Proteins that bind to CD80 and / or CD86 and OX40L

Abstract

The present disclosure generally relates to proteins that inhibit T cell costimulatory signaling, including a polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86, and a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L. In some embodiments, the application provides antibodies that specifically bind to OX40L. In some embodiments, the application also provides therapeutic methods that utilize such proteins in the treatment of autoimmune diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 208,478, filed June 8, 2021, and U.S. Provisional Patent Application No. 63 / 343,268, filed May 18, 2022, the entire disclosures of each of which are incorporated herein by reference for all purposes.

[0002] Declaration of array list This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy was created on May 25, 2022, is named EMD-015WO_SL.txt, and is 872,058 bytes in size.

[0003] The present disclosure relates generally to proteins capable of altering T cell costimulatory signaling and their use in autoimmune diseases / disorders. [Background technology]

[0004] One major therapeutic goal for the treatment of autoimmune diseases is to alter T cell stimulation by specifically targeting costimulatory pathways. T cells are fully activated when they receive two signals: 1) T cell receptor (TCR) activation due to interaction with peptide / MHC complexes on the surface of antigen-presenting cells (APCs); and 2) T cell costimulatory receptor activation due to interaction with a cognate ligand. Stimulating T cells in the absence of a second costimulatory signal leads to the development of unresponsive or tolerant T cell responses.

[0005] There are two major groups of costimulatory molecules: 1) the B7-CD28 superfamily and 2) the tumor necrosis factor receptor (TNFR) superfamily. Costimulatory molecules in the B7-CD28 superfamily include CD28 and ICOS on T cells and their cognate ligands on the surface of APCs, CD80 / CD86 (also known as B7.1 / B7.2) and ICOS-L, respectively. Costimulatory molecules in the TNFR superfamily include 4-1BB, CD40L, OX40, CD27, GITR, and CD30 on T cells and their cognate ligands, 4-1BBL, CD40, OX40L, CD70, GITRL, and CD30L, respectively.

[0006] APCs (e.g., B cells, monocytes, macrophages, and dendritic cells) upregulate the expression of CD80 and CD86 upon activation by antigenic, inflammatory, or pathogen-associated molecular pattern receptors. These ligands engage CD28 on T cells to provide the necessary second costimulatory signal to activate naive T cells and promote T cell proliferation.

[0007] Because CD80 and CD86 have greater affinity for CTLA4 than for CD28, expression of CTLA4 on T cells inhibits CD80 / CD86 binding to CD28 by sequestrating costimulatory ligands and attenuating T cell activation. Abatacept is a soluble recombinant CTLA4-Ig fusion protein that contains the extracellular domain of human CTLA4 and an engineered human immunoglobulin 1 (IgG1) Fc domain. Abatacept selectively binds to CD80 / CD86 on APCs and blocks costimulatory activation of CD28 on T cells, resulting in reduced T cell activation, signaling, and proliferation. Abatacept has demonstrated clinical efficacy in the treatment of a number of autoimmune diseases, including adult rheumatoid arthritis, psoriatic arthritis, and juvenile idiopathic arthritis. However, abatacept has been omitted from clinical endpoints in clinical trials for systemic lupus erythematosus (SLE), lupus nephritis (LN), and primary Sjögren's syndrome (pSS).

[0008] OX40 ligand (OX40L, also known as CD252, gp34, and TNFSF4) is a 34 kDa type II transmembrane protein of the TNF superfamily. Expression of OX40L is induced by APC activation and chronically activated T cells. OX40L is also upregulated in non-lymphoid cells, such as human tonsillar mast cells, bronchial smooth muscle cells, and vascular endothelial cells in inflamed or diseased tissues. As is typical for most TNF family ligands, OX40L is cleaved by proteolytic enzymes and therefore exists in a soluble form. Serum levels of soluble OX40L have been reported to be elevated in patients with autoimmune diseases compared to healthy subjects.

[0009] OX40L on APCs binds to OX40 (CD134, TNFRSF4) expressed on the surface of activated CD4+ and CD8+ T cells. OX40 is not expressed on the surface of resting naive or resting memory T cells. OX40L binds to three OX40 monomers on T cells as a homotrimeric complex. Binding of OX40L to OX40 stimulates T cell proliferation, differentiation, and survival of T effector subsets, including T helper 1 (Th1), T helper 2 (Th2), T follicular helper (Tfh), and T helper 17 (Th17) cells. OX40 signaling is also crucial for the generation, maintenance, and optimal reactivation of memory CD4+ T cells.

[0010] Dysregulation of the OX40L-OX40 pathway has been implicated in a variety of inflammatory diseases, including multiple sclerosis, arthritis, graft-versus-host disease (GVHD), lupus nephritis, and rheumatoid arthritis. TNFSF4 is the gene encoding OX40L and is one susceptibility locus for systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). It has been demonstrated that myeloid and B cells expressing OX40L support the development of Tfh cells and contribute to SLE disease pathogenesis. OX40L-OX40 signaling also regulates the expression of T regulatory cells (Treg or T regsFoxp3+ Tregs contribute to the development of autoimmune diseases by dysregulating overactivated T effector (Teff, T eff , or T エフェクタ OX40L-OX40 signaling inhibits Foxp3 expression in Tregs, negatively affecting their suppressive function in human SLE. Summary of the Invention [Problem to be solved by the invention]

[0011] The present disclosure relates generally to proteins capable of altering T cell costimulatory signaling and their use in autoimmune diseases / disorders. [Means for solving the problem]

[0012] In one aspect of the present disclosure, a protein is provided that includes a polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86 and a polypeptide or a complex of two or more polypeptides that specifically binds to OX40L. In some embodiments, the polypeptide or the complex of two or more polypeptides that specifically binds to CD80 and / or CD86 is a polypeptide comprising an extracellular domain of CTLA4 or a functional fragment thereof; a polypeptide comprising an extracellular domain of CD28 or a functional fragment thereof; an antibody; a Fab; a Fab', a F(ab')2, a single chain variable fragment (scFv); a minibody; or a nanobody (VHH). In some embodiments, the polypeptide or the complex of two or more polypeptides that specifically binds to CD80 and / or CD86 consists of a sequence at least 90% identical to SEQ ID NO:29, SEQ ID NO:173, or SEQ ID NO:174, or ... or a functional fragment thereof. In some embodiments, the polypeptide, or the complex of two or more polypeptides, that specifically binds to CD80 and / or CD86 inhibits CD28 costimulatory signaling in T cells.

[0013] In another aspect of the present disclosure, the polypeptide or the complex of two or more polypeptides that specifically bind to OX40L inhibits the binding of endogenous OX40L to endogenous OX40. In some embodiments, the polypeptide or the complex of two or more polypeptides that specifically bind to OX40L comprises an antigen-binding site. In some embodiments, the antigen-binding site is an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH). In some embodiments, the antigen-binding site is a Fab that comprises a polypeptide comprising the sequence of SEQ ID NO: 336, and is connected to the C-terminus of the heavy chain CH1 domain of the Fab. In other embodiments, the polypeptide or the complex of two or more polypeptides that specifically bind to OX40L is the extracellular domain of OX40 or a functional fragment thereof.

[0014] In some embodiments, the protein of the disclosure further comprises a bridging moiety. In some embodiments, the bridging moiety is functional or non-functional. In some embodiments, the bridging moiety comprises a polypeptide of an immunoglobulin Fc domain or a functional fragment thereof, a human serum albumin (HSA) polypeptide or a functional fragment thereof, a polypeptide linker, or a polypeptide hinge. In some embodiments, the bridging moiety comprises a polypeptide of an Fc domain of a human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the polypeptide of a human IgG1 Fc domain comprises a sequence that is at least 90% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240. In some embodiments, the polypeptide of a human IgG1 Fc domain comprises one or more effector function silencing mutations. In some embodiments, the hinge polypeptide comprises leucine to alanine mutations at positions 234 and 235.

[0015] In some embodiments, the bridging moiety further comprises a hinge polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:242, SEQ ID NO:176, SEQ ID NO:249, SEQ ID NO:177, SEQ ID NO:243, SEQ ID NO:212, SEQ ID NO:244, SEQ ID NO:213, SEQ ID NO:245, SEQ ID NO:247, and SEQ ID NO:248.

[0016] In some embodiments of the protein of the invention, the bridging moiety is connected to the N-terminus or C-terminus of the polypeptide or the complex of two or more polypeptides that specifically binds to CD80 and / or CD86. In some embodiments, the polypeptide or the complex of two or more polypeptides that specifically binds to CD80 and / or CD86 is connected to the N-terminus of the bridging moiety. In some embodiments, the bridging moiety is connected to the N-terminus or C-terminus of the polypeptide or the complex of two or more polypeptides that specifically binds to OX40L. In some embodiments, the polypeptide or the complex of two or more polypeptides that specifically binds to OX40L is connected to the C-terminus of the bridging moiety. In some embodiments, (GGGGS) n A linker polypeptide comprising the sequence (SEQ ID NO:181), where n is 1-12, connects the polypeptide that specifically binds to OX40L, or the complex of two or more polypeptides, to the bridging moiety.

[0017] In some embodiments, the protein of the present disclosure comprises a second polypeptide or a complex of two or more polypeptides that specifically binds to OX40L. In some embodiments, the bridging moiety is a polypeptide of a human IgG1 Fc domain, and the polypeptide or the complex of two or more polypeptides that specifically binds to OX40L is connected to the C-terminus of the polypeptide of the human IgG1 Fc domain, and the second polypeptide or the complex of two or more polypeptides that specifically binds to OX40L is connected to the C-terminus of the second polypeptide of the human IgG1 Fc domain. In some embodiments, the first and / or second polypeptide or the complex of two or more polypeptides that specifically binds to OX40L comprises an antigen binding site (e.g., Fab) or comprises an extracellular domain of OX40 or a functional fragment thereof. In some embodiments, the second polypeptide or the complex of two or more polypeptides that specifically binds to OX40L comprises the amino acid sequence of (GGGGS) n(SEQ ID NO: 181) (wherein n is 1 to 12).

[0018] In some embodiments, a protein of the disclosure comprises an antigen-binding site comprising the amino acid sequence of: (a) ARHRGX1YX2FDX3 (SEQ ID NO: 220), where X1 is S or I, X2 is F or H, and X3 is I or Y; or (b) ARERSX1X2WYPX3DY (SEQ ID NO: 221), where X1 is N or S, X2 is N, D, G, or S, and X3 is I or F.

[0019] In some embodiments, a protein of the disclosure comprises: (a) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence GX1SX2X3X4SX5YY (SEQ ID NO: 222), where X1 is A, G, or V, X2 is V or I, X3 is S or R, X4 is S or T, and X5 is S or G; (b) (1) IX1YX2GST (SEQ ID NO: 223), where X1 is Y or N and X2 is S or G; (2) X1DYSGT (SEQ ID NO: 224), where X1 is I or M; or (3) IGSVDYSGX1 and (c) an antigen binding site comprising a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of: (1) ARHRGX1YX2FDX3 (SEQ ID NO: 220) (wherein X1 is S or I, X2 is F or H, and X3 is I or Y); or (2) ARERSX1X2WYPX3DY (SEQ ID NO: 221) (wherein X1 is N or S, X2 is N, D, G, or S, and X3 is I or F).

[0020] In some embodiments, a protein of the disclosure comprises: (a) a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of (1) X1IENKN (SEQ ID NO: 226), where X1 is N or D; or (2) SX1RX2X3X4 (SEQ ID NO: 227), where X1 is V or L, X2 is R or N, X3 is F or Y, and X4 is F or Y; (b) RDN (SEQ ID NO: 82), GKD (SEQ ID NO: 228), or RDS (SEQ ID NO: 90); and (c) an antigen-binding site comprising a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of (1) QVX1DSX2X3VV (SEQ ID NO:231) (wherein X1 is R or W, X2 is N, T, or A, and X3 is I, T, or A) or (2) NSRDSSGYX1VX2 (SEQ ID NO:232) (wherein X1 is L or H and X2 is L or V).

[0021] In some embodiments, a protein of the disclosure comprises: (i) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence GX1SX2X3X4SX5YY (SEQ ID NO:222), where X1 is A, G, or V, X2 is V or I, X3 is S or R, X4 is S or T, and X5 is S or G; (b) a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence IX1YX2GST (SEQ ID NO:223), where X1 is Y or N, and X2 is S or G; and (c) an amino acid sequence ARHRGX1YX2FDX3 (SEQ ID NO:220), where X1 is S or I, , X2 is F or H, and X3 is I or Y; (ii) an antigen binding site comprising: (a) a heavy chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence X1IENKN (SEQ ID NO:226) (wherein X1 is N or D); (b) a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence RDN (SEQ ID NO:82); and (c) a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence QVX1DSX2X3VV (SEQ ID NO:231) (wherein X1 is R or W, X2 is N, T, or A, and X3 is I, T, or A).

[0022] In some embodiments, a protein of the disclosure comprises: (i) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising: (a) the amino acid sequence GX1SX2X3X4SX5YY (SEQ ID NO: 222), where X1 is A, G, or V, X2 is V or I, X3 is S or R, X4 is S or T, and X5 is S or G; and (b) the amino acid sequence X1DYSGT (SEQ ID NO: 224). (c) a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence ARERSX1X2WYPX3DY (SEQ ID NO: 221) (wherein X1 is N or S, X2 is N, D, G, or S, and X3 is I or F); (ii) (a) a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence ARERSX1X2WYPX3DY (SEQ ID NO: 221) (wherein X1 is N or S, X2 is N, D, G, or S, and X3 is I or F); or N, X3 is F or Y, and X4 is F or Y; (b) a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence GKD (SEQ ID NO: 229); and (c) a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence NSRDSSGYX1VX2 (SEQ ID NO: 232), where X1 is L or H and X2 is L or V.

[0023] In some embodiments, a protein of the disclosure comprises an antigen binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISTSSYY (SEQ ID NO:77), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO:78), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO:79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO:81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO:82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO:83).

[0024] In some embodiments, a protein of the disclosure comprises an antigen binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO: 93), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of MDYSGT (SEQ ID NO: 94), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARERSNNWYPIDY (SEQ ID NO: 95), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SVRRFF (SEQ ID NO: 97), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of GKD (SEQ ID NO: 98), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of NSRDSSGYLVL (SEQ ID NO: 99).

[0025] In some embodiments, a protein of the disclosure comprises an antigen binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GASVSSSSYY (SEQ ID NO: 85), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of INYGGST (SEQ ID NO: 86), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYHFDY (SEQ ID NO: 87), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91).

[0026] In some embodiments, a protein of the disclosure comprises an antigen binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISSSSYY (SEQ ID NO: 101), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IGSVDYSGNT (SEQ ID NO: 102), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91).

[0027] In some embodiments, a protein of the disclosure comprises an antigen-binding site comprising an amino acid sequence having at least 90% identity to SEQ ID NO:76, SEQ ID NO:92, SEQ ID NO:84, or SEQ ID NO:100, and an amino acid sequence having at least 90% identity to SEQ ID NO:80, SEQ ID NO:96, SEQ ID NO:88, or SEQ ID NO:104.

[0028] In some embodiments, a protein of the disclosure comprises an antigen binding site comprising an amino acid sequence having at least 90% identity to SEQ ID NO:76 and an amino acid sequence having at least 90% identity to SEQ ID NO:80.

[0029] In some embodiments, a protein of the disclosure comprises an antigen binding site comprising an amino acid sequence having at least 90% identity to SEQ ID NO:92 and an amino acid sequence having at least 90% identity to SEQ ID NO:96.

[0030] In some embodiments, a protein of the disclosure comprises an antigen-binding site comprising an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:14, or SEQ ID NO:18, and an amino acid sequence having at least 90% identity to SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:13, or SEQ ID NO:17.

[0031] In some embodiments, a protein of the disclosure comprises an antigen-binding site comprising an amino acid sequence having at least 90% identity to SEQ ID NO:8 and an amino acid sequence having at least 90% identity to SEQ ID NO:7.

[0032] In some embodiments, a protein of the disclosure comprises an antigen-binding site comprising an amino acid sequence having at least 90% identity to SEQ ID NO:16 and an amino acid sequence having at least 90% identity to SEQ ID NO:15.

[0033] In some embodiments, the proteins of the disclosure comprise an antigen binding site that binds to positions 60 and / or 83 of the extracellular domain of human OX40L. In some embodiments, the antigen binding site binds to positions 17, 18, 19, 20, 21, 23, 26, 28, 60, 83, 110, 111, 112, 113, and 114 of the extracellular domain of human OX40L. In some embodiments, the antigen binding site binds to positions 58, 59, 60, 61, 62, 63, 81, 82, and 83 of the extracellular domain of human OX40L.

[0034] In some embodiments, a protein of the disclosure comprises: (i) a polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (ii) a bridging portion comprising a sequence comprising SEQ ID NO: 177 and a sequence comprising SEQ ID NO: 179; and (iii) an antigen binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISTSSYY (SEQ ID NO: 77), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83).

[0035] In some embodiments, a protein of the disclosure comprises a first arm and a second arm, the first arm comprising: (i) a first polypeptide comprising an extracellular domain of CTLA4 comprising SEQ ID NO: 174; (ii) a first bridging portion comprising a sequence comprising SEQ ID NO: 177 and a sequence comprising SEQ ID NO: 179; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISTSSYY (SEQ ID NO: 77), a heavy chain variable complementarity determining region 2 (VHCDR3) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78). a first antigen-binding domain comprising a heavy chain variable complementarity determining region 2 (VHCDR2), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83); (ii) a second bridging portion comprising a sequence comprising SEQ ID NO: 177 and a sequence comprising SEQ ID NO: 179; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISTSSYY (SEQ ID NO: 77), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy chain variable complementarity determining region 3 (VHCDR4) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), and a heavy chain variable complementarity determining region 4 (VHCDR5) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 79). and a second antigen-binding site comprising a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO:79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO:81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO:82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO:83), wherein the first arm and the second arm dimerize with each other.

[0036] In some embodiments, a protein of the disclosure comprises: (i) a polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (ii) a bridging portion comprising a sequence comprising SEQ ID NO: 213 and a sequence comprising SEQ ID NO: 216; and (iii) an antigen binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO: 93), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of MDYSGT (SEQ ID NO: 94), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARERSNNWYPIDY (SEQ ID NO: 95), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SVRRFF (SEQ ID NO: 97), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of GKD (SEQ ID NO: 98), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of NSRDSSGYLVL (SEQ ID NO: 99).

[0037] In some embodiments, a protein of the disclosure comprises a first arm and a second arm, the first arm comprising: (i) a first polypeptide comprising an extracellular domain of CTLA4 comprising SEQ ID NO: 174; (ii) a first bridging portion comprising a sequence comprising SEQ ID NO: 213 and a sequence comprising SEQ ID NO: 216; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO: 93), a heavy chain variable complementarity determining region 2 (VHCDR3) comprising the amino acid sequence of MDYSGT (SEQ ID NO: 94). a heavy chain variable complementarity determining region 2 (VHCDR2), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARERSNNWYPIDY (SEQ ID NO: 95), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SVRRFF (SEQ ID NO: 97), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of GKD (SEQ ID NO: 98), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of NSRDSSGYLVL (SEQ ID NO: 99); (ii) a second bridging portion comprising a sequence comprising SEQ ID NO:213 and a sequence comprising SEQ ID NO:216; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO:93), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of MDYSGT (SEQ ID NO:94), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARERSNNWYPIDY (SEQ ID NO:95), a heavy chain variable complementarity determining region 4 (VHCDR4) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO:96), a heavy chain variable complementarity determining region 5 (VHCDR5) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO:97), a heavy chain variable complementarity determining region 6 (VHCDR6) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO:98), a heavy chain variable complementarity determining region 7 (VHCDR8) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO:99), a heavy chain variable complementarity determining region 8 (VHCDR9) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO:99), a heavy chain variable complementarity determining region 9 (VHCDR10) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO:99), a heavy chain variable complementarity determining region 10 (VHCDR11) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO:99), a heavy chain variable complementarity determining region 11 (VHCDR12) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO:99), a heavy chain variable complementarity determining region 12 (VHCDR13) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO:99), a heavy chain variable complementarity determining region 13 ( and a second antigen-binding site comprising a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO:5), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SVRRFF (SEQ ID NO:97), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of GKD (SEQ ID NO:98), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of NSRDSSGYLVL (SEQ ID NO:99), wherein the first arm and the second arm dimerize with each other.

[0038] In some embodiments, a protein of the disclosure comprises: (i) a polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (ii) a bridging portion comprising a sequence comprising SEQ ID NO: 213 and a sequence comprising SEQ ID NO: 214; and (iii) an antigen binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GASVSSSSYY (SEQ ID NO: 85), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of INYGGST (SEQ ID NO: 86), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYHFDY (SEQ ID NO: 87), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91).

[0039] In some embodiments, a protein of the disclosure comprises a first arm and a second arm, the first arm comprising: (i) a first polypeptide comprising an extracellular domain of CTLA4 comprising SEQ ID NO: 174; (ii) a first bridging moiety comprising a sequence comprising SEQ ID NO: 213 and a sequence comprising SEQ ID NO: 214; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GASVSSSSYY (SEQ ID NO: 85), a heavy chain variable complementarity determining region 2 (VHCDR3) comprising the amino acid sequence of INYGGST (SEQ ID NO: 86). a first antigen-binding fragment comprising a heavy chain variable complementarity determining region 2 (VHCDR2), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYHFDY (SEQ ID NO: 87), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91); (ii) a second bridging portion comprising a sequence comprising SEQ ID NO:213 and a sequence comprising SEQ ID NO:214; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GASVSSSSYY (SEQ ID NO:85), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of INYGGST (SEQ ID NO:86), a heavy chain variable complementarity determining region 3 (VHCDR4) comprising the amino acid sequence of ARHRGIYHFDY (SEQ ID NO:87), and a heavy chain variable complementarity determining region 4 (VHCDR5) comprising the amino acid sequence of GASVSSSSYY (SEQ ID NO:88). and a second antigen-binding site comprising a heavy chain variable complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of NIENKN (SEQ ID NO: 87), a light chain variable complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VL CDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VL CDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91), wherein the first arm and the second arm dimerize with each other.

[0040] In some embodiments, a protein of the disclosure comprises: (i) a polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (ii) a bridging portion comprising a sequence comprising SEQ ID NO: 213 and a sequence comprising SEQ ID NO: 216; and (iii) an antigen binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISSSSYY (SEQ ID NO: 101), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of GSVDYSGNT (SEQ ID NO: 102), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91).

[0041] In some embodiments, a protein of the disclosure comprises a first arm and a second arm, the first arm comprising: (i) a first polypeptide comprising an extracellular domain of CTLA4 comprising SEQ ID NO: 174; (ii) a first bridging portion comprising a sequence comprising SEQ ID NO: 213 and a sequence comprising SEQ ID NO: 216; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISSSSYY (SEQ ID NO: 101), a heavy chain variable complementarity determining region 2 (VHCDR3) comprising the amino acid sequence of GSVDYSGNT (SEQ ID NO: 102). a first antigen-binding fragment comprising a heavy chain variable complementarity determining region 2 (VHCDR2), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91); (ii) a second bridging portion comprising a sequence comprising SEQ ID NO:213 and a sequence comprising SEQ ID NO:216; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISSSSYY (SEQ ID NO:101), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of GSVDYSGNT (SEQ ID NO:102), a heavy chain variable complementarity determining region 3 (VHCDR4) comprising the amino acid sequence of ARHRGIYFFDY (SEQ ID NO:103), and a heavy chain variable complementarity determining region 4 (VHCDR5) comprising the amino acid sequence of ARHRGIYFFDY (SEQ ID NO:104). and a second antigen-binding site comprising a heavy chain variable complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of NIENKN (SEQ ID NO: 103), a light chain variable complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VL CDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VL CDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91), wherein the first arm and the second arm dimerize with each other.

[0042] In some embodiments, a protein of the disclosure comprises: (i) a polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (ii) a bridging portion comprising a sequence comprising SEQ ID NO: 213 and a sequence comprising SEQ ID NO: 216; and (iii) an antigen binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISTSSYY (SEQ ID NO: 77), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83).

[0043] In some embodiments, a protein of the disclosure comprises a first arm and a second arm, the first arm comprising: (i) a first polypeptide comprising an extracellular domain of CTLA4 comprising SEQ ID NO: 174; (ii) a first bridging portion comprising a sequence comprising SEQ ID NO: 213 and a sequence comprising SEQ ID NO: 216; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISTSSYY (SEQ ID NO: 77), a heavy chain variable complementarity determining region 2 (VHCDR3) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78). a first antigen-binding domain comprising a heavy chain variable complementarity determining region 2 (VHCDR2), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83); (ii) a second bridging portion comprising a sequence comprising SEQ ID NO:213 and a sequence comprising SEQ ID NO:216; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISTSSYY (SEQ ID NO:77), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO:78), a heavy chain variable complementarity determining region 3 (VHCDR4) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO:79), a heavy chain variable complementarity determining region 4 (VHCDR5) comprising the amino acid sequence of IYYSGST (SEQ ID NO:79), a heavy chain variable complementarity determining region 5 (VHCDR6) comprising the amino acid sequence of IYYSGST (SEQ ID NO:79), a heavy chain variable complementarity determining region 6 (VHCDR7) comprising the amino acid sequence of IYYSGST (SEQ ID NO:79), a heavy chain variable complementarity determining region 7 (VHCDR8) comprising the amino acid sequence of IYYSGST (SEQ ID NO:79), a heavy chain variable complementarity determining region 8 (VHCDR9) comprising the amino acid sequence of IYYSGST (SEQ ID NO:79), a heavy chain variable complementarity determining region 9 (VHCDR10) comprising the amino acid sequence of IYYSGST (SEQ ID NO:79), a heavy chain variable complementarity determining region 10 (VHCDR11) comprising the amino acid sequence of IYYSGST (SEQ ID NO:79), a heavy chain variable complementarity determining region 11 (VHCDR12) comprising the amino acid sequence of IYYSGST (SEQ ID NO:79), a heavy chain variable complementarity determining region 12 (VHCDR13) comprising the amino acid sequence of IYYSGST (SEQ ID NO:79), a heavy chain variable complementarity determining region 13 (VHCDR14) comprising the amino acid sequence of IYYSGST (SEQ and a second antigen-binding site comprising a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO:79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO:81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO:82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO:83), wherein the first arm and the second arm dimerize with each other.

[0044] In some embodiments, a protein of the disclosure comprises: (i) a polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:174, SEQ ID NO:119, SEQ ID NO:215, SEQ ID NO:233, SEQ ID NO:234, and SEQ ID NO:235; (ii) a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248, and a bridging moiety comprising a sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:214; SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240; and (iii) GFTFSNYA( and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QSISSY (SEQ ID NO: 121).

[0045] In some embodiments, a protein of the disclosure comprises a first arm and a second arm, the first arm comprising: (i) a first polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:174, SEQ ID NO:119, SEQ ID NO:215, SEQ ID NO:233, SEQ ID NO:234, and SEQ ID NO:235; (ii) a first polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248; 8, SEQ ID NO:237, SEQ ID NO:214; a first bridging portion comprising a sequence selected from the group consisting of SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GFTFSNYA (SEQ ID NO:133), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of ISGSGGAT (SEQ ID NO:113), and an amino acid sequence of TKDRLIMATVRGPYYYGMDV (SEQ ID NO:114). a first antigen-binding site comprising a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of QSISSY (SEQ ID NO: 121), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of QSISSY (SEQ ID NO: 121), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of AAS (SEQ ID NO: 146), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QQSHSVSFT (SEQ ID NO: 154); (ii) a second polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248, and a second bridging moiety comprising a sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:214; SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240;and (iii) a second antigen-binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GFTFSNYA (SEQ ID NO: 133), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of ISGSGGAT (SEQ ID NO: 113), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of TKDRLIMATVRGPYYYGMDV (SEQ ID NO: 114), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of QSISSY (SEQ ID NO: 121), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of AAS (SEQ ID NO: 146), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QQSHSVSFT (SEQ ID NO: 154), wherein the first arm and the second arm dimerize with each other.;

[0046] In some embodiments, a protein of the disclosure comprises: (i) a polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:174, SEQ ID NO:119, SEQ ID NO:215, SEQ ID NO:233, SEQ ID NO:234, and SEQ ID NO:235; (ii) a bridging moiety comprising a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248, and a sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:214; SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240; and (iii) SYAMS (SEQ ID NO:1 05), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IISGSGGFTYYADSVK (SEQ ID NO: 106), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of DRLVAPGTFDY (SEQ ID NO: 107), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of RASQGISSWLA (SEQ ID NO: 169), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of AASSLQS (SEQ ID NO: 170), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QQYNSYPYT (SEQ ID NO: 171).

