Multispecific proteins and related methods

Abstract

Provided herein are multispecific proteins and compositions (e.g., pharmaceutical compositions) comprising the multispecific proteins, as well as methods for making the multispecific proteins and compositions. The multispecific proteins provided herein are useful in pharmaceutical compositions and methods, including, for example, methods for treating diseases (e.g., cancer), activating T cells, and inducing or enhancing immune responses.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Patent Application No. 63 / 413,765, filed October 6, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to multispecific proteins (and polypeptides thereof) and polynucleotides that encode them. The disclosure further relates to methods of making and using them. [Background technology]

[0003] T cell engager immunotherapy works by activating T cells (or a subset thereof, e.g., CD8+ T cells) to mediate, for example, tumor cell lysis. A subset of T cell engagers is multispecific and aims to target activated T cells to tumor cells by simultaneously binding to a T cell antigen (e.g., CD3) and an antigen expressed on the surface of the tumor cell (e.g., a tumor-associated antigen). Exemplary T cell engagers include bispecific T cell engagers (BiTEs), bifunctional checkpoint inhibitory T cell engagers (CiTEs), simultaneous multiple interacting T cell engagers (SMITEs), trispecific killer engagers (TriKEs), and BiTE-expressing chimeric antigen receptor (CAR) T cells (CART.BiTE cells). Summary of the Invention

[0004] Provided herein, among other things, are multispecific proteins (and polypeptides thereof) and polynucleotides encoding them; methods of making pharmaceutical compositions; and methods of use, including, for example, methods of treating disease (e.g., cancer) and methods of inducing an immune response.

[0005] In one aspect, provided herein is (a) (i) a first light chain comprising, from N-terminus to C-terminus, a light chain variable region (VL) region and a light chain constant region (CL) region; (ii) a first heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable region (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; a full-length antibody comprising, at its C-terminus, a second light chain comprising a VL region and a CL region, wherein the first light chain and the first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and the first heavy chain and the second heavy chain associate to form a dimer; and (b) a first single-chain variable fragment (scFv) operably linked to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody, a first single-chain variable fragment (scFv), the first scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; and (c) a second scFv operably linked to the C-terminus of the CH3 region of a second heavy chain of a full-length antibody, the second scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region, wherein the first antigen-binding domain of the full-length antibody specifically binds to a first human tumor-associated antigen (hTAA); the second antigen-binding domain of the full-length antibody specifically binds to a second hTAA; the first scFv specifically binds to a human T-cell costimulatory antigen (hTCSA) (e.g., hCD28, hCD2); and the second scFv specifically binds to human CD3 (hCD3);and the CH3 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a reference CH3 region, e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions), and the CH3 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions); and one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody are different from one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody; one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody and one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody promote heterodimerization of the first and second heavy chains of the full-length antibody; the CH2 region of the first heavy chain of the full-length antibody is compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions). the CH2 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications; and the one or more amino acid modifications in the CH2 region of the first heavy chain of the full-length antibody and the one or more amino acid modifications in the CH2 region of the second heavy chain of the full-length antibody are compared to the amino acid sequence of a reference heavy chain (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions). and / or FcγRIIIb (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))) as compared to a heavy chain comprising a heavy chain comprising a human FcγRIB, ...

[0006] In some embodiments, the first scFv is operably linked via a peptide bond directly to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody.

[0007] In some embodiments, the first scFv is operably connected to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody through a first peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of a glycine amino acid residue or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319 comprising or consisting of one, two, or three amino acid substitutions.

[0008] In some embodiments, the second scFv is operably linked via a peptide bond directly to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody.

[0009] In some embodiments, the second scFv is operably connected to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody through a second peptide linker. In some embodiments, the amino acid sequence of the second peptide linker comprises or consists of glycine amino acid residues or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the second peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319 comprising or consisting of one, two, or three amino acid substitutions.

[0010] In some embodiments, the first scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; the N-terminus of the VL region of the scFv is operably connected to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody. In some embodiments, the first scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; the N-terminus of the VH region of the scFv is operably connected to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody. In some embodiments, the second scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; the N-terminus of the VL region of the scFv is operably connected to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody. In some embodiments, the second scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; and the N-terminus of the VH region of the scFv is operably connected to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody.

[0011] In some embodiments, the first hTAA and the second hTAA are expressed by (e.g., on the surface of) the same tumor cell. In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first scFv and the second scFv specifically bind to the same epitope of the same hTAA. In some embodiments, the first scFv and the second scFv specifically bind to different epitopes of the same hTAA. In some embodiments, the first tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2.

[0012] In some embodiments, the amino acid sequence of the VH region of the first scFv and the amino acid sequence of the VL region of the first scFv each contain a cysteine ​​amino acid residue capable of forming a disulfide bond. In some embodiments, the amino acid sequence of the VH region of the first scFv contains a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, according to the amino acid numbering system of Kabat; and the amino acid sequence of the VL region of the first scFv contains a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, according to the amino acid numbering system of Kabat. In some embodiments, the amino acid sequence of the VH region of the second scFv comprises a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, amino acid numbering according to Kabat; and the amino acid sequence of the VL region of the second scFv comprises a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, amino acid numbering according to Kabat.

[0013] In some embodiments, the full-length antibody is a human IgG (hIgG) antibody. In some embodiments, the full-length antibody is a hIgG1 or hIgG4 antibody.

[0014] In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises an amino acid substitution at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises a cysteine ​​at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises an amino acid substitution at amino acid position T366, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises a tryptophan at amino acid position T366, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises an amino acid substitution at amino acid position S354, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises a cysteine ​​at amino acid position S354, numbered according to EU index of Kabat.

[0015] In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises an amino acid substitution at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises a cysteine ​​at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises an amino acid substitution at amino acid position T366, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises a tryptophan at amino acid position T366, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises an amino acid substitution at amino acid position S354, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises a cysteine ​​at amino acid position S354, numbered according to EU index of Kabat.

[0016] In some embodiments, the multispecific protein does not substantially mediate ADCC, does not substantially mediate ADCP, does not substantially mediate CDC, and / or does not bind to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

[0017] In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an amino acid substitution at amino acid position L234 and / or an amino acid substitution at amino acid position L235, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG4 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an amino acid substitution at amino acid position L234, an amino acid substitution at amino acid position L235, and / or an amino acid substitution at amino acid position P329, numbered according to the EU index of Kabat.

[0018] In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or an alanine at amino acid position P329, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or a glycine at amino acid position P329, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG4 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position E235, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG4 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position E235, numbered according to the EU index of Kabat.

[0019] In some embodiments, the hTCSA is hCD28, hCD2, hCD137, hCD27, hCD278, hCD134, or hCD40. In some embodiments, the hTCSA is hCD28. In some embodiments, the hTCSA is hCD2.

[0020] In one aspect, provided herein is (a) a full-length antibody comprising: (i) a first light chain comprising, from N-terminus to C-terminus, a VL region and a CL region; (ii) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (iv) a second light chain comprising, from N-terminus to C-terminus, a VL region and a CL region, wherein the first light chain and the first heavy chain associate to form a first antigen-binding domain; and a second heavy chain associated to form a second antigen-binding domain; and a full-length antibody in which the first heavy chain and the second heavy chain associated to form a dimer; (b) a first scFv operably connected to the N-terminus of the first heavy chain of the full-length antibody, the first scFv comprising, from the N-terminus to the C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; (c) a second scFv operably connected to the N-terminus of the second heavy chain of the full-length antibody, the first scFv comprising, from the N-terminus to the C-terminus, (i) a VH region, a peptide linker, and a VL region. and a VL region, or (ii) a second scFv comprising a VL region, a peptide linker, and a VH region, wherein a first antigen-binding domain of the full-length antibody specifically binds to a first hTAA; a second antigen-binding domain of the full-length antibody specifically binds to a second hTAA; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); the second scFv specifically binds to hCD3; and the CH3 region of the first heavy chain of the full-length antibody is a reference CH3 region that does not contain one or more amino acid modifications (e.g., substitutions). the CH3 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101), wherein the CH3 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions); and the one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody are different from the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody;one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody and one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody promote heterodimerization of the first and second heavy chains of the full-length antibody; the CH2 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); the CH2 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); the full-length antibody and one or more amino acid modifications in the CH2 region of the second heavy chain of the full-length antibody reduce or eliminate one or more of the following heavy chain effector functions: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))) compared to a reference heavy chain that does not contain the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., a heavy chain comprising SEQ ID NO: 100);

[0021] In some embodiments, the first scFv is operably linked via a peptide bond directly to the N-terminus of the VH region of the first heavy chain of the full-length antibody.

[0022] In some embodiments, the first scFv is operably connected to the N-terminus of the VH region of the first heavy chain of the full-length antibody through a first peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of a glycine amino acid residue or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319 comprising or consisting of one, two, or three amino acid substitutions.

[0023] In some embodiments, the second scFv is operably linked via a peptide bond directly to the N-terminus of the VH region of the second heavy chain of the full-length antibody.

[0024] In some embodiments, the second scFv is operably connected to the N-terminus of the VH region of the second heavy chain of the full-length antibody through a second peptide linker. In some embodiments, the amino acid sequence of the second peptide linker comprises or consists of a glycine amino acid residue or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the second peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319 comprising or consisting of one, two, or three amino acid substitutions.

[0025] In some embodiments, the first scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; the C-terminus of the VH region of the scFv is operably connected to the N-terminus of the VH region of the first heavy chain of the full-length antibody. In some embodiments, the first scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; the C-terminus of the VL region of the scFv is operably connected to the N-terminus of the VH region of the first heavy chain of the full-length antibody. In some embodiments, the second scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; the C-terminus of the VH region of the scFv is operably connected to the N-terminus of the VH region of the second heavy chain of the full-length antibody. In some embodiments, the second scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; and the C-terminus of the VL region of the scFv is operably connected to the N-terminus of the VH region of the second heavy chain of the full-length antibody.

