CD28 / OX40 dual-specific antibody

Abstract

Provided herein are multispecific binding proteins comprising: (a) a first antigen-binding domain (ABD) comprising an immunoglobulin single variable domain (ISVD) (e.g., VHH) that has binding specificity for CD28, and (b) a second ABD comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) that have binding specificity for OX40. Also provided are methods for treating autoimmune diseases with the multispecific binding proteins.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to European Patent Application No. 22315332.1, filed December 19, 2022, and U.S. Patent Application No. 63 / 543,351, filed October 10, 2023, the disclosures of each of which are incorporated by reference in their entirety.

[0002] Reference to an electronically submitted sequence listing The contents of the Sequence Listing submitted electronically in XML format (Name: SA9-342PC(747938)-2023-12-15.xml; Size: 93,595 bytes; and Creation Date: December 15, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to novel antibodies and antigen-binding fragments thereof that specifically bind to CD28 and OX40, and methods of using the same. [Background technology]

[0004] T cell-mediated autoimmunity has numerous adverse health effects and manifests as any one of a variety of autoimmune diseases. T cell-mediated autoimmunity is mediated, in part, by the activity of the costimulatory T cell receptors CD28 and OX40. Therapeutic methods to antagonize either CD28 or OX40 are currently being tested to treat autoimmune diseases.

[0005] Successful suppression of T cell-mediated autoimmunity will likely require simultaneous intervention at multiple levels, and therefore therapeutic agents that antagonize both CD28 and OX40 are needed. Summary of the Invention [Means for solving the problem]

[0006] The bispecific antigen-binding proteins of the present disclosure can antagonize both CD28 and OX40 on the surface of T cells. The anti-OX40 antigen-binding domain of the bispecific antigen-binding protein 1) inhibits the survival and activation of effector T cells generated from either naive or memory T cells; 2) reduces the Tfh response, which promotes autoantibody production; and 3) increases the selectivity of the bispecific for activated T cells at inflammatory sites. The anti-CD28 antigen-binding domain of the bispecific antigen-binding protein 1) suppresses the generation of effector T cells from naive T cells; and 2) preserves the suppressive function of Tregs by selectively blocking the CD28-B7 interaction while preserving the CTLA4-B7-mediated regulatory mechanism (unlike abatacept and belatacept). These complementary functions of the bispecific antibody can potently and selectively control the proliferation of effector / memory T cells in severe immune-related disorders.

[0007] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: Provided is a multispecific binding protein comprising: (a) a first antigen binding domain (ABD) comprising an immunoglobulin single variable domain (ISVD) (e.g., VHH) that has binding specificity for CD28; and (b) a second ABD comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) that have binding specificity for OX40, wherein when the multispecific binding protein binds to CD28 and OX40, the multispecific binding protein inhibits activated T cells.

[0008] In certain embodiments, the multispecific binding protein inhibits T cell proliferation.

[0009] In certain embodiments, the multispecific binding protein inhibits the expression of one or more pro-inflammatory cytokines.

[0010] In certain embodiments, the proinflammatory cytokines are interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 2 (IL-2), interleukin 5 (IL-5), interleukin 6 (IL-6), and interleukin 10 (IL-10).

[0011] In certain embodiments, the multispecific binding protein further comprises an immunoglobulin Fc domain or a variant thereof.

[0012] In certain embodiments, the Fc domain or variant thereof comprises a first Fc heavy chain and a second Fc heavy chain.

[0013] In certain embodiments, the first ABD binds to a first Fc heavy chain and the second ABD binds to a second Fc heavy chain.

[0014] In certain embodiments, the first ABD is linked to the N-terminus of the second ABD VH.

[0015] In certain embodiments, the first ABD is attached to the C-terminus of the second Fc heavy chain and the second ABD is attached to the N-terminus of the second Fc heavy chain.

[0016] In certain embodiments, the VH binds to a CH1 domain and the VL binds to a constant light chain (CL) domain.

[0017] In certain embodiments, the first ABD is attached to the C-terminus of the CL domain.

[0018] In certain embodiments, the first ABD binds to one or more amino acids E32, E46, V47, C48, Y51, G52, N53, S55, Q57, L58, Q59, V60, Y61, S62, K63, T64, N67, C68, and D69 of SEQ ID NO: 46. In certain embodiments, the first ABD binds to amino acids E32, E46, V47, C48, Y51, G52, N53, S55, Q57, L58, Q59, V60, Y61, S62, K63, T64, N67, C68, and D69 of SEQ ID NO: 46.

[0019] In certain embodiments, the second ABD binds to one or more amino acids V22, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, W55, R64, L67, C68, T69, and A70 of SEQ ID NO: 47. In certain embodiments, the second ABD binds to amino acids V22, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, W55, R64, L67, C68, T69, and A70 of SEQ ID NO: 47.

[0020] In certain embodiments, the second ABD binds to one or more amino acids R16, N19, V22, V32, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, T54, W55, C56, R64, L67, C68, T69, A70, and T74 of SEQ ID NO: 47. In certain embodiments, the second ABD binds to amino acids R16, N19, V22, V32, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, T54, W55, C56, R64, L67, C68, T69, A70, and T74 of SEQ ID NO: 47.

[0021] In certain embodiments, the multispecific binding protein comprises: (a) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of INTDGDFT (SEQ ID NO: 2); and HCDR3 sequence containing the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3) a first antigen-binding domain (ABD) having binding specificity to CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 4); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 5); and HCDR3 sequence containing the amino acid sequence of ARGEAYWYRWAFDY (SEQ ID NO: 6) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 7), an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 8); and LCDR3 sequence containing the amino acid sequence of QQYSDYSYT (SEQ ID NO: 9) and a second ABD having binding affinity to OX40 comprising an immunoglobulin light chain variable domain (VL) comprising:

[0022] In certain embodiments, the multispecific binding protein comprises: (a) HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14), and HCDR3 sequence containing the amino acid sequence of SARIRTSGGGGWSTY (SEQ ID NO: 15) a first antigen-binding domain (ABD) having binding specificity to CD28 comprising an immunoglobulin single variable domain (ISVD) comprising (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 4); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 5); and HCDR3 sequence containing the amino acid sequence of ARGEAYWYRWAFDY (SEQ ID NO: 6) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 7), an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 8); and LCDR3 sequence containing the amino acid sequence of QQYSDYSYT (SEQ ID NO: 9) immunoglobulin light chain variable domain (VL) containing and a second ABD having binding affinity to OX40 comprising:

[0023] In certain embodiments, the multispecific binding protein comprises: (a) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of IINTDGDFT (SEQ ID NO: 2), and HCDR3 sequence containing the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3) a first antigen-binding domain (ABD) having binding specificity to CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GYTFTSYG (SEQ ID NO: 17); an HCDR2 sequence comprising the amino acid sequence of ISAYTGNT (SEQ ID NO: 18), and HCDR3 sequence containing the amino acid sequence of ARDGYPIDY (SEQ ID NO: 19) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 20); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 21); and LCDR3 sequence containing the amino acid sequence of QQYTSYSDT (SEQ ID NO: 22) immunoglobulin light chain variable domain (VL) containing and a second ABD having binding affinity to OX40 comprising:

[0024] In certain embodiments, the multispecific binding protein comprises: (a) HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14), and HCDR3 sequence containing the amino acid sequence of SARIRTSGGGGWSTY (SEQ ID NO: 15) a first antigen-binding domain (ABD) having binding specificity to CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GYTFTSYG (SEQ ID NO: 17); an HCDR2 sequence comprising the amino acid sequence of ISAYTGNT (SEQ ID NO: 18), and HCDR3 sequence containing the amino acid sequence of ARDGYPIDY (SEQ ID NO: 19) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 20); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 21); and LCDR3 sequence containing the amino acid sequence of QQYTSYSDT (SEQ ID NO: 22) immunoglobulin light chain variable domain (VL) containing and a second ABD having binding affinity to OX40 comprising:

[0025] In certain embodiments, the multispecific binding protein comprises: (a) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of INTDGDFT (SEQ ID NO: 2); and HCDR3 sequence containing the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3) a first antigen-binding domain (ABD) having binding specificity to CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 25); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 26); and HCDR3 sequence containing the amino acid sequence of ARGGSGWYNSEFDY (SEQ ID NO: 27) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 28), an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 29); and LCDR3 sequence comprising the amino acid sequence of QQYNDYSYT (SEQ ID NO: 30) immunoglobulin light chain variable domain (VL) containing and a second ABD having binding affinity to OX40 comprising:

[0026] In certain embodiments, the multispecific binding protein comprises: (a) HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14), and HCDR3 sequence containing the amino acid sequence of SARIRTSGGGGWSTY (SEQ ID NO: 15) a first antigen-binding domain (ABD) having binding specificity to CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 25); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 26); and HCDR3 sequence containing the amino acid sequence of ARGGSGWYNSEFDY (SEQ ID NO: 27) An immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 28), an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 29); and LCDR3 sequence comprising the amino acid sequence of QQYNDYSYT (SEQ ID NO: 30) immunoglobulin light chain variable domain (VL) containing and a second ABD having binding affinity to OX40 comprising:

[0027] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10; The VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:11, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:12.

[0028] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 10, and includes the following amino acids compared to SEQ ID NO: 10: Y33, W47, T50, N52, D56, F57, T58, S59, K65, P102, Y103, S104, and R105.

[0029] In certain embodiments, the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 11, and includes amino acids H35, S52, Q54, G56, S57, T58, Y59, Y102, Y104, R105, and W106 compared to SEQ ID NO: 11, and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 12, and includes amino acids W32, D50, and S92 compared to SEQ ID NO: 12.

[0030] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 16; The VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:11, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:12.

[0031] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10; The VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:23, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:24.

[0032] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 16; The VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:23, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:24.

[0033] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10; The VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:31, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:32.

[0034] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 16; The VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:31, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:32.

[0035] In certain embodiments, the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 31, and includes amino acids A33, H35, A50, I51, S52, S53, N54, G55, G56, S57, T58, Y59, Y60, N74, S101, W103, Y104, N105, S106, and E107 compared to SEQ ID NO: 31, and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 32, and includes amino acids W32, Y91, N92, Y94, and Y96 compared to SEQ ID NO: 32.

[0036] In certain embodiments, the first Fc heavy chain comprises a Y349C substitution and the second Fc heavy chain comprises a S354C substitution.

[0037] In certain embodiments, the first Fc heavy chain comprises a Y349C, T366S, L368A, or Y407V substitution, and the second Fc heavy chain comprises a T366W substitution.

[0038] In certain embodiments, at least one Fc heavy chain comprises an H435R and a Y436F substitution.

[0039] In certain embodiments, at least one Fc heavy chain comprises an L234A and an L235A substitution.

[0040] In certain embodiments, the multispecific binding protein comprises (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33, (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 36, and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41.

[0041] In certain embodiments, the multispecific binding protein comprises (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33, (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37, and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42.

[0042] In certain embodiments, the multispecific binding protein comprises (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34, (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 38, and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41.

[0043] In certain embodiments, the multispecific binding protein comprises (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34, (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 39, and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42.

[0044] In certain embodiments, the multispecific binding protein comprises (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 35, (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40, and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41.

[0045] In certain embodiments, the multispecific binding protein comprises (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 35, (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40, and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42.

[0046] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of INTDGDFT (SEQ ID NO: 2); and HCDR3 sequence containing the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3) a first antigen-binding domain (ABD) having binding specificity for CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 4); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 5); and HCDR3 sequence containing the amino acid sequence of ARGEAYWYRWAFDY (SEQ ID NO: 6) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 7), an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 8); and LCDR3 sequence containing the amino acid sequence of QQYSDYSYT (SEQ ID NO: 9) immunoglobulin light chain variable domain (VL) containing a second ABD having binding affinity to OX40, comprising The present invention provides a binding protein comprising:

[0047] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:10.

[0048] In certain embodiments, the VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:11, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:12.

[0049] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14), and HCDR3 sequence containing the amino acid sequence of SARIRTSGGGGWSTY (SEQ ID NO: 15) a first antigen-binding domain (ABD) having binding specificity to CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 4); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 5); and HCDR3 sequence containing the amino acid sequence of ARGEAYWYRWAFDY (SEQ ID NO: 6) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 7), an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 8); and LCDR3 sequence containing the amino acid sequence of QQYSDYSYT (SEQ ID NO: 9) immunoglobulin light chain variable domain (VL) containing a second ABD having binding affinity to OX40, comprising The present invention provides a binding protein comprising:

[0050] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:16.

[0051] In certain embodiments, the VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:11, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:12.

[0052] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of INTDGDFT (SEQ ID NO: 2); and HCDR3 sequence containing the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3) a first antigen-binding domain (ABD) having binding specificity for CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GYTFTSYG (SEQ ID NO: 17); an HCDR2 sequence comprising the amino acid sequence of ISAYTGNT (SEQ ID NO: 18), and HCDR3 sequence containing the amino acid sequence of ARDGYPIDY (SEQ ID NO: 19) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 20); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 21); and LCDR3 sequence containing the amino acid sequence of QQYTSYSDT (SEQ ID NO: 22) immunoglobulin light chain variable domain (VL) containing The present invention provides a binding protein comprising a second ABD that has binding affinity to OX40 comprising:

[0053] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:10.

[0054] In certain embodiments, the VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:23, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:24.

[0055] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14), and HCDR3 sequence containing the amino acid sequence of SARIRTSGGGGWSTY (SEQ ID NO: 15) a first antigen-binding domain (ABD) having binding specificity for CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GYTFTSYG (SEQ ID NO: 17); an HCDR2 sequence comprising the amino acid sequence of ISAYTGNT (SEQ ID NO: 18), and HCDR3 sequence containing the amino acid sequence of ARDGYPIDY (SEQ ID NO: 19) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 20); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 21); and LCDR3 sequence containing the amino acid sequence of QQYTSYSDT (SEQ ID NO: 22) immunoglobulin light chain variable domain (VL) containing a second ABD having binding affinity to OX40, comprising The present invention provides a binding protein comprising:

[0056] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:16.

[0057] In certain embodiments, the VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:23, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:24.

[0058] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of INTDGDFT (SEQ ID NO: 2); and HCDR3 sequence containing the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3) a first antigen-binding domain (ABD) having binding specificity for CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 25); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 26); and HCDR3 sequence containing the amino acid sequence of ARGGSGWYNSEFDY (SEQ ID NO: 27) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 28), an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 29); and LCDR3 sequence comprising the amino acid sequence of QQYNDYSYT (SEQ ID NO: 30) immunoglobulin light chain variable domain (VL) containing a second ABD having binding affinity to OX40, comprising The present invention provides a binding protein comprising:

[0059] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:10.

[0060] In certain embodiments, the VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:31, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:32.

[0061] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14), and HCDR3 sequence containing the amino acid sequence of SARIRTSGGGGWSTY (SEQ ID NO: 15) a first antigen-binding domain (ABD) having binding specificity for CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 25); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 26); and An immunoglobulin heavy chain variable domain (VH) comprising an HCDR3 sequence comprising the amino acid sequence of ARGGSGWYNSEFDY (SEQ ID NO: 27); and (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 28), an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 29); and LCDR3 sequence comprising the amino acid sequence of QQYNDYSYT (SEQ ID NO: 30) immunoglobulin light chain variable domain (VL) containing The present invention provides a binding protein comprising a second ABD that has binding affinity to OX40 comprising:

[0062] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:16.

[0063] In certain embodiments, the VH comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:31, and the VL comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:32.

[0064] In certain embodiments, the binding protein further comprises an immunoglobulin Fc domain or a variant thereof.

[0065] In certain embodiments, the Fc domain or variant thereof comprises a first Fc heavy chain and a second Fc heavy chain.

[0066] In certain embodiments, the first Fc heavy chain comprises a Y349C substitution and the second Fc heavy chain comprises a S354C substitution.

[0067] In certain embodiments, the first Fc heavy chain comprises a Y349C, T366S, L368A, or Y407V substitution, and the second Fc heavy chain comprises a T366W substitution.

[0068] In certain embodiments, at least one Fc heavy chain comprises an H435R and a Y436F substitution.

[0069] In certain embodiments, at least one Fc heavy chain comprises an L234A and an L235A substitution.

[0070] In certain embodiments, the ISVD is V HH , humanized V HH Or Camelization V H or a suitable fragment thereof.

[0071] In one aspect, the present disclosure provides a binding protein comprising a first antigen binding domain (ABD) that has binding specificity for CD28 and a second ABD that has binding affinity for OX40, wherein the binding protein comprises (i) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 33, (ii) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 36, and (iii) a third polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 41.

[0072] In one aspect, the present disclosure provides a binding protein comprising a first antigen binding domain (ABD) that has binding specificity for CD28 and a second ABD that has binding affinity for OX40, wherein the binding protein comprises (i) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 33, (ii) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 37, and (iii) a third polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 42.

[0073] In one aspect, the present disclosure provides a binding protein comprising a first antigen binding domain (ABD) that has binding specificity for CD28 and a second ABD that has binding affinity for OX40, wherein the binding protein comprises (i) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 34, (ii) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 38, and (iii) a third polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 41.

[0074] In one aspect, the present disclosure provides a binding protein comprising a first antigen binding domain (ABD) that has binding specificity for CD28 and a second ABD that has binding affinity for OX40, wherein the binding protein comprises: (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34, (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 39, and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42.

[0075] In one aspect, the present disclosure provides a binding protein comprising a first antigen binding domain (ABD) that has binding specificity for CD28 and a second ABD that has binding affinity for OX40, wherein the binding protein comprises (i) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 35, (ii) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 40, and (iii) a third polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 41.

[0076] In one aspect, the present disclosure provides a binding protein comprising a first antigen binding domain (ABD) that has binding specificity for CD28 and a second ABD that has binding affinity for OX40, wherein the binding protein comprises (i) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 35, (ii) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 40, and (iii) a third polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 42.

[0077] In one aspect, the present disclosure provides a pharmaceutical composition comprising the binding protein described above and a pharmaceutically acceptable carrier.

[0078] In one aspect, the disclosure provides an isolated nucleic acid molecule encoding the binding protein described above.

[0079] In one aspect, the present disclosure provides an expression vector comprising the above-described nucleic acid molecule.

[0080] In one aspect, the present disclosure provides a host cell comprising the above-described expression vector.

[0081] In one aspect, the present disclosure provides a method of treating an autoimmune disease or disorder in a subject, comprising administering to a subject in need thereof a binding protein as described above. In certain embodiments, the autoimmune disease or disorder comprises connective tissue disease-interstitial lung disease (CTD-ILD). In certain embodiments, the autoimmune disease or disorder comprises graft-versus-host disease (GvHD).