[0047] In some embodiments, a protein of the disclosure comprises a first arm and a second arm, the first arm comprising: (i) a first polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:174, SEQ ID NO:119, SEQ ID NO:215, SEQ ID NO:233, SEQ ID NO:234, and SEQ ID NO:235; (ii) a first polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248; and (iii) a second polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:178. , SEQ ID NO:237, SEQ ID NO:214; SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240; and (iii) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:105), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IISGSGSGFTYYADSVK (SEQ ID NO:106), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of DRLVAPGTFDY (SEQ ID NO:107), a first antigen-binding site comprising a light chain variable complementarity determining region 3 (VHCDR3), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of RASQGISSWLA (SEQ ID NO: 169), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of AASSLQS (SEQ ID NO: 170), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QQYNSYPYT (SEQ ID NO: 171); (ii) a second polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248, and a second bridging moiety comprising a sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:214; SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240;and (iii) a second antigen-binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 105), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IISGSGGFTYYADSVK (SEQ ID NO: 106), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of DRLVAPGTFDY (SEQ ID NO: 107), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of RASQGISSWLA (SEQ ID NO: 169), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of AASSLQS (SEQ ID NO: 170), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QQYNSYPYT (SEQ ID NO: 171), wherein the first arm and the second arm dimerize with each other.;

[0048] In some embodiments, the protein of the disclosure comprises: (i) a polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:174, SEQ ID NO:119, SEQ ID NO:215, SEQ ID NO:233, SEQ ID NO:234, and SEQ ID NO:235; (ii) a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248, and a sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:214; SEQ ID NO:216, SEQ ID NO:179 ... and (iii) a bridging portion comprising a sequence selected from the group consisting of SEQ ID NO: 180, SEQ ID NO: 239, and SEQ ID NO: 240; and (iii) an antigen binding site comprising VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, according to the IMGT proprietary numbering system, each comprising an antigen binding site comprising an amino acid sequence corresponding to the sequences of the heavy chain variable domain and light chain variable domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, respectively, listed in Table 3.

[0049] In some embodiments, a protein of the disclosure comprises a first arm and a second arm, the first arm comprising: (i) a first polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:174, SEQ ID NO:119, SEQ ID NO:215, SEQ ID NO:233, SEQ ID NO:234, and SEQ ID NO:235; (ii) a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248; and (iii) a sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:214; and (iii) a first bridging moiety comprising a sequence selected from the group consisting of SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240; and (iii) a first antigen-binding domain comprising VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, each of which comprises an amino acid sequence corresponding to the heavy chain variable domain and light chain variable domain VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, respectively, as listed in Table 3, according to the IMGT proprietary numbering system. and the second arm comprises a site, said second arm comprising: (i) a second polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:174, SEQ ID NO:119, SEQ ID NO:215, SEQ ID NO:233, SEQ ID NO:234, and SEQ ID NO:235; (ii) a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248, and a sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:214; SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239 ... and (iii) a second bridging portion comprising a sequence selected from the group consisting of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, according to the IMGT proprietary numbering system, each comprising an amino acid sequence corresponding to the heavy chain variable domain and light chain variable domain sequences VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, respectively, listed in Table 3, wherein the first arm and the second arm dimerize with each other.

[0050] In some embodiments, the protein of the disclosure comprises: (i) a polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:174, SEQ ID NO:119, SEQ ID NO:215, SEQ ID NO:233, SEQ ID NO:234, and SEQ ID NO:235; (ii) a bridging portion comprising a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248, and a sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:214; SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240; and (iii) an antigen binding site comprising a polypeptide that specifically binds to OX40L.

[0051] In some embodiments, a protein of the disclosure comprises a first arm and a second arm, the first arm comprising: (i) a first polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:174, SEQ ID NO:119, SEQ ID NO:215, SEQ ID NO:233, SEQ ID NO:234, and SEQ ID NO:235; (ii) a first bridging moiety comprising a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248, and a sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:214; SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240; and (iii) a first antigen-binding polypeptide comprising a polypeptide that specifically binds to OX40L. and (iii) a second antigen-binding site comprising a polypeptide that specifically binds to OX40L, wherein the first arm and the second arm dimerize with each other, wherein the second arm comprises: (i) a second polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO:174, SEQ ID NO:119, SEQ ID NO:215, SEQ ID NO:233, SEQ ID NO:234, and SEQ ID NO:235; (ii) a second bridging moiety comprising a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:248, and a sequence selected from the group consisting of SEQ ID NO:178, SEQ ID NO:237, SEQ ID NO:214; SEQ ID NO:216, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:239, and SEQ ID NO:240.

[0052] In some embodiments, the protein of the disclosure is selected from the group consisting of SEQ ID NO:40, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:48, SEQ ID NO:68, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:46, SEQ ID NO:66, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:50, SEQ ID NO:70, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, Column number 38, SEQ ID NO:73, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:37, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273, SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO: No. 278, No. 279, No. 280, No. 281, No. 282, No. 283, No. 284, No. 285, No. 286, No. 287, No. 288, No. 289, No. 290, No. 291, No. 292, No. 293, No. 294, No. 295, No. 296, No. 297, No. 298, No. 299, No. 300, No. 301, No. 302, No. 303, No. 304, No. 305, No. 306, No. 307, No. 308, The amino acid sequence of sequence number 309 is at least 90% identical to the amino acid sequence of SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319, SEQ ID NO:320, SEQ ID NO:321, SEQ ID NO:322, SEQ ID NO:323, SEQ ID NO:324, SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327, SEQ ID NO:328, SEQ ID NO:329, SEQ ID NO:330, SEQ ID NO:331, SEQ ID NO:332, SEQ ID NO:333, SEQ ID NO:334, or SEQ ID NO:335.

[0053] In some embodiments, the proteins of the disclosure are selected from the group consisting of SEQ ID NO:40 and SEQ ID NO:39; SEQ ID NO:62 and SEQ ID NO:61; SEQ ID NO:64 and SEQ ID NO:63; SEQ ID NO:182 and SEQ ID NO:63; SEQ ID NO:183 and SEQ ID NO:63; SEQ ID NO:184 and SEQ ID NO:63; SEQ ID NO:185 and SEQ ID NO:63; SEQ ID NO:186 and SEQ ID NO:63; SEQ ID NO:48 and SEQ ID NO:47; SEQ ID NO:68 and SEQ ID NO:67; SEQ ID NO:187 and SEQ ID NO:67; SEQ ID NO:188 and SEQ ID NO:67; SEQ ID NO:189 and SEQ ID NO:67; SEQ ID NO:190 and SEQ ID NO:67; SEQ ID NO:191 and SEQ ID NO:67; SEQ ID NO:4 6 and SEQ ID NO:45; SEQ ID NO:66 and SEQ ID NO:65; SEQ ID NO:192 and SEQ ID NO:65; SEQ ID NO:193 and SEQ ID NO:65; SEQ ID NO:194 and SEQ ID NO:65; SEQ ID NO:195 and SEQ ID NO:65; SEQ ID NO:196 and SEQ ID NO:65; SEQ ID NO:50 and SEQ ID NO:49; SEQ ID NO:70 and SEQ ID NO:69; SEQ ID NO:197 and SEQ ID NO:69; SEQ ID NO:198 and SEQ ID NO:69; SEQ ID NO:199 and SEQ ID NO:69; SEQ ID NO:200 and SEQ ID NO:69; SEQ ID NO:201 and SEQ ID NO:69; SEQ ID NO:38 and SEQ ID NO:5; SEQ ID NO:73 and SEQ ID NO:5; SEQ ID NO:42 and SEQ ID NO:41; SEQ ID NO:44 and SEQ ID NO:43;SEQ ID NO:52 and SEQ ID NO:51;SEQ ID NO:54 and SEQ ID NO:53;SEQ ID NO:56 and SEQ ID NO:55;SEQ ID NO:58 and SEQ ID NO:57;SEQ ID NO:60 and SEQ ID NO:59;SEQ ID NO:37 and SEQ ID NO:35;SEQ ID NO:256 and SEQ ID NO:63;SEQ ID NO:257 and SEQ ID NO:63;SEQ ID NO:258 and SEQ ID NO:63;SEQ ID NO:259 and SEQ ID NO:63;SEQ ID NO:260 and SEQ ID NO:63;SEQ ID NO:261 and SEQ ID NO:63;SEQ ID NO:262 and SEQ ID NO:63;SEQ ID NO:263 and SEQ ID NO:63;SEQ ID NO:264 and SEQ ID NO:63;SEQ ID NO:265 and SEQ ID NO:63;SEQ ID NO:266 and SEQ ID NO:63;SEQ ID NO:267 and SEQ ID NO:63;SEQ ID NO:268 and SEQ ID NO:63;SEQ ID NO:269 and SEQ ID NO:63;SEQ ID NO:270 and SEQ ID NO:63;SEQ ID NO:271 and SEQ ID NO:63;SEQ ID NO:272 and SEQ ID NO:63;SEQ ID NO:273 and SEQ ID NO:63;SEQ ID NO:274 and SEQ ID NO:63;SEQ ID NO:275 and SEQ ID NO:63;SEQ ID NO:276 and SEQ ID NO:63;SEQ ID NO:277 and SEQ ID NO:63;SEQ ID NO:278 and SEQ ID NO:63;SEQ ID NO:279 and SEQ ID NO:63;SEQ ID NO:280 and SEQ ID NO:63;SEQ ID NO:281 and SEQ ID NO:63;SEQ ID NO:282 and SEQ ID NO:63;SEQ ID NO:283 and SEQ ID NO:63; SEQ ID NO:284 and SEQ ID NO:63; SEQ ID NO:285 and SEQ ID NO:63; SEQ ID NO:286 and SEQ ID NO:63; SEQ ID NO:287 and SEQ ID NO:63; SEQ ID NO:288 and SEQ ID NO:63; SEQ ID NO:289 and SEQ ID NO:63; SEQ ID NO:290 and SEQ ID NO:63; SEQ ID NO:291 and SEQ ID NO:63; SEQ ID NO:292 and SEQ ID NO:63; SEQ ID NO:293 and SEQ ID NO:63; SEQ ID NO:294 and SEQ ID NO:67; SEQ ID NO:295 and SEQ ID NO:67; SEQ ID NO:296 and SEQ ID NO:67; SEQ ID NO:297 and SEQ ID NO:67; SEQ ID NO:298 and SEQ ID NO:67; SEQ ID NO:299 and SEQ ID NO:67; SEQ ID NO:300 and SEQ ID NO:67; SEQ ID NO:301 and SEQ ID NO:67; SEQ ID NO:302 and SEQ ID NO:67; SEQ ID NO:303 and SEQ ID NO:67; SEQ ID NO:304 and SEQ ID NO:67; SEQ ID NO:305 and SEQ ID NO:67; SEQ ID NO:306 and SEQ ID NO:67; SEQ ID NO:307 and SEQ ID NO:67; SEQ ID NO:308 and SEQ ID NO:67; SEQ ID NO:309 and SEQ ID NO:67; SEQ ID NO:310 and SEQ ID NO:67; Sequence number 311 and sequence number 67; sequence number 312 and sequence number 67; sequence number 313 and sequence number 67; sequence number 314 and sequence number 67; sequence number 315 and sequence number 67; sequence number 316 and sequence number 67; sequence number 317 and sequence number 67; sequence number 318 and sequence number 67; sequence number 319 and sequence number 67; sequence number 320 and sequence number 67; sequence number 321 and sequence number 67; sequence number 322 and sequence number 67; sequence number 323 and sequence number 67; sequence number 324 and sequence number 67; sequence and polypeptides comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:335 or SEQ ID NO:255.

[0054] In some embodiments, a protein of the disclosure includes two polypeptides comprising the amino acid sequence of SEQ ID NO:63 and two polypeptides comprising the amino acid sequence of SEQ ID NO:64, or includes two polypeptides comprising the amino acid sequence of SEQ ID NO:68 and two polypeptides comprising the amino acid sequence of SEQ ID NO:67.

[0055] In some embodiments, the proteins of the disclosure have an IC of less than 12 nM, e.g., between 5 nM and 12 nM, or between 5 nM and 9 nM, as measured in an OX40L neutralization / OX40-HEK reporter assay. 50 Have.

[0056] In some embodiments, the proteins of the present disclosure have an IC50 of 2 nM to 12 nM, e.g., 2 nM to 9.5 nM, as measured in a primary T cell activation assay. 50 Have.

[0057] In some embodiments, the proteins of the disclosure have a K of less than 75 nM, e.g., a K of 20 nM to 75 nM, for OX40L binding as measured in a Biacore assay. D Have.

[0058] In some embodiments, the proteins of the disclosure significantly inhibit at least one of IL-2, IFNγ, IL-6, and TNFα production by cells in an in vitro mixed lymphocyte reaction assay compared to cells treated under the same conditions but in the absence of the protein, or in the presence of human CTLA4 extracellular domain fused to the N-terminus of human IgG1 Fc domain (CTLA4-Ig) provided separately and anti-OX40L antibody, or in the presence of a combination of the two proteins provided separately.

[0059] In some embodiments, the proteins of the present disclosure inhibit alloreactive CD4 + T cells and CD8 + T cell proliferation was measured in alloreactive CD4 T cells treated under the same conditions but in the absence of protein or in the presence of CTLA4-Ig and / or anti-OX40L antibodies.+ T cells and CD8 + Significantly more inhibitory than T cells.

[0060] In some embodiments, the proteins of the present disclosure are capable of inhibiting the activity of regulatory T cells (T reg ) proliferation compared with alloreactive CD4 + T cells and CD8 + T cell proliferation was measured in alloreactive CD4 T cells treated under the same conditions but in the absence of protein or in the presence of CTLA4-Ig and / or anti-OX40L antibodies. + T cells and CD8 + Selectively inhibits compared to T cells.

[0061] In some embodiments, the proteins of the present disclosure inhibit T reg The inhibitory function was evaluated in T cells treated under the same conditions but in the absence of protein or in the presence of CTLA4-Ig and / or anti-OX40L antibodies. reg The increase is significant compared to cells.

[0062] In some embodiments, the proteins of the present disclosure inhibit alloreactive CD4 + T cells and CD8 + T cell proliferation was measured using alloreactive CD4 T cells in mice treated under the same conditions but in the absence of protein or in the presence of CTLA4-Ig and / or anti-OX40L antibodies. + T cells and CD8 + Significantly more inhibitory than T cells.

[0063] In some embodiments, the proteins of the disclosure significantly inhibit serum IFNγ levels in adoptive transfer assays in humanized mice compared to mice treated under the same conditions but in the absence of the protein or in the presence of CTLA4-Ig and / or anti-OX40L antibodies.

[0064] In some embodiments, proteins of the present disclosure are not significantly internalized into bone marrow derived dendritic cells (MDDCs).

[0065] In some embodiments, the proteins of the present disclosure cross-react with cynomolgus monkey OX40L and / or do not cross-react with mouse, rabbit, or rat OX40L. In some embodiments, the proteins of the present disclosure cross-react with cynomolgus monkey CD80 and CD86.

[0066] In another aspect, the disclosure provides for utilizing a heavy chain polypeptide disclosed herein to prepare a protein comprising the heavy chain polypeptide and a light chain polypeptide disclosed herein.

[0067] In another aspect, the present disclosure provides a formulation comprising a protein disclosed herein and a pharma- ceutically acceptable carrier.

[0068] In another aspect, the disclosure provides a nucleic acid encoding a protein disclosed herein.

[0069] In yet another aspect, a cell is provided that contains one or more nucleic acids encoding the proteins disclosed herein.

[0070] In another aspect, the present disclosure provides a method for treating an autoimmune disease in a patient, comprising administering to the patient a protein or preparation disclosed herein.For example, the autoimmune diseases that can be treated by the protein disclosed herein include rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, atopic dermatitis, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune testicular disease. inflammation, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital atrioventricular block, Coxsackie cardiomyopathy, CREST syndrome, Crohn's disease, cutaneous lupus, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus erythematosus, Dressler's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein Herpes gestationis or pemphigoid of gestationis (PG), hidradenitis suppurativa (HS) (acne inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, autoimmune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile dermatomyositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, Meniere's disease,Microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mukka-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemorrhage, Polymorphonuclear leukemia (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyendocrine syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing biliary vasculitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome The following diseases are considered to be afflicted by the following conditions: subacute bacterial endocarditis (SBE), Sazak syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undiagnosed connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease. In another aspect, the disclosure provides a method of treating graft-versus-host disease (GVHD) in a patient, comprising administering to a patient a protein or formulation disclosed herein,

[0071] In yet another aspect, the disclosure provides an antibody or functional fragment thereof, comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISTSSYY (SEQ ID NO:77), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO:78), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO:79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO:81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO:82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO:83), according to the IMGT unique numbering scheme.

[0072] In some embodiments, an antibody of the disclosure comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO:76, and a light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO:80.

[0073] In some embodiments, an antibody or functional fragment thereof of the present disclosure comprises a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GVSIRSNGYY (SEQ ID NO: 93), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of MDYSGT (SEQ ID NO: 94), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARERSNNWYPIDY (SEQ ID NO: 95), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SVRRFF (SEQ ID NO: 97), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of GKD (SEQ ID NO: 98), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of NSRDSSGYLVL (SEQ ID NO: 99), according to the IMGT unique numbering scheme.

[0074] In some embodiments, an antibody of the disclosure comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO:92 and a light chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO:96.

[0075] In some embodiments, an antibody or functional fragment thereof of the present disclosure comprises a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GASVSSSSYY (SEQ ID NO: 85), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of INYGGST (SEQ ID NO: 86), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYHFDY (SEQ ID NO: 87), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91), according to the IMGT proprietary numbering scheme.

[0076] In some embodiments, an antibody of the disclosure comprises a heavy chain variable domain comprising an amino acid sequence at least 90% identical to SEQ ID NO:84 and a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to SEQ ID NO:88.

[0077] In some embodiments, an antibody or functional fragment thereof of the present disclosure comprises a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISSSSYY (SEQ ID NO: 101), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IGSVDYSGNT (SEQ ID NO: 102), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91), according to the IMGT proprietary numbering scheme.

[0078] In some embodiments, an antibody of the disclosure comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 100, and a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 104.

[0079] In some embodiments, the antibody or functional fragment thereof of the present disclosure is a human IgG1 antibody.

[0080] In another aspect, the present disclosure provides the use of an antibody, or functional fragment thereof, disclosed herein in the preparation of a protein disclosed herein.

[0081] Further embodiments and details of the present disclosure are provided herein below. [Brief description of the drawings]

[0082] [Figure 1-1] Figures 1A-1W provide schematics of various protein designs of fusion proteins with an Ig Fc constant domain that binds CD80 and / or CD86 and OX40L. Figure 1A is a schematic of one representative fusion protein in which the extracellular domain (ECD) of CD28 is fused to the N-terminus of an Fc domain and the ECD of OX40 is fused to the C-terminus of the same Fc domain. Figure 1B is a schematic of one representative fusion protein in which the ECD of OX40 is fused to the N-terminus of an Fc domain and the ECD of CD28 is fused to the C-terminus of the same Fc domain. Figure 1C is a schematic of one representative fusion protein in which the ECD of CTLA4 is fused to the N-terminus of an Fc domain and the ECD of OX40 is fused to the C-terminus of the same Fc domain. Figure 1D is a schematic of one representative fusion protein in which the ECD of OX40 is fused to the N-terminus of an Fc domain and the ECD of CTLA4 is fused to the C-terminus of the same Fc domain. [Figure 1-2]FIG. 1E is a schematic diagram of a representative fusion protein in which the ECDs of CD28 are each fused in tandem to the N-terminus of the ECD of OX40, which is then fused to the N-terminus of an Fc domain. FIG. 1F is a schematic diagram of a representative fusion protein in which the ECDs of CTLA4 are each fused in tandem to the N-terminus of the ECD of OX40, which is then fused to the N-terminus of an Fc domain. FIG. 1G is a schematic diagram of a representative fusion protein in which the ECDs of OX40 are each fused in tandem to the N-terminus of the ECD of CD28, which is then fused to the N-terminus of an Fc domain. FIG. 1H is a schematic diagram of a representative fusion protein in which the ECDs of OX40 are each fused in tandem to the N-terminus of the ECD of CTLA4, which is then fused to the N-terminus of an Fc domain. [Figure 1-3] FIG. 1I is a schematic diagram of one representative fusion protein in which any tandem ECD combination (here labeled X and Y) from FIGS. 1E-1H is fused to the C-terminus of an Fc domain. FIG. 1J is a schematic diagram of one representative fusion protein in which the ECD of CD28 is fused to the N-terminus of an Fc domain and the binding domain of an anti-OX40L antibody is fused to the C-terminus of the same Fc domain. FIG. 1K is a schematic diagram of one representative fusion protein in which the ECD of CTLA4 is fused to the N-terminus of an Fc domain and the binding domain of an anti-OX40L antibody is fused to the C-terminus of the same Fc domain. FIG. 1L is a schematic diagram of one representative fusion protein in which the ECD of OX40 is fused to the N-terminus of an Fc domain and the binding domains of an anti-CD80 antibody and an anti-CD86 antibody are fused to the C-terminus of an Fc domain. [Figure 1-4]FIG. 1M is a schematic diagram of one representative fusion protein in which the binding domain of an anti-OX40L antibody is fused to the N-terminus of an Fc domain, and the binding domains of an anti-CD80 antibody and an anti-CD86 antibody are fused to the C-terminus of the Fc domain. FIG. 1N is a schematic diagram of one representative fusion protein in which the binding domains of an anti-CD80 antibody and an anti-CD86 antibody are fused to the N-terminus of an Fc domain, and the binding domain of an anti-OX40L antibody is fused to the C-terminus of the Fc domain. FIG. 1O is a schematic diagram of one representative fusion protein in which the binding domains of an anti-CD80 antibody and an anti-CD86 antibody are fused in tandem to the N-terminus of an anti-OX40L antibody binding domain, which is then fused to the N-terminus of an Fc domain. FIG. 1P is a schematic diagram of one representative fusion protein in which the binding domain of an anti-OX40L antibody is fused in tandem to the N-terminus of the binding domains of an anti-CD80 antibody and an anti-CD86 antibody, which are then fused to the N-terminus of an Fc domain. [Figure 1-5] FIG. 1Q is a schematic diagram of a representative fusion protein in which the binding domains of anti-CD80 and anti-CD86 antibodies are fused to the C-terminus of an Fc domain, and the binding domains of anti-CD80 and anti-CD86 antibodies are fused in tandem to the N-terminus of an anti-OX40L antibody binding domain. FIG. 1R is a schematic diagram of a representative fusion protein in which the binding domain of anti-OX40L antibody is fused to the C-terminus of an Fc domain, and the binding domains of anti-CD80 and anti-CD86 antibodies are fused in tandem to each other to the C-terminus of an Fc domain. FIG. 1S is a schematic diagram of a representative fusion protein in which the binding domain of anti-OX40L antibody is fused to the N-terminus of an Fc domain, and the binding domains of anti-CD80 and anti-CD86 antibodies are fused in tandem to each other to the C-terminus of an Fc domain. FIG. 1T is a schematic diagram of one representative fusion protein in which the binding domains of anti-CD80 and anti-CD86 antibodies are fused to each other in tandem and fused to the N-terminus of an Fc domain, and the binding domain of an anti-OX40L antibody is fused to the C-terminus of the Fc domain. [Figure 1-6]Figure 1U is a schematic diagram of one representative fusion protein in which the binding domain of an anti-OX40L antibody is fused to the N-terminus of an Fc domain and the ECD of CD28 is fused to the C-terminus of the same Fc domain. Figure 1V is a schematic diagram of one representative fusion protein in which the binding domain of an anti-OX40L antibody is fused to the N-terminus of an Fc domain and the ECD of CTLA4 is fused to the C-terminus of the same Fc domain. Figure 1W is a schematic diagram of one representative fusion protein in which the ECD of CTLA4 is fused to the N-terminus of a variant IgG1 Fc domain with a mutation that reduces Fc effector function and an anti-OX40L Fab is fused to the C-terminus of the variant IgG1 Fc domain.

[0083] [Figure 2A] FIG. 2A is a flow chart showing the antibody screening cascade. [Figure 2B] FIG. 2B is a flow chart illustrating the candidate characterization cascade.

[0084] [Figure 3-1] 3A-3C are graphs showing neutralization of OX40L by anti-OX40L in IgG format (FIG. 3A), FcFab format (FIG. 3B), and fusion protein format (FIG. 3C) using an OX40 luciferase reporter assay. [Figure 3-2] 3A-3C are graphs showing neutralization of OX40L by anti-OX40L in IgG format (FIG. 3A), FcFab format (FIG. 3B), and fusion protein format (FIG. 3C) using an OX40 luciferase reporter assay.

[0085] [Figure 4]FIG. 4 is a graph showing neutralization of OX40L using CTLA4_anti-OX40L fusion proteins (95B06, 98C01, 98E10, 84E11, 68F03, 67B06, 97G07, 89B09, and Ref4_CTLA4_O13), reference monoclonal anti-OX40L antibodies (Ref1_anti-OX40L, Ref2_anti-OX40L), or isotype controls using an OX40 luciferase reporter assay.

[0086] [Diagram 5] Figures 5A-5B are graphs showing the expression of the pro-inflammatory cytokines IL-2 (Figure 5A) and TNFα (Figure 5B) in the presence of various concentrations of CTLA4_anti-OX40L fusion proteins (89B09, 67B06, 98E10, and 98C01), reference single agents (Ref2_anti-OX40L, Ref3_CTLA4Ig), a combination of monoclonal anti-OX40L antibody and CTLA4-Ig (combination), or an isotype control using an in vitro mixed lymphocyte reaction (MLR) assay.

[0087] [Figure 6] Figures 6A-6B are graphs showing proliferation of alloreactive CD4+OX40+ T cells (Figure 6A) and CD8+OX40+ T cells (Figure 6B) in the presence of CTLA4_anti-OX40L fusion proteins (89B09, 67B06, 98E10, and 98C01), reference single agents (Ref2_anti-OX40L, Ref3_CTLA4Ig), a combination of monoclonal anti-OX40L antibody and CTLA4-Ig (combination), or an isotype control using a monocyte-derived dendritic cell:T cell (MDDC:T) MLR assay.

[0088] [Figure 7]FIG. 7 is a graph showing in vitro Treg induction levels in the presence or absence of CTLA4_anti-OX40L fusion proteins (89B09, 67B06, and 98C01), reference single agents (Ref1_anti-OX40L, Ref2_anti-OX40L, Ref3_CTLA4Ig), or isotype IgG1 control (Iso) using a Treg induction assay.

[0089] [Figure 8] FIG. 8 is a histogram showing Treg:Teff ratios in the presence of CTLA4_anti-OX40L fusion proteins (89B09, 67B06, and 98C01), reference single agents (Ref2_anti-OX40L, Ref3_CTLA4Ig), or isotype controls using the MLR assay.

[0090] [Figure 9] Figure 9A is a graph showing Treg:Teff ratios in the presence of various concentrations of CTLA4_anti-OX40L fusion proteins (89B09, 67B06, and 98C01), reference single agents (Ref2_anti-OX40L, Ref3_CTLA4Ig), monoclonal anti-OX40L antibody and CTLA4-Ig combinations (combinations), rapamycin, voclosporin, or isotype control using an MLR assay. Figure 9B is a graph showing effector T cell proliferation in the presence of CTLA4_anti-OX40L fusion proteins (89B09), reference single agents (Ref2_anti-OX40L, Ref3_CTLA4Ig), monoclonal anti-OX40L antibody and CTLA4-Ig combinations (combinations), or isotype control using an MLR assay.

[0091] [Figure 10-1]Figures 10A-10C are graphs showing IC50 values ​​for IL-2 (Figure 10A), TNFα (Figure 10B), and IL-6 (Figure 10C) in the presence of CTLA4_anti-OX40L fusion proteins (67B06 and 89B09), reference single agents (Ref1_anti-OX40L, Ref3_CTLA4Ig), or a combination of monoclonal anti-OX40L antibody and CTLA4-Ig (combination) using an MLR assay. [Figure 10-2] Figures 10A-10C are graphs showing IC50 values ​​for IL-2 (Figure 10A), TNFα (Figure 10B), and IL-6 (Figure 10C) in the presence of CTLA4_anti-OX40L fusion proteins (67B06 and 89B09), reference single agents (Ref1_anti-OX40L, Ref3_CTLA4Ig), or a combination of monoclonal anti-OX40L antibody and CTLA4-Ig (combination) using an MLR assay.