[0026] In some embodiments, the first hTAA and the second hTAA are expressed by (e.g., on the surface of) the same tumor cell. In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first scFv and the second scFv specifically bind to the same epitope of the same hTAA. In some embodiments, the first scFv and the second scFv specifically bind to different epitopes of the same hTAA. In some embodiments, the first tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2.

[0027] In some embodiments, the amino acid sequence of the VH region of the first scFv and the amino acid sequence of the VL region of the first scFv each contain a cysteine ​​amino acid residue capable of forming a disulfide bond. In some embodiments, the amino acid sequence of the VH region of the first scFv contains a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, according to the amino acid numbering system of Kabat; and the amino acid sequence of the VL region of the first scFv contains a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, according to the amino acid numbering system of Kabat. In some embodiments, the amino acid sequence of the VH region of the second scFv comprises a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, amino acid numbering according to Kabat; and the amino acid sequence of the VL region of the second scFv comprises a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, amino acid numbering according to Kabat.

[0028] In some embodiments, the full-length antibody is a human IgG (hIgG) antibody. In some embodiments, the full-length antibody is a hIgG1 or hIgG4 antibody.

[0029] In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises an amino acid substitution at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises a cysteine ​​at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises an amino acid substitution at amino acid position T366, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises a tryptophan at amino acid position T366, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises an amino acid substitution at amino acid position S354, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises a cysteine ​​at amino acid position S354, numbered according to EU index of Kabat.

[0030] In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises an amino acid substitution at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the second heavy chain of the full length antibody comprises a cysteine ​​at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises an amino acid substitution at amino acid position T366, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises a tryptophan at amino acid position T366, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises an amino acid substitution at amino acid position S354, numbered according to EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequence of the first heavy chain of the full length antibody comprises a cysteine ​​at amino acid position S354, numbered according to EU index of Kabat.

[0031] In some embodiments, the multispecific protein does not substantially mediate ADCC, does not substantially mediate ADCP, does not substantially mediate CDC, and / or does not bind to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

[0032] In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an amino acid substitution at amino acid position L234 and / or an amino acid substitution at amino acid position L235, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG4 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an amino acid substitution at amino acid position L234, an amino acid substitution at amino acid position L235, and / or an amino acid substitution at amino acid position P329, numbered according to the EU index of Kabat.

[0033] In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or an alanine at amino acid position P329, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG1 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or a glycine at amino acid position P329, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG4 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position E235, numbered according to the EU index of Kabat. In some embodiments, the full length antibody is an IgG4 antibody, and the amino acid sequences of the first heavy chain and the second heavy chain each comprise a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position E235, numbered according to the EU index of Kabat.

[0034] In one aspect, there is provided herein: (a) a first Fab comprising (i) a first Fab heavy chain comprising, from N-terminus to C-terminus, a first VH region and a first CH1 region, and (ii) a first light chain comprising, from N-terminus to C-terminus, a first VL region and a first CL region; (b) a first scFv operably connected to the C-terminus of the first CH1 region of the first Fab, the first scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region; or (ii) a VL region, a peptide linker, and a VH region; (c) a first Fc region operably connected to the C-terminus of the first scFv, the first Fc region comprising, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; (d) a second Fab comprising, from the N-terminus to the C-terminus, (i) a second Fab heavy chain comprising, from the N-terminus to the C-terminus, a second VH region and a second CH1 region; and (ii) a second light chain comprising, from the N-terminus to the C-terminus, a second VL region and a first CL region; and (e) a second scFv operably connected to the C-terminus of the second CH1 of the second Fab, comprising, from the N-terminus to the C-terminus, (i) a VH region, a peptide a peptide linker, and a VL region; or (ii) a second scFv comprising a VL region, a peptide linker, and a VH region; and (f) a second Fc region operably connected to the C-terminus of the second scFv, the second Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region, wherein the first Fab specifically binds to a first human hTAA; the second Fab specifically binds to a second hTAA; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); the scFv specifically binds to hCD3; and the CH3 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions), and the CH3 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions);and the one or more amino acid modifications in the CH3 region of the first Fc region are different from the one or more amino acid modifications in the CH3 region of the Fc region; the one or more amino acid modifications in the CH3 region of the first Fc region and the one or more amino acid modifications in the CH3 region of the second Fc region promote heterodimerization of the first and second Fc regions; the CH2 region of the first Fc region comprises one or more amino acid modifications compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); and the CH2 region of the second Fc region comprises one or more amino acid modifications compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions). and wherein the one or more amino acid modifications in the CH2 region of the Fc region and the one or more amino acid modifications in the CH2 region of the second Fc region reduce or eliminate one or more of the following Fc region effector functions: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))) compared to a reference Fc region (e.g., a heavy chain comprising a wild-type CH2 domain, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications.

[0035] In some embodiments, the first Fab is operably linked to the first scFv directly through a peptide bond.

[0036] In some embodiments, the first Fab is operably connected to the first scFv through a first peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of a glycine amino acid residue or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319 comprising or consisting of one, two, or three amino acid substitutions.

[0037] In some embodiments, the second Fab is operably linked to the second scFv directly through a peptide bond.

[0038] In some embodiments, the second Fab is operably connected to the second scFv through a second peptide linker. In some embodiments, the amino acid sequence of the second peptide linker comprises or consists of a glycine amino acid residue or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the second peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319 comprising or consisting of one, two, or three amino acid substitutions.

[0039] In some embodiments, the first scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; the N-terminus of the VL region of the scFv is operably connected to the C-terminus of the CH1 region of the first Fab. In some embodiments, the first scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; the N-terminus of the VH region of the scFv is operably connected to the C-terminus of the CH1 region of the first Fab. In some embodiments, the second scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; the C-terminus of the VH region of the scFv is operably connected to the N-terminus of the CH1 region of the second Fab. In some embodiments, the second scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; the C-terminus of the VL region of the scFv is operably connected to the N-terminus of the CH1 region of the second Fab.

[0040] In some embodiments, the first hTAA and the second hTAA are expressed by (e.g., on the surface of) the same tumor cell. In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first scFv and the second scFv specifically bind to the same epitope of the same hTAA. In some embodiments, the first scFv and the second scFv specifically bind to different epitopes of the same hTAA. In some embodiments, the first tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2.

[0041] In some embodiments, the amino acid sequence of the VH region of the first scFv and the amino acid sequence of the VL region of the first scFv each contain a cysteine ​​amino acid residue capable of forming a disulfide bond. In some embodiments, the amino acid sequence of the VH region of the first scFv contains a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, according to the amino acid numbering system of Kabat; and the amino acid sequence of the VL region of the first scFv contains a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, according to the amino acid numbering system of Kabat. In some embodiments, the amino acid sequence of the VH region of the second scFv comprises a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, amino acid numbering according to Kabat; and the amino acid sequence of the VL region of the second scFv comprises a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, amino acid numbering according to Kabat.

[0042] In some embodiments, the first Fc region and the second Fc region are of the human IgG (hIgG) isotype. In some embodiments, the first Fc region and the second Fc region are of the hIgG1 or hIgG4 isotype.

[0043] In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises a cysteine ​​at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises a tryptophan at amino acid position T366, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position S354, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the second Fc region comprises a cysteine ​​at amino acid position S354, numbered according to the EU index of Kabat.

[0044] In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises a cysteine ​​at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position T366, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises a tryptophan at amino acid position T366, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position S354, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the first Fc region comprises a cysteine ​​at amino acid position S354, numbered according to the EU index of Kabat.

[0045] In some embodiments, the multispecific protein does not substantially mediate ADCC, does not substantially mediate ADCP, does not substantially mediate CDC, and / or does not bind to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

[0046] In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position L234 and / or an amino acid substitution at amino acid position L235, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position L234, an amino acid substitution at amino acid position L235, and / or an amino acid substitution at amino acid position P329, numbered according to the EU index of Kabat.

[0047] In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or an alanine at amino acid position P329, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or a glycine at amino acid position P329, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of the IgG4 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position E235, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of the IgG4 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position E235, numbered according to the EU index of Kabat.

[0048] In some embodiments, the hTCSA is hCD28, hCD2, hCD137, hCD27, hCD278, hCD134, or hCD40. In some embodiments, the hTCSA is hCD28. In some embodiments, the hTCSA is hCD2.

[0049] In one aspect, provided herein is an scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; (b) a Fab comprising, from N-terminus to C-terminus, (i) a Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a CH1 region, and (ii) a light chain comprising, from N-terminus to C-terminus, a VL region and a CL region; (c) a first Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region; (d) a second Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region; (e) an IgM CH2 mFab comprising, from N-terminus to C-terminus, (i) an IgM CH2 mFab heavy chain comprising, from N-terminus to C-terminus, a VH region and an IgM CH2 region, and (ii) an IgM CH2 mFab light chain comprising, from N-terminus to C-terminus, a VL region and an IgM CH2 region. a multispecific protein comprising an IgM CH2 mFab, and an IgM CH2 mFab, wherein the C-terminus of the scFv is operably connected to the N-terminus of the Fab heavy chain of a first Fab; the C-terminus of the Fab heavy chain is operably connected to the N-terminus of a first Fc region; the C-terminus of the mFab heavy chain is operably connected to the N-terminus of a second Fc region; the scFv specifically binds to hTCSA (e.g., hCD28, hCD2); the Fab specifically binds to hCD3; and the IgM CH2 the mFab specifically binds to a human tumor-associated antigen (hTAA); the CH3 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions), and the CH3 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions); and the one or more amino acid modifications in the CH3 region of the first Fc region are different from the one or more amino acid modifications in the CH3 region of the Fc region; the one or more amino acid modifications in the CH3 region of the first Fc region and the one or more amino acid modifications in the CH3 region of the second Fc region promote heterodimerization of the first and second heavy chains;the CH2 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); the CH2 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); provides multispecific proteins in which a plurality of amino acid modifications reduce or eliminate one or more of the following Fc region effector functions: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))) compared to a reference Fc region (e.g., a heavy chain comprising a wild-type CH2 domain, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications;

[0050] In some embodiments, the scFv is operably linked directly to the N-terminus of the Fab heavy chain of the first Fab through a peptide bond.