[0082] In one aspect, the present disclosure provides the binding protein described above for use as a medicament.

[0083] In one aspect, the present disclosure provides the binding protein described above for use in a method for treating an autoimmune disease or disorder. In certain embodiments, the autoimmune disease or disorder comprises connective tissue disease-interstitial lung disease (CTD-ILD). In certain embodiments, the autoimmune disease or disorder comprises graft-versus-host disease (GvHD).

[0084] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: a) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of INTDGDFT (SEQ ID NO: 2); and an HCDR3 sequence comprising the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3); or b) HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14), and HCDR3 sequence containing the amino acid sequence of SARIRTSGGGGWSTY (SEQ ID NO: 15) The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD28, an immunoglobulin single variable domain (ISVD) comprising:

[0085] In certain embodiments, the ISVD comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:16.

[0086] In certain embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0087] In certain embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody.

[0088] In certain embodiments, the bispecific antibody comprises an antigen-binding domain that comprises binding affinity for OX40.

[0089] In certain embodiments, the antibody or antigen-binding fragment thereof is operably linked to an Fc region. In certain embodiments, the Fc region is a human IgG1 Fc region.

[0090] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an antagonist antibody or antigen-binding fragment thereof.

[0091] In one aspect, the present disclosure provides a pharmaceutical composition comprising the above-described antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0092] In one aspect, the disclosure provides an isolated nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof.

[0093] In one aspect, the present disclosure provides an expression vector comprising the above-described nucleic acid molecule.

[0094] In one aspect, the present disclosure provides a host cell comprising the above-described expression vector.

[0095] In one aspect, the disclosure provides a method of treating an autoimmune disease in a subject, the method comprising administering to a subject in need of treatment for the autoimmune disease an antibody or antigen-binding fragment thereof described above.

[0096] In one aspect, the present disclosure provides the above-described antibody or antigen-binding fragment thereof for use as a medicament.

[0097] In one aspect, the present disclosure provides the above-described antibody, or antigen-binding fragment thereof, for use in a method for treating or preventing an autoimmune disease.

[0098] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: a) (a1) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 4); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 5); and HCDR3 sequence containing the amino acid sequence of ARGEAYWYRWAFDY (SEQ ID NO: 6) an immunoglobulin heavy chain variable domain (VH) comprising: (a2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 7), an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 8); and LCDR3 sequence containing the amino acid sequence of QQYSDYSYT (SEQ ID NO: 9) an immunoglobulin light chain variable domain (VL) comprising: b) (b1) an HCDR1 sequence comprising the amino acid sequence of GYTFTSYG (SEQ ID NO: 17); an HCDR2 sequence comprising the amino acid sequence of ISAYTGNT (SEQ ID NO: 18), and HCDR3 sequence containing the amino acid sequence of ARDGYPIDY (SEQ ID NO: 19) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 20); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 21); and LCDR3 sequence containing the amino acid sequence of SYDMS (SEQ ID NO: 22) an immunoglobulin light chain variable domain (VL) comprising: c) (c1) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 25); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 26); and HCDR3 sequence containing the amino acid sequence of ARGGSGWYNSEFDY (SEQ ID NO: 27) an immunoglobulin heavy chain variable domain (VH) comprising: (c2) LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 28), an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 29); and LCDR3 sequence comprising the amino acid sequence of QQYNDYSYT (SEQ ID NO: 30) immunoglobulin light chain variable domain (VL) containing The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to OX40, comprising an immunoglobulin single variable domain (ISVD) comprising:

[0099] In certain embodiments, a) the VH comprises an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 11, and the VL comprises an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 12; b) the VH comprises an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 23, and the VL comprises an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 24; or c) the VH comprises an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 31, and the VL comprises an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 32.

[0100] In certain embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0101] In certain embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody.

[0102] In certain embodiments, the bispecific antibody comprises an antigen-binding domain that comprises binding affinity for CD28.

[0103] In certain embodiments, the antibody or antigen-binding fragment thereof is operably linked to an Fc region.

[0104] In certain embodiments, the Fc region is a human IgG1 Fc region.

[0105] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an antagonist antibody or antigen-binding fragment thereof.

[0106] In one aspect, the present disclosure provides a pharmaceutical composition comprising the above-described antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0107] In one aspect, the disclosure provides an isolated nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof.

[0108] In one aspect, the present disclosure provides an expression vector comprising the above-described nucleic acid molecule.

[0109] In one aspect, the present disclosure provides a host cell comprising the above-described expression vector.

[0110] In one aspect, the disclosure provides a method of treating an autoimmune disease in a subject, the method comprising administering to a subject in need of treatment for the autoimmune disease an antibody or antigen-binding fragment thereof described above.

[0111] In one aspect, the present disclosure provides the above-described antibody or antigen-binding fragment thereof for use as a medicament.

[0112] In one aspect, the present disclosure provides the above-described antibody, or antigen-binding fragment thereof, for use in a method for treating or preventing an autoimmune disease.

[0113] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are administered intravenously to a subject in need of treatment or prevention.

[0114] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are administered intravenously to a subject at a dose of about 0.3 mg to about 30 mg.

[0115] In certain embodiments, an antibody or antigen-binding fragment thereof described herein is administered intravenously to a subject at a dose of about 0.3 mg. In certain embodiments, an antibody or antigen-binding fragment thereof described herein is administered intravenously to a subject at a dose of about 1 mg. In certain embodiments, an antibody or antigen-binding fragment thereof described herein is administered intravenously to a subject at a dose of about 3 mg. In certain embodiments, an antibody or antigen-binding fragment thereof described herein is administered intravenously to a subject at a dose of about 10 mg. In certain embodiments, an antibody or antigen-binding fragment thereof described herein is administered intravenously to a subject at a dose of about 30 mg.

[0116] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are administered subcutaneously to a subject in need of treatment or prevention.

[0117] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are administered subcutaneously to a subject at a dose of about 30 mg to about 500 mg.

[0118] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are subcutaneously administered to a subject at a dose of about 30 mg. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are subcutaneously administered to a subject at a dose of about 60 mg. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are subcutaneously administered to a subject at a dose of about 75 mg. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are subcutaneously administered to a subject at a dose of about 120 mg. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are subcutaneously administered to a subject at a dose of about 150 mg. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are subcutaneously administered to a subject at a dose of about 240 mg. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are subcutaneously administered to a subject at a dose of about 300 mg. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are subcutaneously administered to a subject at a dose of about 500 mg.

[0119] In certain embodiments, an antibody or antigen-binding fragment thereof described herein is administered subcutaneously to a subject once every two weeks (Q2W) at a dose of about 30 mg. In certain embodiments, an antibody or antigen-binding fragment thereof described herein is administered subcutaneously to a subject once every two weeks (Q2W) at a dose of about 60 mg. In certain embodiments, an antibody or antigen-binding fragment thereof described herein is administered subcutaneously to a subject once every two weeks (Q2W) at a dose of about 120 mg. In certain embodiments, an antibody or antigen-binding fragment thereof described herein is administered subcutaneously to a subject once every two weeks (Q2W) at a dose of about 240 mg.

[0120] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0121] [Figure 1A] FIG. 1 is a schematic diagram of several anti-CD28 / OX40 bispecific antibody formats of the present disclosure. [Figure 1B] FIG. 1 is a schematic diagram of several anti-CD28 / OX40 bispecific antibody formats of the present disclosure. [Figure 2A] Figure 1 shows the epitopes of OX40 and CD28 binders and a comparison with their ligand binding sites. Top: Epitope of an exemplary OX40 mAb on OX40 as determined by X-ray crystallography (left); epitopes of the L2 and L5 OX40 arm mAb on OX40 as determined by cryo-EM (center); and the OX40L binding site on OX40 as mapped using PDB 2HEV (right). Bottom: Epitopes of the L4, L6, and L5 CD28 arms on CD28 as determined by X-ray crystallography (left) and modeled on the CD80 binding site on CD28 (based on the CD80-CTLA4 structure; protein database (PDB 1I8L)) (right). The CD28 dimer was modeled on PDB 1YJD. [Figure 2B] 2 shows the crystal structure of L4, L6, and L5 CD28 arm VHH bound to CD28 at 2.9 Å resolution. The positions of the CDRs on the CD28 VHH are indicated. [Figure 3] FIG. 1 shows a comparison of the activity of a bispecific antibody prototype ("BsAb CD28-OX40 prototype") compared to multiple monospecific antibodies and multiple monospecific antibody combinations in a mixed lymphocyte reaction (MLR) assay. [Figure 4A] Figure 1 shows the results of an SPR binding kinetics study of exemplary bispecific antibodies designated L1 through L6, where "L" represents the lead candidate and L1, L2, L3, L4, L5, and L6 represent six lead candidate exemplary bispecific antibodies. [Figure 4A-1] Same as above. [Figure 4B] Figure 1 shows the results of an SPR binding kinetics study of exemplary bispecific antibodies designated L1 through L6, where "L" represents the lead candidate and L1, L2, L3, L4, L5, and L6 represent six lead candidate exemplary bispecific antibodies. [Figure 5A] 1 shows binding of exemplary bispecific antibodies, designated L1-L6, on the HEK293 cell line. [Figure 5B] Binding in various recombinant cells is shown. [Figure 5C] Binding to various primary T cells is shown. [Figure 6A] Figure 1 shows high potency of bispecific compounds with multiple pro-inflammatory cytokines in the MLR assay. [Figure 6B] It exhibits high selectivity for effector T cells. [Figure 6C] It exhibits high selectivity for memory T cells. [Figure 7A] The results of the inhibitory activity of bispecific antibodies in a Derp1 antigen recall assay are shown. In the figure, each bar within a series of data (e.g., #L2, #L1, etc.) represents a serial 1 / 10 dilution ranging from 60 nM to 0.006 nM. [Figure 7B] The results of the inhibitory activity of bispecific antibodies in a Derp1 antigen recall assay are shown. In the figure, each bar within a series of data (e.g., #L2, #L1, etc.) represents a serial 1 / 10 dilution ranging from 60 nM to 0.006 nM. [Figure 7C] 1 shows the results of a CMVpp65 antigen recall assay. [Figure 7D] 1 shows the results of a CMVpp65 antigen recall assay. [Figure 8] Figure 1 shows the ability of bispecific prototype antibodies to enhance the suppressive function of Tregs. [Figure 9A] Figure 9B shows the activity of bispecific prototype antibodies in two independent humanized GvHD models. In Figure 9B, "aOX40" and "aCD28" correspond to monospecific controls. [Figure 9A-1] Same as above. [Figure 9B] Figure 9B shows the activity of bispecific prototype antibodies in two independent humanized GvHD models. In Figure 9B, "aOX40" and "aCD28" correspond to monospecific controls. [Figure 9C] Figure 1 shows survival, GvHD scores, and weight loss, respectively, of mice therapeutically treated with bispecific antibody L4 over a two-week period. [Figure 9D] Figure 1 shows survival, GvHD scores, and weight loss, respectively, of mice therapeutically treated with bispecific antibody L4 over a two-week period. [Figure 9E] Figure 1 shows survival, GvHD scores, and weight loss, respectively, of mice therapeutically treated with bispecific antibody L4 over a two-week period. [Figure 9F] Figure 1 shows survival, GvHD scores, and weight loss, respectively, of mice therapeutically treated with bispecific antibody L4 over a one-week period. [Figure 9G] Figure 1 shows survival, GvHD scores, and weight loss, respectively, of mice therapeutically treated with bispecific antibody L4 over a one-week period. [Figure 9H] Figure 1 shows survival, GvHD scores, and weight loss, respectively, of mice therapeutically treated with bispecific antibody L4 over a one-week period. [Figure 10] PK activity of exemplary bispecific antibodies. [Figure 11A] Figure 1 shows the effect of bispecific antibody treatment on paw swelling in two independent DTH models. [Figure 11B] Figure 1 shows the effect of bispecific antibody treatment on paw swelling in two independent DTH models. [Figure 12A] Figure 1 shows the activity of bispecific antibodies in two independent TDAR models. [Figure 12B] Figure 1 shows the activity of bispecific antibodies in two independent TDAR models. [Figure 13A]Figure 13A (IFN-γ), Figure 13B (IL-10), Figure 13C (IL-2), Figure 13D (IL-6), Figure 13E (TNF-α), and Figure 13F (IL-5) show the lack of agonistic activity of the bispecific antibodies as measured in a MIMIC-based CRA assay. Figure 13G (IFN-γ), Figure 13H (IL-2), Figure 13I (IL-4), and Figure 13J (MIP1-b) show the lack of agonistic activity of the bispecific antibodies as measured in a pre-activated anti-CD3 PBMC assay. Figure 13K (TNF-α), Figure 13L (IL-2), Figure 13M (IL-6), and Figure 13N (IFN-γ) show the lack of agonistic activity of the bispecific antibodies as measured in a high-density PBMC assay. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above. [Figure 13F] Same as above. [Figure 13G] Same as above. [Figure 13H] Same as above. [Figure 13I] Same as above. [Figure 13J] Same as above. [Figure 13K] Same as above. [Figure 13L] Same as above. [Figure 13M] Same as above. [Figure 13N] Same as above. [Figure 14A] Figure 14 shows the lack of detectable ADCC activity induced by the bispecific antibody in Jurkat (Figure 14A) and HEK293 (Figure 14B) assay systems. [Figure 14B] Figure 14 shows the lack of detectable ADCC activity induced by the bispecific antibody in Jurkat (Figure 14A) and HEK293 (Figure 14B) assay systems. [Figure 14C] 1 shows the lack of detectable CDC activity induced in the CD4 and CD8 assay systems. [Figure 14D]1 shows the lack of detectable CDC activity induced in the CD4 and CD8 assay systems. DETAILED DESCRIPTION OF THE INVENTION

[0122] Before the present disclosure is described, it is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. The scope of the present disclosure will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure, exemplary methods and materials are described here. All publications mentioned herein are incorporated by reference as if set forth in their entirety.

[0124] The term "about" or "approximately" means within about 20%, such as within about 10%, within about 5%, or within about 1% or less of a given value or range.

[0125] As used herein, the term "antibody" or "antigen-binding protein" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with an antigen or epitope, and includes both polyclonal and monoclonal antibodies, as well as functional antibody fragments thereof. The term "antibody" or "antigen-binding protein" includes immunoglobulin single variable domain (ISVD or ISV) antibodies (e.g., sdAb, sdFv, Nanobody®, VHH). The term "antibody" also includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, meditope-enabled antibodies, heteroconjugate antibodies (e.g., multispecific antibodies, bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv), and the like.

[0126] As used herein, the term "functional antibody fragment" refers to an antibody fragment that has at least 80%, at least 85%, at least 90%, or at least 95% of the affinity as the antibody of interest from which the fragment is derived.

[0127] The term "multispecific antibody" as used herein refers to bispecific, trispecific, or multispecific antibodies, and antigen-binding fragments thereof. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for epitopes of two or more target polypeptides. A multispecific antibody may be a single multifunctional polypeptide, or it may be a multimeric complex of two or more polypeptides that are covalently or noncovalently bound to each other. The term "multispecific antibody" includes antibodies of the present disclosure that may be linked to or coexpressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be operatively linked (e.g., by chemical bond, genetic fusion, noncovalent bonding, or otherwise) to one or more other molecular entities, such as proteins or fragments thereof, to generate a bispecific or multispecific antibody with a second binding specificity.

[0128] As used herein, "monovalent" with respect to an antibody refers to an antibody having a single antigen recognition site specific for a target antigen. Examples of monovalent antibodies include monovalent immunoglobulin single variable domain antibodies (e.g., VHHs) or monovalent antibody fragments. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single-chain Fv fragments (scFvs). Furthermore, multispecific antibodies can have multiple antigen-binding sites, each of which recognizes a different target antigen. Thus, each antigen-binding site is monovalent with respect to the target antigen.

[0129] As used herein, "multivalent" with respect to an antibody refers to an antibody having multiple (two or more) antigen recognition sites specific for a target antigen.

[0130] As used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (HCDR1, HCDR2, HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, LCDR3). The term "framework region" or "FR" is known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, each heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).

[0131] The precise amino acid sequence boundaries of a given CDR or FR can be determined using the methods described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol, 2003 January;27(1):55-77 ("IMGT" numbering scheme), and Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 June.8;309(3):657-70, (AHo numbering scheme).

[0132] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions accommodated by an insertion letter, e.g., "30a," and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0133] The "CDRs" or "complementarity-determining regions" of a given antibody or a region thereof, such as its variable region, or individual designated CDRs (e.g., "HCDR1," "HCDR2," "HCDR3"), should be understood to encompass complementarity-determining regions defined (or specific) by any of the known schemes. Similarly, the "FRs" or "framework regions" of a given antibody or a region thereof, such as its variable region, or individual designated FRs (e.g., "FR-H1," "FR-H2"), should be understood to encompass framework regions defined (or specific) by any of the known schemes. In some instances, CDRs defined by a scheme for identifying specific CDRs or FRs, such as the IMGT, Kabat, Chothia, AbM, or contact method, are designated. In other cases, the specific amino acid sequences of the CDRs or FRs are provided. Unless otherwise specified, all specific CDR amino acid sequences described in this disclosure are IMGT CDRs. However, alternative CDRs defined by other schemes, such as those determined by the abysis Key Annotation (Website: abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), are also encompassed by the present disclosure. Exemplary CDR sequences for the anti-CD28 and anti-OX40 antibodies described herein are listed in Tables 1 and 2 below.

[0134] [Table 1]

[0135] [Table 2]

[0136] [Table 3]

[0137] [Table 4]

[0138] [Table 5]

[0139] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs are replaced with residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, llama, or non-human primate antibody, that has the desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced with corresponding framework region residues from the donor antibody. Humanized antibodies can also contain residues that are not found in either the recipient or donor antibody. Such modifications can be made to further refine antibody function. A humanized sequence can be identified by its primary sequence and does not necessarily indicate the process by which the antibody was generated.

[0140] As used herein, the terms "specifically binds," "specifically binding," "binding specificity," or "specifically recognized" refer to an antigen-binding protein or antigen-binding fragment thereof that exhibits significant affinity for an antigen (e.g., CD28 antigen or OX40 antigen) and does not exhibit significant cross-reactivity with targets that are neither CD28 nor OX40 proteins. As used herein, the term "affinity" refers to the strength of the interaction between the antigen-binding site of an antigen-binding protein or antigen-binding fragment thereof and the epitope to which it binds. In certain exemplary embodiments, affinity is measured by surface plasmon resonance (SPR), for example, in a Biacore instrument. As will be readily understood by one of skill in the art, antigen-binding protein affinity may be reported as a dissociation constant (KD) in molar concentration (M).

