Anti-TNFR2 antigen-binding protein and uses thereof

Abstract

The present application provides antigen binding proteins (e.g., antibodies, such as single domain antibodies) that specifically bind to tumor necrosis factor receptor 2 (TNFR2). The application also provides fusion proteins and conjugates comprising the antigen binding proteins, polynucleotides and recombinant vectors encoding the antigen binding proteins, and host cells and methods for preparing the antigen binding proteins. The application further provides pharmaceutical compositions comprising the antigen binding proteins, and methods of treating diseases or disorders using the antigen binding proteins.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 437,877, filed January 9, 2023, and U.S. Provisional Patent Application No. 63 / 472,175, filed June 9, 2023, the disclosures of both of which are incorporated herein by reference in their entireties. SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which has been submitted electronically in XML format and is incorporated by reference in its entirety. This XML copy, created on January 5, 2024, has the filename 260525_000029_SL.xml and is 4,669,862 bytes in size. [Technical Field]

[0003] This application relates to antigen binding proteins (e.g., antibodies, such as single domain antibodies) that specifically bind to tumor necrosis factor receptor 2 (TNFR2), methods for their preparation, and uses thereof. [Background technology]

[0004] Regulatory T cells (Tregs) are a subset of T cells that play an important role in peripheral self-tolerance and autoimmunity prevention. Their potent immunosuppressive function makes them a potential target for the treatment of autoimmunity. Current strategies aimed at increasing or modulating Tregs in autoimmune patients are based on ex vivo expansion of Tregs before autologous transplantation. However, a major limitation of current strategies is their inability to stabilize the Treg phenotype to ensure long-lasting immunomodulation.

[0005] Tumor necrosis factor receptor 2 (TNFR2) signaling has been shown to induce proliferation, sustained suppressive function, and FOXP3 promoter demethylation in Tregs (Tseng et al., 2019). TNFR2 signaling also induces the expression of EZH2 (Urbano et al., 2018), a histone methyltransferase involved in suppressing effector transcriptome programs and stabilizing the Treg phenotype (DuPage et al., 2015). TNFR2 signaling, due to its role in Treg biology and FOXP3 promoter demethylation, can be leveraged to induce a stable immunosuppressive phenotype and enhance Treg function to benefit autoimmune diseases. Therefore, there is a need in the art for the development of therapeutic molecules that can effectively activate TNFR2 signaling. Summary of the Invention [Problem to be solved by the invention]

[0006] As noted in the "Background" section above, there is an unmet need in the art to develop therapeutic molecules that can effectively activate TNFR2 signaling. The present application provides compositions and methods to address this and other related needs. [Means for solving the problem]

[0007] In one aspect, the present disclosure provides an antigen binding protein that specifically binds to tumor necrosis factor receptor 2 (TNFR2), comprising: a)(Y / F)YQ(S / A)LS(T / S)(P / A)N(Y / F)GQ(V / T)F(SEQ ID NO: 60); b) AADSDL(S / R)TV(V / T)VGPHDY (SEQ ID NO: 61); c) AKDAG(S / G)WG(T / R)GPFG(Y / F)(E / D)YDY (SEQ ID NO: 62); d) AA(T / A)PSGKAY(T / S)Y (SEQ ID NO: 63); e) ATPGPY(T / S / M)YCAPYGSSWSRGYDY (SEQ ID NO: 64); f) ARV(R / G)G(T / S / A)PY(E / D)Y(N / G)Y (SEQ ID NO: 65); g) (T / A / V)A(S / A)PTGRAF(T / N / A)Y (SEQ ID NO: 66); h) AGSAFDF (SEQ ID NO: 42); i)S(V / M)(V / L)GRDM(M / V)TY(SEQ ID NO: 67); j) AVGDFEGELVLKGDY (SEQ ID NO: 4063); k)AAD(L / V)G(F / V / Y)LY(A / T / V)DYV(P / R)LH(M / T)HHFGS(SEQ ID NO: 4517); l)A(A / G)(T / A)(P / L)(S / T)GKAY(T / S)Y(SEQ ID NO: 4771) The present invention provides an antigen-binding protein comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of:

[0008] In some embodiments, the CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 3, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 4063, 4067, 4071, 4524, 4530, and 4727-4730.

[0009] In some embodiments, the antigen binding protein is: a) GSI(V / F)(R / S)(T / A)(N / D)(S / G / A) (SEQ ID NO: 68); b) GFT(F / L)DD(I / Y)A (SEQ ID NO: 69); c) GFTFS(S / R / G)YA (SEQ ID NO: 70); d) GRTFSDYG (SEQ ID NO: 16); e) G(L / F)TLDYYA (SEQ ID NO: 71); f) GF(T / N)FSMYS (SEQ ID NO: 72); g)GRTF(G / R / S)(N / S)(Y / L)(T / F)(SEQ ID NO: 73); h) GASLS RNA (SEQ ID NO: 40); i) GS(I / T)FRFPP (SEQ ID NO: 74); j) GFTLDDYA (SEQ ID NO: 4061); and k)G(F / V)(S / T)LD(D / Y)(H / Y)T(SEQ ID NO: 4519) and further comprising a CDR1 comprising an amino acid sequence selected from:

[0010] In some embodiments, the antigen binding protein comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 1, 5, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 4061, 4065, 4069, 4520, and 4719-4722.

[0011] In some embodiments, the antigen binding protein is: a) IRSDGF(T / I) (SEQ ID NO: 75); b) I(Y / F)SY(S / G)(S / P)NT (SEQ ID NO: 76); c) I(Y / S)(S / D)DGS(E / D)T (SEQ ID NO: 77); d) INWSN(G / A)RT (SEQ ID NO: 4699); e) I(S / N)(V / T)(S / G)DGST (SEQ ID NO: 78); f) IDT(R / G)GST (SEQ ID NO: 79); g)IR(W / R / Y)(T / P)G(G / L)(S / I)T(SEQ ID NO: 80); h) IYDDGET (SEQ ID NO: 41); i) LTSGGST (SEQ ID NO: 45); j) IFSYSSNT (SEQ ID NO: 4062); and k)I(N / S)SNDG(S / T)(T / V)(SEQ ID NO: 4518) and a CDR2 comprising an amino acid sequence selected from the group consisting of:

[0012] In some embodiments, the antigen binding protein comprises a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 2, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 4062, 4066, 4070, 4527, and 4723-4726.

[0013] In some embodiments, the antigen binding protein is: i) CDR1 comprising the amino acid sequence of SEQ ID NO: 68, CDR2 comprising the amino acid sequence of SEQ ID NO: 75, and CDR3 comprising the amino acid sequence of SEQ ID NO: 60; ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 69, CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR3 comprising the amino acid sequence of SEQ ID NO: 61; iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 70, CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and CDR3 comprising the amino acid sequence of SEQ ID NO: 62; iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 63; v) CDR1 comprising the amino acid sequence of SEQ ID NO: 71, CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and CDR3 comprising the amino acid sequence of SEQ ID NO: 64; vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 72, CDR2 comprising the amino acid sequence of SEQ ID NO: 79, and CDR3 comprising the amino acid sequence of SEQ ID NO: 65; vii) CDR1 comprising the amino acid sequence of SEQ ID NO: 73, CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and CDR3 comprising the amino acid sequence of SEQ ID NO: 66; viii) CDR1 comprising the amino acid sequence of SEQ ID NO: 40, CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or ix) CDR1 comprising the amino acid sequence of SEQ ID NO: 74, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR3 comprising the amino acid sequence of SEQ ID NO: 67; x) CDR1 comprising the amino acid sequence of SEQ ID NO: 4061, CDR2 comprising the amino acid sequence of SEQ ID NO: 4062, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4063; xi) CDR1 comprising the amino acid sequence of SEQ ID NO: 4519, CDR2 comprising the amino acid sequence of SEQ ID NO: 4518, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4517; or xii) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4771 Includes.

[0014] In some embodiments, the antigen binding protein is: a) CDR1 comprising the amino acid sequence of SEQ ID NO: 69, CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR3 comprising the amino acid sequence of SEQ ID NO: 61; b) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 63; c) CDR1 comprising the amino acid sequence of SEQ ID NO: 73, CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or d) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4771 Includes.

[0015] In some embodiments, the antigen binding protein is: i) CDR1 having the amino acid sequence GSI(V / F)(R / S)(A / T)(N / D)(G / A) (SEQ ID NO: 4700), CDR2 comprising the amino acid sequence IRSDGFT (SEQ ID NO: 2), and CDR3 comprising the amino acid sequence YYQ(S / A)LSSPNYGQ(V / T)F (SEQ ID NO: 4701); ii) a CDR1 having the amino acid sequence GFTFDDIA (SEQ ID NO: 8), a CDR2 comprising the amino acid sequence IYSYGPNT (SEQ ID NO: 9), and a CDR3 comprising the amino acid sequence AADSDLSTVV(V / T)GPHDY (SEQ ID NO: 4702); iii) a CDR1 having the amino acid sequence GFTFSRYA (SEQ ID NO: 12), a CDR2 comprising the amino acid sequence ISDDGSDT (SEQ ID NO: 13), and a CDR3 comprising the amino acid sequence AKDAGSWGTGPFGYEYDY (SEQ ID NO: 14); iv) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 16), CDR2 comprising the amino acid sequence INWSN(G / A)RT (SEQ ID NO: 4699), and CDR3 comprising the amino acid sequence AA(T / A)PSGKAYSY (SEQ ID NO: 4703); v) CDR1 having the amino acid sequence GLTLDYYA (SEQ ID NO: 20), CDR2 comprising the amino acid sequence ISTSDGST (SEQ ID NO: 21), and CDR3 comprising the amino acid sequence ATPGPYTYCAPYGSSWSRGYDY (SEQ ID NO: 22); vi) CDR1 having the amino acid sequence GF(T / N)FSMYS (SEQ ID NO: 72), CDR2 comprising the amino acid sequence IDT(R / G)GST (SEQ ID NO: 79), and CDR3 comprising the amino acid sequence ARV(G / R)G(T / A)PYEY(N / G)Y (SEQ ID NO: 4704); vii) CDR1 having the amino acid sequence GRTF(G / S)S(Y / L)(T / F) (SEQ ID NO: 4705), CDR2 comprising the amino acid sequence IR(W / R / Y)(T / P)G(G / L)(S / I)T (SEQ ID NO: 80), and CDR3 comprising the amino acid sequence (A / V)A(A / S)PTGRAF(T / N)Y (SEQ ID NO: 4707); viii) CDR1 having the amino acid sequence GASLSRNA (SEQ ID NO: 40), CDR2 comprising the amino acid sequence IYDDGET (SEQ ID NO: 41), and CDR3 comprising the amino acid sequence AGSAFDF (SEQ ID NO: 42); ix) CDR1 having the amino acid sequence GS(T / I)FRFPP (SEQ ID NO: 4708), CDR2 comprising the amino acid sequence LTSGGST (SEQ ID NO: 45), and CDR3 comprising the amino acid sequence SVLGRDM(M / V)TY (SEQ ID NO: 4706); x) CDR1 having the amino acid sequence GFTLDDYA (SEQ ID NO: 4061), CDR2 comprising the amino acid sequence IFSYSSNT (SEQ ID NO: 4062), and CDR3 comprising the amino acid sequence AVGDFEGELVLKGDY (SEQ ID NO: 4063); xi) a CDR1 having the amino acid sequence GFTLDYYT (SEQ ID NO: 4065), a CDR2 comprising the amino acid sequence ISSNDSVGSV (SEQ ID NO: 4066), and a CDR3 comprising the amino acid sequence AADLGYLYVDYVRLHTHHFGS (SEQ ID NO: 4067); or xii) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 16), CDR2 comprising the amino acid sequence INWSN(G / A)RT (SEQ ID NO: 4699), and CDR3 comprising the amino acid sequence A(A / G)(T / A)(P / L)(S / T)GKAY(T / S)Y (SEQ ID NO: 4771) Includes.

[0016] In some embodiments, the antigen binding protein is: a) CDR1 having the amino acid sequence GFTFDDIA (SEQ ID NO: 8), CDR2 comprising the amino acid sequence IYSYGPNT (SEQ ID NO: 9), and CDR3 comprising the amino acid sequence AADSDLSTVV(V / T)GPHDY (SEQ ID NO: 4702); b) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 16), CDR2 comprising the amino acid sequence INWSN(G / A)RT (SEQ ID NO: 4699), and CDR3 comprising the amino acid sequence AA(T / A)PSGKAYSY (SEQ ID NO: 4703); c) a CDR1 having the amino acid sequence GRTF(G / S)S(Y / L)(T / F) (SEQ ID NO: 4705), a CDR2 comprising the amino acid sequence IR(W / R / Y)(T / P)G(G / L)(S / I)T (SEQ ID NO: 80), and a CDR3 comprising the amino acid sequence (A / V)A(A / S)PTGRAF(T / N)Y (SEQ ID NO: 4707); or d) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 16), CDR2 comprising the amino acid sequence INWSN(G / A)RT (SEQ ID NO: 4699), and CDR3 comprising the amino acid sequence A(A / G)(T / A)(P / L)(S / T)GKAY(T / S)Y (SEQ ID NO: 4771). Includes.

[0017] In some embodiments, the antigen binding protein is: i) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3; ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 5, CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 6; iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10; iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 12, CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR3 comprising the amino acid sequence of SEQ ID NO: 14; v) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR3 comprising the amino acid sequence of SEQ ID NO: 22; vii) CDR1 comprising the amino acid sequence of SEQ ID NO: 24, CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR3 comprising the amino acid sequence of SEQ ID NO: 26; viii) CDR1 comprising the amino acid sequence of SEQ ID NO: 28, CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR3 comprising the amino acid sequence of SEQ ID NO: 30; ix) CDR1 comprising the amino acid sequence of SEQ ID NO: 32, CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR3 comprising the amino acid sequence of SEQ ID NO: 34; x) CDR1 comprising the amino acid sequence of SEQ ID NO: 36, CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR3 comprising the amino acid sequence of SEQ ID NO: 38; xi) CDR1 comprising the amino acid sequence of SEQ ID NO: 40, CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42; xii) CDR1 comprising the amino acid sequence of SEQ ID NO: 44, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR3 comprising the amino acid sequence of SEQ ID NO: 46; xiii) CDR1 comprising the amino acid sequence of SEQ ID NO: 4061, CDR2 comprising the amino acid sequence of SEQ ID NO: 4062, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4063; xiv) CDR1 comprising the amino acid sequence of SEQ ID NO: 4065, CDR2 comprising the amino acid sequence of SEQ ID NO: 4066, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4067; xv) CDR1 comprising the amino acid sequence of SEQ ID NO: 4069, CDR2 comprising the amino acid sequence of SEQ ID NO: 4070, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4071; xvi) CDR1 comprising the amino acid sequence of SEQ ID NO: 4520, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR3 comprising the amino acid sequence of SEQ ID NO: 46; xvii) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4524; xviii) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; xix) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4530; xx) CDR1 comprising the amino acid sequence of SEQ ID NO: 4719, CDR2 comprising the amino acid sequence of SEQ ID NO: 4723, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4727; xxi) CDR1 comprising the amino acid sequence of SEQ ID NO: 4720, CDR2 comprising the amino acid sequence of SEQ ID NO: 4724, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4728; xxii) CDR1 comprising the amino acid sequence of SEQ ID NO: 4721, CDR2 comprising the amino acid sequence of SEQ ID NO: 4725, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4729; or xxiii) CDR1 comprising the amino acid sequence of SEQ ID NO: 4722, CDR2 comprising the amino acid sequence of SEQ ID NO: 4726, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4730 Includes.

[0018] In some embodiments, the antigen binding protein is: a) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; b) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; c) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4530; d) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10; e) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4524; f) CDR1 comprising the amino acid sequence of SEQ ID NO: 4069, CDR2 comprising the amino acid sequence of SEQ ID NO: 4070, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4071; g) CDR1 comprising the amino acid sequence of SEQ ID NO: 4719, CDR2 comprising the amino acid sequence of SEQ ID NO: 4723, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4727; h) CDR1 comprising the amino acid sequence of SEQ ID NO: 4720, CDR2 comprising the amino acid sequence of SEQ ID NO: 4724, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4728; i) CDR1 comprising the amino acid sequence of SEQ ID NO: 4721, CDR2 comprising the amino acid sequence of SEQ ID NO: 4725, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4729; or j) CDR1 comprising the amino acid sequence of SEQ ID NO: 4722, CDR2 comprising the amino acid sequence of SEQ ID NO: 4726, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4730 Includes.

[0019] In some embodiments, the antigen binding protein is a single domain antibody. In some embodiments, the single domain antibody is a VHH, VNAR, or modified VH domain.

[0020] In some embodiments, the VHH is a Camelidae VHH. In some embodiments, the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, 4521, 93-640, 4079-4125, 2805-3363, 4359-4420, and 4605-4628, or a sequence having at least 75% identity thereto. In some embodiments, the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, and 4521, or a sequence having at least 75% identity thereto.

[0021] In some embodiments, the VHH is a humanized VHH. In some embodiments, the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 4076-4078, 4523, 4526, 4529, 4532, 4731-4734, 641-1127, and 4126-4172, or a sequence having at least 75% identity thereto. In some embodiments, the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 4076-4078, 4523, 4526, 4529, 4731-4734, and 4532, or a sequence having at least 75% identity thereto.

[0022] In some embodiments, the antigen binding protein has an agonist effect when bound to TNFR2.

[0023] In some embodiments, the antigen binding protein binds to human TNFR2. In some embodiments, the antigen binding protein is about 3 x 10 -7 K less than M D In some embodiments, the antigen binding protein binds to human TNFR2 at about 1 x 10 -10 ~5×10 -8 K of M DIt binds to human TNFR2.

[0024] In some embodiments, the antigen binding protein binds to cynomolgus monkey TNFR2. In some embodiments, the antigen binding protein binds to about 3 x 10 -7 K less than M D In some embodiments, the antigen binding protein binds to cynomolgus monkey TNFR2 at about 1 x 10 -9 ~2×10 -7 K of M D It binds to cynomolgus monkey TNFR2.

[0025] In some embodiments, the antigen binding protein binds to the same epitope or epitopes as antibody clone MR2-1. In some embodiments, the antigen binding protein does not bind to the same epitope or epitopes as antibody clone MR2-1.

[0026] In some embodiments, the antigen binding protein increases expression of one or more proteins selected from proteins in the NF-kB pathway, FOXP3, HELIOS, EZH2, HLA-DR, ICAM-1, OX-40, ICOS, and CCR8.

[0027] In some embodiments, the antigen binding protein comprises one or more modifications that reduce binding of said antigen binding protein by pre-existing antibodies found in human blood or serum.

[0028] In another aspect, a fusion protein that specifically binds to tumor necrosis factor receptor 2 (TNFR2) is provided, comprising one or more of the antigen binding proteins described herein.

[0029] In some embodiments, the fusion protein comprises two antigen binding proteins described herein. In some embodiments, the fusion protein comprises three antigen binding proteins described herein. In some embodiments, the fusion protein comprises four antigen binding proteins described herein. In some embodiments, the fusion protein comprises five antigen binding proteins described herein. In some embodiments, the fusion protein comprises six antigen binding proteins described herein.

[0030] In some embodiments of the fusion proteins described herein, one or more antigen binding proteins may bind to the same epitope on TNFR2, while in other embodiments, one or more antigen binding proteins may bind to different epitopes on TNFR2.

[0031] In some embodiments of the fusion proteins described herein, the one or more antigen binding proteins are one or more single domain antibodies. In some embodiments, the one or more single domain antibodies are one or more VHHs.

[0032] In some embodiments of the fusion proteins described herein, the fusion protein further comprises an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region, or a variant thereof.

[0033] In some embodiments, the immunoglobulin Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the human IgG1 Fc region comprises one or more mutations selected from L234A, L235A, G237A, D265A, N297A, and / or P329A (EU numbering). In some embodiments, the human IgG1 Fc region comprises: 1) L234A and L235A; 2) L234A, L235A, and P329A; 3) D265A, N297A, and P329A; and 4) L234A, L235A, and G237A The set of mutations is selected from:

[0034] In some embodiments, the immunoglobulin Fc region is a human IgG1 Fc region comprising L234A, L235A, and P329A.

[0035] In some embodiments, the immunoglobulin Fc region is a human IgG4 Fc region or a variant thereof. In some embodiments, the human IgG4 Fc region comprises one or more mutations selected from S228P, L235E, L235A, and / or F234A (EU numbering). In some embodiments, the human IgG4 Fc region comprises: 1) S228P and L235E; 2) S228P, and L235A; 3) S228P, F234A, and L235E; and 4) S228P, F234A, and L235A The set of mutations is selected from:

[0036] In some embodiments, the immunoglobulin Fc region is a human IgG4 Fc region comprising S228P and L235E.

[0037] In some embodiments of the fusion proteins described herein, the fusion protein further comprises a cytokine. In some embodiments, the cytokine is IL-2 or a variant thereof. In one embodiment, the cytokine is an IL-2 variant comprising an N88D mutation.

[0038] In some embodiments of the fusion proteins described herein, the fusion protein further comprises a moiety that binds to serum albumin.

[0039] In some embodiments of the fusion proteins described herein, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 3933-3964, 4483-4513, 4686-4696, 4709-4716, and 4735-4770, or a sequence having at least 75% identity thereto.

[0040] In some embodiments of the fusion proteins described herein, the fusion protein comprises the amino acid sequence of SEQ ID NO: 4483, or a sequence having at least 75% identity thereto.

[0041] In some embodiments of the fusion proteins described herein, the fusion protein comprises the amino acid sequence of SEQ ID NO: 4489, or a sequence having at least 75% identity thereto.

[0042] In another aspect, there is provided a conjugate comprising an antigen binding protein described herein or a fusion protein described herein, wherein the antigen binding protein or fusion protein is conjugated to a second moiety. In some embodiments, the second moiety is selected from a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a chemotherapeutic agent, a diagnostic agent, or a combination thereof.

[0043] In some embodiments of the conjugates described herein, the second moiety is a cytokine. In some embodiments, the cytokine is IL-2 or a variant thereof. In one embodiment, the cytokine is an IL-2 variant comprising an N88D mutation.

[0044] In another aspect, there is provided a polynucleotide molecule encoding an antigen binding protein described herein or a fusion protein described herein. In some embodiments, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 48-59, 4073-4075, 4522, 4525, 4528, 4531, 3364-3922, 4421-4482, and 4629-4652, or a sequence having at least 70% identity thereto. In some embodiments, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 48-59, 4073-4075, 4522, 4525, 4528, and 4531, or a sequence having at least 70% identity thereto.

[0045] In another aspect, there is provided a recombinant vector comprising a polynucleotide molecule described herein.

[0046] In another aspect, there is provided a host cell comprising a polynucleotide molecule described herein or an expression vector described herein.

[0047] In another aspect, a kit is provided comprising an antigen binding protein, fusion protein, conjugate, polynucleotide molecule, or recombinant vector described herein, and optionally instructions and / or packaging for said kit.

[0048] In another aspect, there is provided a pharmaceutical composition comprising an antigen binding protein, fusion protein, conjugate, polynucleotide molecule, or recombinant vector described herein and a pharmaceutically acceptable carrier and / or excipient.

[0049] In another aspect, there is provided a method for preparing an antigen binding protein or fusion protein that specifically binds to tumor necrosis factor receptor 2 (TNFR2), comprising the steps of: (a) culturing a host cell described herein in a culture medium under conditions suitable for expression of the antigen binding protein or fusion protein; and (b) isolating the antigen binding protein or fusion protein from the host cell and / or culture medium. A method is provided, comprising:

[0050] In another aspect, there is provided a method of promoting the expansion, activating and / or enhancing the suppressive function, and / or stabilizing the immunosuppressive phenotype of a population of regulatory T cells (Tregs), comprising contacting the population of regulatory T cells with an antigen binding protein, fusion protein, or conjugate described herein. In some embodiments, the contacting step is performed in vitro. In some embodiments, the contacting step is performed in vivo. In some embodiments where the method is performed in vivo, the method further comprises administering the antigen binding protein, fusion protein, or conjugate to a subject in need thereof.

[0051] In another aspect, methods of treating or preventing a disease or disorder in a subject in need thereof are provided, the method comprising administering to the subject an antigen binding protein, fusion protein, or conjugate described herein. In some embodiments, the disease or disorder is an immune disease, an inflammatory disease, cancer, a cardiovascular disease, or an infertility and pregnancy related disorder.

[0052] In some embodiments, the immune disorder is selected from an autoimmune disease, a neurological condition, an allergy, asthma, macular degeneration, muscle atrophy, a miscarriage-related disorder, atherosclerosis, bone loss, a musculoskeletal disorder, obesity, graft-versus-host disease, and allograft rejection.

[0053] In some embodiments, the autoimmune disease is lupus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia / fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lichen sclerosus, IgG4-related disease, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disorder, pemphigus vulgaris or related bullous skin diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis / dermatomyositis, premature ovarian failure, primary agammaglobulinemia, primary biliary cholangitis, psoriasis, primary ovarian insufficiency, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthritis, stiff-man syndrome syndrome), type 1 diabetes mellitus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (granulomatosis with polyangiitis) or other immune-mediated vasculitis.

[0054] In some embodiments, the lupus is systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, neonatal lupus, or drug-induced lupus. In some embodiments, the cutaneous lupus is acute cutaneous lupus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus (DLE), or subacute cutaneous lupus erythematosus.

[0055] In some embodiments, the autoimmune disease is atopic dermatitis, psoriasis, systemic lupus erythematosus, or arthritis.

[0056] In some embodiments, the neurological condition is selected from brain tumors, brain metastases, spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Parkinson's disease, and stroke.

[0057] In some embodiments, the allergy is selected from food allergies, seasonal allergies, pet allergies, hives, hay fever, allergic conjunctivitis, poison ivy allergies, oak allergies, mold allergies, drug allergies, dust allergies, cosmetic allergies, and chemical allergies.

[0058] In some embodiments, the allograft rejection is selected from skin graft rejection, bone graft rejection, vascularized tissue graft rejection, ligament graft rejection, and organ graft rejection.

[0059] In some embodiments, the ligament graft rejection is selected from cricothyroid ligament graft rejection, posterior cruciate ligament graft rejection, periodontal ligament graft rejection, lens suspensory ligament graft rejection, volar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, elbow medial collateral ligament graft rejection, elbow lateral collateral ligament graft rejection, mammary suspensory ligament graft rejection, anterior sacroiliac ligament graft rejection, posterior sacroiliac ligament graft rejection, sacrotuberous ligament graft rejection, sacrospinous ligament graft rejection, inferior pubic ligament graft rejection, superior pubic ligament graft rejection, anterior cruciate ligament graft rejection, lateral collateral ligament graft rejection, posterior cruciate ligament graft rejection, medial collateral ligament graft rejection, canine anterior cruciate ligament graft rejection, and patellar ligament graft rejection.

[0060] In some embodiments, the organ transplant rejection is selected from heart transplant rejection, lung transplant rejection, kidney transplant rejection, liver transplant rejection, pancreas transplant rejection, intestinal transplant rejection, and thymus transplant rejection.

[0061] In some embodiments, the graft-versus-host disease arises from bone marrow transplantation or from one or more blood cells selected from B cells, T cells, basophils, common myeloid progenitor cells, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.

[0062] In some embodiments, the inflammatory disease is acute or chronic inflammation.

[0063] In some embodiments, the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovary syndrome, inflammatory bowel disease, fibrotic lung disease (or pulmonary fibrosis), and cardiac inflammation.

[0064] In some embodiments, the cancer is selected from the group consisting of adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (bone sarcoma), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing's sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell carcinoma, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal cell carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia and hairy cell leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CLL), leukemia (CML), chronic T-cell lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromesyndrome (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumors of the gastrointestinal tract, neuroendocrine tumors of the lung, neuroendocrine tumors of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian, fallopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary tumors, Selected from pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, soft tissue tumor, skin cancer (non-melanoma), small intestine cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, uterine cancer, vaginal cancer, von Hippel-Lindau disease, vulvar cancer, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), Werner's syndrome, Wilms' tumor, or xeroderma pigmentosum.

[0065] In some embodiments, the cardiovascular disease is selected from atherosclerosis, heart failure, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, right ventricular failure, congestive heart failure, restrictive cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, idiopathic cardiomyopathy, and hypertension.

[0066] In some embodiments, the infertility and pregnancy-related disorder is selected from recurrent pregnancy loss, preeclampsia, preterm labor, fetal growth restriction, or intrauterine growth restriction.

[0067] In another aspect, methods of regenerating a tissue or organ comprising one or more TNFR2+ cells are provided, the method comprising contacting the tissue or organ with an effective amount of an antigen binding protein, fusion protein, or conjugate described herein. In some embodiments, the tissue or organ is selected from pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerve, eye, thymus, tongue, bone, liver, small intestine, large intestine, gastrointestinal tract, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structure, embryo, and testicular tissue. In some embodiments, the contacting step is performed in vitro. In some embodiments, the contacting step is performed in vivo. In some embodiments where the method is performed in vivo, the method further comprises administering the antigen binding protein, fusion protein, or conjugate to a subject in need thereof.

[0068] In various embodiments of the above-described methods, the subject is a mammal. In some embodiments, the mammal is a human.

[0069] In another aspect, there is provided a method for inducing tolerance to and / or preventing or reducing an immune response to a foreign substance in a subject in need thereof, the method comprising administering to the subject an antigen binding protein, fusion protein, or conjugate described herein.