[0051] In some embodiments, the scFv is operably connected to the N-terminus of the Fab heavy chain of the first Fab through a first peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of a glycine amino acid residue or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319 comprising or consisting of one, two, or three amino acid substitutions.

[0052] In some embodiments, the scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; the C-terminus of the VH region of the scFv is operably connected to the N-terminus of the Fab heavy chain of the first Fab.

[0053] In some embodiments, the scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; the C-terminus of the VL region of the scFv is operably connected to the N-terminus of the Fab heavy chain of the first Fab.

[0054] In some embodiments, the tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2.

[0055] In some embodiments, the amino acid sequence of the VH region of the first scFv and the amino acid sequence of the VL region of the first scFv each contain a cysteine ​​amino acid residue capable of forming a disulfide bond. In some embodiments, the amino acid sequence of the VH region of the first scFv contains a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, according to the amino acid numbering system of Kabat; and the amino acid sequence of the VL region of the first scFv contains a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, according to the amino acid numbering system of Kabat. In some embodiments, the amino acid sequence of the VH region of the second scFv comprises a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, amino acid numbering according to Kabat; and the amino acid sequence of the VL region of the second scFv comprises a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, amino acid numbering according to Kabat.

[0056] In some embodiments, the first Fc region and the second Fc region are of the human IgG (hIgG) isotype. In some embodiments, the first Fc region and the second Fc region are of the hIgG1 or hIgG4 isotype.

[0057] In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises a cysteine ​​at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises a tryptophan at amino acid position T366, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position S354, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the second Fc region comprises a cysteine ​​at amino acid position S354, numbered according to the EU index of Kabat.

[0058] In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the second Fc region comprises a cysteine ​​at amino acid position Y349, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position T366, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises a tryptophan at amino acid position T366, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position S354, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the first Fc region comprises a cysteine ​​at amino acid position S354, numbered according to the EU index of Kabat.

[0059] In some embodiments, the multispecific protein does not substantially mediate ADCC, does not substantially mediate ADCP, does not substantially mediate CDC, and / or does not bind to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

[0060] In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position L234 and / or an amino acid substitution at amino acid position L235, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of an IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position L234, an amino acid substitution at amino acid position L235, and / or an amino acid substitution at amino acid position P329, numbered according to the EU index of Kabat.

[0061] In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or an alanine at amino acid position P329, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or a glycine at amino acid position P329, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of the IgG4 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position E235, numbered according to the EU index of Kabat. In some embodiments, the first Fc region and the second Fc region are of the IgG4 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position E235, numbered according to the EU index of Kabat.

[0062] In some embodiments, the tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2.

[0063] In some embodiments, the hTCSA is hCD28, hCD2, hCD137, hCD27, hCD278, hCD134, or hCD40. In some embodiments, the hTCSA is hCD28. In some embodiments, the hTCSA is hCD2.

[0064] In one aspect, the present specification provides a polynucleotide that encodes the multispecific protein described herein or one or more polypeptides thereof.In some embodiments, the polynucleotide is RNA (e.g., mRNA) or DNA.In some embodiments, the polynucleotide is codon-optimized.

[0065] In one aspect, provided herein is an expression vector comprising a polynucleotide described herein. In some embodiments, the expression vector is a viral vector or a plasmid.

[0066] In one aspect, provided herein is a host cell comprising a multispecific protein described herein, a polynucleotide described herein, or an expression vector described herein.

[0067] In one aspect, provided herein is a carrier comprising a multispecific protein described herein, a polynucleotide described herein, or an expression vector described herein.

[0068] In some embodiments, the carrier is a lipid nanoparticle, liposome, lipoplex, or nanoliposome.

[0069] In one aspect, provided herein is a pharmaceutical composition comprising a multispecific protein described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a carrier described herein, and a pharmaceutically acceptable excipient.

[0070] In one aspect, provided herein is a kit comprising a multispecific protein described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein.

[0071] In one aspect, provided herein is a method of making a multispecific protein as described herein, the method comprising introducing a polynucleotide as described herein or a vector as described herein into an in vitro or ex vivo cell population, culturing the cell population under conditions sufficient for the cell population to express the multispecific protein; and optionally isolating and / or purifying the multispecific protein.

[0072] In one aspect, provided herein is a method of delivering a multispecific protein, polynucleotide, expression vector, host cell, carrier, or pharmaceutical composition to a subject, the method comprising administering a multispecific protein described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein, thereby delivering the multispecific protein, polynucleotide, expression vector, host cell, carrier, or pharmaceutical composition to the subject.

[0073] In one aspect, provided herein is a method of inducing an immune response in a subject, the method comprising administering to the subject a multispecific protein described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein, thereby inducing an immune response in the subject.

[0074] In one aspect, provided herein is a method of activating a T cell or population of T cells in a subject, the method comprising administering to the subject a multispecific protein described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein, thereby activating the T cell or population of T cells in the subject.

[0075] In one aspect, provided herein is a method for preventing or treating cancer in a subject, the method comprising administering a multispecific protein described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein to a subject in need thereof, thereby preventing or treating cancer in the subject. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is breast cancer, ovarian cancer, endometrial cancer, uterine cancer, cervical cancer, anal cancer, prostate cancer, rectal cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, pancreatic cancer, thyroid cancer, thymus cancer, lung cancer, bronchial cancer, skin cancer, brain cancer, spinal cancer, head cancer, neck cancer, lip cancer, or oral cavity cancer. [Brief explanation of the drawings]