[0141] Specific binding can be determined according to any art-recognized means for determining such binding. In some embodiments, specific binding is determined by competitive binding assays (e.g., ELISA) or SPR (Biacore) assays. In certain embodiments, the assays are performed at about 20°C, 25°C, 30°C, or 37°C.

[0142] The term "agonist," as used herein with respect to an antibody, means that the antibody, upon binding to a target protein expressed on the surface of a cell, stimulates or activates signal transduction through the target protein.

[0143] The term "antagonist," as used herein with respect to an antibody, means that the antibody inhibits signal transduction through a target protein upon binding to the target protein expressed on the surface of a cell.

[0144] As used herein, "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance (e.g., an antibody provided herein) to a patient, such as, but not limited to, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease or its symptoms are being managed or treated, administration of the substance typically occurs after the onset of the disease or its symptoms. When a disease or its symptoms are being prevented, administration of the substance typically occurs before the onset of the disease or its symptoms and may be continued chronically to postpone or reduce the appearance or magnitude of disease-related symptoms. The route of administration may be intravenous administration. The route of administration may be subcutaneous administration.

[0145] "Effective amount" means an amount of an active pharmaceutical agent (e.g., an isolated binding polypeptide of the present disclosure) sufficient to achieve a desired physiological outcome in an individual in need thereof. The effective amount may vary from individual to individual depending on the health and condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, an assessment of the individual's medical condition, and other relevant factors.

[0146] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, as used herein, the term "subject" refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, feral animals, farm animals, sport animals, and pets. A subject may be a human.

[0147] As used herein, the term "therapy" refers to any protocol, method, and / or agent that can be used in the prevention, management, treatment, and / or amelioration of a disease or its associated symptoms. In some embodiments, the terms "therapy" and "therapeutic method" refer to biological therapy, supportive therapy, and / or other therapies known to those of skill in the art, such as healthcare professionals, that are useful in the prevention, management, treatment, and / or amelioration of a disease or its associated symptoms.

[0148] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or symptoms associated therewith resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, e.g., an isolated binding polypeptide provided herein). As used herein, the term "treating" can also refer to altering the course of the disease in the subject being treated. The therapeutic effect of a treatment includes, but is not limited to, prevention of the onset or recurrence of the disease, alleviation of symptoms, reduction of the direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0149] As used herein, "autoimmune disease" refers to disease states and conditions in which an individual's immune response is directed against the individual's own components, resulting in an undesirable and often debilitating condition. As used herein, "autoimmune disease" is intended to further include autoimmune conditions, syndromes, and the like.

[0150] Immunoglobulin Single Variable Domains (ISVDs) The term "immunoglobulin single variable domain" (ISV or ISVD), used interchangeably with "single variable domain," defines an immunoglobulin molecule in which the antigen-binding site is present on and formed by a single immunoglobulin domain. This term distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, dis-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. In this case, V H and V L Both complementarity-determining regions (CDRs) of the nucleotides contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0151] In view of the above definition, the antigen-binding domain of a conventional four-chain antibody or an Fab fragment, an F(ab')2 fragment, an Fv fragment such as a disulfide-linked Fv, or an scFv fragment, or a diabody derived from such a conventional four-chain antibody (all known in the art), is not usually considered to be an immunoglobulin single variable domain, because in these cases, binding to each epitope of an antigen is usually not by one (single) immunoglobulin domain, but by a pair of (related) immunoglobulin domains, such as a light and heavy chain variable domain, i.e., the V of immunoglobulin domains which jointly bind to the respective epitope of the antigen. H -V L This is because they arise in pairs.

[0152] In contrast, an immunoglobulin single variable domain can specifically bind to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain consists of a single V H , a single V HH , or a single VL Formed by domains.

[0153] Therefore, a single variable domain may be used with any light chain variable domain sequence (e.g., V), as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit). L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., V H -sequence or V HH sequence) or a suitable fragment thereof.

[0154] Immunoglobulin single variable domains (ISVs) can be, for example, heavy chain ISVs, e.g., camelized V H or humanized V HH Contains V H , V HH In one embodiment, this may be a camelized V H or humanized V HH Contains V HH The heavy chain ISV can be derived from a traditional four-chain antibody or from a heavy chain antibody.

[0155] For example, immunoglobulin single variable domains can be single domain antibodies (or amino acid sequences suitable for use as single domain antibodies), "dAbs" or dAbs (or amino acid sequences suitable for use as dAbs), Nanobody® ISVs (as defined herein, V HH (including but not limited to), other single variable domains, or any suitable fragment of any one of these.

[0156] In particular, the immunoglobulin single variable domains are Nanobody® ISVs (e.g., humanized V HH Or Camelization V H Contains V HH ) or a suitable fragment thereof. [Note: Nanobody® and Nanobodies® are registered trademarks of Ablynx NV.]

[0157] "V HH Domain" is V HH , V HH Antigen fragments, and V HH Also known as antibodies, they were originally described as the antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains"; Hamers-Casterman et al. Nature 363:446-448, 1993). HH The term "variable domain" refers to these variable domains, as compared to the heavy chain variable domains (herein referred to as "V" domains) present in conventional four-chain antibodies. H domain) present in conventional four-chain antibodies and the light chain variable domain (referred to herein as "V L The domain name was chosen to distinguish it from the domains referred to as "domains." HH For further discussion, see the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0158] The generation of immunoglobulin sequences such as VHHs has been widely described in various published literature, including WO 94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001). In these methods, camelids are immunized with a target antigen to induce an immune response against the target antigen. The repertoire of VHHs obtained from this immunization is further screened for VHHs that bind to the target antigen.

[0159] In these instances, antibody generation requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0160] Immunoglobulin sequences of different origins can be used herein, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. Also, fully human, humanized, or chimeric sequences can be used in the methods described herein. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs as described by Ward et al. (e.g., WO 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996), can be used in the methods described herein. Furthermore, ISVs can be fused to form multivalent and / or multispecific constructs (one or more V HH For multivalent and multispecific polypeptides containing domains and their preparation, see Conrath et al., J. Biol. Chem., Vol. 276, 10, 7346-7350, 2001, as well as, for example, WO 96 / 34103 and WO 99 / 23221).

[0161] "Humanized V HH " is a naturally occurring V HH corresponding to the amino acid sequence of the naturally occurring V HH One or more amino acid residues in the amino acid sequence (and particularly in the framework sequences) of the V H The term "humanized" includes amino acid sequences that have been "humanized" by substituting one or more amino acid residues (e.g., as shown above) present at the corresponding positions in the domain. This can be carried out in a manner known per se, for example, based on the prior art (e.g., WO 2008 / 020079), as will be clear to those skilled in the art. Furthermore, such humanized V HHIt should be noted that can be obtained in any suitable manner known per se and is therefore not strictly limited to polypeptides obtained using naturally occurring VHH domain-containing polypeptides as starting material.

[0162] "Camelization V H " is a naturally occurring V H corresponding to the amino acid sequence of the naturally occurring V domain from a conventional four-chain antibody. H One or more amino acid residues in the amino acid sequence of the domain are replaced by the V HH The term "camelized" includes amino acid sequences that have been "camelized" by substituting one or more amino acid residues present at the corresponding positions in the V domain. This can be carried out in a manner known per se, as will be clear to those skilled in the art, for example as described in the prior art (e.g., Davies and Riechman (1994 and 1996), supra). Such "camelized" substitutions are, as defined herein, H -V L The amino acids are inserted at positions that form and / or are present at interfaces and / or so-called camelid hallmark residues (see, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996), supra). In one embodiment, camelized V H V, which is used as a starting material or starting point for generating or designing H The sequence is V from mammals H Sequences, e.g., V of human origin H Array, e.g. V H 3 sequence. However, such camelized V H can be obtained in any suitable manner known per se, and therefore can be obtained without using naturally occurring V as starting material. H It should be noted that the polypeptide obtained using the polypeptide containing the domain is not strictly limited.

[0163] The structure of an immunoglobulin single variable domain sequence can be considered to consist of four framework regions ("FRs"), which are referred to in the art and herein as "framework region 1" ("FR1"); "framework region 2" ("FR2"); "framework region 3" ("FR3"); and "framework region 4" ("FR4"), respectively, interrupted by three complementarity-determining regions ("CDRs"), which are referred to in the art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively.

[0164] In such immunoglobulin sequences, the framework regions may be any suitable framework sequence, and examples of suitable framework sequences will be clear to the skilled person based on, for example, standard handbooks and the further disclosure and prior art referred to herein.

[0165] The framework sequences are immunoglobulin framework sequences or (suitable combinations of) framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization). For example, the framework sequences may be those of a light chain variable domain (e.g., V L sequence) and / or heavy chain variable domain (e.g., V H Array or V HH In a particular embodiment, the framework sequences may be derived from V HH -framework sequences derived from conventional V sequences (in which the framework sequences may optionally be partially or fully humanized) or camelized (as defined herein) H It can be either an array.

[0166] In particular, the framework sequences present in the ISV sequences described herein may include one or more Hallmark residues (defined herein), and the ISV sequences may be, for example, humanized V HHor Camelization V H Contains V HH Non-limiting examples of (suitable combinations of) such framework sequences will be apparent from the disclosure herein.

[0167] V H Domain and V HH The total number of amino acid residues in a domain will usually be in the range of 110 to 120, often 112 to 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0168] It should be noted, however, that the ISVs described herein are not limited with respect to the origin of the ISV sequence (or the nucleotide sequence used to express it), nor are they limited with respect to the manner in which the ISV sequence or nucleotide sequence is generated or obtained. Thus, the ISV sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In one specific, but non-limiting embodiment, the ISV sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including, but not limited to, a "humanized" (as defined herein) immunoglobulin sequence (e.g., a partially or fully humanized mouse or rabbit immunoglobulin sequence, and particularly a partially or fully humanized VHV sequence). HH sequences), "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized V H sequences), as well as ISVs obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, joining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar immunoglobulin sequence engineering techniques well known to those skilled in the art, or any suitable combination of any of the foregoing.

[0169] Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and may be, for example, a sequence isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (and in particular an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using techniques for DNA synthesis known per se.

[0170] Generally, Nanobody® ISVs (especially (partially) humanized V HH Sequence and camelized V H V containing arrays HH A Nanobody® ISV may be characterized by the presence of one or more "hallmark residues" (also as further described herein) in one or more of the framework sequences (as described herein). Thus, in general, a Nanobody® ISV may be defined as an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein FR1-FR4 refer to framework regions 1-4, respectively, CDR1-CDR3 refer to complementarity determining regions 1-3, respectively, and one or more of the hallmark residues are as further defined herein).

[0171] In particular, a Nanobody® ISV can be an immunoglobulin sequence with the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and framework sequences are as further defined herein).

[0172] More specifically, a Nanobody® ISV can be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (In the formula, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the hallmark residues set out in Table A below).

[0173] [Table 6]

[0174] [Table 7]

[0175] CD28 The term "CD28," as used herein, refers to a transmembrane costimulatory signaling protein expressed on T cells. CD28 is involved in T cell activation, proliferation, cytokine production, and survival. An exemplary wild-type human CD28 amino acid sequence can be NCBI Reference Sequence: NP_006130.1; and UniProt Reference Number: P10747-1 v1.

[0176] As used herein, a "CD28-binding polypeptide" or "anti-CD28 antibody" refers to any antigen-binding protein having at least one antigen-binding site that specifically binds to CD28. This encompasses bivalent forms of antibodies (such as native immunoglobulin molecules or F(ab)'2 fragments) having two CD28-binding sites, as well as monovalent forms of antibodies having a single CD28-binding site. As used herein, a CD28-binding polypeptide is typically an immunoglobulin single variable domain (ISVD) antibody (e.g., a VHH)-containing polypeptide having at least one immunoglobulin single variable domain (e.g., a VHH domain) that specifically binds to CD28. In certain embodiments, an anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain selected from any one of the VHH amino acid sequences in Table 3.

[0177] [Table 8]

[0178] The CD28-binding polypeptides provided herein include monovalent and multivalent (e.g., bivalent) constructs. In some embodiments, the CD28-binding polypeptides provided herein contain one or two immunoglobulin single variable domains (e.g., VHH domains), each of which individually binds to CD28.

[0179] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:16.

[0180] In certain embodiments, an anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, an anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain set forth in SEQ ID NO: 10.

[0181] In certain embodiments, an anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, an anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain set forth in SEQ ID NO:16.

[0182] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 regions that are at least about 90%, 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 more identical to any one of the HCDR1, HCDR2, or HCDR3 amino acid sequences listed in Table 1.

[0183] In certain embodiments, an anti-CD28 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 regions that are at least about 90%, 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 more identical to the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In certain embodiments, an anti-CD28 antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0184] In certain embodiments, an anti-CD28 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 regions that are at least about 90%, 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 more identical to the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In certain embodiments, an anti-CD28 antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0185] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain that is at least about 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 10 and further comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NOs: 1, 2, and 3 (i.e., a VHH domain that is at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10 outside the HCDR1, HCDR2, and HCDR3 regions of SEQ ID NOs: 1-3, respectively).

[0186] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain that is at least about 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 16 and further comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 (i.e., a VHH domain that is at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16 outside the HCDR1, HCDR2, and HCDR3 regions of SEQ ID NOs: 13-15, respectively).

[0187] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

[0188] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0189] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a monospecific antibody.

[0190] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a bispecific antibody.

[0191] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is a multispecific antibody. In certain embodiments, the multispecific antibody comprises at least one Fab domain. In certain embodiments, the VH and VL domains of the Fab are replaced with any one of the VHH amino acid sequences of SEQ ID NO: 10 or SEQ ID NO: 16. In certain embodiments, the multispecific antibody comprises at least one Fab domain. In certain embodiments, the VH and VL domains of the Fab are replaced with any one of the VHH amino acid sequences of SEQ ID NO: 10. In certain embodiments, the multispecific antibody comprises at least one Fab domain. In certain embodiments, the VH and VL domains of the Fab are replaced with any one of the VHH amino acid sequences of SEQ ID NO: 16. The Fab domain may function as a specific heterodimerization scaffold to which additional binding domains may be attached. The additional binding domain may be present in several different formats, including, but not limited to, another Fab domain, an scFv, or an sdAb (e.g., a VHH).

[0192] In one aspect, the disclosure provides an anti-CD28 antibody, or antigen-binding fragment thereof, comprising a VHH domain, wherein the VHH domain binds to one or more of amino acids E32, E46, V47, C48, Y51, G52, N53, S55, Q57, L58, Q59, V60, Y61, S62, K63, T64, N67, C68, and D69 of SEQ ID NO: 46. In certain embodiments, the VHH domain binds to amino acids E32, E46, V47, C48, Y51, G52, N53, S55, Q57, L58, Q59, V60, Y61, S62, K63, T64, N67, C68, and D69 of SEQ ID NO: 46. In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain set forth in SEQ ID NO: 10.

[0193] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 10, wherein the VHH domain comprises amino acids Y33, W47, T50, N52, D56, F57, T58, S59, K65, P102, Y103, S104, and R105 (positions according to the numbering of SEQ ID NO: 10).

[0194] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises a VHH domain comprising the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, wherein the VHH domain comprises amino acids W47, T50, S59, and K65 (positions according to the numbering of SEQ ID NO:10).

[0195] OX40 As used herein, the term "OX40" refers to a member of the tumor necrosis factor receptor superfamily that functions as a secondary costimulatory immune checkpoint molecule on the surface of T cells. An exemplary wild-type human OX40 amino acid sequence can be NCBI Reference Sequence: NP_001397638.1; and UniProt Reference Number: P43489 v1.

[0196] As used herein, an "OX40-binding polypeptide" or "anti-OX40 antibody" refers to any antigen-binding protein having at least one antigen-binding site that specifically binds to OX40. This encompasses bivalent forms of antibodies (such as native immunoglobulin molecules or F(ab)'2 fragments) having two OX40-binding sites, as well as monovalent forms of antibodies having a single OX40-binding site. As used herein, an OX40-binding polypeptide is typically a VH / VL pair that specifically binds to OX40. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VH and VL domain selected from any one of the VH and VL amino acid sequences in Table 4.

[0197] [Table 9]

[0198] [Table 10]

[0199] The OX40-binding polypeptides provided herein include monovalent and multivalent (e.g., bivalent) constructs. In some embodiments, the OX40-binding polypeptides provided herein contain VH and VL domains that form an antigen-binding domain that binds to OX40.

[0200] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises a VL domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO:12, SEQ ID NO:24, or SEQ ID NO:32.

[0201] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VL domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 12. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VL domain set forth in SEQ ID NO:12.

[0202] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VL domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 24. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VL domain set forth in SEQ ID NO:24.

[0203] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VL domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 32. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VL domain set forth in SEQ ID NO:32.

[0204] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:23, or SEQ ID NO:31.

[0205] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain set forth in SEQ ID NO:11.

[0206] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 23. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain set forth in SEQ ID NO:23.

[0207] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 31. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain set forth in SEQ ID NO:31.

[0208] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain set forth in SEQ ID NO:11 and a VL domain set forth in SEQ ID NO:12.

[0209] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain set forth in SEQ ID NO:23 and a VL domain set forth in SEQ ID NO:24.

[0210] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain set forth in SEQ ID NO:31 and a VL domain set forth in SEQ ID NO:32.

[0211] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 regions that are at least about 90%, 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 more identical to any one of the HCDR1, HCDR2, or HCDR3 amino acid sequences listed in Table 2.

[0212] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 regions that are at least about 90%, 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 more identical to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0213] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 regions that are at least about 90%, 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 more identical to the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0214] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 regions that are at least about 90%, 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 more identical to the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

[0215] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises an LCDR1 region, an LCDR2 region, and an LCDR3 region that are at least about 90%, 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 more identical to any one of the LCDR1, LCDR2, or LCDR3 amino acid sequences listed in Table 2.

[0216] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2, and LCDR3 regions that are at least about 90%, 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 more identical to the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0217] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2, and LCDR3 regions that are at least about 90%, 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 more identical to the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

[0218] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2, and LCDR3 regions that are at least about 90%, 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 more identical to the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

[0219] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain that is at least about 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 11, and further comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; and a VL domain that is at least about 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 12, and further comprises the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0220] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain that is at least about 90%, 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 more identical to the amino acid sequence of SEQ ID NO:23, and further comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; and a VL domain that is at least about 90%, 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 more identical to the amino acid sequence of SEQ ID NO:24, and further comprises the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0221] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises a VH domain that is at least about 90%, 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 more identical to the amino acid sequence of SEQ ID NO:31, and further comprising the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; and a VL domain that is at least about 90%, 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 more identical to the amino acid sequence of SEQ ID NO:32, and further comprising the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0222] In certain embodiments, the anti-XO40 antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

[0223] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0224] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof is a monospecific antibody.