[0092] [Figure 11A] FIG. 11A is a graph showing IL-2 concentration levels from CD80 and OX40L receptor occupancy assays. [Figure 11B] FIG. 11B is a graph showing the occupancy of human OX40L from a receptor occupancy assay of CD80 and OX40L. [Figure 11C] FIG. 11C is a graph showing human CD80 occupancy from a CD80 and OX40L receptor occupancy assay.

[0093] [Figure 12] FIG. 12 is a graph showing internalization of CTLA4-Ig (Ref3_CTLA4Ig), anti-OX40L (Ref1_anti-OX40L), CTLA4_anti-OX40L fusion protein (67B06), IgG isotype controls (isotype Ctl, isotype IgG1), and non-internalizing anti-CD20 antibodies by bone marrow-derived dendritic cells (MDDCs) in real time.

[0094] [Figure 13-1]13A-13D are graphs showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference single agents (Ref1_anti-OX40L, Ref3_CTLA4Ig), or a combination of monoclonal anti-OX40L antibody and CTLA4-Ig (combination) on the expression levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) (FIG. 13A), IL13 (FIG. 13B), granzyme B (GZMB) (FIG. 13C), and IFNγ (FIG. 13D) in PBMCs from systemic lupus erythematosus (SLE) patients. [Figure 13-2] 13A-13D are graphs showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference single agents (Ref1_anti-OX40L, Ref3_CTLA4Ig), or a combination of monoclonal anti-OX40L antibody and CTLA4-Ig (combination) on the expression levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) (FIG. 13A), IL13 (FIG. 13B), granzyme B (GZMB) (FIG. 13C), and IFNγ (FIG. 13D) in PBMCs from systemic lupus erythematosus (SLE) patients.

[0095] [Figure 14] Figure 14A is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06 and 89B09) or isotype control on suppression of IFNγ responses in a xenograft-versus-host disease (xeno-GVHD) mouse model. Figure 14B is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference single agent 67B06 (IgG format), Ref3_CTLA4Ig, or isotype control on suppression of IFNγ responses in a xenograft-versus-host disease (xeno-GVHD) mouse model.

[0096] [Figure 15]Figure 15A is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference monoclonal anti-OX40L antibody (Ref1_anti-OX40L), or isotype control on weight loss in a xenogeneic GVHD mouse model. Figure 15B is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference monoclonal anti-OX40L antibody (Ref1_anti-OX40L), or isotype control on IFNγ production in a xenogeneic GVHD mouse model. Statistical analysis (one-way ANOVA) was performed in Prism Graphpad 8 ****p≦0.0001, *p=0.0175, ***p=0.0002).

[0097] [Figure 16] Figure 16A is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference CTLA4Ig (Ref3_CTLA4Ig), or isotype control on weight loss in a xenogeneic GVHD mouse model. Figure 16B is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference CTLA4Ig (Ref3_CTLA4Ig), or isotype control on IFNγ production in a xenogeneic GVHD mouse model. Statistical analysis (one-way ANOVA) was performed in Prism Graphpad 8 *p=0.0455, **p=0.0086).

[0098] [Figure 17-1] FIG. 17A is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference CTLA4Ig (Ref3_CTLA4Ig), anti-OX40L (Ref1_anti-OX40L), combination (Ref3 and Ref1), or isotype control on weight loss at day 41 in a xenogeneic GVHD mouse model. [Figure 17-2]Figure 17B is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference CTLA4Ig (Ref3_CTLA4Ig), anti-OX40L (Ref1_anti-OX40L), combination (Ref3 and Ref1), or isotype control on IFNγ production at day 41 in a xenogeneic GVHD mouse model. Statistical analysis (one-way ANOVA) was performed in Prism Graphpad 8 (*p=0.0110, **p=0.0084). Figure 17C is a graph showing the effect of CTLA4_anti-OX40L fusion protein (89B09) or isotype control on IFNγ production at day 12 in a xenogeneic GVHD mouse model. Statistical analysis (unpaired t-test) was performed in Prism Graphpad 8 (**p=0.0036). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] The present application provides proteins that include a polypeptide that specifically binds to CD80 and / or CD86 and a polypeptide that specifically binds to OX40L and inhibits T cell costimulatory signaling. In some embodiments, the present application provides antibodies that specifically bind to OX40L. In some embodiments, the present application also provides therapeutic methods that utilize such proteins in the treatment of autoimmune diseases. Various aspects of the proteins described in the present application are presented in sections below; however, aspects of the proteins described in one particular section are not limited to any particular section.

[0100] definition To facilitate the understanding of this application, a number of terms and expressions are defined below.

[0101] The term "a" or "an" is used herein to mean "one or more" and includes the plural unless the context is inappropriate.

[0102] As used herein, the term "antigen-binding site" refers to that portion of an immunoglobulin (Ig) molecule that is involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues from the N-terminal variable ("V") domains of the heavy ("H") and light ("L") chains. Three highly variable stretches within the V regions of the heavy and light chains are called "hypervariable regions" and are sandwiched between more conserved adjacent stretches known as "framework regions" or "FRs". Thus, the term "FR" refers to the amino acid sequences naturally found between or adjacent to the hypervariable regions in immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional plane in which the antigen-binding site specifically binds to the antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs". In some animals (such as camelids and cartilaginous fish), the antigen-binding site is formed by a single antibody chain providing a "single domain antibody", which can be present as an intact antibody, as an antigen-binding fragment of an antibody that retains the antigen-binding surface (e.g., Fab, Fab', F(ab')2), or as a recombinant polypeptide (such as scFv) that connects the heavy chain variable domain to the light chain variable domain using a peptide linker in a single polypeptide, minibody, or nanobody (VHH).

[0103] As used herein, the term "functional fragment thereof" refers to a portion of a protein or polypeptide that retains the ability to carry out the biological function of the whole protein or polypeptide. For example, a functional fragment of a polypeptide or protein of the present application retains the ability to bind to a corresponding cognate binding partner or ligand.

[0104] As used herein, the terms "subject" and "patient" refer to organisms treated by the methods and compositions described herein, preferably including, but not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably including, humans.

[0105] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a protein of the present application) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or doses, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treat" includes any effect that results in, for example, alleviating, reducing, altering, or eliminating a condition, disease, disorder, or the like, thereby improving it or ameliorating its symptoms.

[0106] As used herein, CTLA4 (also known as CD152) refers to the protein of SEQ ID NO: 172 and related isoforms and orthologues.

[0107] As used herein, OX40L (also known as TNFSF4 or CD252) refers to the protein of SEQ ID NO:2 and related isoforms and orthologues.

[0108] As used herein, OX40 (also known as TNFRSF4 or CD134) refers to the protein of SEQ ID NO:1 and related isoforms and orthologues. Amino acid sequence of human OX40: LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 1)

[0109] As used herein, CD28 (also known as TP44) refers to the protein of SEQ ID NO: 230 and related isoforms and orthologues. CD28 amino acid sequence (underlined amino acids represent the signal peptide): MLRLLLALNLFPSIQVTG NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 230)

[0110] As used herein, CD80 (also known as B7.1) refers to the protein of SEQ ID NO:3 and related isoforms and orthologues. Amino acid sequence of human CD80 VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 3)

[0111] As used herein, CD86 (also known as B7.2) refers to the protein of SEQ ID NO: 4 and related isoforms and orthologues. Amino acid sequence of human CD86 APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPPWITVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF (SEQ ID NO: 4)

[0112] Throughout the description, where a protein is described as having, comprising or including certain components, or where processes and methods are described as having, comprising or including certain steps, it is additionally considered that there are proteins according to the present application that consist essentially of or consist of the components mentioned, and that there are processes and methods according to the present application that consist essentially of or consist of the processing steps mentioned.

[0113] I. Protein The present application provides a protein that specifically binds to CD80, CD86, and OX40L expressed on the surface of an antigen-presenting cell (APC). Binding of the protein to CD80 / CD86 and OX40L inhibits T cell activation by blocking costimulatory signaling downstream of CD28 and OX40, respectively. The protein of the present disclosure comprises two arms, each arm comprising two components (e.g., a polypeptide, or a complex of two or more polypeptides), one component that specifically binds to CD80 and / or CD86, and another component that specifically binds to OX40L. In some embodiments, the component that binds to either CD80 and / or CD86, or OX40L can comprise a single polypeptide. In some embodiments, the component that binds to either CD80 and / or CD86, or OX40L can comprise a complex of two or more polypeptides. In some embodiments, the complex of two or more polypeptides of the polypeptide complex can be connected by one or more covalent interactions (e.g., disulfide bonds) and / or one or more non-covalent interactions (e.g., ionic or hydrophobic interactions). Further descriptions of representative proteins are provided below.

[0114] The protein of the present disclosure comprises a first component, which is a polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86, which may include, but is not limited to, the extracellular domain of CTLA4 or a functional fragment thereof. In some embodiments, the first component can be, but is not limited to, the extracellular domain of CD28 or a functional fragment thereof. In some embodiments, the first component can be, but is not limited to, one or more antigen binding sites, such as an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH), as non-limiting examples.

[0115] The protein of the present disclosure comprises a second component, which is a polypeptide that specifically binds to OX40L, or a complex of two or more polypeptides.In some embodiments, the non-limiting example of the second component can be an antigen binding site (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)).In some embodiments, the non-limiting example of the second component can be the extracellular domain of OX40 or its functional fragment.

[0116] In some embodiments, the protein of the present disclosure further comprises a bridging moiety, non-limiting examples of which may include an immunoglobulin Fc domain polypeptide or a functional fragment thereof, a human serum albumin (HSA) polypeptide or a functional fragment thereof, or a polypeptide linker. In some embodiments, the bridging moiety is an immunoglobulin Fc domain polypeptide. In some embodiments, the protein of the present disclosure further comprises a polypeptide hinge immediately N-terminus of the immunoglobulin Fc domain polypeptide or a functional fragment thereof.

[0117] In some embodiments, the proteins of the present disclosure further comprise a linker polypeptide that connects a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L to a bridging moiety. In some embodiments, the proteins of the present disclosure further comprise a linker polypeptide that connects a polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86 to a bridging moiety.

[0118] The proteins described herein can take a variety of formats. For example, one protein format comprises (i) a first arm comprising, from N-terminus to C-terminus, a first polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, a first polypeptide of an immunoglobulin Fc domain, and a first polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, and (ii) a second arm comprising, from N-terminus to C-terminus, a second polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, a second polypeptide of an immunoglobulin Fc domain, and a second polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, wherein the first and second polypeptides of the immunoglobulin Fc domain dimerize. In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 include the extracellular domain of CD28 or a functional fragment thereof (Figure 1A and Figure 1J); the extracellular domain of CTLA4 or a functional fragment thereof (Figure 1C, Figure 1K, and Figure 1W); or the first polypeptide, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 is an antigen binding site that specifically binds to CD80 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)), and the second polypeptide, or a complex of two or more polypeptides, that specifically binds to CD80 and / or CD86 is an antigen binding site that specifically binds to CD86 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (Figure 1N).

[0119] In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to OX40L include the extracellular domain of OX40 or a functional fragment thereof (FIGS. 1A and 1C); or an antigen binding site (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (FIGS. 1J, 1K, and 1N). In some embodiments, the first and second complex of two or more polypeptides that specifically bind to OX40L includes a Fab, in which a partial hinge (e.g., a polypeptide having a sequence of SEQ ID NO: 336) is connected to the C-terminus of the Fab heavy chain (HC) CH1 domain and forms a disulfide bond with the Fab light chain (LC).

[0120] Proteins of this format can optionally include a polypeptide hinge in each of the first and second arms that connects the C-terminus of a first or second polypeptide, or a complex of two or more polypeptides, that specifically binds CD80 and / or CD86, to the N-terminus of a first or second polypeptide, respectively, of an immunoglobulin Fc domain. Proteins of this format can also optionally include a linker polypeptide in one or both of the first and / or second arms that connects the C-terminus of a first and / or second polypeptide, or a complex of two or more polypeptides, respectively, of an immunoglobulin Fc domain, to the N-terminus of a first and / or second polypeptide, or two or more polypeptides, that specifically bind OX40L.

[0121] Another form of the protein comprises (i) a first arm comprising, from N-terminus to C-terminus, a first polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, a first polypeptide of an immunoglobulin Fc domain, and a first polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86; and (ii) a second arm comprising, from N-terminus to C-terminus, a second polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, a second polypeptide of an immunoglobulin Fc domain, and a second polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, wherein the first and second polypeptides of the immunoglobulin Fc domain dimerize. In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to OX40L include the extracellular domain of OX40 or a functional fragment thereof (FIG. 1B, FIG. 1D, and FIG. 1L); or an antigen binding site (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (FIG. 1M, FIG. 1U, and FIG. 1V). In some embodiments, the first and second complex of two or more polypeptides that specifically bind to OX40L includes a Fab, in which a partial hinge (e.g., a polypeptide having a sequence of SEQ ID NO: 336) is connected to the C-terminus of the Fab heavy chain (HC) CH1 domain and forms a disulfide bond with the Fab light chain (LC).In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 include the extracellular domain of CD28 or a functional fragment thereof (FIG. 1B and FIG. 1U); the extracellular domain of CTLA4 or a functional fragment thereof (FIG. 1D and FIG. 1V); or the first polypeptide, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 is an antigen binding site that specifically binds to CD80 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)), and the second polypeptide, or a complex of two or more polypeptides, that specifically binds to CD80 and / or CD86 is an antigen binding site that specifically binds to CD86 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (FIG. 1L and FIG. 1M). Proteins of this format can optionally include a polypeptide hinge in each of the first and second arms that connects the C-terminus of a first or second polypeptide, or a complex of two or more polypeptides, that specifically binds OX40L to the N-terminus of a first or second polypeptide, respectively, of an immunoglobulin Fc domain. Proteins of this format can also optionally include a linker polypeptide in one or both of the first and / or second arms that connects the C-terminus of a first and / or second polypeptide, or a complex of two or more polypeptides, respectively, of an immunoglobulin Fc domain to the N-terminus of a first and / or second polypeptide, or a complex of two or more polypeptides, that specifically binds CD80 and / or CD86.

[0122] In another example, the protein format includes (i) a first arm comprising, from N-terminus to C-terminus, a first polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, a first polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, and a first polypeptide of an immunoglobulin Fc domain; and (ii) a second arm comprising, from N-terminus to C-terminus, a second polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, a second polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, and a second polypeptide of an immunoglobulin Fc domain, wherein the first and second polypeptides of the immunoglobulin Fc domain dimerize. In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 include the extracellular domain of CD28 or a functional fragment thereof (FIG. 1E); the extracellular domain of CTLA4 or a functional fragment thereof (FIG. 1F); or the first polypeptide, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 is an antigen binding site that specifically binds to CD80 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)), and the second polypeptide, or a complex of two or more polypeptides, that specifically binds to CD80 and / or CD86 is an antigen binding site that specifically binds to CD86 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (FIG. 1O). In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to OX40L comprise the extracellular domain of OX40 or a functional fragment thereof (Figures 1E and 1F); or an antigen binding site (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (Figure 1O).In some embodiments, the first and second complexes of two or more polypeptides that specifically bind to OX40L include a Fab, with a partial hinge (e.g., a polypeptide having a sequence of SEQ ID NO: 336) connected to the C-terminus of the Fab heavy chain (HC) CH1 domain to form a disulfide bond with the Fab light chain (LC). Proteins of this format can optionally include a polypeptide hinge in each of the first and second arms that connects the C-terminus of the first or second polypeptide, or complex of two or more polypeptides that specifically bind to OX40L, to the N-terminus of the first or second polypeptide of the immunoglobulin Fc domain, respectively. Proteins of this format can also optionally include a linker polypeptide in the first and / or second arms that connects the C-terminus of the first and / or second polypeptide, or complex of two or more polypeptides that specifically bind to CD80 and / or CD86, to the N-terminus of the first and / or second polypeptide, or complex of two or more polypeptides, respectively.

[0123] Another protein format comprises (i) a first arm comprising, from N-terminus to C-terminus, a first polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, a first polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, and a first polypeptide of an immunoglobulin Fc domain; and (ii) a second arm comprising, from N-terminus to C-terminus, a second complex of two or more polypeptides that specifically binds to OX40L, a second polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, and a second polypeptide of an immunoglobulin Fc domain, wherein the first and second polypeptides of the immunoglobulin Fc domain dimerize. In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to OX40L include the extracellular domain of OX40 or a functional fragment thereof (FIG. 1G and FIG. 1H); or an antigen binding site (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (FIG. 1P). In some embodiments, the first and second complex of two or more polypeptides that specifically bind to OX40L includes a Fab, in which a partial hinge (e.g., a polypeptide having a sequence of SEQ ID NO: 336) is connected to the C-terminus of the Fab heavy chain (HC) CH1 domain and forms a disulfide bond with the Fab light chain (LC).In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 include the extracellular domain of CD28, or a functional fragment thereof (FIG. 1G); the extracellular domain of CTLA4, or a functional fragment thereof (FIG. 1H); or the first polypeptide, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 is CD80 and has an antigen binding site that specifically binds to CD80 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) and / or the second polypeptide, or a complex of two or more polypeptides, that specifically binds to CD86 is an antigen binding site that specifically binds to CD86 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (FIG. 1P). Proteins of this format can optionally include a polypeptide hinge in the first and second arms that connects the C-terminus of a first or second polypeptide, or a complex of two or more polypeptides, that specifically binds CD80 and / or CD86 to the N-terminus of the first or second polypeptide, respectively, of an immunoglobulin Fc domain. Proteins of this format can also optionally include a linker polypeptide in the first and / or second arms that connects the C-terminus of a first and / or second polypeptide, or a complex of two or more polypeptides, that specifically binds OX40L to the N-terminus of a first and / or second polypeptide, or a complex of two or more polypeptides, respectively, that specifically binds CD80 and / or CD86.

[0124] In another example, the protein format comprises (i) a first arm comprising, from N-terminus to C-terminus, a first polypeptide of an immunoglobulin Fc domain, a first polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, and a first polypeptide or a complex of two or more polypeptides that specifically binds to OX40L; and (ii) a second arm comprising, from N-terminus to C-terminus, a second polypeptide of an immunoglobulin Fc domain, a second polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, and a second polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, wherein the first and second polypeptides of the immunoglobulin Fc domain dimerize. In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 include the extracellular domain of CD28 or a functional fragment thereof (FIG. 1I); the extracellular domain of CTLA4 or a functional fragment thereof; or the first polypeptide, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 is an antigen binding site that specifically binds to CD80 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)), and the second polypeptide, or a complex of two or more polypeptides, that specifically binds to CD80 and / or CD86 is an antigen binding site that specifically binds to CD86 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (FIG. 1Q). In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to OX40L comprise the extracellular domain of OX40 or a functional fragment thereof (Figure 1I); or an antigen binding site (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (Figure 1Q).In some embodiments, the complex of two or more polypeptides, the first and second, that specifically bind to OX40L, comprises a Fab, and a partial hinge (e.g., a polypeptide having a sequence of SEQ ID NO: 336) is connected to the C-terminus of the Fab heavy chain (HC) CH1 domain and forms a disulfide bond with the Fab light chain (LC). This format of the protein can optionally include a polypeptide hinge in each of the first and second arm N-terminus of each of the first or second polypeptides of the immunoglobulin Fc domain. This format of the protein can also optionally include a linker polypeptide in one or both of the first and / or second arms that connects the C-terminus of the first and / or second polypeptide of the immunoglobulin Fc domain to the N-terminus of each of the first and / or second polypeptides or complex of two or more polypeptides that specifically bind to CD80 and / or CD86. Proteins of this format can also optionally include a linker polypeptide in one or both of the first and / or second arms that connects the C-terminus of a first and / or second polypeptide that specifically binds CD80 and / or CD86, or a complex of two or more polypeptides, to the N-terminus of a first and / or second polypeptide, respectively, that specifically binds OX40L, or a complex of two or more polypeptides.

[0125] Another protein format comprises (i) a first arm comprising, from N-terminus to C-terminus, a first polypeptide of an immunoglobulin Fc domain, a first polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, and a first polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86; and (ii) a second arm comprising, from N-terminus to C-terminus, a second polypeptide of an immunoglobulin Fc domain, a second polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, and a second polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, wherein the first and second polypeptides of the immunoglobulin Fc domain dimerize. In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to OX40L include the extracellular domain of OX40 or a functional fragment thereof (FIG. 1I); or an antigen-binding site (e.g., an antibody, Fab, Fab', F(ab')2, single-chain variable fragment (scFv), minibody, or nanobody (VHH)) (FIG. 1R). In some embodiments, the first and second complex of two or more polypeptides that specifically bind to OX40L includes a Fab, in which a partial hinge (e.g., a polypeptide having a sequence of SEQ ID NO: 336) is connected to the C-terminus of the Fab heavy chain (HC) CH1 domain and forms a disulfide bond with the Fab light chain (LC).In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 include the extracellular domain of CD28 or a functional fragment thereof (FIG. 1I); the extracellular domain of CTLA4 or a functional fragment thereof; or the first polypeptide, or a complex of two or more polypeptides, that specifically bind to CD80 and / or CD86 is an antigen binding site that specifically binds to CD80 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)), and the second polypeptide, or a complex of two or more polypeptides, that specifically binds to CD80 and / or CD86 is an antigen binding site that specifically binds to CD86 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (FIG. 1R). Proteins of this format can optionally include a polypeptide hinge in each of the first and second arm N-terminus of each of the first and / or second polypeptides of the immunoglobulin Fc domain. Proteins of this format can also optionally include a linker polypeptide in one or both of the first and / or second arms that connects the C-terminus of the first and / or second polypeptide of the immunoglobulin Fc domain to the N-terminus of the first and / or second polypeptide that specifically binds OX40L, or a complex of two or more polypeptides, respectively. Proteins of this format can also optionally include a linker polypeptide that connects the C-terminus of the first and / or second polypeptide that specifically binds OX40L, or a complex of two or more polypeptides, respectively, to the N-terminus of the first and / or second polypeptide that specifically binds CD80 and / or CD86, or a complex of two or more polypeptides, respectively.

[0126] Another protein format comprises: (i) a first arm comprising, from N-terminus to C-terminus, a first polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, a first polypeptide of an immunoglobulin Fc domain, and a first polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86; and (ii) a second polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, a second polypeptide of an immunoglobulin Fc domain, and a second polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, wherein the first and second polypeptides of the immunoglobulin Fc domain dimerize. In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to OX40L comprise the extracellular domain of OX40 or a functional fragment thereof; or an antigen binding site (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (Figure 1S). In some embodiments, the first and second complex of two or more polypeptides that specifically bind to OX40L comprises a Fab, in which a partial hinge (e.g., a polypeptide having a sequence of SEQ ID NO: 336) is connected to the C-terminus of the Fab heavy chain (HC) CH1 domain and forms a disulfide bond with the Fab light chain (LC).In this format, a first and a second polypeptide, or a complex of two or more polypeptides, that specifically bind CD80 and / or CD86 may be comprised of an antigen-binding moiety that specifically binds CD80 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) linked with or without a linker polypeptide to an antigen-binding moiety that specifically binds CD86 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)). or an antigen binding site that specifically binds CD86 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) linked, with or without a linker polypeptide, to an antigen binding site that specifically binds CD80 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (FIG. 1S). Proteins of this format can optionally include a polypeptide hinge in each of the first and second arms that connects the C-terminus of the first or second polypeptide, or a complex of two or more polypeptides that specifically bind OX40L, to the N-terminus of the first or second polypeptide, respectively, of the immunoglobulin Fc domain. Proteins of this format can also optionally include a linker polypeptide in one or both of the first and / or second arms that connects the C-terminus of the first and / or second polypeptide of an immunoglobulin Fc domain to the N-terminus of the first and / or second polypeptide, respectively, that specifically binds CD80 and / or CD86, or a complex of two or more polypeptides.

[0127] Another protein format comprises (i) a first arm comprising, from N-terminus to C-terminus, a first polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, a first polypeptide of an immunoglobulin Fc domain, and a first polypeptide or a complex of two or more polypeptides that specifically binds to OX40L; and (ii) a second arm comprising, from N-terminus to C-terminus, a second polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, a second polypeptide of an immunoglobulin Fc domain, and a second polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, wherein the first and second polypeptides of the immunoglobulin Fc domain dimerize. In this format, the first and second polypeptides, or a complex of two or more polypeptides, that specifically bind to OX40L comprise the extracellular domain of OX40 or a functional fragment thereof; or an antigen binding site (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)) (Figure 1T). In some embodiments, the first and second complex of two or more polypeptides that specifically bind to OX40L comprises a Fab, in which a partial hinge (e.g., a polypeptide having a sequence of SEQ ID NO: 336) is connected to the C-terminus of the Fab heavy chain (HC) CH1 domain and forms a disulfide bond with the Fab light chain (LC).In this format, a polypeptide or complex of two or more polypeptides that specifically binds to CD80 and / or CD86 comprises an antigen binding site that specifically binds CD80 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv) minibody, or nanobody (VHH)) connected with or without a linker polypeptide to an antigen binding site that specifically binds CD86 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv) minibody, or nanobody (VHH)) (FIG. 1T), or an antigen binding site that specifically binds CD86 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv) minibody, or nanobody (VHH)) connected with or without a linker polypeptide to an antigen binding site that specifically binds CD80 (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv) minibody, or nanobody (VHH)). Proteins in this format can optionally include a polypeptide hinge in each of the first and second arms that connects the C-terminus of a first or second polypeptide, or a complex of two or more polypeptides, that specifically binds CD80 and / or CD86, to the N-terminus of a first or second polypeptide of an immunoglobulin Fc domain, respectively. Proteins in this format can also optionally include a linker polypeptide in one or both of the first and / or second arms that connects the C-terminus of a first and / or second polypeptide, or a complex of two or more polypeptides, that specifically binds OX40L, respectively. In some embodiments, the first and / or second polypeptide can be a single polypeptide chain. In some embodiments, the first and / or second polypeptide can be a complex of two or more polypeptide chains.

[0128] The individual components of the protein are described in more detail below.

[0129] IA A polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86 A polypeptide, or a complex of two or more polypeptides, that specifically binds to CD80 and / or CD86 on antigen presenting cells (APCs) (such as B cells, monocytes, macrophages, and dendritic cells) blocks endogenous CD80 and / or CD86 from binding to CD28 on T cells. Polypeptides that specifically bind to CD80 and / or CD86 can inhibit T cell costimulatory receptor signaling by blocking the interaction between CD80 / CD86 and CD28, resulting in reduced T cell activation, proliferation, and induction of unresponsive, immunosuppressive, or immunoresponsive T cell responses.

[0130] As used herein, a polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86 is a polypeptide or a complex of two or more polypeptides that binds to a protein having the sequence of SEQ ID NO:3, and related isoforms and orthologues, and / or a protein having the sequence of SEQ ID NO:4, and related isoforms and orthologues.

[0131] In some embodiments, non-limiting examples of a polypeptide or complex of two or more polypeptides that specifically bind to CD80 and / or CD86 include the CTLA4 extracellular domain or a functional fragment thereof, an antibody, Fab, Fab', F(ab')2, a single chain variable fragment (scFv), a minibody, or a nanobody (VHH).

[0132] For example, in some embodiments, a polypeptide that specifically binds to CD80 and / or CD86 is the extracellular domain of CTLA4 or a functional fragment thereof. In some embodiments, the extracellular domain of CTLA4 can comprise a sequence derived from the wild-type sequence of human CTLA4 (e.g., Genbank Accession No. NP_005205). Alternatively, the CTLA4 polypeptide sequence can be a variant CTLA4 sequence as described in Larsen et al. Am J Transplant. 2005 Mar;5(3):443-53.;Xu et al. J Immunol. 2012 Nov 1;189(9):4470-7.;Bernett et al., MAbs. 2013 May-Jun;5(3):384-96.;Oshima et al. Protein Eng Des Sel. 2016 May;29(5):159-67.;Douthwaite et al. J Immunol. 2017 Jan 1;198(1):528-537. Erratum: J Immunol. 2017 Sep 1;199(5):1943. For example, the CTLA4 sequence can include one or more mutations selected from mutations at positions 16, 24, 25, 27, 28, 29, 30, 32, 49, 50, 51, 53, 54, 55, 56, 58, 61, 64, 65, 70, 80, 85, 93, 96, and 104 (numbering relative to SEQ ID NO: 174).