[0076] [Figure 1] 1 is a graphical representation of a representative format of the BCA405 format of a multispecific protein described herein. In the specific embodiment shown, the multispecific protein comprises: (i) a full-length antibody that specifically binds to a first human tumor-associated antigen (hTAA) and a second hTAA; (ii) a first scFv operably linked to the C-terminus of a first heavy chain of the full-length antibody, the first scFv specifically binding to human CD3 (hCD3); and a second scFv operably linked to the C-terminus of a second heavy chain of the full-length antibody, the second scFv specifically binding to a human T-cell costimulatory antigen (hTCSA) (e.g., human CD28 (hCD28), human CD2 (hCD2)). In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first scFv is operably connected to the C-terminus of the first heavy chain of the full-length antibody via a peptide linker; and the second scFv is operably connected to the C-terminus of the second heavy chain of the full-length antibody via a peptide linker. [Figure 2]Figure 2 is a graphic representation of a representative format of the BCA406 format of a multispecific protein described herein. In the specific embodiment shown, the multispecific protein comprises: (i) a full-length antibody that specifically binds to a first human hTAA and a second hTAA; (ii) a first scFv operably linked to the N-terminus of a first heavy chain of the full-length antibody, the first scFv specifically binding to hCD3; and a second scFv operably linked to the N-terminus of a second heavy chain of the full-length antibody, the second scFv specifically binding to an hTCSA (e.g., hCD28, hCD2). In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first scFv is operably linked to the N-terminus of the first heavy chain of the full-length antibody via a peptide linker; and the second scFv is operably linked to the N-terminus of the second heavy chain of the full-length antibody via a peptide linker. [Figure 3] 3 is a graphic representation of a representative format of the BCA424 format of the multispecific protein described herein. In the specific embodiment shown, the multispecific protein comprises a first Fab operably connected to a first scFv operably connected to a first Fc region; and a second Fab operably connected to a second scFv operably connected to a second Fc region; the first Fab specifically binds to a first hTAA, the second Fab specifically binds to a second hTAA, the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2), and the second scFv specifically binds to hCD3. In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first Fab is operably connected to the first scFv via a peptide linker, and the second Fab is operably connected to the second scFv via a peptide linker. [Figure 4]4 is a graphic representation of a representative format of the BCA418 format of the multispecific protein described herein. In the specific embodiment shown, the multispecific protein comprises a first scFv operably connected to a VH region of a first Fab operably connected to a first Fc region; and a first IgM CH2 mFab operably connected to a second Fc region; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2), the first Fab specifically binds to hCD3, and the first IgM CH2 mFab specifically binds to an hTAA. In some embodiments, the first Fab is operably connected to the VL region of the scFv. In some embodiments, the first Fab is operably connected to the VH region of the scFv. In some embodiments, the first Fab is operably connected to the first scFv via a peptide linker. [Figure 5] Figure 5A is a line graph showing ELISA-based HER2 binding of the indicated multispecific proteins or controls at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows EC50 values ​​based on n=2 for BCA401 and n=3 for BCA405, BCA406, and BCA424. Figure 5B is a line graph showing ELISA-based HER2 binding of the indicated multispecific proteins or controls at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows EC50 values ​​based on n=2 for BCA418 and BCA401, and n=3 for BCA418. [Figure 6] Figure 6 is a line graph showing EGFR binding in A431 cells with the indicated concentrations of the indicated multispecific proteins (BCA405.EG, BCA406.EG, BCA424.EG, which are in the same format as shown in Figures 1, 2, and 3, with anti-EGFR as the TAA targeting arm) or control (cetuximab, a positive control for EGFR binding). The table below the graph shows EC50 (nM) values. [Figure 7]Figure 7A is a line graph showing ELISA-based CD3 binding of the indicated multispecific proteins or controls at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows EC50 values ​​based on n=3 for BCA405, BCA406, and BCA424. Figure 7B is a line graph showing ELISA-based CD3 binding of the indicated multispecific proteins or controls at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows EC50 values ​​based on n=2 for BCA418. [Figure 8] Figure 8 is a line graph showing CD3 binding on CD28 knockout Jurkat cells at the indicated concentrations of the indicated multispecific proteins or controls. The graph shows representative data from two independent experiments. The table below the graph shows a qualitative ranking based on the curve shift compared to BCA403 (an anti-CD3 mAb). The qualitative ranking is based on the curve shift, with +, ++, and +++ representing low, moderate, and high binding compared to BCA403. [Figure 9] Figure 9A is a line graph showing ELISA-based CD28 binding of the indicated multispecific proteins or controls at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows EC50 values ​​based on n=3 for BCA405, BCA406, and BCA424. Figure 9B is a line graph showing CD28 binding of the indicated multispecific proteins or controls at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows EC50 values ​​based on n=2 for BCA418. [Figure 10]Figure 10 is a line graph showing CD28 binding on CD3 knockout Jurkat cells by the indicated multispecific proteins or controls at the indicated concentrations. The graph shows representative data from two independent experiments. The table below the graph shows a qualitative ranking based on the curve shift compared to BCA402 (an anti-CD28 mAb). The qualitative ranking is based on the curve shift, with +, ++, and +++ representing low, moderate, and high binding compared to BCA402. [Figure 11] Figure 11 is a line graph showing the percent cytotoxicity mediated by the indicated multispecific proteins or controls at the indicated concentrations in an SKBR3-PBMC coculture assay. Target cells (tumor cells, T) were cocultured with human PBMCs (E) at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was assessed using a luminescence readout (BioGlo). The formula used was: % cytotoxicity = [100 - (RLUN / RLU untreated SKBR3-Luc) x 100], where RLUN = RLU total group - RLU unstimulated PBMC. RLU = relative luminescence units. Each point represents the mean ± SD of duplicate values ​​in an experiment. The graph shows representative data from at least two independent experiments using two PBMC donors. A human IgG isotype control antibody (hIgG) was used as a negative control. The table below the graph shows EC50 (nM) values. [Figure 12] Figure 12 is a line graph showing the percent cytotoxicity mediated by the indicated multispecific proteins or controls at the indicated concentrations in a BxPC3-PBMC coculture assay. Target cells were cocultured with human PBMCs at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was assessed using a luminescence readout (BioGlo). The formula used was: % cytotoxicity = [100 - (RLUN / RLU untreated SKBR3-Luc) x 100], where RLUN = RLU total groups - RLU unstimulated PBMCs. RLU = relative luminescence units. Percent cytotoxicity is shown normalized to the hIgG control. Each point represents the mean ± SD of duplicate values ​​within an experiment and is representative of two independent experiments. [Figure 13-1]Figure 13A is a bar graph showing the levels of IL-2 and IFNγ released from SKBR3luc-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. Data are plotted as the mean ± SD of duplicate wells from the experiment. Figure 13B is a bar graph showing the levels of granzyme B released from SKBR3luc-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. Data are plotted as the mean ± SD of duplicate wells from the experiment. [Figure 13-2] Figure 13C is a bar graph showing the levels of IL-6 and TNFα released from SKBR3luc-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. Data are plotted as the mean ± SD of duplicate wells from an experiment. Figure 13D is a bar graph showing the levels of IL-2 and IFNγ released from PBMC cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. [Figure 13-3] Figure 13E is a bar graph showing the levels of granzyme B released from PBMC cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. Data are plotted as the mean ± SD of duplicate wells from an experiment. Figure 13F is a bar graph showing the levels of IL-6 and TNFα released from PBMC cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. [Figure 14-1]Figure 14A is a bar graph showing the frequency of activated T cells (CD3+ CD25+ cells) in PBMC cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. The graph is representative of n=3 experiments for BCA405, BCA406, BCA424, and BCA410, and n=2 experiments for BCA418, for two PBMC donors. Figure 14B is a bar graph showing the frequency of Bcl-xL-expressing T cells (CD3+ Bcl-xL+ cells) in PBMC cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. The graph is representative of n=3 experiments for BCA405, BCA406, BCA424, and BCA410, and n=2 experiments for BCA418, for two PBMC donors. [Figure 14-2] Figure 14C is a bar graph showing the frequency of activated T cells (CD3+ CD25+ cells) in SKBR3-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. The graph is representative of two PBMC donors, n=3 for BCA405, BCA406, BCA424, and BCA410, and n=2 for BCA418. Figure 14D is a bar graph showing the frequency of Bcl-xL-expressing T cells (CD3+ Bcl-xL+ cells) in SKBR3-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. The graph is representative of two PBMC donors, n=3 for BCA405, BCA406, BCA424, and BCA410, and n=2 for BCA418. [Figure 15] Figure 15A is a bar graph showing the frequency of Bcl-xL-expressing T cells (CD3+ Bcl-xL+ cells) in PBMC cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. Figure 15B is a bar graph showing the frequency of Bcl-xL-expressing T cells (CD3+ Bcl-xL+ cells) in FaDu-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. [Figure 16]Figure 16A is a bar graph showing the frequency of Bcl-xL-expressing T cells (CD3+ Bcl-xL+ cells) in PBMC cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. Figure 16B is a bar graph showing the frequency of Bcl-xL-expressing T cells (CD3+ Bcl-xL+ cells) in SKBR3-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. [Figure 17-1] Figure 17A is a line graph showing the percent cytotoxicity mediated by the indicated multispecific proteins or controls at the indicated concentrations in an SKBR3-PBMC coculture assay. Target cells were cocultured with human PBMCs at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was assessed using a luminescence readout (BioGlo). The formula used was: % cytotoxicity = [100 - (RLUN / RLU untreated SKBR3-Luc) x 100], where RLUN = RLU total group - RLU unstimulated PBMC. RLU = relative luminescence units. Each point represents the mean ± SD of duplicate values ​​in an experiment. Figure 17B is a line graph showing the percent cytotoxicity mediated by the indicated multispecific proteins or controls at the indicated concentrations in an SKBR3-PBMC coculture assay. Target cells were cocultured with human PBMCs at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was assessed using a luminescence readout (BioGlo). The formula used was: % cytotoxicity = [100 - (RLUN / RLU untreated SKBR3-Luc) x 100], where RLUN = RLU all groups - RLU unstimulated PBMC. RLU = relative luminescence units. Each point represents the mean ± SD of duplicate values ​​in an experiment. [Figure 17-2]Figure 17C is a line graph showing the percent cytotoxicity mediated by the indicated multispecific proteins or controls at the indicated concentrations in an SKBR3-PBMC coculture assay. Target cells were cocultured with human PBMCs at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was assessed using a luminescence readout (BioGlo). The formula used was: % cytotoxicity = [100 - (RLUN / RLU untreated SKBR3-Luc) x 100], where RLUN = RLU total group - RLU unstimulated PBMC. RLU = relative luminescence units. Each point represents the mean ± SD of duplicate values ​​in an experiment. Figure 17D is a line graph showing the percent cytotoxicity mediated by the indicated multispecific proteins or controls at the indicated concentrations in an SKBR3-PBMC coculture assay. Target cells were cocultured with human PBMCs at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was assessed using a luminescence readout (BioGlo). The formula used was: % cytotoxicity = [100 - (RLUN / RLU untreated SKBR3-Luc) x 100], where RLUN = RLU all groups - RLU unstimulated PBMC. RLU = relative luminescence units. Each point represents the mean ± SD of duplicate values ​​in an experiment. [Figure 18-1] Figure 18A is a bar graph showing the percentage of activated CD4+ T cells (CD4+ CD25+ cells) in FaDu-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations, and Figure 18B is a bar graph showing the percentage of activated CD8+ T cells (CD8+ CD25+ cells) in FaDu-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. [Figure 18-2] Figure 18C is a bar graph showing the percentage of activated CD4+ T cells (CD4+ CD25+ cells) in PBMC cultures treated with the indicated multispecific proteins or controls at the indicated concentrations, and Figure 18D is a bar graph showing the percentage of activated CD8+ T cells (CD8+ CD25+ cells) in PBMC cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. [Figure 19-1]Figure 19A is a line graph showing the percentage of activated CD4+ CD25+ memory T cells (CD45RO and CCR7 (naive CD45RO-CCR7+), (TEM CD45RO+CCR7-), (TCM CD45RO+CCR7+), (DN CD45RO-CCR7-) in FaDu-PBMC cocultures treated with the indicated controls at the indicated concentrations. Figure 19B is a line graph showing the percentage of activated CD8+ CD25+ memory T cells (CD45RO and CCR7 (naive CD45RO-CCR7+), (TEM CD45RO+CCR7-), (TCM CD45RO+CCR7+), (DN CD45RO-CCR7-) in FaDu-PBMC cocultures treated with the indicated controls at the indicated concentrations. [Figure 19-2] Figure 19C is a line graph showing the percentage of activated CD4+ CD25+ memory T cells (CD45RO and CCR7 (naive CD45RO-CCR7+), (TEM CD45RO+CCR7-), (TCM CD45RO+CCR7+), (DN CD45RO-CCR7-)) in FaDu-PBMC cocultures treated with the indicated multispecific proteins at the indicated concentrations. Figure 19D is a line graph showing the percentage of activated CD8+ CD25+ memory T cells (CD45RO and CCR7 (naive CD45RO-CCR7+), (TEM CD45RO+CCR7-), (TCM CD45RO+CCR7+), (DN CD45RO-CCR7-)) in FaDu-PBMC cocultures treated with the indicated multispecific proteins at the indicated concentrations. [Figure 20-1] Figure 20A is a line graph showing the levels of IFNγ and IL-2 released from FaDu-PBMC co-cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. Figure 20B is a line graph showing the levels of IL-2 released from FaDu-PBMC co-cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. [Figure 20-2]Figure 20C is a line graph showing the levels of TNF and IL-6 released from FaDu-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. Figure 20D is a line graph showing the levels of IL-6 released from FaDu-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. [Figure 20-3] Figure 20E is a line graph showing the levels of IL-4 and IL-10 released from FaDu-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. Figure 20F is a line graph showing the levels of IL-10 released from FaDu-PBMC cocultures treated with the indicated multispecific proteins or controls at the indicated concentrations. [Figure 20-4] FIG. 20G is a line graph showing the levels of IL-17A released from FaDu-PBMC co-cultures treated with the indicated multispecific proteins or controls at the indicated concentrations. [Figure 21-1] Figure 21A is a graphic representation of the structure of multispecific antibody BCA605. The multispecific structure is similar to that of BCA405 (described in Figure 1). The anti-TAA Fab of BCA605 specifically binds to MSLN. In some embodiments, the anti-CD3 and anti-hTCSA (e.g., CD28, CD2) scFv arms contain engineered disulfide bonds (represented by lines in the graphic). Figure 21B is a graphic representation of the structure of multispecific antibody BCA606. The multispecific structure is similar to that of BCA406 (described in Figure 2). The anti-TAA Fab of BCA605 specifically binds to MSLN. In some embodiments, the anti-CD3 and anti-hTCSA (e.g., CD28, CD2) scFv arms contain engineered disulfide bonds (represented by lines in the graphic). [Figure 21-2]Figure 21C is a graphical representation of the structure of multispecific antibody BCA624. The multispecific structure is similar to that of BCA424 (described in Figure 3). The anti-TAA Fab of BCA605 specifically binds to MSLN. In some embodiments, the anti-CD3 and anti-hTCSA (e.g., CD28, CD2) scFv arms contain engineered disulfide bonds (represented by lines in the graphic). [Figure 22] Figure 22A is a line graph showing binding of the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or controls (BCA429(MOR), hIgG) at the indicated concentrations to MSLN expressed on the surface of OVACAR-3 / CMV Luc cells. Figure 22B is a line graph showing binding of the indicated multispecific proteins (BCA605(FB6), BCA606(FB6), BCA624(FB6)) or controls (hIgG) at the indicated concentrations to MSLN expressed on the surface of OVACAR-3 / CMV Luc cells. [Figure 23] Figure 23A is a line graph showing the binding of the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or controls (BCA402, BCA403, hIgG) at the indicated concentrations to CD28 expressed on the surface of CD3eKO Jurkat cells as measured by flow cytometry. Figure 23B is a line graph showing the binding of the indicated multispecific proteins (BCA605(FB6), BCA606(FB6), BCA624(FB6)) or controls (BCA402, BCA403, hIgG) at the indicated concentrations to CD28 expressed on the surface of CD3eKO Jurkat cells as measured by flow cytometry. [Figure 24-1]Figure 24A is a line graph showing binding of the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or controls (BCA402, BCA403, hIgG) at the indicated concentrations to CD3 expressed on the surface of CD28eKO Jurkat cells as measured by flow cytometry. Figure 24B is a line graph showing binding of the indicated multispecific proteins (BCA605(FB6), BCA606(FB6), BCA624(FB6)) or controls (BCA402, BCA403, hIgG) at the indicated concentrations to CD3 expressed on the surface of CD28eKO Jurkat cells as measured by flow cytometry. [Figure 24-2] Figure 24C is a line graph showing binding of the indicated multispecific proteins (BCA605(FB6), BCA606(FB6), BCA624(FB6)) or control (BCA403) at the indicated concentrations to CD3 as measured by ELISA. [Figure 25] Figure 25A is a line graph showing the percent cytotoxicity mediated by the indicated concentrations of the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR), BCA605(FB6), BCA606(FB6), BCA624(FB6)) in an OVCAR3-PBMC (1:10) co-culture assay. Figure 25B is a line graph showing the percent cytotoxicity mediated by the indicated concentrations of the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR), BCA605(FB6), BCA606(FB6), BCA624(FB6)) in a SCOV3-PBMC (1:10) co-culture assay. [Figure 26-1]Figure 26A is a bar graph showing IL-2 release from tumor-PBMC cocultures at the indicated concentrations with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or control (BCA429(MOR), hIgG, unstimulated coculture (target + effector (T+E))). Figure 26B is a bar graph showing IL-6 release from tumor-PBMC cocultures at the indicated concentrations with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or control (BCA429(MOR), hIgG, unstimulated coculture (target + effector (T+E))). [Figure 26-2] Figure 26C is a bar graph showing IFNγ release from tumor-PBMC cocultures at the indicated concentrations with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or control (BCA429(MOR), hIgG, unstimulated coculture (target + effector (T+E))). Figure 26D is a bar graph showing TNF release from tumor-PBMC cocultures at the indicated concentrations with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or control (BCA429(MOR), hIgG, unstimulated coculture (target + effector (T+E))). [Figure 26-3] Figure 26E is a bar graph showing IL-10 release from tumor-PBMC cocultures at the indicated concentrations with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or control (BCA429(MOR), hIgG, unstimulated coculture (target + effector (T+E))). Figure 26F is a bar graph showing IL-17 release from tumor-PBMC cocultures at the indicated concentrations with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or control (BCA429(MOR), hIgG, unstimulated coculture (target + effector (T+E))). [Figure 26-4]Figure 26G is a bar graph showing IL-4 release from tumor-PBMC cocultures at the indicated concentrations with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or controls (BCA429(MOR), hIgG, unstimulated coculture (target + effector (T+E))). [Figure 27] Figure 27A is a bar graph showing IFNγ release from PBMCs treated with either soluble or tethered forms of the indicated multispecific antibodies (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or controls (BCA403(MOR), hIgG). Data from two PBMC lots are shown. Figure 27B is a bar graph showing IFNγ release from PBMCs treated with either soluble or tethered forms of the indicated multispecific antibodies (BCA605(FB6), BCA606(FB6), BCA624(FB6)) or controls (BCA403(MOR), hIgG). Data from two PBMC lots are shown. [Figure 28] FIG. 28 shows a graphical representation of the format of a control multispecific protein, referred to herein as BCA410. [Figure 29] Figure 29 shows a graphical representation of the overall structure of a naturally occurring full-length antibody. This structure is utilized for several control antibodies described herein, including, for example, BCA401 (full-length anti-HER2 antibody), BCA402 (full-length anti-CD28 antibody), BCA403 (full-length anti-EGFR antibody), BCA403 (full-length αCD3ε antibody), and cetuximab (full-length anti-EGFR antibody). DETAILED DESCRIPTION OF THE INVENTION