[0070] In some embodiments, the exogenous substance is a therapeutic protein, peptide, vector, biochemical vector, lipid, carbohydrate, nucleic acid, sperm, oocyte, or embryo. In some embodiments, the vector is a viral vector, bacterial vector, or fungal vector. In some embodiments, the viral vector is a DNA or RNA vector. [Brief explanation of the drawings]

[0071] [Figure 1]An example of a general panning strategy for isolating tumor necrosis factor (TNF) receptor type 2 (TNFR2)-specific heavy chain variable domain (VHH) antibodies, also referred to herein as V-bodies (Vb), is shown. Binders against human and rodent TNFR2 were enriched from a VHH immune library by two rounds of phage display. BM stands for bone marrow. [Figure 2] Selection of VHH immune libraries for next-generation sequencing (NGS) throughout the phage display process is shown. Three initial libraries, 12 samples from the first round of panning, and 36 samples from the second round of panning were sequenced at 20 million, 2 million, and 2 million reads, respectively. Comparing the enrichment of V-bodies from the initial libraries to the first and second rounds of panning allowed us to identify potential V-body candidates. [Figure 3] A schematic diagram of an exemplary NGS workflow is shown. Following phage display, the VHH regions of the phage eluate were amplified by polymerase chain reaction (PCR). Next, unique and sample-specific barcodes were fused (barcode fusion) before NGS was performed using the Illumina NovaSeq platform (Genewiz). Raw data were demultiplexed and then processed through the NGS analysis pipeline. Forward and reverse sequence pairs were merged via overlapping regions, and VHHs containing complementarity-determining regions (CDRs) were annotated. Clustering of V-body sequences based on CDR3 identity enabled detailed analysis of, for example, V-body enrichment during phage display, sequence diversity, CDR3 length distribution, and cluster abundance. Based on this analysis, over 600 candidates were selected for DNA synthesis (Twist) and further characterization. [Figure 4A]Figure 4 shows the binding validation of human TNFR2 (hTNFR2) V-bodies at a fixed V-body concentration of 1 μM. The bar graph (Figure 4A) shows the percentage of Alexa488-positive cells for all VHHs tested. In the bar graph, the black dotted line indicates background staining (approximately 5%), and the gray dotted line indicates twice the background level. V-bodies with a signal-to-noise ratio of >2 are considered "binders." [Figure 4B] Figure 4B shows the results of validation of human TNFR2 (hTNFR2) V-body binding at a fixed V-body concentration of 1 μM. The table (Figure 4B) shows the percentage of Alexa488-positive cells for all VHHs tested. Grey shading in the table indicates binders to hTNFR2. Italicized text in the table indicates various cell passage numbers and experimental dates. [Figure 5A] Figure 5A shows the results of validation of human TNFR2 (hTNFR2) V-body binding at a fixed V-body concentration of 100 nM. The bar graph (Figure 5A) shows the percentage of Alexa488-positive cells for all VHHs tested. In the bar graph, the black dotted line indicates background staining (approximately 5%), and the gray dotted line indicates twice the background level. V-bodies with a signal-to-noise ratio of >2 are considered "binders." [Figure 5B] Figure 5B shows the binding results of human TNFR2 (hTNFR2) V-bodies at a fixed V-body concentration of 100 nM. The table (Figure 5B) shows the percentage of Alexa488-positive cells for all VHHs tested. Gray shading in the table indicates binders to hTNFR2. [Figure 6A] Figure 6A shows the cross-specificity of V-body binding to mouse TNFR2 (mTNFR2) (Figure 6A) at a fixed concentration of 100 nM. The bar graph shows the percentage of Alexa488-positive cells for all VHHs tested. In the bar graph, the black dotted line indicates background staining, and the gray dotted line indicates twice the background level. [Figure 6B]Figure 6B shows the cross-specificity of V-body binding to cynomolgus monkey TNFR2 (cTNFR2) (Figure 6B) at a fixed concentration of 100 nM. The bar graph shows the percentage of Alexa488-positive cells for all VHHs tested. In the bar graph, the black dotted line indicates background staining, and the gray dotted line indicates twice the background level. [Figure 6C] Figure 6C shows the cross-specificity of V-body binding to mouse TNFR2 (mTNFR2) and cynomolgus monkey TNFR2 (cTNFR2) at a fixed concentration of 100 nM. The table (Figure 6C) shows the percentage of Alexa488 positive cells for all VHHs tested. [Figure 7] Figure 1 shows the results of binding studies of human TNFR2 V-bodies over a range of concentrations: V-bodies ODY-31D6, ODY-35A10, ODY-31G3, ODY-31G11, ODY-33D4, and ODY-37C7. V-bodies were tested at molar concentrations of 100 nM, 50 nM, 12.5 nM, 6.25 nM, 3.12 nM, and 1.55 nM. [Figure 8] Figure 1 shows a schematic diagram (left panel) of an exemplary experimental setup for determining the binding affinity of V-bodies to their respective targets by surface plasmon resonance (SPR) and a corresponding table (right panel) describing the V-body candidates analyzed. The figure discloses SEQ ID NO: 4717. [Figure 9A] Figure 1 shows surface plasmon resonance (SPR) sensorgrams of VHH binding to human, cynomolgus monkey, and mouse TNFR2, including fitted binding curves and calculated dissociation constants (KD). [Figure 9B] Figure 1 shows surface plasmon resonance (SPR) sensorgrams of VHH binding to human, cynomolgus monkey, and mouse TNFR2, including fitted binding curves and calculated dissociation constants (KD). [Figure 9C]Figure 1 shows surface plasmon resonance (SPR) sensorgrams of VHH binding to human, cynomolgus monkey, and mouse TNFR2, including fitted binding curves and calculated dissociation constants (KD). [Figure 9D] Figure 1 shows surface plasmon resonance (SPR) sensorgrams of VHH binding to human, cynomolgus monkey, and mouse TNFR2, including fitted binding curves and calculated dissociation constants (KD). [Figure 9E] Figure 1 shows surface plasmon resonance (SPR) sensorgrams of VHH binding to human, cynomolgus monkey, and mouse TNFR2, including fitted binding curves and calculated dissociation constants (KD). [Figure 9F] Figure 1 shows surface plasmon resonance (SPR) sensorgrams of VHH binding to human, cynomolgus monkey, and mouse TNFR2, including fitted binding curves and calculated dissociation constants (KD). [Figure 10] Figure 1 shows a summary of the binding affinities of 16 selected anti-TNFR2 V-bodies to human, cynomolgus monkey, and mouse TNFR2. Cyno stands for cynomolgus monkey. [Figure 11] These results indicate that some humanized anti-TNFR2 V-bodies target the epitope recognized by the MR2-1 bivalent agonist. N1365hu1 and N1409hu1 may recognize the same epitope as MR2-1. Binding of MR2-1 enhanced the binding of N1402hu1, N1425hu1, and N1277hu1 to TNFR2. [Figure 12A] Figure 12 shows TNFR2 agonism by multivalent V-body fusion constructs. The agonism of bivalent anti-TNFR2 constructs (Figure 12A) was characterized on NF-κB reporter HEK293 cells stably expressing TNFR2. Dot plots show the dose-dependent response of anti-TNFR2 VHHs compared to control VHHs (Ctrl). RLU stands for relative luminescence unit. [Figure 12B]Figure 12B shows TNFR2 agonism by multivalent V-body fusion constructs. The agonism of tetravalent (Figure 12B) anti-TNFR2 constructs was characterized on NF-κB reporter HEK293 cells stably expressing TNFR2. Dot plots show the dose-dependent response of anti-TNFR2 VHHs compared to control VHHs (Ctrl). RLU stands for relative luminescence unit. RLU stands for relative luminescence unit. [Figure 12C] Figure 12C shows TNFR2 agonism by multivalent V-body fusion constructs. The agonism of tetravalent (Figure 12C) anti-TNFR2 constructs was characterized on NF-κB reporter HEK293 cells stably expressing TNFR2. Dot plots show the dose-dependent response of anti-TNFR2 VHHs compared to control VHHs (Ctrl). RLU stands for relative luminescence unit. [Figure 12D] Figure 12 shows TNFR2 agonism by multivalent V-body fusion constructs. The agonism of IL-2 N88D-fused (Figure 12D) anti-TNFR2 constructs was characterized on NF-κB reporter HEK293 cells stably expressing TNFR2. Dot plots show the dose-dependent response of anti-TNFR2 VHHs compared to control VHHs (Ctrl). RLU stands for relative luminescence unit. [Figure 12E] Figure 12B shows TNFR2 agonism by multivalent V-body fusion constructs. Dot plots show the dose-dependent response of anti-TNFR2 VHHs compared to control VHHs (Ctrl). Figure 12C shows that the activity of WIL_33D4_2xVHH-Fc and IL-2 muteins was demonstrated in reporter cell lines specific for each signaling pathway. [Figure 13]HEK293 TNFR2 NF-κB (Luc) reporter gene assay control. The anti-hTNFR2 agonist MR2-1 monoclonal antibody was tested against the NF-κB reporter (Luc) HEK293 reporter cell line (clone 25) stably expressing TNFR2 and the parental cell line (PCL). RLU represents relative light units. [Figure 14A] HEK293 TNFR2 NF-κB(Luc) reporter gene assay samples and assay controls are shown. A description of the reporter gene assay samples and assay controls is shown in Figure 14A. [Figure 14B] HEK293 TNFR2 NF-κB(Luc) reporter gene assay samples and assay controls are shown. A bar graph showing the protein concentration (mg / mL) of V-body constructs and each control is shown in Figure 14B. [Figure 14C] HEK293 TNFR2 NF-κB(Luc) reporter gene assay samples and assay controls are shown. A bar graph showing the V-body constructs tested in PCL controls and the RLU for each control is shown in Figure 14C. [Figure 15A] Concentration range curve data generated using the MR2-1 control (Figure 15A) for four assay plates is shown. [Figure 15B] Concentration range curve data generated using a TNFα control (FIG. 15B) for four assay plates is shown. [Figure 16A] An exemplary dot plot of RLU measured with increasing concentrations (mol / L) of a control (Control 12) is shown. [Figure 16B] An exemplary dot plot of RLU measured at increasing concentrations (mol / L) of a tetravalent V-body fusion construct comprising four V-bodies mounted on the fragment crystallizable (Fc) region of an IgG4 variant containing S228P, L235E, and P329G mutations is shown. [Figure 16C]An exemplary dot plot of RLU measured at increasing concentrations (mol / L) of a tetravalent V-body fusion construct comprising four V-bodies mounted on the fragment crystallizable (Fc) region of an IgG4 variant containing S228P, L235E, and P329G mutations is shown. [Figure 17A] An exemplary dot plot of RLU measured with increasing concentrations (mol / L) of a control (Control 10) is shown. [Figure 17B] Figure 1 shows an exemplary dot plot of RLU measured at increasing concentrations (mol / L) of another design of a tetravalent V-body fusion construct comprising four V-bodies mounted on the Fc region of an IgG4 variant containing S228P, L235E, and P329G mutations. [Figure 17C] Figure 1 shows an exemplary dot plot of RLU measured at increasing concentrations (mol / L) of another design of a tetravalent V-body fusion construct comprising four V-bodies mounted on the Fc region of an IgG4 variant containing S228P, L235E, and P329G mutations. [Figure 17D] Figure 1 shows an exemplary dot plot of RLU measured at increasing concentrations (mol / L) of another design of a tetravalent V-body fusion construct comprising four V-bodies mounted on the Fc region of an IgG4 variant containing S228P, L235E, and P329G mutations. [Figure 17E] Figure 1 shows an exemplary dot plot of RLU measured at increasing concentrations (mol / L) of another design of a tetravalent V-body fusion construct comprising four V-bodies mounted on the Fc region of an IgG4 variant containing S228P, L235E, and P329G mutations. [Figure 17F] Figure 1 shows an exemplary dot plot of RLU measured at increasing concentrations (mol / L) of another design of a tetravalent V-body fusion construct comprising four V-bodies mounted on the Fc region of an IgG4 variant containing S228P, L235E, and P329G mutations. [Figure 18A]An exemplary dot plot of RLU measured at increasing concentrations (mol / L) of a control (Control 2) and a bivalent V-body fusion construct is shown. The limit of detection is expressed as LOD. [Figure 18B] An exemplary dot plot of RLU measured with increasing concentrations (mol / L) of bivalent V-body fusion constructs is shown. [Figure 18C] An exemplary dot plot of RLU measured with increasing concentrations (mol / L) of bivalent V-body fusion constructs is shown. [Figure 19A] 1 shows an exemplary dot plot of RLU measured with increasing concentrations (mol / L) of a control (Control 13) and an IL-2 N88D V-body fusion construct. The limit of detection is expressed as LOD. [Figure 19B] An exemplary dot plot of RLU measured with increasing concentrations (mol / L) of IL-2 N88D V-body fusion construct is shown. [Figure 19C] 1 shows an exemplary dot plot of RLU measured with increasing concentrations (mol / L) of IL-2 N88D V-body fusion construct. [Figure 20] Figure 1 shows a comparison of RLU measured at increasing concentrations (mol / L) of monospecific construct 10 (tetravalent Fc) and construct 12 (Vb-Fc-Vb) tested in the NF-κB reporter (Luc) HEK293 reporter cell line stably expressing TNFR2 (clone 8). [Figure 21] FIG. 1 shows an exemplary experimental timeline of TNFR2 stimulation with multivalent V-body fusion constructs (e.g., tetravalent Fc, Vb-Fc-Vb, rigid bivalent no Fc) in primary human peripheral blood mononuclear cells (PBMCs) and cluster of differentiation 4 positive (CD4+) CD25+ CD127dim regulatory T cells (Tregs). [Figure 22]A bar graph summarizing the in-assay concentrations (nM) of multivalent V-body fusion construct first wave conjugates is shown. The concentrations (nM) of VHH constructs are also shown. [Figure 23] An exemplary gating strategy applied to Treg markers is shown. Treg donor 1 is shown as an example, and the same strategy was used for Treg donors 2 and 3. Live cell and CD4 gating was performed based on determining the cutoff point between background fluorescence and the positive cell population using a fluorescence minus one (FMO) control. Gating for forkhead box P3 (FoxP3), human leukocyte antigen, DR isotype (HLA-DR), chemokine motif (CC motif) receptor 8 (CCR8), and OX-40 was performed based on the CD4 subset of an IgG control-stained sample from the same donor. For FoxP3, the gate was set at approximately 0.2%. For OX-40, HLA-DR, and CCR8, the gate was set at approximately 2%. [Figure 24A] Figure 24A shows that multivalent anti-TNFR2 V-body fusion constructs increased the expression of the Treg suppressive markers HLA-DR and CCR8. Histogram showing the expression of the Treg suppressive marker HLA-DR in specific 37C7 conjugates compared to the control format (Figure 24A). Fluorescein isothiocyanate is represented by FITC, and phycoerythrin is represented by PE. [Figure 24B] Figure 24B shows that multivalent anti-TNFR2 V-body fusion constructs increased the expression of Treg suppressive markers HLA-DR and CCR8. Density plot showing the expression of Treg suppressive markers HLA-DR and CCR8 in specific 37C7 binders compared to the control format (Figure 24B). Fluorescein isothiocyanate is represented by FITC, and phycoerythrin is represented by PE. [Figure 25A]Figure 1 shows that tetravalent anti-TNFR2 V-body fusion constructs significantly increased the expression of the Treg suppressive marker HLA-DR on FoxP3+ Tregs. The bar graph shows the HLA-DR mean fluorescent intensity (MFI) measured for each of the tetravalent Fc, Vb-Fc-Vb, and rigid bivalent Fc-less V-body fusion formats compared to the control format. [Figure 25B] Figure 1 shows that tetravalent anti-TNFR2 V-body fusion constructs significantly increased the expression of the Treg suppressive marker HLA-DR on FoxP3+ Tregs. The bar graph shows the HLA-DR mean fluorescent intensity (MFI) measured for each of the tetravalent Fc, Vb-Fc-Vb, and rigid bivalent Fc-less V-body fusion formats compared to the control format. [Figure 26] Dose-response curves based on HLA-DR MFI values ​​of CD4+FoxP3+ Tregs are shown for construct 37C7 from donor 2 and control 10. [Figure 27] Dose-dependent induction of expression of the Treg suppressive marker HLA-DR across various concentrations of tetravalent anti-TNFR2 V-body Fc fusion construct 10 from donor 1 (top panel) and donor 2 (bottom panel) is shown. [Figure 28] Dose-dependent induction of expression of the Treg suppressive marker HLA-DR across various concentrations of rigid bivalent anti-TNFR2 V-body fusion construct 2 from donor 1 (top panel) and donor 2 (bottom panel) is shown. [Figure 29] 1 shows the dose-dependent induction of expression of the Treg suppressive marker HLA-DR across various concentrations of tetravalent anti-TNFR2 V-body Vb-Fc-Vb fusion construct 12 from donor 1 (top panel) and donor 2 (bottom panel). [Figure 30]

[0023] Figure 1 shows an exemplary design of a multivalent anti-TNFR2 V-body fusion construct. Anti-TNFR2 V-bodies are shown as ellipses, flexible linkers (e.g., GS linkers) are shown as curved lines, rigid linkers (e.g., proline linkers) are shown as straight lines, and the Fc domain is shown as a dimeric bar. The figure discloses SEQ ID NO: 4718. [Figure 31] Figure 1 shows the evaluation of the activity of tetravalent-Fc VHHs against naive CD4+CD25+CD45RA+ human Tregs. HLA-DR and CCR8 expression in CD4+FOXP3+ cells and proliferation after 5 days of stimulation with anti-CD3 / IL-2 and VHHs or MR2-1 are shown. [Figure 32A] Figure 1 shows the ability of TNFR2 VHHs to stabilize Tregs. Naive CD4+CD25+CD45RA+ human Tregs derived from healthy donors were stimulated with IL-2 and anti-CD3 for 5 days in the presence of TNFR2 agonist VHHs or the TNFR2 monoclonal agonist MR2-1. [Figure 32B] Figure 1 shows the effect of TNFR2 VHHs on early markers of Treg stability. Naive CD4+CD25+CD45RA+ human Tregs derived from healthy donors were stimulated with IL-2 and anti-CD3 for 5 days in the presence of TNFR2 agonist VHHs or IL-2 muteins. [Figure 33A] Further results of in vitro treatment of human Tregs (CD4+FOXP3+) with VHH WIL_33D4_2xVHH-Fc or IL-2 muteins in the presence of anti-CD3 and IL-2 are shown. WIL_33D4_2xVHH-Fc induced and expanded a Treg population with elevated levels of FOXP3, EZH2 (a marker of stability), CCR8, and HLA-DR (a biomarker of tissue homing and Treg immunosuppressive function). [Figure 33B]Further results of in vitro treatment of human Tregs (CD4+FOXP3+) with VHH WIL_33D4_2xVHH-Fc or IL-2 muteins in the presence of anti-CD3 and IL-2 are shown. WIL_33D4_2xVHH-Fc induced and expanded a Treg population with elevated levels of FOXP3, EZH2 (a marker of stability), CCR8, and HLA-DR (a biomarker of tissue homing and Treg immunosuppressive function). T-test: *p<0.05; **p<0.01; n=3. [Figure 34A] The effect of TNFR2 VHH on Treg stability under inflammatory conditions is shown. Human Tregs were expanded with IL-2 muteins or TNFR2 VHHs in the presence of anti-CD3 and IL-2 for 5 days, then cultured with proinflammatory cytokines (IL-1b, IL-21, and IL-23 + / - TGFb) for 11–12 days. IL-17A or IFNγ production after PMA / ionomycin stimulation was assessed by flow cytometry with FOXP3. In vitro conversion of human Tregs to Th1 / 17 cytokine (IFNγ / IL-17A)-producing cells, triggered by the indicated inflammatory cytokines, was inhibited by costimulation with TNFR2 VHHs but not with IL-2 muteins. Fc = human IgG4 mutant Fc. [Figure 34B]The effect of TNFR2 VHH on Treg stability under inflammatory conditions is shown. Human Tregs were expanded with IL-2 muteins or TNFR2 VHHs in the presence of anti-CD3 and IL-2 for 5 days, then cultured with proinflammatory cytokines (IL-1b, IL-21, and IL-23 + / - TGFb) for 11–12 days; IL-17A or IFNγ production after PMA / ionomycin stimulation was assessed by flow cytometry with FOXP3. In vitro conversion of human Tregs to Th1 / 17 cytokine (IFNγ / IL-17A)-producing cells, triggered by the indicated inflammatory cytokines, was inhibited by costimulation with TNFR2 VHHs but not by costimulation with IL-2 muteins. Fc = human IgG4 mutant Fc. [Figure 34C] The effect of TNFR2 VHH on Treg stability under inflammatory conditions is shown. Human Tregs were expanded with IL-2 muteins or TNFR2 VHHs in the presence of anti-CD3 and IL-2 for 5 days, followed by culture with proinflammatory cytokines (IL-1b, IL-21, and IL-23 + / - TGFb) for 11–12 days. IL-17A or IFNγ production after PMA / ionomycin stimulation was assessed by flow cytometry with FOXP3. In vitro conversion of human Tregs to Th1 / 17 cytokine (IFNγ / IL-17A)-producing cells, triggered by the indicated inflammatory cytokines, was inhibited by costimulation with TNFR2 VHHs but not with IL-2 muteins. P values ​​indicate paired t-test results; * = p < 0.05; ** = p < 0.01; Fc = human IgG4 mutant Fc. [Figure 35]Figure 1 shows the evaluation of Treg function in response to TNFR2 agonism. Naive Tregs were stimulated for 7 days with anti-CD3 / IL-2+TNFR2 VHH (WIL_33D4_2xVHH-Fc), control VHH, MR2-1, control IgG, or IL-2 mutein. After 7 days, the stimulation was removed, and the cells were incubated with cell tracer-labeled autologous responder cells (naive CD4+ T cells). The bar graph shows the proliferation of effector CD4+ cells, measured as a percentage of dividing CD4+FOXP3- cells. The FACS histogram shows the dilution of cell tracer at various Treg:CD4 (responder) ratios for one of four donors. WIL_33D4_2xVHH-Fc induces Tregs that can better restrict the proliferation of effector CD4+ cells than IL-2 mutein. [Figure 36-1] Figure 1 shows the effect of TNFR2 agonist VHHs on the size of the Treg population in mice. An exemplary design of the experimental procedure is shown. [Figure 36-2] Figure 1 shows the effect of TNFR2 agonist VHH on the size of the Treg population in mice. Shown is the expansion of CD4+FOXP3+ Treg in the spleen of mice 5 days after a single injection of 2.5 mg / kg of control VHH or TNFR2-specific VHH WIL_33D4_2xVHH-Fc. [Figure 37]We demonstrate that TNFR2 agonist VHHs activate Tregs in vivo. CCR8 is a chemokine receptor expressed on strong suppressive Tregs and involved in cell migration (Whiteside et al., Immunol 2021;163:512). The ICAM-1 surface adhesion molecule is required for Treg function (Gottrand et al., Immunol 2015;146(4):657). The ICOS costimulatory molecule is upregulated upon Treg activation and maintains FOXP3 expression (Landuyt et al., J Immunol 2019;202(4):1039). The percentage of splenic Tregs (CD4+CD25+FOXP3+) expressing activation markers (CCR8, ICAM-1, or ICOS) 5 days after a single injection of control VHH or TNFR2-specific VHH WIL_33D4_2xVHH-Fc is shown. One-way ANOVA was performed between control VHH and WIL_33D4_2xVHH-Fc; only significant differences are shown; ****=p<0.0001. [Figure 38] Figure 2 shows that TNFR2 agonist VHH selectively expands Tregs in the spleen. Cell subsets are shown as the percentage of CD45+ cells in the spleen 5 days after a single injection of TNFR2-specific VHH WIL_33D4_2xVHH-Fc or control VHH. [Figure 39] TNFR2 agonist VHHs increase serum levels of IL-10. IL-10 is an important anti-inflammatory cytokine (Saraiva et al., J Exp Med 2020;217(1):e20190418). Serum cytokine concentrations are shown 5 days after a single injection of TNFR2-specific VHH WIL_33D4_2xVHH-Fc or control VHH. One-way ANOVA was performed between control VHH and WIL_33D4_2xVHH-Fc; only significant differences are shown; ****=p<0.0001. [Figure 40A]The frequency of Tregs (CD4+FOXP3+) among total immune cells (CD45+) in the spleen and blood of human TNFR2 knock-in mice 5 days after a single injection of ODY-520. T-test: ***p<0.001; ****p<0.0001; n=4. [Figure 40B] The frequency of Tregs (CD4+FOXP3+) among total immune cells (CD45+) in the colon and lung of human TNFR2 knock-in mice 5 days after a single injection of ODY-520. T-test: ***p<0.001; ****p<0.0001; n=4. [Figure 41A] Compared with IL-2N88D, an active mutein in mice, ODY-520 selectively increases the Treg population in the spleen 5 days after a single administration. WIL_33D4_2xVHH-Fc is highly selective for Tregs, inducing high levels of FOXP3 and surface markers (FOXP3, ICAM-1, OX-40, ICOS, and CCR8), indicating superior function and stability. One-way ANOVA: ****p<0.0001; n=4. [Figure 41B] Compared with IL-2N88D, an active mutein in mice, ODY-520 selectively increases the Treg population in the spleen 5 days after a single administration. WIL_33D4_2xVHH-Fc is highly selective for Tregs, inducing high levels of FOXP3 and surface markers (FOXP3, ICAM-1, OX-40, ICOS, and CCR8), indicating superior function and stability. One-way ANOVA: ****p<0.0001; n=4. [Figure 41C] Compared with IL-2N88D, an active mutein in mice, ODY-520 selectively increases the Treg population in the spleen 5 days after a single administration. WIL_33D4_2xVHH-Fc is highly selective for Tregs, inducing high levels of FOXP3 and surface markers (FOXP3, ICAM-1, OX-40, ICOS, and CCR8), indicating superior function and stability. One-way ANOVA: ****p<0.0001; n=4. [Figure 41D]Compared with IL-2N88D, an active mutein in mice, ODY-520 selectively increases the Treg population in the spleen 5 days after a single administration. WIL_33D4_2xVHH-Fc is highly selective for Tregs, inducing high levels of FOXP3 and surface markers (FOXP3, ICAM-1, OX-40, ICOS, and CCR8), indicating superior function and stability. One-way ANOVA: ****p<0.0001; n=4. [Figure 41E] Compared with IL-2N88D, an active mutein in mice, ODY-520 selectively increases the Treg population in the spleen 5 days after a single administration. WIL_33D4_2xVHH-Fc is highly selective for Tregs, inducing high levels of FOXP3 and surface markers (FOXP3, ICAM-1, OX-40, ICOS, and CCR8), indicating superior function and stability. One-way ANOVA: ****p<0.0001; n=4. [Figure 42A] We show that the expression of Tregs in the spleen and increased expression of FOXP3 were associated with Treg stability and function, and Treg activation as indicated by upregulation of ICAM-1 and ICOS. [Figure 42B] We show that the expression of Tregs in the spleen and increased expression of FOXP3 were associated with Treg stability and function, and Treg activation as indicated by upregulation of ICAM-1 and ICOS. [Figure 43A] 1 shows the reduction of arthritis as measured by paw volume and arthritis score upon treatment with TNFR2 agonists ODY-520 and ODY-781 in a collagen antibody-induced arthritis model. [Figure 43B] 1 shows the reduction of arthritis as measured by paw volume and arthritis score upon treatment with TNFR2 agonists ODY-520 and ODY-781 in a collagen antibody-induced arthritis model. [Figure 43C]1 shows the reduction of arthritis as measured by paw volume and arthritis score upon treatment with TNFR2 agonists ODY-520 and ODY-781 in a collagen antibody-induced arthritis model. [Figure 44A] Figure 2 shows that TNFR2 agonists expand Tregs without inducing proinflammatory cytokines. [Figure 44B] Figure 2 shows that TNFR2 agonists expand Tregs without inducing proinflammatory cytokines. DETAILED DESCRIPTION OF THE INVENTION

[0072] Regulatory T cells (Tregs) are a population of lymphocytes with immunosuppressive functions. Activation and expansion of Tregs is an attractive therapeutic approach for autoimmune diseases that is currently being clinically evaluated. In addition to inducing Treg activation and proliferation, effective Treg-targeted therapy requires generating cells with a stable immunosuppressive phenotype that are resistant to conversion to T effector function under inflammatory conditions. Nevertheless, current clinical approaches to stimulating Tregs to treat autoimmunity expand Tregs by increasing their homeostatic proliferation (e.g., with IL-2 muteins) without improving Treg stability. Although clinical trials testing IL-2-based approaches to treat autoimmune diseases by enhancing Treg function have demonstrated safety and the ability to expand Tregs across multiple diseases, Treg specificity, Treg stability, and therapeutic efficacy have been suboptimal (PNAS 2010;107(45):19402; clinicaltrials.gov / study / NCT03943550; clinicaltrials.gov / study / NCT04433585). Optimal Treg therapy requires (1) expanding the size of the Treg population with cells that (2) migrate to disease-affected tissues and (3) exert immunosuppressive effects while (4) limiting their transformation into proinflammatory Th1 / 17 cells. Therefore, improved therapeutic approaches to promote and stabilize the immunosuppressive activity of Tregs are needed.

[0073] TNFR2 agonism is an alternative approach to enhancing Treg function, which is expected to address all objectives required for optimal therapy, including the generation of stable Treg cells that limit Th1 / 17 conversion (Front Immunol 2022;13:888274; Sci Rep 2023;13(1):13762; PNAS 2019;116(43):21666; J Immunol 2013;190:1076; Arthritis Rheumatol 2020;72(4):576). In one aspect, the present invention provides TNFR2 agonists for enhancing the immunosuppressive activity of Tregs using a single-domain antibody (e.g., VHH) platform, as described herein.

[0074] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For purposes of interpretation of this specification, the following explanations of terms shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety into this application. In the event that any explanation of a term provided conflicts with any document incorporated by reference into this application, the explanation of the term provided below shall prevail.

[0075] As used herein, the term "about," when used in connection with a particular numerical value, means that the value can vary by up to 5% from the stated value. For example, as used herein, the expression "about 100" includes 95 and 105, and all values ​​therebetween (e.g., 96, 97, 98, 99, etc.).

[0076] The term "antigen" encompasses any agent (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portion thereof, or combination thereof) to which a particular product of humoral or cellular immunity, such as an antibody molecule or a T-cell receptor, can specifically bind. In various embodiments of the present disclosure, the antigen described herein is TNFR2, including human, cynomolgus monkey, and / or mouse TNFR2.

[0077] The term "epitope" refers to an antigenic determinant on the surface of an antigen to which an antibody molecule binds. A single antigen may have two or more epitopes. Thus, different antibodies may bind to different regions of an antigen, resulting in different biological effects (e.g., agonist or antagonist effects). Epitopes may be structural or linear. A structural epitope is formed by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues within a polypeptide chain. In some cases, non-peptide moieties on the antigen, such as sugars, phosphoryl groups, or sulfonyl groups, may be included.

[0078] The term "antigen-binding protein," in its broadest sense, refers to a protein that specifically binds to an antigen (e.g., TNFR2). In certain embodiments, the antigen-binding protein is an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody, a camelid antibody, a chimeric antibody, a recombinant antibody, a heavy-chain antibody, a single-domain antibody (e.g., VHH), a single-chain antibody (e.g., single-chain fragment variable (scFv)), a diabody, a triabody, a tetrabody, a Fab fragment, a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody, and fragments thereof. The term "antigen-binding protein" also encompasses alternative protein scaffolds or artificial scaffolds, e.g., onto which CDRs or CDR derivatives are grafted. Such scaffolds include, but are not limited to, antibody-derived scaffolds, e.g., containing mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic scaffolds, e.g., comprising biocompatible polymers. Additionally, peptide antibody mimics and antibody mimic-based scaffolds utilizing fibronectin components (eg, fibronectin type III domain (FN3)) as scaffolds can also be used.