[0133] Polypeptides that specifically bind to CD80 and / or CD86 polypeptides of the invention can comprise an amino acid sequence selected from any wild-type or variant sequence listed in Table 1, or a functional fragment thereof.

[0134] [Table 1]

[0135] In some embodiments, the polypeptide that specifically binds to CD80 and / or CD86 is the extracellular domain of CTLA4 or a functional fragment thereof comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to a sequence listed in Table 1.

[0136] In some embodiments, the polypeptide that specifically binds to CD80 and / or CD86 is the extracellular domain of CD28 (SEQ ID NO: 236) or a functional fragment thereof. CD28 (The underlined portion represents the signal peptide sequence.) MLRLLLALNLFPSIQVTGN KILVKQSPMLVAYDNAVNLSWKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 236)

[0137] In some embodiments, the polypeptide or complex of two or more polypeptides that specifically bind to CD80 and / or CD86 includes an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, and nanobody (VHH).

[0138] In some embodiments of the proteins of the invention, the heavy chain of a polypeptide that specifically binds CD80 and / or CD86, or a complex of two or more polypeptides, can be connected to a heavy chain of a polypeptide that specifically binds OX40L (e.g., a polypeptide comprising an amino acid sequence listed in Table 3), or a complex of two or more polypeptides, to form a continuous polypeptide chain. In some embodiments of the proteins of the invention, the polypeptide that specifically binds CD80 and / or CD86, or a complex of two or more polypeptides, is connected to a polypeptide that specifically binds OX40L, or a complex of two or more polypeptides, via a bridging moiety (e.g., a polypeptide comprising an amino acid sequence listed in Table 4), which connects the C-terminus of the heavy chain of the polypeptide that specifically binds CD80 and / or CD86, or a complex of two or more polypeptides, to the N-terminus of the heavy chain of the polypeptide that specifically binds OX40L, or a complex of two or more polypeptides, to form a continuous polypeptide chain. In some embodiments, the heavy chain of a polypeptide that specifically binds CD80 and / or CD86, or a complex of two or more polypeptides, is connected to a bridging moiety via a hinge polypeptide (e.g., a polypeptide comprising an amino acid sequence listed in Table 5), which connects the C-terminus of the heavy chain of the polypeptide that specifically binds CD80 and / or CD86, or a complex of two or more polypeptides, to the N-terminus of the bridging moiety of the bridging moiety to form a continuous polypeptide chain.

[0139] IB A polypeptide or a complex of two or more polypeptides that specifically bind to OX40L A polypeptide, or a complex of two or more polypeptides, that specifically binds to OX40L on the surface of antigen-presenting cells (APCs) (such as B cells, monocytes, macrophages, and dendritic cells) blocks binding of T cells to endogenous OX40 on the surface. Polypeptides that specifically bind to OX40L can inhibit T cell costimulatory receptor signaling by blocking the interaction between OX40L and OX40, resulting in reduced T cell activation, proliferation, and induction of unresponsive, immunosuppressive, or immunoresponsive T cell responses.

[0140] As used herein, a polypeptide, or a complex of two or more polypeptides, that specifically binds to OX40L is a polypeptide that binds to the protein of Genbank Accession No. NP_003317 (SEQ ID NO: 2), and related isoforms and orthologs. OX40L (The underlined parts represent the transmembrane domain, and the bolded parts represent the first position of the extracellular domain.) MERVQPLEENVGNAARPRFERNK LLLVASVIQGLGLLLCFTYICLHFSAL QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL (SEQ ID NO: 2)

[0141] In some embodiments, a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L described herein binds to positions 17, 18, 19, 20, 21, 23, 26, 28, 60, 83, 110, 111, 112, 113, and 114 of the extracellular domain of human OX40L. In other embodiments, a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L described herein binds to positions 58, 59, 60, 61, 62, 63, 81, 82, and 83 of the extracellular domain of human OX40L. In some embodiments, a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L binds to positions 60 and / or 83 of the extracellular domain of human OX40L.

[0142] In some embodiments, non-limiting examples of a polypeptide or complex of two or more polypeptides that specifically bind to OX40L include an antigen binding site (e.g., an antibody, Fab, Fab', F(ab')2, single chain variable fragment (scFv), minibody, or nanobody (VHH)); or the extracellular domain of OX40 or a functional fragment thereof.

[0143] In some embodiments, a polypeptide or complex of two or more polypeptides that specifically binds to OX40L comprises an antigen-binding site. In some embodiments, the antigen-binding site is a Fab. Table 2 lists the complementarity determining regions (CDRs) of consensus heavy chain variable domains (VH) and light chain variable domains (VL) that can, in combination, specifically bind to OX40L. In some embodiments, the heavy chain variable domains and light chain variable domains are in a Fab format with VH and VL CDR sequences selected from the consensus VH and VL sequences in Table 2. In some embodiments, a partial hinge (e.g., a polypeptide having a sequence of SEQ ID NO: 336) is connected to the C-terminus of the Fab heavy chain (HC) CH1 domain and forms a disulfide bond with the Fab light chain (LC).

[0144] Unless otherwise stated, the CDR sequences shown in Table 2 are determined under the IMGT proprietary numbering scheme.

[0145] [Table 2]

[0146] In certain embodiments, an antigen binding site that specifically binds to OX40L comprises an amino acid sequence selected from the consensus sequence of SEQ ID NO:220 or SEQ ID NO:221.

[0147] In some embodiments, the antigen-binding site that specifically binds to OX40L comprises a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SEQ ID NO: 222; a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of SEQ ID NO: 223, SEQ ID NO: 224, or SEQ ID NO: 225; and a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO: 220 or SEQ ID NO: 221.

[0148] In some embodiments, the antigen binding site that specifically binds to OX40L comprises a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO:226 or SEQ ID NO:227; a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of SEQ ID NO:82, SEQ ID NO:228, or SEQ ID NO:90; and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO:231 or SEQ ID NO:232.

[0149] In some embodiments, an antigen-binding site that specifically binds to OX40L comprises a VHCDR1 comprising the amino acid sequence of SEQ ID NO: 222; a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 223; a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 220; a VLCDR1 comprising the amino acid sequence of SEQ ID NO: 226; a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 82; and a VLCDR3 comprising the amino acid sequence of SEQ ID NO: 231.

[0150] In some embodiments, an antigen binding site that specifically binds to OX40L comprises a VHCDR1 comprising the amino acid sequence of SEQ ID NO: 222; a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 224; a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 221; a VLCDR1 comprising the amino acid sequence of SEQ ID NO: 227; a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 228; and a VLCDR3 comprising the amino acid sequence of SEQ ID NO: 232.

[0151] In some embodiments, an antigen binding site that specifically binds to OX40L comprises VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, according to the IMGT proprietary numbering scheme, each of which comprises an amino acid sequence corresponding to the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 sequences of the VHCDR and VLCDR consensus sequences in Table 2, respectively.

[0152] In some embodiments, a polypeptide or complex of two or more polypeptides that specifically binds to OX40L comprises an antigen binding site. In some embodiments, the antigen binding site is a Fab. Table 3 lists heavy chain variable domain (VH) and light chain variable domain (VL) polypeptide sequences that, in combination, specifically bind OX40L. In some embodiments, the heavy chain variable domain and light chain variable domain are in a Fab format with VH and VL sequences selected from the VH and VL sequences in Table 3. Table 3 additionally lists heavy chain (HC) and light chain (LC) polypeptide sequences that, in combination, can specifically bind OX40L. In some embodiments, the heavy and light chains are in a Fab format with HC and LC sequences selected from the HC and LC sequences in Table 3.

[0153] Unless otherwise stated, the CDR sequences shown in Table 3 are determined under the IMGT proprietary numbering scheme.

[0154] [Table 3-1]

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

[0155] In one embodiment, an antigen-binding site that specifically binds to OX40L comprises an antibody heavy chain (HC) that comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the HC of an antibody disclosed in Table 3, and an antibody light chain (LC) that comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the LC of the same antibody disclosed in Table 3. In one embodiment, an antigen-binding site that specifically binds to OX40L comprises an antibody heavy chain variable domain (VH) that comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH of an antibody disclosed in Table 3, and an antibody light chain variable domain (VL) that comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL of the same antibody disclosed in Table 3.In one embodiment, the antigen binding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of the VH and VL sequences disclosed in Table 3, as determined by the IMGT proprietary numbering scheme, Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745), or any other method of CDR determination known in the art.

[0156] In one embodiment, the antigen-binding site that specifically binds to OX40L comprises a heavy chain complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISTSSYY (SEQ ID NO: 77), a heavy chain complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy chain complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light chain complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light chain complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light chain complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83).

[0157] In some embodiments, an antigen binding site that specifically binds to OX40L comprises a VHCDR1 having an amino acid sequence of GVSIRSNGYY (SEQ ID NO: 93), a VHCDR2 having an amino acid sequence of MDYSGT (SEQ ID NO: 94), a VHCDR3 having an amino acid sequence of ARERSNNWYPIDY (SEQ ID NO: 95), a VLCDR1 having an amino acid sequence of SVRRFF (SEQ ID NO: 97), a VLCDR2 having an amino acid sequence of GKD (SEQ ID NO: 98), and a VLCDR3 having an amino acid sequence of NSRDSSGYLVL (SEQ ID NO: 99).

[0158] In some embodiments, an antigen binding site that specifically binds to OX40L comprises a VHCDR1 comprising the amino acid sequence of GASVSSSSYY (SEQ ID NO: 85), a VHCDR2 comprising the amino acid sequence of INYGGST (SEQ ID NO: 86), a VHCDR3 comprising the amino acid sequence of ARHRGIYHFDY (SEQ ID NO: 87), a VLCDR1 comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a VLCDR2 comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a VLCDR3 comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91).

[0159] In some embodiments, an antigen binding site that specifically binds to OX40L comprises a VHCDR1 comprising the amino acid sequence of GGSISSSSYY (SEQ ID NO: 101), a VHCDR2 comprising the amino acid sequence of IGSVDYSGNT (SEQ ID NO: 102), a VHCDR3 comprising the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a VLCDR1 comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a VLCDR2 comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a VLCDR3 comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91).

[0160] In some embodiments, an antigen binding site that specifically binds to OX40L comprises a VHCDR1 comprising the amino acid sequence of GFTFSNYA (SEQ ID NO: 133), a VHCDR2 comprising the amino acid sequence of ISGSGGAT (SEQ ID NO: 113), a VHCDR3 comprising the amino acid sequence of TKDRLIMATVRGPYYYGMDV (SEQ ID NO: 114), a VLCDR1 comprising the amino acid sequence of QSISSY (SEQ ID NO: 121), a VLCDR2 comprising the amino acid sequence of AAS (SEQ ID NO: 146), and a VLCDR3 comprising the amino acid sequence of QQSHSVSFT (SEQ ID NO: 154).

[0161] In some embodiments, an antigen binding site that specifically binds to OX40L comprises a VHCDR1 having the amino acid sequence of SYAMS (SEQ ID NO: 105), a VHCDR2 having the amino acid sequence of IISGSGGFTYYADSVK (SEQ ID NO: 106), a VHCDR3 having the amino acid sequence of DRLVAPGTFDY (SEQ ID NO: 107), a VLCDR1 having the amino acid sequence of RASQGISSWLA (SEQ ID NO: 169), a VLCDR2 having the amino acid sequence of AASSLQS (SEQ ID NO: 170), and a VLCDR3 having the amino acid sequence of QQYNSYPYT (SEQ ID NO: 171).

[0162] In one embodiment, the antigen binding site that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 76, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 80.

[0163] In one embodiment, an antigen-binding site that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 92, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 96.

[0164] In one embodiment, an antigen-binding site that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 84, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 88.

[0165] In one embodiment, an antigen-binding site that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 100, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 104.

[0166] In one embodiment, an antigen-binding site that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 30.

[0167] In one embodiment, the antigen binding site that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 33, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 32.

[0168] In one embodiment, the antigen binding site that specifically binds to OX40L comprises an HC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:8, and an LC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:7.

[0169] In one embodiment, the antigen binding site that specifically binds to OX40L comprises an HC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16, and an LC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 15.

[0170] In one embodiment, the antigen-binding site that specifically binds to OX40L comprises an HC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 14, and an LC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 13.

[0171] In one embodiment, the antigen binding site that specifically binds to OX40L comprises an HC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, and an LC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17.

[0172] In one embodiment, the antigen-binding site that specifically binds to OX40L comprises an HC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 252, and an LC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 253.

[0173] In one embodiment, an antigen-binding site that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 254, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 255.

[0174] IC cross-linked part In some embodiments, the proteins of the present disclosure further comprise a bridging moiety. In certain embodiments, the bridging moiety can be non-functional, i.e., it serves merely as a structural connection and / or attachment and exerts no biological function or has no biological purpose. In other embodiments, the bridging moiety is functional and has a biological function in the context of the protein.

[0175] In some embodiments, the N-terminus of the bridging moiety is connected to the C-terminus of a polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86, and the C-terminus of the bridging moiety is connected to the N-terminus of a polypeptide or a complex of two or more polypeptides that specifically binds to OX40L. In some embodiments, the bridging moiety connects a heavy chain of a polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86 with a heavy chain of a polypeptide or a complex of two or more polypeptides that specifically binds to OX40L to form a continuous polypeptide chain.

[0176] In some embodiments, the bridging moiety is connected to the C-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L. In other embodiments, the bridging moiety is connected to the C-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86. In some embodiments, the bridging moiety is connected to the N-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L. In other embodiments, the bridging moiety is connected to the N-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86.

[0177] In some embodiments, non-limiting examples of bridging moieties include a polypeptide of an immunoglobulin Fc domain or a functional fragment thereof, a human serum albumin (HSA) polypeptide or a functional fragment thereof, a polypeptide linker, or a polypeptide hinge. In an embodiment, the bridging moiety comprises a polypeptide of an immunoglobulin Fc domain. For example, in some embodiments, the bridging moiety is a polypeptide of an IgG, IgM, IgA, IgD, or IgE Fc domain. In an embodiment, the bridging moiety is a polypeptide of an IgG1, IgG2, IgG3, or IgG4 Fc domain. In an embodiment, the bridging moiety is a polypeptide of an IgG1 Fc domain. In an embodiment, the bridging moiety is a polypeptide of a human IgG1 Fc domain. In an embodiment, the protein of the present disclosure comprises a first polypeptide of an immunoglobulin Fc domain and a second polypeptide of an immunoglobulin Fc domain, which dimerize with each other.

[0178] Human IgG Fc Within the polypeptides of the Fc domain, the binding of CD16 is through the hinge region and the CH2 domain. For example, within human IgG1, the interaction with CD16 is mainly focused on the amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239 within the CH2 domain, numbered according to the EU index as in Kabat, and the carbohydrate residue N-acetyl-D-glucosamine (see Sondermann et al., Nature, 406 (6793):267-273). Mutations can be selected to enhance or reduce the binding affinity to CD16, such as by using a phage display library or a yeast surface display cDNA library based on known domains, or designed based on known three-dimensional structures of the interaction. Thus, in certain embodiments, the polypeptides of the immunoglobulin Fc domain or portions thereof comprise a hinge polypeptide and a CH2 domain.

[0179] In certain embodiments, the bridging moiety is a polypeptide of a human IgG1 Fc domain comprising one or more mutations that reduce binding to an Fcγ receptor (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB) or a complement component (e.g., C1q) in the first and / or second polypeptide of the human IgG1 Fc domain. Such mutations are useful for reducing effector function. For example, proteins of the disclosure comprise LALA (L234A and L235A) mutations, LALAPA (L234A, L235A, and P329A) mutations, LALAPG (L234A, L235A, and P329G) mutations, or LALEGAASPS (L234A, L235E, G237A, A330S, and P331S) mutations. In some embodiments, the terminal lysine residue of the human IgG1 Fc domain is mutated (K447A) or deleted (K447Δ). In some embodiments, amino acids at any one or more of positions 322, 330, 331, 355, and 358 may be mutated. Unless otherwise noted, all positions of amino acid substitutions are numbered according to the EU index as in Kabat.

[0180] Representative wild-type and mutant polypeptides of human immunoglobulin Fc domains are listed in Table 4. In some embodiments, the cross-linking moiety comprises an amino acid sequence selected from any one of the human immunoglobulin Fc sequences listed in Table 4.

[0181] [Table 4]

[0182] In certain embodiments, a protein of the disclosure comprises a polypeptide of a human IgG Fc domain cross-linking portion that comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to a wild-type or variant human IgG Fc domain sequence listed in Table 4 and numbered according to the EU index as in Kabat.

[0183] In certain embodiments, a protein of the disclosure comprises a polypeptide of a human IgG1 Fc domain cross-linking portion comprising an amino acid sequence at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:178.

[0184] In certain embodiments, a protein of the disclosure comprises a polypeptide of a human IgG1 Fc domain cross-linking portion comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:237.

[0185] In certain embodiments, a protein of the disclosure comprises a polypeptide of a human IgG1 Fc domain cross-linking portion comprising an amino acid sequence at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:238.

[0186] In certain embodiments, a protein of the disclosure comprises a polypeptide of a human IgG1 Fc domain cross-linking portion comprising an amino acid sequence at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:214.

[0187] In certain embodiments, a protein of the disclosure comprises a polypeptide of a human IgG1 Fc domain cross-linking portion comprising an amino acid sequence at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:216.

[0188] In certain embodiments, a protein of the disclosure comprises a polypeptide of a human IgG1 Fc domain cross-linking portion comprising an amino acid sequence at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:179.

[0189] In certain embodiments, a protein of the disclosure comprises a polypeptide of a human IgG1 Fc domain cross-linking portion comprising an amino acid sequence at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:180.

[0190] In certain embodiments, a protein of the disclosure comprises a polypeptide of a human IgG1 Fc domain cross-linking portion comprising an amino acid sequence at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:239.

[0191] In certain embodiments, a protein of the disclosure comprises a polypeptide of a human IgG1 Fc domain cross-linking portion comprising an amino acid sequence at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:240.

[0192] Hinge Polypeptide In some embodiments, the bridging moiety comprises at least a portion of a hinge polypeptide. The hinge polypeptide can be derived from a heavy chain of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, or other class). Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, or IgG4. More preferably, the hinge region is derived from a human IgG1 heavy chain.

[0193] In some embodiments, at least a portion of the hinge polypeptide is connected to the N-terminus of the heavy chain constant domain 2 (CH2) of a polypeptide of a human immunoglobulin Fc domain. In some embodiments, the hinge polypeptide connects the C-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86 to the N-terminus of the CH2 domain of a polypeptide of a human immunoglobulin Fc domain. In other embodiments, the hinge polypeptide connects the C-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L to the N-terminus of the CH2 domain of a polypeptide of a human immunoglobulin Fc domain. In some embodiments, a partial hinge (e.g., a polypeptide having a sequence of SEQ ID NO: 336) is connected to the C-terminus of the CH1 domain of a Fab heavy chain (HC) that specifically binds to OX40L and forms a disulfide bond with a Fab light chain (LC) that specifically binds to OX40L. In some embodiments, a partial hinge (e.g., a polypeptide having the sequence of SEQ ID NO: 336) is attached to the C-terminus of the CH1 domain of a Fab heavy chain (HC) that specifically binds CD80 or CD86, and forms a disulfide bond with a Fab light chain (LC) that specifically binds CD80 or CD86, respectively.

[0194] In some embodiments, amino acids located at any one or more of positions C220, E233, L234, or L235 may be mutated in the hinge region of human IgG1. Unless otherwise noted, all positions of amino acid substitutions are numbered according to the EU index as in Kabat.

[0195] Representative wild-type and mutant human immunoglobulin hinge polypeptides are listed in Table 5. In some embodiments, the bridging moiety comprises an amino acid sequence selected from any one of the human immunoglobulin Fc hinge polypeptide sequences listed in Table 5.

[0196] [Table 5]

[0197] In some embodiments, a bridging moiety of the disclosure comprises a hinge polypeptide comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to a wild-type or variant human immunoglobulin Fc hinge sequence listed in Table 5 and numbered according to the EU index as in Kabat.

[0198] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 175.

[0199] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 242.

[0200] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 176.

[0201] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 249.

[0202] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 177.

[0203] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 243.

[0204] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 212.

[0205] In one embodiment, a bridging portion of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 244.

[0206] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 213.

[0207] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 245.

[0208] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 247.

[0209] In certain embodiments, a bridging moiety of the present disclosure comprises a hinge polypeptide comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to number 248.

[0210] Human serum albumin In some embodiments, the cross-linking moiety of the present disclosure comprises a human serum albumin (HSA) polypeptide of a functional fragment thereof. For example, the cross-linking moiety of the present disclosure can comprise an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:219. (query number 219)

[0211] ID Linker In some embodiments, the N-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L is connected to the C-terminus of a bridging moiety via a linker polypeptide. In other embodiments, the N-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86 is connected to the C-terminus of a bridging moiety via a linker polypeptide. In some embodiments, the N-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86 is connected to the C-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L via a linker polypeptide. In other embodiments, the N-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L is connected to the C-terminus of a polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86 via a linker polypeptide.

[0212] With respect to the amino acid composition of the linker polypeptide, the linker polypeptide sequence is selected to have properties that confer flexibility and minimize interference with the structure and function of other domains and / or with the polypeptides of the protein described in this application. The linker polypeptide sequence is also selected to be resistant to proteolytic cleavage. For example, glycine and serine residues generally provide protease resistance.

[0213] In certain embodiments, the proteins described herein include a (GlyGlyGlyGlySer)4 ((G4S)4) linker (SEQ ID NO: 111). The length of the linker (e.g., flexible linker) can be "short", e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, or "long", e.g., at least 13 amino acid residues. In certain embodiments, the linker is 10-50, 10-40, 10-30, 10-25, 10-20, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30, or 20-25 amino acid residues in length.

[0214] In certain embodiments, the proteins of the disclosure comprise one or more polypeptide linkers that comprise or consist of an amino acid sequence listed in Table 6.

[0215] [Table 6]

[0216] IE Representative proteins Listed below are examples of proteins of the invention that contain a polypeptide or a complex of two or more polypeptides that specifically bind to CD80 and / or CD86, and a polypeptide or a complex of two or more polypeptides that specifically bind to OX40L.

[0217] For example, a polypeptide that specifically binds to CD80 and / or CD86 can comprise a wild-type or variant extracellular domain of CTLA4 selected from any one of the sequences listed in Table 1. Alternatively, a polypeptide, or a complex of two or more polypeptides, that specifically binds to CD80 and / or CD86 can comprise a wild-type or variant extracellular domain of CD28, or a functional fragment thereof, or an antigen binding site (e.g., an antibody, Fab; Fab', F(ab')2, single chain variable fragment (scFv); minibody; or nanobody (VHH)).

[0218] In some embodiments, a polypeptide or complex of two or more polypeptides that specifically binds to OX40L is an antigen-binding site (e.g., an antibody, Fab; Fab', F(ab')2, single chain variable fragment (scFv); minibody; or nanobody (VHH)). For example, an antigen-binding site that specifically binds to OX40L can include a Fab that includes a VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 sequence selected from any of the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 sequences listed in Table 2, the VH and VL sequences listed in Table 3, and / or the HC and LC sequences listed in Table 3. In other embodiments, a polypeptide that specifically binds to OX40L can include the extracellular domain of OX40 or a functional fragment thereof.

[0219] As described above, the protein of the present invention can include a bridging moiety. In some embodiments, a polypeptide or a complex of two or more polypeptides that specifically binds to CD80 and / or CD86 is connected at its C-terminus to the N-terminus of a bridging moiety, and a polypeptide or a complex of two or more polypeptides that specifically binds to OX40L is connected at its N-terminus to the C-terminus of a bridging moiety. As described above, the bridging moiety can be a wild-type human IgG1 Fc domain polypeptide that optionally includes one or more mutations and includes a sequence selected from any one of the sequences listed in Table 4. The bridging moiety can also include a hinge polypeptide (such as a sequence selected from any one of the sequences listed in Table 5) at its N-terminus, optionally including one or more mutations.

[0220] As also described above, a polypeptide that specifically binds to OX40L, or a complex of two or more polypeptides, can be connected to the C-terminus of the bridging moiety via a linker polypeptide. For example, the linker polypeptide can include a polypeptide that includes a sequence selected from any one of the sequences listed in Table 6.

[0221] The proteins of the invention can include heavy and light chains. For example, the heavy chains of the invention can include, from N-terminus to C-terminus, the extracellular domain of CTLA4 or a functional fragment thereof, a bridging portion including CH2 and CH3 domains of a polypeptide of human IgG1 Fc with at least a portion of a hinge polypeptide at the N-terminus; a linker polypeptide; and a heavy chain of an anti-OX40L Fab including a VH domain and a CH1 domain. The heavy chain can additionally include a partial IgG1 hinge sequence (e.g., a polypeptide including the amino acid sequence of SEQ ID NO: 336) at the C-terminus of the CH1 domain that includes a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue in the light chain constant domain (CL). Table 7 lists representative heavy chain sequences of the invention.

[0222] A light chain of the invention can comprise, from N-terminus to C-terminus, the VL and CL domains of an anti-OX40L Fab. Table 7 lists representative light chain sequences of the invention.

[0223] In combination, the heavy and light chains listed in Table 7 can associate, for example linked by disulfide bonds, and specifically bind to CD80 and CD86, and OX40L. As contemplated in the proteins of the invention, the hinge polypeptide and CH3 domain of the human IgG1 Fc polypeptide can facilitate dimerization of the two heavy chains, resulting in a protein that contains two heavy chains and two light chains.

[0224] [Table 7-1] [Table 7-2]

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

Table 7-20

Table 7-21

Table 7-22

Table 7-23

Table 7-24

Table 7-25

Table 7-26

Table 7-27

Table 7-28

Table 7-29

Table 7-30

Table 7-31

Table 7-32

Table 7-33

Table 7-34

Table 7-35

Table 7-36

[0225] Also provided herein is the use of a heavy chain selected from any of the amino acid sequences listed in Table 7 in the preparation of a protein, in which the heavy chain associates (e.g., via a disulfide bond) with a light chain selected from any of the amino acid sequences listed in Table 7.

[0226] Also contemplated in this disclosure is the nucleic acid that encodes the protein of the present invention.The nucleic acid that encodes the protein disclosed herein can be codon optimized for optimal expression using standard bioinformatics methods.Also contemplated is the cell that contains one or more nucleic acids that encode the protein of the present invention, which can be produced by standard transfection or transduction methods (e.g., electroporation, calcium chloride transfection, lipofection, lentivirus delivery, or adeno-associated virus delivery).

[0227] Characteristics of IE proteins The proteins of the present invention can specifically bind to CD80 and / or CD86 and OX40L. In some embodiments, the proteins of the present invention bind to CD80 and / or CD86 and OX40L with high affinity as measured by any one of a variety of assays known in the art. The binding activity and immunosuppressive activity of the proteins of the present invention can be compared to CTLA4-Ig and / or anti-OX40L reference molecules. CTLA4-Ig (Ref3_CTLA4Ig) AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34) CTLA4-IgG_variants AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 71) IgG1 Fc variants -CTLA4EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDS (SEQ ID NO: 72) Ref2_Anti_OX40L Heavy chain sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFNSYAMSWVRQAPGKGLEWVSIISGSGGFTYYADSVKGRFTISRDNSRTTLYLQMNSLRAEDTAVYYCAKDRLVAPGTFDYWGQGA LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 36) Light chain sequence DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 35) United States Patent No. 9,139,653 (Ref1_Anti-OX40L) Heavy chain variable domain (VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGLEWVSTISGSGGATRYADSVKGRFTISRDNSRNTVYLQMNSLRVEDTAVFYCTKDRLIMATVRGPYYYGMDVWGQGTTVTVSS (SEQ ID NO: 31) Light chain variable domain (VL) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPNLLIYAASSLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSHSVSFTFGPGTKVDIK (SEQ ID NO: 30)

[0228] For example, the proteins described herein can specifically bind and neutralize OX40L on the surface of APCs, thereby inhibiting T cell costimulatory signaling and activation. In some embodiments, the proteins of the invention have an IC of less than 12 nM as measured in the OX40L neutralization / OX40-HEK reporter assay described in Example 3 below. 50 In some embodiments, the proteins of the invention have an IC of 5 nM-12 nM, 5 nM-11.5 nM, 5 nM-11 nM, 5 nM-11.5 nM, 5 nM-10 nM, 5 nM-10.5 nM, or 5 nM-9 nM when measured in an OX40L neutralization / OX40-HEK reporter assay. 50 Have.