[0077] T cell-engaging immunotherapy works by targeting T cells (or subsets thereof, e.g., CD8+ T cells) to tumors. For example, bispecific T cell-engaging single-chain antibodies (also known as BiTEs) are single-chain proteins that simultaneously bind to antigens on tumor cells and antigens expressed on the surface of T cells to activate T cells and induce tumor lysis. Despite the promising efficacy of T cell engagers in clinical trials, they also exhibit severe dose-limiting adverse events, including cytokine release syndrome (CRS) and neurotoxicity. CRS is an uncontrolled systemic inflammatory response characterized by elevated levels of pro-inflammatory cytokines (e.g., IL-6) induced by T cell activation. CRS mediated by T cell engagers results in peripheral toxicity, particularly through the activation of peripheral T cells outside the tumor microenvironment. The present inventors have discovered novel multispecific protein formats that specifically bind to hCD3, hTCSA (e.g., hCD28, hCD2), and hTAA, among others, and exhibit low or no peripheral toxicity. Thus, the novel multispecific proteins described herein are good candidates for the treatment of diseases (e.g., cancer). As such, the present disclosure provides novel multispecific proteins for use in pharmaceutical compositions for the treatment of diseases (e.g., cancer), among others.

[0078] 5.1 Definition The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0079] 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 the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any claimed subject matter.

[0080] In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Furthermore, the use of the term "including" and other forms such as "include," "includes," and "included" is not limiting.

[0081] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and "consisting essentially of" are also provided.

[0082] The term "and / or," as used herein, should be construed as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or," when used herein in phrases such as "A and / or B," is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" when used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0083] As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof (e.g., tenths and hundredths of integers), where appropriate, unless otherwise indicated.

[0084] The term "about" refers to a value or composition that is within an acceptable error range of the particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. When a particular value or composition is provided in this application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range of that particular value or composition.

[0085] Unless otherwise indicated or apparent from the context, the use of terms such as "first and second," or "(a) and (b)," or "(i) and (ii)," and similar terms herein are used to identify multiple components of a composition or method, and do not refer to an order or direction. Where these terms are intended to refer to an order or direction, it will be apparent to one of skill in the art from the context.

[0086] Where proteins and / or polypeptides are described herein, it is understood that the polynucleotides (e.g., RNA (e.g., mRNA) or DNA polynucleotides) that encode the proteins or polypeptides are also provided herein.

[0087] Where proteins, polypeptides, polynucleotides, cells, expression vectors, etc. are described herein, it is understood that isolated forms of the proteins, polypeptides, polynucleotides, cells, expression vectors, etc. are also provided herein.

[0088] Where proteins, polypeptides, polynucleotides, etc. are described herein, it is understood that recombinant forms of the proteins, polypeptides, polynucleotides, etc. are also provided herein.

[0089] Where a polypeptide or set of polypeptides is described herein, it is understood that a protein comprising the polypeptide or set of polypeptides folded into that three-dimensional structure (i.e., tertiary or quaternary structure) is also provided herein, and vice versa.