[0079] The terms "antibody" and "immunoglobulin" or "Ig" are used interchangeably and are used in their broadest sense to encompass individual monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), single domain antibodies (e.g., VHHs), single-chain antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of antibodies, e.g., as described below. Antibodies may be human, humanized, camelized, recombinantly produced, chimeric, synthetic, affinity-de-matured, and / or affinity-matured antibodies, as well as antibodies from other species, e.g., mouse, camel, llama, rabbit, etc. In specific embodiments, the specific target antigens to which the antibodies provided herein can bind include TNFR2 polypeptides, TNFR2 fragments, or TNFR2 epitopes. An "antigen-binding fragment" generally refers to a portion of an antibody heavy and / or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of antigen-binding fragments include single-domain antibodies (e.g., VHHs), single-chain Fvs (scFvs), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies, or chemically modified derivatives thereof. In particular, the antibodies provided herein include immunoglobulin molecules and molecules containing one or more immunologically active portions of immunoglobulin molecules, such as one or more complementarity-determining regions (CDRs) of an antibody that binds to TNFR2.Such antibody fragments are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publishers, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Plueckthun and Skerra, Meth. Enzymol., 178:497-515 (1989), and Day, ED, Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990). The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.

[0080] As used herein, the term "single-domain antibody" or "sdAb" refers to an antibody or antibody fragment that contains a single antibody variable domain capable of binding only to a specific antigen and does not require a separate antibody variable domain. The complementarity-determining regions (CDRs) of a single-domain antibody are part of the single antibody variable domain. Examples of single-domain antibodies include, but are not limited to, heavy chain antibodies, antibodies that naturally lack light chains, single-domain antibodies derived from traditional four-chain antibodies, engineered antibodies, variable domains derived from the above-mentioned antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, shark, goat, rabbit, and / or cow. In some embodiments, a single-domain antibody as used herein is a naturally occurring single-domain antibody known as a heavy-chain antibody without a light chain. For clarity, variable domains derived from heavy-chain antibodies that naturally lack light chains are referred to herein as VHHs to distinguish them from the VHs of traditional four-chain immunoglobulins. Such VHH molecules may be derived from antibodies produced in Camelidae species, such as camels, llamas, dromedaries, alpacas, and guanacos. Non-Camelidae species may also naturally produce heavy chain antibodies that lack light chains, and these are within the scope of the present invention. For example, cartilaginous fish, such as sharks, can produce immunoglobulin-like structures known as VNARs. In some embodiments, single domain antibodies can be derived from Camelidae VH domains. In some embodiments, single domain antibodies can be derived from human VHs by camelization. For a review of single domain antibodies, see Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608, the disclosure of which is incorporated herein by reference.

[0081] As used herein, the term "specifically binds" means that an antigen-binding protein forms a complex with a target antigen that is relatively stable under physiological conditions. Specific binding is characterized by a binding affinity of approximately 1×10 -6 M or less (e.g., 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10 -9 Less than M or 10 -10 Dissociation constant (K) D ). Methods for determining the binding affinity of an antigen-binding protein, such as an antibody or antibody fragment, to a target antigen are known in the art and include, for example, surface plasmon resonance (e.g., a BIACORE® assay), biolayer interferometry, ligand-binding assays (e.g., enzyme-linked immunosorbent assay (ELISA)), equilibrium dialysis, fluorescent-activated cell sorting (FACS), or flow cytometry-based binding assays. Specific binding to a particular target antigen from a particular species does not exclude that the antigen-binding protein may also specifically bind to a similar target from a different species. For example, specific binding to human TNFR2 does not exclude that the antigen-binding protein may also specifically bind to TNFR2 from cynomolgus monkeys ("cyno").

[0082] The term "isolated," when used in the context of antigen-binding proteins (e.g., antibodies, such as single domain antibodies), polypeptides, polynucleotides, and vectors, means that the antigen-binding proteins (e.g., antibodies, such as single domain antibodies), polypeptides, polynucleotides, and vectors are at least partially free of other biological molecules derived from the cell or cell culture in which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other substances such as cell debris and growth medium. An isolated antigen-binding protein may also be at least partially free from components of the expression system, such as biological molecules derived from the host cell, or its growth medium. In general, the term "isolated" is not meant to refer to the complete absence of such biological molecules (e.g., although trace or insignificant amounts of impurities may remain), nor is it meant to refer to the absence of water, buffers, or salts, or to refer to the absence of components of a pharmaceutical formulation that comprises the antigen-binding protein (e.g., an antibody, such as a single domain antibody).

[0083] As used herein, the term "operably linked" can refer to a functional relationship between two or more regions of a polypeptide chain such that the two or more regions join to produce a functional polypeptide.

[0084] As used herein, the terms "variant," "derivative," or "derived from," in the context of a protein or polypeptide (e.g., an antigen-binding protein or domain thereof), mean: (a) a polypeptide that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the polypeptide of which it is a variant or derivative; (b) a polypeptide that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the nucleotide sequence encoding the polypeptide of which it is a variant or derivative; 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity; (c) polypeptides that contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid mutations (i.e., additions, deletions, and / or substitutions) relative to the polypeptide of which they are a variant or derivative. (d) a polypeptide encoded by a nucleic acid capable of hybridizing under high, medium, or typical stringency hybridization conditions to a nucleic acid encoding a polypeptide of which the polypeptide is a variant or derivative; (e) a polypeptide encoded by a nucleotide sequence capable of hybridizing under high, medium, or typical stringency hybridization conditions to a nucleotide sequence encoding a fragment of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, or at least 150 contiguous amino acids of a polypeptide of which the polypeptide is a variant or derivative;or (f) a fragment of a polypeptide of which the fragment is a variant or derivative. These terms also encompass fusion proteins or polypeptides, including fusion proteins or polypeptides of which the fusion protein or polypeptide is a variant or derivative;

[0085] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicate that when optimally aligned, with appropriate nucleotide insertions or deletions, with another nucleic acid (or its complementary strand), there is nucleotide sequence identity over at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any known sequence identity algorithm, such as FASTA, BLAST, or Gap, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0086] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned using programs such as GAP or BESTFIT with default gap weighting, share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, the disclosure of which is incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256: 1443-1445, the disclosure of which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0087] Sequence similarity between polypeptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their muteins. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA, a program within GCG Version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0088] The terms "enhance" or "promote" or "increase" or "expand" or "improve" generally refer to the ability of a composition contemplated herein to generate, induce, or cause an increased physiological response (i.e., downstream effect) relative to the response caused by a vehicle or control molecule / composition. Measurable physiological responses include increased immune cell proliferation, activation, effector function, persistence, and / or increased tumor cell killing capacity, among others that are apparent from understanding in the art and the description herein. In certain embodiments, an "increased" or "enhanced" amount can be a "statistically significant" amount and can include a 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or greater (e.g., 500-fold, 1000-fold) increase (e.g., 1.5, 1.6, 1.7, 1.8, etc., including all integers and decimals therebetween and greater than 1) over the response produced by a vehicle or control composition.

[0089] The terms "decrease" or "lower" or "lessen" or "reduce" or "abate" generally refer to the ability of a composition contemplated herein to produce, induce, or cause a physiological response (i.e., a downstream effect) that is less than the response caused by a vehicle or control molecule / composition. In certain embodiments, the amount "decreased" or "reduced" may be a "statistically significant" amount and may include a 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or greater (e.g., 500-fold, 1000-fold) reduction (e.g., 1.5, 1.6, 1.7, 1.8, etc., including all integers and decimals therebetween and greater than 1) of the response produced by a vehicle or control composition (reference response).

[0090] The term "treat" a state, disorder, or condition, or "treatment" thereof, includes: (1) preventing, delaying, or reducing the likelihood of the occurrence and / or appearance of at least one clinical or subclinical symptom of the state, disorder, or condition in a subject who may be suffering from or predisposed to the state, disorder, or condition, but who has not yet experienced or displayed clinical or subclinical symptoms of the state, disorder, or condition; or (2) inhibiting the state, disorder, or condition, i.e., arresting, reducing, or delaying the progression of the disease or its recurrence, or at least one clinical or subclinical symptom thereof; or (3) palliating the disease, i.e., causing regression of the state, disorder, or condition, or at least one clinical or subclinical symptom thereof. The benefit to a treated subject is statistically significant or at least perceptible to the patient or physician.

[0091] The term "effective amount" or "therapeutically effective amount" refers to an amount and / or concentration of a composition containing an active ingredient (e.g., an anti-TNFR2 antigen binding protein) that, when administered to a patient alone (i.e., as monotherapy) or in combination with an additional therapeutic agent, results in a significant reduction in disease progression, for example, by ameliorating or eliminating the symptoms and / or pathogenesis of the disease. An effective amount may be an amount that relieves, reduces, or alleviates at least one symptom or physiological response or effect associated with a disease or disorder, prevents the progression of a disease or disorder, or improves the patient's physical function. A therapeutically effective amount of a composition containing an active agent may vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the active agent to elicit a desired response in the individual's body. A therapeutically effective amount is also an amount such that any toxic or detrimental effects of the active agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic and / or prophylactic result.

[0092] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to animals, e.g., mammals. These terms include humans and veterinary subjects. In some embodiments, methods are provided for treating mammals, including, but not limited to, humans, rodents, apes, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock animals, mammalian sport animals, and mammalian pets. The subject may be male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some embodiments, the subject may be one in need of treatment for a disease or disorder. In certain embodiments, the subject is human.

[0093] Anti-TNFR2 antigen-binding protein The present disclosure provides antigen binding proteins (e.g., antibodies, such as single domain antibodies) that bind to tumor necrosis factor receptor 2 (TNFR2).

[0094] TNFR2 is a single-pass type 1 membrane protein belonging to the TNFR superfamily. It consists of an extracellular domain with four cysteine-rich domains (CRDs) and an intracellular domain involved in signal transduction. The cysteine-rich domains contain a total of 10 disulfide bonds, which stabilize the elongated structure of the protein. Unlike TNFR1, which is widely expressed, TNFR2 expression is restricted to immune cells, including Tregs, myeloid cells, CD8 and NK cells, as well as glial cells, endothelial cells, and fibroblasts (Medler and Wajant, 2019).

[0095] In some embodiments, the human TNFR2 protein is encoded by the human TNF receptor superfamily member 1B (TNFRSF1B) gene (NCBI Gene ID: 7133); MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCA LSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGS TGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS (UniProtKB Accession No. P20333) (SEQ ID NO: 4028).

[0096] In some embodiments, the cynomolgus monkey TNFR2 protein is encoded by the Cyno TNF receptor superfamily member 1B (TNFRSF1B) gene (Gene ID: 102144224); (UniProtKB accession number A0A2K5VET2) (SEQ ID NO: 4029).

[0097] In some embodiments, the mouse TNFR2 protein is encoded by the mouse TNF receptor superfamily member 1B (Tnfrsf1b) gene (Gene ID: 21938); MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQISQEYYDRKAQMCCAKCPPGQYVKHFCNKTSDTVCADCEASMYTQVWNQFRTCLSCSSSCTTDQVEIRACTKQQNRVCACEAGRYCALK THSGSCRQCMRLSKCGPGFGVASSRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDAVCAPESPTLSAIPRTLYVSQPEPTRSQPLDQEPGPSQTPSILTSLGSTPIIEQSTKGGISL PIGLIVGVTSLGLLMLGLVNCIILVQRKKKPSCLQRDAKVPHVPDEKSQDAVGLEQQHLLTTAPSSSSSSLESSASAGDRRAPPGGHPQARVMAEAQGFQEARASSRISDSSHGSHGTHVNVTCIVNVCSSSDHSSQCSSQASATVGDPDAKPSASPKDEQVPFSQEECPSQSPCETTETLQSHEKPLPLGVPDMGMKPSQAGWFDQIAVKVA (UniProtKB Accession No. P25119) (SEQ ID NO: 4030).

[0098] In various embodiments, the antigen binding proteins of the present disclosure have an agonistic effect upon binding to TNFR2. Without wishing to be bound by theory, agonistic TNFR2 binders can promote or increase the activity of TNFR2 and / or enhance one or more signaling pathways mediated by TNFR2. For example, agonistic TNFR2 binders can promote or increase the proliferation of Treg cell populations. Agonistic TNFR2 binders can promote or increase TNFR2 activation by binding to TNFR2 and, for example, inducing a conformational change that confers biological activity to the receptor. For example, agonistic TNFR2 binders can induce TNFR2-mediated signaling by nucleating the trimerization of TNFR2 in a manner similar to the interaction between TNFR2 and its cognate ligand, tumor necrosis factor (TNF). In some embodiments, agonistic TNFR2 binding proteins of the disclosure may induce the proliferation of Treg cells (e.g., CD4+, CD25+, FOXP3+ Treg cells). Agonistic TNFR2 binding proteins of the disclosure may also suppress the proliferation of cytotoxic T lymphocytes (e.g., CD8+ T cells), for example, by activating immunoregulatory Treg cells or by directly binding to TNFR2 on the surface of autoreactive cytotoxic T cells and inducing apoptosis.

[0099] In some embodiments, an antigen binding protein of the present disclosure, after binding to TNFR2, does not inhibit the binding of its cognate ligand, tumor necrosis factor (TNF), to TNFR2. In some embodiments, an antigen binding protein of the present disclosure does not have an epitope that overlaps with TNF. In some embodiments, an antigen binding protein of the present disclosure, after binding to TNFR2, promotes or facilitates TNFR2 oligomerization (in the presence or absence of TNF, respectively). In some embodiments, an antigen binding protein of the present disclosure, after binding to TNFR2, induces intracellular signaling by multimerizing (e.g., dimerizing) TNFR2 trimers.

[0100] In some embodiments, the antigen binding proteins of the disclosure bind to human TNFR2. In some embodiments, the antigen binding proteins (e.g., antibodies, such as single domain antibodies) of the disclosure bind to human TNFR2 at concentrations of about 1 x 10 -6 Less than M, e.g., about 5 × 10 -7 Less than M, approximately 3 x 10 -7 Less than M, approximately 1 x 10 -7 Less than M, approximately 8 x 10 -8 Less than M, approximately 5 x 10 -8 Less than M, approximately 3 x 10 -8 Less than M, approximately 1 x 10 -8 Less than M, approximately 8 x 10 -9 Less than M, approximately 5 x 10 -9 Less than M, approximately 3 x 10 -9 Less than M or about 1 x 10 -9 Less than M or about 1 x 10 -10 ~1×10 -9 M, 1 x 10 -10 ~5×10 -9 M, about 1 x 10 -10 ~1×10 -8 M, about 1 x 10 -10 ~5×10 -8 M, about 1 x 10 -9 ~1×10 -8 M, about 1 x 10 -9 ~5×10 -8 M, about 1 x 10 -9 ~1×10 -7M, or approximately 1 x 10 -8 ~1×10 -7 K of M D and may bind to human TNFR2.

[0101] In some embodiments, the antigen binding proteins of the disclosure bind to cynomolgus monkey ("cyno") TNFR2. In some embodiments, the antigen binding proteins (e.g., antibodies, such as single domain antibodies) of the disclosure bind to about 1 x 10 -6 Less than M, e.g., about 5 × 10 -7 Less than M, approximately 3 x 10 -7 Less than M, approximately 1 x 10 -7 Less than M, approximately 8 x 10 -8 Less than M, approximately 5 x 10 -8 Less than M, approximately 3 x 10 -8 Less than M, approximately 1 x 10 -8 Less than M, approximately 8 x 10 -9 Less than M, approximately 5 x 10 -9 Less than M, approximately 3 x 10 -9 Less than M or about 1 x 10 -9 Less than M or about 1 x 10 -10 ~1×10 -9 M, 1 x 10 -10 ~5×10 -9 M, about 1 x 10 -10 ~1×10 -8 M, about 1 x 10 -10 ~5×10 -8 M, about 1 x 10 -9 ~1×10 -8 M, about 1 x 10 -9 ~5×10 -8 M, about 1 x 10 -9 ~1×10 -7 M, about 1 x 10 -9 ~2×10 -7 M, about 1 x 10 -9 ~5×10 -7 M, about 1 x 10 -8 ~1×10 -7 M, about 1 x 10 -8 ~2×10 -7 M, about 1 x 10 -8 ~5×10 -7 M, or approximately 1 x 10 -8 ~1×10 -6 K of M Dand may bind to cynoTNFR2.

[0102] In some embodiments, the antigen binding proteins of the disclosure bind to mouse TNFR2. In some embodiments, the antigen binding proteins of the disclosure bind to mouse TNFR2 at a concentration of about 1 x 10 -6 Less than M, e.g., about 5 × 10 -7 Less than M, approximately 3 x 10 -7 Less than M, approximately 1 x 10 -7 Less than M, approximately 8 x 10 -8 Less than M, approximately 5 x 10 -8 Less than M, approximately 3 x 10 -8 Less than M, approximately 1 x 10 -8 Less than M, approximately 8 x 10 -9 Less than M, approximately 5 x 10 -9 Less than M, approximately 3 x 10 -9 Less than M or about 1 x 10 -9 Less than M or about 1 x 10 -10 ~1×10 -9 M, 1 x 10 -10 ~5×10 -9 M, about 1 x 10 -10 ~1×10 -8 M, about 1 x 10 -10 ~5×10 -8 M, about 1 x 10 -9 ~1×10 -8 M, about 1 x 10 -9 ~5×10 -8 M, about 1 x 10 -9 ~1×10 -7 M, about 1 x 10 -9 ~2×10 -7 M, about 1 x 10 -9 ~5×10 -7 M, about 1 x 10 -8 ~1×10 -7 M, about 1 x 10 -8 ~2×10 -7 M, about 1 x 10 -8 ~5×10 -7 M, or approximately 1 x 10 -8 ~1×10 -6 K of M D In some embodiments, the antigen binding proteins of the disclosure do not bind to mouse TNFR2.

[0103] In some embodiments, an anti-TNFR2 antigen binding protein of the present disclosure can specifically bind to TNFR2 without exhibiting specific binding to other receptors in the tumor necrosis factor receptor (TNFR) superfamily.

[0104] The binding affinity of a molecular interaction between two molecules can be measured by various techniques, such as surface plasmon resonance (SPR), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescence-activated cell sorting (FACS), or flow cytometry binding assays. Surface plasmon resonance is a biosensor technique that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and another molecule passes over the immobilized molecule under flow conditions (see, e.g., Ober et al. 2001, Intern. Immunology 13: 1551-1559). SPR can be performed, for example, using a BIACORE® system or a Carterra LSA system. Another biosensor technique that can be used to determine the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377: 209-217). Biolayer interferometry is a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer and a layer of immobilized proteins at the biosensor tip (a reference beam and a signal beam, respectively). Changes in the number of molecules bound to the biosensor tip result in a shift in the interference pattern, which is reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Because interactions can be measured in real time, association and dissociation rates, as well as affinity, can be determined. BLI can be performed, for example, using Octet® Systems. Alternatively, affinity can be measured by Kinetic Exclusion Assay (KinExA), a solution-based method for measuring true equilibrium binding affinity and kinetics of unmodified molecules (see, e.g., Drake et al. 2004, Anal. Biochem., 328: 35-43).An equilibrated solution of antibody / antigen complexes is passed through a column containing beads pre-coated with the antigen (or antibody), causing the free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is achieved by a fluorescently labeled protein that binds to the antibody (or antigen).

[0105] Antigen-binding proteins of the present disclosure can include antibodies or antigen-binding fragments of antibodies, such as human antibodies, humanized antibodies; camelid antibodies; chimeric antibodies; recombinant antibodies; heavy chain antibodies; single domain antibodies (e.g., VHH); single chain antibodies (e.g., single chain fragment variable (scFv)); diabodies; triabodies; tetrabodies; Fab fragments; F(ab')2 fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies, and fragments thereof.

[0106] In some embodiments, the antigen binding protein that binds to TNFR2 is a single domain antibody (also referred to as an "sdAb"). The single domain antibodies of the present disclosure may be derived from a number of sources, including, but not limited to, VHHs, VNARs, or VH domains (naturally occurring or engineered VH domains). VHHs can be generated from camelid heavy chain-only antibodies and libraries thereof. VNARs can be generated from cartilaginous fish heavy chain-only antibodies and libraries thereof. Various methods, including interfacing and selection of specific germline families, have been implemented to generate monomeric sdAbs from traditional heterodimeric VH and VL domains. In some embodiments, the sdAbs of the present invention are human or humanized sdAbs.

[0107] In some embodiments, the single domain antibodies described herein are VHH fragments (also known as nanobodies). VHH fragments are also referred to as "V-bodies" in the present disclosure. In some embodiments, the VHH is a camelid VHH, a humanized VHH, or a camelized VH. In some embodiments, the single domain antibodies described herein are VH domains. In some embodiments, the single domain antibodies described herein are naturally occurring VH domains or engineered VH domains.

[0108] The variable domain of an antigen-binding protein (e.g., an antibody such as a single-domain antibody) of the present disclosure comprises at least three complementarity-determining regions (CDRs), which determine its binding specificity. Preferably, within a variable domain, the CDRs are distributed between framework regions. A variable domain typically comprises four framework FR regions separated by three CDR regions, resulting in the following typical antibody variable domain structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs and / or FRs of the single-domain antibody of the present invention may be fragments or derivatives derived from naturally occurring antibody variable domains, or may be synthetic.

[0109] Sequence identifiers corresponding to exemplary anti-TNFR2 VHH antibodies provided herein are listed in Table 1. Table 1-1 shows the sequence identifiers for the amino acid sequences of the complementarity determining regions (CDR1, CDR2, and CDR3), the amino acid and DNA sequences of full-length Camelidae VHH antibodies, and the amino acid sequences of the corresponding humanized VHH antibodies. Further exemplary anti-TNFR2 VHH antibodies and the amino acid sequences of the corresponding humanized VHH antibodies are provided in Table 1-2.

[0110] [Table 1-1]

[0111] [Table 1-2] TIFF2026503077000003.tif237169TIFF2026503077000004.tif235166TIFF2026503077000005.tif237166TIFF2026503077000006.t if236168TIFF2026503077000007.tif236167TIFF2026503077000008.tif237167TIFF2026503077000009.tif237167TIFF2026503077 000010.tif236166TIFF2026503077000011.tif236169TIFF2026503077000012.tif237167TIFF2026503077000013.tif234167TIFF20 26503077000014.tif235167TIFF2026503077000015.tif237167TIFF2026503077000016.tif234165TIFF2026503077000017.tif81166

[0112] In some embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the present disclosure comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the following (the amino acids listed in parentheses represent the possible amino acids at that particular position): a) GSI(V / F)(R / S)(T / A)(N / D)(S / G / A) (SEQ ID NO: 68); b) GFT(F / L)DD(I / Y)A (SEQ ID NO: 69); c) GFTFS(S / R / G)YA (SEQ ID NO: 70); d) GRTFSDYG (SEQ ID NO: 16); e) G(L / F)TLDYYA (SEQ ID NO: 71); f) GF(T / N)FSMYS (SEQ ID NO: 72); g)GRTF(G / R / S)(N / S)(Y / L)(T / F)(SEQ ID NO: 73); h) GASLS RNA (SEQ ID NO: 40); i) GS(I / T)FRFPP (SEQ ID NO: 74); j) GFTLDDYA (SEQ ID NO: 4061); and k)G(F / V)(S / T)LD(D / Y)(H / Y)T (SEQ ID NO: 4519).

[0113] In some embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the present disclosure comprises a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the following (the amino acids listed in parentheses represent the possible amino acids at that particular position): a) IRSDGF(T / I) (SEQ ID NO: 75); b) I(Y / F)SY(S / G)(S / P)NT (SEQ ID NO: 76); c) I(Y / S)(S / D)DGS(E / D)T (SEQ ID NO: 77); d) INWSN(G / A)RT (SEQ ID NO: 4699); e) I(S / N)(V / T)(S / G)DGST (SEQ ID NO: 78); f) DT(R / G)GST (SEQ ID NO: 79); g)IR(W / R / Y)(T / P)G(G / L)(S / I)T(SEQ ID NO: 80); h) IYDDGET (SEQ ID NO: 41); i) LTSGGST (SEQ ID NO: 45); j) IFSYSSNT (SEQ ID NO: 4062); and k)I(N / S)SNDG(S / T)(T / V) (SEQ ID NO: 4518).

[0114] In some embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the disclosure comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the following (the amino acids listed in parentheses represent the possible amino acids at that particular position): a)(Y / F)YQ(S / A)LS(T / S)(P / A)N(Y / F)GQ(V / T)F(SEQ ID NO: 60); b) AADSDL(S / R)TV(V / T)VGPHDY (SEQ ID NO: 61); c) AKDAG(S / G)WG(T / R)GPFG(Y / F)(E / D)YDY (SEQ ID NO: 62); d) AA(T / A)PSGKAY(T / S)Y (SEQ ID NO: 63); e) ATPGPY(T / S / M)YCAPYGSSWSRGYDY (SEQ ID NO: 64); f) ARV(R / G)G(T / S / A)PY(E / D)Y(N / G)Y (SEQ ID NO: 65); g) (T / A / V)A(S / A)PTGRAF(T / N / A)Y (SEQ ID NO: 66); h) AGSAFDF (SEQ ID NO: 42); i)S(V / M)(V / L)GRDM(M / V)TY(SEQ ID NO: 67); j) AVGDFEGELVLKGDY (SEQ ID NO: 4063); k)AAD(L / V)G(F / V / Y)LY(A / T / V)DYV(P / R)LH(M / T)HHFGS(SEQ ID NO: 4517); l) A(A / G)(T / A)(P / L)(S / T)GKAY(T / S)Y (SEQ ID NO: 4771).

[0115] In certain embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the disclosure: i) CDR1 comprising the amino acid sequence of SEQ ID NO: 68, CDR2 comprising the amino acid sequence of SEQ ID NO: 75, and CDR3 comprising the amino acid sequence of SEQ ID NO: 60; ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 69, CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR3 comprising the amino acid sequence of SEQ ID NO: 61; iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 70, CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and CDR3 comprising the amino acid sequence of SEQ ID NO: 62; iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 63; v) CDR1 comprising the amino acid sequence of SEQ ID NO: 71, CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and CDR3 comprising the amino acid sequence of SEQ ID NO: 64; vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 72, CDR2 comprising the amino acid sequence of SEQ ID NO: 79, and CDR3 comprising the amino acid sequence of SEQ ID NO: 65; vii) CDR1 comprising the amino acid sequence of SEQ ID NO: 73, CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and CDR3 comprising the amino acid sequence of SEQ ID NO: 66; viii) CDR1 comprising the amino acid sequence of SEQ ID NO: 40, CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or ix) CDR1 comprising the amino acid sequence of SEQ ID NO: 74, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR3 comprising the amino acid sequence of SEQ ID NO: 67; x) CDR1 comprising the amino acid sequence of SEQ ID NO: 4061, CDR2 comprising the amino acid sequence of SEQ ID NO: 4062, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4063; xi) CDR1 comprising the amino acid sequence of SEQ ID NO: 4519, CDR2 comprising the amino acid sequence of SEQ ID NO: 4518, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4517; or xii) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4771 Includes.

[0116] In certain embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the disclosure: a) CDR1 comprising the amino acid sequence of SEQ ID NO: 69, CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR3 comprising the amino acid sequence of SEQ ID NO: 61; b) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 63; c) CDR1 comprising the amino acid sequence of SEQ ID NO: 73, CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or d) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4771 Includes.

[0117] In certain embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the disclosure: i) CDR1 having the amino acid sequence GSI(V / F)(R / S)(A / T)(N / D)(G / A) (SEQ ID NO: 4700), CDR2 comprising the amino acid sequence IRSDGFT (SEQ ID NO: 2), and CDR3 comprising the amino acid sequence YYQ(S / A)LSSPNYGQ(V / T)F (SEQ ID NO: 4701); ii) a CDR1 having the amino acid sequence GFTFDDIA (SEQ ID NO: 8), a CDR2 comprising the amino acid sequence IYSYGPNT (SEQ ID NO: 9), and a CDR3 comprising the amino acid sequence AADSDLSTVV(V / T)GPHDY (SEQ ID NO: 4702); iii) a CDR1 having the amino acid sequence GFTFSRYA (SEQ ID NO: 12), a CDR2 comprising the amino acid sequence ISDDGSDT (SEQ ID NO: 13), and a CDR3 comprising the amino acid sequence AKDAGSWGTGPFGYEYDY (SEQ ID NO: 14); iv) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 16), CDR2 comprising the amino acid sequence INWSN(G / A)RT (SEQ ID NO: 4699), and CDR3 comprising the amino acid sequence AA(T / A)PSGKAYSY (SEQ ID NO: 4703); v) CDR1 having the amino acid sequence GLTLDYYA (SEQ ID NO: 20), CDR2 comprising the amino acid sequence ISTSDGST (SEQ ID NO: 21), and CDR3 comprising the amino acid sequence ATPGPYTYCAPYGSSWSRGYDY (SEQ ID NO: 22); vi) CDR1 having the amino acid sequence GF(T / N)FSMYS (SEQ ID NO: 72), CDR2 comprising the amino acid sequence IDT(R / G)GST (SEQ ID NO: 79), and CDR3 comprising the amino acid sequence ARV(G / R)G(T / A)PYEY(N / G)Y (SEQ ID NO: 4704); vii) CDR1 having the amino acid sequence GRTF(G / S)S(Y / L)(T / F) (SEQ ID NO: 4705), CDR2 comprising the amino acid sequence IR(W / R / Y)(T / P)G(G / L)(S / I)T (SEQ ID NO: 80), and CDR3 comprising the amino acid sequence (A / V)A(A / S)PTGRAF(T / N)Y (SEQ ID NO: 4707); viii) CDR1 having the amino acid sequence GASLSRNA (SEQ ID NO: 40), CDR2 comprising the amino acid sequence IYDDGET (SEQ ID NO: 41), and CDR3 comprising the amino acid sequence AGSAFDF (SEQ ID NO: 42); ix) CDR1 having the amino acid sequence GS(T / I)FRFPP (SEQ ID NO: 4708), CDR2 comprising the amino acid sequence LTSGGST (SEQ ID NO: 45), and CDR3 comprising the amino acid sequence SVLGRDM(M / V)TY (SEQ ID NO: 4706); x) CDR1 having the amino acid sequence GFTLDDYA (SEQ ID NO: 4061), CDR2 comprising the amino acid sequence IFSYSSNT (SEQ ID NO: 4062), and CDR3 comprising the amino acid sequence AVGDFEGELVLKGDY (SEQ ID NO: 4063); xi) CDR1 having the amino acid sequence GFTLDYYT (SEQ ID NO: 4065), CDR2 comprising the amino acid sequence ISSNDSVGSV (SEQ ID NO: 4066), and CDR3 comprising the amino acid sequence AADLGYLYVDYVRLHTHHFGS (SEQ ID NO: 4067); xii) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 4719), CDR2 comprising the amino acid sequence INWSNGRT (SEQ ID NO: 4723), and CDR3 comprising the amino acid sequence AATPTGKAYTY (SEQ ID NO: 4727); xiii) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 4720), CDR2 comprising the amino acid sequence INWSNGRT (SEQ ID NO: 4724), and CDR3 comprising the amino acid sequence AATPTGKAYTY (SEQ ID NO: 4728); xiv) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 4721), a CDR2 comprising the amino acid sequence INWSNGRT (SEQ ID NO: 4725), and a CDR3 comprising the amino acid sequence AGTLSGKAYTY (SEQ ID NO: 4729); or xv) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 4722), CDR2 comprising the amino acid sequence INWSNGRT (SEQ ID NO: 4726), and CDR3 comprising the amino acid sequence AGTLSGKAYTY (SEQ ID NO: 4730) Includes.

