregs, and wherein the Tregs are defined as optionally CD3+CD4+CD25+FOXP3+.

19. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 18, wherein the RelA phosphorylation induced and / or increased by said anti-TNFR2 has a highest activity of at least: a) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4- fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25- fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold. 8-fold, 8.25-fold, 8.5- fold, 8.75-fold. 9-fold. 9.25-fold. 9.5-fold. 9.75-fold, 10-fold. 10.25-fold. 10.5-fold,10.75-fold, 11 -fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold,12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold,14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold,16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold,18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold,20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than an isotype control; and b) about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1 -fold to 2.5 -fold, 1.1 -fold to 5 -fold, 1.1 -fold to 10-fold, 1.1 -fold to 15-fold, 1.1 -fold to 20-fold, 1.1 -fold to 22-fold, 2-fold to 2.5-fold. 2-fold to 5-fold, 2-fold to 7.5-fold. 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22- fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold. 7-fold to 18-fold, 7-fold to 22-fold, 8- fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12- fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11-fold to 14-fold, 11-fold to 18-fold, 11-fold to 22- fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold. 13- fold to 18-fold. 13-fold to 22-fold. 14-fold to 16-fold. 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21 -fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22- fold, 20-fold to 22 -fold, or 21 -fold to 22 -fold higher than an isotype control.

20. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 11-19, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is of the IgG4 subclass and has a S228P mutation, and further wherein the anti-TNFR2 antibody or antigen-binding fragment thereof has an enhanced ability to activate NFKB signaling in a larger fraction of Tregs as compared to IgGl Fc-based variants, and wherein the enhanced ability of NFKB signaling activation is characterized by an increase of RelA phosphorylation (pRelA) in primary human Tregs, and wherein the Tregs are optionally defined as CD3+CD4+CD25+FOXP3+.

21. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 20, wherein the RelA phosphorylation (pRelA) in primary' human Tregs induced and / or increased by said anti-TNFR2 antibody or antigen-binding fragment thereof comprising an IgG4 having a S228P mutation is at least:a) about 1.1-fold. 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold. 3.5-fold, 3.75-fold, 4- fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25- fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold, 8-fold, 8.25-fold, 8.5- fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold,10.75-fold, 11 -fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold,12.75-fold. 13-fold, 13.25-fold, 13.5-fold, 13.75-fold. 14-fold, 14.25-fold, 14.5-fold,14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold,16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold,18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold,20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than IgGl Fc- based variants; and b) about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1 -fold to 5-fold,1.1 -fold to 10-fold, 1.1 -fold to 15-fold, 1.1 -fold to 20-fold, 1.1 -fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold. 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold. 4-fold to 6-fold, 4-fold to 10-fold. 4-fold to 16-fold. 4-fold to 22- fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold. 7-fold to 18-fold, 7-fold to 22 -fold, 8- fold to 11-fold. 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12- fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold, 10-fold to 18-fold, 10-fold to 22-fold, 11-fold to 14-fold, 11-fold to 18-fold, 11-fold to 22- fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22 -fold, 13-fold to 14-fold, 13- fold to 18-fold. 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22- fold, 20-fold to 22-fold, or 21-fold to 22 -fold higher than IgGl Fc-based variants.

22. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 6-10 and 14-19, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof (i) comprises an IgGl Fc region having a P329G mutation, and (ii) has an enhanced ability to activate NFKB signaling of Tregs, wherein the enhanced ability ofNFKB signaling activation is characterized by an increase of RelA phosphorylation (pRelA) in primary human Tregs, and wherein the Tregs are defined as optionally CD3+CD4+CD25+FOXP3+.

23. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 22, wherein the RelA phosphorylation (pRelA) in primary human Tregs induced and / or increased by said anti-TNFR2 antibody or antigen-binding fragment thereof comprising an IgGl Fc region having a P329G mutation is at least: a) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4- fold, 4.25-fold, 4.5-fold, 4.75-fold. 5-fold, 5.25-fold, 5.5-fold. 5.75-fold, 6-fold, 6.25- fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold. 7.5-fold, 7.75-fold. 8-fold. 8.25-fold, 8.5- fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold,10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold,12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold,14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold,16.75-fold. 17-fold, 17.25-fold, 17.5-fold. 17.75-fold. 18-fold, 18.25-fold, 18.5-fold.18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold,20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold, as compared to a control without the substitution; and b) about 1.1-fold to 1.2-fold. 1.1-fold to 1.5-fold, 1.1 -fold to 2.5 -fold. 1.1 -fold to 5 -fold,1 .1 -fold to 10-fold, 1.1 -fold to 15-fold, 1.1 -fold to 20-fold, 1.1 -fold to 22-fold, 2-fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold. 4-fold to 22- fold, 5-fold to 8-fold. 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8- fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12- fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold. 10-fold to 18-fold, 10-fold to 22-fold, 11-fold to 14-fold, 11-fold to 18-fold, 11-fold to 22- fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22 -fold, 13-fold to 14-fold, 13- fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18-fold,16-fold to 20-fold, 16-fold to 22 -fold, 17-fold to 19-fold, 17-fold to 21 -fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22- fold, 20-fold to 22-fold, or 21 -fold to 22-fold higher, as compared to a control without the substitution.

24. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 11-21, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof comprising an IgG4 constant heavy region having a S228P mutation has an enhanced ability to activate NFKB signaling as compared to IgGl Fc-based variants, and wherein the enhanced ability to activate NFKB signaling is characterized by an increase of alkaline phosphatase secretion level from an engineered reporter cell line.

25. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1 -24, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof preferentially binds to Tregs over non-Treg cells and cytotoxic T cells, wherein the Tregs are defined as optionally CD3+CD4+CD25+FoxP3+.

26. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-25, wherein a maximum response resulting from the anti-TNFR2 antibody or antigen-binding fragment thereof binding to CD3+CD4+CD25+FoxP3+ Tregs cells is at least: c) about 10%, 20%, 30%, 40%, 50%. 60%. 70%. 80%. 90%. 100%, 125%, 150%, 175%, 200%, 225%. 250%, 275%, 300%. 325%, 350%, 375%. 400%, 425%, 450%. 475%, 500%, 525%, 550%, 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000% higher than the maximum response resulting from the binding to non-Treg cells or to cytotoxic T cells; and d) about 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%. 55% to 100%, 58% to 100%, 60% to 100%. 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%. 73% to 100%, 75% to 100%, 78% to 100%. 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, 100% to 400%, 110% to 400%, 120% to 400%, 130% to 400%. 140% to 400%, 150% to 400%, 160% to 400%, 170% to 400%, 180%to 400%, 190% to 400%. 100% to 500%, 110% to 500%, 120% to 500%, 130% to 500%, 140% to 500%, 150% to 500%. 160% to 500%, 170% to 500%, 180% to500%, 190% to 500%, 100% to 600%, 110% to 600%, 120% to 600%, 130% to600%, 140% to 600%, 150% to 600%, 160% to 600%, 170% to 600%, 180% to600%, 190% to 600%, 100% to 700%, 110% to 700%, 120% to 700%, 130% to700%, 140% to 700%, 150% to 700%. 160% to 700%, 170% to 700%, 180% to700%, 190% to 700%, 100% to 800%. 110% to 800%, 120% to 800%, 130% to800%, 140% to 800%, 150% to 800%, 160% to 800%, 170% to 800%, 180% to800%, 190% to 800%, 100% to 900%, 110% to 900%, 120% to 900%, 130% to900%, 140% to 900%, 150% to 900%, 160% to 900%, 170% to 900%, 180% to900%, 190% to 900% 110% to 1000%, 120% to 1000%, 130% to 1000%, 140% to 1000%, 150% to 1000%, 160% to 1000%, 170% to 1000%, 180% to 1000%, 190% to 1000%, 200% to 1000%, 250% to 1000%, 300% to 1000%, 350% to 1000%, 400% to 1000%, 450% to 1000%, 500% to 1000%, 600% to 1000%, 700% to 1000%, 800% to 1000%. 900% to 1000%. or 950% to 1000% higher than the maximum response resulting from the binding to non-Treg cells or to cytotoxic T cells.

27. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-26, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is characterized by a preferential binding to Tregs over non-Treg cells.

28. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 27, wherein the Tregs comprise CD4+ Tregs, CD4+CD25+FoxP3+ Tregs, FoxP3+ CD4+ Tregs, CD25+FoxP3+ Tregs, or a combination of any one or more of the foregoing.

29. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 28, wherein the FoxP3-expressing cells comprise a subpopulation of FoxP3 high-expressing cells and a subpopulation of FoxP3 low-expressing cells, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof exhibits a higher level of binding to the FoxP3 high-expressing cells than to the FoxP3 low-expressing cells.

30. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 25-29, wherein the non-Treg cells comprise conventional CD4+ T cells (CD4+ Tconv), conventional CD8+ T cells (CD8+ T conv), NK cells, monocytes and / or neutrophils, or a combination of any one or more of the foregoing.

1. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 25-30, wherein the preferential binding is measured by a percent of binding positive cells, wherein the percent of binding positive cells in Tregs is at least:(c) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4- fold, 4.25-fold, 4.5-fold, 4.75-fold. 5-fold, 5.25-fold, 5.5-fold. 5.75-fold, 6-fold, 6.25- fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold. 7.5-fold, 7.75-fold. 8-fold. 8.25-fold, 8.5- fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold,10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold,12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold,14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold,16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold,18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold,20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than the percent of binding positive cells to non-Treg cells, or(d) about or 1.1-fold to 1.2-fold. 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold. 1.1-fold to 5- fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1 -fold to 22-fold, 2- fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5- fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold. 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6- fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22- fold, 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold. 10-fold to 14-fold, 10- fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold,13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15 -fold to 22-fold, 16-fold to 18- fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold. 17- fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22 -fold, or 21 -fold to 22-fold higher than the percent of binding positive cells to non-Treg cells.

2. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 25-31, wherein the preferential binding is measured by a total median fluorescence intensity, wherein the total median fluorescence intensity of Tregs is at least: a) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4- fold, 4.25-fold, 4.5-fold, 4.75-fold. 5-fold, 5.25-fold, 5.5-fold. 5.75-fold, 6-fold, 6.25- fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold. 7.5-fold, 7.75-fold. 8-fold. 8.25-fold, 8.5- fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold,10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold,12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold,14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold,16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold,18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold,20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than the total median fluorescence intensity of non-Treg cells; and b) about or 1.1 -fold to 1.2-fold, 1.1-fold to 1.5-fold. 1.1 -fold to 2.5-fold, 1.1-fold to 5- fold, 1.1 -fold to 10-fold, 1.1 -fold to 15-fold, 1.1 -fold to 20-fold, 1.1 -fold to 22-fold, 2- fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5- fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold. 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6- fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22- fold, 8-fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold. 10-fold to 14-fold, 10- fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold,13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15 -fold to 22-fold, 16-fold to 18- fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold. 17- fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22 -fold, or 21 -fold to 22-fold higher than the total median fluorescence intensity' of non-Treg cells.

33. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-32, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is characterized by specific binding to TNFR2 over a tumor necrosis factor 1 (TNFR1) protein.

34. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 33, wherein the specific binding is measured by a signal-to-noise ratio (SNR) value indicating an interaction between the anti-TNFR2 antibody or antigen-binding fragment thereof and a binding protein, wherein the SNR value indicating the interaction of the anti-TNFR2 antibody or antigen-binding fragment thereof with a TNFR2 receptor is at least: a) about 1.1-fold. 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold. 3-fold, 3.25-fold. 3.5-fold, 3.75-fold, 4- fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25- fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold, 8-fold, 8.25-fold, 8.5- fold, 8.75-fold, 9-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold,10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold,12.75-fold. 13-fold, 13.25-fold, 13.5-fold. 13.75-fold. 14-fold, 14.25-fold, 14.5-fold.14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold,16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold,18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold,20.75-fold. 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than the SNR value indicating the interaction with TNFR1 ; and b) about or 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1-fold to 5- fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1-fold to 20-fold. 1.1 -fold to 22-fold, 2- fold to 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5- fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6- fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22- fold, 8-fold to 11-fold, 8-fold to 15-fold. 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold, 10- fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22-fold, 13-fold to 14-fold,13-fold to 18-fold, 13-fold to 22 -fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15 -fold to 22-fold, 16-fold to 18- fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17- fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22 -fold, or 21 -fold to 22-fold higher than the SNR value indicating the interaction with TNFR1.

35. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-34, wherein the TNFR2 receptor is TNFR isoform 1.

36. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-34, wherein the TNFR2 receptor is TNFR isoform 2.

37. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1-36, wherein the agonistic activity toward TNFR2 is characterized by an increase in expression of Treg functional markers associated with immunosuppressive ability, wherein the functional markers are selected from the group consisting of CD25, FoxP3, TIGIT, and Ki67, and combinations of any one or more of the foregoing.

38. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 37, wherein the increase of Treg functional marker expression induced and / or increased by said anti-TNFR2 antibody or antigen-binding fragment thereof is at least: a) about 1.1-fold. 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold. 3-fold, 3.25-fold. 3.5-fold. 3.75-fold, 4- fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25- fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold, 8-fold, 8.25-fold, 8.5- fold, 8.75-fold, 9-fold, 9.25-fold. 9.5-fold, 9.75-fold, 10-fold, 10.25-fold, 10.5-fold,10.75-fold, 11-fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold,12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold,14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold,16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold,18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold,20.75-fold. 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher as compared to an isotype control; and b) about 1.1-fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1-fold to 2.5-fold, 1.1 -fold to 5 -fold,1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1 -fold to 20-fold, 1.1-fold to 22-fold, 2-foldto 2.5-fold, 2-fold to 5-fold, 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5-fold, 2-fold to 20-fold. 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold. 4-fold to 22- fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6-fold to 22-fold, 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22-fold, 8- fold to 11-fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to 12- fold, 9-fold to 16-fold, 9-fold to 19-fold. 9-fold to 22-fold, 10-fold to 14-fold. 10-fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22- fold, 12-fold to 15-fold, 12-fold to 18-fold, 12-fold to 22 -fold, 13-fold to 14-fold, 13- fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold. 15-fold to 22-fold. 16-fold to 18-fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17-fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22- fold, 20-fold to 22 -fold, or 21 -fold to 22 -fold higher as compared to an isotype control.

39. The anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 11-21 , wherein the anti-TNFR2 antibody or antigen-binding fragment thereof comprising an IgG4 constant heavy region having a S228P mutation has an enhanced ability to induce and / or increase expression of Treg functional markers, as compared to IgGl Fc-based variants.

40. The anti-TNFR2 antibody or antigen-binding fragment thereof according to claim 39, wherein the expression of Treg functional markers induced and / or increased by the anti- TNFR2 antibody or antigen-binding fragment thereof comprising an IgG4 constant region having a S228P mutation are at least: a) about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4- fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6-fold, 6.25- fold, 6.5-fold, 6.75-fold, 7-fold, 7.25-fold, 7.5-fold, 7.75-fold. 8-fold, 8.25-fold, 8.5- fold, 8.75-fold. 9-fold. 9.25-fold. 9.5-fold. 9.75-fold, 10-fold, 10.25-fold. 10.5-fold,10.75-fold, 11 -fold, 11.25-fold, 11.5-fold, 11.75-fold, 12-fold, 12.25-fold, 12.5-fold,12.75-fold, 13-fold, 13.25-fold, 13.5-fold, 13.75-fold, 14-fold, 14.25-fold, 14.5-fold,14.75-fold, 15-fold, 15.25-fold, 15.5-fold, 15.75-fold, 16-fold, 16.25-fold, 16.5-fold,16.75-fold, 17-fold, 17.25-fold, 17.5-fold, 17.75-fold, 18-fold, 18.25-fold, 18.5-fold,18.75-fold, 19-fold, 19.25-fold, 19.5-fold, 19.75-fold, 20-fold, 20.25-fold, 20.5-fold,20.75-fold, 21-fold, 21.25-fold, 21.5-fold, 21.75-fold, or 22-fold higher than IgGl Fc- based variants; and b) about or 1.1 -fold to 1.2-fold, 1.1-fold to 1.5-fold, 1.1 -fold to 2.5-fold, 1.1-fold to 5- fold, 1.1-fold to 10-fold, 1.1-fold to 15-fold, 1.1-fold to 20-fold, 1.1 -fold to 22-fold, 2- fold to 2.5-fold, 2-fold to 5-fold. 2-fold to 7.5-fold, 2-fold to 10-fold, 2-fold to 12.5- fold, 2-fold to 20-fold, 4-fold to 6-fold, 4-fold to 10-fold, 4-fold to 16-fold, 4-fold to 22-fold, 5-fold to 8-fold, 5-fold to 12-fold, 5-fold to 14-fold, 5 fold to 18-fold, 5-fold to 22-fold, 6-fold to 8-fold, 6-fold to 10-fold, 6-fold to 14-fold, 6-fold to 18-fold, 6- fold to 22-fold. 7-fold to 10-fold, 7-fold to 14-fold, 7-fold to 18-fold, 7-fold to 22- fold, 8-fold to 11 -fold, 8-fold to 15-fold, 8-fold to 18-fold, 8-fold to 22-fold, 9-fold to12-fold, 9-fold to 16-fold, 9-fold to 19-fold, 9-fold to 22-fold, 10-fold to 14-fold, 10- fold to 18-fold, 10-fold to 22-fold, 11 -fold to 14-fold, 11 -fold to 18-fold, 11 -fold to 22-fold, 12-fold to 15-fold, 12-fold to 18-fold. 12-fold to 22-fold, 13-fold to 14-fold,13-fold to 18-fold, 13-fold to 22-fold, 14-fold to 16-fold, 14-fold to 18-fold, 14-fold to 22-fold, 15-fold to 18-fold, 15-fold to 20-fold, 15-fold to 22-fold, 16-fold to 18- fold, 16-fold to 20-fold, 16-fold to 22-fold, 17-fold to 19-fold, 17-fold to 21-fold, 17- fold to 22-fold, 18-fold to 20-fold, 18-fold to 22-fold, 19-fold to 20-fold, 19-fold to 22-fold, 20-fold to 22-fold, or 21-fold to 22-fold higher than IgGl Fc-based variants.