[0090] As used herein, the term "administering" refers to the physical introduction of an agent, e.g., a therapeutic agent (or a precursor of a therapeutic agent that is metabolized or modified in the subject's body to produce the therapeutic agent in vitro), into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Administering can be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0091] As used herein, the term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to an immune mechanism that results in the lysis of target cells coated with an antibody (or Fc region-containing polypeptide or protein) (e.g., an Ig Fc-containing fusion protein or polypeptide described herein) by immune effector cells (e.g., NK cells). As used herein, "reduced ADCC" and similar terms refer to either a reduction in the number of target cells lysed at a given concentration of antibody (or Ig Fc region-containing polypeptide or protein) (e.g., an Fc region-containing fusion protein or polypeptide described herein) in the medium surrounding the target cells in a given period of time by the mechanism of ADCC as defined above, and / or an increase in the concentration of antibody (or Fc region-containing polypeptide or protein) (e.g., an Fc-containing fusion protein or polypeptide described herein) required to achieve lysis of a given number of target cells in the medium surrounding the target cells in a given period of time by the mechanism of ADCC as defined above. The reduction in ADCC is relative to the ADCC mediated by the same antibody (or Fc region-containing polypeptide or protein) (e.g., an Fc-containing fusion protein or polypeptide described herein) produced by the same type of host cell using the same standard production, purification, formulation, and storage methods (known to those of skill in the art), but unaltered (e.g., not containing one or more amino acid modifications, e.g., amino acid substitutions, that mediate reduced ADCC). For example, the reduction in ADCC mediated by an antibody (or Fc region-containing polypeptide or protein) (e.g., an Fc-containing fusion protein or polypeptide described herein) that contains an amino acid substitution in its Fc region that reduces ADCC is relative to the ADCC mediated by the same antibody (or Fc region-containing polypeptide or protein) (e.g., an Fc-containing fusion protein or polypeptide described herein) that does not have said amino acid substitution in the Fc region.

[0092] As used herein, the term "affinity" refers to the strength of binding between one protein (e.g., an antibody) and another protein (e.g., an antigen). Protein affinity is measured by the dissociation constant, Kd, ​​defined as [antibody] x [antigen] / [antibody-antigen], where [antibody-antigen] is the molar concentration of the antibody-antigen complex, [antibody] is the molar concentration of unbound antibody, and [ligand] is the molar concentration of unbound antigen. The affinity constant, Ka, is defined by 1 / Kd. Standard methods for measuring affinity are known to those of skill in the art. Exemplary methods for measuring affinity are described herein, see, e.g., § 5.2.5.

[0093] As used herein, the term "antibody" or "antibodies" is used in the broadest sense and encompasses a variety of immunoglobulin (Ig) (e.g., human Ig (hIg)) structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific (e.g., bispecific, trispecific) antibodies, and antibody fragments (i.e., antigen-binding fragments or variants) so long as they exhibit the desired antigen-binding activity. Thus, the term antibody includes, for example, full-length antibodies; antigen-binding fragments of full-length antibodies; molecules comprising antibody CDRs, VH regions, and / or VL regions; and antibody-like scaffold structures (e.g., fibronectin). Examples of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelized antibodies, intrabodies, affibodies, diabodies, tribodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies (e.g., VHH, (VHH)2), single chain antibodies, single chain Fv (scFv; (scFv)2), Fab fragments (e.g., Fab, single chain Fab (scFab), F(ab')2 fragments, disulfide fragments, Antibodies include combined Fvs (sdFvs), Fc fusions (e.g., Fab-Fc, scFv-Fc, VHH-Fc, (scFv)2-Fc, (VHH)2-Fc), and antigen-binding fragments of any of the above, as well as conjugates or fusion proteins comprising any of the above. Antibodies may be of any Ig isotype (e.g., IgG, IgE, IgM, IgD, or IgA), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass of Ig (e.g., IgG 2a or IgG 2b)). In certain embodiments, the antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In some embodiments, the antibody is a human, humanized, or chimeric IgG1 or IgG4 monoclonal antibody. In some embodiments, the term antibody refers to a monoclonal antibody or a polyclonal antibody population. The antibodies described herein can be produced by any standard method known in the art, e.g., recombinant production in a host cell, e.g., see §5.3, or synthetic production.

[0094] As used herein, the term "antibody-like scaffold" refers to non-Ig-based antigen binding domains.Various antibody-like scaffolds are known in the art.For example, the tenth type III domain of fibronectin (e.g., AdNectins®) and designed ankyrin repeat proteins (e.g., DARPins®) have been used as alternative scaffolds for antigen binding domains, see, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13:695-701 (2008), the entire contents of each of which are incorporated herein by reference for all purposes. Exemplary antibody-like scaffold structures include, but are not limited to, lipocalins (see, e.g., U.S. Pat. No. 7,250,297) (e.g., Anticalin®), protein A-derived molecules, such as the z-domain of protein a (see, e.g., U.S. Pat. No. 5,831,012) (e.g., Affibody®), A-domains of membrane receptors stabilized by disulfide bonds and Ca2+ (see, e.g., U.S. Pat. No. 7,803,907) (e.g., Avimer / Maxibody®), serum transmembrane domains (see, e.g., U.S. Pat. No. 6,111,162) (e.g., Avimer / Maxibody®), and the like. Spherins (see, e.g., U.S. Patent Application Publication No. 2004023334) (e.g., Transbody®); designed ankyrin repeat proteins (see, e.g., U.S. Patent Application Publication No. 7417130) (e.g., DARPin®), fibronectin (see, e.g., U.S. Patent Application Publication No. 6818418) (e.g., AdNectin®), C-type lectin domains (see, e.g., U.S. Patent Application Publication No. 2004132094) (e.g., Tetranectin®);Human gamma-crystallin or ubiquitin (see, e.g., U.S. Pat. No. 7,838,629) (e.g., Affilin®); Kunitz-type domains of human protease inhibitors (see, e.g., U.S. Patent Application Publication No. 2004209243), C-type lectins (see, e.g., U.S. Patent Application Publication No. 2004132094) (e.g., Tetranectins®), cysteine ​​knots or knottins (see, e.g., U.S. Pat. No. 7,186,524) see detailed description) (e.g., Microbodies®), nucleic acid aptamers (see, e.g., U.S. Pat. No. 5,475,096), thioredoxin A scaffolds (see, e.g., U.S. Pat. No. 6,004,746) (peptide aptamers), and the tenth type III domain of fibronectin (see, e.g., U.S. Pat. No. 6,818,418) (e.g., AdNectins®), and cystine-dense peptides (see, e.g., WO 2023023031). Additional exemplary antibody-like scaffolds are known in the art and are described, for example, in Storz U. Intellectual property protection: strategies for antibody inventions. MAbs. 2011;3(3):310-317. doi:10.4161 / mabs.3.3.15530. The entire contents of each of the foregoing references are incorporated herein by reference for all purposes. Antibody-like scaffolds include, for example, naturally occurring antigen binders, variants (e.g., functional variants) of naturally occurring antigen binders, fragments (e.g., functional fragments) of naturally occurring antigen binders, and synthetic antigen binders (i.e., non-naturally occurring antigen binders);

[0095] The term "antigen-binding domain" refers to a polypeptide or protein, or a portion of a polypeptide or protein, that is capable of specifically binding to an antigen. Exemplary antigen-binding domains include, but are not limited to, single-domain antibodies (e.g., VHH, (VHH)2), single-chain Fvs (e.g., scFv; (scFv)2), Fab fragments (e.g., Fab, single-chain Fab (scFab), F(ab')), and the like. 2) Examples of antigen-binding domains include Fvs, disulfide-linked Fvs (sdFvs), and disulfide-linked Fvs (sdFvs). An antigen-binding domain can be part of a larger polypeptide or protein, such as a full-length antibody or an Fc fusion. In some embodiments, the antigen-binding domain is part of a full-length antibody. In some embodiments, the antigen-binding domain is operably linked to an Fc region. When an antigen-binding domain refers to the use of a target protein or polypeptide, the term "antigen" may be replaced with the name of the target protein or antigen. For example, an antigen-binding domain that specifically binds to hCD3 may be referred to herein as an "hCD3-binding domain."

[0096] The terms "cancer" and "tumor" are used interchangeably herein to refer to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth leads to the formation of malignant tumors that can invade adjacent tissues and can also metastasize to distant parts of the body, for example, through the lymphatic system or bloodstream.

[0097] As used herein, the term "CDR" or "complementarity-determining region" refers to the discontinuous antigen-binding sites found in the variable regions of both heavy and light chain polypeptides. These specific regions are described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), the entire contents of each of which are incorporated herein by reference for all purposes. Unless otherwise specified, the term "CDR" refers to the CDR defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991).

[0098] The terms "CH1" and "CH1 region" are used interchangeably herein to refer to the first constant region of an immunoglobulin heavy chain. An exemplary reference amino acid sequence of a hIgG1 CH1 region is set forth in SEQ ID NO:98; and an exemplary reference amino acid sequence of a hIgG4 CH1 region is set forth in SEQ ID NO:111.

[0099] The terms "CH2" and "CH2 region" are used interchangeably herein to refer to the second constant region of an immunoglobulin heavy chain. An exemplary reference amino acid sequence of a hIgG1 CH2 region is set forth in SEQ ID NO: 100; and an exemplary reference amino acid sequence of a hIgG4 CH2 region is set forth in SEQ ID NO: 113.

[0100] The terms "CH3" and "CH3 region" are used interchangeably herein to refer to the third constant region of an immunoglobulin heavy chain. An exemplary reference amino acid sequence of a hIgG1 CH3 region is set forth in SEQ ID NO: 101; and an exemplary reference amino acid sequence of a hIgG4 CH3 region is set forth in SEQ ID NO: 113.

[0101] The terms "constant region" and "constant domain" are used interchangeably herein and refer to the carboxyl-terminal portions of the light and / or heavy chains of a full-length antibody that are not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interaction with Ig Fc receptors (e.g., Fc gamma receptors). The constant regions of Ig molecules generally have more conserved amino acid sequences compared to Ig variable domains.

[0102] As used herein, the term "derived" with reference to a polynucleotide refers to a polynucleotide having at least 70% (e.g., at least 85%) sequence identity with a reference polynucleotide (e.g., a naturally occurring polynucleotide) or a fragment thereof. The term "derived" with reference to a polypeptide or protein refers to a polypeptide or protein comprising an amino acid sequence that has at least 70% (e.g., at least 85%) sequence identity with the amino acid sequence of a reference polypeptide or protein (e.g., a naturally occurring polypeptide or protein). The term "derived" as used herein does not refer to any particular process or method for obtaining the polynucleotide, polypeptide, or protein. For example, a polynucleotide, polypeptide, or protein can be recombinantly produced or chemically synthesized.