[0118] In certain embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the disclosure: a) CDR1 having the amino acid sequence GFTFDDIA (SEQ ID NO: 8), CDR2 comprising the amino acid sequence IYSYGPNT (SEQ ID NO: 9), and CDR3 comprising the amino acid sequence AADSDLSTVV(V / T)GPHDY (SEQ ID NO: 4702); b) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 16), CDR2 comprising the amino acid sequence INWSN(G / A)RT (SEQ ID NO: 4699), and CDR3 comprising the amino acid sequence AA(T / A)PSGKAYSY (SEQ ID NO: 4703); c) a CDR1 having the amino acid sequence GRTF(G / S)S(Y / L)(T / F) (SEQ ID NO: 4705), a CDR2 comprising the amino acid sequence IR(W / R / Y)(T / P)G(G / L)(S / I)T (SEQ ID NO: 80), and a CDR3 comprising the amino acid sequence (A / V)A(A / S)PTGRAF(T / N)Y (SEQ ID NO: 4707); or d) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4771 Includes.

[0119] Provided herein are anti-TNFR2 antigen binding proteins (e.g., antibodies, such as single domain antibodies) comprising a CDR1 (CDR1) comprising an amino acid sequence selected from any of the CDR1 amino acid sequences listed in Table 1-1 or Table 5, or an analogous sequence of said amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0120] In some embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 1, 5, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 4061, 4065, 4069, 4520, 4719-4722, 1128-1686, 4173-4234, and 4533-4556, or an analogous sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0121] Provided herein are anti-TNFR2 antigen binding proteins (e.g., antibodies, such as single domain antibodies) comprising a CDR2 (CDR2) comprising an amino acid sequence selected from any of the CDR2 amino acid sequences listed in Table 1-1 or Table 5, or an analogous sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0122] In some embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 2, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 4062, 4066, 4070, 4527, 4723-4726, 1687-2245, 4235-4296, and 4557-4580, or an analogous sequence thereto having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0123] Provided herein are anti-TNFR2 antigen binding proteins (e.g., antibodies, such as single domain antibodies) comprising a CDR3 (CDR3) comprising an amino acid sequence selected from any of the CDR3 amino acid sequences listed in Table 1-1 or Table 5, or an analogous sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0124] In some embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 3, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 4063, 4067, 4071, 4524, 4530, 4727-4730, 2246-2804, 4297-4358, and 4581-4604, or an analogous sequence thereto having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0125] Provided herein are anti-TNFR2 antigen binding proteins (e.g., antibodies such as single domain antibodies) that comprise a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained in any of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, or Table 5. In certain embodiments, the anti-TNFR2 antigen binding proteins (e.g., antibodies such as single domain antibodies) of the present disclosure comprise: i) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 3; ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 5, CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 6; iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10; iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 12, CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR3 comprising the amino acid sequence of SEQ ID NO: 14; v) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR3 comprising the amino acid sequence of SEQ ID NO: 22; vii) CDR1 comprising the amino acid sequence of SEQ ID NO: 24, CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR3 comprising the amino acid sequence of SEQ ID NO: 26; viii) CDR1 comprising the amino acid sequence of SEQ ID NO: 28, CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR3 comprising the amino acid sequence of SEQ ID NO: 30; ix) CDR1 comprising the amino acid sequence of SEQ ID NO: 32, CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR3 comprising the amino acid sequence of SEQ ID NO: 34; x) CDR1 comprising the amino acid sequence of SEQ ID NO: 36, CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR3 comprising the amino acid sequence of SEQ ID NO: 38; xi) CDR1 comprising the amino acid sequence of SEQ ID NO: 40, CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42; xii) CDR1 comprising the amino acid sequence of SEQ ID NO: 44, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR3 comprising the amino acid sequence of SEQ ID NO: 46; xiii) CDR1 comprising the amino acid sequence of SEQ ID NO: 4061, CDR2 comprising the amino acid sequence of SEQ ID NO: 4062, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4063; xiv) CDR1 comprising the amino acid sequence of SEQ ID NO: 4065, CDR2 comprising the amino acid sequence of SEQ ID NO: 4066, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4067; xv) CDR1 comprising the amino acid sequence of SEQ ID NO: 4069, CDR2 comprising the amino acid sequence of SEQ ID NO: 4070, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4071; xvi) CDR1 comprising the amino acid sequence of SEQ ID NO: 4520, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR3 comprising the amino acid sequence of SEQ ID NO: 46; xvii) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4524; xviii) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; xix) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4530; xx) CDR1 comprising the amino acid sequence of SEQ ID NO: 4719, CDR2 comprising the amino acid sequence of SEQ ID NO: 4723, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4727; xxii) CDR1 comprising the amino acid sequence of SEQ ID NO: 4720, CDR2 comprising the amino acid sequence of SEQ ID NO: 4724, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4728; xxiii) CDR1 comprising the amino acid sequence of SEQ ID NO: 4721, CDR2 comprising the amino acid sequence of SEQ ID NO: 4725, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4729; or xxiv) CDR1 comprising the amino acid sequence of SEQ ID NO: 4722, CDR2 comprising the amino acid sequence of SEQ ID NO: 4726, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4730 Includes.

[0126] In some embodiments, the anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) is: a) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; b) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; c) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4530; d) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10; e) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4524; f) CDR1 comprising the amino acid sequence of SEQ ID NO: 4069, CDR2 comprising the amino acid sequence of SEQ ID NO: 4070, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4071; g) CDR1 comprising the amino acid sequence of SEQ ID NO: 4719, CDR2 comprising the amino acid sequence of SEQ ID NO: 4723, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4727; h) CDR1 comprising the amino acid sequence of SEQ ID NO: 4720, CDR2 comprising the amino acid sequence of SEQ ID NO: 4724, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4728; i) CDR1 comprising the amino acid sequence of SEQ ID NO: 4721, CDR2 comprising the amino acid sequence of SEQ ID NO: 4725, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4729; or j) CDR1 comprising the amino acid sequence of SEQ ID NO: 4722, CDR2 comprising the amino acid sequence of SEQ ID NO: 4726, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4730 Includes.

[0127] In a related embodiment, provided herein is an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained in a VHH amino acid sequence defined by any of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, or Table 5. For example, provided herein is an antibody, or an antigen-binding fragment thereof, comprising a set of CDR1-CDR2-CDR3 amino acid sequences contained in a VHH amino acid sequence selected from SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, 4521, 81 to 92, 93 to 640, 4079 to 4125, 2805 to 3363, 4359 to 4420, 4605 to 4628, 5426, 4529, 4532, 4078, 4523, 4076, 4077, 4078, and 4731 to 4734.

[0128] In some embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the disclosure: a) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4; b) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 7; c) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 11; d) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 15; e) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 19; f) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 23; g) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 27; h) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 31; i) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 35; j) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 39; k) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 43; l) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 47; m) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4064; n) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4068; o) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4072; p) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4521; q) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4526; r) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4529; s) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4532; t) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4078; u) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4731; v) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4732; w) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4733; x) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4734; may include:

[0129] In some embodiments, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the disclosure: a) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 19; b) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4072; c) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4526; d) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4529; e) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4532; f) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4731; g) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4732; h) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4733; i) a variable domain comprising CDR1, CDR2, and CDR3 contained in a VHH comprising the amino acid sequence of SEQ ID NO: 4734; may include:

[0130] In one embodiment provided herein, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the disclosure can comprise a VHH amino acid sequence selected from SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, 4521, 93-640, 4079-4125, 2805-3363, 4359-4420, 4605-4628, and 4653-4685, or an analogous sequence of said VHH amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0131] In one embodiment provided herein, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the disclosure can comprise a VHH amino acid sequence selected from SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, 4521, and 4653-4685, or an analogous sequence of said VHH amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0132] In one embodiment provided herein, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the present disclosure can comprise a humanized VHH amino acid sequence selected from SEQ ID NOs: 81-92, 4076-4078, 4523, 4526, 4529, 4532, 4731-4734, 641-1127, and 4126-4172, or an analogous sequence of the humanized VHH amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0133] In one embodiment provided herein, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the present disclosure can comprise a VHH amino acid sequence selected from SEQ ID NOs: 19, 4072, 4078, 4526, 4529, 4532, and 4653-4685, or an analogous sequence of said VHH amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0134] In some embodiments, the present disclosure also provides anti-TNFR2 antigen binding proteins (e.g., antibodies such as single domain antibodies) that compete for binding to TNFR2 with any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, or Table 5.

[0135] In some embodiments, the present disclosure also provides anti-TNFR2 antigen binding proteins (e.g., antibodies such as single domain antibodies) that bind to the same epitope on TNFR2 as any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, or Table 5.

[0136] Single Domain Antibodies Single domain antibodies (e.g., VHHs) can be obtained by immunizing dromedaries, camels, llamas, alpacas, or sharks with the desired antigen and then isolating the mRNA encoding the heavy chain antibody. The antigen can be purified from natural sources or during recombinant production. Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens. Genetic libraries of single domain antibodies containing millions of clones can be generated by reverse transcription and polymerase chain reaction (PCR). Screening techniques such as phage display, yeast display, and ribosome display are useful for identifying clones that bind to the antigen. Methods for producing heavy chain antibody fragments are described, for example, in WO 94 / 04678; Hamers-Casterman et al. 1993; Muyldermans et al. 2001; and Arbabi Ghahroudi, M. et al. (1997). FEBS Letters 414 (3): 521-526, each of which is incorporated herein by reference in its entirety.

[0137] Alternatively, gene libraries derived from animals that have not been previously immunized may be used. Such naive libraries typically contain only antibodies with low affinity to the desired antigen, and therefore require an additional step of affinity maturation by random mutagenesis. See, for example, Saerens, D.; et al. (2008). "Single-domain antibodies as building blocks for novel therapeutics". Current Opinion in Pharmacology 8 (5): 600-608.

[0138] Affinity maturation strategies can be categorized as targeted / rational approaches or non-targeted / random approaches. Targeted approaches require information about the VHH of interest, such as affinity maturation hotspots or structural information about the VHH:antigen complex, while non-targeted approaches do not require prior information. Targeted approaches applicable to the affinity maturation of VHHs include site-directed in vitro mutagenesis and in silico / computational approaches. Common non-targeted approaches used for the affinity maturation of VHHs include random in vitro mutagenesis, CDR swapping, and autonomous hypermutation yeast surface display, the latter two being novel, emerging, and extremely time-efficient techniques. Common to most of these strategies is the application of a specific randomization strategy to generate a mutant library, which can then be screened to select the best binders by employing standard display techniques such as yeast, phage, or ribosome display. The choice of display system often depends on the size of the library to be displayed, with yeast display typically reaching approximately 10 7 ~10 9 , phage display is about 10 8 ~10 10 , ribosome display is about 10 12 ~10 13 Library sizes of up to 1000kb can be handled (Chan and Groves, 2021). In particular, the number of highly interactive residues, such as aromatic amino acids, increases within the CDR regions during affinity maturation. Selected affinity-matured clones can be further evaluated by developability assessment to test for undesirable properties, such as nonspecific binding to off-targets or VHH instability.

[0139] Targeted in vitro mutagenesis involves mutating a selected set of residues within the CDRs of VHHs (Tiller et al., 2017; Yau et al., 2005). Preselection of these residues can be performed by identifying mutation hotspot residues using alanine scanning or by using structural data of antigen:VHH complexes to identify positions to mutate. These sites can then be subjected to saturation mutagenesis, where a specific site is substituted with all possible amino acids or subjected to specific amino acid substitutions to obtain several small libraries. After mutagenesis, binders can be displayed and the best mature candidates selected. Targeted mutagenesis is typically performed multiple times using separate sublibraries to obtain combinations of distinct mutations that, in concert, result in increased binding affinity.

[0140] Computer-assisted / in silico methods are often used to guide targeted in vitro mutagenesis. Homology modeling or docking of the target:VHH complex can be used to identify mutation hotspots, which are then subjected to in vitro mutagenesis (Bert Schepens et al., 2021; Cheng et al., 2019; Inoue et al., 2013; Mahajan et al., 2018). Furthermore, in silico methods allow the creation of virtual libraries (approximately 10 components). 40 All designed variants in a library can be searched relatively quickly to identify a reasonable number of promising candidates for experimental testing. These techniques can be particularly useful when structural data on the drug-target interaction are available.

[0141] Non-targeted / random affinity maturation strategies that can be applied for VHH affinity maturation include random in vitro mutagenesis, CDR shuffling / swapping, and in vivo affinity maturation by yeast display. Random in vitro mutagenesis randomly mutates the sequence of the entire VHH or only the CDRs (Chen et al., 2021; Ye et al., 2021; Zupancic et al., 2021). The most commonly used technique is error-prone PCR, which uses a DNA polymerase lacking proofreading activity and PCR conditions that further increase the polymerase's error rate. This technique can be applied without additional structural knowledge or information regarding the importance of residues contributing to antigen:VHH interactions. The resulting mutation library can then be displayed to select the best mature candidates. This technique can be combined with NGS sequencing of the display eluate to perform an exhaustive readout of all resulting candidates, thereby identifying promising clones that are less abundant (Chen et al., 2021).

[0142] In some embodiments, CDR shuffling or swapping is applied to VHH affinity maturation, as described in Zupancic et al., 2021. In CDR swapping, the enriched library can be used as input material for PCR reactions to individually amplify the CDRs of VHHs. The PCR products can then be mixed and reassembled using overlap PCR to generate whole plasmids for further display to select the best mature binders. One limitation of this approach is that, as with synthetic libraries, it can only be used for VHHs containing the same framework.

[0143] In some embodiments, in vivo affinity maturation by yeast display is applied to VHH affinity maturation, as described in Wellner et al., 2021. This method is based on autonomous hypermutation yeast surface display (AHEAD), which mimics somatic hypermutation during VHH selection using engineered yeast strains. The error-prone orthogonal DNA replication system of yeast can generate novel variants during plasmid replication by randomly introducing mutations. These novel variants can then be displayed and selected using yeast surface display to identify the best binders. This allows for the generation of high-affinity clones in a very short time (approximately two weeks), significantly shorter than traditional affinity maturation procedures. This method can be applied using synthetic or immune libraries, non-enriched libraries, enriched libraries, or subsets of preselected clones.

[0144] If intermediate affinity binders are needed and the affinity of identified candidates needs to be reduced, as in the case of anti-TNFR2 V-bodies, very similar techniques can be applied. For example, the same targeted or non-targeted approaches described for affinity maturation can be used to introduce affinity-reducing mutations. Subsequent selection can be adapted accordingly. If larger libraries are generated and need to be screened by display techniques, the selection strategy can be adapted to enrich for intermediate affinity binders while eliminating high affinity candidates. This can be done, for example, by pre-panning with phage display at low antigen concentrations to remove all high affinity candidates, followed by selection at high antigen concentrations to obtain intermediate affinity VHHs. With library sizes up to 1000 candidates, characterization of kinetic dissociation rates can be used to obtain immediate information on the kinetic behavior of the candidates.

[0145] Once the most potent clones are identified, their DNA sequences can be optimized to improve their stability against enzymes, for example. Another goal is humanization to prevent immune responses in the human organism against the antibody. Humanization can be achieved based on homology between camelid VHH and human VH fragments, as described in more detail below. Finally, optimized single domain antibodies can be translated and expressed in a suitable organism, such as E. coli or Saccharomyces cerevisiae.

[0146] Single-domain antibodies may be derived from conventional antibodies. In some embodiments, single-domain antibodies can be generated from conventional four-chain mouse or human IgG. The process is similar and involves gene libraries from immunized or naive donors and display techniques to identify the most specific antigens. However, the binding region of conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate due to their lipophilicity. Monomerization can be achieved by replacing lipophilic amino acids with hydrophilic amino acids. (See, e.g., Borrebaeck, CAK; Ohlin, M. (2002). "Antibody evolution beyond Nature". Nature Biotechnology 20 (12): 1189-90.) If affinity can be maintained after monomerization, the single-domain antibody can also be produced in Escherichia coli, Saccharomyces cerevisiae, or other suitable organisms.

[0147] A "humanized antibody" refers to a genetically engineered chimeric antibody in which amino acid sequences (typically CDRs) from an antibody (donor antibody), e.g., a camelid antibody, have been grafted onto a human antibody (acceptor antibody). A humanized antibody thus typically comprises CDRs from the donor antibody and variable region frameworks and (if present) constant regions from a human antibody. A "humanized VHH" thus comprises CDRs that correspond to the CDRs of a naturally occurring VHH domain (e.g., a camelid VHH), but which have been "humanized." Humanized VHHs can be prepared by substituting one or more amino acid residues in the amino acid sequence (particularly in the framework sequences) of a naturally occurring VHH sequence with one or more amino acid residues occurring at the corresponding positions in a VH domain derived from a conventional four-chain human antibody. Such humanized VHHs can be obtained by any suitable method known to those skilled in the art and therefore not strictly limited to the methods described herein.

[0148] Humanization of VHHs can be achieved using resurfacing or CDR-grafting. Resurfacing strategies are described, for example, in Conrath et al., 2005 J Mol Biol; Kazemi-Lomedasht et al., 2018; Vincke et al., 2009 J Biol Chem, and CDR-grafting strategies are described, for example, in ben Abderrazek et al., 2011; van Faassen et al., 2020 FASEB; Li et al., 2018; Vaneycken et al., 2010; Vincke et al., 2009 J Biol Chem; and Yu et al., 2017 (each of which is incorporated herein by reference in its entirety).

[0149] To humanize camelid VHHs using the resurfacing approach, a human germline reference sequence most similar to the camelid germline sequence of the selected VHH can be identified. The majority of isolated camelid VHHs in the literature belong to the camelid IGHV3 subfamily 2 (Nguyen et al., 2000, EMBO J), and DP-47 / VH3-23 from the IGHV3 family is commonly used as a human reference. The framework of the camelid VHH can then be compared to the human reference sequence. Surface-exposed residues are likely to contribute less to protein stability and are therefore replaced with the corresponding human residues. However, buried residues are likely to contribute more to overall VHH stability and therefore remain of camelid origin. While humanization of framework regions 1, 3, and 4 typically does not affect the biochemical properties of the VHH, global humanization of framework 2 significantly increases the local hydrophobicity. Residues H37, H44, H45, and H47 (Chothia numbering), the so-called tetrade or hallmark residues in framework 2, are partially buried in human VHs (VGLWs) and involved in VH / VL pairing, resulting in some hydrophobicity. However, in camelid VHHs, these residues are partially charged (FERG), significantly increasing the solubility of VHHs and inhibiting camelid VL pairing (Soler et al., 2021, Biomolecule; Conrath et al., 2005 J Mol Biol). Furthermore, residues H37 and H47 are known to contribute to antigen-binding affinity by interacting with the CDR-H3 loop of many VHHs to stabilize their conformation. Furthermore, a significant number of VHHs utilize framework 2 residues H44, H45, and H47 for antigen binding (Zavrtanik et al., 2018, J Mol Biol). Therefore, full humanization of these residues often results in reduced solubility or aggregation of the VHHs, and reduced or complete loss of binding affinity to the target antigen (van Faassen et al., 2020, Vincke et al., 2009).Consequently, when humanizing VHHs, all or at least some of these hallmark residues within framework 2 remain of Camelidae origin.

[0150] Another approach that can be applied to humanize VHHs is CDR grafting. The CDRs of a selected VHH can be grafted onto a partially or fully humanized universal VHH framework (Saerens et al., 2009 J Biol Chem, Soler et al., 2021, Vincke et al., 2009 J Biol Chem). While CDR grafting has been successful in some cases, it has failed in other cases, resulting in VHHs often losing their ability to bind to the desired antigen and / or becoming structurally unstable and prone to aggregation (van Faassen et al., 2020, FASEB). This is primarily due to interactions between CDR3 and specific residues in framework 2 that are important for CDR3 conformation, overall VHH stability, and overall hydrophobicity, which are compromised by this approach. Camelid back mutations can also be introduced into the framework to compensate for these effects (van Faassen et al., 2020, FASEB).

[0151] An alternative strategy that alleviates the need to humanize selected VHH sequences is to use libraries of fully or partially humanized synthetic VHHs instead of Camelidae immune libraries for VHH discovery (Moutel et al. 2016, eLife; McMahon, 2018, NSMB; Zimmermann et al., 2018, eLife). In many of these libraries, the hallmark residues are still of Camelidae origin, for the reasons mentioned above.

[0152] Other suitable humanizing substitutions are described in WO 09 / 138519 and WO 08 / 020079, as well as Tables A-3 to A-8 of WO 08 / 020079 (lists of possible humanizing substitutions), each of which is incorporated herein by reference in its entirety. Non-limiting examples of such humanizing substitutions include Q108L and A14P. Such humanizing substitutions may be appropriately combined with one or more other mutations described herein (e.g., with one or more mutations that reduce binding by a pre-existing antibody).

[0153] In some embodiments, the humanized VHH sequence still retains residues associated with binding to Protein A. In some embodiments, engineering can be performed during humanization to confer Protein A binding properties to VHHs that did not interact with Protein A (Graille et al., 2000, PNAS).

[0154] Similar to a "humanized antibody," a "camelized antibody" refers to an antibody having amino acid sequences (typically CDRs) derived from a donor antibody, e.g., a human antibody, and variable region framework and (if present) constant regions derived from a camelid antibody. A "camelized VH" thus comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but which has been "camelized." A camelized VH can be prepared by substituting one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional four-chain antibody with one or more of the amino acid residues occurring at the corresponding positions in a VHH domain of a heavy-chain antibody. This can be done, for example, as described in WO 2008 / 020079. Such "camelizing" substitutions are typically inserted at amino acid positions forming and / or present at the VH-VL interface and / or at the so-called Camelidae hallmark residues, e.g., F37, E44, R45, and F47 (see, e.g., WO 94 / 04678; Davies and Riechmann (1994 and 1996)). In one embodiment, the VH sequence used as starting material or starting point for the generation or design of a camelized VH is a VH sequence of mammalian origin or a VH sequence of a human antibody. However, such a camelized VH can be obtained by any suitable method known to those skilled in the art and is therefore not strictly limited to polypeptides obtained using as starting material a polypeptide comprising a naturally occurring VH domain.

[0155] The amino acid residues of single-domain antibodies can be numbered according to the general numbering scheme for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelidae described by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see, e.g., Figure 2 of this publication). The total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. For example, one or more positions according to the Kabat numbering may be unoccupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering. Consequently, the Kabat numbering may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain or VHH domain is typically 110 to 120, and often 112 to 115. However, shorter and longer sequences may also be suitable for the purposes described herein.

[0156] The CDR regions of single-domain antibodies can be determined using the methods described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, January 2003; 27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun. 8; 309(3):657-70 ("Aho" numbering scheme); and Martin et al.This can be accomplished using a variety of methods, including those described in "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272 ("AbM" numbering scheme) (each reference cited herein is incorporated by reference in its entirety).

[0157] The boundaries of a given CDR or framework (FR) may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia numbering schemes are based on the length of the most common antibody region sequence, with insertions addressed by inserted letters, e.g., "30a," and deletions found in some antibodies. The two schemes place certain insertions and deletions ("indels") in different locations, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions and is based on the definitions used by Oxford Molecular's AbM antibody modeling software.

[0158] In some embodiments, CDRs can be defined according to any of the Kabat numbering scheme, the Chothia numbering scheme, a combination of Kabat and Chothia, the AbM numbering scheme, and / or the Contact numbering scheme. VHHs typically contain three CDRs, designated CDR1, CDR2, and CDR3. Tables 1-3 below list exemplary boundary positions for CDR-H1, CDR-H2, and CDR-H3, as identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbers are indicated using both the Kabat and Chothia numbering schemes. FRs are located between the CDRs; for example, FR-H1 precedes CDR-H1, FR-H2 is located between CDR-H1 and CDR-H2, and FR-H3 is located between CDR-H2 and CDR-H3, etc. Note that in the Kabat numbering scheme shown, the insertions are located at H35A and H35B, so the ends of the Chothia CDR-H1 loop numbered using the Kabat numbering rules shown vary between H32 and H34 depending on the length of the loop. [Table 1-3]

[0159] Thus, unless otherwise specified, a "CDR" or "complementarity determining region" or a designated individual CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of an antibody or a region thereof, such as a variable region, is understood to encompass a (specific) CDR defined by any of the above-described schemes. For example, when a specific CDR (e.g., CDR-H3) is described as comprising the amino acid sequence of a corresponding CDR in a VHH amino acid sequence, such CDR is understood to have the sequence of the corresponding CDR (e.g., CDR-H3) in the VHH, as defined by any of the above-described schemes. In some embodiments, specific CDR sequences are specified. While exemplary CDR sequences of the provided antibodies are described using various numbering schemes (see, e.g., Tables 1-3), it is understood that the provided antibodies may comprise CDRs described according to any other of the above-described numbering schemes or other numbering schemes known to those of skill in the art.

[0160] In the single domain antibody sequences of the present disclosure, the framework sequences may be any suitable framework sequences. For example, the framework sequences may be framework sequences from a heavy chain variable domain (e.g., a VH sequence or a VHH sequence). In some embodiments, the framework sequences are framework sequences from a VHH sequence (in which case the framework sequences may optionally be partially or fully humanized) or conventional VH sequences (in which case the framework sequences may optionally be partially or fully camelized).

[0161] Antigen-binding fragments (or combinations of fragments) of any of the single domain antibodies described herein, for example fragments comprising one or more CDR sequences and suitably flanked and / or linked via one or more framework sequences, are also encompassed within the scope of the present disclosure.

[0162] However, it should be noted that the present disclosure is not limited by the origin of the single domain antibody (or of the nucleotide sequence used to express it), nor by the method for producing or obtaining said single domain antibody or nucleotide sequence. Thus, antigen binding proteins of the present disclosure may comprise naturally occurring sequences (derived from a suitable species), recombinant sequences, or synthetic or semi-synthetic sequences. Similarly, nucleotide sequences encoding antigen binding proteins of the present disclosure may comprise naturally occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (e.g., sequences prepared by PCR or sequences isolated from a library).

[0163] Anti-TNFR2 antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) of the present disclosure may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy chain variable domain compared to the exemplary antibody sequences provided herein. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. An antigen-binding molecule of the present disclosure may comprise an antigen-binding domain derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework and / or CDR regions are mutated to one or more corresponding residues in the germline sequence from which the antibody is derived, or to one or more corresponding residues in another germline sequence, or to conservative amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and / or CDR residues in the VHH domain are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of one or more framework and / or CDR residues are mutated to the corresponding one or more residues in a different germline sequence (i.e., a different germline sequence from the germline sequence from which the antigen-binding domain was originally derived).

[0164] Furthermore, an antigen-binding domain may comprise any combination of two or more germline mutations in the framework and / or CDR regions, e.g., individual specific residues are mutated to the corresponding residue in a particular germline sequence, while other specific residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once an antigen-binding domain containing one or more germline mutations is obtained, it can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced biological properties (e.g., agonist effect), reduced immunogenicity, etc. Antigen-binding proteins comprising one or more antigen-binding domains obtained by such general methods are encompassed within the scope of the present disclosure.

[0165] Provided herein are anti-TNFR2 antigen binding proteins comprising variants of any of the VHH and / or CDR amino acid sequences disclosed herein having one or more amino acid substitutions. For example, the present disclosure includes anti-TNFR2 antigen binding proteins having VHH and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, 3 or less, 2, or 1 amino acid substitutions relative to any of the VHH and / or CDR amino acid sequences set forth in Tables 1-1 and 1-2 herein. Amino acid substitutions may be introduced into the antigen binding protein of interest, and the resulting variants can be screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or reduced ADCC or CDC.

[0166] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. In some embodiments, the amino acid substitutions are conservative, meaning that an amino acid is replaced with another amino acid of the same class. In some embodiments, amino acid substitutions can also include non-conservative substitutions, meaning that an amino acid is replaced with an amino acid of a different class. Other exemplary amino acid substitutions are shown in Tables 1-4. [Table 1-4]

[0167] In some embodiments, single domain antibodies (e.g., VHHs) of the present disclosure comprise one or more modifications that reduce binding of the single domain antibody (e.g., VHHs) to pre-existing antibodies found in human blood or serum. In some embodiments, single domain antibodies (e.g., VHHs) of the present disclosure are modified by a mutation at amino acid position 11, e.g., Leu11Glu (L11E), Leu11Lys (L11K), or Leu11Val (L11V). In one embodiment, a single domain antibody (e.g., VHH) of the present disclosure may comprise a valine (V) at amino acid position 11 (according to the Kabat numbering) and a leucine (L) at amino acid position 89. As another example, a single domain antibody (e.g., VHH) of the present disclosure may comprise a 1-5 (naturally occurring) amino acid extension, such as a single alanine (A) extension, at the C-terminus of the single domain antibody (e.g., VHH). The C-terminus of a VHH is typically VTVSS (SEQ ID NO: 4031). In one embodiment, a single domain antibody (e.g., a VHH) of the disclosure comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering). In another embodiment, a single domain antibody (e.g., a VHH) of the disclosure comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering). Thus, the C-terminus of a single domain antibody (e.g., a VHH) can be any one of VKVSS (SEQ ID NO: 4032), VQVSS (SEQ ID NO: 4033), VTVKS (SEQ ID NO: 4034), VTVQS (SEQ ID NO: 4035), VKVKS (SEQ ID NO: 4036), VKVQS (SEQ ID NO: 4037), VQVKS (SEQ ID NO: 4038), or VQVQS (SEQ ID NO: 4039).In another embodiment, a single domain antibody (e.g., VHH) of the disclosure comprises a valine (V) at amino acid position 11 (according to Kabat numbering), a leucine (L) at amino acid position 89, and optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering), and one to five (naturally occurring) amino acid extensions, such as a single alanine (A) extension, at the C-terminus of the single domain antibody (e.g., VHH) (thus the C-terminus of a single domain antibody (e.g., VHH) has, for example, the sequence VTVSSA (SEQ ID NO: 4040), VKVSSA (SEQ ID NO: 4041), or VQVSSA (SEQ ID NO: 4042)). In a further embodiment, a single domain antibody (e.g., VHH) of the disclosure is modified by altering the carboxy-terminal region, for example to a terminal sequence having the sequence GQGTLVTVKPGG (SEQ ID NO: 4043) or GQGTLVTVEPGG (SEQ ID NO: 4044), or to a variant thereof. Further modifications to reduce binding by pre-existing antibodies in human serum can be found, for example, in WO 2012 / 175741, WO 2015 / 173325, WO 2016 / 150845, WO 2011 / 003622, WO 2013 / 024059; U.S. Pat. No. 11,426,468, U.S. Pat. No. 10,526,397, each of which is incorporated herein by reference in its entirety.