[0229] Inhibition of T cell activation can also be measured using the primary T cell activation assay described below in Example 3. For example, in some embodiments, the proteins of the invention have an IC of 2 nM to 12 nM, 2 nM to 11.5 nM, 2 nM to 11 nM, 2 nM to 10.5 nM, 2 nM to 10 nM, or 2 nM to 9.5 nM when measured in a primary T cell activation assay. 50 Have.

[0230] In some embodiments, a protein of the invention has a K for OX40L of less than 75 nM as measured in a Biacore assay (described in Example 3 below). DFor example, in some embodiments, OX40L binding has a K of 20 nM to 75 nM, 20 nM to 70 nM, 20 nM to 65 nM, 20 nM to 60 nM, 25 nM to 75 nM, 25 nM to 70 nM, 25 nM to 65 nM, 25 nM to 60 nM, 30 nM to 75 nM, 30 nM to 70 nM, 30 nM to 65 nM, and 30 nM to 60 nM as measured in a Biacore assay. D Have.

[0231] Blocking T cell costimulatory signaling inhibits T cell activation and the production of proinflammatory mediators. In some embodiments, the proteins described herein significantly inhibit the production of at least one or more proinflammatory cytokines, non-limiting examples of which include IL-2, IFNγ, IL-6, or TNFα. Inhibition of proinflammatory cytokine production can be measured using an in vitro mixed lymphocyte reaction assay by comparing cells treated with the proteins described herein with cells treated under the same conditions but in the absence of the proteins, or in the presence of CTLA4-Ig (the extracellular domain of human CTLA4 fused to the N-terminus of human IgG1 Fc domain; SEQ ID NO: 34) and anti-OX40L antibodies (SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 30 and SEQ ID NO: 31) provided separately, or in the presence of a combination of the two proteins provided separately. For example, in some embodiments, the proteins described herein inhibit production of at least one pro-inflammatory cytokine by cells in a mixed lymphocyte reaction assay by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% compared to cells treated under the same conditions but in the absence of the protein.

[0232] Blocking T cell costimulation also results in inhibition of T cell proliferation. In some embodiments, the proteins described herein inhibit alloreactive CD4 + T cells and CD8 +The proliferation of T cells was measured using alloreactive CD4 T cells treated under the same conditions but in the absence of proteins, or in the presence of CTLA4-Ig (the extracellular domain of human CTLA4 fused to the N-terminus of the human IgG1 Fc domain; SEQ ID NO: 34) and anti-OX40L antibodies (SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 30 and SEQ ID NO: 31) provided individually, or in the presence of a combination of the two proteins provided separately. + T cells and CD8 + For example, in some embodiments, the proteins described herein inhibit CD4 T cells in a mixed lymphocyte reaction assay. + T cells and CD8 + The proliferation of T cells is inhibited by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to cells treated under the same conditions but in the absence of the protein.

[0233] In some embodiments, the proteins described herein inhibit the proliferation and proliferation of T cells (T reg or Treg) proliferation compared to alloreactive CD4 + T cells and CD8 + The proliferation of T cells was measured using alloreactive CD4 T cells treated under the same conditions but in the absence of proteins, or in the presence of CTLA4-Ig (the extracellular domain of human CTLA4 fused to the N-terminus of the human IgG1 Fc domain; SEQ ID NO: 34) and anti-OX40L antibodies (SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 30 and SEQ ID NO: 31) provided individually, or in the presence of a combination of the two proteins provided separately. + T cells and CD8 + For example, in some embodiments, mixed lymphocyte reaction assays treated with a protein described herein selectively inhibit T cells compared to cells treated under the same conditions but in the absence of the protein. reg :T エフェクタ The cell ratio is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%.

[0234] Costimulation by inhibiting activation of CD28 and OX40 reg In some embodiments, the proteins described herein can promote differentiation of T cells in an in vitro mixed lymphocyte reaction assay. reg The inhibitory function was evaluated in T cells treated under the same conditions but in the absence of the proteins, or in the presence of CTLA4-Ig (the extracellular domain of human CTLA4 fused to the N-terminus of the human IgG1 Fc domain; SEQ ID NO: 34) and anti-OX40L antibodies (SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 30 and SEQ ID NO: 31) provided individually, or in the presence of a combination of the two proteins provided separately. reg For example, in some embodiments, mixed lymphocyte reaction assays treated with the proteins described herein significantly enhance T reg Proliferating T in co-culture with エフェクタ (Teff or T eff ) reduces the number of cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% compared to cells treated under the same conditions but in the absence of the protein.

[0235] In some embodiments, the proteins of the invention inhibit alloreactive CD4 + T cells and CD8 + T cell proliferation was compared with alloreactive CD4 T cells in mice treated under the same conditions but in the absence of proteins, or in the presence of CTLA4-Ig (the extracellular domain of human CTLA4 fused to the N-terminus of the human IgG1 Fc domain; SEQ ID NO: 34) and anti-OX40L antibodies (SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 30 and SEQ ID NO: 31) provided individually, or in the presence of a combination of the two proteins provided separately. + T cells and CD8 + For example, in some embodiments, mice treated with a protein described herein significantly inhibit proliferation of alloreactive CD4 T cells in adoptive transfer assays compared to mice treated under the same conditions but in the absence of the protein. + T cells and CD8+ The number of T cells is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%.

[0236] In some embodiments, the proteins described herein significantly inhibit serum IFNγ levels in adoptive transfer assays in humanized mice compared to mice treated under the same conditions but in the absence of the protein, or in the presence of CTLA4-Ig (the extracellular domain of human CTLA4 fused to the N-terminus of a human IgG1 Fc domain; SEQ ID NO:34) and anti-OX40L antibodies (SEQ ID NO:35 and SEQ ID NO:36; SEQ ID NO:30 and SEQ ID NO:31) provided separately, or in the presence of a combination of the two proteins provided separately. For example, in some embodiments, mice treated with the proteins described herein reduce serum IFNγ levels by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% in adoptive transfer assays compared to mice treated under the same conditions but in the absence of the protein.

[0237] In some embodiments of the present invention, the proteins described herein are not significantly internalized into bone marrow derived dendritic cells (MDDCs).

[0238] In some embodiments, the proteins described herein cross-react with cynomolgus monkey OX40L. For example, the proteins of the invention can bind to cynomolgus monkey OX40L and have a K Dis 20nM to 120nM, 40nM to 120nM, 60nM to 120nM, 80nM to 120nM, 20nM to 100nM, 40nM to 100nM, 60nM to 100nM, 80nM to 100nM, 20nM to 80nM, 40nM to 80nM, or 60nM to 80nM. In one embodiment, the protein of the present invention does not cross-react with mouse, rabbit, or rat OX40L. For example, the protein of the present invention has a K D is greater than 200 nM, greater than 250 nM, or greater than 500 nM. In some embodiments, the proteins of the invention cross-react with cynomolgus monkey CD80 and CD86. For example, the proteins of the invention can cross-react with cynomolgus monkey CD80 and CD86 and have a K D is 20nM to 120nM, 40nM to 120nM, 60nM to 120nM, 80nM to 120nM, 20nM to 100nM, 40nM to 100nM, 60nM to 100nM, 80nM to 100nM, 20nM to 80nM, 40nM to 80nM, or 60nM to 80nM.

[0239] II Antibodies Also provided herein is an antibody that specifically binds to OX40L. In some embodiments, the antibody that specifically binds to OX40L comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the sequences listed in Table 2.

[0240] In some embodiments, the heavy and light chain variable domains of the antibodies described herein comprise VH and VL CDR sequences selected from the consensus VH and VL sequences listed in Table 2.

[0241] Unless otherwise noted, the CDR sequences shown in Table 2 are determined under the IMGT proprietary numbering scheme.

[0242] In some embodiments, the antibody of the present invention can be IgG, IgM, IgA, IgD, or IgE. In some embodiments, the antibody described herein is IgG1, IgG1, IgG3, or IgG4. In some embodiments, the antibody of the present invention is a human IgG1 antibody.

[0243] In certain embodiments, an antibody that specifically binds to OX40L comprises a VHCDR3 sequence selected from the consensus sequence of SEQ ID NO:220 or SEQ ID NO:221.

[0244] In some embodiments, an antibody that specifically binds to OX40L comprises a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SEQ ID NO: 222; a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of SEQ ID NO: 223, SEQ ID NO: 224, or SEQ ID NO: 225; and a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO: 220 or SEQ ID NO: 221.

[0245] In some embodiments, an antibody that specifically binds to OX40L comprises a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO:226 or SEQ ID NO:227; a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of SEQ ID NO:82, SEQ ID NO:228, or SEQ ID NO:90; and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO:231 or SEQ ID NO:232.

[0246] In some embodiments, an antibody that specifically binds to OX40L comprises a VHCDR1 comprising the amino acid sequence of SEQ ID NO: 222; a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 223; a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 220; a VLCDR1 comprising the amino acid sequence of SEQ ID NO: 226; a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 82; and a VLCDR3 comprising the amino acid sequence of SEQ ID NO: 231.

[0247] In some embodiments, an antibody that specifically binds to OX40L comprises a VHCDR1 comprising the amino acid sequence of SEQ ID NO: 222; a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 224; a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 221; a VLCDR1 comprising the amino acid sequence of SEQ ID NO: 227; a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 228; and a VLCDR3 comprising the amino acid sequence of SEQ ID NO: 232.

[0248] In some embodiments, an antibody that specifically binds to OX40L comprises VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, according to the IMGT proprietary numbering scheme, each of which comprises the sequences of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, respectively, of the consensus sequences of VHCDR and VLCDR in Table 2.

[0249] In one embodiment, an antibody that specifically binds to OX40L comprises an antibody heavy chain (HC) that contains an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the antibody HC of an antibody disclosed in Table 3, and an antibody light chain (LC) that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the antibody LC of the same antibody disclosed in the amino acid sequence Table 3. In one embodiment, an antibody that specifically binds to OX40L comprises an antibody heavy chain variable domain (VH) that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the antibody heavy chain variable domain (VH) of an antibody disclosed in Table 3, and an antibody light chain variable domain (VL) that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the antibody light chain variable domain (VL) of the same antibody disclosed in Table 3.In one embodiment, the antibody comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of the VH and VL sequences disclosed in Table 3 (as determined under the IMGT proprietary numbering scheme, Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J. Mol. Biol. 196:901-917), MacCallum (see MacCallum RM et al., (1996) J. Mol. Biol. 262:732-745), or any other CDR determination method known in the art).

[0250] In one embodiment, an antibody that specifically binds to OX40L comprises a heavy chain complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISTSSYY (SEQ ID NO: 77), a heavy chain complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy chain complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light chain complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light chain complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light chain complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83).

[0251] In some embodiments, an antibody that specifically binds to OX40L comprises a VHCDR1 having an amino acid sequence of GVSIRSNGYY (SEQ ID NO: 93), a VHCDR2 having an amino acid sequence of MDYSGT (SEQ ID NO: 94), a VHCDR3 having an amino acid sequence of ARERSNNWYPIDY (SEQ ID NO: 95), a VLCDR1 having an amino acid sequence of SVRRFF (SEQ ID NO: 97), a VLCDR2 having an amino acid sequence of GKD (SEQ ID NO: 98), and a VLCDR3 having an amino acid sequence of NSRDSSGYLVL (SEQ ID NO: 99).

[0252] In some embodiments, an antibody that specifically binds to OX40L comprises a VHCDR1 having the amino acid sequence of GASVSSSSYY (SEQ ID NO: 85), a VHCDR2 having the amino acid sequence of INYGGST (SEQ ID NO: 86), a VHCDR3 having the amino acid sequence of ARHRGIYHFDY (SEQ ID NO: 87), a VLCDR1 having the amino acid sequence of NIENKN (SEQ ID NO: 89), a VLCDR2 having the amino acid sequence of RDS (SEQ ID NO: 90), and a VLCDR3 having the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91).

[0253] In some embodiments, an antibody that specifically binds to OX40L comprises a VHCDR1 having the amino acid sequence of GGSISSSSYY (SEQ ID NO: 101), a VHCDR2 having the amino acid sequence of IGSVDYSGNT (SEQ ID NO: 102), a VHCDR3 having the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a VLCDR1 having the amino acid sequence of NIENKN (SEQ ID NO: 89), a VLCDR2 having the amino acid sequence of RDS (SEQ ID NO: 90), and a VLCDR3 having the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91).

[0254] In some embodiments, an antibody that specifically binds to OX40L comprises a VHCDR1 having the amino acid sequence of GFTFSNYA (SEQ ID NO: 133), a VHCDR2 having the amino acid sequence of ISGSGGAT (SEQ ID NO: 113), a VHCDR3 having the amino acid sequence of TKDRLIMATVRGPYYYGMDV (SEQ ID NO: 114), a VLCDR1 having the amino acid sequence of QSISSY (SEQ ID NO: 121), a VLCDR2 having the amino acid sequence of AAS (SEQ ID NO: 146), and a VLCDR3 having the amino acid sequence of QQSHSVSFT (SEQ ID NO: 154).

[0255] In some embodiments, an antibody that specifically binds to OX40L comprises a VHCDR1 having the amino acid sequence of SYAMS (SEQ ID NO: 105), a VHCDR2 having the amino acid sequence of IISGSGGFTYYADSVK (SEQ ID NO: 106), a VHCDR3 having the amino acid sequence of DRLVAPGTFDY (SEQ ID NO: 107), a VLCDR1 having the amino acid sequence of RASQGISSWLA (SEQ ID NO: 169), a VLCDR2 having the amino acid sequence of AASSLQS (SEQ ID NO: 170), and a VLCDR3 having the amino acid sequence of QQYNSYPYT (SEQ ID NO: 171).

[0256] In one embodiment, an antibody that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:76, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence SEQ ID NO:80.

[0257] In one embodiment, an antibody that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:92, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence SEQ ID NO:96.

[0258] In one embodiment, an antibody that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:84, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence SEQ ID NO:88.

[0259] In one embodiment, an antibody that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 100, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence SEQ ID NO: 104.

[0260] In one embodiment, an antibody that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:31, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence SEQ ID NO:30.

[0261] In one embodiment, an antibody that specifically binds to OX40L comprises a VH comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 33, and a VL comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence SEQ ID NO: 32.

[0262] In one embodiment, an antibody that specifically binds to OX40L comprises a HC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:8, and a LC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence SEQ ID NO:7.

[0263] In one embodiment, an antibody that specifically binds to OX40L comprises a HC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16, and a LC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence SEQ ID NO: 15.

[0264] In one embodiment, an antibody that specifically binds to OX40L comprises a HC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 14, and a LC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence SEQ ID NO: 13.

[0265] In one embodiment, an antibody that specifically binds to OX40L comprises a HC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, and a LC comprising an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence SEQ ID NO: 17.

[0266] III. Therapeutic Applications The present disclosure also provides a pharmaceutical formulation containing a therapeutically effective amount of the protein disclosed herein. The pharmaceutical formulation includes one or more excipients and is maintained at a pH. The term "excipient" as used herein means any non-therapeutic agent added to a formulation to impart a desired physical or chemical property (e.g., pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissociation or release, adsorption, or penetration).

[0267] The present application provides a method of treating an autoimmune disease using the proteins described herein and / or the pharmaceutical formulations described herein. The method can be used to treat a variety of autoimmune diseases, including, but not limited to, rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, atopic dermatitis, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune ovarian failure, and autoimmune ovarian failure. Focal inflammation, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital atrioventricular block, Coxsackie cardiomyopathy, CR EST syndrome, Crohn's disease, cutaneous lupus, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus erythematosus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schön syndrome Line's purpura (HSP), herpes gestationis or pemphigoid of gestationis (PG), hidradenitis suppurativa (HS) (acne inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, autoimmune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile dermatomyositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, Meniere's disease,Microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mukka-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemorrhage, Polymorphonuclear leukemia (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyendocrine syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing biliary vasculitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome These include subacute bacterial endocarditis (SPS), subacute bacterial endocarditis (SBE), Sazak syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undiagnosed connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

[0268] The present application further provides a method of treating graft-versus-host disease (GVHD) in a patient, the method comprising administering a protein or pharmaceutical formulation described herein. EXAMPLES

[0269] The present disclosure, which will now be generally described, will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the present disclosure, and are not intended to limit the scope of the disclosure in any way.

[0270] Example 1: Preparation of Reagents, Immunization Methods, and Screening Assays This Example describes the reagents, immunization procedures, and screening assays used to generate the data presented herein.

[0271] 1.1 Preparation of reagents and screening assays 1.1.1 Generation of OX40-HEK reporter cells The plasmid vector was transfected into the HEK293 EBNA-NFkb-Luc cell line to stably express full-length human OX40 (SEQ ID NO: 250). Stable mini-pools were selected using hygromycin and screened for induction of luciferase activity after incubating cells with recombinant His-tagged human OX40L protein (R&D systems). Human OX40 (the underlined part represents the signal peptide) MCVGARRLGRGPCAALLLLGLGLSTVTG LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 250)

[0272] 1.1.2 Generation of multivalent human OX40L recombinant protein The human OX40L extracellular domain (ECD) (amino acids 51-183 of SEQ ID NO:2) was expressed fused to an Avi-GST-6His-tag domain ("6His" disclosed as SEQ ID NO:338), purified through the GST domain, and further purified by size-exclusion chromatography. The resulting OX40L ECD fusion protein "AVI-GST-His6-hOX40L" ("His6" disclosed as SEQ ID NO:338) is a polyvalent soluble protein reagent with respect to the OX40L ECD.

[0273] 1.1.3 Generation of CHO-S cells expressing human, cynomolgus, or mouse OX40L CHO-S cells were transfected with plasmid vectors to stably express full-length human OX40L (SEQ ID NO:2), full-length cynomolgus monkey OX40L (SEQ ID NO:337), or full-length mouse OX40L (SEQ ID NO:251). Stable mini-pools were selected using hygromycin, and cells were screened and selected for surface expression of OX40L protein by flow cytometry. Amino acid sequence of cynomolgus monkey OX40L MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHQYPRIQSIKVQFTEYKKEEGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL (SEQ ID NO: 337) Amino acid sequence of mouse OX40L MEGEGVQPLDENLENGSRPRFKWKKTLRLVVSGIKGAGMLLCFIYVCLQLSSSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCDGLYIIYLKGSFFQEVKIDLHFREDHNPISIPMLNDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEHLQINDGELIVVQLTPGYCAPEGSYHSTVNQVPL (SEQ ID NO: 251)

[0274] 1.1.4 Flow cytometry assays to screen for binding of human, cynomolgus, or mouse OX40L CHO-S cells expressing OX40L (human, cynomolgus, or mouse) on their surface, as prepared in 1.1.2, were stained with a dilution series prepared from serum of immunized rats or with recombinant anti-OX40L antibodies. Binding of antibodies to CHO-S-OX40L cells was detected using fluorescently labeled anti-rat IgG or anti-human IgG Fc secondary antibodies as appropriate and measured by flow cytometry.

[0275] 1.1.5 ELISA screens for binding to human, cynomolgus, or mouse recombinant OX40L ELISA assay plates were coated with recombinant human (Novoprotein Catalog No. CJ45), cynomolgus monkey (Novoprotein Catalog No. CP72), or mouse OX40L (R&D systems Catalog No. 1236-OX) protein and then incubated with a dilution series prepared from immunized rat serum or recombinant anti-OX40L antibody. Peroxidase-labeled anti-rat IgG or IgM, or anti-human IgG Fc secondary antibodies, as appropriate, were used to detect antibody binding to the coated ELISA plates containing recombinant OX40L.

[0276] 1.1.6 ELISA Screen to Find Anti-OX40L Antibodies That Block the OX40L:OX40 Protein Binding Interaction Recombinant OX40-Fc fusion protein (R&D Systems) was coated onto 384-well ELISA assay plates, which were then blocked with BSA. Soluble recombinant OX40L-His protein (R&D Systems or homemade) was added to the OX40-Fc-coated assay plates, and after incubation and washing, binding of soluble OX40L-His to the coated OX40-Fc was detected with a peroxidase-labeled anti-His antibody. This condition gives the maximum signal for OX40L binding to OX40 without inhibition. To test the anti-OX40L antibody for inhibition of OX40L:OX40 binding, soluble OX40L-His protein was mixed with anti-OX40L antibody, incubated, and added to the OX40-Fc-coated assay plates, and binding of OX40L to OX40 was detected as described above.

[0277] 1.1.7 Steady-state affinity assay of human and cynomolgus OX40L by surface plasmon resonance (SPR) Recombinant OX40L protein (human OX40L protein, R&D systems, catalogue no. 1054-OX; cynomolgus OX40L, Novoprotein catalogue no. CP72) was immobilized on a Biacore CM5 sensor chip and anti-OX40L antibodies were bound to this surface at a range of concentrations. Steady-state responses were measured using a Biacore T200 instrument (GE Healthcare), plotted against antibody concentration and fitted to a 1:1 binding model. The steady-state KD was calculated as the maximum response (R max ) was calculated as the antibody concentration at which the response was equal to half the total antibody concentration.

[0278] 1.1.8 Steady-State Affinity Assays of Human and Cynomolgus Monkey CD80 and CD86 by Surface Plasmon Resonance (SPR) or Biolayer Interferometry (BLI) Experimental CTLA4_anti-OX40L fusion proteins or CTLA4-Ig reference protein were captured through their Fc region using goat anti-human IgG Fc (Jackson ImmunoResearch goat anti-human IgG Fc antibody, catalog no. 109-005-098) immobilized on a Biacore CM5 sensor chip. Recombinant CD80 protein (human CD80, Novoprotein catalog no. CK61; cynomolgus monkey CD80, Novoprotein catalog no. CP54), and CD86 protein (human CD86, Novoprotein catalog no. C404) were bound to this surface at a range of concentrations. Steady-state responses were measured using a Biacore T200 instrument (GE Healthcare), plotted against antibody concentration, and fitted to a 1:1 binding model. Steady-state KD was calculated as the maximum response (R max ) was calculated as the antibody concentration at which the response was equal to half the total antibody concentration.

[0279] Steady-state affinity of cynomolgus CD86 (Novoprotein catalog number CP41) was measured using an Octet RED96 instrument (ForteBio) in a BLI assay. An anti-human IgG Fc capture (AHC) biosensor (ForteBio, catalog number 18-5060) was used to capture the Fc region of the experimental CTLA4_anti-OX40L fusion protein or the CTLA4-Ig reference protein. Recombinant cynomolgus CD86 was bound to this surface at a range of concentrations. Steady-state responses were plotted against recombinant protein concentration and fitted to a 1:1 binding model. Steady-state K D was determined as the concentration of recombinant protein at a response equal to half the maximum response (Rmax).

[0280] 1.1.9 OX40L Neutralization Assay Screen HEK293 NFkB-Luc hOX40 reporter cells (described in 1.1.1) were cultured at 0.5 × 10 per mL in serum-free medium (Life Technologies: DMEM, hygromycin B, blasticidin, and L-glutamine). 6Cells were seeded at 50,000 cells / 60 μl / well in tissue culture treated clear bottom 96 well plates (Falcon) and incubated overnight at 37°C and 5% CO2. Dilution series from immunized rat serum or anti-OX40L antibodies were prepared in medium in 96 well plates (NUNC). 30 μl of titration series were transferred to 96 well plates (NUNC) containing 30 μL of 10 μg / mL recombinant human OX40L (described in 1.1.2) in each well. The antibody and recombinant OX40L mixture was incubated for 30 min at room temperature and 20 μL of the mixture / well was added to the reporter cells and incubated overnight at 37°C and 5% CO2. Each condition was performed in duplicate. 100 μL of SteadyGlo substrate (Promega, #E2520) was added to each well, the plate was covered and protected from light, and incubated at room temperature for 30 minutes. Plates were read on an Envision plate reader (Perkin Elmer) using the ultra-sensitive luminescence program (emission 0.1 seconds).

[0281] 1.1.10 Human OX40L-induced IL-2 in primary T cell activation assays CD4 T cells were enriched using the RosetteSep Human T Cell Enrichment Kit (StemCell Technologies, 15062). + T cells were isolated. 96-well plates were coated with 5 μg / mL anti-CD3 (BD ​​Biosciences, 555338) and 5 μg / mL anti-CD28 (BD Biosciences, 555725) for 1 h at room temperature. Plates were washed twice with 200 μL 1×PBS. Antibodies were titrated from 100 nM in a 1:3.5 series and then incubated with 10 μg / mL recombinant human OX40L (R&D Systems, 1054-OX) for 30 min at 37°C, 5% CO2. Cells were incubated at 1×10 6Cells were resuspended at 1000x cells / mL and 600 μL of cells were added to 0.5 mL 96-deep well plates with 120 μL of antibody / rhOX40L mix. 200 μL of cells / antibody / rhOX40L mix was added to each well of the anti-CD3 / anti-CD28 coated plates. Plates were incubated at 37°C, 5% CO2 for 48 hours. After incubation, 50 μL of supernatant was collected and probed using a human IL-2 AlphaLISA (Perkin Elmer, AL221C). Plates were read using the AlphaScreen protocol on an Envision plate reader (Perkin Elmer).

[0282] Example 2: Identification, generation, and characterization of anti-OX40L antibodies This example describes the identification, generation, and characterization of anti-OX40L antibodies. To generate anti-OX40L antibodies, OmniRat® rodents (transgenic animals expressing immunoglobulins with fully human variable regions, purchased from Open Monoclonal Technology, Inc., Palo Alto, Calif., a subsidiary of Ligand Pharmaceuticals, Inc., San Diego, Calif.) were first immunized with a human OX40L DNA expression vector, followed by an immunization round with his-tagged recombinant human OX40L extracellular domain (ECD) protein. Sera from immunized OmniRats® were screened for binding to CHO-S cells expressing OX40L protein on the cell surface using flow cytometry as described in Example 1, 1.1.4. Sera from immunized OmniRats® were also screened for binding to recombinant OX40L protein using ELISA as described in Example 1, 1.1.5. After multiple rounds of immunization with human OX40L antigen by DNA expression vector and recombinant protein immunization, OmniRats® were identified by serum immune response for binding to human and cynomolgus OX40L. Sera were then screened for neutralizing activity against soluble recombinant OX40L in the blocking ELISA assay described in Example 1, 1.1.6 and the human OX40L neutralization assay described in Example 1, 1.1.9. Rats whose sera showed good neutralizing activity and high titers for binding to cell surface OX40L and soluble recombinant OX40L were selected. Cells were harvested from the spleens and lymph nodes of the selected rats, followed by direct B cell cloning, and then cloning followed by B cell culture and screening.

[0283] 2.1 B cell selection, B cell culture, and cloning of anti-OX40L antibodies Single B cell sorting was performed from lymphocytes collected from selected immunized rats that had high serum immune responses and showed OX40L neutralizing activity and binding to OX40L targets. Briefly, cells collected from the spleens and / or lymph nodes of selected rats were first incubated with anti-rat CD32 (clone D34-485, BD Biosciences) at 4°C to block Fc receptors on these cells, and then incubated with multivalent human OX40L recombinant protein (prepared as described in Example 1, 1.1.2). Cells were then washed and incubated with a mixture of FITC-labeled goat anti-rat IgM (SouthernBiotech Cat. No. 3020-02), PE-Cy7-labeled mouse anti-rat CD45R (clone HIS 24, eBioscience), APC-labeled mouse anti-His (clone AD1.1.10R, R&D), and, depending on the selected OmniRat® genetic background and sorting strategy, PE-labeled mouse anti-human Ig lambda (clone 1-155-2, BD Biosciences) or V450-labeled mouse anti-human Ig kappa (clone G20-193, BD Biosciences). Single OX40L-binding B cells were sorted with a FACS Aria III (BD Biosciences) into each well of a 96-well plate. For direct B cell cloning, single OX40L-binding B cells were directly selected and placed into wells containing lysis buffer (0.1 M DTT, 40 U / ml RNase inhibitor, Invitrogen, Cat. No. 10777-019), and the plates were frozen on dry ice immediately after sealing and stored at -80°C. For B cell culture and screening, OX40L-binding B cells were selected and placed into wells filled with B cell medium containing cytokines and irradiated homemade feeder cells. After incubating these B cell culture plates at 37°C in a 5% CO2 incubator for 7 days, the supernatant from each well was screened for binding to human and cynomolgus OX40L by ELISA as described in Example 1, 1.1.5. The selected OX40L-binding B cell culture clones were collected from the wells and placed into lysis buffer.