[0225] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof is a bispecific antibody.

[0226] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof is a multispecific antibody.

[0227] In certain embodiments, a multispecific antibody comprises at least one Fab domain. In certain embodiments, the VH of the Fab comprises the amino acid sequence of SEQ ID NO: 11, and the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the VH of the Fab comprises the amino acid sequence of SEQ ID NO: 23, and the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the VH of the Fab comprises the amino acid sequence of SEQ ID NO: 31, and the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 32. The Fab domain may function as a specific heterodimerization scaffold to which additional binding domains may be linked. The additional binding domain may exist in several different formats, including, but not limited to, another Fab domain, an scFv, or an sdAb (e.g., a VHH).

[0228] In one aspect, the disclosure provides an anti-OX40 antibody or antigen-binding fragment thereof comprising a VH domain and a VL domain, wherein the anti-OX40 antibody or antigen-binding fragment thereof binds to one or more of amino acids H13, E14, C15, R16, P17, G18, N19, T31, V32, C33, R34, P35, C36, G37, P38, G39, F40, Y41, V45, L67, C68, T69, A70, and T71 of SEQ ID NO:47. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof binds to amino acids H13, E14, C15, R16, P17, G18, N19, T31, V32, C33, R34, P35, C36, G37, P38, G39, F40, Y41, V45, L67, C68, T69, A70, and T71 of SEQ ID NO: 47. In one aspect, the disclosure provides an anti-OX40 antibody or antigen-binding fragment thereof comprising a VH domain and a VL domain, wherein the anti-OX40 antibody or antigen-binding fragment thereof binds to one or more of amino acids V22, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, W55, R64, L67, C68, T69, and A70 of SEQ ID NO: 47. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof binds to amino acids V22, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, W55, R64, L67, C68, T69, and A70 of SEQ ID NO: 47. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises the VH domain of SEQ ID NO: 11 and the VL domain of SEQ ID NO: 12.

[0229] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises (i) a VH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 11, wherein the VH domain is comprised of amino acids H35, S52, Q54, G56, S57, T58, Y59, Y102, Y104, R105, and W106. (ii) a VL domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 12, wherein the VL domain comprises W32, D50, and S92 (positions according to the numbering of SEQ ID NO: 12).

[0230] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises (i) a VH domain comprising the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and (ii) a VL domain comprising the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, wherein the VH domain comprises amino acids H35 and Y59 (positions according to the numbering of SEQ ID NO:11). In certain embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO:12.

[0231] In one aspect, the disclosure provides an anti-OX40 antibody or antigen-binding fragment thereof comprising a VH domain and a VL domain, wherein the anti-OX40 antibody or antigen-binding fragment thereof binds one or more of a plurality of amino acids to one or more amino acids R16, N19, V22, V32, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, T54, W55, C56, R64, L67, C68, T69, A70, and T74 of SEQ ID NO:47. In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof binds to amino acids R16, N19, V22, V32, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, T54, W55, C56, R64, L67, C68, T69, A70, and T74 of SEQ ID NO:47.

[0232] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, and the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30. In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises the VH domain of SEQ ID NO: 31 and the VL domain of SEQ ID NO: 32.

[0233] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises (i) a VH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 31, wherein the VH domain is comprised of the following amino acids: A33, H35, A50, I51, S52, S53, N54, G55, G56, S57, T58, Y59, Y60, N74, S101, W103, Y104, N and (ii) a VL domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 32, wherein the VL domain comprises W32, Y91, N92, Y94, and Y96 (positions according to the numbering of SEQ ID NO: 32).

[0234] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises (i) a VH domain comprising the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, and (ii) a VL domain comprising the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, wherein the VH domain comprises amino acids H35, A50, Y59, Y60, and N74 (positions according to the numbering of SEQ ID NO:31). In certain embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO:32.

[0235] In one aspect, the disclosure provides an anti-OX40 antibody or antigen-binding fragment thereof comprising a VH domain and a VL domain, wherein the anti-OX40 antibody or antigen-binding fragment thereof binds one or more of a plurality of amino acids to one or more of amino acids G20, M21, V22, R24, P35, C26, G37, P38, G39, F40, Y41, S46, K48, P49, C50, K51, P52, C53, T54, and W55 of SEQ ID NO: 47. In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof binds to amino acids G20, M21, V22, R24, P35, C26, G37, P38, G39, F40, Y41, S46, K48, P49, C50, K51, P52, C53, T54, and W55 of SEQ ID NO: 47.

[0236] In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, and the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102. In certain embodiments, an anti-OX40 antibody or antigen-binding fragment thereof comprises the VH domain of SEQ ID NO: 103 and the VL domain of SEQ ID NO: 104.

[0237] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises: (i) a VH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 103, wherein the VH domain is comprised of the following amino acids: D30, D31, T33, L50, S52, W53, D54, S57, Y59, K65, L101, W102, Y103, and (ii) a VL domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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 more identical to the amino acid sequence of SEQ ID NO: 104, wherein the VL domain comprises W92 and T97 (positions according to the numbering of SEQ ID NO: 104).

[0238] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises (i) a VH domain comprising the HCDR1, HCDR2, and HCDR3 regions set forth in SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, and (ii) a VL domain comprising the LCDR1, LCDR2, and LCDR3 regions set forth in SEQ ID NO:100, SEQ ID NO:101, and SEQ ID NO:102, wherein the VH domain comprises amino acids L50, Y59, and K65 (positions according to the numbering of SEQ ID NO:103). In certain embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO:104.

[0239] Multispecific antigen-binding proteins The multispecific binding proteins (e.g., bispecific binding proteins) described herein comprise: (a) a first antigen binding domain (ABD) comprising a single immunoglobulin heavy chain variable domain (ISVD) (e.g., VHH) that has binding specificity for CD28 (such as the anti-CD28 antibodies described herein); and (b) a second ABD comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) that has binding specificity for OX40 (such as the anti-CD40 antibodies described herein).

[0240] Multispecific binding proteins can antagonize the activity of both CD28 and OX40 on the surface of T cells, thereby suppressing CD28 and OX40 costimulatory signals.

[0241] In certain embodiments, when the multispecific binding protein binds to CD28 and OX40, the multispecific binding protein inhibits activated T cells. In certain embodiments, the multispecific binding protein inhibits T cell proliferation. In certain embodiments, the multispecific binding protein inhibits the expression of one or more proinflammatory cytokines. In certain embodiments, the proinflammatory cytokines are interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 2 (IL-2), interleukin 5 (IL-5), interleukin 6 (IL-6), and interleukin 10 (IL-10). In certain embodiments, the proinflammatory cytokine is interferon gamma (IFNγ). In certain embodiments, the proinflammatory cytokine is tumor necrosis factor alpha (TNFα). In certain embodiments, the proinflammatory cytokine is interleukin 2 (IL-2). In certain embodiments, the proinflammatory cytokine is interleukin 5 (IL-5). In certain embodiments, the proinflammatory cytokine is interleukin 6 (IL-6). In certain embodiments, the pro-inflammatory cytokine is interleukin-10 (IL-10).

[0242] The multispecific binding proteins (e.g., bispecific binding proteins) described herein comprise a hybrid format of an anti-CD28 VHH domain and an anti-OX40 Fab domain comprising a VH and VL domain. One of several hybrid formats can be used. In one embodiment, the multispecific binding protein comprises the format shown in Figure 1A. In other embodiments, the multispecific binding protein comprises one of the three formats shown in Figure 1B.

[0243] In each format, the multispecific binding protein comprises an immunoglobulin Fc domain or a variant thereof. In certain embodiments, the Fc domain is a human IgG1 Fc domain. In certain embodiments, the Fc domain or variant thereof comprises a first Fc heavy chain and a second Fc heavy chain.

[0244] In certain embodiments, a first ABD (e.g., an anti-CD28 VHH) binds to a first Fc heavy chain, and a second ABD (e.g., an anti-OX40 VH / VL) binds to a second Fc heavy chain.

[0245] In certain embodiments, a first ABD (e.g., an anti-CD28 VHH) binds to the N-terminus of a first Fc heavy chain, and a second ABD (e.g., an anti-OX40 VH / VL) binds to the N-terminus of a second Fc heavy chain.

[0246] In certain embodiments, the first ABD is linked to the N-terminus of the second ABD VH.

[0247] In certain embodiments, the first ABD is attached to the C-terminus of the second Fc heavy chain and the second ABD is attached to the N-terminus of the second Fc heavy chain.

[0248] In certain embodiments, the VH binds to the CH1 domain and the VL binds to the CL domain. In certain embodiments, the first ABD binds to the C-terminus of the CL domain.

[0249] In other aspects, the present disclosure provides multispecific binding proteins (e.g., bispecific binding proteins) comprising: (a) a first antigen-binding domain (ABD) comprising an immunoglobulin single variable domain (ISVD) (e.g., VHH) that has binding specificity for a first target antigen; (b) a second ABD comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) that has binding specificity for a second target antigen; and (c) an immunoglobulin Fc domain or variant thereof.

[0250] In certain embodiments, the Fc domain is a human IgG1 Fc domain. In certain embodiments, the Fc domain or variant thereof comprises a first Fc heavy chain and a second Fc heavy chain.

[0251] In certain embodiments, the first ABD binds to a first Fc heavy chain and the second ABD binds to a second Fc heavy chain.

[0252] In certain embodiments, the first ABD is linked to the N-terminus of the second ABD VH.

[0253] In certain embodiments, the first ABD is attached to the C-terminus of the second Fc heavy chain and the second ABD is attached to the N-terminus of the second Fc heavy chain.

[0254] In certain embodiments, the VH binds to the CH1 domain, the VL binds to the CL domain, and the first ABD binds to the C-terminus of the CL domain.

[0255] In certain embodiments, the first target antigen comprises a tumor-associated antigen (TAA). In certain embodiments, the second target antigen comprises a tumor-associated antigen (TAA).

[0256] In certain embodiments, the first target antigen is a target associated with an autoimmune disease. In certain embodiments, the second target antigen is a target associated with an autoimmune disease.

[0257] Fc domain As used herein, the term "Fc domain" refers to an immunoglobulin hinge region (which naturally has a first binding site for an FcγR), a CH2 domain (which naturally has a second binding site for an FcγR), and a CH3 domain of an immunoglobulin (e.g., of an IgG, IgA, or IgD immunoglobulin), and / or, where applicable, a CH4 domain of an immunoglobulin (e.g., for IgM and IgE). An Fc domain or variant thereof comprises a first Fc heavy chain polypeptide and a second Fc heavy chain polypeptide, which dimerize to form the Fc domain.

[0258] As used herein, the term "Fc variant" refers to a molecule or sequence that is modified from a native Fc. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues, or in which one or more Fc sites or residues have been modified, that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, (7) antibody-dependent cellular cytotoxicity (ADCC), and / or heterodimerization. The term "Fc variant" also encompasses Fc domains that have been clipped at the C-terminus, such as by clipping or removing the C-terminal lysine from the Fc domain CH3 region. C-terminal Fc clipping is described in further detail in Faid et al. (Eur J Pharm Sci. 2021.159: 105730), which is incorporated herein by reference.

[0259] In one embodiment of the present disclosure, multispecific binding proteins comprising a first Fc heavy chain polypeptide and a second Fc heavy chain polypeptide are heterodimerized by knob-in-hole pairing (KiH). This dimerization technique utilizes protrusions ("knobs") and cavities ("holes") engineered into the interface of the CH3 domains. If a properly positioned and appropriately sized knob or hole is present at the interface of either the first or second CH3 domain, it is only necessary to engineer a corresponding hole or knob, respectively, into the adjacent interface, thereby facilitating and strengthening Fc domain pairing at the CH3 / CH3 domain interface. A "knob" refers to at least one amino acid side chain, typically a relatively large side chain, that protrudes from the interface of the CH3 portion of the first Fc domain. The protrusion creates a "knob" that is complementary to the CH3 portion of the second Fc domain and can be received by a "hole" therein. The "hole" is at least one amino acid side chain, usually a relatively small side chain, that is recessed from the interface of the CH3 portion of the second Fc domain. This technology is described, for example, in U.S. Pat. No. 5,821,333; Ridgway et al., Protein Engineering 9:617-621 (1996); and Carter P., J. Immunol. Methods 248:7-15 (2001).

[0260] Exemplary amino acid residues that can serve as knobs include arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). An existing amino acid residue within the CH3 domain can be exchanged or substituted with a knob amino acid residue. Preferred amino acids that can be substituted include any amino acid with a small side chain, such as alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), or valine (V).

[0261] Exemplary amino acid residues that can serve as holes include alanine (A), serine (S), threonine (T), and valine (V). An existing amino acid residue in the CH3 domain can be exchanged or substituted with a hole amino acid residue. Preferred amino acids that can be substituted include any amino acid with a large or bulky side chain, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W).

[0262] The CH3 domain can be derived from a human IgG1 antibody. Exemplary amino acid substitutions in the CH3 domain include T366Y, T366W, F405A, F405W, Y407T, Y407A, Y407V, T394S, and combinations thereof. One particular exemplary combination is a knob mutation in the first CH3 domain (T366 or T366W) and a hole mutation in the second CH3 domain (Y407T or Y407V).

[0263] In certain embodiments, the first Fc heavy chain comprises a Y349C substitution according to Eu numbering, and the second Fc heavy chain comprises a S354C substitution according to Eu numbering.

[0264] In certain embodiments, the first Fc heavy chain comprises a Y349C, T366S, L368A, or Y407V substitution according to Eu numbering, and the second Fc heavy chain comprises a T366W substitution according to Eu numbering.

[0265] In certain embodiments, at least one Fc heavy chain comprises H435R and Y436F substitutions according to Eu numbering.

[0266] In certain embodiments of the present disclosure, a multispecific binding protein comprising a first Fc heavy chain polypeptide and a second Fc heavy chain polypeptide is heterodimerized by Fab arm exchange (FAE). A human IgG1 with a P228S hinge mutation can contain an F405L or K409R CH3 domain mutation. Mixing the two antibodies with a reducing agent results in FAE. This technique is described in U.S. Pat. No. 9,212,230 and Labrijn AF, Proc Natl Acad Sci USA 110(13):5145-5150 (2013).

[0267] In certain embodiments of the present disclosure, a multispecific binding protein comprising a first Fc heavy chain polypeptide and a second Fc heavy chain polypeptide is heterodimerized by electrostatic steering. This dimerization technique utilizes electrostatic steering to promote and strengthen Fc domain pairing at the CH3 / CH3 domain interface. The charge complementarity between the two CH3 domains is altered to favor heterodimerization (pairing of opposite charges) over homodimerization (pairing of same charges). In this method, electrostatic repulsion prevents homodimerization. Exemplary amino acid residue substitutions include K409D, K392D, and / or K370D in the first CH3 domain and D399K, E356K, and / or E357K in the second CH3 domain. This technology is described in U.S. Patent Application Publication No. 2014 / 0154254 A1 and Gunasekaran K., J Biol Chem 285(25):19637-19646(2010).

[0268] In certain embodiments of the present disclosure, a multispecific binding protein comprising a first Fc heavy chain polypeptide and a second Fc heavy chain polypeptide is heterodimerized due to hydrophobic interaction effects. This dimerization technique utilizes hydrophobic interactions instead of electrostatic interactions to promote and strengthen Fc domain pairing at the CH3 / CH3 domain interface. Exemplary amino acid residue substitutions can include K409W, K360E, Q347E, Y349S, and / or S354C in the first CH3 domain and D399V, F405T, Q347R, E357W, and / or Y349C in the second CH3 domain. Preferred pairs of amino acid residue substitutions between the first and second CH3 domains include K409W:D399V, K409W:F405T, K360E:Q347R, Y349S:E357W, and S354C:Y349C. This technology is described in U.S. Patent Application Publication No. 2015 / 0307628 A1.

[0269] In certain embodiments of the present disclosure, a multispecific binding protein comprising a first Fc heavy chain polypeptide and a second Fc heavy chain polypeptide is heterodimerized by a leucine zipper fusion. A leucine zipper domain fused to the C-terminus of each CH3 domain of the antibody chain allows for heterodimerization. This technique is described in Wranik B., J Biol Chem 287(52):43331-43339(2012).

[0270] In certain embodiments of the present disclosure, multispecific binding proteins comprising a first Fc heavy chain polypeptide and a second Fc heavy chain polypeptide are heterodimerized using a chain exchange engineered domain (SEED). CH3 domains derived from IgG and IgA formats drive heterodimerization. This technique is described in Muda M., Protein Eng. Des. Sel. 24(5):447-454 (2011)).

[0271] Unless otherwise specified, all antibody constant region numbering used herein corresponds to the EU numbering scheme described in Edelman et al., Proc. Natl. Acad. Sci. USA 63(1):78-85 (1969). Further methods for heterodimerization of heavy and / or light chains and for the production and purification of asymmetric antibodies are well known in the art. See, e.g., Klein C., mABs 4(6):653-663 (2012) and U.S. Pat. No. 9,499,634, each of which is incorporated herein by reference.

[0272] Additional Fc domain mutations that alter Fc function are contemplated, including, but not limited to, altered antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0273] In certain embodiments, the multispecific binding protein comprises an IgG1 constant domain comprising the L234A and L235A amino acid substitutions according to Eu numbering.

[0274] Expression of antigen-binding proteins In one aspect, nucleic acid molecules encoding the antibodies and antigen-binding fragments thereof disclosed herein are provided. Methods of making binding proteins comprising expressing these nucleic acid molecules are also provided.

[0275] Nucleic acid molecules encoding the antibodies disclosed herein are typically inserted into expression vectors for introduction into host cells, which can be used to produce desired quantities of the antibody. Thus, in certain aspects, the disclosure provides expression vectors comprising the nucleic acid molecules disclosed herein, as well as host cells comprising these vectors and nucleic acid molecules.

[0276] The term "vector" or "expression vector" is used herein to mean a vector used in accordance with the present disclosure as a vehicle for introducing and expressing a desired gene in a cell. As known to those skilled in the art, such vectors may be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the present disclosure will contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0277] Numerous expression vector systems can be utilized for the purposes of this disclosure. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Furthermore, cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for selection of transfected host cells. Markers can provide prototrophy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or can be introduced into the same cell by cotransformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include signal sequences, splice signals, and transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with a heavy chain constant region gene (eg, a human constant region gene) synthesized as discussed above.