[0168] In one embodiment, a single domain antibody (e.g., a VHH) of the disclosure comprises the amino acid sequence VAGG (SEQ ID NO: 4697) or VPAG (SEQ ID NO: 4698) at its carboxy terminus starting at position 111 according to Chothia. In one embodiment, a single domain antibody (e.g., a VHH) of the disclosure comprises the amino acid sequence VAGG (SEQ ID NO: 4697) at its carboxy terminus starting at position 111 according to Chothia. In one embodiment, a single domain antibody (e.g., a VHH) of the disclosure comprises the amino acid sequence VPAG (SEQ ID NO: 4698) at its carboxy terminus starting at position 111 according to Chothia.

[0169] In some embodiments, a single domain antibody (e.g., a VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, 4521, 93-640, 4079-4125, 2805-3363, 4359-4420, and 4605-4628, or a sequence with at least 75% identity thereto, wherein the amino acid sequence comprises VAGG (SEQ ID NO: 4697) or VPAG (SEQ ID NO: 4698) at the carboxy terminus starting at position 111 according to Chothia.

[0170] In some embodiments, a single domain antibody (e.g., a VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, and 4521, or a sequence with at least 75% identity thereto, wherein the amino acid sequence comprises VAGG (SEQ ID NO: 4697) or VPAG (SEQ ID NO: 4698) at the carboxy terminus starting at position 111 according to Chothia.

[0171] In some embodiments, a single domain antibody (e.g., a VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 4076-4078, 4523, 4526, 4529, 4532, 4731-4734, 641-1127, and 4126-4172, or a sequence having at least 75% identity thereto, wherein the amino acid sequence comprises VAGG (SEQ ID NO: 4697) or VPAG (SEQ ID NO: 4698) at the carboxy terminus starting at position 111 according to Chothia.

[0172] In some embodiments, a single domain antibody (e.g., a VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 4076-4078, 4523, 4526, 4529, 4731-4734, and 4532, or a sequence having at least 75% identity thereto, wherein the amino acid sequence comprises VAGG (SEQ ID NO: 4697) or VPAG (SEQ ID NO: 4698) at the carboxy terminus starting at position 111 according to Chothia.

[0173] In some embodiments, single-domain antibodies (e.g., VHHs) of the present disclosure are modified to enhance binding to Staphylococcal protein A (SpA) or Streptococcal protein G (SpG). The binding of SpA and SpG to antibodies or antibody fragments can be useful in the manufacturing process of the antibodies or antibody fragments. The high-affinity interaction between the IgG Fc region and SpA and SpG has been widely utilized and is the gold standard for monoclonal antibody purification (Bjoerck and Kronvall, 1984). Other non-Fc-containing antibody fragments, such as VHHs and Fabs, do not have the ability to bind SpA or SpG via their Fc regions. However, these Fc-free antibody fragments have been demonstrated to interact with SpA in a sequence-dependent manner (Graille et al., 2000; Henry et al., 2016). This feature avoids the potential use of affinity tags fused to drug candidates for affinity chromatography, which have sequence-related disadvantages. This is because it may affect the immunogenicity of the protein, as well as the structure and stability of the protein, potentially compromising functionality. The interaction of single-domain antibodies (e.g., VHH) with SpA relies on a different binding mode, with affinities of 1-5 μM, comparable to the 0.2-3 μM measured for VH-SpA interactions (To et al., JBC, 2005; Henry et al., Plos One, 2016).

[0174] In some embodiments, a single domain antibody (e.g., a VHH) of the present disclosure contains or is engineered to contain an SpA-binding motif. For example, the VHH-SpA interface has been mapped to 13 residues clustered within the framework of the dorsal side of the V-body, away from the CDRs (Graille et al., 2000, Henry et al., 2016). In the absence of a VHH-SpA co-structure, the binding mode can be visualized by superimposing the SpA-Fab crystal structure with the VHH. Based on structural and functional analyses, 13 residues at the VHH-SpA interface have been characterized as either intolerant to substitutions (residues Gly15, Arg19, Tyr59, Gly65, and Arg66), tolerant to specific substitutions (residues Thr / Lys / Arg57, Thr68, Gln81, Asn82a, and Ser82b), or tolerant to a variety of substitutions overall (residues Ser17, Lys64, and Ser70) (all residue positions refer to Kabat numbering) (Henry et al., Plos One, 2016). Thus, the SpA-binding motif contained in a single domain antibody (e.g., a VHH) of the present disclosure may comprise one or more, or all, of these 13 residues.

[0175] In some embodiments, single domain antibodies (e.g., VHHs) of the present disclosure comprise one or more modifications at the N-terminus to prevent pyroglutamic acid formation and product heterogeneity, in one embodiment, the amino acid residue Glu at the first position of the single domain antibody (e.g., VHH) is substituted with Asp (E1D).

[0176] In some embodiments, a single domain antibody (e.g., a VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, 4521, 93-640, 4079-4125, 2805-3363, 4359-4420, and 4605-4628, or a sequence with at least 75% identity thereto, wherein the amino acid residue Glu at the first position of the single domain antibody (e.g., a VHH) is substituted with Asp (E1D).

[0177] In some embodiments, a single domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, and 4521, or a sequence with at least 75% identity thereto, wherein the amino acid residue Glu at the first position of the single domain antibody (e.g., VHH) is substituted with Asp (E1D).

[0178] In some embodiments, a single domain antibody (e.g., a VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 4076-4078, 4523, 4526, 4529, 4532, 4731-4734, 641-1127, and 4126-4172, or a sequence having at least 75% identity thereto, wherein the amino acid residue Glu at the first position of the single domain antibody (e.g., a VHH) is substituted with Asp.

[0179] In some embodiments, a single domain antibody (e.g., a VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 4076-4078, 4523, 4526, 4529, 4731-4734, and 4532, or a sequence having at least 75% identity thereto, wherein the amino acid residue Glu at the first position of the single domain antibody (e.g., a VHH) is substituted with Asp (E1D).

[0180] Alternative protein scaffolds In some embodiments, the anti-TNFR2 antigen binding proteins of the present disclosure may also employ alternative protein scaffolds. Such alternative protein scaffolds may be single-chain polypeptide frameworks, optionally reduced in size (e.g., less than about 200 amino acids), that comprise a highly structured core associated with variable domains with high conformational tolerance, allowing for insertions, deletions, or other substitutions. Such antigen binding proteins can be generated by grafting the CDRs or variable regions described herein onto a suitable protein scaffold. The structure of the alternative scaffolds can vary, but preferably is of human origin for those developed as therapeutics.

[0181] The alternative protein scaffolds of the present disclosure can be based on traditional immunoglobulin (Ig) scaffolds or can be derived from entirely unrelated proteins, whose variable domains can be engineered to form novel binding interfaces for any target antigen. In some embodiments, alternative protein scaffolds of the disclosure may be derived from Protein A, e.g., its Z domain (affibody), ImmE7 (immunity protein), BPTI / APPI (Kunitz domain), Ras-binding protein AF-6 (PDZ-domain), charybdotoxin (scorpion venom), CTLA-4, Min-23 (knottin), lipocalin (anticalin), neocarzinostatin, fibronectin domain (used in "adnectins"), ankyrin repeat (AR) domain (used in "DARPins"), avidity multimers (also known as "avimers"), or thioredoxin (Skerra, A., Curr. Opin. Biotechnol. 18:295-304 (2005); Hosse et al., Protein Sci. 15:14-27 (2006); Nicaise et al., Protein Sci. 13:1882-1891 (2004); Nygren and Uhlen, Curr. Opin. Struc. Biol. 7:463-469 (1997) (all of which are incorporated by reference in their entireties into this application).

[0182] Anticalins are a type of non-Ig-based alternative scaffold suitable for use in the antigen-binding molecules of the present disclosure. Anticalins are a class of engineered ligand-binding proteins based on the lipocalin scaffold. Lipocalins are a family of proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have limited sequence homology but share a common tertiary structure architecture based on eight antiparallel β-barrels. Lipocalins contain four exposed loops built on a rigid β-barrel structure. An exemplary anticalin protein commonly used is approximately 180 amino acids in size and approximately 20 kDa in mass.

[0183] DARPins are another suitable non-Ig-based alternative scaffold that can be used in the antigen-binding molecules of the present disclosure. DARPins are engineered antibody-mimetic proteins that typically exhibit high specificity and high affinity target protein binding. They are derived from natural ankyrin repeat (AR) proteins, which typically contain a 33-amino acid protein motif consisting of two α-helices separated by a loop, which mediates protein-protein interactions. DARPins can be generated using combinatorial AR libraries constructed based on the 33-amino acid AR motif with seven randomized positions. DARPin libraries can be screened using ribosome display, and library members typically produce well in Escherichia coli, do not aggregate, and exhibit high thermodynamic stability. Preferably, DARPins contain two to four of these motifs, flanked by N- and C-terminal capping motifs to shield hydrophobic regions and improve solubility.

[0184] Avimers can also be used as protein scaffolds to generate suitable non-Ig-based alternative scaffolds. Avimers typically consist of two or more peptide sequences, each 30–35 amino acids long, connected by a peptide linker. Each sequence is derived from the A domain of various membrane receptors and possesses a rigid structure stabilized by disulfide bridges and calcium. Each A domain can bind to a specific epitope on a target protein. Combining domains that bind different epitopes of the same protein increases affinity for that protein, an effect known as avidity.

[0185] Proteins derived from the fibronectin III (FN3) domain can also be used to generate suitable non-Ig-based alternative scaffolds (also known as "monobodies"). For example, the 10th fibronectin type III domain (FN10) of human fibronectin corresponds to a β-sandwich with seven β-strands and three connecting loops, exhibiting structural homology to Ig domains without disulfide bridges. In some cases, the connecting loops of FN10, each approximately 15-21 amino acids in length, can be randomized and the domain displayed on both phage and yeast to select scaffolds with desired properties. Adnectin™ is an exemplary scaffold generated using the 10th FN3 domain randomized and displayed in this manner. Another exemplary scaffold containing the FN3 domain is Centrytin™. Centrytin™ contains the consensus sequence of the FN3 domain of human tenascin C (TNC), which is found in the extracellular matrix of various tissues. The Centritin™ scaffold is a small (approximately 10 kDa), simple, and highly stable single-domain protein with loops that share structural homology with antibody variable domains (i.e., CDR1, CDR2, and CDR3) and contain no cysteines, disulfides, or glycosylated residues. Centritin™ has excellent biophysical properties, including stability to heat, pH, denaturants, and organic solvents, reversible unfolding, and monodispersity. Another recent exemplary FN3-based scaffold that can be used in the present disclosure is the fluctuation-regulated affinity protein (FLAP), as described in See et al., 2020. Biotechnology Journal 15(12):e2000078, incorporated herein by reference in its entirety.

[0186] Fusion Proteins and Conjugates In certain aspects, provided herein are fusion proteins and conjugates comprising at least one anti-TNFR2 antigen binding protein (e.g., an antibody, such as a single-domain antibody) directly or indirectly linked to one or more additional domains or moieties. In some embodiments, the fusion proteins or conjugates of the present disclosure comprise a single polypeptide. In other embodiments, the fusion proteins or conjugates of the present disclosure comprise two or more polypeptides. In some embodiments, the fusion proteins or conjugates of the present disclosure comprise two polypeptides.

[0187] In some embodiments, a fusion protein or conjugate of the present disclosure comprises at least one anti-TNFR2 antigen binding protein (e.g., an antibody, such as a single domain antibody) described herein. In some embodiments, the fusion protein or conjugate is multivalent. For example, a fusion protein or conjugate of the present disclosure can be at least bivalent, but can also be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc. The terms "bivalent," "trivalent," "tetravalent," "pentavalent," or "hexavalent" are all included within the term "multivalent" and indicate the presence of two, three, four, five, or six binding units (e.g., VHHs), respectively.

[0188] In certain embodiments, the fusion protein or conjugate is multispecific. For example, in some cases, the one or more additional domains or moieties can be one or more additional binding domains that bind to one or more additional antigens or proteins. The fusion proteins or conjugates of the present disclosure can be, for example, bispecific, trispecific, tetraspecific, pentaspecific, etc. The terms "bispecific," "trispecific," "tetraspecific," "pentaspecific," etc. are all encompassed by the term "multispecific" and refer to binding to two, three, four, five, etc. different target molecules, respectively.

[0189] When two or more anti-TNFR2 antigen-binding proteins are included in a fusion protein or conjugate, the two or more anti-TNFR2 antigen-binding proteins may comprise the same sequence or different sequences. In such embodiments, the two or more anti-TNFR2 antigen-binding proteins may bind to the same epitope on TNFR2, or may bind to different epitopes on TNFR2. For example, a fusion protein or conjugate of the present disclosure may be biparatopic, e.g., when two VHHs bind to two different epitopes on TNFR2.

[0190] An exemplary design of a multivalent anti-TNFR2 fusion construct comprising two or more anti-TNFR2 binding units (eg, VHHs) is shown in FIG.

[0191] Fusion or conjugation to the Fc region In some embodiments, a fusion protein or conjugate of the disclosure comprises at least one anti-TNFR2 antigen binding protein (e.g., an antibody, such as a single-domain antibody) provided herein operably linked to a dimerization domain, such as an immunoglobulin Fc region. The immunoglobulin Fc region may be indirectly or directly linked to at least one anti-TNFR2 antigen binding protein (e.g., an antibody, such as a single-domain antibody). In some embodiments, a fusion protein or conjugate of the disclosure comprises one, two, three, four, five, six, or even more anti-TNFR2 antigen binding proteins provided herein operably linked to Fc regions.

[0192] As used herein, "Fc region" refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, when the Fc region comprises a hinge, the hinge can mediate dimerization between two Fc-containing polypeptides. In various embodiments, the Fc region included in the fusion protein or conjugate of the present disclosure is a human immunoglobulin Fc region or is derived from a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is of the IgG, IgE, IgM, IgD, IgA, or IgY isotype. In some embodiments, the immunoglobulin Fc region is an IgG isotype, such as an IgG1, IgG2, IgG3, or IgG4 subclass. The immunoglobulin Fc region may comprise a variant or fragment of a native IgG Fc region.

[0193] Native Fc regions typically possess effector functions, including, but not limited to, Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Such effector functions generally require combining the Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0194] In some embodiments, a fusion protein or conjugate of the present disclosure can comprise a dimer of an Fc region. In some embodiments, the Fc region mediates dimerization of TNFR2-binding units under physiological conditions, such as when expressed from a cell, thereby forming a dimer that doubles the number of TNFR2-binding units. For example, a fusion polypeptide comprising one VHH domain that binds to TNFR2 and an Fc region is monovalent as a monomer, but the Fc region can mediate dimerization, resulting in a bivalent fusion protein (i.e., two anti-TNFR2 VHH domains per molecule). Similarly, in some embodiments, two anti-TNFR2 VHH domains (2x) are fused to a single IgG Fc region, and dimerization results in a tetravalent fusion protein (i.e., four anti-TNFR2 VHH domains per molecule). In some embodiments, three anti-TNFR2 VHH domains (3x) are fused to one IgG Fc region, resulting in a pentavalent fusion protein upon dimerization (i.e., six anti-TNFR2 VHH domains per molecule).

[0195] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each having the following structure: (anti-TNFR2 VHH)n-linker-Fc, where n can be any integer (e.g., 1, 2, 3, 4, 5, etc.). When n≧2, each anti-TNFR2 VHH may optionally be operably linked to another anti-TNFR2 VHH via a linker.

[0196] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each having the following structure: (anti-TNFR2 VHH)n-linker-Fc-(anti-TNFR2 VHH)m, where n and m can independently be any integer (e.g., 1, 2, 3, 4, 5, etc.). When n≧2 or m≧2, each anti-TNFR2 VHH may optionally be operably linked to another anti-TNFR2 VHH via a linker.

[0197] In some embodiments, a fusion protein or conjugate of the present disclosure is bivalent. In some embodiments, a bivalent fusion protein or conjugate of the present disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-TNFR2 VHH)-linker-Fc.

[0198] In some embodiments, a fusion protein or conjugate of the present disclosure is tetravalent. In some embodiments, a tetravalent fusion protein or conjugate of the present disclosure comprises two polypeptide chains, each having the following structure: (anti-TNFR2 VHH)-linker-(anti-TNFR2 VHH)-linker-Fc. In some embodiments, a tetravalent fusion protein or conjugate of the present disclosure comprises two polypeptide chains, each having the following structure: (anti-TNFR2 VHH)-linker-Fc-linker-(anti-TNFR2 VHH). The linkers used in the fusion protein are not necessarily identical.

[0199] In some embodiments, a fusion protein or conjugate of the present disclosure is hexavalent. In some embodiments, a hexavalent fusion protein or conjugate of the present disclosure comprises two polypeptide chains, each having the following structure: (anti-TNFR2 VHH)-linker-(anti-TNFR2 VHH)-linker-(anti-TNFR2 VHH)-linker-Fc. In some embodiments, a hexavalent fusion protein or conjugate of the present disclosure comprises two polypeptide chains, each having the following structure: (anti-TNFR2 VHH)-linker-(anti-TNFR2 VHH)-linker-Fc-linker-(anti-TNFR2 VHH). In some embodiments, a hexavalent fusion protein or conjugate of the present disclosure comprises two polypeptide chains, each having the following structure: (anti-TNFR2 VHH)-linker-Fc-linker-(anti-TNFR2 VHH)-linker-(anti-TNFR2 VHH). The linkers used in the fusion protein are not necessarily identical.

[0200] In some embodiments, the CH3 domain of the Fc region can be used as a homodimerization domain, and the resulting fusion protein can be formed from two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated to enable heterodimerization. For example, incorporating a heterodimerization domain into the fusion protein can make the construct a heterodimeric fusion protein.

[0201] When dimers of Fc regions are used in the fusion proteins or conjugates of the present disclosure, the first and second Fc regions can be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second Fc regions can be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0202] In some embodiments, the Fc region included in the fusion protein or conjugate of the present disclosure can be mutated or modified. In some embodiments, the mutations include one or more amino acid substitutions to reduce the effector function of the Fc region. Various examples of mutations to the Fc region to alter, e.g., reduce, effector function are known and include any of those described below. Generally, the numbering of residues in an immunoglobulin heavy chain or portion thereof, such as the Fc region, is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0203] In some embodiments, the human IgG Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Non-limiting examples of amino acid modifications that can alter ADCC and / or CDC are described in Alegre et al, 1992 J Immunol, 148: 3461-3468; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Shields et al., 2001 JBC, 276(9): 6591-6604; Lazar et al., 2006 PNAS, 103(11): 4005-4010; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Moore et al., 2010 mAbs, 2(2): 181-189; and Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11, each of which is incorporated herein by reference in its entirety.

[0204] In some embodiments, the Fc region included in the fusion proteins or conjugates of the present disclosure exhibits reduced effector function (e.g., CDC and ADCC). Various in vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the fusion protein construct and / or its truncated components lack binding to FcγR (and thus likely lack ADCC activity) but retain FcRn binding ability. The primary cells mediating ADCC are NK cells, which express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described, for example, in U.S. Patent No. 5,500,362; U.S. Patent No. 5,821,337; Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986); and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987). Alternatively, non-radioactive assays, such as the ACTI™ non-radioactive cytotoxicity assay for flow cytometry or the CytoTox96™ non-radioactive cytotoxicity assay, may be employed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).C1q binding assays may be performed to confirm that the fusion protein construct, or its truncated components, is unable to bind C1q and therefore lacks CDC activity (see, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402). CDC assays may be performed to assess complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of binding to FcRn and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0205] Examples of mutations that enhance ADCC include mutations at Ser239 and Ile332, such as Ser239Asp and Ile332Glu (S239D, 1332E). Examples of mutations that enhance CDC include mutations at Lys326 and Glu333. In some embodiments, the Fc region is mutated at one or both of these positions, for example, Lys326Ala and / or Glu333Ala (K326A and E333A) using the Kabat numbering system.

[0206] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325), or Ala327 (A327), or Pro329 (P329). For example, Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A), or Pro329Gly (P329G), Asn325Glu (N325E), or Ala327Ser (A327S). In some embodiments, modifications within the Fc region reduce binding to Fc receptor gamma receptors (FcγR) while minimally affecting binding to neonatal Fc receptors (FcRn).

[0207] In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (Kabat numbering) to alter Fc receptor interaction, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat numbering) to alter Fc receptor interaction, e.g., Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat numbering) to alter Fc receptor interaction, e.g., Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A / L235A), or Leu234Val and Leu235Ala (L234V / L235A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro329Ala (L234A / L235A / P329A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Asp265 (Kabat numbering) to alter Fc receptor interactions, e.g., Asp265Ala (D265A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat numbering) to alter Fc receptor interactions, e.g., Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fc region of the fusion protein is modified at both amino acids 265 and 329.For example, Asp265Ala and Pro329Ala (D265A / P329A), or Asp265Ala and Pro329Gly (D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 265, for example, Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, for example, Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, 265, and 329. For example, Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding. For example, Gly235 is deleted from the fusion protein. In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 to enhance interaction with CD32A. For example, Gly236Ala (G236A). In some embodiments, the human IgG1 Fc region lacks Lys447 (Kabat et al 1991 Sequences of Proteins of Immunological Interest, EU index).

[0208] In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, e.g., Leu234Gly, Leu235Ser, Gly236Arg (L234G / L235S / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, e.g., Leu234Ser, Leu235Thr, Gly236Arg (L234S / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, e.g., Leu234Ser, Leu235Val, Gly236Arg (L234S / L235V / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, e.g., Leu234Thr, Leu235Gln, Gly236Arg (L234T / L235Q / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236, e.g., Leu234Thr, Leu235Thr, Gly236Arg (L234T / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329, e.g., Leu234Thr, Leu235Thr, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 252, 254, and 256. For example, Met252Tyr, Ser254Thr, Thr256Glu (M252Y / S254T / T256E).

[0209] In some embodiments, the Fc region of the fusion protein lacks amino acids at one or more of the following positions: Glu233 (E233), Leu234 (L234), or Leu235 (L235), thereby reducing binding to Fc receptors. In some embodiments, the Fc region of the fusion protein lacks amino acids at one or more of the following positions: Glu233 (E233), Leu234 (L234), or Leu235 (L235), and is modified at one or more of Asp265 (D265), Asn297 (N297), or Pro329 (P329), thereby reducing binding to Fc receptors. For example, the Fc region included in the TNFR2-binding polypeptide is derived from a human Fc domain and includes a deletion of three amino acids in the lower hinge corresponding to IgG1 E233, L234, and L235. In some embodiments, such Fc polypeptides do not bind to FcγR and are therefore referred to as "effector silent" or "effector null." For example, Fc deletion of these three amino acids reduces binding to the complement protein C1q. In some embodiments, polypeptides having an Fc region with an Fc deletion of these three amino acids retain binding to FcRn, thereby increasing half-life and transcytosis associated with FcRn-mediated recycling.

[0210] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000020.tif40170

[0211] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000021.tif40170

[0212] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000022.tif38169

[0213] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000023.tif41170

[0214] In one embodiment, the immunoglobulin Fc region of the multispecific antigen binding protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000024.tif47169

[0215] In one embodiment, the immunoglobulin Fc region of the multispecific antigen binding protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000025.tif48170

[0216] In one embodiment, the immunoglobulin Fc region of the multispecific antigen binding protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000026.tif49170

[0217] In one embodiment, the immunoglobulin Fc region of the multispecific antigen binding protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000027.tif48170

[0218] In one embodiment, the immunoglobulin Fc region of the multispecific antigen binding protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000028.tif47170

[0219] In one embodiment, the immunoglobulin Fc region of the multispecific antigen binding protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000029.tif48170

[0220] In one embodiment, the immunoglobulin Fc region of the multispecific antigen binding protein is a variant of a human IgG1 Fc region having the following amino acid sequence: TIFF2026503077000030.tif48170

[0221] In some embodiments, the human IgG Fc region is modified to enhance binding to FcRn. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (Kabat numbering, Dall'Acqua et al. 2006, J. Biol Chem Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al. 2010 Nature Biotech, Vol. 28(2) 157-159), or Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L, respectively) (Kabat et al. 1991 EU Index of Sequences of Proteins of Immunological Interest).

[0222] In some embodiments, the Fc region lacks or has reduced fucose attached to the N-linked glycan chain at N297. There are numerous methods to prevent fucosylation, including, but not limited to, production in FUT8-deficient cell lines; addition of inhibitors, such as castanospermine, to mammalian cell culture media; and metabolic engineering of the production cell line.

[0223] In some embodiments, the Fc domain included in the fusion protein or conjugate of the present disclosure is derived from a human Fc domain and comprises the mutations M252Y and M428V. In some embodiments, the mutated or modified Fc polypeptide comprises the following mutations: M252Y and M428L, using the Kabat numbering system. In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5) but lose detectable binding at neutral pH (about 7.2), allowing for enhanced recycling via FcRn and extended half-life.

[0224] In some embodiments, the Fc domain included in the fusion protein or conjugate is derived from a human Fc domain and contains mutations to induce heterodimerization. In some embodiments, such mutations include those referred to as "knob" and "hole" mutations. For example, if a CH3 domain contains an amino acid modification at Thr366, substitution with a bulkier amino acid, such as Try (T366W), allows this CH3 domain to preferentially pair with a second CH3 domain containing less bulky amino acids at Thr366, Leu368, and Tyr407, such as Ser, Ala, and Val, respectively (T366S / L368A / Y407V). In some embodiments, a "knob" Fc domain contains the mutation T366W. In some embodiments, a "hole" Fc domain contains the mutations T366S, L368A, and Y407V. Heterodimerization through CH3 modifications can be further stabilized by introducing disulfide bonds, such as changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) in opposing CH3 domains (as described in Carter, 2001 Journal of Immunological Methods, 248:7-15). In some embodiments, the Fc domain used for heterodimerization contains an additional mutation, such as the S354C mutation on the first member of the heterodimeric Fc pair, which forms an asymmetric disulfide bond with the corresponding Y349C mutation on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair contains the modification H435R or H435K to prevent binding to Protein A while maintaining binding to FcRn. In some embodiments, one member of the heterodimeric Fc pair comprises the modification H435R or H435K, and the second member of the heterodimeric Fc pair is unmodified at H435. In various embodiments, the hole Fc domain comprises the modification H435R or H435K (referred to as "hole R" in some instances where the modification is H435R), and the knob Fc domain does not comprise the modification.In some instances, the hole R mutation improves purification of the heterodimer over any homodimeric hole Fc domain that may be present.

[0225] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the fusion protein of the present disclosure is monomeric. For example, modification of residue Thr366 to a charged residue, such as Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0226] In some embodiments, the immunoglobulin Fc region of the fusion protein is of the human IgG3 isotype or a variant thereof. In one embodiment, the IgG3 Fc region is modified at amino acid Asn297 (Kabat numbering) to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG3 Fc region is modified at amino acid 435 to extend half-life, e.g., Arg435His (R435H). In some embodiments, the human IgG3 Fc region lacks Lys447 (EU index of Kabat et al. 1991).

[0227] In some embodiments, the immunoglobulin Fc region of the fusion protein is of the human IgG4 isotype or a variant thereof. In one embodiment, the human IgG4 Fc region is modified at amino acid 235 to alter Fc-receptor interaction, e.g., Leu235Glu (L235E). In some embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (Kabat numbering) to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG4 Fc region lacks Lys447 (EU index of Kabat et al. 1991).

[0228] In some embodiments, the IgG4 Fc region of the fusion protein is altered at amino acids 228 and 235, for example, Ser228Pro, Leu235Glu, or Leu235Ala (S228P / L235E, or S228P / L235A). In some embodiments, the IgG4 Fc region of the fusion protein is altered at amino acids 228, 234, and 235, for example, Ser228Pro, Phe234Ala, Leu235Glu, or Leu235Ala (S228P / F234A / L235E, or S228P / F234A / L235A). In some embodiments, the IgG4 Fc region of the fusion protein is altered at amino acids 228, 235, and 329. For example, Ser228Pro, Leu235Glu, and P329G (S228P / L235E / P329G).

[0229] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of a human IgG4 Fc region having the following amino acid sequence: TIFF2026503077000031.tif38170

[0230] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of a human IgG4 Fc region having the following amino acid sequence: TIFF2026503077000032.tif39170

[0231] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of a human IgG4 Fc region having the following amino acid sequence: TIFF2026503077000033.tif39170

[0232] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of a human IgG4 Fc region having the following amino acid sequence: TIFF2026503077000034.tif39170

[0233] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of a human IgG4 Fc region having the following amino acid sequence: TIFF2026503077000035.tif39170

[0234] Additional IgG4 heavy chain modifications suitable for use in the fusion proteins or conjugates of the present disclosure include those described in Tables 1 and 2 of Dumet et al., mAbs, 11:8, 1341-1350, which is incorporated herein by reference in its entirety.

[0235] In some embodiments, the fusion protein or conjugate comprises an immunoglobulin hinge region. In some embodiments, the hinge region functions as a linker connecting one or more TNFR2 binding units (e.g., VHH) to the Fc region. In other embodiments, the fusion protein can comprise a linker in addition to the hinge region connecting one or more TNFR2 binding units (e.g., VHH) to the Fc region. The hinge region can also be selected from any of the human IgG subclasses. For example, the fusion protein can comprise a modified IgG1 hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO: 3923), in which Cys220, which typically forms a disulfide bond with the C-terminal cysteine ​​of the light chain, has been mutated to serine, e.g., Cys220Ser (C220S). In other embodiments, the fusion protein comprises a truncated hinge having the sequence DKTHTCPPC (SEQ ID NO: 3924).