41. A composition comprising the anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1 -40 and a pharmaceutically acceptable carrier.

42. Use of the anti-TNFR2 antibody or antigen-binding fragment thereof according to any one of claims 1 -40 or the composition according to claim 41 for the treatment of an autoimmune disease.

43. The use according to claim 42, wherein said autoimmune disease is an autoimmune disease of the skin, lungs, gastrointestinal tract, central nervous system, endocrine system, or kidneys.

44. The use according to either claim 42 or claim 43, wherein said autoimmune disease is selected from the group consisting of acute febrile mucocutaneous lymph node syndrome, amyloidosis, asthma such as allergic asthma, autoimmune hepatitis, celiac disease, chronic obstructive pulmonary disease, Crohn’s disease, dermatitis, diabetes mellitus Wpe1. glomerulonephritis including rapidly progressive glomerulonephritis, graft versus host disease, immunoglobulin a vasculitis, an inflammatory bowel disease, interstitial cystitis, lupus erythematosus, lupus nephritis, membranous nephropathy, multiple sclerosis including relapsing remitting multiple sclerosis, myocarditis, neuromyelitis optica, optic neuritis, pancreatitis, pemphigus, psoriasis, rheumatoid arthritis, scleroderma, Sjogren’s syndrome, ulcerative colitis, uveitis, vasculitis, vitiligo including vitiligo-associated multiple autoimmune disease susceptibility 1.

45. The use according to any one of claims 42-44, wherein said autoimmune disease is selected from multiple sclerosis, vitiligo, and ulcerative colitis.

46. The use according to any one of claims 42-45, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is capable of inducing and / or enhancing the immunosuppressive ability of a population of Treg cells, (e.g, levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by at least: a) about 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%,0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%. 0.0008%, 0.0009%, 0.001%, 0.002%. 0.003%, 0.004%. 0.005%. 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%. 4.0%, 5.0%, 6.0%. 7.0%, 8.0%, 9.0%. 10.0%. 20.0%. 30.0%. 40.0%. 50.0%. 60.0%, 70.0%, 80.0%, 90.0%, or 100% as compared to a control or untreated subject; and b) about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%. 0.00005% to 0.009%, 0.000055% to 0.01%. 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%. 0.002% to 2%. 0.003% to 3%. 0.004% to 4%. 0.005% to 5%. 0.006% to 6%.0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%. 68% to 100%, 70% to 100%, 73% to 100%. 75% to 100%, 78% to 100%. 80% to 100%, 83% to 100%, 85% to 100%. 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subj ect.

47. The use according to any one of claims 42-46, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is capable of inducing and / or enhancing the proliferation of a population of Treg cells, (e.g., levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by at least: a) about 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%,0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%. 0.0008%, 0.0009%, 0.001%, 0.002%. 0.003%, 0.004%. 0.005%. 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%. 4.0%, 5.0%, 6.0%. 7.0%, 8.0%, 9.0%. 10.0%. 20.0%. 30.0%. 40.0%. 50.0%. 60.0%, 70.0%, 80.0%, 90.0%, or 100%, as compared to a control or untreated subject; and b) about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%. 0.00005% to 0.009%, 0.000055% to 0.01%. 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%. 0.002% to 2%. 0.003% to 3%. 0.004% to 4%. 0.005% to 5%. 0.006% to 6%.0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%. 68% to 100%, 70% to 100%, 73% to 100%. 75% to 100%, 78% to 100%. 80% to 100%, 83% to 100%, 85% to 100%. 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subj ect.