[0284] Ig V gene cloning was performed from each sorted single B cell using a protocol modified from Tiller et al., J Immunol Methods, 2008, 1; 329 (1-2). Briefly, total RNA was reverse transcribed from the sorted single B cells using a final amount / concentration of 150ng random hexamer primer (pd(N)6, AppliedBiosystems, P / N N808-0127) and 50U of Superscript IV reverse transcriptase (Invitrogen, Cat. No. 18090050) according to the manufacturer's protocol in a final volume of 14μl / well of the original 96-well sorting plate filled with nuclease-free water (Invitrogen, Cat. No. AM9935). Primers were modified based on previous publications (Wardemann et al, Science, 2003, 301:1374-1377) and / or designed by consulting published Ig gene compartment nucleotide sequences from IMGT®, the International Immunogenetic Information System (http: / / www.imgt.org; (Lefranc et al., 2009) and NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) databases. Human Igh, IgK, and IgL V gene transcripts were independently amplified by two rounds of nested (IgH, IgK, and IgL) PCR starting from 5 μl of cDNA as template. The first round of PCR used Platinum Multiplex PCR Reaction (Invitrogen, Cat. No. 4464269), followed by nested PCR using AccuPrime Taq DNA Polymerase High Purification Kit (Invitrogen, Cat. No. 4464269). All PCR reactions were performed in a total volume of 50 μl / well in 96-well plates using the Fidelity kit, (Invitrogen, Cat. No. 12346-094) according to the manufacturer's protocol. The first round of PCR was carried out at 95°C for 2 min, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 40 s, and a final incubation at 72°C for 5 min.

[0285] A second round of nested PCR was carried out with 5 μl of unpurified first round PCR product at 95°C for 2 min, followed by five cycles of 94°C for 30 s, 42°C for 30 s, and 72°C for 45 s, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s, with a final incubation at 72°C for 5 min.

[0286] V from 360 OX40L-binding B cells from direct B cell sorting and cloning H Antibody sequence and V L Obtain antibody sequence pairs and identify 54 V H Antibody sequence and V L Antibody sequence pairs were obtained from B cell culture, screening, and cloning. H Arrays and V L These pairs of sequences were cloned into an IgG expression vector for expressing the recombinant antibodies as standard format IgG1 antibodies and for further screening, characterization, and DNA sequence identification and analysis. Diverse antibody gene sequences were captured from the selected B cell clones and classified into multiple clonal sequence clusters.

[0287] 2.2 Screening and selection of anti-OX40L antibody clones IgG format: V obtained through the direct B cell cloning and B cell culture approach described in section 2.1 of this example. H Arrays and V LRecombinant anti-OX40L antibodies were generated using Ig expression vectors constructed using pairs of sequences. Correctly paired heavy and light chain vectors derived from B cell clones were used for small-scale transient transfection of Expi293F cells, and then cell culture supernatants were harvested after 7 days of incubation. Cell culture supernatants containing recombinant anti-OX40L antibody clones were screened by flow cytometry as described in Example 1, 1.1.4 for binding to CHO-S cells expressing OX40L protein on the cell surface, by ELISA as described in Example 1, 1.1.5 for binding to recombinant OX40L protein, by ELISA competition assay as described in Example 1, 1.1.6 for blocking binding of OX40L protein to OX40, and by OX40 reporter cell assay as described in Example 1, 1.1.9 for neutralization of OX40L.

[0288] Based on screening data for recombinant anti-OX40L clones in cell culture supernatants and analysis of anti-OX40L clone sequences, a total of 85 anti-OX40L antibodies were selected, produced at a 15 ml scale from transient transfection of ExpiCHO cells, and affinity purified using Protein A beads to allow more detailed characterization and comparison between the clones and similarly produced reference proteins. After comparing these purified anti-OX40L antibodies based on binding to CHO-S cells expressing cell surface OX40L protein by flow cytometry as described in Example 1, 1.1.4, binding to recombinant OX40L protein by ELISA as described in Example 1, 1.1.5, blocking binding of OX40L protein to OX40 by ELISA competition assay as described in Example 1, 1.1.6, neutralization of OX40L by OX40 reporter cell assay as described in Example 1, 1.1.9, and neutralization of OX40L-induced IL-2 in a primary T cell activation assay as described in Example 1, 1.1.10, the 24 candidates were then compared by steady-state K for binding to OX40L protein by surface plasmon resonance (SPR) as described in Example 1, 1.1.7. D The measurements were compared. The purified candidate anti-OX40L antibodies were compared as described above, and clone sequence analysis, neutralization assay IC 50 <20 nM (reporter cell assays and primary cell assays), and steady-state SPR binding K DFurther selection was performed using criteria including <60 nM. All selected candidates bound to human and cynomolgus OX40L, and none cross-reacted with respect to binding to mouse OX40L. Twenty-two of the 85 candidates met the above criteria. The antibody sequences of these 22 candidates were analyzed, and 12 candidate clones that showed diverse antibody clone sequences were selected for further study. Among these 12 anti-OX40L antibodies, clones 97G07 (IgG), 67B06 (IgG), 68F03 (IgG), 98E10 (IgG), 89B09 (IgG), 98C01 (IgG), 88B06 (IgG), and 85F10 (IgG) bound to human OX40L. D is 15-31.9 nM, and the binding K of the reference anti-OX40L antibody is D The remaining clones bound human OX40L and exhibited steady-state K D was greater than 32 nM. Selected data for these 12 anti-OX40L antibody clones are summarized in Table 8.

[0289] 2.3 Characterization of selected anti-OX40L antibody clones 2.3.1 OX40L Neutralization Assay The OX40L blocking function of anti-OX40L antibody clones was examined in an OX40L neutralization assay. Briefly, test antibodies, isotype controls, and known anti-OX40L reference antibodies (positive controls) were serially titrated in culture medium, incubated with recombinant human OX40L, and added to HEK-luciferase reporter cells expressing the OX40 receptor, as described in Example 1, 1.1.9. Data were plotted using GraphPad Prism 8 software. Normalization was performed with luciferase activity from each experimental plate being 0% for unstimulated cells and 100% for stimulation with OX40L in the absence of antibody. The IC of anti-OX40L antibodies was calculated by curve fitting using the relationship between log(inhibitor) and normalized response-variable slope using GraphPad Prism. 50Eleven anti-OX40L IgG antibody clones neutralized OX40L in the HEK reporter assay with varying potency. Clones 97G07 (IgG), 67B06 (IgG), 68F03 (IgG), 98E10 (IgG), 89B09 (IgG), 98C01 (IgG), and 95B06 (IgG) had IC values ​​of 3.6–7.5 nM, comparable to the reference anti-OX40L antibodies. 50 Clones 30F02 (IgG), 88B06 (IgG), 85F10 (IgG), and 84E11 (IgG) had IC values ​​ranging from 17.7 to 72.7 nM. 50 Table 8 summarizes the data from this reporter assay.

[0290] 2.3.2 Primary T cell activation assay Ten anti-OX40L antibody clones (clones 85F12 and 85F10 were not tested in this assay) were additionally tested in the primary T cell activation assay described in 1.1.10. Briefly, CD4 + T cells were stimulated with anti-CD3, anti-CD28, and recombinant human OX40L, and anti-OX40L antibody clones were tested in serial dilutions in this assay. OX40L-mediated T cell activation results in high levels of IL-2 cytokines that can be reduced with increasing concentrations of anti-OX40L IgG antibodies, thus indicating neutralizing activity. Data were plotted using GraphPad Prism 8 software. Normalization was performed such that the IL-2 response from each experimental plate was 0% for unstimulated T cells and 100% for stimulation with OX40L in the absence of antibody. IC was calculated by curve fitting using the relationship between log(inhibitor) and normalized response-variable slope using GraphPad Prism. 50 Table 8 shows the IC values ​​from two experiments. 50 The IC values ​​(units are nM ± SEM) are summarized. Most of the anti-OX40L antibody clones examined showed IC 50 It showed OX40L neutralizing activity of <20 nM.

[0291] 2.3.3 Steady-state affinity assays A steady state affinity assay was performed as described in section 1.1.7 of Example 1. Table 8 summarizes the data from this assay. [Table 8]

[0292] Figure 2A is a flow chart providing the antibody screening cascade for anti-OX40L antibodies. The flow chart shows the assays used to screen anti-OX40L antibody clones to select the final candidates, which yielded 12 candidates. Figure 2B is a flow chart providing the candidate characterization cascade.

[0293] Example 3: Generation and characterization of anti-OX40L FcFab antibodies and CTLA_anti-OX40L fusion proteins FcFab and CTLA4_anti-OX40L fusion protein formats: This example describes how 12 anti-OX40L clones were generated in FcFab and CTLA4 fusion formats, followed by characterization assays.

[0294] 3.1 Generation of anti-OX40L FcFab antibody and CTLA_anti-OX40L ("CTLA4-Fc-anti-OX40L") fusion protein The Fab domains of 12 anti-OX40L clones (described in Example 2, section 2.2) were generated in two engineered protein formats: "FcFab" antibody and "CTLA4-Fc-anti-OX40L". In both protein formats, the antibody Fab domain was attached to the C-terminus of a variant IgG1 Fc domain protein. Briefly, the N-terminus of the antibody clone heavy chain consisting of the VH-CH1- and partial IgG1 hinge (EPKSC) sequence was fused to the C-terminus of a 16 amino acid linker sequence fused to the C-terminus of the Fc polypeptide. These protein formats of the heavy chain polypeptides were expressed together with the antibody clone light chain polypeptides associated with the heavy chain polypeptides of the Fab, and disulfide bonds were formed between the light chain and the partial IgG1 hinge at the C-terminus of the heavy chain.

[0295] The anti-OX40L FcFab antibody heavy chain N-terminus began at the first amino acid E216 (Kabat EU index numbering) of the IgG1 hinge element as shown in the sequence of SEQ ID NO: 73. The amino acid sequences of the heavy and light chains of the 12 CTLA4_anti-OX40L fusion proteins are shown in SEQ ID NOs: 37-60.

[0296] Heavy chain DNA expression vectors for each of the anti-OX40L FcFab antibodies were generated by PCR amplification of the anti-OX40L antibody VH sequence and the PCR product was cloned into the FcFab heavy chain expression vector using the restriction enzyme sites 5'BamHI and 3'NheI, which are adjacent to the VH sequence and part of the codon sequence for the 16-amino acid linker and CH1 domain, respectively. The FcFab heavy chain expression vectors were generated by gene synthesis (Quintara Biosciences) and the gene synthesis product was then cloned into the transient expression vector pTT5 (Durocher et al., Nucleic Acids Res. 2002 Jan 15;30(2):E9.). The CTLA4_anti-OX40L heavy chain expression vector was generated by gene synthesis (Genewiz) and the gene synthesis product was then cloned into the transient expression vector pTT5. For each of the 12 CTLA4_anti-OX40L heavy chain vectors, a clone-specific VH sequence was cloned between the restriction enzyme sites 5'BamHI and 3'NheI, which are adjacent to the VH sequence and are part of the codon sequence for the 16-amino acid linker and the CH1 domain, respectively. Representative DNA sequences constructed in these CTLA4_anti-OX40L expression vectors are shown in SEQ ID NO: 74 (light chain sequence) and SEQ ID NO: 75 (heavy chain sequence). DNA sequence of 97G07 heavy chain DNA sequence of the 97G07 light chain tactatgagctgactcagccactctcagtgtcagtggccctgggacagacggccaggattccctgtgggggaaacaacattgaaaataaaaatgtgcactggtaccaacagaagccaggccaggcccctgtactggtcatctatagggatagcaaccggccctctgggatccctgagcgattctctggctccaactcggggaacacggccaccctgtccatcaacagagcccaagccggggatgaggctgactattactgtcaggtgtgggacagcaacactgtgatattcggcggagggaccaaggtcaccgtcctaggacagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagtcccacaaaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca (SEQ ID NO: 74)

[0297] Anti-OX40L FcFab antibody and CTLA4_anti-OX40L protein were produced by transient transfection of the correctly paired light and heavy chain DNA expression vectors detailed above. Anti-OX40L FcFab antibody was produced at 15-ml scale from transient transfection of ExpiCHO cells, affinity purified using Protein A beads, and characterized. CTLA4_anti-OX40L protein was produced at 1000-ml scale from transient transfection of ExpiCHO cells, affinity purified on a Protein A column (Hi-Trap MAbSelect SuRe, GE Healthcare) on an Akta Avant 25 chromatography system (GE Healthcare). Final protein concentration was measured and the protein was characterized by analytical size-exclusion chromatography (SEC), SDS-PAGE gel, and checked for endotoxin before being used in the planned studies.

[0298] Because there is no guarantee that any anti-OX40L IgG will retain the OX40L binding and neutralization characteristics of the original anti-OX40L IgG when converted into an anti-OX40L FcFab or CTLA4_anti-OX40L fusion protein format, the FcFab and fusion protein formats were examined following the same screening assay cascade (Figure 2A) as utilized for the IgG format. A positive reference control, anti-OX40L IgG, was used in every assay.

[0299] 3.2 Characterization of anti-OX40L FcFab antibodies 3.2.1 OX40L Neutralization Assay The OX40L blocking function of anti-OX40L FcFAb antibody clones was examined by OX40L neutralization assay. Briefly, as described in Example 1, 1.1.9, test antibodies, isotype controls, and known anti-OX40L reference antibodies (positive control) were serially titrated in culture medium, incubated with recombinant human OX40L, and added to HEK-luciferase reporter cells expressing OX40 receptor. Data was plotted using GraphPad Prism 8 software. Normalization was performed by setting the luciferase activity from each experimental plate as 0% for unstimulated cells and 100% for stimulation with OX40L in the absence of antibody.

[0300] The OX40L blocking activity of FcFab antibody clones 97G07 (FcFab), 67B06 (FcFab), 68F03 (FcFab), 98E10 (FcFab), 89B09 (FcFab), and 98C01 (FcFab) was measured based on their IC 50 As can be seen from the IC values, these are similar to their corresponding IgG counterparts, suggesting that the format conversion to FcFab did not significantly affect their neutralization potency. However, the FcFab antibody clones 84E11 (FcFab), 88B06 (FcFab), 95B06 (FcFab), 30F02 (FcFab), 85F12 (FcFab), and 85F10 (FcFab) showed a significant decrease in potency, with IC values ​​lower than their corresponding IgG counterparts. 50 The IC of the anti-OX40L FcFab antibody was calculated by curve fitting using the relationship between log(inhibitor) and normalized response-variable slope using GraphPad Prism. 50 Values ​​were calculated. Table 9 summarizes the data from duplicates from one experiment.

[0301] 3.2.2 Primary T cell activation assay Eleven anti-OX40L FcFab antibody clones (clone 98C01 was not tested in this assay) were additionally tested in the primary T cell activation assay described in 1.1.10. Briefly, CD4 +T cells were stimulated with anti-CD3, anti-CD28, and recombinant human OX40L, and anti-OX40L FcFAb antibody clones were tested at serial dilutions in this assay. Data were plotted using GraphPad Prism 8 software. Normalization was performed to set the IL-2 response from each experimental plate to 0% for unstimulated T cells and 100% for stimulation with OX40L in the absence of antibody. IC was calculated by curve fitting using the relationship between log(inhibitor) and normalized response-variable slope using GraphPad Prism. 50 Table 9 shows the IC values ​​from two experiments. 50 The IC values ​​(units are nM ± SEM) are summarized. 97G07 (FcFab), 67B06 (FcFab), 98E10 (FcFab), 89B09 (FcFab), 30F02 (FcFab), and 85F10 (FcFab) retained similar potency to the reference control anti-OX40L despite the format conversion. 50 =5.7~9.3nM).

[0302] 3.2.3 Steady-state affinity assays Similar to the IgG clones, all 12 FcFabs were subjected to binding affinity determination for human OX40L using the steady-state affinity assay described in Example 1, section 1.1.7. Most FcFabs retained binding to the target and showed a range of affinities, K D = 20.3-64.1 nM, with the exception of 84E11 (FcFab), which completely lost binding activity. Table 9 summarizes the data from this assay.

[0303] [Table 9]

[0304] 3.3 Characterization of CTLA4_anti-OX40L fusion protein 3.3.1 OX40L Neutralization Assay The OX40L blocking function of CTLA4_anti-OX40L fusion proteins was examined in an OX40L neutralization assay. Briefly, as described in Example 1, 1.1.9, test fusion proteins, isotype controls, and known anti-OX40L reference antibodies (positive control) were serially titrated in culture medium, incubated with recombinant human OX40L, and added to HEK-luciferase reporter cells expressing the OX40 receptor. Data were plotted using GraphPad Prism 8 software. Normalization was performed by setting the luciferase activity from each experimental plate as 0% for unstimulated cells and 100% for stimulation with OX40L in the absence of fusion proteins. Data from two experiments are summarized in Table 10. Figure 4 is a graph showing neutralization of OX40L using an OX40 luciferase reporter assay for CTLA4_anti-OX40L fusion proteins 95B06, 98C01, 98E10, 84E11, 68F03, 67B06, 97G07, 89B09, and Ref4_CTLA4_O13, for reference anti-OX40L monoclonal antibodies Ref1_anti-OX40L and Ref2_anti-OX40L, or for an isotype control. As shown in Figure 4, seven CTLA4_anti-OX40L fusion proteins (97G07, 68F03, 67B06, 98E10, 89B09, 98C01, 95B06) retained similar potency as the reference anti-OX40L monoclonal antibody. Five fusion proteins showed a significant reduction in potency or loss of their neutralizing capacity compared to their IgG counterparts: 84E11 (IgG) IC 50 =17nM→CTLA4_anti-OX40L IC 50 =50.2nM;88B06(IgG), 30F02(IgG), 85F12(IgG), 85F10(IgG)IC 50 =19~72.7nM→CTLA4_anti-OX40L IC 50 ≧100 nM). The data are shown in Table 8.

[0305] 3.3.2 Primary T cell activation assay Only eight fusion proteins were evaluated for neutralization of human OX04L in the primary human T cell activation assay (described in Example 1, 1.1.10) since 88B06, 30F02, 85F12, and 85F10 showed weak or no inhibition. Data were plotted using GraphPad Prism 8 software. Normalization was performed with IL-2 response from each experimental plate set at 0% for unstimulated T cells and 100% for stimulation with OX40L in the absence of fusion protein. IC was calculated by curve fitting using the relationship between log(inhibitor) and normalized response-variable slope using GraphPad Prism. 50 The IC values ​​are shown in Table 10. 50 (Units are nM ± SEM) are summarized.

[0306] As shown in Table 10, fusion proteins 97G07, 67B06, 68F03, 84E11, 98E10, 89B09, and 98C01 retained similar potency as their IgG counterparts and the reference control anti-OX40L despite the format conversion. 50 =5.7~9.3nM).

[0307] 3.3.3 Steady-state affinity for OX40L, CD80, and CD86 The binding affinity for human OX40L was determined for all 12 CTLA4_anti-OX40L fusion proteins using the steady-state affinity assay described in section 1.1.7 of Example 1. Similar to the FcFab format, the majority of the CTLA4_anti-OX40L fusion proteins retained binding to the target and showed a range of binding affinities, K D =27.6-106.1 nM. As expected, the fusion protein 84E11, like its FcFab counterpart, lost binding affinity to human OX40L. Table 10 summarizes the data from this assay.

[0308] Additionally, CTLA4_anti-OX40L fusion proteins 89B09, 67B06, and 98C01, as well as a reference anti-OX40L antibody, were evaluated for binding to human and cynomolgus OX40L expressed on the surface of CHO-S cells by flow cytometry as described in Example 1, section 1.1.4. 50 , 89B09(CHO-S-huOX40L EC 50 = 4.62 and CHO-S-cynoOX40L EC 50 =4.37nM), 67B06(CHO-S-huOX40L EC 50 =1.85 and CHO-S-cynoOX40L EC 50 = 2.77 nM), and 98C01 (CHO-S-huOX40L EC 50 = 2.79 and CHO-S-cynoOX40L EC 50 = 3.45 nM) with reference anti-OX40L (CHO-S-huOX40L EC 50 = 1.14 and CHO-S-cynoOX40L EC 50 = 0.95 nM), indicating excellent cross-reactivity between human and cynomolgus OX40L. The binding affinity for cynomolgus OX40L was determined for fusion proteins 67B06 and 89B09 and compared to a reference anti-Ox40L antibody. The K D is the K of the reference anti-OX40L antibody D was found to be similar (57 nM).

[0309] The binding affinity of the CTLA4_anti-OX40L fusion protein and the reference CTLA4-Ig protein to human and cynomolgus CD80 and CD86 was determined using the steady state affinity assay described in Example 1, section 1.1.8. The steady state binding KDs were comparable to the CTLA4-Ig reference protein (human CD80 / human CD86=450nM / 663nM; cynomolgus CD80 / cynomolgus CD86=433nM / 910nM) for 67B06 (human CD80 / human CD86=538nM / 670nM; cynomolgus CD80 / cynomolgus CD86=486nM / 770nM) and 89B09 (human CD80 / human CD86=540nM / 654nM; cynomolgus CD80 / cynomolgus CD86=474nM / 750nM). Thus, fusing the anti-OX40L Fab domain to the C-terminus of the CTLA4-Ig protein to generate the CTLA4_anti-OX40L fusion protein did not significantly alter the binding affinity of the N-terminal CTLA4 domain to CD80 or CD86. In conclusion, the CTLA4_anti-OX40L fusion protein retained binding to human and cynomolgus monkey OX40L, CD80, and CD86, and showed good cross-reactivity between them.

[0310] The seven CTLA4_anti-OX40L fusion proteins were ranked and four clonally diverse sequences were selected based on highly potent neutralizing activity and closest affinity to a monoclonal anti-OX40L IgG reference antibody.

[0311] [Table 10]

[0312] Comparison of the three formats To directly compare data from the three different formats (anti-OX40L (IgG), anti-OX40L (FcFab), and CTLA4_anti-OX40L fusion protein), data were plotted from the neutralization assay. Figures 3A-3C are graphs showing neutralization of OX40L by anti-OX40L (97G07 and 67B06) in IgG (Figure 3A), FcFab (Figure 3B), and fusion protein (Figure 3C) formats utilizing an OX40 luciferase reporter assay. Monoclonal anti-OX40L antibodies (Ref1_anti-OX40L and / or Ref2_anti-OX40L) were used as reference controls. Anti-HEL IgG1 was used as an isotype control (isotype-IgG1).

[0313] Example 4: Mechanism of action of CTLA4_anti-OX40L fusion protein using in vitro assays In this example, an in vitro assay is used to elucidate the mechanism of action of the CTLA4_anti-OX40L fusion protein.

[0314] 4.1 Allogeneic MDDC:T Mixed Lymphocyte Reaction (MLR) Assay The CTLA4_anti-OX40L fusion protein, which maintained good neutralization and binding of human OX40L, was examined for its bifunctional activity. To determine whether the fusion protein could block proinflammatory cytokines produced by T cells receiving costimulatory signals through both the CD80 / CD86-CD28 and OX40L-OX40 pathways, a human allogeneic MDDC:T mixed lymphocyte reaction (MLR) was performed. This assay utilized activated primary monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells expressing all three targets, CD80, CD86, and OX40L, allowing the evaluation of both the CD28 and OX40 pathways contributing to T cell proinflammatory cytokine release and T proliferation.

[0315] Briefly, freshly isolated monocytes were cultured for 5 days in the presence of GM-CSF and IL-4 (in AIM-V medium containing 5% human AB serum, 50 U / ml penicillin, and 50 μg / ml streptomycin) to generate immature monocyte-derived dendritic cells (iMDDCs). iMDDCs were then treated with 1 μg / ml prostaglandin E2 (PGE2), 20 ng / ml TNFα, 10 ng / mL IL-1b, and 20 ng / mL IL-6 for 2 days to generate mature MDDCs. Cell surface expression of CD86, CD80, and OX40L on mature MDDCs was confirmed by flow cytometry. To perform the mixed lymphocyte reaction, fresh PBMCs were isolated from the buffy coat using a Ficoll gradient. Total T cells were isolated from PBMCs using a pan T cell isolation kit (Miltenyi Biotec, 130-096-535). In flat-bottom 96-well tissue culture plates, 20,000 MDDCs and 80,000 T cells were seeded in each well, mixed thoroughly, and treated with various concentrations of either medium control, isotype control, or test agent. CTLA4_anti-OX40L fusion proteins 89B09, 67B06, 98E10, and 98C01 were treated at three concentrations (0.8 nM, 6.25 nM, and 50 nM) and compared with equimolar concentrations of single reference agents (Ref2_anti-OX40L, Ref3_CTLA4Ig), and a combination of monoclonal anti-OX40L antibody and CTLA4-Ig (combination). Cells were then cultured at 37°C and 5% CO2 for 4 days, after which cytokines released in the supernatant were analyzed. Levels of IL-2, IFNγ, TNFα, and IL-6 were assessed by AlphaLISA assay (Perkin Elmer).

[0316] As shown in Figures 5A-5B, CTLA4-Ig alone (Ref3-CTLA4Ig) or anti-OX40L alone (Ref2_anti-OX40L) showed a concentration-dependent reduction in cytokines. Performance decreased at lower concentrations, demonstrating that the assay can measure effects from either pathway blockade. In contrast, fusion proteins (67B06, 98E10, and 98C01) demonstrated greater inhibition of cytokines IL-2 (Figure 5A) and TNFα (Figure 5B) compared to the single reference agents at all concentrations examined. Surprisingly, the fusion proteins inhibited these cytokines more potently than the combination of CTLA4-Ig and anti-OX40L. This suggests a synergistic effect of the bifunctional fusion proteins in combination compared to the single agents. Data shown are normalized from the four donor MLR pairs. The IC of all fusion proteins examined was determined by curve fitting using the relationship between log(inhibitor) and normalized response-variable slope. 50 Table 11 shows the IC values ​​from 4 to 6 donor pairs. 50 (Units are nM ± SEM) All 67B06 variants containing different cross-linking moieties were similar in their inhibition of IL-2 and TNFα (data not shown).

[0317] Fusion proteins 67B06 and 89B09 were serially titrated in subsequent assays to determine the IC 50 Values ​​were determined for IL-2 (Figure 10A), TNFα (Figure 10B), and IL-6 (Figure 10C). Figures 10A-10C show that 89B09 and 67B06 were consistently the most potent agents in suppressing the proinflammatory cytokines IL-2, TNFα, and IL-6 compared to single agent controls or the combination, with the IC values ​​of each agent for each cytokine being 0.01 and 0.02. 50 (nM) are plotted as mean ± SEM. Data shown are summary of four different MDDC:T MLR pairs and statistical significance was examined using one-way ANOVA, *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, ****p ≤ 0.00005.

[0318] Anti-OX40L treatment alone produced measurable IC in two of four MLR experiments 50 This suggests that the fusion protein had a more robust and reliable effect across many experiments and many donors.

[0319] To determine whether CTLA4_anti-OX40L fusion proteins altered alloreactive proliferation, isotype control fusion proteins, single agents, and combinations were examined in the MDDC:T MLR assay. For proliferation readout, T cells were labeled with Cell Trace Violet dye (Invitrogen) and cultured with mature MDDCs as described above with the addition of 0.35 μL / mL of anti-CD3 beads (StemCell, 10309). Fusion proteins (89B09, 67B06, 98E10, and 98C01), CTLA4-Ig (Ref3_CTLA4Ig), anti-OX40L (Ref2_anti-OX40L), or a combination of CTLA4-Ig and anti-OX40L (combination) were examined at equimolar concentrations of 6.25 nM or 50 nM. Cells were then cultured for 4 days at 37°C and 5% CO2 and stained with fluorescently labeled antibodies against the markers CD3, CD4, and OX40 for FACS analysis on a BD LSR Fortessa X-20 to identify proliferating CD4 + OX40 + Cells and proliferating CD4 - OX40 + Cells or CD8 + OX40 + The cell percentages were analyzed by FlowJo.

[0320] As shown in Figures 6A-6B, the fusion proteins (89B09, 67B06, 98E10, and 98C01) inhibited alloreactive CD4 + OX40 + Cells and CD8 + OX40 +The combination of CTLA4-Ig and anti-OX40L reduced cell proliferation more potently than the combination of CTLA4-Ig and anti-OX40L (combination), and was statistically significant. Results from the two experiments were plotted as percentages in GraphPad PRISM8 and statistical significance was examined using one-way ANOVA, *p≦0.05. This was significant because treatment with the fusion protein, but not the single-agent reference control or the combination, reduced memory T cells and CD8 + OX40 + T cells, suggesting that simultaneous dual blockade of both the CD28 and OX40 pathways was required to achieve this effect.