[0278] In other embodiments, antibodies can be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as antibody heavy and light chains, can be produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to produce relatively high levels of polypeptides in eukaryotic host cells. Suitable IRES sequences are described in U.S. Patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will understand that such expression systems can be used to effectively produce the full range of polypeptides disclosed in the present application.

[0279] More generally, once a vector or DNA sequence encoding an antibody or fragment thereof has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Plasmid introduction into host cells can be accomplished by a variety of techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See Ridgway, AAG, "Mammalian Expression Vectors," Chapter 24.2, pp. 470-472, in Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Plasmid introduction into the host can also be by electroporation. Transformed cells are grown under conditions appropriate for the production of light and heavy chains, and assayed for the synthesis of heavy and / or light chain proteins. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0280] As used herein, the term "transformation" refers broadly to the introduction of DNA into a recipient host cell, resulting in a change in the genotype.

[0281] Similarly, a "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA techniques and encoding at least one heterologous gene. In describing the process of isolating a polypeptide from a recombinant host, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody, unless clearly specified otherwise. In other words, recovery of polypeptide from the "cells" can mean either from spun down whole cells, from the supernatant of a lysed cell culture, or from the cell culture fluid containing both the medium and suspended cells.

[0282] In one embodiment, the host cell line used for antibody expression is of mammalian origin. One skilled in the art can determine the particular host cell line most suitable for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 carrying the SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HEK (human kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). In one embodiment, the cell line provides altered glycosylation, e.g., afucosylation, of the antibody expressed therefrom (e.g., PER.C6® (Crucell) or a FUT8-knockout CHO cell line (POTELLIGENT® cells) (Biowa, Princeton, NJ)). In one embodiment, NSO cells may be used. CHO cells are particularly useful. Host cell lines are typically available from commercial services, e.g., the American Tissue Culture Collection, or from authors in the published literature.

[0283] In vitro production allows for scale-up to obtain large quantities of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogenous suspension culture in airlift reactors or continuous stirred reactors, or culturing cells immobilized or entrapped, for example, in hollow fibers, in microcapsules, on agarose microbeads, or on ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immuno)affinity chromatography.

[0284] Genes encoding the antibodies featured in this disclosure can also be expressed in non-mammalian cells, such as bacterial cells or yeast cells or plant cells. In this regard, it will be understood that various unicellular microorganisms other than mammals, such as bacteria, can also be transformed, i.e., can be grown in culture or fermentation. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as Escherichia coli or strains of Salmonella, Bacillaceae family, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. It will further be appreciated that when expressed in bacteria, the binding proteins may become part of inclusion bodies. In some embodiments, the binding proteins are subsequently isolated, purified, and assembled into functional molecules. In some embodiments, the binding proteins of the present disclosure are expressed in bacterial host cells. In some embodiments, the bacterial host cells are transformed with an expression vector comprising a nucleic acid molecule encoding a binding protein of the present disclosure.

[0285] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although many other strains are commonly available. For expression in Saccharomyces, plasmid YRp7, for example (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)), is commonly used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow on tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0286] Methods of Administering Antigen-Binding Proteins Methods for preparing antigen-binding proteins and administering antigen-binding proteins to subjects are well known to, or easily determined by, those of skill in the art. The route of administration of the antigen-binding proteins of the present disclosure can be oral, parenteral, inhalation, or topical. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. The route of administration can be intravenous. The route of administration can be subcutaneous. While all of these administration forms are clearly contemplated within the scope of the present disclosure, the administration form will be an injectable solution, particularly a solution for intravenous or intraarterial injection or infusion. Typically, a suitable injectable pharmaceutical composition can include a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), and optionally a stabilizer (e.g., human albumin). However, in other methods consistent with the teachings herein, the modified antibodies can be delivered directly to the site of harmful cell populations, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0287] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. In the compositions and methods of the present disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases. More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0288] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0289] In either case, sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide alone or a modified binding polypeptide in combination with other active agents) in the required amount in an appropriate solvent, with one or a combination of ingredients enumerated herein, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods typically include vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof. Preparations for injection are processed and filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under aseptic conditions according to methods known in the art. Furthermore, preparations can be packaged and sold in the form of kits, such as those described in U.S. Patent Application Publication No. 20020102208 and U.S. Patent No. 6,994,840, each of which is incorporated herein by reference. Such articles of manufacture may include a label or package insert indicating that the associated composition is useful for treating a subject suffering from or predisposed to an autoimmune or neoplastic disorder.

[0290] The effective dose of the compositions of the present disclosure for treating the above conditions will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other pharmaceutical agents administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, although non-human mammals, including transgenic mammals, can also be treated. Treatment dosages can be titrated to optimize safety and efficacy using routine methods known to those skilled in the art.

[0291] As previously discussed, the antigen binding proteins, immunoreactive fragments or recombinant forms thereof of the present disclosure may be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders. In this regard, it will be understood that the disclosed antigen binding proteins are formulated to facilitate administration and to promote stability of the active agent.

[0292] Pharmaceutical compositions according to the present disclosure typically comprise a pharmaceutically acceptable, non-toxic, sterile carrier, such as saline, non-toxic buffers, preservatives, etc. For purposes of this application, a pharmaceutically effective amount of a modified antigen-binding protein, immunoreactive fragment, or recombinant thereof, conjugated or unconjugated to a therapeutic agent, shall be held to mean an amount sufficient to achieve effective binding to the antigen and achieve a benefit, e.g., to ameliorate the symptoms of a disease or disorder, or to detect a substance or cell. In the case of tumor cells, the modified binding polypeptide will typically be able to interact with a selected immunoreactive antigen on tumor cells or immunoreactive cells, resulting in increased death of those cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

[0293] In accordance with the scope of the present disclosure, antigen-binding proteins of the present disclosure may be administered to humans or other animals in accordance with the aforementioned treatment methods in an amount sufficient to produce a therapeutic or prophylactic effect. The antigen-binding proteins of the present disclosure may be administered to such humans or other animals in conventional dosage forms prepared by combining an antibody of the present disclosure with a conventional pharmaceutically acceptable carrier or diluent in accordance with known techniques. It will be recognized by those skilled in the art that the form and characteristics of the pharmaceutically acceptable carrier or diluent will be dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables. Those skilled in the art will further appreciate that cocktails comprising one or more binding polypeptides described in the present disclosure may prove particularly effective.

[0294] The biological activity of the pharmaceutical compositions defined herein can be measured by T cell activation assays, for example, through the detection of pro- or anti-inflammatory cytokine expression. "Efficacy" or "in vivo efficacy," as used herein, refers to a response to therapy with a pharmaceutical composition of the present disclosure, for example, using standardized NCI efficacy criteria. The success or in vivo efficacy of therapy using a pharmaceutical composition of the present disclosure refers to the effectiveness of the composition for its intended purpose, i.e., the ability of the composition to cause its desired effect, i.e., depletion of pathological cells, e.g., tumor cells, or suppression of activated immune cells. In vivo efficacy can be monitored by established standard methods for each disease entity, including, but not limited to, white blood cell count, differential, fluorescently activated cell sorting, and bone marrow aspirate. Additionally, various disease-specific clinical chemistry parameters and other established standard methods can be used.

[0295] Treatment method The antigen binding proteins described herein (e.g., multispecific binding proteins with specificity for CD28 and OX40) are useful in treating or preventing diseases associated with activated T cells (e.g., autoimmune diseases). The targets CD28 and OX40 are costimulatory receptors on the surface of T cells that are responsible, in part, for promoting T cell activity and proliferation. Thus, antagonistic anti-CD28 and anti-OX40 antibodies, and antagonistic multispecific antibodies having anti-CD28 and anti-OX40 antigen-binding domains, can suppress the costimulatory activity of CD28 and OX40 in T cells.

[0296] In certain embodiments, the multispecific binding proteins of the present disclosure inhibit activated T cells. In certain embodiments, the multispecific binding proteins of the present disclosure inhibit the proliferation of T cells. In certain embodiments, the multispecific binding proteins of the present disclosure inhibit the expression of one or more proinflammatory cytokines. In certain embodiments, the proinflammatory cytokines include, but are not limited to, interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 2 (IL-2), interleukin 5 (IL-5), interleukin 6 (IL-6), and interleukin 10 (IL-10). In certain embodiments, the proinflammatory cytokine is interferon gamma (IFNγ). In certain embodiments, the proinflammatory cytokine is tumor necrosis factor alpha (TNFα). In certain embodiments, the proinflammatory cytokine is interleukin 2 (IL-2). In certain embodiments, the proinflammatory cytokine is interleukin 5 (IL-5). In certain embodiments, the proinflammatory cytokine is interleukin 6 (IL-6). In certain embodiments, the pro-inflammatory cytokine is interleukin-10 (IL-10).

[0297] In one aspect, the disclosure provides a method of treating an autoimmune disease or disorder in a subject, the method comprising administering to a subject in need thereof a multispecific binding protein comprising: (a) a first antigen binding domain (ABD) comprising an immunoglobulin single variable domain (ISVD) (e.g., VHH) that has binding specificity for CD28, and (b) a second ABD comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) that have binding specificity for OX40.

[0298] In certain embodiments, when the multispecific binding protein binds to CD28 and OX40, the multispecific binding protein inhibits activated T cells.

[0299] In another aspect, the disclosure provides a multispecific binding protein comprising: (a) a first antigen binding domain (ABD) comprising an immunoglobulin single variable domain (ISVD) (e.g., VHH) that has binding specificity for CD28, and (b) a second ABD comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) that have binding specificity for OX40, for use in a method of treating an autoimmune disease or disorder.

[0300] In certain embodiments, the autoimmune disease is connective tissue disease-interstitial lung disease (CTD-ILD). CTD-ILD (also called connective tissue disease-associated interstitial lung disease) is a lung disease that can occur in subjects with connective tissue disease. CTD-ILD causes inflammation and scarring (fibrosis) in the lungs.

[0301] In certain embodiments, the autoimmune disease is graft-versus-host disease (GvHD). [Example]

[0302] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions featured in this disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. While attempts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0303] Example 1. Transfection, expression, and purification Transfection for expression FREESTYLE HEK-293-FS cells were transfected with high-quality plasmid preparations according to the manufacturer's instructions. Expression was examined 48 or 144 hours posttransfection by flow cytometry or Octet. Supernatants were collected and purified on a HiTrap MabSelect SuRe (Cytiva) column according to the manufacturer's instructions. Affinity-purified samples were polished by size-exclusion chromatography (SEC) using a Superdex200 26 / 60 (Cytiva) column.

[0304] Fab-like construction, production, and purification After amplification by polymerase chain reaction, the complementary DNA of the VHH was cloned in frame with either the human CL domain or the human IgG1 CH1 domain fused to a human influenza hemagglutinin and a 6-His tag into a proprietary mammalian expression vector. Plasmids were purified using a NucleoBond Macherey-Nagel kit and Sanger sequenced. Bispecific (bsFab) or bivalent Fab-like (bvFab) antibodies were produced by cotransfecting FREESTYLE HEK293-FS cells with a mixture of two plasmids encoding two distinct (bsFab) or two identical (bvFab) VHHs fused to each of the Fab constant domains. Supernatants were collected after 7 days, purified on a nickel affinity column, and analyzed on a CALIPER CXII (Perkin Elmer).

[0305] Example 2. Anti-CD28 / OX40 bispecific mAb Origin of anti-CD28 human VHH Anti-human CD28 VHHs were generated by phage display selection on the human CD28 antigen using a VHH library constructed from a llama immunized with human CD28. The cynomolgus CD28 sequence is 100% identical to that of the human, so there was no need to perform specific selection on the cynomolgus CD28 antigen.

[0306] The 42 VHHs were characterized in bivalent and monovalent Fab-like formats for: i) binding to cell lines expressing human CD28; ii) binding to human CD28 recombinant protein by ELISA; iii) epitope binding to and competition with the natural ligand CD80 and benchmark antibodies (TGN1412 and 9.3); iv) sequence clustering; and v) antagonist activity in MLRs.

[0307] Eighteen antagonist VHHs were then selected for detailed functional investigation: i) antagonist efficacy in reporter assays; ii) agonist activity in T cell activation assays (TCA) as a counterscreen; iii) superior agonist activity in cytokine release assays as a counterscreen; iv) antagonist efficacy in MLR.

[0308] Five sequence-diverse, functionally potent VHHs were selected based on sequence-activity correlation (antagonist activity without agonist activity). The five hit VHHs showed comparable or better antagonist activity in MLRs compared with clinically validated anti-CD28 benchmark molecules.

[0309] Determination of anti-CD28 VHH epitopes and paratopes The epitopes and paratopes of the L4, L6, and L5 CD28 arm VHHs were determined by generating crystal structures of the VHHs in complex with human CD28. CD28-His (amino acids 19-136 of human CD28) was expressed in Expi293 cells. CD28-His was purified using HisTrap and SEC purification. The purified CD28-His and VHH-His were mixed and then SEC-purified to prepare CD28-L4, L6, and L5 CD28 arm VHH complexes. Crystal structures were then generated at 2.9 Å resolution (Figure 2B). The epitopes of the L4, L6, and L5 CD28 arm VHHs are shown below, with the epitope amino acids indicated in bold and underlined text. [ka]

[0310] The epitopes of the L4, L6 and L5 CD28 arm VHHs are amino acid positions E32, E46, V47, C48, Y51, G52, N53, S55, Q57, L58, Q59, V60, Y61, S62, K63, T64, N67, C68, and D69 (numbering according to SEQ ID NO: 46).

[0311] The paratopes of the L4, L6, and L5 CD28 arm VHHs are amino acid positions Y33, W47, T50, N52, D56, F57, T58, S59, K65, P102, Y103, S104, and R105.

[0312] Determination of anti-OX40 Fab epitopes and paratopes The epitopes and paratopes of the L1 and L4 OX40 arm Fabs were determined by generating a crystal structure of the Fab in complex with human OX40. Human OX40 (amino acids 32-109 of human OX40) was used. The cryo-EM structure of human OX40 in complex with the L1 and L4 OX40 arms was determined at 3.25 Å resolution. The epitopes of the L1 and L4 OX40 arm Fabs are shown below, with the epitope amino acids in bold and underlined text. [ka]

[0313] The epitopes of the L1 and L4 OX40 arm Fabs are amino acid positions V22, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, W55, R64, L67, C68, T69, and A70 (numbering according to SEQ ID NO: 47).

[0314] The paratopes of the L1 and L4 OX40 arm Fabs are amino acid positions [light chain] W32, D50, S92 and amino acid positions [heavy chain] H35, S52, Q54, G56, S57, T58, Y59, Y102, Y104, R105, and W106.

[0315] The epitopes and paratopes of the L2 and L5 OX40 arm Fabs were determined by generating cryo-EM crystal structures of the Fabs in complex with human OX40. Human OX40 (amino acids 29-170 of human OX40) was used. The cryo-EM structure of human OX40 in complex with the L2 and L5 arms was determined at 3.3 Å resolution. The epitopes of the L2 and L5 OX40 arm Fabs are shown below, with the epitope amino acids in bold and underlined text. [ka]

[0316] The epitopes of the L2 and L5 OX40 arm Fabs are at amino acid positions R16, N19, V22, V32, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, T54, W55, C56, R64, L67, C68, T69, A70, and T74 (numbering according to SEQ ID NO: 47).

[0317] The paratopes of the L2 and L5 OX40 arm Fabs are at amino acid positions [light chain] W32, Y91, N92, Y94, and Y96, and amino acid positions [heavy chain] A33, H35, A50, I51, S52, S53, N54, G55, G56, S57, T58, Y59, Y60, N74, S101, W103, Y104, N105, S106, and E107.

[0318] The epitope and paratope of the prototype OX40 arm Fab were determined by generating a cryo-EM crystal structure of the Fab in complex with human OX40. Human OX40 (amino acids 29-170 of human OX40) was used. The crystal structure of human OX40 in complex with the prototype arm was determined at 3.35 Å resolution. The epitope of the prototype OX40 arm Fab is shown below, with the epitope amino acids in bold and underlined text. [ka]

[0319] The epitope of the prototype OX40 arm Fab is at amino acid positions G20, M21, V22, R24, P35, C26, G37, P38, G39, F40, Y41, S46, K48, P49, C50, K51, P52, C53, T54, and W55 (numbering according to SEQ ID NO: 47).

[0320] The paratopes of the prototype OX40 arm Fab are amino acid positions [light chain] W92 and T97, and amino acid positions [heavy chain] D30, D31, T33, L50, S52, W53, D54, S57, Y59, K65, L101, W102, Y104, and L105.

[0321] Screening in a dual-specific format Prior to finalization of the monoblock screening, three anti-OX40 mAbs, two anti-OX40 VHHs, and two anti-CD28 VHHs were selected for bispecific format evaluation. The bispecific formats were designed to evaluate the following criteria: i) the effect of N- / C-terminal fusion of the building blocks, ii) the required physical distance between the two paratopes, and iii) the valency requirements of the anti-OX40 building blocks.

[0322] In total, six mAb-VHH hybrid formats, ten VHH-Fc fusion formats, and six VHH-only formats were screened. A total of 137 bispecific molecules and 40 control molecules were produced, and their biophysical and functional characteristics were investigated. The best formats were analyzed directly in the MLR assay at 0.1 nM. A clear advantage was observed for the hybrid format over the VHH-Fc fusion and VHH-only constructs. Therefore, an antibody-like mAb-VHH hybrid format was selected as the lead format (see Figure 1A for the lead format and Figure 1B for several variations of the lead format). Importantly, bispecific molecules in this hybrid format showed greater inhibition of the MLR at lower concentrations compared to clinically validated anti-CD28 or anti-OX40 benchmark molecules and abatacept, as well as building block combinations.

[0323] Cleaning Monoblock Hits Hit building blocks (nine anti-OX40 mAbs and five anti-CD28 VHHs (plus one positive control with clear agonist activity in TCA)) were combined in a bispecific hybrid format. Biophysical and functional characterization of these 70 molecules (including controls) allowed the selection of six bispecific molecules by combining anti-OX40 L2 and L5 OX40 arms, L1 and L4 OX40 arms, and L3 and L6 OX40 arm mAbs with anti-CD28 L1, L3, and L2 CD28 arms and L4, L6, and L5 CD28 arm VHHs.

[0324] Immunogenicity prediction and optimization anti-CD28 VHH In silico T cell epitope prediction (NetMHCII pan, Metapredictor) showed improved antigenicity scores for the sequence-optimized variants compared to the parental clone. Two VHH hits were found to be within the Metapredictor range predicted for the VHH.