48. The use according to any one of claims 42-47, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is effective in suppressing, reducing, and / or decreasing antigen-induced skin inflammation, wherein the skin inflammation is suppressed and / or reduced and / or decreased by a) about 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%,0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%. 0.0008%, 0.0009%, 0.001%, 0.002%. 0.003%, 0.004%. 0.005%. 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%. 4.0%, 5.0%, 6.0%. 7.0%, 8.0%, 9.0%. 10.0%. 20.0%. 30.0%. 40.0%. 50.0%. 60.0%, 70.0%, 80.0%, 90.0%, or 100%, as compared to a control or untreated subject; and b) about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%. 0.00005% to 0.009%, 0.000055% to 0.01%. 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%, 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%. 0.002% to 2%. 0.003% to 3%. 0.004% to 4%. 0.005% to 5%. 0.006% to 6%.0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%. 68% to 100%, 70% to 100%, 73% to 100%. 75% to 100%, 78% to 100%. 80% to 100%, 83% to 100%, 85% to 100%. 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subj ect.

49. The use according to any one of claims 42-48, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is effective in increasing and / or enhancing survival rate of subjects with GvHD, wherein the survival rate is increased and / or enhanced by at least: a) about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%,200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, 525%. 550%, 575%, 600%. 625%, 650%, 675%. 700%, 725%, 750%. 775%, 800%, 825%. 850%. 875%, 900%. 925%, 950%, 975%. or 1000%. as compared to a control or untreated subject; and b) 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to100%, 25% to 100%. 28% to 100%, 30% to 100%, 33% to 100%. 35% to 100%, 38% to 100%, 40% to 100%, 43% to 100%. 45% to 100%, 48% to 100%, 50% to 100%. 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 1 0%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%. 88% to 100%, 90% to 100%, 93% to 100%. 95% to 100%, 98% to 100%. 100% to 400%, 110% to 400%, 120% to 400%. 130% to 400%, 140% to 400%, 150% to 400%, 160% to 400%, 170% to 400%, 180% to 400%, 190% to 400%, 100% to 500%, 110% to 500%, 120% to 500%, 130% to 500%, 140% to 500%, 150% to 500%, 160% to 500%, 170% to 500%, 180% to 500%, 190% to 500%, 100% to 600%. 110% to 600%, 120% to 600%, 130% to 600%, 140% to 600%, 150% to 600%. 160% to 600%, 170% to 600%, 180% to 600%, 190% to 600%, 100% to 700%, 110% to 700%, 120% to 700%, 130% to 700%, 140% to 700%, 150% to 700%, 160% to 700%, 170% to 700%, 180% to 700%, 190% to 700%, 100% to 800%, 110% to 800%, 120% to 800%, 130% to800%, 140% to 800%, 150% to 800%, 160% to 800%, 170% to 800%, 180% to800%, 190% to 800%, 100% to 900%. 110% to 900%, 120% to 900%, 130% to900%, 140% to 900%, 150% to 900%, 160% to 900%, 170% to 900%, 180% to900%, 190% to 900% 110% to 1000%, 120% to 1000%, 130% to 1000%, 140% to 1000%, 150% to 1000%, 160% to 1000%, 170% to 1000%, 180% to 1000%, 190% to 1000%. 200% to 1000%. 250% to 1000%. 300% to 1000%, 350% to 1000%, 400% to 1000%. 450% to 1000%. 500% to 1000%. 600% to 1000%. 700% to 1000%. 800% to 1000%, 900% to 1000%, or 950% to 1000%, as compared to a control or untreated subject.

50. A method of treating an autoimmune disease in a subject, comprising administering a composition according to claim 41 to a subject in need thereof.

51. The method of treatment according to claim 50, wherein said autoimmune disease is an autoimmune disease of the skin, lungs, gastrointestinal tract, central nervous system, endocrine system, or kidneys.

52. The method of treatment according to either claim 50 or claim 51, wherein said autoimmune disease is selected from the group consisting of acute febrile mucocutaneous lymph node syndrome, amyloidosis, asthma such as allergic asthma, autoimmune hepatitis, celiac disease, chronic obstructive pulmonary disease, Crohn's disease, dermatitis, diabetes mellitus type 1, glomerulonephritis including rapidly progressive glomerulonephritis, graft versus host disease, immunoglobulin a vasculitis, an inflammatory bowel disease, interstitial cystitis, lupus erythematosus, lupus nephritis, membranous nephropathy, multiple sclerosis including relapsing remitting multiple sclerosis, myocarditis, neuromyelitis optica, optic neuritis, pancreatitis, pemphigus, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, ulcerative colitis, uveitis, vasculitis, and vitiligo including vitiligo-associated multiple autoimmune disease susceptibility 1.