[0321] 4.2 Treg induction assay Soluble OX40L has been shown to inhibit Treg induction in vitro. Fusion proteins (89B09, 67B06, and 98C01) were evaluated for OX40L neutralizing function and ability to restore Treg generation in a Treg induction assay compared to reference anti-OX40L (Ref1_anti-OX40L and Ref2_anti-OX40L) and CTLA4-Ig (Ref3_CTLA4Ig) controls.

[0322] Naive T cells were freshly isolated from human blood using a human naive CD4 T cell isolation kit (Miltenyi). Complete RPMI 1640 medium (supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 50 IU penicillin / 50 ug / ml streptomycin, 1 mM Na pyruvate, 55 μM β-mercaptoethanol, and 0.01 M HEPES) was used for in vitro T cell culture. 100,000 naive T cells were seeded into each well of a 96-well tissue culture plate and stimulated with 5 ng / ml TGF-β and 50 U / ml IL-2 in addition to plate-bound anti-CD3 (5 ug / ml) and soluble anti-CD28 (1 ug / ml). Cells were cultured at 37°C in a CO2 incubator for 5 days.

[0323] Recombinant soluble OX40L (1 μg / ml, R&D Systems) was added to most conditions except the control. Either isotype control IgG, fusion protein, or reference control treatment (50 nM) was added to each well on day 0. At the end of the experiment, cells were harvested and intracellular FoxP3 staining was performed using the manufacturer's instructions (eBioscience) for "Staining Intracellular FoxP3". FACS analysis was performed using a BD LSR Fortessa X-20. Data analysis was performed using FlowJo software. Treg frequencies were normalized to those of isotype-treated conditions and statistical significance was examined using one-way ANOVA, *p≦0.05. As shown in FIG. 7, iTreg generation was significantly reduced 3-fold in induction conditions containing soluble OX40L. However, the presence of equimolar CTLA4_anti-OX40L fusion proteins (89B09, 67B06, and 98C01) or reference anti-OX40L antibodies (Ref1_anti-OX40L and Ref2_anti-OX40L) neutralized the effect of soluble OX40L and fully restored Treg induction in vitro. As expected, CTLA4-Ig (Ref3_CTLA4Ig) did not bind OX40L and had no effect on Treg recovery.

[0324] 4.3 MDDC:Treg:Teff MLR Tripartite Assay A MDDC:Treg:Teff MLR tripartite assay was utilized to demonstrate whether fusion CTLA4_anti-OX40L fusion protein acts synergistically with Tregs to further suppress Teff proliferation.

[0325] Freshly isolated monocytes were cultured (in AIM-V medium containing 5% human AB serum, 50 U / ml penicillin, and 50 μg / ml streptomycin) in the presence of 50 ng / ml GM-CSF and 50 ng / ml IL-4 for 5 days to become immature monocyte-derived dendritic cells, and were treated with 1 μg / ml PGE2, 20 ng / ml TNFα, 20 ng / ml IL-6, and 10 ng / ml IL-1β for 2 days to become mature MDDCs (MDDCs). Cell surface expression of CD86, CD80, and OX40L was confirmed by FACS.

[0326] To perform the mixed lymphocyte reaction, fresh PBMCs were isolated from the buffy coat using a Ficoll gradient. CD4 T cell isolation kit (Miltenyi) was used. + T cells were isolated from PBMCs. Freshly isolated CD4 + T cells were then analyzed by flow cytometry to identify CD4 + CD25 高 CD127 低 Treg and non-Treg CD4 + T (or Teff, T eff , or T エフェクタ The selected Tregs were labeled with 2 μM CellTrace Violet and were then sorted into Teff CD4 + T cells were labeled with 2 μM CellTrace CFSE. 25,000 MDDCs and 50,000 Teff CD4 T cells were cultured in flat-bottom 96-well tissue culture plates. + T cells were seeded into each well and cultured in complete AIM-V medium (supplemented with 5% human AB serum, soluble anti-CD3 (0.6 μl / ml) (StemCell), 50 IU penicillin / 50 μg / ml streptomycin). In the Treg condition, 12,500 Tregs were added. Cultured cells were also treated with either media control, isotype control Ab, CTLA4-Ig, anti-OX40L reference antibody, or CTLA4_anti-OX40L fusion protein (89B09, 67B06, and 98C01) at 10 nM.

[0327] In a separate experiment, fusion protein 89B09 and relevant controls were tested at 10 nM in the presence of various Treg:Teff ratio conditions. Cells were then cultured at 37°C, 5% CO2 for 4-5 days and then analyzed on a BD LSR Fortessa X-20. Effector T cell proliferation was revealed by CFSE dilution. Histograms were analyzed by FlowJo and the ratio of Treg to Teff and absolute cell numbers of proliferating Teff cells were plotted using Graphpad PRISM8.

[0328] As shown in Figure 8, it was demonstrated that the fusion proteins (89B09, 67B06, 98C01) could suppress Teff proliferation to a greater extent than the control drug in the presence of Tregs (1:4 Treg:Teff condition). This result indicated that the fusion proteins could attenuate Teff more effectively and could act in combination with Tregs to further affect Teff suppression.

[0329] Rapamycin is an mTOR inhibitor and a common T cell immunosuppressant used in transplantation to increase Tregs and effectively suppress Teff by increasing the Treg:Teff ratio. Voclosporin, a second generation cyclosporine, is a calcineurin inhibitor and is known as a T cell immunosuppressant that was recently FDA approved to treat lupus nephritis. The same MLR assay described above was performed with varying concentrations of fusion proteins, single agents, combinations, and 100 nM rapamycin or vorcosporin. As shown in Figure 9A, CTLA4_anti-OX40L fusion proteins (89B09, 67B06, 98C01) increased the Treg:Teff ratio in a concentration-dependent manner compared to single agents or combinations. Rapamycin or voclosporin did not increase the Treg:Teff ratio at clinically effective concentrations in this in vitro assay.

[0330] Since Tregs also require CD28 signaling for activation and proliferation, it was essential to address whether the fusion protein could also inhibit Tregs and negatively affect their suppressive function on Teffs. We also examined the effect of fusion protein treatment on inhibition of T effector proliferation in the presence of increasing Tregs by varying the Treg:Teff ratio in the tripartite MLR cultures described above. As shown in Figure 9B, cultures treated with isotype control, increasing Treg:Teff ratios reduced Teff proliferation as expected. CTLA4-Ig treatment did not additionally alter Teff suppression in addition to Tregs, whereas combination with anti-OX40L moderately altered Teff proliferation. Strikingly, the fusion protein 89B09 inhibited Teff proliferation more than the other treatments in the presence of increasing Tregs. This indicates that the fusion protein did not negatively affect Treg function but acted synergistically with Tregs to more efficiently attenuate Teff proliferation. The observation that the fusion protein, but not the single-agent combination, acted synergistically with Tregs to selectively reduce Teff proliferation was a surprising finding and revealed a potentially intriguing mechanistic difference between treatment with the CTLA4_anti-OX40L fusion protein with dual costimulatory blocking properties and the single-agent combination. [Table 11]

[0331] Example 5: Characterization of CTLA4_anti-OX40L fusion protein This example describes the facts that contribute to the superior potency of the CTLA4_anti-OX40L fusion protein. The unexpected aspect of the 67B06 fusion protein showing much greater potency (by several orders of magnitude) compared to combination treatments indicated that the superior potency was one defining feature of a single bifunctional protein compared to a combination of two separate molecules. To explore which factors may contribute to the superior potency for cytokine attenuation seen with the fusion protein, receptor occupancy of the fusion protein, single agent, and combination was compared to the target ligands (CD80 and OX40L) and IL-2 production was measured in the MDDC:T MLR assay.

[0332] 5.1 Receptor occupancy assays for CD80 and OX40L Receptor occupancy of CD80 and OX40L by CTLA4_anti-OX40L fusion proteins or single active reference controls in allogeneic MDDC:T MLR assays was assessed by flow cytometry on day 4. Briefly, cells were washed with PBS, stained with anti-CD11c (clone 3.9, Biolegend), anti-CD80 (clone 2D10, Biolegend), anti-CD86 (clone FUN-1, BD), and anti-OX40L (clone ik-1, BD), and assessed by FACS on a BD Fortessa to determine CD80, CD86, and OX40L expression on MDDC. Mean fluorescence intensity was determined for CD80, CD86, and OX40L, and dose-response curves were fitted using GraphPad PRISM 8. Conversion of data to occupancy was performed using the following formula (where MFI NS was set to 100% inhibition, and MDDC:T co-cultures in the absence of antibody (0 CPDΔMFI), indicated as "M+T only" in the legend of Figure 11, were set to minimal inhibition: % Inhibition for sample A = 100 - (AΔMFI / 0CPDΔMFI) x 100 MFI T = Median total fluorescence intensity of the PE channel for the population in question MFI NS = Median nonspecific fluorescence intensity of PE in incubated duplicate samples ΔMFI=MFIT -MFI NS = Background corrected MFI 0CPDΔMFI=ΔMFI of samples dosed with 0 mg / mL of compound

[0333] As shown in Figures 11A-11C, the increased potency of the CTLA4_anti-OX40L fusion protein is due to increased receptor occupancy of CD80, not OX40L. The 67B06 fusion protein potently inhibited IL-2 production in this assay compared to the single reference agents or their combinations (Figure 11A). This effect was demonstrated by 67B06 occupancy of the target CD80, and was observed at 50% receptor occupancy (RO 50 ) is CTLA4-Ig (RO 50 =3.5nM) or combination (RO 50 = 1.03 nM) compared to 0.019 nM. The results showed that the potency of 67B06 in binding to CD80 was increased 184-fold over CTLA4-Ig and 54-fold over the combination (Figure 11B). 67B06 occupies OX40L and its potency is increased by 1.08-fold over anti-OX40L (RO 50 =0.02nM) or combination treatment (RO 50 Combination 0.01nM) compared to RO 50 = 0.003 nM, a 6-fold and 3-fold increase in potency against OX40L, respectively (Figure 11C). Thus, the stronger effect of 67B06 in attenuating cytokines is most likely attributable to stronger CD80 occupancy due to the avidity of the protein by engaging its other corresponding target, OX40L.

[0334] 5.2 Internalization assay To examine whether other mechanisms (such as physical internalization or clearance of the test agent) might affect its potency in the MDDC:T MLR assay, an internalization assay was performed using real-time live-cell imaging technology.

[0335] Using a live-cell imaging system Celldiscoverer7 (Zeiss), real-time internalization of CTLA4-Ig, anti-OX40L, and CTLA4_anti-OX40L 67B06 fusion proteins was compared on MDDCs over a 20-hour period. Briefly, activated MDDCs were incubated with 100 nM of pHrodo-labeled antibodies. By utilizing pHrodo dyes that fluoresce at low pH when internalized into the endocytic compartment of the cells, internalization of 67B06, CTLA4-Ig, and anti-OX40L agents was tracked. Negative controls for internalization (including pHrodo-labeled isotype controls and the anti-CD20 antibody Rituxumab) were run in the same assay. The time course of internalization of the test agents and the detected pHrodo signal was tracked and recorded. CTLA4-Ig has been reported to be internalized upon binding to its corresponding targets, which may be an efficient way to block targets CD80 and CD86 by removing them from the cell surface, although target-mediated clearance of the drug may affect its efficacy. As shown in Figure 12, CTLA4-Ig (Ref3_CTLA4Ig) was rapidly internalized within 6 hours and continuously internalized over a 20-hour period as indicated by the increased pHrodo signal in MDDCs (Figure 12). In contrast, the anti-CD20 antibody Rituxumab (anti-CD20-IgG1), isotype controls (isotype-Ctl and isotype-IgG1), or anti-OX40L reference control (Ref1_anti-OX40L) were not internalized. Although the CTLA4_anti-OX40L fusion protein (67B06) bound to the same targets CD80 and CD86 as CTLA4-Ig, strikingly it did not share the same internalization profile and in fact showed limited internalization into MDDCs over a 20-hour period (Figure 12). This may enable the fusions to block targets CD80, CD86, and OX40L for extended periods without internalization or excretion, which may explain the enhanced potency compared to single-agent controls in the MDDC:T MLR assay.

[0336] 5.3 MDDC:SLE PBMC MLR Assay To evaluate the enhanced potency of the CTLA4_anti-OX40L fusion protein in suppressing additional pro-inflammatory cytokines, an MDDC:SLE PBMC assay was developed. A similar assay setup to the allogeneic MDDC:T MLR assay in section 3.1 was performed to evaluate the effect of the fusion protein in PBMCs derived from patients with systemic lupus erythematosus (SLE). The assay conditions were similar to 3.1, except that patient PBMCs were used instead of T cells from healthy volunteers. On day 4, supernatants were collected from the co-cultures and analyzed for pro-inflammatory cytokines (GM-CSF, granzyme B, IFN gamma, IL-2, IL-4, IL-10, IL-13, IL-17A, TNFalpha, IL-9, IL-17 / IL-25, IL-17F, IL-21, IL-22, MIP-3alpha) using MSD Immuno-Oncology Group 1 and Biomarker Group 1 kits. Data from triplicate donor pairs were plotted using GraphPad PRISM 8. For ELISPOT readout, MDDC:SLE PBMC assays required the addition of anti-CD3. Cell cultures were plated directly onto IFNγ ELISPOT plates and probed on day 4 according to the manufacturer's instructions (human IFNγ, R&D Systems). Spots were counted on a CTL ImmunoSpot S6 Analyzer and data analysis was performed using GraphPad PRISM 8.

[0337] As shown in Figures 13A-13C, lupus PBMCs induced elevated levels of IL-2 (data not shown), TNFα (data not shown), GMCSF (Figure 13A), IL-13 (Figure 13B), and GZMB (Figure 13C) in the culture supernatant when cultured with MDDCs, and these cytokines could be potently suppressed by 1 nM fusion protein (67B06) compared to single agents or the combination. At 50 nM, both fusion and combination treatments were similarly effective in reducing these cytokines. Alloreactive IFNγ producing cells were significantly reduced by treatment with 50 nM fusion protein and combination compared to single agents. Figures 13A-13C show normalized data from three donors.

[0338] Example 6: In vivo pharmacodynamic effects of CTLA4_anti-OX40L fusion protein This example describes the in vivo pharmacodynamic effects of CTLA4_anti-OX40L fusion protein using a humanized MLR PD model.

[0339] 6.1 In vivo humanized MLR PD model As none of the CTLA4_anti-OX40L fusion proteins cross-react with murine OX40L, a humanized in vivo pharmacodynamics (PD) model was developed to evaluate the fusion proteins. Briefly, monocyte-derived dendritic cells were activated as previously described in the in vitro MDDC:T MLR assay in section 3.1. On day -1, NOD.Cg-Prkdc scid Il2rg tm1Wjl 10 / SzJ or NSG mice (Jackson Labs) were treated i.v. with various concentrations of fusion proteins (67B06, 89B09), single-arm control, or isotype control, followed by infusion of mixed activated MDDCs (1 × 10 6 cells) and freshly isolated allogeneic T cells (1 × 10 7Cells) were transferred ip on day 0. Serum was collected 6 days later for human cytokine measurements using an MSD kit (Human u-Plex Pro-inflammatory Combo Kit, Catalog No. K15049K-2). IFNγ levels and statistical analysis (one-way ANOVA) were performed using GraphPad PRISM 8.

[0340] As shown in Figure 14A, both 67B06 and 89B09 dose-dependently reduced human IFNγ production compared to the isotype control, demonstrating in vivo efficacy in mice. In a separate study, mice were treated once with equimolar (2.88 nmol / kg or 0.58 nmol / kg) doses of 67B06 fusion protein, the respective single agent controls CTLA4-Ig, or anti-OX40L 67B06 (IgG) to assess whether the enhanced potency of the fusion protein observed in vitro compared to the isotype control was reflected in vivo. As shown in Figure 14B, 2.88 nmol / kg (equivalent to 0.5 mg / kg) of the fusion protein 67B06 suppressed human IFNγ production more than the comparative control at the same equimolar concentrations examined.

[0341] Example 7: In vivo efficacy of CTLA4_anti-OX40L fusion protein This example describes the in vivo efficacy of CTLA4_anti-OX40L fusion protein utilizing xenograft, acute and chronic graft-versus-host disease (xeno-GVHD) models.

[0342] The xenogeneic GVHD model is a well-characterized model that relies on injected human PBMCs to generate a strong xenoreactive response against a host mouse lacking the mouse immune system. Xenogeneic GVHD models have well-known limitations in immune subsets that are present in mice and drive disease. For example, human B cells and myeloid cells do not survive in mice for more than 7-10 days due to lack of survival factors. However, human T cells survive and proliferate well in mice, so that by day 7, the majority of the transplanted cells in the mouse are T cells. The expanded T cells are xenoreactive and infiltrate target organs (e.g., liver, lung, and intestine) where they cause tissue damage and increase in inflammatory cytokines (e.g., IFNγ), leading to weight loss. Mice die of GVHD if untreated. This model has therefore previously been useful to evaluate a number of biologics that target T cells and can suppress T cells and alleviate GVHD by assessing their effect on reducing GVHD-mediated weight loss and IFNγ production.

[0343] In the first experiment, CTLA4_anti-OX40L fusion protein (67B06) and anti-OX40L (Ref1_anti-OX40L) were evaluated. Briefly, female NSG mice (n=8 / group) were irradiated (200rad) and administered 200μg / mouse (equivalent to 10mg / kg) of fusion protein, single agent reference control, or isotype control IgG intraperitoneally three times per week (Q3W) starting on day -1. On day 0, mice received 10 million human peripheral blood mononuclear cells (PBMCs). On day 7, mice received 10 million human CD45 + Cell phenotype was characterized by flow cytometry. The study was terminated on day 25 to measure endpoints of survival, weight loss, and serum IFNγ. Statistical analysis (one-way ANOVA) was performed using Prism GraphPad 8.

[0344] Figure 15A is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference monoclonal anti-OX40L antibody (Ref1_anti-OX40L), or isotype control on weight loss in a xenogeneic GVHD mouse model. Figure 15B is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference monoclonal anti-OX40L antibody (Ref1_anti-OX40L), or isotype control on IFNγ production in a xenogeneic GVHD mouse model. As shown in Figures 15A-15B, both drugs rescued mice from weight loss, but only 67B06 fusion protein showed a reduction in IFNγ compared to anti-OX40L treatment alone (Figure 15B ANOVA: ****p≦0.0001, *p=0.0175, ***p=0.0002).

[0345] In a second experiment, performed and evaluated substantially as described above, CTLA4_anti-OX40L fusion protein (67B06) was compared to CTLA4-Ig. CTLA4_anti-OX40L fusion protein (67B06), CTLA4-Ig (Ref3_CTLA4Ig), or isotype control were administered intraperitoneally once a week (Q1W) at doses of 20 μg / mouse or 1 μg / mouse (equivalent to 1 mg / kg or 0.05 mg / kg, respectively).

[0346] Figure 16A is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference CTLA4Ig (Ref3_CTLA4Ig), or isotype control administered ip at 1 mg / kg or 0.05 mg / kg Q1W on weight loss in a xenogeneic GVHD mouse model. Figure 16B is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06), reference CTLA4Ig (Ref3_CTLA4Ig), or isotype control on IFNγ production in a xenogeneic GVHD mouse model. As shown in Figures 16A-16B, both agents rescued mice from weight loss, but only 67B06 fusion protein showed a reduction in IFNγ compared to CTLA4Ig treatment alone (Figure 16B ANOVA: *p=0.0455, **p=0.0086).

[0347] In further experiments with this model, the effect of CTLA4_anti-OX40L fusion protein (67B06) was compared to that of CTLA4Ig and anti-OX40L used in combination. CTLA4_anti-OX40L fusion protein (67B06), Ref3_CTLA4Ig, Ref1_anti-OX40L, and the combination of Ref3_CTLA4Ig and Ref1_anti-OX40L were administered intraperitoneally once a week (Q1W) at a dose of 20 μg / mouse (1 mg / kg). The study was terminated on day 41.

[0348] Figure 17A is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06) reference CTLA4Ig (Ref3_CTLA4Ig), anti-OX40L (Ref1_anti-OX40L), combination (Ref3 and Ref1), or isotype control on weight loss in a xenogeneic GVHD mouse model. Figure 17B is a graph showing the effect of CTLA4_anti-OX40L fusion protein (67B06) reference CTLA4Ig (Ref3_CTLA4Ig), anti-OX40L (Ref1_anti-OX40L), combination (Ref3 and Ref1), or isotype control on IFNγ production in a xenogeneic GVHD mouse model. As shown in Figures 17A-17B, both the fusion protein (67B06), the reference CTLA4Ig (Ref3_CTLA4Ig), and the combination (Ref3_CTLA4Ig and Ref1_anti-OX40L) rescued mice from weight loss, but only the fusion protein 67B06 or the combination showed a reduction in IFNγ more than CTLA4Ig or anti-OX40L treatment alone (Figure 17B ANOVA: *p=0.0110, **p=0.0084).

[0349] In a separate experiment, a different CTLA4_anti-OX40L fusion protein (89B09) is tested and compared to an isotype control. Briefly, starting on day -1, the fusion protein (89B09) or isotype control is administered intraperitoneally once a week (Q1W) at a dose of 20 μg / mouse (1 mg / kg). As with the experiment with fusion protein 67B06, mice are monitored for weight loss and IFNγ production is compared to the isotype treatment alone on day 41 of treatment.

[0350] Figure 17C is a graph showing the effect of CTLA4_anti-OX40L fusion protein (89B09) or isotype control on IFNγ production in a xenogeneic GVHD mouse model at day 12. The graph shows a significant reduction in IFNγ production by the fusion protein (ANOVA: **p=0.0036).

[0351] In a further acute GVHD model, severe combined immunodeficiency (SCID) mice (5-10 weeks old) were injected with 20 μg of rat anti-mouse IL-2Rβ antibody to deplete endogenous NK cells. The next day, mice were given 2.5 Gy of irradiation using a cesium source. Four hours later, each mouse was given 10 million total human PBMCs by intraperitoneal (IP) injection, immediately followed by intravenous (IV) injection of various doses of CTLA4_anti-OX40L fusion protein, reference monoclonal anti-OX40L antibody, or isotype control. Alternatively, IV injection of various doses of CTLA4_anti-OX40L fusion protein, reference monoclonal anti-OX40L antibody, or isotype control could be delayed until the third or sixth day after IP injection of PBMCs. Mice were weighed every 3-4 days and given anti-IL-2Rβ antibody weekly. On day 12, mice were euthanized for gross pathology assessment, flow cytometric analysis of splenocyte, liver, and intestinal histology, and serum collection for cytokine and antibody analysis.

[0352] Incorporation by Reference The entire disclosure of each patent document and journal article referred to herein is incorporated by reference for all purposes.

[0353] Equivalent The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments should therefore be considered in all respects as illustrative rather than limiting of the disclosure set forth herein. The various structural elements of the different embodiments and various steps of the disclosed methods may be utilized in various combinations and permutations, and all such variations are considered to be aspects of the present disclosure. The scope of the present disclosure is therefore indicated by the appended claims rather than by the above description, and all modifications within the meaning and range of equivalence of the claims are intended to be embraced in the present disclosure.

Claims

**Claim 1**: A protein comprising an extracellular domain of CTLA4 or a functional fragment thereof, and a polypeptide or a complex of two or more polypeptides that specifically binds to OX40L, wherein the polypeptide or the complex of two or more polypeptides that specifically binds to OX40L comprises an antigen-binding site. **Claim 2** The protein according to claim 1, wherein the extracellular domain of CTLA4 or the functional fragment thereof comprises a sequence that is at least 90% identical to SEQ ID NO: 29, SEQ ID NO: 173, or SEQ ID NO:

174. **Claim 3** The protein according to claim 2, wherein the antigen-binding site is a Fab or an antibody. **Claim 4** The protein according to claim 3, wherein the Fab comprises a polypeptide comprising the sequence of SEQ ID NO: 336 connected to the C-terminus of the CH1 domain of the heavy chain of the Fab. **Claim 5** Further comprising a cross-linking moiety, wherein the cross-linking moiety comprises a polypeptide of the human IgG1 Fc domain, and optionally the polypeptide of the human IgG1 Fc domain comprises a sequence that is at least 90% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 178, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 239, and SEQ ID NO: 240, the protein according to claim 1. **Claim 6** The protein according to claim 5, wherein the cross-linking moiety further comprises a hinge polypeptide, and the hinge polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 175, SEQ ID NO: 242, SEQ ID NO: 176, SEQ ID NO: 249, SEQ ID NO: 177, SEQ ID NO: 243, SEQ ID NO: 212, SEQ ID NO: 244, SEQ ID NO: 213, SEQ ID NO: 245, SEQ ID NO: 247, and SEQ ID NO:

248. **Claim 7** The protein according to claim 6, wherein: (a) The extracellular domain of CTLA4 or a functional fragment thereof is linked to the N-terminus of the cross-linking portion; or (b) A polypeptide specifically binding to OX40L or a complex of two or more polypeptides is linked to the C-terminus of the cross-linking portion; A protein that satisfies any one of the following conditions, and optionally a linker polypeptide connects the polypeptide specifically binding to OX40L or the complex of two or more polypeptides to the cross-linking portion.

8. Further comprising a second polypeptide specifically binding to OX40L or a complex of two or more polypeptides, wherein the cross-linking portion is a polypeptide of the human IgG1 Fc domain, and the polypeptide specifically binding to OX40L or the complex of two or more polypeptides is connected to the C-terminus of the polypeptide of the human IgG1 Fc domain, and the second polypeptide specifically binding to OX40L or the complex of two or more polypeptides is connected to the C-terminus of the second polypeptide of the human IgG1 Fc domain. The protein according to claim 7.

9. The second polypeptide specifically binding to OX40L or the complex of two or more polypeptides is connected to the cross-linking portion by a linker polypeptide, and optionally the linker polypeptide is (GGGGGS) ｎ (SEQ ID NO: 181) (where n is 1 to 12). The protein according to claim 8.

10. The antigen-binding site is (a) The heavy chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence GX １ SX ２ X ３ X ４ SX ５ YY (SEQ ID NO: 222) (where X １ is A, G, or V, X ２ is V or I, X ３ is S or R, X ４ is S or T, X ５ is S or G); (b) (1) IX １ YX ２ GST (SEQ ID NO: 223) (where X １ is Y or N, X ２ is S or G) (2) X １ DYSGT (SEQ ID NO: 224) (where X １ is I or M), and (3) IGSVDYSGX １ T (SEQ ID NO: 225) (where X １ is N, A, or S) A heavy chain variable complementarity determining region 2 (VHCDR2) comprising an amino acid sequence selected from the group consisting of; (c) (1) ARHRGX １ YX ２ FDX ３ (SEQ ID NO: 220) (where X １ is S or I, X ２ is F or H, X ３ is I or Y) and (2) ARERSX １ X ２ WYPX ３ DY (SEQ ID NO: 221) (where X １ is N or S, X ２ is N, D, G, or S, X ３ is I or F) A heavy chain variable complementarity determining region 3 (VHCDR3) comprising an amino acid sequence selected from the group consisting of; (d) (1) X １ IENKN (SEQ ID NO: 226) (where X １ is N or D) and (2) SX １ RX ２ X ３ X ４ (SEQ ID NO: 227) (where X １ is V or L, X ２ is R or N, X ３ is F or Y, X ４ is F or Y) and comprising a light chain variable complementarity determining region 1 (VLCDR1) selected from the group consisting of amino acid sequences; (e) a light chain variable complementarity determining region 2 (VLCDR2) comprising an amino acid sequence selected from the group consisting of RDN, GKD, and RDS; and (f) (1) QVX １ DSX ２ X ３ VV (SEQ ID NO: 231) (where X １ is R or W, X ２ is N, T, or A, X ３ is I, T, or A) (2) NSRDSYXX １ VX ２ (SEQ ID NO: 232) (where X １ is L or H, X ２ is L or V) and comprising a light chain variable complementarity determining region 3 (VLCDR3) selected from the group consisting of amino acid sequences; The protein according to claim 3, comprising

11. The antigen-binding site is: (i) (a) comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence GGGSISTSSYY (SEQ ID NO: 77), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence IYYSGST (SEQ ID NO: 78), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence ARHRGSYFFDI (SEQ ID NO: 79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence DIENKN (SEQ ID NO: 81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence RDN (SEQ ID NO: 82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence QVRDSNIVV (SEQ ID NO: 83); (b) comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence GVSIRSNGYY (SEQ ID NO: 93), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence MDYSGT (SEQ ID NO: 94), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence ARERSNNWYPIDY (SEQ ID NO: 95), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence SVRRFF (SEQ ID NO: 97), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence GKD (SEQ ID NO: 98), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence NSRDSSGYLVL (SEQ ID NO: 99); (c) comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence GASVSSSSYY (SEQ ID NO: 85), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence INYGGSST (SEQ ID NO: 86), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence ARHRGIYHFDY (SEQ ID NO: 87), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence QVWDSNTVV (SEQ ID NO: 91); (d) a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSSISSSSYY (SEQ ID NO: 101), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IGSVDYSGNT (SEQ ID NO: 102), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91); or (e) according to the IMGT unique numbering scheme, comprising VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, each of which comprises an amino acid sequence corresponding to the sequences of the heavy chain variable domain and the light chain variable domain VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 respectively listed in Table 3; and / or (ii) (a) an amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 84, or SEQ ID NO: 100, and an amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 80, SEQ ID NO: 96, SEQ ID NO: 88, or SEQ ID NO: 104; (b) an amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 76, and an amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 80; (c) an amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 92, and an amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 96; or (d) according to the IMGT unique numbering scheme, comprising a heavy chain variable domain and a light chain variable domain, each of which comprises an amino acid sequence corresponding to the heavy chain variable domain sequence and the light chain variable domain sequence of the heavy chain variable domain and the light chain variable domain respectively listed in Table 3; and / or (iii) (a) An amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 14, or SEQ ID NO: 18, and an amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 13, or SEQ ID NO: 17; (b) An amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 8, and an amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 7; (c) An amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 16, and an amino acid sequence having a sequence that is at least 90% identical to SEQ ID NO: 15; or (d) According to the IMGT numbering scheme, comprising a heavy chain and a light chain, each of which comprises an amino acid sequence corresponding to the heavy chain sequence and the light chain sequence of the heavy chain and the light chain listed in Table 3; The protein according to claim 3.