[0103] As used herein, the term "diagnosing" or "diagnosis" refers to the determination of the presence, absence, severity, or course of treatment of a disease (e.g., cancer). The term "diagnosing" encompasses the initial determination as well as subsequent determinations (e.g., monitoring) after the initial determination.

[0104] As used herein, the term "disease" refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, disorder, condition, malaise, state, or symptom in which physiological function is impaired, regardless of the nature of the etiology.

[0105] The terms "hinge" or "hinge region" are used interchangeably herein to refer to the hinge region of an immunoglobulin heavy chain. An exemplary reference hIgG1 hinge region amino acid sequence is set forth in SEQ ID NO: 99; and an exemplary reference hIgG4 hinge region amino acid sequence is set forth in SEQ ID NO: 112.

[0106] The terms "DNA" and "polydeoxyribonucleotide" are used interchangeably herein to refer to a polymer comprising multiple deoxyribonucleotides polymerized through phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0107] The term "effector function," when used in reference to an antibody, refers to those biological activities attributable to the Fc region of the antibody and therefore vary depending on the antibody isotype. Antibody effector functions include, but are not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), Fc receptor binding (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa)), and Clq binding.

[0108] As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85(1969), and Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 5th edition, 1991, the entire contents of each of which are incorporated herein by reference for all purposes.

[0109] As used herein, the term "Fab" refers to an antigen-binding domain comprising, from the N-terminus to the C-terminus, a Fab heavy chain comprising a VH region and a CH1 region; and, from the N-terminus to the C-terminus, a light chain comprising a VL region and a CL region, wherein the Fab heavy chain and light chain associate to form the antigen-binding domain.

[0110] The term "Fab-Fc," as used herein, refers to an antibody comprising a Fab operably linked to an Fc region. For example, a full-length antibody comprises a first Fab operably linked to a first Fc region and a second Fab operably linked to a second Fc region.

[0111] As used herein, the term "Fc region" refers to the C-terminal region of an hIg heavy chain comprising, from N-terminus to C-terminus, at least a CH2 region operably connected to a CH3 region. In some embodiments, the Fc region comprises an Ig hinge region or at least a portion of an Ig hinge region operably connected to the N-terminus of the CH2 region. In some embodiments, the Fc region is modified (e.g., comprises one or more amino acid modifications) compared to a reference Fc region; see, e.g., §5.2.7.1. Additional examples of proteins with modified Fc regions can be found in Saunders 2019 (KO Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life," 2019, Frontiers in Immunology, Vol. 10, Art. 1296, pp. 1-20, the entire contents of which are incorporated herein by reference for all purposes). In some embodiments, the CH3 region comprises a deletion of one or more C-terminal amino acid residues compared to a wild-type CH3 region (eg, C-terminal lysine; C-terminal glycine-lysine).

[0112] As used herein, the term "Fc-modified fusion protein or polypeptide" refers to a fusion polypeptide or protein comprising an Fc region in which the Fc region has been modified (e.g., contains one or more amino acid modifications (e.g., one or more amino acid substitutions, deletions, or additions)) compared to a reference Fc region.

[0113] As used herein, the terms "first" and "second," such as in reference to an Fc region, are used for convenience to distinguish when more than one of each type of moiety is present. The use of these terms is not intended to confer a particular order or orientation within the fusion protein unless expressly stated as such.

[0114] As used herein, the term "framework region" or "FR region" refers to amino acid residues that are part of the variable region of an antibody but are not part of the CDRs (e.g., using the Kabat definition of a CDR).

[0115] As used herein, the term "full length antibody" refers to an antibody having a structure substantially similar to that of a native antibody. For example, an antibody comprising: (i) a first Ig light chain comprising, from N-terminus to C-terminus, a light chain variable region (VL) region and a light chain constant region (CL) region; (ii) a first Ig heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable region (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second Ig heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (iv) a second Ig light chain comprising, from N-terminus to C-terminus, a VL region and a VH region, wherein the first light chain and the first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and the first heavy chain and the second heavy chain associate to form a dimer. In some embodiments, the two heavy chains comprise substantially identical amino acid sequences; and the two light chains comprise substantially identical amino acid sequences. In some embodiments, the two heavy chains comprise substantially identical amino acid sequences, except for one or more amino acid modifications that promote heterodimerization of the correct heavy chains (e.g., as described herein); and the two light chains comprise substantially identical amino acid sequences. Antibody chains can be substantially identical, but may not be completely identical if they differ by post-translational modifications, such as C-terminal truncation of lysine residues, alternative glycosylation patterns, etc.

[0116] The term "functional variant," as used herein with reference to a polypeptide or protein, refers to a polypeptide or protein that contains at least one, but not more than 15%, not more than 12%, not more than 10%, not more than 8% amino acid variation (e.g., substitution, deletion, addition) compared to the amino acid sequence of a reference polypeptide or protein, and that retains at least one specific function of the reference polypeptide or protein. A functional variant of a protein need not retain all of the functions of a reference polypeptide or protein (e.g., wild-type). In some examples, one or more functions are selectively reduced or eliminated. In some embodiments, the reference polypeptide or protein is a wild-type protein.

[0117] The term "functional fragment," as used herein in reference to a polypeptide or protein, refers to a fragment of a reference polypeptide or protein that retains at least one specific function. A functional fragment of a polypeptide or protein does not necessarily retain all of the functions of the reference polypeptide or protein. In some instances, one or more functions are selectively reduced or eliminated. In some embodiments, the reference polypeptide or protein is a wild-type protein.

[0118] As used herein, the term "fused" and its grammatical equivalents refer to an operative connection between at least a first polypeptide and a second polypeptide, where the first and second polypeptides are not found operatively connected together in nature. For example, the first and second polypeptides are derived from different proteins. The term fused encompasses both a direct connection of at least two polypeptides through a peptide bond and an indirect connection through a linker (e.g., a peptide linker).

[0119] As used herein, the term "fusion protein" and its grammatical equivalents refer to a protein comprising at least one polypeptide operably linked to another polypeptide, wherein the first and second polypeptides are different and are not naturally found operably linked together. For example, the first and second polypeptides of a fusion protein are each derived from different proteins. The at least two polypeptides of a fusion protein may be operably linked directly through a peptide bond; or indirectly through a linker (e.g., a peptide linker). Thus, for example, the term "fusion polypeptide" encompasses embodiments in which polypeptide A is operably linked directly to polypeptide B through a peptide bond (polypeptide A-polypeptide B) and embodiments in which polypeptide A is operably linked to polypeptide B through a peptide linker (polypeptide A-peptide linker-polypeptide B).

[0120] As used herein, the term "heavy chain" typically refers to a portion of an immunoglobulin (e.g., human Ig) comprising, from N- to C-terminus, a heavy chain variable region (VH), CH1 region, hinge region, CH2 region, and CH3 region. The constant region of the heavy chain (i.e., the CH1 region, hinge region, CH2 region, and CH3 region) can be of any distinct isotype, e.g., human alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to the hIgA, hIgD, IgE, hIgG, and hIgM classes of human antibodies, respectively, including subclasses of hIgG, e.g., hIgG1, hIgG2, hIgG3, and hIgG4. As used herein, the term "heavy chain," when used in reference to a human antibody, can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to the human IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of human IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0121] As used herein, the term "half-life extending moiety" refers to a moiety (e.g., a small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., polymer of PEG), etc.) that, when conjugated or otherwise operably connected (e.g., fused) to a polypeptide or protein (a polypeptide or protein of interest), increases the half-life of the polypeptide or protein of interest in vivo upon administration to a subject (e.g., a human subject). The pharmacokinetic properties of a polypeptide or protein can be assessed utilizing in vitro and in vivo models known in the art.

[0122] As used herein, the term "half-life extending polypeptide" or "half-life extending protein" refers to a polypeptide that, when operably linked to another polypeptide (a polypeptide or protein of interest), increases the half-life of the polypeptide of interest in vivo upon administration to a subject (e.g., a human subject). The pharmacokinetic properties of a polypeptide or protein can be assessed using in vitro and in vivo models known in the art.

[0123] As used herein, the term "heterologous," when used to describe a first element with reference to a second element, means that the first element and the second element do not naturally occur in the described arrangement. For example, a polypeptide comprising a "heterologous moiety" refers to a polypeptide linked to a moiety (e.g., a small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., polymer of PEG), etc.) that is not naturally linked to the polypeptide. For example, a non-limiting example of a heterologous moiety is a heterologous polypeptide (as defined herein).

[0124] As used herein, the term "heterologous signal peptide" refers to a signal peptide that is not naturally operably linked to a polypeptide or protein of interest. For example, in reference to a polypeptide comprising the signal peptide from human IL-2 (hIL-2) operably linked to hIL-12p40, the hIL-2 signal peptide constitutes a heterologous signal peptide.

[0125] As used herein, the term "cognate signal peptide" refers to a signal peptide that is naturally operably linked to a polypeptide or protein of interest. For example, in reference to a polypeptide comprising a signal peptide from human IL-2 operably linked to hIL-2, the hIL-2 signal peptide constitutes the cognate signal peptide.

[0126] As used herein, the term "human T cell costimulatory antigen" or "hTCSA" refers to a protein expressed on the surface of human T cells that is not part of the T cell receptor complex and that enhances T cell activation upon binding of its cognate ligand. Exemplary hTCSAs are described herein and include, for example, hCD28, hCD2, and h41BB.

[0127] As used herein, the term "human CD28" or "hCD28" refers to the human CD28 protein. hCD28 is a transmembrane protein expressed on T cells that provides a costimulatory signal necessary for T cell activation and survival. The amino acid sequence of an exemplary reference mature hCD28 protein is set forth in SEQ ID NO:2.