[0325] Anti-OX40 Fab (9 hits) In silico analysis was performed using EpiVax and Metapredictor. Six of the nine compounds showed scores within the expected range in both the EpiVax and Metapredictor tools. Clones G3, J9, and CL-154787 showed higher scores. The three clones selected for bispecificity have no non-embryonic residues and show acceptable antigenicity scores.

[0326] Example 3. Target Binding Affinity SPR binding to recombinant target antigen Target binding affinity measurements (KD (nM)) of the six bispecific antibodies to human and cynomolgus monkey OX40, and human CD28 were performed by surface plasmon resonance (SPR) analysis using a Biacore 8K instrument (Cytiva).

[0327] Measurements were performed using HBS-EP+ (Cytiva BR1006-69) as the running buffer. Anti-human Fc antibody (Human Antibody Capture Kit, Cytiva BR-1008-39) was covalently coupled onto a sensor chip CM5 (Cytiva 29149603).

[0328] First, all eight flow cells were activated with a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide mixture (75–11.5 mg / mL) for 420 seconds using a flow rate of 5 μL / min (amine coupling kit, Cytiva LifeSciences, BR100050). Anti-human Fc antibodies were diluted to 25 μg / mL in 10 mM acetate, pH 5.0, and coupled for 420 seconds using a flow rate of 5 μL / min. Unbound sites were inactivated by injecting 1 M methanolamine (pH 8.5) for 420 seconds using a flow rate of 10 μL / min. After surface preparation, the bispecific molecules were diluted to 0.15 μg / mL in running buffer and captured as ligands for 90 seconds at 10 μL / min.

[0329] The corresponding target antigens (human CD28 (CD28-3910H, Creative Bio Mart), human OX40 (10481-H08H, Sino Biological), and cynomolgus monkey OX40 (internally produced)) were then injected as analytes in multi-cycle kinetic experiments at concentrations of 100, 50, 25, 12.5, 6.3, 3.1, and 1.6 nM. Analyte association was monitored for 400 s at 30 μL / min, followed by an 800 s dissociation phase at 30 μL / min using running buffer as the injectate. All analyte concentrations were performed in duplicate, including a buffer control for double referencing.

[0330] The surface was regenerated with a 60-second burst of 3 M MgCl using a flow rate of 30 μL / min. To analyze the binding kinetics, a mass-transfer-limited 1:1 binding model was used within Insight evaluation software (Cytiva).

[0331] As shown in Figures 4A and 4B, six bispecific antibodies (L1 to L6) showed binding to CD28, human OX40, and cynomolgus monkey OX40.

[0332] SPR binding to recombinant human FcRn The binding affinity (KD (nM)) of the six bispecific antibodies to human FcRn (Immunitrack, ITF01-200) was measured by surface plasmon resonance (SPR) analysis using a Biacore 8K instrument (Cytiva). A Series S sensor chip CAP was prepared according to the manufacturer's instructions (Cytiva, 28920234). After surface preparation, human FcRn was diluted to 0.5 μg / mL in running buffer and captured as a ligand at 10 μL / min for 90 seconds.

[0333] The bispecific samples were then injected as analytes in multi-cycle kinetic experiments at concentrations of 1600, 800, 400, 200, 100, 50, 25, and 12.5 nM, respectively, using running buffer (pH 6.0).

[0334] Analyte association was monitored for 60 seconds at 30 μL / min, followed by a 120-second dissociation phase at 30 μL / min using running buffer as the injection fluid. To demonstrate the absence of bispecific binding to FcRn at pH 7.4, a single analyte injection in HBS-EP + running buffer (pH 7.4) was performed. All analyte concentrations were run in duplicate, including a buffer control for double referencing.

[0335] The surface was regenerated with a 60-second burst of GdnHCl / NaOH using a flow rate of 10 μL / min. The equilibrium constant, KD, was calculated using the steady-state evaluation tool in Biacore Insight evaluation software (Cyvita). KD values ​​for binding to CD28, human OX40, cynomolgus monkey OX40, and FcRn are listed in Table 5 below.

[0336] [Table 11]

[0337] Binding to recombinant cell lines The ability of bispecific antibodies L1 to L6 to bind to cells expressing CD28 or OX40 on their surface was tested. The cell lines used were HEK293 cells expressing human OX40 or cynomolgus monkey OX40, Jurkat cells, and Jurkat cells engineered to express human OX40. Flow cytometry binding assays were performed on a BD FACSCelesta™ flow cytometer or a BD FACSCanto™ II flow cytometer (Becton Dickinson) using 96-well U-bottom Cellstar plates (Greiner, 650185). Frozen target cells were thawed and 5 × 10 4 cells / well.

[0338] For live / dead cells, differentiated cells were stained using the LIVE / DEAD™ Fixable Yellow Dead Cell Stain Kit (Molecular Probes, L34959) according to the manufacturer's instructions.

[0339] For dose-response curves, cells were incubated with serial dilutions of bispecific antibodies at the indicated concentrations in staining buffer FBS (Becton Dickinson, 554656) for 30 min at 4° C. After a washing step, cells were incubated with secondary detection antibody (mouse anti-human IgG FC PE, Southern Biotech, 9040-09) for 15 min at 4° C., followed by a washing step. 4 The number of viable cells / well was measured using a flow cytometer.

[0340] Analysis was performed using FlowJo™ software (Becton Dickinson). Cells were first gated by FSC / SSC. From the resulting cell population, single cells and subsequent viable cells were gated. Binding was analyzed as the median fluorescence intensity (MFI) of the secondary antibody. Curves and plots were generated using GraphPad Prism software (Domatics).

[0341] Binding to human primary cells The binding of bispecific antibodies to human T cells was also tested. Briefly, human PBMCs isolated from two different healthy donors were plated at 200,000 cells / well in U-bottom 96-well culture plates (NUNC #163320) precoated with OKT3 (Invitrogen 16-0037-85, used at 5 μg / mL, diluted in PBS, overnight at 4°C). As a control condition, PBMCs were cultured in an additional plate without OKT3 activation. Cells were cultured in X-VIVO™ 15 with phenol red (Lonza, #BE02-060Q) for 48 hours and washed with FACS buffer (PBS, 2 mM ETA, 2% FCS). The activated PBMCs were then incubated with serially diluted bispecific antibodies ranging from 600 nM to 0.003 nM for 30 minutes at 4°C. After incubation, cells were centrifuged and labeled with 5 μg / mL PE-conjugated mouse anti-human IgG Fab secondary antibody (Invitrogen, #MA1-10377) diluted in FACS buffer. The stained cells were incubated for 45 minutes at 4°C, washed, and then incubated with 25 μL of FACS buffer containing a mixture of anti-CD4 BV711 (BD Biosciences #563028), anti-CD8 BV786 (BD Biosciences #563823), and CD25 BV421 (Biolegend #356114) for 30 minutes at 4°C. Finally, the stained cells were washed with FACS buffer and incubated with 50 μL of the viability dye eFluor™ 780 diluted 1 / 3000 in PBS for 20 minutes in the dark at 4°C. After the final washing step, the cells were analyzed on a Fortessa X-20 flow cytometer (BD Biosciences). For analysis, cells were gated on FSC / SSC and on either CD4 or CD8 populations. Binding was analyzed as the median fluorescence intensity (MFI) of the secondary antibody. Curves and plots of associated EC50 values ​​were generated using GraphPad Prism software.

[0342] As shown in Figures 5A, 5B, and 5C, each of the bispecific antibodies L1 to L6 was able to bind either CD28 or OX40 on the surface of target cells.

[0343] Example 4. In vitro functional data Allogeneic mixed leukocyte reaction (MLR) Human CD14+ monocytes were isolated from PBMCs and cultured with GM-CSF and IL-4 cytokines for 5 days. The resulting immature dendritic cells were activated for 3 days with a cytokine cocktail consisting of GM-CSF, IL-4, IL-1b, IL-6, TNFα, and PGE2, and incubated with 1×10 dendritic cells (1×10 dendritic cells) with or without negative and positive controls or test compounds, used at 6 nM at the start of the assay. 4 Carboxyfluorescein succinimidyl ester (CFSE)-labeled allogeneic PBMCs (1 × 10 4 Mature DCs were co-cultured with 2 × 10 mature DCs and 2 × 10 PBMCs. After 4–7 days, cytokine levels were measured in collected supernatants, and T cell proliferation was measured using CFSE dilution. In parallel, samples were collected at different time points from day 0 to day 7 and stained with the viability dye eFluor 506 (eBioscience), anti-CD3 BUV805 (BD Bioscience), anti-CD4 BUV395 (BD Biosciences), anti-CD8 BUV3496 (BD Biosciences), anti-CD25 BV421 (BD Biosciences), anti-CD45RA FITC (eBioscience), and anti-CCR7 PE (eBioscience) for 30 min at 4 °C. Samples were then washed, diluted in PBS containing 10% Scintillator Bright beads (Invitrogen #C36950), and analyzed on a Fortessa X-20 flow cytometer (BD Biosciences). Natural memory T cells and central memory T cell subpopulations were identified as CD45RA+CD25 low and CD45RA-CCR7 hi It was defined as follows.

[0344] Dose-dependent inhibition of cytokine production (e.g., IL-2, IL-5, IL-13, GMCSF) was measured by the bispecific antibody (12 concentrations of test compound). High potency (subnanomolar EC50) was found for the bispecific antibody (Figure 6A). The efficacy was superior to that of competitors abatacept, belatacept, and the benchmark anti-CD28 molecule (Figure 6A). Furthermore, the bispecific antibody selectively targeted alloreactive memory T cell proliferation compared to depletion of the anti-CD52 benchmark antibody (Figure 6B).

[0345] Derp1 allergen assay The bispecific antibodies were evaluated in the MRC5-PBMC coculture system. MRC5 cells were grown in EMEM medium (Sigma 4655) supplemented with 10% FCS, HEPES, NEAA, and 1 mM pyruvate. For experiments, MRC5 cells were dissociated using activase stempro (Thermofischer), washed, and cultured for 5.10 e in AIMV medium containing 5% serum replacement (Gibco) in 96-well flat-bottom cell culture plates. +5 The cells were thawed and replated overnight at 10e6 cells / mL. In parallel, frozen house dust mite-susceptible PBMCs from four different donors were thawed, stained with 5 μM CFSE, and diluted in XVIVO15 medium at 4.10e6 cells / mL. CFSE-stained PBMCs (50 μL) were added to MRC5 cells (50 μL) in the presence of 50 μL of Dermatophagoides pteronyssinus (DerP, Indoor Biotechnologies: final concentration of 3 μg / mL) and 50 μL of treatment antibody (4X). After 7 days, culture supernatants were collected for cytokine measurements (e.g., IL-5, IL-13), and cells were harvested and stained with anti-CD4 and CD8 antibodies to measure the percentage of CFSE-labeled CD4+ and CD8+ T cell proliferation. As shown in Figures 7A and 7B, each of the bispecific antibodies L1 to L6 was able to suppress the production of IL-5 and IL-13.

[0346] Antigen (CMV) Recall Assay Bispecific antibodies were tested in an antigen recall assay using the CMVpp65 antigen. Frozen PBMCs from a minimum of five donors were thawed and diluted in XVIVO15 medium containing 5 μM CTV. After a 10-minute incubation (37°C, 5% CO), the cells were centrifuged (400g, 5 minutes) and plated at 2 x 10 cells per well in a 96-well U-bottom culture dish (Thermofisher) in the presence of 50 μL of CMVpp65 (diluted 1 / 250, Miltenyi) and 50 μL of treatment antibody (6 nM final concentration). 5 The cells were seeded at 100 μL / well. After 5 days, the culture supernatant was collected for cytokine measurement, and the cells were harvested on the 7th day.

[0347] As shown in Figures 7C and 7D, each of the bispecific antibodies L1 to L6 was able to suppress IFNγ production and T cell proliferation.

[0348] T cell activation assay Each arm and the monoblocks for the bispecific antibodies were tested in T cell activation cells in the presence of anti-CD3 Ab. U-bottom 384-well plates were coated overnight at 4°C with 5 μg / mL anti-human CD3 antibody (eBiosciences, 15288347, OKT3 clone). The plates were washed with PBS, and 50,000 T cells were added in complete X-VIVO15 culture medium (Lonza, BE02-060F) in the presence of 10, 30, and 100 nM negative and positive control antibodies or test compounds and incubated at 37°C in a 5% CO2 incubator. After 6 days of incubation, supernatants were collected and stored at -20°C until cytokine measurement. CELLTITER-GLO reagent was added to the cells for cell counting. Cytokine levels were measured using a homogeneous time-resolved fluorescent human IFNγ / tumor necrosis factor (TNF)α cytokine kit according to the manufacturer's instructions (Cisbio). Samples were read on a PHERAstar FSX multimode reader (BMG Labtech). Data were expressed as a percentage of effect compared to the isotype control. No enhancement was measured in either T cell proliferation or cytokine secretion (IFNg, TNFα).

[0349] MIMIC-based cytokine release assay (MIMIC-CRA) A MIMIC-based CRA assay was used for its sensitivity in detecting CD28 agonist activity and was performed as described by Dhir et al. (J Immunotoxicol. 2012.9(1):34-42). Reconstituted leukocytes and autologous platelet-deficient plasma were derived from 10 independent donors. Test articles at six different concentrations and controls were added 24 hours before supernatants were collected for multiplexed cytokine analysis by Luminex.

[0350] As shown in Figures 13A-13F, bispecific antibodies L1-L6 did not stimulate the production of the pro-inflammatory cytokines TNFα, IFNγ, IL-2, IL-5, IL-6, or IL-10, respectively.

[0351] Anti-CD3 pre-activated PBMC-based CRA assay (PBMC-CRA) OX40 expression was induced using a PBMC-based CRA assay preactivated with anti-CD3, allowing not only CD28 but also OX40 agonist activity to be detected in the same assay. Cryopreserved PBMCs from 12 independent donors were cultured at 2 × 10 cells per 96-well plate in serum-free medium containing 500 ng / mL anti-CD3 antibody (clone UCHT1) for 24 hours. 6 Test articles at six different concentrations and controls were added for a further 24 hours before supernatants were collected for multiplexed cytokine analysis by Luminex.

[0352] As shown in Figures 13G-N, no significant reduction in pro-inflammatory cytokines was observed for any of the bispecific antibodies L1-L6 compared to the anti-CD3 control.

[0353] High-density PBMC assay To evaluate the ability of bispecific antibodies to induce cytokine release through their anti-CD28 moieties, we used the high-density PBMC assay (HDPBMC) described by Romer et al. in 2011 (Blood). Briefly, frozen PBMCs from a minimum of eight healthy donors were thawed and pre-cultured in complete RPMI 1640 L-glutamine medium (RPMI 1640 L-glutamine + 10% FCS, penicillin / streptomycin, non-essential amino acids, sodium pyruvate) at 10 x 10 cells / mL per well in 24-well culture dishes for 2 days. The cells were then harvested, centrifuged (5 min, 400 g, 4°C), and cultured in complete AIMV medium at 200,000 cells / well in 96-well flat-bottom cell culture dishes (TPP, 150 μL cells per well) in the presence of 50 μL of bispecific antibody or control antibody diluted in complete AIMV medium at a final concentration of 60 or 6 nM. OKT3 and the superagonist anti-CD28 TGN1412 were used as positive control mAbs, and mouse IgG2a, human IgG1, and IgG4 mAbs were used as isotype controls. After one day, culture supernatants were collected and analyzed for the presence of cytokines (IL-2, IL-6, IL-10, IFNγ, and TNFα) using a BD™ Cytometric Bead Array Flex according to the manufacturer's instructions.

[0354] Treg-mediated suppression assay The ability of bispecific prototype antibodies to enhance the suppressive function of Treg cells was assessed in a suppression assay in which isolated Tregs, CD4+ cells, and mature monocyte-derived DCs were co-cultured for 6 days.

[0355] Human mononuclear cell-derived DCs were generated and matured for 3 days as described in Example 4 (MLR). Frozen Treg and CD4+ Teff cells (previously isolated from human PBMCs) were thawed and labeled with 5 μM CFSE and Cell Trace Violet, respectively. After 10 minutes of incubation at 37°C (5% CO), the labeled cells were washed and counted. MoDCs and CD4+ Teff cells were co-cultured in 96-well round-bottom plates at a 1:40 ratio (1 × 10 DCs to 4 × 10 CD4+ Teffs) in the presence of Treg cells and 60 nM bispecific prototype or isotype control, seeded at various CD4+ Teff:Treg ratios.

[0356] After 6 days of culture, cells were harvested and the purification of CD4+ Teff cells was examined by CFSE dilution by flow cytometry. As shown in Figure 8, the bispecific prototype antibody enhanced the suppressive function of Treg cells.

[0357] The bispecific antibody prototypes were also compared to multiple monospecific antibodies (alone) and to a combination of multiple monospecific antibodies in a mixed lymphocyte reaction (MLR) assay. As shown in Figure 3, the bispecific antibody prototypes resulted in a reduction in IL-15 expression compared to multiple monospecific antibodies (alone) or a combination of multiple monospecific antibodies.

[0358] The bispecific antibody prototypes were also compared to multiple monospecific antibodies (alone) and a combination of multiple monospecific antibodies in a humanized GvHD model. As shown in Figures 9A and 9B, the bispecific antibody prototypes resulted in reduced GvHD scores compared to multiple monospecific antibodies (alone) or a combination of multiple monospecific antibodies.

[0359] As shown in Figures 9C-9H, treatment with the bispecific L4 antibody for either 1 week (Figures 9C-9E) or 2 weeks (Figures 9F-9H) was sufficient to reduce the progression of GVHD for each of the endpoints measured.

[0360] ADCC and CDC activity ADCC ADCC assays were performed using Jurkat and HEK293 cells stably expressing huOX40 as target cells and human PBMCs as effector cells. HEK-OX40 and Jurkat OX40 cells (target cells) were diluted in a calcein-containing solution (RPMI1640 WO Red Phenol, 1% FCS, and 10 μg / mL calcein (Invitrogen, C3100MP)) and incubated at 37°C for 10 minutes. The cells were then washed and incubated with serially diluted bispecific antibodies or control antibodies in ADCC medium (RPMI1640 WO Phenol Red, 1% FCS, and 1X probenecid) for 30 minutes at 4°C. Calcein-labeled cells were then seeded into 96-well round-bottom culture dishes containing PBMCs pre-activated overnight with 20 ng / mL recombinant huIL-15 (1 x 10 target cells + 2.5 x 10 PBMCs / well). After 4 hours of incubation at 37°C in 5% CO, culture supernatants were collected and calcein fluorescence was measured using an Envision reader (PerkinElmer).