53. The method of treatment according to any one of claims 50-52, wherein said autoimmune disease is selected from multiple sclerosis, vitiligo, and ulcerative colitis.

54. The method of treatment according to any one of claims 50-53. wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is capable of inducing and / or enhancing the immunosuppressive ability7of a population of Treg cells, (e.g, levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by at least:a) about 0.00001%, 0.00002%, 0.00003%. 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%. 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%,0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%. 4.0%, 5.0%, 6.0%. 7.0%, 8.0%, 9.0%. 10.0%. 20.0%. 30.0%. 40.0%. 50.0%. 60.0%, 70.0%, 80.0%, 90.0%, or 100%, as compared to a control or untreated subject; and b) about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%. 0.00005% to 0.009%, 0.000055% to 0.01%. 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%. 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%. 0.002% to 2%. 0.003% to 3%. 0.004% to 4%. 0.005% to 5%. 0.006% to 6%. 0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to 100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%. 38% to 100%, 40% to 100%, 43% to 100%. 45% to 100%, 48% to 100%. 50% to 100%, 53% to 100%, 55% to 100%. 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subject.

55. The method of treatment according to any one of claims 50-54. wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is capable of inducing and / or enhancing the proliferation of a population of Treg cells, (e.g, levels of CD3+, CD4+, CD25+, and / or FOXP3+ T cells) by at least:a) about 0.00001, 0.00002%. 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%. 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%,0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%. 4.0%, 5.0%, 6.0%. 7.0%, 8.0%, 9.0%. 10.0%. 20.0%. 30.0%. 40.0%. 50.0%. 60.0%, 70.0%, 80.0%, 90.0%, or 100%, as compared to a control or untreated subject; and b) about 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%. 0.00005% to 0.009%, 0.000055% to 0.01%. 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%. 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%. 0.002% to 2%. 0.003% to 3%. 0.004% to 4%. 0.005% to 5%. 0.006% to 6%. 0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to 100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%. 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%. 50% to 100%, 53% to 100%, 55% to 100%. 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subject.

56. The method of treatment according to any one of claims 50-55. wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is effective in suppressing, reducing, and / or decreasing antigen-induced skin inflammation, wherein the skin inflammation is suppressed and / or reduced and / or decreased by at least:a) about 0.00001%, 0.00002%, 0.00003%. 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%. 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%,0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%. 4.0%, 5.0%, 6.0%. 7.0%, 8.0%, 9.0%. 10.0%. 20.0%. 30.0%. 40.0%. 50.0%. 60.0%, 70.0%, 80.0%, 90.0%, or 100%, as compared to a control or untreated subject; and b) 0.00001% to 0.001%, 0.000015% to 0.002%, 0.00002% to 0.003%, 0.000025% to 0.004%, 0.00003% to 0.005%, 0.000035% to 0.006%, 0.00004% to 0.007%, 0.000045% to 0.008%. 0.00005% to 0.009%, 0.000055% to 0.01%. 0.00006% to 0.02%, 0.000065% to 0.03%, 0.00007% to 0.04%, 0.000075% to 0.05%, 0.00008% to 0.06%, 0.000085% to 0.07%, 0.00009% to 0.08%, 0.000095% to 0.09%, 0.0001% to 0.1%, 0.0002% to 0.2%, 0.0003% to 0.3%, 0.0004% to 0.4%, 0.0005% to 0.5%, 0.0006% to 0.6%, 0.0007% to 0.7%. 0.0008% to 0.8%, 0.0009% to 0.9%, 0.001% to 1%. 0.002% to 2%. 0.003% to 3%. 0.004% to 4%. 0.005% to 5%. 0.006% to 6%. 0.007% to 7%, 0.008% to 8%, 0.009% to 9%, 0.01% to 10%, 0.02% to 20%, 0.03% to 30%, 0.04% to 40%, 0.05% to 50%, 0.06% to 60%, 0.07% to 70%, 0.08% to 80%, 0.09% to 90%, 0.1% to 100%, 0.2% to 100%, 0.3% to 100%, 0.4% to 100%, 0.5% to 100%, 0.6% to 100%, 0.7% to 100%, 0.8% to 100%, 0.9% to 100%, 1% to 100%, 3% to 100%, 5% to 100%, 7% to 100%, 10% to 100%, 13% to 100%, 15% to 100%, 18% to 100%, 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%, 30% to 100%, 33% to 100%, 35% to 100%. 38% to 100%, 40% to 100%, 43% to 100%. 45% to 100%, 48% to 100%. 50% to 100%, 53% to 100%, 55% to 100%. 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%, 73% to 100%, 75% to 100%, 78% to 100%, 80% to 100%, 83% to 100%, 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, or 98% to 100%, as compared to a control or untreated subject.