[0236] In some embodiments, the fusion protein or conjugate has a modified hinge from IgG4, which has the sequence ESKYGPPCPPC (SEQ ID NO: 3925), modified to prevent or reduce strand exchange (e.g., Ser228Pro (S228P)).

[0237] In alternative embodiments, the fusion proteins or conjugates of the present disclosure may include sequences other than the Fc region to achieve multimerization (e.g., dimerization). For example, the inclusion of an amino acid sequence containing at least one cysteine ​​residue can promote dimerization of two polypeptides by forming a disulfide bond between the two polypeptides. In some embodiments, such multimerization domains may include one or more cysteine ​​residues or short cysteine-containing peptides. Other multimerization domains include peptides or polypeptides that comprise or consist of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0238] Suitable mutations for use in the fusion proteins disclosed herein are also discussed, for example, in Wilkinson et al., Fc-engineered antibodies with immune effector functions completely abolished. PLoS One. 2021; WO 2021234402; U.S. Pat. No. 8,969,526; EP 3692065; and U.S. Pat. No. 7,083,784, each of which is incorporated herein by reference.

[0239] Fusion or conjugation to a half-life extending moiety In some embodiments, the fusion proteins or conjugates of the present disclosure may include one or more other moieties that provide the fusion protein or conjugate with increased (in vivo) half-life. By increased in vivo half-life is meant that the fusion protein or conjugate has an increased half-life within the body of a mammal, such as a human subject, after administration.

[0240] Non-limiting examples of half-life extending moieties suitable for use in the present disclosure include polyethylene glycol (PEG) molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc portions, and small proteins or peptides capable of binding to serum proteins.

[0241] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise a binding moiety capable of binding to a serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG. In one embodiment, a fusion protein or conjugate of the present disclosure may comprise a binding moiety capable of binding to human serum albumin. In one embodiment, the binding moiety is a single domain antibody (e.g., a VHH).

[0242] For example, but not by way of limitation, albumin conjugates described in, for example, WO 04 / 041865, WO 06 / 122787, WO 2012 / 175400, WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, WO 2018 / 134234 (each of which is incorporated herein by reference in its entirety) can be used in the fusion proteins or conjugates of the present disclosure.

[0243] Fusion or conjugation to cytokines In some embodiments, the fusion proteins or conjugates of the present disclosure may comprise one or more cytokine molecules. Non-limiting examples of cytokine molecules that can be conjugated include interleukin-2 (IL-2), transforming growth factor beta (TGF-β), thymic stromal lymphopoietin (TSLP), or variants or combinations thereof.

[0244] The cytokine IL-2 plays an important role in the activation and function of Tregs. Incorporation of IL-2 into the anti-TNFR2 fusion proteins or conjugates of the present disclosure can enhance the ability of the anti-TNFR2 antigen binding proteins to promote the expansion and stabilization of Tregs.

[0245] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each having the following structure: (anti-TNFR2 VHH)n-linker-Fc-(IL-2)m, where n and m are independently any integer (e.g., 1, 2, 3, 4, 5, etc.). When n≧2, each anti-TNFR2 VHH may optionally be operably linked to another anti-TNFR2 VHH via a linker. When m≧2, each anti-TNFR2 VHH may optionally be operably linked to another IL-2 via a linker.

[0246] In one embodiment, a fusion protein or conjugate of the present disclosure comprises two polypeptide chains, each having the following structure: (anti-TNFR2 VHH)-linker-Fc-(IL-2).

[0247] IL-2 fusion proteins can be prepared, for example, as described in U.S. Pat. No. 10,174,091, WO 2014 / 023752, and WO 2019 / 246404, each of which is incorporated herein by reference in its entirety.

[0248] In one embodiment, the IL-2 molecule used in the fusion protein or conjugate of the present disclosure has the amino acid sequence: TIFF2026503077000036.tif32170 and wild-type IL-2.

[0249] In some embodiments, the IL-2 molecule used in a fusion protein or conjugate of the present disclosure is a variant of IL-2 having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3926.

[0250] In one embodiment, the IL-2 molecule used in the fusion protein or conjugate of the present disclosure has the amino acid sequence: It is a variant of IL-2 with TIFF2026503077000037.tif34170.

[0251] Other suitable IL-2 variants that can be used in the fusion proteins or conjugates of the present disclosure include those described in U.S. Pat. Nos. 10,174,091, 10,174,092, 11,091,526, 11,091,527, WO 2016 / 164937, 9,580,486, 7,105,653, 9,616,105, 9,428, 567, U.S. Patent Application Publication No. 2017 / 0051029, U.S. Patent Application Publication No. 2014 / 0286898A1, WO 2014 / 153111, WO 2010 / 085495, WO 2016 / 014428, WO 2016 / 025385, and U.S. Patent Application Publication No. 2006 / 0269515 (each of which is incorporated herein by reference in its entirety).

[0252] Fusion or conjugation to other moieties The anti-TNFR2 antigen binding proteins (e.g., antibodies such as single domain antibodies) provided herein may be operably linked, directly or indirectly, to a second moiety, such as, but not limited to, a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulator, a cytokine, a cytotoxic agent, a small molecule drug, a chemotherapeutic agent, a therapeutic agent, a diagnostic agent, or a combination thereof.

[0253] In some embodiments, the conjugates of the present disclosure comprise a label, which is capable of generating a detectable signal. Such conjugates can be used for research or diagnostic purposes, such as in vivo detection of cancer. Preferably, the label is capable of directly or indirectly generating a detectable signal. For example, the label can be a radiopaque or radioactive isotope (such as 3H, 14C, 32P, 35S, 123I, 125I, or 131I); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound (such as fluorescein isothiocyanate, rhodamine, or luciferin); an enzyme (such as β-galactosidase, alkaline phosphatase, or horseradish peroxidase); an imaging agent; or a metal ion. In some embodiments, the label is a radioactive atom, such as 99Tc or 123I, for scintigraphic studies, or a spin label, such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron, for nuclear magnetic resonance (NMR) imaging. Zirconium-89 can also be complexed with various metal chelators and conjugated to antibodies, e.g., for PET imaging (WO 2011 / 056983).

[0254] Anti-TNFR2 antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) of the present disclosure may be conjugated to other moieties, such as epitope tags, for example, for purposes of purification or detection. Examples of such molecules useful for protein purification include molecules that display a structural epitope that can be recognized by a second molecule. This is commonly employed for protein purification by affinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule that can bind to the immobilized compound. Non-limiting examples of epitope tag molecules that can be conjugated to anti-TNFR2 antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) of the present disclosure, for example, for molecular recognition, include polyhistidine tags (His tags), myc tags, human influenza hemagglutinin (HA) tags, FLAG tags, maltose-binding protein, glutathione-S-transferase, biotin, and streptavidin. Conjugates containing the epitopes presented by these molecules can be recognized by complementary molecules such as maltose, glutathione, nickel-containing complexes, anti-FLAG antibodies, anti-myc antibodies, anti-HA antibodies, streptavidin, or biotin, respectively. For example, anti-TNFR2 antigen-binding proteins of the present disclosure conjugated to an epitope tag can be purified from complex mixtures of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc.) by treating the mixture with a solid-phase resin containing a complementary molecule that can selectively recognize and bind to the epitope tag of the TNFR2 antibody or fragment thereof. Examples of solid-phase resins include agarose beads, which are suitable for purification in aqueous solutions.

[0255] In some embodiments, a conjugate of the present disclosure may comprise one or more anti-TNFR2 VHH domains described herein conjugated to a therapeutic agent, which may be cytotoxic, cytostatic, or provide some therapeutic benefit. In some embodiments, the cytotoxic agent is a drug, chemotherapeutic agent, growth inhibitory agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragment thereof), or radioisotope (e.g., a radioconjugate). Such conjugates may be applicable, for example, for the treatment or prevention of diseases associated with autoreactive cytotoxic T cell activity. In some embodiments, the antibody-drug conjugates described herein may enable targeted delivery of the drug moiety to a target tissue (e.g., a tumor).

[0256] In some embodiments, the conjugates of the present disclosure comprise a toxin. In some embodiments, the toxin may be, for example, a bacterial toxin such as diphtheria toxin, a plant toxin such as ricin, a small molecule toxin such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993). Toxins can exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Examples of other therapeutic agents that can be conjugated to the anti-TNFR2 antigen binding proteins of the disclosure are described herein (see the "Methods of Treatment and Other Uses" section).

[0257] In some embodiments, anti-TNFR2 antigen binding proteins (e.g., antibodies such as single domain antibodies) of the present disclosure may be fused or conjugated to one or more moieties that facilitate delivery to the central nervous system (CNS) / brain. Moieties that can facilitate delivery of anti-TNFR2 antigen binding proteins to the CNS / brain may be, for example, peptides, polypeptides, small molecules, lipids, or synthetic polymers. Various approaches for delivering single domain antibodies to the brain are described in Pothin et al., Pharmaceutics 2020, 12(10), 937 (incorporated herein by reference in its entirety).

[0258] As a non-limiting example, an anti-TNFR2 antigen binding protein (e.g., an antibody, such as a single domain antibody) of the present disclosure may be fused or conjugated to a moiety (e.g., an antibody) that binds to the transferrin receptor (TfR) or the insulin receptor. The transferrin receptor (TfR) is highly expressed by brain capillary endothelial cells (BCECs), which form the blood-brain barrier (BBB), and has been used as a target for drug delivery to the brain. Monoclonal antibodies that bind to TfR, such as clone Ri7, have been shown to be internalized by BCECs in vivo. As another example, an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody) of the present disclosure may be conjugated to a hydrophobic fatty acid moiety, such as a C18 fatty acid (stearic acid), a C16 fatty acid (palmitic acid), or a C8 fatty acid (octanoic acid) moiety; or an amphiphilic block copolymer moiety, such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (Pluronic® or poloxamer) or poly(2-oxazoline). Various fatty acid moieties and block copolymer moieties available for delivery of proteins to the brain are described, for example, in Yi and Kabanov, J Drug Target. 2013; 21(10): 940-955, which is incorporated herein by reference in its entirety.

[0259] Exemplary methods for attaching moieties such as labels to binding proteins include those described in Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel and Meares, Elsevier, NY (1983); and Colcher et al., Meth. Enzymol., 121:802-16 (1986). Additional suitable methods for preparing the conjugates of the present disclosure include those described, for example, in WO 2009 / 067800, WO 2011 / 133886, and U.S. Patent Application Publication No. 2014322129, which are incorporated herein by reference in their entireties.

[0260] In some embodiments, the bond between the anti-TNFR2 antigen binding protein and the second moiety can be covalent or non-covalent, for example, via a non-covalent biotin-streptavidin interaction. In some embodiments, the second moiety can be attached to the anti-TNFR2 antigen binding protein using any of a variety of molecular biological or chemical conjugation and linking methods known in the art and described below. In some embodiments, the second moiety can be linked or conjugated to the anti-TNFR2 antigen binding protein described herein using a linker, such as a peptide linker, a cleavable linker, a non-cleavable linker, or a linker that aids in the conjugation reaction.

[0261] In some embodiments, an anti-TNFR2 antigen-binding protein (e.g., an antibody, such as a single-domain antibody) is conjugated to, for example, about 1 to about 20 moieties per molecule, optionally via linkers. In some embodiments, the one or more second moieties can be the same or different. The linker can be composed of one or more linker components. For covalent attachment of the antibody to the second moiety, the linker typically has two reactive functional groups, i.e., is bivalent from a reactive perspective. Bivalent linker reagents useful for attaching two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups, are described, for example, in Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242.

[0262] In some embodiments, linkers used in conjugates of the present disclosure may include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthiopentanoate" ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-I carboxylate ("SMCC"), or N-succinimidyl (4-iodoacetyl)aminobenzoate ("STAB"), or combinations thereof.

[0263] In some embodiments, the linker used in the conjugates of the present disclosure may comprise an amino acid residue. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues used in amino acid linker components can include naturally occurring amino acids, as well as minor amino acids and non-naturally occurring amino acid analogs such as citrulline. Amino acid linker components can be designed and optimized for selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0264] Conjugates of anti-TNFR2 antigen binding proteins (e.g., antibodies such as single domain antibodies) and a second moiety (e.g., a cytotoxic agent) can be made using a variety of bifunctional protein linking agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl substrates), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0265] The conjugates of the present disclosure can be prepared by a variety of methods, including: (1) reacting a nucleophilic group on a VHH domain with a bivalent linker reagent to form a covalent VHH-linker, which is then reacted with a drug moiety; or (2) reacting a nucleophilic group on a drug moiety with a bivalent linker reagent to form a covalent drug-linker, which is then reacted with a nucleophilic group on a VHH domain.

[0266] Nucleophilic groups on proteins, including antibodies (e.g., VHH domains), include, but are not limited to: (i) N-terminal amine groups, (ii) side-chain amine groups (e.g., lysine), (iii) side-chain thiol groups (e.g., cysteine), and (iv) sugar hydroxyl or amino groups on which the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can form covalent bonds with electrophilic groups on linker moieties and linker reagents, including: (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups. Additional nucleophilic groups can be introduced into proteins (e.g., antibodies, such as VHH domains) by reacting lysine with 2-iminothiolane (Traut's reagent), which converts the amine to a thiol. Reactive thiol groups can be introduced into proteins (eg antibodies such as VHH domains) by introducing one, two, three, four or even more cysteine ​​residues.

[0267] Conjugates, such as antibody-drug conjugates, can also be prepared by modifying antibodies, such as VHH domains, to introduce electrophilic moieties that can react with nucleophilic substituents on linker reagents or drugs. The sugars of glycosylated antibodies can be oxidized, for example, with periodate oxidation reagents to form aldehyde or ketone groups, which can react with amine groups on linker reagents or drug moieties. The resulting imine Schiff base groups can form stable bonds or can be reduced, for example, with borohydride reagents to form stable amine linkages. In one embodiment, the carbohydrate moiety of a glycosylated antibody can be reacted with galactose oxidase or sodium metaperiodate to generate carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on a drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can be reacted with sodium metaperiodate to generate an aldehyde in place of the first amino acid. This aldehyde can be reacted with a nucleophile on the drug moiety or linker.

[0268] Similarly, nucleophilic groups on drug moieties include, but are not limited to, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups, which can form covalent bonds with electrophilic groups on linker moieties and linker reagents, including: (i) activated esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups.

[0269] Alternatively, a fusion protein containing a VHH domain and a cytotoxic agent can be produced, for example, by recombinant DNA techniques or peptide synthesis. The DNA sequence can be engineered to contain regions encoding the two portions of the fusion protein, either adjacent to each other or separated by a region encoding a linker peptide that does not impair the desired properties of the fusion protein. This DNA sequence can then be transfected into a host cell that expresses the fusion protein. The fusion protein can be recovered and purified from the cell culture using techniques known in the art.

[0270] Linker In some embodiments, one or more polypeptides of a fusion protein of the present disclosure are operably linked via a peptide linker, which can be from 2 to 60 or more amino acids in length, and in certain aspects, the peptide linker is 3 to 50, 4 to 30, 5 to 25, 10 to 25, 10 to 60, 12 to 20, 20 to 50, or 25 to 35 amino acids in length.

[0271] In some embodiments, the peptide linker, e.g., the peptide linker separating two VHH domains or separating a VHH domain and a heavy chain constant region, is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, or at most 60 amino acids in length.

[0272] In some embodiments, the linker is 5 to 50 amino acids in length, e.g., 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acids in length. In other embodiments, the linker is 6 to 50 amino acids in length, e.g., 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acids in length. In yet other embodiments, the linker is 7 to 50 amino acids in length, e.g., 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids in length.

[0273] In some embodiments, charged linkers (e.g., charged hydrophilic linkers) and / or flexible linkers are used. Examples of flexible linkers that can be used in the fusion proteins of the present disclosure include those described in Chen et al., 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers include repeats of glycine and serine, e.g., G n S (SEQ ID NO: 4013) or SG n (SEQ ID NO: 4014), where n is an integer between 1 and 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is a repeat of G4S (SEQ ID NO: 3969), e.g., (GGGGS) n (SEQ ID NO: 4015).

[0274] Polyglycine linkers can be suitably used in the fusion proteins of the present disclosure. In some embodiments, peptide linkers used herein include two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 4016), five consecutive glycines (5Gly) (SEQ ID NO: 4017), six consecutive glycines (6Gly) (SEQ ID NO: 4018), seven consecutive glycines (7Gly) (SEQ ID NO: 4019), eight consecutive glycines (8Gly) (SEQ ID NO: 4020), or nine consecutive glycines (9Gly) (SEQ ID NO: 4021).

[0275] In some embodiments, a GS linker used herein comprises an amino acid sequence selected from GGSGGS, i.e., (GGS)2 (SEQ ID NO: 4022); GGSGGSGGS, i.e., (GGS)3 (SEQ ID NO: 4023); GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 4024); and GGSGGSGSGGSGGGS, i.e., (GGS)5 (SEQ ID NO: 4025). In some embodiments, the fusion protein can comprise a combination of a GS linker and a glycine linker.

[0276] In one embodiment, two or more VHHs are linked via a GGGGSGGGSGGGGS (SEQ ID NO: 3970) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGS (SEQ ID NO: 4026) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGSESKYGPPCPSCP (SEQ ID NO: 4008) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGS (SEQ ID NO: 3969) linker.

[0277] In some embodiments, one or more polypeptides of a fusion protein of the present disclosure are operably linked via a "rigid" peptide linker. Such a peptide linker may comprise a proline-rich peptide. In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAP (SEQ ID NO: 4009). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 4012). In one embodiment, a rigid peptide linker comprises A(EAAAK)nA (SEQ ID NO: 4027), where n is any integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0278] Other exemplary peptide linkers that can be used in the fusion proteins described herein are shown in Table 2. [Table 2] TIFF2026503077000039.tif55170

[0279] Exemplary Fusion Proteins of the Disclosure Non-limiting examples of fusion proteins (e.g., bivalent or tetravalent constructs with or without Fc regions, IL-2 fusion constructs) are disclosed in the "Examples" and "Sequence Listing" sections below.

[0280] In various embodiments, the fusion protein of the present disclosure comprises any one of SEQ ID NOs: 3933-3964, 4483-4513, 4686-4696, 4709-4716, and 4735-4770, or an analogous sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0281] In some embodiments, the fusion protein of the present disclosure comprises any one of SEQ ID NOs: 4483-4513, 4686-4696, 4709-4716, and 4735-4770, or an analogous sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0282] In some embodiments, the fusion protein of the present disclosure comprises SEQ ID NO: 4483, or an analogous sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0283] In some embodiments, the fusion protein of the present disclosure comprises SEQ ID NO: 4489, or an analogous sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0284] In some embodiments, the fusion protein of the present disclosure comprises any one of SEQ ID NOs: 4709-4716, or an analogous sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0285] In some embodiments, the fusion protein of the present disclosure comprises any one of SEQ ID NOs: 4735-4770, or an analogous sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0286] In some embodiments, provided herein are fusion proteins that specifically bind to TNFR2, the fusion protein comprising two polypeptides, each polypeptide comprising two anti-TNFR2 antigen binding proteins described herein operably linked to each other, one of the antigen binding proteins further operably linked to a dimerization domain (e.g., an immunoglobulin Fc region). The two polypeptides dimerize in the presence of the dimerization domain to form a tetravalent molecule (i.e., four anti-TNFR2 antigen binding proteins per molecule).

[0287] In some embodiments, the two antigen-binding proteins are operably linked to each other via a peptide linker. In one embodiment, the peptide linker is a (GS)n (SEQ ID NO: 4015) linker. In one embodiment, the peptide linker is a GGGGSGGGSGGGGS linker (SEQ ID NO: 3970).

[0288] In some embodiments, one of the two antigen binding proteins is further operably linked to an immunoglobulin Fc region via a peptide linker. In one embodiment, the peptide linker is a (GS)n (SEQ ID NO: 4015) linker. In one embodiment, the peptide linker is a GGGGS linker (SEQ ID NO: 3969).

[0289] In some embodiments, the fusion proteins described herein further comprise an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region, or a variant thereof.

[0290] In some embodiments, the immunoglobulin Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the human IgG1 Fc region comprises one or more mutations selected from L234A, L235A, G237A, D265A, N297A, and / or P329A (EU numbering). In some embodiments, the human IgG1 Fc region comprises: 1) L234A and L235A; 2) L234A, L235A, and P329A; 3) D265A, N297A, and P329A; and 4) L234A, L235A, and G237A The set of mutations is selected from:

[0291] In some embodiments, the immunoglobulin Fc region is a human IgG1 Fc region comprising L234A, L235A, and P329A.

[0292] In some embodiments, the immunoglobulin Fc region is a human IgG4 Fc region or a variant thereof. In some embodiments, the human IgG4 Fc region comprises one or more mutations selected from S228P, L235E, L235A, and / or F234A (EU numbering). In some embodiments, the human IgG4 Fc region comprises: 1) S228P and L235E; 2) S228P, and L235A; 3) S228P, F234A, and L235E; and 4) S228P, F234A, and L235A The set of mutations is selected from:

[0293] In some embodiments, the immunoglobulin Fc region is a human IgG4 Fc region comprising S228P and L235E.

[0294] Although the exemplary fusion proteins described herein contain non-humanized VHH amino acid sequences, it will be understood that such non-humanized VHH amino acid sequences can be replaced with any of the humanized VHH amino acid sequences described herein (e.g., in Tables 1-1 and 1-2).

[0295] In some embodiments, the fusion proteins described herein may further comprise a signal sequence at its N-terminus. The signal sequence may be present in a precursor molecule of the fusion protein and may be removed after the protein is secreted from the host cell during production. In some embodiments, the signal sequence is MAVMAPRTLVLLLSGALALTQTWA (SEQ ID NO: 3928), or a fragment or variant thereof. In some embodiments, the signal sequence is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 3929), or a fragment or variant thereof.

[0296] Polynucleotide molecules In another aspect, provided herein are polynucleotide molecules that encode the anti-TNFR2 antigen binding proteins (e.g., antibodies, including single domain antibodies) or fusion proteins described herein. Polynucleotide molecules that encode one or more polynucleotide portions of the conjugates of this disclosure are also within the scope of this disclosure.

[0297] In some embodiments, a polynucleotide molecule of the present disclosure encodes an anti-TNFR2 VHH amino acid sequence selected from SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, 4521, 93-640, 4079-4125, 2805-3363, 4359-4420, and 4605-4628, or an analog thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0298] In some embodiments, a polynucleotide molecule of the present disclosure encoding an anti-TNFR2 VHH comprises the nucleotide sequence of any one of SEQ ID NOs: 48-59, 4073-4075, 4522, 4525, 4528, 4531, 3364-3922, 4421-4482, and 4629-4652, or an analogous sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0299] In one embodiment provided herein, the polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence selected from SEQ ID NOs: 81-92, 4076, 4078, 4523, 4526, 4529, 4532, 4731-4734, 641-1127, and 4126-4172, or an analogous sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0300] In one embodiment provided herein, a polynucleotide molecule of the disclosure encodes the humanized VHH amino acid sequence of SEQ ID NO: 4526, or an analogous sequence thereof with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In one embodiment provided herein, a polynucleotide molecule encoding the humanized VHH amino acid sequence of SEQ ID NO: 4526 comprises the nucleotide sequence of SEQ ID NO: 4525, or an analogous sequence thereof with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0301] In one embodiment provided herein, a polynucleotide molecule of the disclosure encodes the humanized VHH amino acid sequence of SEQ ID NO: 4529, or an analogous sequence thereof with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In one embodiment provided herein, a polynucleotide molecule encoding the humanized VHH amino acid sequence of SEQ ID NO: 4529 comprises the nucleotide sequence of SEQ ID NO: 4528, or an analogous sequence thereof with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0302] In one embodiment provided herein, a polynucleotide molecule of the disclosure encodes the humanized VHH amino acid sequence of SEQ ID NO: 4532, or an analogous sequence thereof with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In one embodiment provided herein, a polynucleotide molecule encoding the humanized VHH amino acid sequence of SEQ ID NO: 4532 comprises the nucleotide sequence of SEQ ID NO: 4531, or an analogous sequence thereof with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0303] In one embodiment provided herein, the polynucleotide molecule of the present disclosure encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 3933-3964, and 4483-4513, 4686-4696, 4709-4716, and 4735-4770, or an analogous sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0304] Polynucleotide molecules can be used to transform / transfect host cells or host organisms for expression and / or production of the polypeptides. Suitable hosts or host cells for production of the anti-TNFR2 polypeptides described herein include any suitable fungal cell or cell line, prokaryotic cell or cell line, or eukaryotic cell or cell line, or any suitable fungal, prokaryotic, or eukaryotic organism. Hosts or host cells comprising polynucleotide molecules encoding the anti-TNFR2 antigen binding protein polypeptides or fusion proteins described herein are also encompassed by the present disclosure.

[0305] The polynucleotide molecule may be, for example, DNA, RNA, or a hybrid thereof, and may also include (e.g., chemically) modified nucleotides, such as locked nucleic acid (LNA) or peptide nucleic acid (PNA). In some embodiments, the polynucleotide is single-stranded. In some embodiments, the polynucleotide is double-stranded. In one embodiment, the polynucleotide is in the form of double-stranded DNA (e.g., a plasmid). In some embodiments, the polynucleotide is in the form of single-stranded RNA (e.g., mRNA).

[0306] Techniques for generating polynucleotides can include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof); introducing mutations that lead to the expression of truncated expression products; introducing one or more restriction sites (e.g., to create cassettes and / or regions that can be easily digested and / or ligated using suitable restriction enzymes); and / or introducing mutations by PCR reactions using one or more "mismatch" primers. Alternatively, polynucleotides of the present disclosure can be isolated from suitable natural sources. Polynucleotide sequences encoding naturally occurring (poly)peptides can be subjected to, for example, site-directed mutagenesis to generate polynucleotide molecules encoding polypeptides with sequence diversity.

[0307] vector Also provided herein are vectors comprising polynucleotide molecules encoding the anti-TNFR2 antigen binding proteins (e.g., antibodies, including single domain antibodies), fusion proteins, or other related polypeptides of the present disclosure. As used herein, a "vector" is a vehicle suitable for delivering genetic material to a host cell. Vectors can include nucleic acid vectors, such as plasmids or mRNA, or nucleic acids embedded in larger structures, such as liposomes or viral vectors.

[0308] A vector may contain one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters, enhancers, terminators) that control the expression of a polypeptide of interest, and / or one or more selectable marker genes (e.g., drug resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). In the case of DNA-based vectors, this usually includes the presence of elements for transcription (e.g., promoters and polyA signals) and translation (e.g., Kozak sequences). In some embodiments, the vector is an expression vector, i.e., a vector suitable for expressing an encoded polypeptide or construct under suitable conditions in a host cell.

[0309] To express an anti-TNFR2 antigen binding protein or fusion protein (or fragment thereof) of the present disclosure, a polynucleotide encoding a partial or full-length polypeptide chain (e.g., VHH, VHH-Fc), obtained, for example, as described above, can be inserted into an expression vector such that the gene is operably linked to one or more transcriptional and translational control sequences. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. Polynucleotides encoding two or more polypeptide chains (which, if present, are different from each other) of an anti-TNFR2 antigen binding protein or fusion protein of the present disclosure can be inserted into separate vectors, or, optionally, can be incorporated into the same expression vector.

[0310] In addition to a polynucleotide encoding one or more polypeptide chains of an anti-TNFR2 antigen-binding protein or fusion protein, a recombinant expression vector of the invention may contain regulatory sequences that control the expression of the gene encoding the one or more polypeptide chains in a host cell. The design of an expression vector, including the selection of regulatory sequences, can depend on the choice of host cell to be transformed and / or the desired level of protein expression, among other factors. For example, suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Further examples of viral regulatory elements and sequences thereof include those described in, for example, U.S. Pat. Nos. 168,062; 4,510,245; and 4,968,615, the disclosures of each of which are incorporated herein by reference.

[0311] The recombinant expression vectors of the present disclosure may contain additional sequences, such as sequences regulating replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, the disclosures of each of which are incorporated herein by reference in their entireties). For example, selectable marker genes typically confer resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin, or cytotoxic drugs such as G418, puromycin, blasticidin, hygromycin, or methotrexate, on the host cells into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-deficient host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0312] The vectors of the present disclosure may further contain sequence elements that enhance the rate of translation of these genes or improve the stability or nuclear transport of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, an internal ribosomal entry site (IRES), and a polyadenylation signal site to induce efficient transcription of the genes carried on the expression vector.

[0313] Viral vectors can be used to efficiently deliver foreign genes into the genome of cells (e.g., eukaryotic or prokaryotic cells). Viral vectors are particularly useful for gene delivery because the polynucleotides contained in such genomes are typically integrated into target cells by general or specific transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of suitable viral vectors include retroviruses; adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48); parvoviruses (e.g., adeno-associated viruses (AAVs) such as AAV2, AAV8, and AAV9); negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), and paramyxoviruses (e.g., measles and Sendai virus); positive-strand RNA viruses such as picornaviruses and alphaviruses; and adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus, and leukemia virus). Examples of viruses that can be used to deliver polynucleotides encoding the polypeptides of the present disclosure include double-stranded DNA viruses such as rabies, baculovirus, coronavirus, and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses that can be used to deliver polynucleotides encoding the polypeptides of the present disclosure include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include, but are not limited to, avian leukosis-sarcoma viruses, mammalian type C, type B, and type D viruses, the HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, JM 1996. Fundamental Virology, DMKDN Fields, P.M. Howley, eds. (Philadelphia, Lippincott-Raven Publishers): 763-843, the disclosure of which is incorporated herein by reference).Other examples of viral genomes that can be used in the compositions and methods of the present disclosure include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline sarcoma virus, feline leukemia virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0314] host cell In certain aspects, the present disclosure also provides host cells or host organisms comprising polynucleotides or vectors encoding anti-TNFR2 antigen binding proteins (e.g., antibodies, including single-domain antibodies), fusion proteins, or other related polypeptides described herein. Suitable host cells or host organisms can be any suitable fungal cell or cell line, prokaryotic cell or cell line, or eukaryotic cell or cell line, or any suitable fungal, prokaryotic, or eukaryotic organism. A host cell includes the progeny of a single host cell, which may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell can also include cells transfected in vivo with one or more polynucleotides or vectors provided herein.

[0315] Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris); plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), COS cells, SP2 / 0 cells, and 293 and CHO cells, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Exemplary prokaryotic cells include bacterial cells, such as E. coli.

[0316] Preparation method The present disclosure also provides methods of producing the anti-TNFR2 antigen binding proteins (eg, antibodies, including single domain antibodies), fusion proteins, or conjugates described herein.