[0361] CDC Human whole blood was collected from various donors in heparinized tubes, and PBMCs were isolated by density gradient centrifugation. These PBMCs were then washed (without disruption, by centrifugation at 350 g for 30 minutes at room temperature in RPMI 1640 10% FCS) and incubated with or without 50% serum prepared from allogeneic whole blood (50 μL PBMCs + 50 μL serum, or medium + 5 μL of 10 treatments) in the presence of various treatments at 37°C and 5% CO2 for 3.5 hours. Cells were then washed twice in PBS (by centrifugation at 450 g for 5 minutes), stained with Fixable Viability Dye eFluor (BD Biosciences) and anti-CD3, anti-CD4, and anti-CD8 antibodies (BD Biosciences), and fixed in 4% PFA solution for 30 minutes at 4°C. The fixed cells were then diluted in PBS containing 10% shining bright beads (Invitrogen #C36950), washed, and acquired using a BD Fortessa LSR X20 flow cytometer. The absolute numbers of viable CD4+ and CD8+ T cells were calculated.

[0362] As shown in Figures 14A and 14B, the bispecific antibody lacked detectable ADCC activity in Jurkat (Figure 14A) and HEK293 (Figure 14B) assay systems. As shown in Figures 14C and 14D, the bispecific antibody lacked detectable CDC activity induced in CD4 and CD8 assay systems.

[0363] Example 5. In vivo activity of bispecific antibodies Delayed-type hypersensitivity (DTH) is a short-term model for evaluating the effects of compounds on cellular immune responses. This type of immune response primarily involves T cells, not antibodies. hCD28 / hOX40 double knock-in mice were immunized with methylated bovine serum albumin (mBSA) emulsified in complete Freund's adjuvant (CFA). Five days after immunization, the mice were boosted with soluble mBSA into one of their hind paw pads. Paw thickness was measured and the amount of swelling was determined immediately before and 24 hours after the boost. Subcutaneous administration of the bispecific antibody on days 3 and 5 showed a significant reduction in paw swelling compared to the isotype control (Figures 11A and 11B).

[0364] The effect of test compounds on antibody production was evaluated using T cell-dependent antibody responses (TDAR). hCD28 / hOX40 double knock-in mice were immunized with keyhole limpet hemocyanin (KLH). 14 days after the first immunization, the mice received a second immunization with KLH. Starting 5 days before the first immunization, animals were administered bispecific antibodies or an isotype control twice a week. 21 days after the first immunization, serum levels of anti-KLH IgG were measured as the primary endpoint. Mice treated with bispecific antibodies (e.g., L1, L3, or L4) showed significantly lower serum levels of anti-KLH IgG compared with the isotype control (Figures 12A and 12B).

[0365] GvHD was used as a POM model. Fresh human PBMCs were transferred into NSG mice. The bispecific antibody was administered IP. The bispecific prototype antibody showed in vivo activity in this model when tested at a fixed concentration (0.012 mg). The effect was observed in survival, total GVHD score, and a reduction in the memory CD4+ T cell population (Figure 9A). This therapeutic effect of the bispecific was superior to the clinical benchmark abatacept, or anti-CD28 or anti-OX40 monotherapy, as well as combined anti-CD28 and anti-OX40 treatments used at equimolar concentrations (Figure 9B).

[0366] Other GvHD studies were performed to examine the protective efficacy of bispecific antibody L4 when administered therapeutically twice weekly ip at a dose of 0.12 mg for only 1 or 2 weeks. Data from these dose-duration studies indicate that treatment with L4 for either 1 or 2 weeks was sufficient to reduce GvHD progression for each of the endpoints measured, providing more effective protection from GvHD disease than abatacept or the bispecific clinical benchmark molecule targeting two costimulatory receptors (Figures 9C-H).

[0367] Example 6. PK characterization of bispecific antibodies The pharmacokinetic profile of the bispecific antibody was characterized in hFcRn Tg32 mice, where the bispecific antibody exhibited slow clearance (CL) and a long half-life (T), within the range of expected PK parameters for a mAb / Fc asset (Figure 10, Table 6).

[0368] [Table 12]

[0369] Example 7. A randomized, double-blind, placebo-controlled study of the safety, tolerability, pharmacokinetics, and pharmacodynamics of ascending single and multiple doses of antigen-binding protein L4 in healthy adult participants A Phase 1 clinical trial is planned to investigate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ascending single and multiple doses of antigen-binding protein L4 in healthy female and male participants aged 18-55 years.

[0370] Intervention Description: Formulation: Powder for injection and infusion solution supplied in single-use glass vials. Each vial contains 100 mg of antigen-binding protein L4. To prevent the antigen-binding protein L4 from adsorbing to the bag, the infusion bag is coated with a pre-coating solution supplied with the antigen-binding protein L4. Route of administration: Intravenous (IV) infusion or subcutaneous (SC) infusion.

[0371] Dosage regimen: Part 1: The single ascending dose (SAD) study will include up to nine cohorts treated with ascending doses, each with a single dose, with five cohorts receiving IV infusions in the expected dose range of 0.3 to 30 mg IV infusion, followed by up to four cohorts receiving SC infusions in the expected dose range of 75 to 500 mg or less.

[0372] Part 2: The multiple ascending dose (MAD) study will include up to four cohorts treated with ascending SC doses, with projected doses of up to 30, 60, or 120 mg. Dosing will be by SC infusion Q2W, for a total of three doses. In any MAD cohort (MAD4), any dose lower or higher than those in MAD1-MAD3 can be selected, as long as the resulting projected exposure is less than the maximum exposure in the SAD study.

[0373] SC administration will be used for cohorts 6 and above in Parts 1 and 2, as this will be the administration method later in clinical development. Regarding safety characteristics, cohorts 1-5 in Part 1 are planned to be administered by IV infusion. The infusion duration is planned to be approximately 60 minutes, with an opportunity to stop the infusion if an acute AE is observed.

[0374] Doses in the SAD and MAD studies may vary based on emerging safety data and PK (pharmacokinetics), PD (pharmacodynamics), and ADA (anti-drug antibody) results. Participants in the same group will receive only one part. Nurses will administer the doses.

[0375] Control treatment: Placebo IV: Formulation: Isotonic saline. Route of administration: Intravenous (IV) injection. Dosing regimen: For the final formulation of IMP, at the same frequency and volume as antigen binding protein L4, and with the same posture requirements, in the corresponding cohort.

[0376] Placebo SC: Formulation: Isotonic saline solution for injection, supplied in single-use glass vials. Each vial contains 8 mL of placebo. Route of administration: Subcutaneous (SC) injection. Dosing regimen: For the final formulation of IMP (Investigational Medicinal Product), same frequency and same volume as antigen binding protein L4, and same posture requirements and options as antigen binding protein L4, in the corresponding cohort.

[0377] Outcomes: Primary outcomes: Number of participants with adverse events (AEs) / treatment-emergent adverse events (TATEs), including reactions at the injection site: by assessment of vital signs (heart rate, systolic and diastolic blood pressure [using automated BP monitoring, in sitting and standing positions], temperature [using ear thermometer], clinical laboratory assessments (hematology, biochemistry, coagulation, urinalysis) assessed using blood samples, and 12-lead ECG).

[0378] time Part 1 Cohorts 1-2: Baseline to End of Study (Day 85): Vital signs investigated at baseline, D1 (pre-dose, 0.5, 1, 4, 8 hours post-dose), D2, D3, D4, D5, D6, D8, D15, D22, D29, D36, D43, D57, and D85. -Blood samples collected at baseline, D2, D6, D8, D15, D22, D29, D36, D43, D57, and D85. - 12-lead ECG performed at baseline, D1 (0.5, 1, 4, 8 hours post-dose), D2, D3, D4, D5, D6, D8, D15, D22, D29, D36, D43, D57, and D85.

[0379] Part 1 Cohorts 3-9: Vital signs investigated at baseline, D-12, D1 (pre-dose, 0.5, 1, 4, 8 hours post-dose), D2, D3, D4, D5, D6, D8, D15, D22, D29, D36, D43, D57, and D85. Blood samples collected at baseline, D2, D6, D8, D15, D22, D29, D36, D43, D57, and D85 - 12-lead ECG performed at baseline, D1 (0.5, 1, 4, 8 hours post-dose), D2, D3, D4, D5, D6, D8, D15, D22, D29, D36, D43, D57, and D85.

[0380] Part 2: Baseline to End of Study (Day 113) Vital signs taken at baseline, D1 (before the first dose and 8 hours after the first dose), D2, D3, D4, D8, D9, D15 (before the second dose and 8 hours after the second dose), and D16, D17, D18, D22, D23, D29 (before the third dose and 8 hours after the third dose), D30, D31, D32, D36, D43, D50, D57, D71, D85, and D113. -Blood samples collected at baseline and on D2, D3, D4, D8, D14, D16, D17, D18, D22, D28, D30, D31, D32, D36, D43, D50, D57, D71, D85, and D113. - 12 reads performed on D1 (before the first dose, 8 hours after the dose), D2, D3, D4, D8, D15 (before the second dose, 8 hours after the dose), D18, D29 (before the third dose, 8 hours after the dose), D30, D31, D32, D36, D43, D50, D57, D71, D85, and D113.

[0381] Secondary Outcome 1: Part 1: Investigation of pharmacokinetic (OK) parameters using blood samples: IV:Cmax, Tmax, AUClast, AUC, t1 / 2z, CL, Vss SC:Cmax, tmax, AUClast, AUC, F, t1 / 2z, CL / F, Vz / F Part 2: Investigation of pharmacokinetic (OK) parameters using blood samples: D1AUCtau, tmax, Cmax) D15 (Ctrough) ·D29(Cmax, tmax, AUCtau, Ctrough, t1 / 2z) Time point for secondary outcome 1 Part 1: Baseline to End of Study (Day 85) Cohorts 1-5 (IV administration): Blood samples collected at: baseline, D1 (0.5, 1, 4, 8 hours post-dose), D2, D3, D4, D5, D6, D8, D15, D22, D29, D36, D43, and D85. Cohorts 6-9 (SC administration): Blood samples collected at: baseline, D1 (8 hours post-dose), D2, D3, D4, D5, D6, D8, D15, D22, D29, D36, D43, and D85. Part 2: Baseline to End of Study (Day 113) Blood samples were collected on the following days: D1 (before the first dose, 8 hours after administration), D2, D3, D4, D8, D15 before the second dose, D29 (before the third dose, 8 hours after administration), and D30, D31, D32, D36, D43, D50, D57, D71, D85, and D112.

[0382] Secondary Outcome 2: Anti-drug antibodies against antigen-binding protein L4 using blood samples. Secondary outcome 2 time point Part 1: Baseline to End of Study (Day 85) Blood samples collected at: D1, D15, D29 and D85. Part 2: Baseline to End of Study (Day 113) Blood samples collected on: D1, D15 (before the second dose), D29 (before the third dose), and D113.

[0383] Secondary Outcome 3: Circulating immunoglobulin G antibodies against KLH (keyhole limpet hemocyanin) using blood samples. Time point for secondary outcome 3 Part 1: Blood samples collected on the following days: D-12 before KLH administration, D-5, D1 before administration, D3 before KLH injection, D10, D17 (before ID injection of KLH), D22, D29, and D36. Part 2: Blood samples were collected on the following days: D1 pre-dose, D8 (before KLH injection), D15 (before second dose), D22 (before KLH injection), D29 (before third dose), D36 (before KLH injection), D43, D50, and D57.

[0384] Secondary Outcome 4: Skin erythema measured photographically. Time point for secondary outcome 4 Cohorts 3-9 in Part 1 Performed on: D17 (before KLH ID injection), D18, and D19. Part 2: Performed on: D36 (before KLH injection), D37, and D38.

[0385] Secondary Outcome 5: Skin blood perfusion measured by laser speckle contrast imaging (LSCI). Secondary outcome 5 time point Cohorts 3-9 in Part 1 Performed on: D17 (before KLH ID injection), D18, and D19. Part 2: Performed on: D36 (before KLH injection), D37, and D38.

[0386] Eligibility: Main inclusion criteria Participants were apparently healthy as determined by medical evaluation including medical / surgical history, physical examination, laboratory tests, and cardiac monitoring. For men, weight was 50.0–110.0 kg (inclusive), and for women, BMI was 40.0–90.0 kg (inclusive) and 18.0–32.0 kg / m2 (inclusive).

[0387] Female participants who are not pregnant or breastfeeding and for whom one of the following conditions applies: - Are women of non-childbearing potential (WONCBP), or -A woman of childbearing potential (WOCBP) and agrees to use highly effective contraception. - WOCBP must have a negative high-sensitivity pregnancy test (urine or serum as required by local regulations) within 36 hours prior to the first dose of study intervention.

[0388] A male participant agrees that: -Refrain from donating sperm; -Agree to abstain from heterosexual intercourse and alcohol (long-term and sustained abstinence) and remain abstinent as part of a normal, desirable lifestyle; or -Must agree to use contraception / barrier. age: 18 to 55 years old. sex: Men and women

[0389] Key exclusion criteria: Participants were excluded from the study if any of the following criteria applied: -Any history or presence of clinically relevant cardiovascular, pulmonary, gastrointestinal, hepatic, renal, metabolic, hematological, neurological, muscular, articular, psychiatric, systemic, ophthalmological, gynecological (if female), immunological or infectious disease, or signs of acute illness. -Current or past history of drug hypersensitivity or allergic disease diagnosed and treated by a physician. -Anaphylaxis of any cause. -Known hypersensitivity to any component of the IMP formulation. -Prior exposure to KLH or hypersensitivity to shellfish. -History of mild, controlled allergies may be included at the investigator's discretion. Any immunization with a non-live vaccine, including against COVID-19, within 4 weeks of enrollment (SAD study cohorts 1 and 2, MAD study) or within 2 weeks of enrollment (SAD study cohorts 3 and above). - Any immunization with a live vaccine within 3 months of enrollment. - Symptomatic herpes zoster within 3 months prior to screening. - History of recurrent oral or genital herpes. -Evidence of active or latent TB documented by medical history and examination, chest x-ray or positive QuantiFERON-TB Gold Plus test. - History of invasive opportunistic infections, regardless of recovery, including histoplasmosis, listeriosis, coctidioidomycosis, candidiasis, pneumocystis jirovecii, and aspergillosis.

[0390] Dosage scheme:

[0391] [Table 13]

[0392] [Table 14]

[0393] [Table 15]

[0394] [Table 16]

[0395] [Table 17]

[0396] Table 18

[0397] Table 19

[0398] Table 20

[0399] Table 21

[0400] Table 22

Claims

1. 1. A multispecific binding protein comprising: (a) a first antigen-binding domain (ABD) comprising an immunoglobulin single variable domain (ISVD) with binding specificity for CD28; and (b) a second ABD comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the second ABD has binding specificity for OX40; A multispecific binding protein, wherein when said multispecific binding protein binds to CD28 and OX40, said multispecific binding protein inhibits activated T cells.

2. 10. The multispecific binding protein of claim 1, wherein the multispecific binding protein inhibits T cell proliferation.

3. 3. The multispecific binding protein of claim 1 or 2, wherein the multispecific binding protein inhibits the expression of one or more pro-inflammatory cytokines.

4. 4. The multispecific binding protein of claim 3, wherein the proinflammatory cytokines are interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 2 (IL-2), interleukin 5 (IL-5), interleukin 6 (IL-6), and interleukin 10 (IL-10).

5. 5. The multispecific binding protein of claim 1, further comprising an immunoglobulin Fc domain or variant thereof, wherein said Fc domain or variant thereof comprises a first Fc heavy chain and a second Fc heavy chain.

6. 7. The multispecific binding protein of claim 6, wherein the first ABD binds to the first Fc heavy chain and the second ABD binds to the second Fc heavy chain.

7. 7. The multispecific binding protein of claim 1, wherein the first ABD is linked to the N-terminus of the second ABD VH.

8. 8. The multispecific binding protein of any one of claims 5 to 7, wherein the first ABD binds to the C-terminus of the second Fc heavy chain and the second ABD binds to the N-terminus of the second Fc heavy chain.

9. 9. The multispecific binding protein of any one of claims 5 to 8, wherein the VH binds to a CH1 domain, the VL binds to a constant light (CL) domain, and the first ABD binds to the C-terminus of the CL domain.

10. 10. The multispecific binding protein of any one of claims 1 to 9, wherein the first ABD binds to one or more amino acids E32, E46, V47, C48, Y51, G52, N53, S55, Q57, L58, Q59, V60, Y61, S62, K63, T64, N67, C68, and D69 of SEQ ID NO:

46.

11. 11. The multispecific binding protein of any one of claims 1 to 10, wherein the first ABD binds to amino acids E32, E46, V47, C48, Y51, G52, N53, S55, Q57, L58, Q59, V60, Y61, S62, K63, T64, N67, C68, and D69 of SEQ ID NO:

46.

12. 12. The multispecific binding protein of any one of claims 1 to 11, wherein the second ABD binds to one or more amino acids V22, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, W55, R64, L67, C68, T69, and A70 of SEQ ID NO:

47.

13. 13. The multispecific binding protein of any one of claims 1 to 12, wherein the second ABD binds to amino acids V22, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, W55, R64, L67, C68, T69, and A70 of SEQ ID NO:

47.

14. (a) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of INTDGDFT (SEQ ID NO: 2); and HCDR3 sequence comprising the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3) a first antigen-binding domain (ABD) having binding specificity for CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 4); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 5); and HCDR3 sequence comprising the amino acid sequence of ARGEAYWYRWAFDY (SEQ ID NO: 6) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) an LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 7); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 8); and LCDR3 sequence comprising the amino acid sequence of QQYSDYSYT (SEQ ID NO: 9) a second ABD having binding affinity to OX40 comprising an immunoglobulin light chain variable domain (VL) comprising:

14. The multispecific binding protein of any one of claims 1 to 13, comprising:

15. 15. The multispecific binding protein of any one of claims 1 to 14, wherein the ISVD comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 10, and includes the following amino acids compared to SEQ ID NO: 10: Y33, W47, T50, N52, D56, F57, T58, S59, K65, P102, Y103, S104, and R105.

16. 16. The multispecific binding protein of any one of claims 1 to 15, wherein the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 11 and comprises amino acids H35, S52, Q54, G56, S57, T58, Y59, Y102, Y104, R105, and W106 compared to SEQ ID NO: 11; and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 12 and comprises amino acids W32, D50, and S92 compared to SEQ ID NO:

12.