12. The antigen-binding site is: (i) Position 60 and / or 83 of the extracellular domain of human OX40L; (ii) Positions 17, 18, 19, 20, 21, 23, 26, 28, 60, 83, 110, 111, 112, 113, and 114 of the extracellular domain of human OX40L; or (iii) Positions 58, 59, 60, 61, 62, 63, 81, 82, and 83 of the extracellular domain of human OX40L; The protein according to claim 3, which binds to.

13. The protein according to claim 3, wherein: (i) (a) A polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (b) A bridging portion comprising a sequence comprising SEQ ID NO: 177 and a sequence comprising SEQ ID NO: 179; and (c) A heavy-chain variable complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence of GGGSISTSSYY (SEQ ID NO: 77), a heavy-chain variable complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy-chain variable complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light-chain variable complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light-chain variable complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light-chain variable complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83); or (ii) A first arm and a second arm, wherein the first arm (a) A first polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (b) A first cross-linking moiety comprising the sequence comprising SEQ ID NO: 177 and the sequence comprising SEQ ID NO: 179; and (c) A heavy-chain variable complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence of GGGSISTSSYY (SEQ ID NO: 77), a heavy-chain variable complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy-chain variable complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light-chain variable complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light-chain variable complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light-chain variable complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83) in a first antigen-binding site; and the second arm is: (a) A second polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (b) A second cross-linking moiety comprising the sequence comprising SEQ ID NO: 177 and the sequence comprising SEQ ID NO: 179; and (c) A second antigen-binding site comprising a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GGGSISTSSYY (SEQ ID NO: 77), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of DIENKN (SEQ ID NO: 81), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of RDN (SEQ ID NO: 82), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83); A protein comprising the above and wherein the first arm and the second arm dimerize with each other.

14. The protein according to claim 3, wherein: (i) (a) A polypeptide comprising the extracellular domain of CTLA4 containing SEQ ID NO: 174; (b) A bridging portion comprising the sequence containing SEQ ID NO: 213 and the sequence containing SEQ ID NO: 216; and (c) An antigen-binding site comprising a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GVSIRSNGYY (SEQ ID NO: 93), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of MDYSGT (SEQ ID NO: 94), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARERSNNWYPIDY (SEQ ID NO: 95), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of SVRRFF (SEQ ID NO: 97), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of GKD (SEQ ID NO: 98), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of NSRDSSGYLVL (SEQ ID NO: 99); (ii) The first arm and the second arm, wherein the first arm is (a) A first polypeptide comprising the extracellular domain of CTLA4 containing SEQ ID NO: 174; (b) A first bridging portion comprising the sequence containing SEQ ID NO: 213 and the sequence containing SEQ ID NO: 216; and (c) A first antigen-binding site comprising a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GVRSNGYY (SEQ ID NO: 93), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of MDYSGT (SEQ ID NO: 94), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARERSNNWYPIDY (SEQ ID NO: 95), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of SVRRFF (SEQ ID NO: 97), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of GKD (SEQ ID NO: 98), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of NSRDSGSYLVL (SEQ ID NO: 99); comprising, wherein the second arm (a) A second polypeptide comprising the extracellular domain of CTLA4 containing SEQ ID NO: 174; (b) A second cross-linking moiety comprising the sequence containing SEQ ID NO: 213 and the sequence containing SEQ ID NO: 216; and (c) A second antigen-binding site comprising a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GVRSNGYY (SEQ ID NO: 93), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of MDYSGT (SEQ ID NO: 94), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARERSNNWYPIDY (SEQ ID NO: 95), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of SVRRFF (SEQ ID NO: 97), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of GKD (SEQ ID NO: 98), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of NSRDSGSYLVL (SEQ ID NO: 99) comprising, wherein the first arm and the second arm dimerize with each other; (iii) (a) A polypeptide comprising the extracellular domain of CTLA4 containing SEQ ID NO: 174; (b) A cross-linking moiety comprising the sequence containing SEQ ID NO: 213 and the sequence containing SEQ ID NO: 214; and (c) A heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GASVSSSSYY (SEQ ID NO: 85), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of INYGGST (SEQ ID NO: 86), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARHRGIYHFDY (SEQ ID NO: 87), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of NIENKN (SEQ ID NO: 89), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of RDS (SEQ ID NO: 90), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91); (iv) A first arm and a second arm, wherein the first arm (a) A first polypeptide containing the extracellular domain of CTLA4 containing SEQ ID NO: 174; (b) A first cross-linking portion containing the sequence containing SEQ ID NO: 213 and the sequence containing SEQ ID NO: 214; and (c) A first antigen-binding site containing a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GASVSSSSYY (SEQ ID NO: 85), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of INYGGST (SEQ ID NO: 86), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARHRGIYHFDY (SEQ ID NO: 87), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of NIENKN (SEQ ID NO: 89), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of RDS (SEQ ID NO: 90), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91); and the second arm (a) A second polypeptide containing the extracellular domain of CTLA4 containing SEQ ID NO: 174; (b) A second cross-linking portion containing the sequence containing SEQ ID NO: 213 and the sequence containing SEQ ID NO: 214; and (c) A second antigen-binding site comprising a heavy-chain variable complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence of GASVSSSSYY (SEQ ID NO: 85), a heavy-chain variable complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence of INYGGST (SEQ ID NO: 86), a heavy-chain variable complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYHF DY (SEQ ID NO: 87), a light-chain variable complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light-chain variable complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light-chain variable complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91); comprising, wherein the first arm and the second arm dimerize with each other; (v) (a) A polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (b) A bridging portion comprising the sequence comprising SEQ ID NO: 213 and the sequence comprising SEQ ID NO: 216; and (c) An antigen-binding site comprising a heavy-chain variable complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence of GGSISSSSYY (SEQ ID NO: 101), a heavy-chain variable complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence of GSV DYSGNT (SEQ ID NO: 102), a heavy-chain variable complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a light-chain variable complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), a light-chain variable complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence of RDS (SEQ ID NO: 90), and a light-chain variable complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91) (vi) The first arm and the second arm, wherein the first arm (a) A first polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (b) A first bridging portion comprising the sequence comprising SEQ ID NO: 213 and the sequence comprising SEQ ID NO: 216; and (c) A first antigen-binding site comprising a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GGSSISSSSYY (SEQ ID NO: 101), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of GSVDSGSNT (SEQ ID NO: 102), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of NIENKN (SEQ ID NO: 89), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of RDS (SEQ ID NO: 90), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91); comprising, wherein the second arm (a) A second polypeptide comprising the extracellular domain of CTLA4 containing SEQ ID NO: 174; (b) A second cross-linking moiety comprising the sequence containing SEQ ID NO: 213 and the sequence containing SEQ ID NO: 216; and (c) A second antigen-binding site comprising a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GGSSISSSSYY (SEQ ID NO: 101), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of GSVDSGSNT (SEQ ID NO: 102), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of NIENKN (SEQ ID NO: 89), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of RDS (SEQ ID NO: 90), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91); comprising, wherein the first arm and the second arm dimerize with each other (vii) (a) A polypeptide comprising the extracellular domain of CTLA4 containing SEQ ID NO: 174; (b) A cross-linking moiety comprising the sequence containing SEQ ID NO: 213 and the sequence containing SEQ ID NO: 216; and (c) A heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGGSISTSSYY (SEQ ID NO: 77), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83); (viii) A first arm and a second arm, wherein the first arm (a) A first polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (b) A first cross-linking portion comprising the sequence comprising SEQ ID NO: 213 and the sequence comprising SEQ ID NO: 216; and (c) A first antigen-binding site comprising a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGGSISTSSYY (SEQ ID NO: 77), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83); and the second arm (a) A second polypeptide comprising the extracellular domain of CTLA4 comprising SEQ ID NO: 174; (b) A second cross-linking portion comprising the sequence comprising SEQ ID NO: 213 and the sequence comprising SEQ ID NO: 216; and (c) A second antigen-binding site comprising a heavy-chain variable complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence of GGGSISTSSYY (SEQ ID NO: 77), a heavy-chain variable complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy-chain variable complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light-chain variable complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence of DIENKN (SEQ ID NO: 81), a light-chain variable complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence of RDN (SEQ ID NO: 82), and a light-chain variable complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence of QVRDSNIVV (SEQ ID NO: 83); comprising, wherein the first arm and the second arm dimerize with each other; (ix) (a) A polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO: 174, SEQ ID NO: 119, SEQ ID NO: 215, SEQ ID NO: 233, SEQ ID NO: 234, and SEQ ID NO: 235; (b) A sequence selected from the group consisting of SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 212, SEQ ID NO: 213, and SEQ ID NO: 248, and a cross-linking portion comprising a sequence selected from the group consisting of SEQ ID NO: 178, SEQ ID NO: 237, SEQ ID NO: 214; SEQ ID NO: 216, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 239, and SEQ ID NO: 240; and (c) An antigen-binding site comprising VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, each of which, according to the IMGT numbering system, comprises an amino acid sequence corresponding to the sequence of each of the heavy-chain variable domain and the light-chain variable domains VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 listed in Table 3; (x) The first arm and the second arm, wherein the first arm (a) A first polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO: 174, SEQ ID NO: 119, SEQ ID NO: 215, SEQ ID NO: 233, SEQ ID NO: 234, and SEQ ID NO: 235; (b) a first cross-linking moiety comprising a sequence selected from the group consisting of SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 212, SEQ ID NO: 213, and SEQ ID NO: 248 and a sequence selected from the group consisting of SEQ ID NO: 178, SEQ ID NO: 237, SEQ ID NO: 214; SEQ ID NO: 216, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 239, and SEQ ID NO: 240; and (c) a first antigen-binding site comprising VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 according to the IMGT numbering system, each comprising an amino acid sequence corresponding to the sequences of the heavy-chain variable domain and the light-chain variable domains VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 respectively as listed in Table 3 and comprising, wherein the second arm (a) a second polypeptide comprising an extracellular domain of CTLA4 comprising a sequence selected from the group consisting of SEQ ID NO: 174, SEQ ID NO: 119, SEQ ID NO: 215, SEQ ID NO: 233, SEQ ID NO: 234, and SEQ ID NO: 235; (b) a second cross-linking moiety comprising a sequence selected from the group consisting of SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 212, SEQ ID NO: 213, and SEQ ID NO: 248 and a sequence selected from the group consisting of SEQ ID NO: 178, SEQ ID NO: 237, SEQ ID NO: 214; SEQ ID NO: 216, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 239, and SEQ ID NO: 240; and (c) a second antigen-binding site comprising VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 according to the IMGT numbering system, each comprising an amino acid sequence corresponding to the sequences of the heavy-chain variable domain and the light-chain variable domains VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 respectively as listed in Table 3, and wherein the first arm and the second arm dimerize with each other A protein comprising any of the above. **Claim 15**: The protein according to claim 14, wherein: (i) the antigen-binding site further comprises a partial hinge polypeptide comprising the sequence of SEQ ID NO: 336; or (ii) both the first antigen-binding site and the second antigen-binding site further comprise a partial hinge polypeptide comprising the sequence of SEQ ID NO: 336; A protein. **Claim 16**: The protein according to claim 3, wherein: (i) an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 48, SEQ ID NO: 68, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 46, SEQ ID NO: 66, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 50, SEQ ID NO: 70, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 38, SEQ ID NO: 73, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 37, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, and SEQ ID NO: 335; (ii) SEQ ID NO: 40 and SEQ ID NO: 39; SEQ ID NO: 62 and SEQ ID NO: 61; SEQ ID NO: 64 and SEQ ID NO: 63; SEQ ID NO: 182 and SEQ ID NO: 63; SEQ ID NO: 183 and SEQ ID NO: 63; SEQ ID NO: 184 and SEQ ID NO: 63; SEQ ID NO: 185 and SEQ ID NO: 63; SEQ ID NO: 186 and SEQ ID NO: 63; SEQ ID NO: 48 and SEQ ID NO: 47; SEQ ID NO: 68 and SEQ ID NO: 67; SEQ ID NO: 187 and SEQ ID NO: 67; SEQ ID NO: 188 and SEQ ID NO: 67; SEQ ID NO: 189 and SEQ ID NO: 67; SEQ ID NO: 190 and SEQ ID NO: 67; SEQ ID NO: 191 and SEQ ID NO: 67; SEQ ID NO: 46 and SEQ ID NO: 45; SEQ ID NO: 66 and SEQ ID NO: 65; SEQ ID NO: 192 and SEQ ID NO: 65; SEQ ID NO: 193 and SEQ ID NO: 65; SEQ ID NO: 194 and SEQ ID NO: 65; SEQ ID NO: 195 and SEQ ID NO: 65; SEQ ID NO: 196 and SEQ ID NO: 65; SEQ ID NO: 50 and SEQ ID NO: 49; SEQ ID NO: 70 and SEQ ID NO: 69; SEQ ID NO: 197 and SEQ ID NO: 69; SEQ ID NO: 198 and SEQ ID NO: 69; SEQ ID NO: 199 and SEQ ID NO: 69; SEQ ID NO: 200 and SEQ ID NO: 69; SEQ ID NO: 201 and SEQ ID NO: 69; SEQ ID NO: 38 and SEQ ID NO: 5; SEQ ID NO: 73 and SEQ ID NO: 5; SEQ ID NO: 42 and SEQ ID NO: 41; SEQ ID NO: 44 and SEQ ID NO: 43; SEQ ID NO: 52 and SEQ ID NO: 51; SEQ ID NO: 54 and SEQ ID NO: 53; Array number 56 and array number 55; Array number 58 and array number 57; Array number 60 and array number 59; Array number 37 and array number 35; Array number 256 and array number 63; Array number 257 and array number 63; Array number 258 and array number 63; Array number 259 and array number 63; Array number 260 and array number 63; Array number 261 and array number 63; Array number 262 and array number 63; Array number 263 and array number 63; Array number 264 and array number 63; Array number 265 and array number 63; Array number 266 and array number 63; Array number 267 and array number 63; Array number 268 and array number 63; Array number 269 and array number 63; Array number 270 and array number 63; Array number 271 and array number 63; Array number 272 and array number 63; Array number 273 and array number 63; Array number 274 and array number 63; Array number 275 and array number 63; Array number 276 and array number 63; Array number 277 and array number 63; Array number 278 and array number 63; Array number 279 and array number 63; Array number 280 and array number 63; Array number 281 and array number 63; Array number 282 and array number 63; Array number 283 and array number 63; Array number 284 and array number 63; Array number 285 and array number 63; Array number 286 and array number 63; Array number 287 and array number 63; Array number 288 and array number 63; Array number 289 and array number 63; Array number 290 and array number 63; Array number 291 and array number 63; Array number 292 and array number 63; Array number 293 and array number 63; Array number 294 and array number 67; Array number 295 and array number 67; Array number 296 and array number 67; Array number 297 and array number 67; Array number 298 and array number 67; Array number 299 and array number 67; Array number 300 and array number 67; Array number 301 and array number 67; Array number 302 and array number 67; Array number 303 and array number 67; Array number 304 and array number 67; Array number 305 and array number 67; Array number 306 and array number 67; Array number 307 and array number 67; Array number 308 and array number 67; Array number 309 and array number 67; Array number 310 and array number 67; Array number 311 and array number 67; Array number 312 and array number 67; Array number 313 and array number 67; Array number 314 and array number 67; Array number 315 and array number 67; Array number 316 and array number 67; Array number 317 and array number 67; Array number 318 and array number 67; Array number 319 and array number 67; Array number 320 and array number 67; Array number 321 and array number 67; Array number 322 and array number 67; Array number 323 and array number 67; Array number 324 and array number 67; Array number 325 and array number 67; Array number 326 and array number 67; Array number 327 and array number 67; Array number 328 and array number 67; Array number 329 and array number 67; Array number 330 and array number 67; Array number 331 and array number 67; Array number 332 and array number 253; Array number 333 and array number 255; Array number 334 and array number 253; or Array number 335 and array number 255; A polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of; A protein comprising any of.

17. The protein according to claim 3, wherein: (i) A polypeptide comprising the amino acid sequences of array number 62 and array number 61; (ii) Two polypeptides comprising the amino acid sequence of array number 62 and two polypeptides comprising the amino acid sequence of array number 61; (iii) A polypeptide comprising the amino acid sequences of array number 63 and array number 64; (iv) Two polypeptides comprising the amino acid sequence of array number 63 and two polypeptides comprising the amino acid sequence of array number 64; (v) A polypeptide comprising the amino acid sequences of array number 68 and array number 67; or (vi) Two polypeptides comprising the amino acid sequence of array number 68 and two polypeptides comprising the amino acid sequence of array number 67; A protein comprising.

18. The protein according to claim 1, wherein: (i) the protein has an IC of less than 12 nM when measured in an OX40L neutralization / OX40-HEK reporter assay ５０ and optionally the protein has an IC of 5 nM to 12 nM when measured in an OX40L neutralization / OX40-HEK reporter assay ５０ and further optionally the protein has an IC of 5 nM to 9 nM when measured in an OX40L neutralization / OX40-HEK reporter assay ５０ ; and / or (ii) the protein has an IC of 2 nM to 12 nM when measured in a primary T cell activation assay ５０ and optionally the protein has an IC of 2 nM to 9.5 nM when measured in a primary T cell activation assay ５０ ; and / or (iii) the protein has a K of less than 75 nM for OX40L binding when measured in a Biacore assay Ｄ and optionally the protein has a K of 20 nM to 75 nM for OX40L binding when measured in a Biacore assay Ｄ ; and / or (iv) the protein significantly inhibits the production of at least one of IL-2, IFNγ, IL-6, and TNFα by cells in an in vitro mixed lymphocyte reaction assay under the same conditions but in the absence of the protein, or in the presence of the human CTLA4 extracellular domain fused to the N-terminus of the human IgG1 Fc domain (CTLA4-Ig) provided individually, or in the presence of an anti-OX40L antibody, or in the presence of a combination of the two proteins provided separately; and / or (v) the protein significantly inhibits the proliferation of alloreactive CD4 ＋ T cells and CD8 ＋ T cells in an in vitro mixed lymphocyte reaction assay under the same conditions but in the absence of the protein, or in the presence of CTLA4-Ig and / or an anti-OX40L antibody, compared to alloreactive CD4 ＋ T cells and CD8 ＋ T cells; and / or (vi) when the protein selectively inhibits, in an in vitro mixed lymphocyte reaction assay, the proliferation of alloreactive CD4 ｒｅｇ T cells and CD8 ＋ T cells as compared to the proliferation of regulatory T cells (T ＋ ) under the same conditions but in the absence of the protein or in the presence of CTLA4-Ig and / or anti-OX40L antibody; and / or ＋ CD4 ＋ T cells and CD8 (vii) when the protein significantly enhances, in an in vitro mixed lymphocyte reaction assay, the inhibitory function as compared to T ｒｅｇ cells under the same conditions but in the absence of the protein or in the presence of CTLA4-Ig and / or anti-OX40L antibody; ｒｅｇ (viii) when the protein significantly inhibits, in an adoptive transfer assay in humanized mice, the proliferation of alloreactive CD4 T cells and CD8 ＋ T cells as compared to the proliferation of alloreactive CD4 ＋ T cells and CD8 ＋ T cells of mice treated under the same conditions but in the absence of the protein or in the presence of CTLA4-Ig and / or anti-OX40L antibody; and / or ＋ (ix) when the protein significantly inhibits, in an adoptive transfer assay in humanized mice, the serum IFNγ level as compared to mice treated under the same conditions but in the absence of the protein or in the presence of CTLA4-Ig and / or anti-OX40L antibody; and / or (x) when the protein is not significantly internalized into bone marrow-derived dendritic cells (MDDCs); and / or (xi) when the protein cross-reacts with cynomolgus monkey OX40L; and / or (xii) when the protein does not cross-react with mouse, rabbit, or rat OX40L; and / or (xiii) when the protein cross-reacts with cynomolgus monkey CD80 and CD86; (xiv) the protein. Protein. Claim 19 A preparation comprising the protein according to claim 1 and a pharmaceutically acceptable base. Claim 20 A nucleic acid encoding the protein according to claim 1. Claim 21 A cell comprising one or more nucleic acids encoding the protein according to claim 1. Claim 22 A method for treating an autoimmune disease in a patient, the method comprising administering the protein according to claim 1 to the patient. Claim 23 The selection of the autoimmune disease is rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, atopic dermatitis, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid antibody syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal neuropathy (AMAN), Barlow's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis with polyangiitis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital atrioventricular block, coxsackie myocarditis, CREST syndrome, Crohn's disease, cutaneous lupus, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus erythematosus, Dressler's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid gestationis (PG), hidradenitis suppurativa (HS) (acne inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile dermatomyositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucosa - Harbermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus,A method according to claim 22, which consists of a group consisting of neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Persistent-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, multiple endocrine neoplasia type I, type II, type III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren syndrome, sperm and testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura (TTP), thyroid eye disease (TED), Troxler-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

24. A method for treating a patient's graft-versus-host disease (GVHD), the method comprising administering to the patient the protein according to claim 1.

25. An antibody or a functional fragment thereof, wherein: (i) According to the IMGT unique numbering scheme, it includes a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GGSSISTSSYY (SEQ ID NO: 77), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of IYYSGST (SEQ ID NO: 78), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARHRGSYFFDI (SEQ ID NO: 79), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of DIENKN (SEQ ID NO: 81), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of RDN (SEQ ID NO: 82), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of QVRDSNIVVV (SEQ ID NO: 83); or (ii) According to the IMGT unique numbering scheme, it includes a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GVSIRSNGYY (SEQ ID NO: 93), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of MDYSGT (SEQ ID NO: 94), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARERSNNWYPIDY (SEQ ID NO: 95), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of SVRRFF (SEQ ID NO: 97), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of GKD (SEQ ID NO: 98), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of NSRDSSSGYLVL (SEQ ID NO: 99); or (iii) According to the IMGT unique numbering scheme, it includes a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GASVSSSSYY (SEQ ID NO: 85), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of INYGGSST (SEQ ID NO: 86), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARHRGIYHFDY (SEQ ID NO: 87), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of NIENKN (SEQ ID NO: 89), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of RDS (SEQ ID NO: 90), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91); (iv) According to the IMGT unique numbering scheme, it includes a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GGSSISSSSYY (SEQ ID NO: 101), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of IGSVDYSGNT (SEQ ID NO: 102), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of NIENKN (SEQ ID NO: 89), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of RDS (SEQ ID NO: 90), and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of QVWDSNTVV (SEQ ID NO: 91); or (v) According to the IMGT unique numbering scheme, a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GGSSISSSSYY (SEQ ID NO: 109), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of IGSVDYSGNT (SEQ ID NO: 110), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), a light-chain variable complementarity-determining region 1 (VLCDR1) containing the amino acid sequence of NIENKN (SEQ ID NO: 89), a light-chain variable complementarity-determining region 2 (VLCDR2) containing the amino acid sequence of RDS, and a light-chain variable complementarity-determining region 3 (VLCDR3) containing the amino acid sequence of QVWDSNTVI (SEQ ID NO: 115); or (vi) According to the IMGT unique numbering scheme, a heavy-chain variable complementarity-determining region 1 (VHCDR1) containing the amino acid sequence of GGSIDTSSQY (SEQ ID NO: 117), a heavy-chain variable complementarity-determining region 2 (VHCDR2) containing the amino acid sequence of IYFSGST (SEQ ID NO: 118), a heavy-chain variable complementarity-determining region 3 (VHCDR3) containing the amino acid sequence of ARHRGIYFFDY (SEQ ID NO: 103), The light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), the light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDT, and the light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSNTVI (SEQ ID NO: 123); or (vii) According to the IMGT unique numbering scheme, the heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSSISSSGSFY (SEQ ID NO: 125), the heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGST (SEQ ID NO: 126), the heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARERSSSWYPFDY (SEQ ID NO: 127), the light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SLRNYF (SEQ ID NO: 129), the light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of GKN, and the light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of NSRDSSGYHVVV (SEQ ID NO: 131); or (viii) According to the IMGT unique numbering scheme, the heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GFTFSNYA (SEQ ID NO: 133), the heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of ISGSGDST (SEQ ID NO: 134), the heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of AKDRTPVYGLDV (SEQ ID NO: 135), the light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of KIGRKN (SEQ ID NO: 137), the light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of GDS, and the light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSSTV (SEQ ID NO: 139); or (ix) According to the IMGT unique numbering scheme, a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GFTSFSYA (SEQ ID NO: 141), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of ISGSGGST (SEQ ID NO: 142), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of AKDLGFYSTWDTDEY (SEQ ID NO: 143), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of QGIRND (SEQ ID NO: 145), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of AAS, and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of LQHNTYPWT (SEQ ID NO: 147); or (x) According to the IMGT unique numbering scheme, a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GASISSSNHYWG (SEQ ID NO: 149), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of IYYSGNT (SEQ ID NO: 150), a heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARHRGSYFFDH (SEQ ID NO: 151), a light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIGDKN (SEQ ID NO: 153), a light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS, and a light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSSTVV (SEQ ID NO: 155); or (xi) According to the IMGT unique numbering scheme, a heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GDSVSSNSAV (SEQ ID NO: 157), a heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of TDYRSKWNN (SEQ ID NO: 158), A heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARGDSGMASFDY (SEQ ID NO: 159), A light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of KLGDKY (SEQ ID NO: 161), A light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of QDR, and A light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QTWDRRTAV (SEQ ID NO: 163); or (xii) According to the IMGT unique numbering scheme, A heavy chain variable complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of GGSFSDYY (SEQ ID NO: 165), A heavy chain variable complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of INHSGST (SEQ ID NO: 166), A heavy chain variable complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of ARKRGANFFDD (SEQ ID NO: 167), A light chain variable complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of NIENKN (SEQ ID NO: 89), A light chain variable complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of RDS, and A light chain variable complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of QVWDSSTVV (SEQ ID NO: 155); An antibody or a functional fragment thereof, optionally wherein the antibody is a human IgG1 antibody. **Claim 26** An antibody according to claim 25, wherein: (i) The heavy chain variable domain (VH) has an amino acid sequence that is 90% or more identical to SEQ ID NO: 76, and the light chain variable domain (VL) has an amino acid sequence that is 90% or more identical to SEQ ID NO: 80; or (ii) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 92, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 96; or (iii) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 84, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 88; or (iv) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 100, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 104; or (v) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 108, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 112; or (vi) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 116, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 120; or (vii) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 124, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 128; or (viii) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 132, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 136; or (ix) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 140, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 144; or (x) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 148, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 152; or (xi) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 156, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 160; or (xii) VH has an amino acid sequence that is 90% or more identical to SEQ ID NO: 164, and VL has an amino acid sequence that is 90% or more identical to SEQ ID NO: 168; and optionally the antibody is a human IgG1 antibody.

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