[0317] In some embodiments, methods may comprise transforming / transfecting a host cell or host organism with a polypeptide encoding an anti-TNFR2 antigen binding protein (e.g., an antibody, such as a single domain antibody), fusion protein, or one or more other polypeptides of interest described herein; expressing the anti-TNFR2 antigen binding protein (e.g., an antibody, such as a single domain antibody), fusion protein, or one or more other polypeptides of interest in the host; and optionally one or more subsequent isolation and / or purification steps.

[0318] A recombinant expression vector encoding one or more polypeptides of an anti-TNFR2 antigen binding protein (e.g., an antibody, such as a single domain antibody), fusion protein, or conjugate of the present disclosure is introduced into mammalian host cells, and the host cells are cultured for a period of time sufficient to express the one or more proteins or polypeptides within the host cells or to secrete the one or more proteins or polypeptides into the culture medium in which the host cells are growing. The one or more proteins or polypeptides can be recovered from the culture medium using standard protein purification methods. The host cells can also be used to produce portions of an entire antibody, such as a VHH domain.

[0319] After a protein or polypeptide of the disclosure is produced by recombinant expression, it can be purified by any method known in the art for purifying proteins or polypeptides, such as chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly by affinity for TNFR2 after selection of Protein A or Protein G), and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Additionally, proteins or polypeptides of the disclosure can be fused to heterologous polypeptide sequences described herein (e.g., His tags) or heterologous polypeptides known in the art to facilitate purification or to generate therapeutic conjugates, as described below. After isolation, proteins or polypeptides of the disclosure can be further purified, if desired, by, for example, high performance liquid chromatography or gel filtration chromatography, for example, on a Superdex™ column.

[0320] Pharmaceutical Compositions and Formulations The present disclosure also provides a composition comprising an anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody), fusion protein, or conjugate of the present technology, at least one polynucleotide molecule encoding the same, at least one vector comprising the polynucleotide molecule, or at least one host cell comprising the polynucleotide molecule or vector. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent, or excipient and / or adjuvant, and may optionally comprise one or more additional polypeptides and / or compounds having pharmaceutical activity.

[0321] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. Supplementary active compounds can also be incorporated into the compositions.

[0322] Examples of suitable formulations include, but are not limited to, solutions, suspensions, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Phdomain Sci Technol 52:238-311.

[0323] Pharmaceutical compositions of the present disclosure may be formulated according to the intended route of administration. Examples of suitable routes of administration include, for example, intravenous, subcutaneous, intratumoral, oral (e.g., buccal, sublingual), intranasal, inhalation, intraocular, intramuscular, intradermal, transdermal (i.e., topical), intraperitoneal, transmucosal, vaginal, and rectal administration, or injection into the CNS / brain (e.g., intraspinal, intracerebral, or intrathecal administration). Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: a sterile diluent such as water for injection, saline, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a fixed oil; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as a phosphate, acetate, or citrate salt; and a tonicity adjuster such as sodium chloride or glucose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of plastic or glass.

[0324] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include, for example, physiological saline, sterile water, Cremophor EL®, or phosphate buffered saline (PBS). The composition is preferably sterile and has suitable fluidity. In most embodiments, the composition is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial contamination can be achieved by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, sodium chloride, etc. in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0325] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent containing one or a combination of the above-mentioned components as needed, and then sterilizing by filtration.Generally, it is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and the other necessary components listed above.For the preparation of sterile powder for the preparation of sterile injectable solution, the preparation method includes vacuum drying and / or freeze-drying, which allows the powder of active ingredient and any desired additional ingredients to be obtained from the solution that has been previously sterile-filtered.

[0326] Oral compositions may include an inert diluent or an edible carrier. These may be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be combined with an excipient and used in the form of a tablet, troche, capsule, or liquid. Tablet and liquid formulations can be used for protease-insensitive VHHs. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied to the mouth and swished before being expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0327] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0328] Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salve, gel, or cream, as is generally known in the art.

[0329] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0330] For brain delivery, the compounds of the present disclosure may be formulated to facilitate passage through the blood-brain barrier. For example, the anti-TNFR2 antigen binding protein (e.g., an antibody such as a single domain antibody), fusion protein, or conjugate of the present disclosure may be encapsulated in brain-targeting liposomes, lipid nanoparticles, lipid microparticles, or lipid macrocapsules for delivery to the brain. An exemplary liposome delivery system is described in Pothin et al., Pharmaceutics 2020, 12(10), 937 (the entirety of which is incorporated herein by reference).

[0331] In some embodiments, the active compounds are prepared with carriers that can protect the compounds from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 (incorporated herein by reference in its entirety).

[0332] For ease of administration and uniform dosage, it is particularly advantageous to prepare oral or parenteral compositions into unit dosage form.As used herein, unit dosage form refers to a physically separate unit that is adapted to be a unit dosage for the subject to be treated, and each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect together with desired pharmaceutical carrier.The specification of unit dosage form of the present disclosure depends on the unique characteristics of active compound and the specific therapeutic effect to be achieved, and the inherent limitations of the technology of compounding active compound as described above for individual treatment.

[0333] The pharmaceutical compositions (or components thereof) can be included in a kit, container, pack, or dispenser together with instructions for administration. These pharmaceutical compositions can be included in diagnostic kits together with instructions for use.

[0334] The pharmaceutical composition is administered in an amount effective for treating or preventing a particular indication. A therapeutically effective amount typically depends on the weight of the subject being treated, the subject's physical condition or health, the severity of the condition being treated, or the age of the subject being treated. In some embodiments, the pharmaceutical composition may be administered in an amount of about 50 μg / kg to about 50 mg / kg of body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount of about 100 μg / kg to about 50 mg / kg of body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount of about 100 μg / kg to about 20 mg / kg of body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount of about 0.5 mg / kg to about 20 mg / kg of body weight per dose. The frequency and duration of treatment can be adjusted depending on the severity of the condition. The effective dosage and schedule for administering the pharmaceutical composition of the present disclosure can be determined empirically, for example, by monitoring the patient's progress through periodic evaluation, and the dosage can be adjusted accordingly. Furthermore, interspecies dosage scaling can be performed using methods known in the art (e.g., Mordenti et al., 1991, Phdomainaceut. Res. 8:1351).

[0335] In some embodiments, the pharmaceutical composition may be administered in an amount of about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount of about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount of about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount of about 20 mg to about 200 mg per dose.

[0336] In some embodiments in which the antigen binding proteins of the disclosure are administered as a viral vector (e.g., AAV), the dosage ranges and frequency of administration of the viral vectors described herein may vary depending on the nature and medical condition of the viral vector, as well as the parameters of the particular patient and route of administration used. In some embodiments, the viral vector composition is administered in a dose of about 1 x 10 5 Plaque forming unit (pfu) ~ approximately 1 x 10 15 The viral vector composition can be administered to a subject at a dose ranging from about 1 x 10 pfu. 8 pfu~approx. 1×10 15 pfu, or approximately 1 x 10 10 pfu~approx. 1×10 15 pfu, or approximately 1 x 10 8 pfu~approx. 1×10 12 The dose can be in the range of 1×10 pfu. The more precise dose can also depend on the subject receiving the dose. For example, if the subject is a young person, a relatively low dose may be required, and if the subject is an adult subject, a relatively high dose may be required. In certain embodiments, the more precise dose can depend on the body weight of the subject. In certain embodiments, for example, a young human subject can receive a dose of about 1×10 8 pfu~approx. 1×10 10 pfu can be administered to adult human subjects, while approximately 1 × 10 10 pfu~approx. 1×10 12 A dose of pfu can be administered.

[0337] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, including encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, intraspinal, intracerebral, intrathecal, and oral routes. The compositions can be administered by any convenient route, such as infusion or bolus injection, absorption through epithelial or inner mucocutaneous layers (e.g., oral, rectal, and intestinal mucosa), and may be administered in conjunction with other bioactive agents. Administration can be systemic or local.

[0338] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, pen delivery devices are readily adapted to deliver the pharmaceutical compositions of the present disclosure. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge is fully administered and the cartridge is emptied, the empty cartridge can be simply discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Instead, disposable pen delivery devices are provided pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0339] Under certain circumstances, pharmaceutical compositions can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are described in the review by Langer, 1990, Science 249:1527-1533.

[0340] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intramuscular, intratumoral, intraperitoneal, intraspinal, intracerebral, and intrathecal injections, infusions, and the like. In one embodiment, injectable preparations can be prepared by, for example, dissolving, suspending, or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, physiological saline and isotonic solutions containing glucose and other adjuvants, which can be used in combination with an appropriate solubilizer such as alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (a polyoxyethylene alkyl ether (50 mol) adduct of hydrogenated castor oil), and the like. Oily media include, for example, sesame oil, soybean oil, and the like, which can be used in combination with an appropriate solubilizer such as benzyl benzoate, benzyl alcohol, and the like. The injectable preparations prepared in this manner are preferably filled into appropriate ampoules.

[0341] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in a unit dose form adapted to fit the dose of the active ingredient. Examples of such unit dose forms include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antigen-binding protein described herein may be about 5 to about 500 mg per unit dose form; particularly in the form of an injection, the antigen-binding protein described herein may be contained in an amount of about 5 to about 100 mg, and in other dosage forms, about 10 to about 250 mg.

[0342] The pharmaceutical composition can be administered to a subject as needed. In some embodiments, an effective dose of the pharmaceutical composition is administered to a subject one or more times. In various embodiments, an effective dose of the pharmaceutical composition is administered to a subject once a month, or less than once a month, for example, every two months, every three months, or every six months. In other embodiments, an effective dose of the pharmaceutical composition is administered more than once a month, for example, every two weeks, every week, twice a week, three times a week, daily, or multiple times a day. An effective amount of the pharmaceutical composition is administered to a subject at least once. In some embodiments, an effective amount of the pharmaceutical composition can be administered multiple times, including over a period of at least one month, at least six months, or at least one year. In some embodiments, administering the pharmaceutical composition to a subject as needed alleviates one or more symptoms of a medical condition.

[0343] In some embodiments, the pharmaceutical compositions of the present disclosure may be administered to a subject at a level lower than that required to achieve the desired therapeutic effect, and the dosage may be gradually increased until the desired effect is achieved. Alternatively, the pharmaceutical compositions of the present disclosure may be administered at a high dose, followed by gradually decreasing doses until the therapeutic effect is achieved. Generally, a suitable daily dose of an antigen-binding protein of the present invention is the amount of antibody that is the lowest dose effective to produce a therapeutic effect.

[0344] Pharmaceutical compositions of the present disclosure may optionally contain two or more active agents. For example, compositions of the present disclosure may contain an anti-TNFR2 antigen binding protein conjugated to, mixed with, or administered separately from another pharmaceutically active molecule, such as Treg cells or an additional agent useful for inducing T cell proliferation. For example, an anti-TNFR2 antigen binding protein may be mixed with one or more additional active agents to treat an immune disease, a disorder described herein. Alternatively, pharmaceutical compositions of the present disclosure may be formulated for simultaneous or sequential administration with one or more additional active agents that can be used to attenuate the growth of CD8+ T cells. Examples of additional active agents that can be used to attenuate the proliferation of cytotoxic T cells and that can be conjugated to, mixed with, or administered separately from an anti-TNFR2 antigen binding protein of the present disclosure include cytotoxic agents, such as those described herein.

[0345] Treatment and Other Uses In certain aspects, provided herein are methods of stimulating the expansion of a population of regulatory T (Treg) cells (e.g., CD4+, CD25+, FOXP3+ Treg cells) using anti-TNFR2 antigen binding proteins, fusion proteins, or conjugates of the present disclosure. This response can also have the effect of reducing the population of cytotoxic T lymphocytes (e.g., CD8+ T cells), which are often associated with the generation of inappropriate immune responses that can lead to immune disorders. Furthermore, the anti-TNFR2 antigen binding proteins, fusion proteins, or conjugates of the present disclosure can exhibit synergistic effects with existing Treg growth factors, such as IL-2 and TNFα.

[0346] Also provided herein are methods of activating and / or enhancing the suppressive function of a population of Treg cells (e.g., inhibiting the function or proliferation of effector T / B cells or the function of antigen-presenting cells) using the anti-TNFR2 antigen binding proteins, fusion proteins, or conjugates of the disclosure.

[0347] Also provided herein are methods of stabilizing the immunosuppressive phenotype of a population of Treg cells (e.g., including stable expression of FOXP3, HELIOS, CTLA-4) using the anti-TNFR2 antigen binding proteins, fusion proteins, or conjugates of the disclosure.

[0348] In various embodiments of the above-described methods, the methods may include contacting a population of regulatory T cells with an anti-TNFR2 antigen binding protein, fusion protein, or conjugate described herein. The methods may be performed in vitro or in vivo. When the method is performed in vivo, the method further includes administering to a subject an anti-TNFR2 antigen binding protein, fusion protein, or conjugate described herein.

[0349] Tregs are a subset of T cells that play an important role in peripheral self-tolerance and autoimmunity prevention. Historically, Tregs were identified as a CD4+ subset that specifically expresses the high-affinity IL-2 receptor α chain, CD25 (Sakaguchi et al., 1995). Subsequently, the FOXP3 transcription factor was identified as a master regulator of CD4+ Tregs (Hori et al., 2003). Indeed, deficiency of FOXP3 leads to systemic autoimmunity in both mice and humans, which is caused by immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome due to Treg deficiency and dysregulation of effector T cell function (Bennett et al., 2001). CD4 Tregs can differentiate during T cell development (thymic "tTregs") or in the periphery (peripheral "pTregs") under non-inflammatory T cell receptor stimulation (Wing et al., 2019). Numerous subsets have been described, including naive and memory Tregs (Sakaguchi et al., 2020), Th-like Tregs (Halim et al., 2017), and CD8 Tregs (Mishra et al., 2021; Niederlova et al., 2021). CD4 Tregs regulate immune responses through diverse mechanisms, including secretion of regulatory cytokines (e.g., IL-10, IL-35, TGF-β), scavenging of IL-2, adenosine production, direct cytotoxicity, and modulation of dendritic cells (Vignali et al., 2008). As used herein, the term "regulatory T cells" refers to Tregs that regulate immune responses through diverse mechanisms, including secretion of regulatory cytokines (e.g., IL-10, IL-35, TGF-β), scavenging of IL-2, adenosine production, direct cytotoxicity, and modulation of dendritic cells (Vignali et al., 2008). The term "regulatory T cell" or "Treg" is intended to encompass all of the above-mentioned subsets of regulatory T cells.

[0350] Tregs have enhanced affinity for MHC II-presented self-antigen peptides and possess TCR repertoires that do not overlap with effector CD4 T cells (Fazilleau et al., 2007; Hsieh et al., 2006; Pacholczyk et al., 2006). Therefore, peripheral self-antigen recognition can induce tTreg activation (Moran et al., 2011). Importantly, however, once activated, Tregs can suppress effector cells with distinct antigen specificities through bystander suppression by regulating antigen-presenting cells or soluble factors (Thornton and Shevach, 2000; Yeh et al., 2017; Yu et al., 2005).

[0351] It has been shown that Tregs can maintain a certain degree of plasticity and lose FOXP3 expression over time. These so-called "ex-Tregs" have elevated levels of FOXP3 promoter methylation compared to Tregs, express less FOXP3, and acquire effector function (Zhou et al., 2009). In Tregs, demethylation of the FOXP3 promoter, particularly in the "Treg-specific demethylated region" (TSDR) (Huehn et al., 2009), stabilizes gene expression. Similarly, human Tregs exposed to IL-2 plus inflammatory cytokines have been shown to lose FOXP3 expression while upregulating RORg and IL-17, characteristics associated with TH17 cells. The instability of the Treg phenotype in the presence of inflammatory cytokines can be termed "Treg fragility," and is crucial for the treatment of autoimmune diseases. Indeed, to induce long-term therapeutic benefits, it is important to stabilize the phenotype and function of Tregs to prevent their transformation into pathogenic cells that lead to disease exacerbation.

[0352] In some embodiments, an anti-TNFR2 antigen binding protein, fusion protein, or conjugate of the present disclosure may stimulate the expansion of a population of Treg cells by 1% to 100% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) relative to untreated cells, as measured, for example, by fluorescence-activated cell sorting (FACS) analysis. In certain embodiments, an anti-TNFR2 antigen binding protein, fusion protein, or conjugate of the present disclosure may reduce the expansion of a population of CD8+ T cells by, for example, about 10% to about 200% (e.g., 10%, 20%, 30%, 40%, 50%, 75%, 100%, 125%, 150%, 175%, or 200%) relative to untreated cells.

[0353] In some embodiments, anti-TNFR2 antigen binding proteins of the present disclosure can be used to enhance the immunomodulatory activity of Treg cells by promoting the expansion of these cell populations. Accordingly, anti-TNFR2 antigen binding proteins of the present disclosure can be used to attenuate aberrant cell-mediated or humoral immune responses associated with a variety of human diseases, such as autoimmune disorders, asthma, allergic reactions, and diseases associated with allograft tolerance. Administration of anti-TNFR2 antigen binding proteins of the present disclosure can suppress the activity of cytotoxic T cells and B cells, thereby attenuating a subject's response to self-antigens or benign antigens. Administration of anti-TNFR2 antigen binding proteins of the present disclosure to a mammalian subject, such as a human, can attenuate aberrant immune responses, such as responses to self-antigens or non-threatening antigens. Alternatively, anti-TNFR2 antigen binding proteins of the present disclosure can be used to ex vivo expand a population of Treg cells extracted, for example, from a patient or an MHC-matched donor. These Treg cells can be induced to expand in culture by contact with an anti-TNFR2 antigen binding protein of the present disclosure, and then administered to a subject, for example, using adoptive cell transfer techniques known in the art or described herein. In this manner, the anti-TNFR2 antigen binding proteins of the present disclosure can synergize with existing techniques for suppressing humoral and cell-mediated immune responses as a treatment for patients suffering from a variety of immune disorders.

[0354] In some embodiments, anti-TNFR2 antigen binding proteins of the present disclosure can interact with TNFR2 and promote TNFR2-mediated signaling events. Anti-TNFR2 antigen binding proteins of the present disclosure may induce a conformational change in TNFR2 that leads to receptor trimerization. This spatial organization has been shown to activate TNFR2 for MAPK / TRAF2 / 3 signaling, which leads to activation of NF-κB-mediated transcription of genes involved in Treg cell growth and evasion of apoptosis (Faustman, et al., Nat Rev Drug Discov. 9:482-493 (2010) (incorporated herein by reference)).

[0355] In some embodiments, anti-TNFR2 antigen binding proteins of the present disclosure may be capable of increasing the transcription and / or expression of various genes. For example, anti-TNFR2 antigen binding proteins of the present disclosure may induce the expression of one or more of Akt, clAP2, Etk, TRAF2, VEGFR2, P13K, genes encoding proteins involved in the angiogenesis pathway, IKK complex, RIP, NIK, MAP3K, genes encoding proteins involved in the NF-κB pathway, NIK, JNK, AP-1, MEK (e.g., MEK1, MEK7), MKK3, NEMO, IL2R, Foxp3, IL2, TNF, and lymphotoxin (e.g., lymphotoxin a and lymphotoxin β). Increased expression of these genes can be detected using established molecular biology techniques known in the art, such as by detecting elevated mRNA levels by Northern blot analysis or reverse-transcription PCR (RT-PCR), or by detecting elevated protein levels by immunoblot analysis or ELISA-based techniques. In some embodiments, anti-TNFR2 antigen binding proteins of the present disclosure may promote the activity of one or more proteins associated with the TNFR2 signaling pathway (or related signaling pathways activated as a result of TNFR2 signaling). For example, anti-TNFR2 antigen binding proteins of the present disclosure may promote increased phosphorylation of one or more proteins, such as Akt, clAP2, Etk, TRAF2, VEGFR2, P13K, proteins involved in the angiogenesis pathway, IKK complex, RIP, NIK, MAP3K, proteins involved in the NF-κB pathway, NIK, JNK, AP-1, MEK (e.g., MEK1, MEK7), MKK3, NEMO, IL2R, Foxp3, IL2, TNF, and lymphotoxin (e.g., lymphotoxin α and lymphotoxin β). Increased phosphorylation of one or more proteins, e.g., as a result of treating a subject or treating a sample of cells isolated from a subject, can be detected using standard molecular biology techniques known in the art, such as by immunoblot analysis or ELISA-based techniques.

[0356] In some embodiments, the antigen binding proteins of the disclosure increase the expression of one or more proteins selected from proteins of the NF-kB pathway, FOXP3, HELIOS, EZH2, HLA-DR, ICAM-1, OX-40, ICOS, and CCR8.

[0357] In another aspect, a method of inhibiting an immune response mediated by B cells or CD8+ T cells in a subject is provided, the method comprising administering to the subject an anti-TNFR2 antigen binding protein (e.g., an antibody, such as a single domain antibody), fusion protein, conjugate, polynucleotide molecule, vector, or host cell described herein.

[0358] In another aspect, the anti-TNFR2 antigen binding proteins (e.g., antibodies such as single domain antibodies), fusion proteins, conjugates, polynucleotide molecules, vectors, and / or host cells, or pharmaceutical compositions thereof, described herein are useful for the treatment (prophylactic or therapeutic) of a wide variety of diseases or disorders. Accordingly, the present technology provides anti-TNFR2 antigen binding proteins (e.g., antibodies such as single domain antibodies), fusion proteins, conjugates, polynucleotide molecules, vectors, or host cells for use as pharmaceuticals. Also provided are methods for treating (prophylactic and / or therapeutic) diseases or disorders, comprising administering to a subject in need thereof a pharmaceutically active amount of an anti-TNFR2 antigen binding protein (e.g., antibody such as single domain antibody), fusion protein, conjugate, polynucleotide molecule, vector, or host cell described herein.

[0359] Diseases or disorders that can be treated using the compositions and methods described herein include, but are not limited to, immune diseases (e.g., autoimmune diseases), inflammatory diseases, cancer, and cardiovascular diseases (e.g., atherosclerosis, heart failure, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, right heart failure, congestive heart failure, restrictive cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, idiopathic cardiomyopathy, hypertension), infertility and pregnancy-related diseases (e.g., recurrent pregnancy loss, preeclampsia, preterm labor, fetal growth restriction, intrauterine growth restriction).

[0360] Examples of immune disorders that can be treated using the compositions and methods described herein include, but are not limited to, autoimmune disorders, allergies, asthma, neurological disorders, metabolic disorders (e.g., diabetes), macular disorders (e.g., macular degeneration), muscle atrophy, disorders associated with miscarriage, vascular disorders (e.g., atherosclerosis), disorders associated with bone loss (e.g., bone loss as a result of menopause or osteoporosis), blood disorders (e.g., hemophilia), musculoskeletal disorders, disorders associated with growth receptor expression or activity, obesity, graft-versus-host disease (GVHD), or allograft rejection.

[0361] In some embodiments, the compositions and methods described herein are used to treat an autoimmune disease, including lupus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac disease dermatitis, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-associated fibromyalgia, and fibromyalgia-associated fibromyalgia. Fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lichen sclerosus, IgG4-related disease, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disorder, pemphigus vulgaris or related bullous skin diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome syndrome), polymyalgia rheumatica, polymyositis / dermatomyositis, premature ovarian failure, primary agammaglobulinemia, primary biliary cholangitis, psoriasis, primary ovarian insufficiency, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, stiff-man syndrome, type 1 diabetes mellitus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (granulomatosis with polyangiitis) or other immune-mediated vasculitis.

[0362] In some embodiments, the compositions and methods described herein are used to treat lupus, which in some embodiments is systemic lupus erythematosus (SLE), cutaneous lupus (including acute cutaneous lupus, chronic cutaneous lupus, or discoid lupus erythematosus (DLE), and subacute cutaneous lupus erythematosus), lupus nephritis, neonatal lupus, or drug-induced lupus.

[0363] In some embodiments, the compositions and methods described herein are used to treat allergies, which in some embodiments are allergic conjunctivitis, chemical allergies, cosmetic allergies, drug allergies, dust allergies, food allergies, hay fever, hives, mold allergies, pet allergies, poison ivy allergies, oak allergies, or seasonal allergies.

[0364] In some embodiments, the compositions and methods described herein are used to treat a neurological condition, hi some embodiments, the neurological condition is a brain tumor, brain metastasis, spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Parkinson's disease, or stroke.

[0365] In some embodiments, the compositions and methods described herein are used to treat transplant rejection. Without wishing to be bound by theory, the anti-TNFR2 antigen binding proteins of the present disclosure can treat transplant rejection, for example, by binding to TNFR2 receptors on the surface of autoreactive CD8+ T cells that bind to antigens presented on the surface of the transplant and inducing apoptosis in these CD8+ T cells or by inducing the proliferation of Treg cells, which can subsequently eliminate the autoreactive CD8+ T cells. Examples of transplant rejection that can be treated using the compositions and methods described herein include, but are not limited to, skin graft rejection, bone graft rejection, vascularized tissue graft rejection, ligament graft rejection (e.g., anterior cruciate ligament graft rejection, anterior sacroiliac ligament graft rejection, posterior cruciate ligament graft rejection, canine anterior cruciate ligament graft rejection, cricothyroid ligament graft rejection, dorsal radiocarpal ligament graft rejection, inferior pubic ligament graft rejection, lateral collateral ligament graft rejection, and medial collateral ligament graft rejection). Rejection of organ transplants (e.g., heart, lung, kidney, liver, pancreas, intestine, and thymus) may include transplant rejection of organs such as: volar radiocarpal ligament graft rejection, patellar ligament graft rejection, periodontal ligament graft rejection, posterior cruciate ligament graft rejection, posterior sacroiliac ligament graft rejection, lateral collateral ligament of elbow graft rejection, sacrospinous ligament graft rejection, sacrotuberous ligament graft rejection, superior pubic ligament graft rejection, mammary suspensory ligament graft rejection, lentisus suspensory ligament graft rejection, medial collateral ligament of elbow graft rejection, and organ transplant rejection (e.g., heart, lung, kidney, liver, pancreas, intestine, and thymus graft rejection).

[0366] In some embodiments, the compositions and methods described herein are used to treat graft-versus-host disease, which in some embodiments arises from bone marrow transplantation or from one or more blood cells, such as B cells, T cells, basophils, common myeloid progenitor cells, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.

[0367] In some embodiments, the compositions and methods described herein are used to treat an inflammatory disease. The inflammatory disease can be acute or chronic inflammation. In some embodiments, the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovary syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.

[0368] In some embodiments, the compositions and methods described herein are used to treat cancer, including adenoid cystic carcinoma, adrenal tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (bone sarcoma), brainstem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, pediatric cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, pediatric tumor, ependymoma, esophageal cancer, Ewing's sarcoma, eye tumor, eyelid tumor, familial adenomatous polyposis, familial GIST, familial idiopathic pulmonary fibrosis, familial urinary tract cancer, ... Family melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumors (including childhood germ cell tumors), gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell carcinoma, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal cell carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumors, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B Cellular prolymphocytic leukemia and hairy cell leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic T-cell lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), Hodgkin's lymphoma, non-Hodgkin's lymphoma, Lynch syndrome, mastocytosis, medulloblastoma (including childhood medulloblastoma), melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-related Polyposis, myelodysplastic syndrome (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma (including childhood neuroblastoma), neuroendocrine tumors of the gastrointestinal tract, neuroendocrine tumors of the lung, neuroendocrine tumors of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian, fallopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary tumors, pleuropulmonary blastoma, prostate cancer,Retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, soft tissue tumor, skin cancer (non-melanoma), small intestine cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, uterine cancer, vaginal cancer, von Hippel-Lindau disease, vulvar cancer, primary macroglobulinemia (lymphoplasmacytic lymphoma), Werner syndrome, Wilms' tumor, or xeroderma pigmentosum.

[0369] In some embodiments, the anti-TNFR2 antigen binding proteins of the present disclosure can also be used to treat patients in need of organ repair or regeneration, for example, by inducing proliferation of cells within damaged tissues or organs. Without wishing to be bound by any theory, it is believed that agonistic TNFR2 antibodies can stimulate organ repair or regeneration, for example, by binding to TNFR2 on the surface of cells within damaged tissues and inducing TRAF2 / 3- and / or NF-κB-mediated cell proliferation. Examples of tissues and organs in which regeneration can be induced using the anti-TNFR2 antigen binding proteins of the present disclosure include blood vessels, including the aorta, bone, cranial nerves, ear, eye, embryonic structures, heart, cardiac, hematopoietic system, kidney, small intestine, large intestine, liver, lung, nerves, olfactory gland, pancreas, pituitary gland, peripheral nervous system, central nervous system, spinal cord, salivary glands, head structures, testes, thymus, and tongue.

[0370] Additional diseases that can be treated with the compositions and methods of the present disclosure include genetic diseases with immunological phenotypes. Exemplary genetic diseases with immunological phenotypes are described, for example, in Table S2 of Tangye et al., Journal of Clinical Immunology volume 42, pages 1473-1507 (2022), which is incorporated herein by reference in its entirety.

[0371] In some embodiments, patients receiving an anti-TNFR2 treatment of the present disclosure can be monitored for responsiveness to the treatment. For example, a physician may monitor a mammalian subject's (e.g., a human's) response to treatment with an anti-TNFR2 antigen binding protein of the present disclosure by analyzing the amount of IFNγ secreted by CD8+ T cells in a particular patient. For example, a composition of the present disclosure may reduce IFNγ secretion by 1% to 100% (e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%). Alternatively, a physician may monitor a mammalian subject's (e.g., a human's) responsiveness to treatment with a composition of the present disclosure by analyzing the Treg cell population in the lymph of a particular subject. For example, a physician may obtain a blood sample from a mammalian subject (e.g., a human) and determine the quantity or density of a population of Treg cells (e.g., CD4+CD25+FOXP3+ Treg cells or CD17+ Treg cells) using established procedures, such as FACS analysis. In such embodiments, a high number of Treg cells may be indicative of effective therapy, while a low number of Treg cells may indicate that the patient needs to be prescribed or administered a higher dosage of an anti-TNFR2 antigen binding protein of the present disclosure, e.g., until an ideal number of Treg cells is achieved. Furthermore, a physician skilled in the art may monitor the effectiveness of treatment with a composition of the present disclosure to a subject suffering from an immune disorder, such as an autoimmune disease described herein, by analyzing the amount of autoreactive CD8+ T cells in a lymph sample isolated from the patient. The anti-TNFR2 antigen binding proteins of the present invention may attenuate the proliferation of autoreactive T cells, e.g., by binding to TNFR2 on the surface of autoreactive T cells and inducing apoptosis, and / or by stimulating the proliferation of Treg cells, which can subsequently eliminate the autoreactive T lymphocytes. Treatment with an anti-TNFR2 antigen binding protein can result in a decrease in the amount of autoreactive T cells in lymph isolated from patients receiving treatment, and a rapid decrease in the population of autoreactive T cells in lymph samples isolated from such patients can indicate that the treatment is effective.If lymph samples isolated from a patient show numbers of autoreactive T cells that are not reduced in response to agonistic TNFR2 antibody therapy, a physician may pr...