17. the ISVD comprises the amino acid sequence of SEQ ID NO: 10; and The VH comprises the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO:

12. The multispecific binding protein of any one of claims 1 to 16.

18. (a) HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14); and HCDR3 sequence comprising the amino acid sequence of SARIRTSGGGGGWSTY (SEQ ID NO: 15) a first antigen-binding domain (ABD) having binding specificity for CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 4); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 5); and HCDR3 sequence comprising the amino acid sequence of ARGEAYWYRWAFDY (SEQ ID NO: 6) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) an LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 7); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 8); and LCDR3 sequence comprising the amino acid sequence of QQYSDYSYT (SEQ ID NO: 9) An immunoglobulin light chain variable domain (VL) comprising a second ABD having binding affinity to OX40 comprising 14. The multispecific binding protein of any one of claims 1 to 13, comprising:

19. 20. The multispecific binding protein of any one of claims 1 to 13 and 18, wherein the ISVD comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO:

16.

20. 20. The multispecific binding protein of any one of claims 1 to 13, 18, and 19, wherein the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 11 and includes amino acids H35, S52, Q54, G56, S57, T58, Y59, Y102, Y104, R105, and W106 compared to SEQ ID NO: 11; and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 12 and includes amino acids W32, D50, and S92 compared to SEQ ID NO:

12.

21. the ISVD comprises the amino acid sequence of SEQ ID NO: 16; and The VH comprises the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO:

12.

21. The multispecific binding protein of any one of claims 1 to 13 and 18 to 20.

22. 10. The multispecific binding protein of any one of claims 1 to 9, wherein the second ABD binds to one or more amino acids R16, N19, V22, V32, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, T54, W55, C56, R64, L67, C68, T69, A70, and T74 of SEQ ID NO:

47.

23. 23. The multispecific binding protein of any one of claims 1-9 or 22, wherein the second ABD binds to amino acids R16, N19, V22, V32, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, T54, W55, C56, R64, L67, C68, T69, A70, and T74 of SEQ ID NO:

47.

24. (a) HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14); and HCDR3 sequence comprising the amino acid sequence of SARIRTSGGGGGWSTY (SEQ ID NO: 15) a first antigen-binding domain (ABD) having binding specificity for CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 25); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 26); and HCDR3 sequence comprising the amino acid sequence of ARGGSGWYNSEFDY (SEQ ID NO: 27) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) an LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 28); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 29); and an LCDR3 sequence comprising the amino acid sequence of QQYNDYSYT (SEQ ID NO: 30); An immunoglobulin light chain variable domain (VL) comprising a second ABD having binding affinity to OX40 comprising:

24. The multispecific binding protein of any one of claims 1 to 9, 22, or 23, comprising:

25. 25. The multispecific binding protein of any one of claims 1 to 9 and 22 to 24, wherein the ISVD comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO:

16.

26. 26. The multispecific binding protein of any one of claims 1 to 9 and 22 to 25, wherein the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 31 and includes the amino acids A33, H35, A50, 151, S52, S53, N54, G55, G56, S57, T58, Y59, Y60, N74, S101, W103, Y104, N105, S106, and E107 compared to SEQ ID NO: 31; and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 32 and includes the amino acids W32, Y91, N92, Y94, and Y96 compared to SEQ ID NO:

32.

27. the ISVD comprises the amino acid sequence of SEQ ID NO: 16; and The VH comprises the amino acid sequence of SEQ ID NO: 31, and the VL comprises the amino acid sequence of SEQ ID NO:

32.

27. The multispecific binding protein of any one of claims 1 to 9 and 22 to 26.

28. (a) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of INTDGDFT (SEQ ID NO: 2); and HCDR3 sequence comprising the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3) a first antigen-binding domain (ABD) having binding specificity for CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 25); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 26); and an immunoglobulin heavy chain variable domain (VH) comprising an HCDR3 sequence comprising the amino acid sequence of ARGGSGWYNSEFDY (SEQ ID NO: 27); and (b2) an LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 28); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 29); and LCDR3 sequence comprising the amino acid sequence of QQYNDYSYT (SEQ ID NO: 30) An immunoglobulin light chain variable domain (VL) comprising a second ABD having binding affinity to OX40 comprising 24. The multispecific binding protein of any one of claims 1 to 11, 22, and 23, comprising:

29. 30. The multispecific binding protein of any one of claims 1-11, 22, 23, and 28, wherein the ISVD comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 10, and includes the following amino acids compared to SEQ ID NO: 10: Y33, W47, T50, N52, D56, F57, T58, S59, K65, P102, Y103, S104, and R105.

30. 30. The multispecific binding protein of any one of claims 1 to 11, 22, 23, 28, and 29, wherein the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 31 and comprises the amino acids A33, H35, A50, 151, S52, S53, N54, G55, G56, S57, T58, Y59, Y60, N74, S101, W103, Y104, N105, S106, and E107 compared to SEQ ID NO: 31; and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 32 and comprises the amino acids W32, Y91, N92, Y94, and Y96 compared to SEQ ID NO:

32.

31. the ISVD comprises the amino acid sequence of SEQ ID NO: 10; and The VH comprises the amino acid sequence of SEQ ID NO: 31, and the VL comprises the amino acid sequence of SEQ ID NO:

32. The multispecific binding protein of any one of claims 1 to 11, 22, 23, and 28 to 30.

32. (a) an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of INTDGDFT (SEQ ID NO: 2); and HCDR3 sequence comprising the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3) a first antigen-binding domain (ABD) having binding specificity to CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GYTFTSYG (SEQ ID NO: 17); an HCDR2 sequence comprising the amino acid sequence of ISAYTGNT (SEQ ID NO: 18); and HCDR3 sequence comprising the amino acid sequence of ARDGYPIDY (SEQ ID NO: 19) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) an LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 20); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 21); and LCDR3 sequence comprising the amino acid sequence of QQYTSSYSDT (SEQ ID NO: 22) An immunoglobulin light chain variable domain (VL) comprising a second ABD having binding affinity to OX40 comprising 12. The multispecific binding protein of any one of claims 1 to 11, comprising:

33. 33. The multispecific binding protein of any one of claims 1 to 11 and 32, wherein the ISVD comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 10, and includes the following amino acids compared to SEQ ID NO: 10: Y33, W47, T50, N52, D56, F57, T58, S59, K65, P102, Y103, S104, and R105.

34. 34. The multispecific binding protein of any one of claims 1 to 11, 32, and 33, wherein the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 23 and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO:

24.

35. the ISVD comprises the amino acid sequence of SEQ ID NO: 10; and The VH comprises the amino acid sequence of SEQ ID NO: 23, and the VL comprises the amino acid sequence of SEQ ID NO:

24.

35. The multispecific binding protein of any one of claims 1 to 11 and 32 to 34.

36. (a) HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14); and an HCDR3 sequence comprising the amino acid sequence of SARIRTSGGGGWSTY (SEQ ID NO: 15); a first antigen-binding domain (ABD) having binding specificity for CD28 comprising an immunoglobulin single variable domain (ISVD) comprising: (b) (b1) an HCDR1 sequence comprising the amino acid sequence of GYTFTSYG (SEQ ID NO: 17); an HCDR2 sequence comprising the amino acid sequence of ISAYTGNT (SEQ ID NO: 18); and HCDR3 sequence comprising the amino acid sequence of ARDGYPIDY (SEQ ID NO: 19) an immunoglobulin heavy chain variable domain (VH) comprising: (b2) an LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 20); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 21); and LCDR3 sequence comprising the amino acid sequence of QQYTSSYSDT (SEQ ID NO: 22) An immunoglobulin light chain variable domain (VL) comprising a second ABD having binding affinity to OX40 comprising 10. The multispecific binding protein of any one of claims 1 to 9, comprising:

37. 37. The multispecific binding protein of any one of claims 1 to 9 and 36, wherein the ISVD comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO:

16.

38. 38. The multispecific binding protein of any one of claims 1-9, 36, and 37, wherein the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO:23 and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO:

24.

39. the ISVD comprises the amino acid sequence of SEQ ID NO: 16; and The VH comprises the amino acid sequence of SEQ ID NO: 23, and the VL comprises the amino acid sequence of SEQ ID NO:

24.

39. The multispecific binding protein of any one of claims 1 to 9 and 36 to 38.

40. 12. The multispecific binding protein of any one of claims 1 to 11, wherein the second ABD binds to one or more amino acids G20, M21, V22, R24, P35, C26, G37, P38, G39, F40, Y41, S46, K48, P49, C50, K51, P52, C53, T54, and W55 of SEQ ID NO:

47.

41. 41. The multispecific binding protein of any one of claims 1 to 11 and 40, wherein the second ABD binds to amino acids G20, M21, V22, R24, P35, C26, G37, P38, G39, F40, Y41, S46, K48, P49, C50, K51, P52, C53, T54, and W55 of SEQ ID NO:

47.

42. 42. The multispecific binding protein of any one of claims 5 to 41, wherein said first Fc heavy chain comprises a Y349C substitution and said second Fc heavy chain comprises a S354C substitution according to Eu numbering.

43. 43. The multispecific binding protein of any one of claims 5 to 42, wherein the first Fc heavy chain comprises a Y349C, T366S, L368A, or Y407V substitution and the second Fc heavy chain comprises a T366W substitution according to Eu numbering.

44. 44. The multispecific binding protein of any one of claims 5 to 43, wherein said at least one Fc heavy chain comprises H435R and Y436F substitutions according to Eu numbering.

45. 45. The multispecific binding protein of any one of claims 5 to 44, wherein said at least one Fc heavy chain comprises L234A and L235A substitutions according to Eu numbering.

46. The binding protein is (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33; and (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 36; and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41; and 10. The multispecific binding protein of any one of claims 1 to 9, comprising:

47. The binding protein is (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33; and (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37; and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42; and 10. The multispecific binding protein of any one of claims 1 to 9, comprising:

48. The binding protein is (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34; and (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 38; and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41; and 10. The multispecific binding protein of any one of claims 1 to 9, comprising:

49. The binding protein is (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34; and (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 39; and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42; and 10. The multispecific binding protein of any one of claims 1 to 9, comprising:

50. The binding protein is (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 35; (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40; and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41; and 10. The multispecific binding protein of any one of claims 1 to 9, comprising:

51. The binding protein is (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 35; (ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40; and (iii) a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42; and 10. The multispecific binding protein of any one of claims 1 to 9, comprising:

52. The ISVD is V ＨＨ , humanized V ＨＨ Or Camelization V Ｈ 52. The multispecific binding protein of any one of claims 1 to 51, which is

53. 53. A pharmaceutical composition comprising the binding protein of any one of claims 1 to 52 and a pharmaceutically acceptable carrier.

54. 53. An isolated nucleic acid molecule encoding a binding protein according to any one of claims 1 to 52.

55. 53. A method of treating an autoimmune disease or disorder in a subject, comprising administering to a subject in need of treatment for said autoimmune disease or disorder the binding protein of any one of claims 1-52.

56. 56. The method of claim 55, wherein the autoimmune disease or disorder comprises connective tissue disease-interstitial lung disease (CTD-ILD).

57. 56. The method of claim 55, wherein the autoimmune disease or disorder comprises graft-versus-host disease (GvHD).

58. 1. A binding protein comprising an immunoglobulin single variable domain (ISVD) with binding specificity for CD28, wherein the ISVD binds to one or more amino acids E32, E46, V47, C48, Y51, G52, N53, S55, Q57, L58, Q59, V60, Y61, S62, K63, T64, N67, C68, and D69 of SEQ ID NO:

46.

59. 59. The binding protein of claim 58, wherein the ISVD binds to amino acids E32, E46, V47, C48, Y51, G52, N53, S55, Q57, L58, Q59, V60, Y61, S62, K63, T64, N67, C68, and D69 of SEQ ID NO:

46.

60. an HCDR1 sequence comprising the amino acid sequence of GFTFSSYY (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of INTDGDFT (SEQ ID NO: 2); and HCDR3 sequence comprising the amino acid sequence of ARARGPYSRGSQGHDY (SEQ ID NO: 3) 60. The binding protein of claim 58 or 59, comprising:

61. 61. The binding protein of any one of claims 58-60, wherein the ISVD comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 10, and includes the following amino acids compared to SEQ ID NO: 10: Y33, W47, T50, N52, D56, F57, T58, S59, K65, P102, Y103, S104, and R105.

62. 62. The binding protein of any one of claims 58 to 61, wherein the ISVD comprises the amino acid sequence of SEQ ID NO:

10.

63. 1. A binding protein comprising an immunoglobulin single variable domain (ISVD) that has binding specificity for CD28, HCDR1 sequence comprising the amino acid sequence of GSFFSIDT (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of VTSGGLT (SEQ ID NO: 14); and HCDR3 sequence comprising the amino acid sequence of SARIRTSGGGGGWSTY (SEQ ID NO: 15) A binding protein comprising:

64. 64. The binding protein of claim 63, wherein the ISVD comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO:

16.

65. 1. A binding protein having binding specificity for OX40, comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the binding protein binds to one or more amino acids V22, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, W55, R64, L67, C68, T69, and A70 of SEQ ID NO:

47.

66. 66. The binding protein of claim 65, wherein the binding protein binds to amino acids V22, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, W55, R64, L67, C68, T69, and A70 of SEQ ID NO:

47.

67. HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 4); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 5); and an HCDR3 sequence comprising the amino acid sequence of ARGEAYWYRWAFDY (SEQ ID NO: 6); and an LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 7); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 8); and LCDR3 sequence comprising the amino acid sequence of QQYSDYSYT (SEQ ID NO: 9) 67. The binding protein of claim 65 or 66, comprising:

68. 68. The binding protein of any one of claims 65 to 67, wherein the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 11, and comprises amino acids H35, S52, Q54, G56, S57, T58, Y59, Y102, Y104, R105, and W106 compared to SEQ ID NO: 11; and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 12, and comprises amino acids W32, D50, and S92 compared to SEQ ID NO:

12.

69. 69. The binding protein of any one of claims 65 to 68, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

12.

70. 1. A binding protein having binding specificity to OX40, comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the binding protein binds to one or more amino acids R16, N19, V22, V32, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, T54, W55, C56, R64, L67, C68, T69, A70, and T74 of SEQ ID NO:

47.

71. 71. The binding protein of claim 70, wherein the binding protein binds to amino acids R16, N19, V22, V32, C33, R34, P35, C36, G37, P38, G39, F40, P49, C50, C53, T54, W55, C56, R64, L67, C68, T69, A70, and T74 of SEQ ID NO:

47.

72. HCDR1 sequence comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 25); an HCDR2 sequence comprising the amino acid sequence of ISSQGGST (SEQ ID NO: 26); and an HCDR3 sequence comprising the amino acid sequence of ARGGSGWYNSEFDY (SEQ ID NO: 27); and an LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 28); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 29); and LCDR3 sequence comprising the amino acid sequence of QQYNDYSYT (SEQ ID NO: 30) 72. The binding protein of claim 70 or 71, comprising:

73. 73. The binding protein of any one of claims 70-72, wherein the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 31 and includes the amino acids A33, H35, A50, 151, S52, S53, N54, G55, G56, S57, T58, Y59, Y60, N74, S101, W103, Y104, N105, S106, and E107 compared to SEQ ID NO: 31; and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 32 and includes the amino acids W32, Y91, N92, Y94, and Y96 compared to SEQ ID NO:

32.

74. 75. The binding protein of any one of claims 70 to 74, wherein the VH comprises the amino acid sequence of SEQ ID NO: 31 and the VL comprises the amino acid sequence of SEQ ID NO:

32.

75. an HCDR1 sequence comprising the amino acid sequence of GYTFTSYG (SEQ ID NO: 17); an HCDR2 sequence comprising the amino acid sequence of ISAYTGNT (SEQ ID NO: 18); and HCDR3 sequence comprising the amino acid sequence of ARDGYPIDY (SEQ ID NO: 19) an immunoglobulin heavy chain variable domain (VH) comprising: an LCDR1 sequence comprising the amino acid sequence QSISSW (SEQ ID NO: 20); an LCDR2 sequence comprising the amino acid sequence of DAS (SEQ ID NO: 21); and LCDR3 sequence comprising the amino acid sequence of QQYTSSYSDT (SEQ ID NO: 22) An immunoglobulin light chain variable domain (VL) comprising A binding protein having binding specificity to OX40, comprising:

76. 76. The binding protein of claim 75, wherein the VH comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 23, and the VL comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO:

24.

77. 77. The binding protein of claim 75 or 76, wherein the VH comprises the amino acid sequence of SEQ ID NO: 23 and the VL comprises the amino acid sequence of SEQ ID NO:

24.

78. 1. A binding protein having binding specificity to OX40, comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the binding protein binds to one or more amino acids G20, M21, V22, R24, P35, C26, G37, P38, G39, F40, Y41, S46, K48, P49, C50, K51, P52, C53, T54, and W55 of SEQ ID NO:

47.

79. 79. The binding protein of claim 78, wherein the binding protein binds to amino acids G20, M21, V22, R24, P35, C26, G37, P38, G39, F40, Y41, S46, K48, P49, C50, K51, P52, C53, T54, and W55 of SEQ ID NO:

47.

80. 80. The binding protein of any one of claims 65 to 79, wherein the VH binds to a CH1 domain and the VL binds to a constant light (CL) domain.

81. 81. The binding protein of any one of claims 58 to 80, further comprising an immunoglobulin Fc domain or a variant thereof, wherein said Fc domain or variant thereof comprises a first Fc heavy chain and a second Fc heavy chain.

82. 82. The binding protein of claim 81 , wherein said first Fc heavy chain comprises a Y349C substitution and said second Fc heavy chain comprises a S354C substitution according to Eu numbering.

83. 83. The binding protein of claim 81 or 82, wherein the first Fc heavy chain comprises a Y349C, T366S, L368A, or Y407V substitution and the second Fc heavy chain comprises a T366W substitution, according to Eu numbering.

84. 84. The binding protein of any one of claims 81 to 83, wherein at least one Fc heavy chain comprises H435R and Y436F substitutions according to Eu numbering.

85. 85. The binding protein of any one of claims 81 to 84, wherein at least one Fc heavy chain comprises a L234A and a L235A substitution according to Eu numbering.

86. 86. A pharmaceutical composition comprising the binding protein of any one of claims 58 to 85 and a pharmaceutically acceptable carrier.

87. 86. An isolated nucleic acid molecule encoding a binding protein according to any one of claims 58 to 85.

88. 86. A method of treating an autoimmune disease or disorder in a subject, comprising administering to a subject in need of treatment for said autoimmune disease or disorder the binding protein of any one of claims 58-85.

89. 89. The method of claim 88, wherein the autoimmune disease or disorder comprises connective tissue disease-interstitial lung disease (CTD-ILD).

90. 89. The method of claim 88, wherein the autoimmune disease or disorder comprises graft-versus-host disease (GvHD).

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