57. The method of treatment according to any one of claims 50-56. wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is effective in increasing survival rate of subjects with GvHD, wherein the survival rate is increased and / or enhanced by at least:a) about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%. 125%, 150%, 175%,200%, 225%. 250%, 275%, 300%. 325%, 350%, 375%. 400%, 425%, 450%. 475%, 500%, 525%, 550%, 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000%, as compared to a control or untreated subject; and b) about 10% to 100%. 13% to 100%, 15% to 100%, 18% to 100%. 20% to 100%, 23% to 100%, 25% to 100%, 28% to 100%. 30% to 100%, 33% to 100%, 35% to 100%. 38% to 100%, 40% to 100%, 43% to 100%, 45% to 100%, 48% to 100%, 50% to 100%, 53% to 100%, 55% to 100%, 58% to 100%, 60% to 100%, 63% to 100%, 65% to 100%, 68% to 100%, 70% to 100%. 73% to 100%, 75% to 100%, 78% to 100%. 80% to 100%, 83% to 100%. 85% to 100%, 88% to 100%, 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, 100% to 400%, 110% to 400%, 120% to 400%, 130% to 400%, 140% to 400%, 150% to 400%, 160% to 400%, 170% to 400%, 180% to 400%, 190% to 400%, 100% to 500%, 110% to 500%, 120% to 500%, 130% to 500%, 140% to 500%, 150% to 500%. 160% to 500%, 170% to 500%, 180% to500%, 190% to 500%. 100% to 600%. 110% to 600%, 120% to 600%. 130% to600%, 140% to 600%, 150% to 600%, 160% to 600%, 170% to 600%, 180% to600%, 190% to 600%, 100% to 700%, 110% to 700%, 120% to 700%, 130% to700%, 140% to 700%, 150% to 700%. 160% to 700%, 170% to 700%, 180% to700%, 190% to 700%, 100% to 800%. 110% to 800%, 120% to 800%, 130% to800%, 140% to 800%, 150% to 800%, 160% to 800%, 170% to 800%, 180% to800%, 190% to 800%, 100% to 900%, 110% to 900%, 120% to 900%, 130% to900%, 140% to 900%, 150% to 900%, 160% to 900%, 170% to 900%, 180% to900%, 190% to 900% 110% to 1000%, 120% to 1000%, 130% to 1000%, 140% to 1000%, 150% to 1000%, 160% to 1000%, 170% to 1000%, 180% to 1000%, 190% to 1000%, 200% to 1000%, 250% to 1000%, 300% to 1000%, 350% to 1000%, 400% to 1000%, 450% to 1000%, 500% to 1000%, 600% to 1000%, 700% to 1000%, 800% to 1000%. 900% to 1000%. or 950% to 1000%, as compared to a control or untreated subject.

58. A nucleic acid composition comprising: a) a first nucleic acid encoding a heavy chain variable region comprising a VHCDR1, VHCDR2, VHCDR3 and optionally a heavy chain constant region, of an anti-TNFR2antibody or antigen-binding fragment thereof according to any one of claims 2-40; and b) a second nucleic acid encoding a light chain variable region comprising a VLCDR1, VLCDR2 and VLCDR3, of an anti-TNFR2 antibody or antigen-binding fragment thereof according to any of claims 2-40.

59. An expression vector composition comprising: a) a first expression vector comprising said first nucleic acid of claim 58; and b) a second expression vector comprising said second nucleic acid of claim 58.

60. An expression vector composition comprising an expression vector comprising said first nucleic acid and said second nucleic acid of claim 58.

61. A host cell comprising the nucleic acid composition of claim 58.

62. A host cell comprising the expression vector composition of either claim 58 or claim 59.

63. A method of making an anti TNFR2 antibody or antigen-binding fragment thereof comprising: a) culturing the host cell of either claim 61 or claim 62 under conditions wherein the antibody or antigen-binding fragment thereof is expressed; and b) recovering the antibody or antigen-binding fragment thereof.

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