Claims

1. 1. An antigen binding protein that specifically binds to tumor necrosis factor receptor 2 (TNFR2): a) (Y / F)YQ(S / A)LS(T / S)(P / A)N(Y / F)GQ(V / T)F (SEQ ID NO: 60); b) AADSDL(S / R)TV(V / T)VGPHDY (SEQ ID NO: 61); c) AKDAG(S / G)WG(T / R)GPFG(Y / F)(E / D)YDY (SEQ ID NO: 62); d) AA(T / A)PSGKAY(T / S)Y (SEQ ID NO: 63); e) ATPGPY(T / S / M)YCAPYGSSWSRGYDY (SEQ ID NO: 64); f) ARV(R / G)G(T / S / A)PY(E / D)Y(N / G)Y (SEQ ID NO: 65); g) (T / A / V)A(S / A)PTGRAF(T / N / A)Y (SEQ ID NO: 66); h) AGSAFDF (SEQ ID NO: 42); i) S(V / M)(V / L)GRDM(M / V)TY (SEQ ID NO: 67); j) AVGDFEGELVLKGDY (SEQ ID NO: 4063); k) AAD(L / V)G(F / V / Y)LY(A / T / V)DYV(P / R)LH(M / T)HHFGS (SEQ ID NO: 4517); and l) A(A / G)(T / A)(P / L)(S / T)GKAY(T / S)Y (SEQ ID NO: 4771) An antigen binding protein comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from:

2. 2. The antigen binding protein of claim 1, wherein the CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 3, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 4063, 4067, 4071, 4524, 4530, and 4727-4730.

3. a) GSI(V / F)(R / S)(T / A)(N / D)(S / G / A) (SEQ ID NO: 68); b) GFT(F / L)DD(I / Y)A (SEQ ID NO: 69); c) GFTFS(S / R / G)YA (SEQ ID NO: 70); d) GRTFSDYG (SEQ ID NO: 16); e) G(L / F)TLDYYA (SEQ ID NO: 71); f) GF(T / N)FSMYS (SEQ ID NO: 72); g) GRTF(G / R / S)(N / S)(Y / L)(T / F) (SEQ ID NO: 73); h) GASLSRNA (SEQ ID NO: 40); i) GS(I / T)FRFPP (SEQ ID NO: 74); j) GFTLDDYA (SEQ ID NO: 4061); and k) G(F / V)(S / T)LD(D / Y)(H / Y)T (SEQ ID NO: 4519) 3. The antigen-binding protein of claim 1 or 2, further comprising a CDR1 comprising an amino acid sequence selected from:

4. 4. The antigen binding protein of claim 3, wherein the CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1, 5, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 4061, 4065, 4069, 4520, and 4719-4722.

5. a) IRSDGF(T / I) (SEQ ID NO: 75); b) I(Y / F)SY(S / G)(S / P)NT (SEQ ID NO: 76); c) I(Y / S)(S / D)DGS(E / D)T (SEQ ID NO: 77); d) INWSN(G / A)RT (SEQ ID NO: 4699); e) I(S / N)(V / T)(S / G)DGST (SEQ ID NO: 78); f) IDT(R / G)GST (SEQ ID NO: 79); g) IR(W / R / Y)(T / P)G(G / L)(S / I)T (SEQ ID NO: 80); h) IYDDGET (SEQ ID NO: 41); i) LTSGGST (SEQ ID NO: 45); j) IFSYSSNT (SEQ ID NO: 4062); and k) I(N / S)SNDG(S / T)(T / V) (SEQ ID NO: 4518) 5. The antigen-binding protein of claim 1, further comprising a CDR2 comprising an amino acid sequence selected from:

6. 6. The antigen binding protein of claim 5, wherein the CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 2, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 4062, 4066, 4070, 4527, and 4723-4726.

7. The antigen binding protein comprises: i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 68, a CDR2 comprising the amino acid sequence of SEQ ID NO: 75, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 60; ii) CDR1 comprising the amino acid sequence of SEQ ID NO: 69, CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR3 comprising the amino acid sequence of SEQ ID NO: 61; iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 70, CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and CDR3 comprising the amino acid sequence of SEQ ID NO: 62; iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 63; v) CDR1 comprising the amino acid sequence of SEQ ID NO: 71, CDR2 comprising the amino acid sequence of SEQ ID NO: 78, CDR3 comprising the amino acid sequence of SEQ ID NO: 64; vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 72, CDR2 comprising the amino acid sequence of SEQ ID NO: 79, and CDR3 comprising the amino acid sequence of SEQ ID NO: 65; vii) CDR1 comprising the amino acid sequence of SEQ ID NO: 73, CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and CDR3 comprising the amino acid sequence of SEQ ID NO: 66; viii) CDR1 comprising the amino acid sequence of SEQ ID NO: 40, CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42; ix) CDR1 comprising the amino acid sequence of SEQ ID NO: 74, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR3 comprising the amino acid sequence of SEQ ID NO: 67; x) CDR1 comprising the amino acid sequence of SEQ ID NO: 4061, CDR2 comprising the amino acid sequence of SEQ ID NO: 4062, CDR3 comprising the amino acid sequence of SEQ ID NO: 4063; xi) CDR1 comprising the amino acid sequence of SEQ ID NO: 4519, CDR2 comprising the amino acid sequence of SEQ ID NO: 4518, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4517; or xii) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4771 6. The antigen-binding protein of any one of claims 1, 3, and 5, comprising:

8. The antigen binding protein comprises: a) CDR1 comprising the amino acid sequence of SEQ ID NO: 69, CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR3 comprising the amino acid sequence of SEQ ID NO: 61; b) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 63; c) a CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or d) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4699, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4771 8. The antigen-binding protein of claim 7, comprising:

9. The antigen binding protein comprises: i) a CDR1 having the amino acid sequence GSI(V / F)(R / S)(A / T)(N / D)(G / A) (SEQ ID NO: 4700), a CDR2 comprising the amino acid sequence IRSDGFT (SEQ ID NO: 2), and a CDR3 comprising the amino acid sequence YYQ(S / A)LSSPNYGQ(V / T)F (SEQ ID NO: 4701); ii) a CDR1 having the amino acid sequence GFTFDDIA (SEQ ID NO: 8), a CDR2 comprising the amino acid sequence IYSYGPNT (SEQ ID NO: 9), and a CDR3 comprising the amino acid sequence AADSDLSTVV(V / T)GPHDY (SEQ ID NO: 4702); iii) a CDR1 having the amino acid sequence GFTFSRYA (SEQ ID NO: 12), a CDR2 comprising the amino acid sequence ISDDGSDT (SEQ ID NO: 13), and a CDR3 comprising the amino acid sequence AKDAGSWGTGPFGYEYDY (SEQ ID NO: 14); iv) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 16), CDR2 comprising the amino acid sequence INWSN(G / A)RT (SEQ ID NO: 4699), and CDR3 comprising the amino acid sequence AA(T / A)PSGKAYSY (SEQ ID NO: 4703); v) a CDR1 having the amino acid sequence GLTLDYYA (SEQ ID NO: 20), a CDR2 comprising the amino acid sequence ISTSDGST (SEQ ID NO: 21), and a CDR3 comprising the amino acid sequence ATPGPYTYCAPYGSSWSRGYDY (SEQ ID NO: 22); vi) a CDR1 having the amino acid sequence GF(T / N)FSMYS (SEQ ID NO: 72), a CDR2 comprising the amino acid sequence IDT(R / G)GST (SEQ ID NO: 79), and a CDR3 comprising the amino acid sequence ARV(G / R)G(T / A)PYEY(N / G)Y (SEQ ID NO: 4704); vii) a CDR1 having the amino acid sequence GRTF(G / S)S(Y / L)(T / F) (SEQ ID NO: 4705), a CDR2 comprising the amino acid sequence IR(W / R / Y)(T / P)G(G / L)(S / I)T (SEQ ID NO: 80), and a CDR3 comprising the amino acid sequence (A / V)A(A / S)PTGRAF(T / N)Y (SEQ ID NO: 4707); viii) a CDR1 having the amino acid sequence GASLSRNA (SEQ ID NO: 40), a CDR2 comprising the amino acid sequence IYDDGET (SEQ ID NO: 41), and a CDR3 comprising the amino acid sequence AGSAFDF (SEQ ID NO: 42); ix) CDR1 having the amino acid sequence GS(T / I)FRFPP (SEQ ID NO: 4708), CDR2 comprising the amino acid sequence LTSGGST (SEQ ID NO: 45), and CDR3 comprising the amino acid sequence SVLGRDM(M / V)TY (SEQ ID NO: 4706); x) a CDR1 having the amino acid sequence GFTLDDYA (SEQ ID NO: 4061), a CDR2 comprising the amino acid sequence IFSYSSNT (SEQ ID NO: 4062), and a CDR3 comprising the amino acid sequence AVGDFEGELVLKGDY (SEQ ID NO: 4063); xi) a CDR1 having the amino acid sequence GFTLDYYT (SEQ ID NO: 4065), a CDR2 comprising the amino acid sequence ISSNDSVGSV (SEQ ID NO: 4066), and a CDR3 comprising the amino acid sequence AADLGYLYVDYVRLHTHHFGS (SEQ ID NO: 4067); or xii) a CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising the amino acid sequence INWSN(G / A)RT (SEQ ID NO: 4699), and a CDR3 comprising the amino acid sequence A(A / G)(T / A)(P / L)(S / T)GKAY(T / S)Y (SEQ ID NO: 4771); 8. The antigen-binding protein of any one of claims 1, 3, 5, and 7, comprising:

10. The antigen binding protein comprises: a) a CDR1 having the amino acid sequence GFTFDDIA (SEQ ID NO: 8), a CDR2 comprising the amino acid sequence IYSYGPNT (SEQ ID NO: 9), and a CDR3 comprising the amino acid sequence AADSDLSTVV(V / T)GPHDY (SEQ ID NO: 4702); b) a CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising the amino acid sequence INWSN(G / A)RT (SEQ ID NO: 4699), and a CDR3 comprising the amino acid sequence AA(T / A)PSGKAYSY (SEQ ID NO: 4703); c) a CDR1 having the amino acid sequence GRTF(G / S)S(Y / L)(T / F) (SEQ ID NO: 4705), a CDR2 comprising the amino acid sequence IR(W / R / Y)(T / P)G(G / L)(S / I)T (SEQ ID NO: 80), and a CDR3 comprising the amino acid sequence (A / V)A(A / S)PTGRAF(T / N)Y (SEQ ID NO: 4707); or d) CDR1 having the amino acid sequence GRTFSDYG (SEQ ID NO: 16), CDR2 comprising the amino acid sequence INWSN(G / A)RT (SEQ ID NO: 4699), and CDR3 comprising the amino acid sequence A(A / G)(T / A)(P / L)(S / T)GKAY(T / S)Y (SEQ ID NO: 4771).

10. The antigen-binding protein of claim 8 or 9, comprising:

11. The antigen binding protein comprises: i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; ii) CDR1 comprising the amino acid sequence of SEQ ID NO:5, CDR2 comprising the amino acid sequence of SEQ ID NO:2, and CDR3 comprising the amino acid sequence of SEQ ID NO:6; iii) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10; iv) CDR1 comprising the amino acid sequence of SEQ ID NO: 12, CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR3 comprising the amino acid sequence of SEQ ID NO: 14; v) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; vi) CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR3 comprising the amino acid sequence of SEQ ID NO: 22; vii) CDR1 comprising the amino acid sequence of SEQ ID NO: 24, CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR3 comprising the amino acid sequence of SEQ ID NO: 26; viii) CDR1 comprising the amino acid sequence of SEQ ID NO: 28, CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR3 comprising the amino acid sequence of SEQ ID NO: 30; ix) CDR1 comprising the amino acid sequence of SEQ ID NO: 32, CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and CDR3 comprising the amino acid sequence of SEQ ID NO: 34; x) CDR1 comprising the amino acid sequence of SEQ ID NO: 36, CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR3 comprising the amino acid sequence of SEQ ID NO: 38; xi) CDR1 comprising the amino acid sequence of SEQ ID NO: 40, CDR2 comprising the amino acid sequence of SEQ ID NO: 41, CDR3 comprising the amino acid sequence of SEQ ID NO: 42; xii) CDR1 comprising the amino acid sequence of SEQ ID NO: 44, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR3 comprising the amino acid sequence of SEQ ID NO: 46; xiii) CDR1 comprising the amino acid sequence of SEQ ID NO: 4061, CDR2 comprising the amino acid sequence of SEQ ID NO: 4062, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4063; xiv) CDR1 comprising the amino acid sequence of SEQ ID NO: 4065, CDR2 comprising the amino acid sequence of SEQ ID NO: 4066, CDR3 comprising the amino acid sequence of SEQ ID NO: 4067; xv) CDR1 comprising the amino acid sequence of SEQ ID NO: 4069, CDR2 comprising the amino acid sequence of SEQ ID NO: 4070, CDR3 comprising the amino acid sequence of SEQ ID NO: 4071; xvi) CDR1 comprising the amino acid sequence of SEQ ID NO: 4520, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, CDR3 comprising the amino acid sequence of SEQ ID NO: 46; xvii) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4524; xviii) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; xix) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4530; xx) CDR1 comprising the amino acid sequence of SEQ ID NO: 4719, CDR2 comprising the amino acid sequence of SEQ ID NO: 4723, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4727; xxi) CDR1 comprising the amino acid sequence of SEQ ID NO: 4720, CDR2 comprising the amino acid sequence of SEQ ID NO: 4724, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4728; xxii) CDR1 comprising the amino acid sequence of SEQ ID NO: 4721, CDR2 comprising the amino acid sequence of SEQ ID NO: 4725, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4729; or xxiii) CDR1 comprising the amino acid sequence of SEQ ID NO: 4722, CDR2 comprising the amino acid sequence of SEQ ID NO: 4726, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4730 10. The antigen-binding protein of any one of claims 1 to 7 and 9, comprising:

12. The antigen binding protein comprises: a) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; b) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; c) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 4527, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4530; d) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10; e) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4524; f) CDR1 comprising the amino acid sequence of SEQ ID NO: 4069, CDR2 comprising the amino acid sequence of SEQ ID NO: 4070, CDR3 comprising the amino acid sequence of SEQ ID NO: 4071; g) CDR1 comprising the amino acid sequence of SEQ ID NO: 4719, CDR2 comprising the amino acid sequence of SEQ ID NO: 4723, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4727; h) CDR1 comprising the amino acid sequence of SEQ ID NO: 4720, CDR2 comprising the amino acid sequence of SEQ ID NO: 4724, CDR3 comprising the amino acid sequence of SEQ ID NO: 4728; i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4721, a CDR2 comprising the amino acid sequence of SEQ ID NO: 4725, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 4729; or j) CDR1 comprising the amino acid sequence of SEQ ID NO: 4722, CDR2 comprising the amino acid sequence of SEQ ID NO: 4726, and CDR3 comprising the amino acid sequence of SEQ ID NO: 4730 12. The antigen-binding protein of any one of claims 1 to 11, comprising:

13. The antigen-binding protein of any one of claims 1 to 12, wherein the antigen-binding protein is a single domain antibody.

14. 14. The antigen binding protein of claim 13, wherein the single domain antibody is a VHH, VNAR, or modified VH domain.

15. 15. The antigen binding protein of claim 14, wherein the VHH is a Camelidae VHH.

16. 16. The antigen-binding protein of claim 15, wherein the VHH comprises an amino acid sequence selected from SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, 4521, 93-640, 4079-4125, 2805-3363, 4359-4420, and 4605-4628, or a sequence with at least 75% identity thereto.

17. 17. The antigen-binding protein of claim 15 or 16, wherein the VHH comprises an amino acid sequence selected from SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 4064, 4068, 4072, and 4521, or a sequence with at least 75% identity thereto.

18. The antigen-binding protein of claim 14, wherein the VHH is a humanized VHH.

19. 19. The antigen-binding protein of claim 18, wherein the humanized VHH comprises an amino acid sequence selected from SEQ ID NOs: 81-92, 4076-4078, 4523, 4526, 4529, 4532, 4731-4734, 641-1127, and 4126-4172, or a sequence with at least 75% identity thereto.

20. 20. The antigen-binding protein of claim 19, wherein the humanized VHH comprises an amino acid sequence selected from SEQ ID NOs: 81-92, 4076-4078, 4523, 4526, 4529, 4731-4734, and 4532, or a sequence having at least 75% identity thereto.

21. 21. The antigen-binding protein of any one of claims 1 to 20, wherein the antigen-binding protein has an agonist effect when bound to TNFR2.

22. 22. The antigen-binding protein of any one of claims 1 to 21, wherein the antigen-binding protein binds to human TNFR2.

23. The antigen-binding protein is about 3×10 －７ K less than M Ｄ 23. The antigen-binding protein of claim 22, which binds to human TNFR2 at

24. The antigen-binding protein is about 1 x 10 －１０ ~5 x 10 －８ K of M Ｄ 24. The antigen-binding protein of claim 23, which binds to human TNFR2 at

25. 11. The antigen-binding protein of any one of claims 1 to 10, wherein the antigen-binding protein binds to cynomolgus monkey TNFR2.

26. The antigen-binding protein is about 3×10 －７ K less than M Ｄ 26. The antigen-binding protein of claim 25, which binds to cynomolgus monkey TNFR2 at

27. The antigen-binding protein is about 1 x 10 －９ ~2 x 10 －７ K of M Ｄ 27. The antigen-binding protein of claim 26, which binds to cynomolgus monkey TNFR2 at

28. The antigen-binding protein of any one of claims 1 to 27, wherein the antigen-binding protein binds to the same epitope or epitopes as antibody clone MR2-1.

29. 28. The antigen-binding protein of any one of claims 1 to 27, wherein the antigen-binding protein does not bind to the same epitope or epitopes as antibody clone MR2-1.

30. 30. The antigen binding protein of any one of claims 1 to 29, wherein the antigen binding protein increases expression of one or more proteins selected from proteins in the NF-kB pathway, FOXP3, HELIOS, EZH2, HLA-DR, ICAM-1, OX-40, ICOS, and CCR8.

31. 31. The antigen binding protein of any one of claims 1 to 30, wherein the antigen binding protein comprises one or more modifications that reduce binding of the antigen binding protein by pre-existing antibodies found in human blood or serum.

32. A fusion protein that specifically binds to tumor necrosis factor receptor 2 (TNFR2), comprising one or more of the antigen-binding proteins of any one of claims 1 to 31.

33. 33. The fusion protein of claim 32, comprising two of the antigen-binding proteins.

34. 33. The fusion protein of claim 32, comprising four of said antigen-binding proteins.

35. The fusion protein of any one of claims 32 to 34, wherein the one or more antigen binding proteins bind to the same epitope on TNFR2.

36. The fusion protein of any one of claims 32 to 34, wherein the one or more antigen binding proteins bind to different epitopes on TNFR2.

37. The fusion protein of any one of claims 32 to 36, wherein the one or more antigen-binding proteins are one or more single-domain antibodies.

38. 38. The fusion protein of claim 37, wherein the one or more single domain antibodies are one or more VHHs.

39. The fusion protein of any one of claims 32 to 38, further comprising an immunoglobulin Fc region.

40. 40. The fusion protein of claim 39, wherein the immunoglobulin Fc region is a human immunoglobulin Fc region.

41. 41. The fusion protein of claim 40, wherein the immunoglobulin Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region, or a variant thereof.

42. 42. The fusion protein of claim 41, wherein the immunoglobulin Fc region is a human IgG1 Fc region or a variant thereof.

43. 43. The fusion protein of claim 42, wherein the Fc region of human IgG1 comprises one or more mutations selected from L234A, L235A, G237A, D265A, N297A, and / or P329A according to EU numbering.

44. The Fc region of human IgG1: 1) L234A and L235A; 2) L234A, L235A, and P329A; 3) D265A, N297A, and P329A; and 4) L234A, L235A, and G237A 44. The fusion protein of claim 43, comprising a set of mutations selected from:

45. 42. The fusion protein of claim 41, wherein the immunoglobulin Fc region is a human IgG4 Fc region or a variant thereof.

46. 46. ​​The fusion protein of claim 45, wherein the Fc region of human IgG4 comprises one or more mutations selected from S228P, L235E, L235A, and / or F234A according to EU numbering.

47. The Fc region of human IgG4: 1) S228P and L235E; 2) S228P and L235A; 3) S228P, F234A, and L235E; and 4) S228P, F234A, and L235A 47. The fusion protein of claim 46, comprising a set of mutations selected from:

48. The fusion protein of any one of claims 32 to 47, further comprising a cytokine.

49. 49. The fusion protein of claim 48, wherein the cytokine is IL-2 or a variant thereof.

50. 50. The fusion protein of claim 49, wherein the cytokine is an IL-2 variant comprising an N88D mutation.

51. 51. The fusion protein of any one of claims 32 to 50, further comprising a moiety that binds to serum albumin.

52. 33. The fusion protein of claim 32, comprising the amino acid sequence of any one of SEQ ID NOs: 3933-3964, 4483-4513, 4686-4696, 4709-4716, and 4735-4770, or a sequence having at least 75% identity thereto.

53. 53. A conjugate comprising an antigen-binding protein according to any one of claims 1 to 31 or a fusion protein according to any one of claims 32 to 52, wherein the antigen-binding protein or the fusion protein is conjugated to a second moiety.

54. 54. The conjugate of claim 53, wherein the second moiety is selected from a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulator, a cytokine, a cytotoxic agent, a chemotherapeutic agent, a diagnostic agent, or a combination thereof.

55. 55. The conjugate of claim 54, wherein the second moiety is a cytokine.

56. 56. The conjugate of claim 55, wherein the cytokine is IL-2 or a variant thereof.

57. 57. The conjugate of claim 56, wherein the cytokine is an IL-2 variant comprising an N88D mutation.

58. A polynucleotide molecule encoding the antigen-binding protein of any one of claims 1 to 31 or the fusion protein of any one of claims 32 to 52.

59. 59. The polynucleotide molecule of claim 58, wherein the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 48-59, 4073-4075, 4522, 4525, 4528, 4531, 3364-3922, 4421-4482, and 4629-4652, or a sequence having at least 70% identity thereto.

60. 60. The polynucleotide molecule of claim 59, wherein the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 48-59, 4073-4075, 4522, 4525, 4528, and 4531, or a sequence having at least 70% identity thereto.

61. A recombinant vector comprising the polynucleotide molecule of any one of claims 58 to 60.

62. A host cell comprising the polynucleotide molecule of any one of claims 58 to 60 or the recombinant vector of claim 61.

63. 62. A kit comprising an antigen binding protein according to any one of claims 1 to 31, a fusion protein according to any one of claims 32 to 52, a conjugate according to any one of claims 53 to 57, a polynucleotide molecule according to any one of claims 58 to 60, or a recombinant vector according to claim 61, and optionally instructions and / or packaging for said kit.

64. 62. A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1 to 31, the fusion protein of any one of claims 32 to 52, the conjugate of any one of claims 53 to 57, the polynucleotide molecule of any one of claims 58 to 60, or the recombinant vector of claim 61, together with a pharmaceutically acceptable carrier and / or excipient.

65. 1. A method for preparing an antigen binding protein or fusion protein that specifically binds to tumor necrosis factor receptor 2 (TNFR2), comprising the steps of: (a) culturing the host cell of claim 62 in a culture medium under conditions suitable for expression of said antigen binding protein or said fusion protein; and (b) isolating the antigen binding protein or the fusion protein from the host cell and / or the culture medium. A method comprising:

66. 58. A method of promoting the expansion, activating and / or enhancing the suppressive function and / or stabilizing the immunosuppressive phenotype of a population of regulatory T cells (Treg), said method comprising contacting said population of regulatory T cells with an antigen binding protein of any one of claims 1 to 31, a fusion protein of any one of claims 32 to 52, or a conjugate of any one of claims 53 to 57.

67. 67. The method of claim 66, wherein the contacting step is performed in vitro.

68. 67. The method of claim 66, wherein the contacting step is performed in vivo.

69. 69. The method of claim 68, wherein the method further comprises administering the antigen binding protein, the fusion protein, or the conjugate to a subject in need thereof.

70. 100. A method of treating or preventing a disease or disorder in a subject in need thereof, said method comprising administering to said subject an antigen binding protein of any one of claims 1 to 31, a fusion protein of any one of claims 32 to 52, or a conjugate of any one of claims 53 to 57.

71. 71. The method of claim 70, wherein the disease or disorder is an immune disease, an inflammatory disease, cancer, a cardiovascular disease, or an infertility and pregnancy-related disorder.

72. 72. The method of claim 71, wherein the immune disorder is selected from an autoimmune disease, a neurological condition, an allergy, asthma, macular degeneration, muscle atrophy, a miscarriage-related disorder, atherosclerosis, bone loss, a musculoskeletal disorder, obesity, graft-versus-host disease, and allograft rejection.

73. The autoimmune diseases include lupus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia / fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, and the like. thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lichen sclerosus, IgG4-related disease, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disorder, pemphigus vulgaris or related bullous skin diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome 73. The method of claim 72, wherein the disease is selected from the group consisting of: rheumatic syndrome, polymyalgia rheumatica, polymyositis / dermatomyositis, premature ovarian failure, primary agammaglobulinemia, primary biliary cholangitis, psoriasis, primary ovarian insufficiency, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, stiff-man syndrome, type 1 diabetes mellitus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis with polyangiitis or other immune-mediated vasculitis.

74. 74. The method of claim 73, wherein the lupus is systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, neonatal lupus, or drug-induced lupus.

75. 75. The method of claim 74, wherein the cutaneous lupus is acute cutaneous lupus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus (DLE), or subacute cutaneous lupus erythematosus.

76. 73. The method of claim 72, wherein the neurological condition is selected from brain tumors, brain metastases, spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Parkinson's disease, and stroke.

77. 73. The method of claim 72, wherein the allergy is selected from a food allergy, a seasonal allergy, a pet allergy, hives, hay fever, allergic conjunctivitis, poison ivy allergy, oak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy, and chemical allergy.

78. 73. The method of claim 72, wherein the allograft rejection is selected from skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection, and organ graft rejection.

79. 73. The method of claim 72, wherein the ligament graft rejection is selected from cricothyroid ligament graft rejection, posterior cruciate ligament graft rejection, periodontal ligament graft rejection, lens suspensory ligament graft rejection, volar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, elbow medial collateral ligament graft rejection, elbow lateral collateral ligament graft rejection, mammary suspensory ligament graft rejection, anterior sacroiliac ligament graft rejection, posterior sacroiliac ligament graft rejection, sacrotuberous ligament graft rejection, sacrospinous ligament graft rejection, inferior pubic ligament graft rejection, superior pubic ligament graft rejection, anterior cruciate ligament graft rejection, lateral collateral ligament graft rejection, posterior cruciate ligament graft rejection, medial collateral ligament graft rejection, canine anterior cruciate ligament graft rejection, and patellar ligament graft rejection.

80. 73. The method of claim 72, wherein the organ transplant rejection is selected from heart transplant rejection, lung transplant rejection, kidney transplant rejection, liver transplant rejection, pancreas transplant rejection, intestinal transplant rejection, and thymus transplant rejection.

81. 73. The method of claim 72, wherein the graft-versus-host disease arises from a bone marrow transplant or from one or more blood cells selected from B cells, T cells, basophils, common myeloid progenitor cells, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.

82. 73. The method of claim 72, wherein the inflammatory disease is acute or chronic inflammation.

83. 73. The method of claim 72, wherein the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovary syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.

84. The cancers include adenoid cystic carcinoma, adrenal tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer, and the like. cancer, cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (bone sarcoma), brainstem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing's sarcoma, eye tumor, eyelid tumor, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor tumor (GIST), germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell carcinoma, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal cell carcinoma, HIV / AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia and hairy cell leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (chronic myeloid leukemia), myeloid leukemia (CML), chronic T-cell lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromesyndrome (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumors of the gastrointestinal tract, neuroendocrine tumors of the lung, neuroendocrine tumors of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian, fallopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary tumor, pleuropulmonary blastoma, 73. The method of claim 72, wherein the cancer is selected from prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, soft tissue tumors, skin cancer (non-melanoma), small intestine cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, uterine cancer, vaginal cancer, von Hippel-Lindau disease, vulvar cancer, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), Werner's syndrome, Wilms' tumor, or xeroderma pigmentosum.

85. 73. The method of claim 72, wherein the cardiovascular disease is selected from atherosclerosis, heart failure, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, right heart failure, congestive heart failure, restrictive cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, idiopathic cardiomyopathy, and hypertension.

86. 73. The method of claim 72, wherein the infertility and pregnancy related disorder is selected from recurrent pregnancy loss, preeclampsia, preterm labor, fetal growth restriction, or intrauterine growth restriction.

87. 100. A method of regenerating a tissue or organ comprising one or more TNFR2+ cells, said method comprising contacting said tissue or organ with an effective amount of an antigen binding protein of any one of claims 1 to 31, a fusion protein of any one of claims 32 to 52, or a conjugate of any one of claims 53 to 57.

88. 88. The method of claim 87, wherein the tissue or organ is selected from pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerve, heart, aorta, olfactory gland, ear, nerve, eye, thymus, tongue, bone, liver, small intestine, large intestine, gastrointestinal tract, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structure, embryo, and testicular tissue.

89. 89. The method of claim 87 or 88, wherein the contacting step is performed in vitro.

90. 89. The method of claim 87 or 88, wherein the contacting step is performed in vivo.

91. 91. The method of claim 90, wherein the method further comprises administering the antigen binding protein, the fusion protein, or the conjugate to a subject in need thereof.

92. 58. A method for inducing tolerance to and / or preventing or reducing an immune response to a foreign substance in a subject in need thereof, said method comprising administering to said subject an antigen-binding protein of any one of claims 1 to 31, a fusion protein of any one of claims 32 to 52, or a conjugate of any one of claims 53 to 57.

93. 93. The method of claim 92, wherein the foreign substance is a therapeutic protein or peptide, a viral vector, a bacterial vector, a fungal vector, a biochemical vector, a lipid, a carbohydrate, a nucleic acid, a sperm, an oocyte, or an embryo.

94. 94. The method of claim 93, wherein the viral vector is a DNA or RNA vector.

95. 95. The method of any one of claims 69 to 83 and 91 to 94, wherein the subject is a mammal.

96. 96. The method of claim 95, wherein the mammal is a human.

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