[0128] As used herein, the term "human CD2" or "hCD2" refers to the human CD2 protein. hCD2 is a transmembrane protein expressed on T cells and NK cells that provides a costimulatory signal. The amino acid sequence of an exemplary reference mature hCD2 protein is set forth in SEQ ID NO: 38.

[0129] As used herein, the term "human CD3" or "hCD3" refers to the human CD3 protein complex. The human CD3 protein complex comprises four distinct proteins: hCD3ε, hCD3ζ, hCD3γ, and hCD3δ. Thus, as used herein, the term "CD3" encompasses hCD3ε, hCD3ζ, hCD3γ, and hCD3δ. When a specific hCD3 protein of the CD3 complex is intended, the specific protein (hCD3ε, hCD3ζ, hCD3γ, or hCD3δ) is designated. For example, reference to an "antigen-binding domain that specifically binds hCD3" and similar references encompasses an antigen-binding domain that specifically binds to any one of hCD3ε, hCD3ζ, hCD3γ, or hCD3δ. On the other hand, reference to an "antigen-binding domain that specifically binds hCD3ε" and similar references refers to an antigen-binding domain that specifically binds to hCD3ε but not to hCD3ζ, hCD3γ, or hCD3δ. The amino acid sequence of an exemplary reference mature hCD3ε protein is set forth in SEQ ID NO: 22. The amino acid sequence of an exemplary reference mature hCD3δ protein is set forth in SEQ ID NO: 28. The amino acid sequence of an exemplary reference mature hCD3γ protein is set forth in SEQ ID NO: 26. The amino acid sequence of an exemplary reference mature hCD3ζ protein is set forth in SEQ ID NO: 24.

[0130] As used herein, the term "human tumor-associated antigen" or "hTAA" refers to a protein expressed on the surface of human cancer cells that enables the recruitment of a multispecific protein described herein to the human cancer cells. In some embodiments, the tumor-associated antigen is expressed by both normal cells and cancer cells. In some embodiments, the tumor-associated antigen is overexpressed by cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold or more overexpression compared to normal cells. In some embodiments, the tumor-associated antigen is a protein that is inappropriately synthesized by cancer cells and contains amino acid modifications (e.g., amino acid deletions, additions, and / or substitutions) compared to proteins expressed by normal cells. In some embodiments, the tumor-associated antigen is expressed only by cancer cells and is not expressed at detectable levels by normal cells. Methods for identifying and validating tumor-associated proteins are known to those skilled in the art and are described in the literature (see, for example, Bornstein, AAPS J. (2015), vol. 17(3), pp. 525-534; Hong et al., BMC Syst Biol. (2018), vol. 12(Suppl 2), p. 17, the entire contents of each of which are incorporated herein by reference for all purposes).

[0131] As used herein, the term "hIgM CH2 mFab" refers to a modified Fab comprising a hIgM CH2 mFab heavy chain and a hIgM CH2 mFab light chain, wherein the hIgM CH2 mFab heavy chain and the hIgM CH2 mFab light chain associate to form an antigen-binding domain.

[0132] As used herein, the term "hIgM CH2 mFab heavy chain" comprises, from N- to C-terminus, a VH region and a hIgM CH2 region. The amino acid sequence of an exemplary hIgM CH2 region is set forth in SEQ ID NO: 126.

[0133] As used herein, the term "hIgM CH2 mFab light chain" comprises, from N- to C-terminus, a VL region and a hIgM CH2 region. The amino acid sequence of an exemplary hIgM CH2 region is set forth in SEQ ID NO: 126.

[0134] As used herein, the term "isolated" with reference to a polypeptide, protein, polynucleotide, vector (and the like) refers to a polypeptide, protein, polynucleotide, vector (or the like) that is substantially free from other cellular components with which it is naturally associated.

[0135] As used herein, the term "light chain" refers to a portion of an immunoglobulin (e.g., a human immunoglobulin) that comprises, from N- to C-terminus, a light chain variable region (VL) operably connected to a light chain constant region (CL). The CL can be of any distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the CL. In some embodiments, the multispecific proteins described herein comprise one or more light chains.

[0136] As used herein, the term translatable RNA refers to any RNA that encodes at least one peptide or protein and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded peptide or protein. In some embodiments, the translatable RNA is mRNA. The translatable RNA can be linear or circular.

[0137] As used herein, the term "modified" when referring to a polynucleotide refers to a polynucleotide that contains at least one substitution, alteration, inversion, addition, or deletion of a nucleotide (e.g., one or more amino acid substitutions) compared to a reference polynucleotide. A modification may include the inclusion of a non-naturally occurring nucleotide residue. As used herein, the term "modified" when referring to an amino acid sequence refers to an amino acid sequence that contains at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference amino acid sequence. A modification may include the inclusion of a non-naturally occurring amino acid residue. Naturally occurring amino acid derivatives are not considered modified amino acids for purposes of determining the percent identity of two amino acid sequences. For example, a naturally occurring modification of a glutamate amino acid residue to a pyroglutamate amino acid residue is not considered an amino acid modification for purposes of determining the percent identity of two amino acid sequences. Furthermore, for example, a naturally occurring modification of a glutamate amino acid residue to a pyroglutamate amino acid residue is not considered an amino acid "modification" as defined herein.

[0138] A "modification that promotes heterodimerization of a first Fc region and a second Fc region" (or similar phrases) refers to a manipulation of the peptide backbone or a post-translational modification of the Fc region that reduces or prevents a polypeptide comprising an Fc region from associating with the same polypeptide to form a homodimer. As used herein, a modification that promotes association specifically includes individual modifications made to each of two Fc regions (i.e., a first Fc region and a second Fc region) that are desired to associate, where the modifications are complementary to each other to promote association of the two Fc regions. For example, a modification that promotes association may alter the structure or charge of one or both of the Fc regions to make the association sterically or electrostatically favorable, respectively. In this manner, heterodimerization occurs between a polypeptide comprising a first Fc region and a polypeptide comprising a second Fc region, which may be non-identical in the sense that the additional components (e.g., antigen-binding domains) fused to each of the Fc regions are not the same. In some embodiments, a modification that promotes association includes an amino acid mutation, specifically an amino acid substitution, in the Fc region. In certain embodiments, the association-promoting modifications comprise individual amino acid mutations, specifically one or more amino acid substitutions in each of the first and second Fc regions. See, e.g., §5.2.7.2.

[0139] As used herein, the term "moiety" is used generically to describe any macromolecule or small molecule that may be operably linked to a polypeptide or protein described herein. Exemplary moieties include, but are not limited to, small molecules, polypeptides, proteins, polynucleotides (e.g., DNA, RNA), carbohydrates, lipids, and synthetic polymers (e.g., polymers of PEG).

[0140] As used herein, the term "multispecific," when referring to a protein or polypeptide (e.g., a protein or polypeptide described herein), refers to the protein or polypeptide comprising at least two antigen-binding domains that bind to different antigens. Thus, the term multispecific includes, for example, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc. In some embodiments, a multispecific antibody is trispecific. In some embodiments, a multispecific antibody is trispecific and tetravalent. In some embodiments, a multispecific antibody is trispecific and trivalent.

[0141] As used herein, the term "operably connected" refers to two moieties (e.g., two polypeptides or two polynucleotides) linked in a functional relationship. For example, a polypeptide is operably connected to another polypeptide if they are linked in-frame (either directly or indirectly via a peptide linker) such that both polypeptides are functional (e.g., a fusion protein or polypeptide described herein). Or, for example, a transcriptional regulatory polynucleotide, such as a promoter, enhancer, or other expression control element, is operably linked to a polynucleotide encoding a protein if it affects the transcription of the polynucleotide encoding the protein. The term "operably connected" can also refer to the conjugation of a moiety, for example, a polynucleotide or polypeptide (e.g., the conjugation of a PEG polymer to a protein or polypeptide).

[0142] The determination of "percent identity" between two sequences (e.g., peptides or proteins (amino acid sequences) or polynucleotides (nucleic acid sequences)) can be achieved using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified by Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, each of which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, for example, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with XBLAST program parameters set to, for example, score 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used, as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, the entire contents of which are incorporated herein by reference. Alternatively, PSI BLAST can be used to perform an iterated search to detect distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) World Wide Web at ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without gaps allowed. When calculating percent identity, typically only exact matches are counted.

[0143] As used herein, the term "pharmaceutical composition" means a composition suitable for administration to an animal, e.g., a human subject, comprising a therapeutic agent and a pharmaceutically acceptable carrier or diluent. A "pharmaceutically acceptable carrier or diluent" means a material for use in contact with the tissues of humans and / or non-human animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable therapeutic benefit / risk ratio.

[0144] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of DNA or RNA. Nucleic acid molecules can be single- or double-stranded; can contain natural, non-natural, or modified nucleotides; and can contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, instead of the phosphodiester linkages found between nucleotides in unmodified nucleic acid molecules. Nucleic acid molecules include all nucleic acid molecules obtained by any means available in the art, including, but not limited to, recombinant means, such as cloning nucleic acid molecules from recombinant libraries or cellular genomes using conventional cloning techniques and the polymerase chain reaction, and synthetic means. Those skilled in the art will recognize that, unless otherwise noted, the nucleic acid sequences set forth in this application recite thymidine (T) in representative DNA sequences, but where the sequence represents RNA (e.g., mRNA), thymidine (T) is substituted with uracil (U). Thus, any RNA polynucleotide encoded by a DNA identified by a particular sequence identification number may also include the corresponding RNA (e.g., mRNA) sequence encoded by that DNA in which each thymidine (T) in the DNA sequence is replaced with uracil (U).

[0145] As used herein, the term "polypeptide" refers to a polymer of at least two (e.g., at least five) amino acids linked by peptide bonds. The term "polypeptide" does not refer to a specific length of the polymer chain of amino acids. It is common in the art to refer to shorter polymers of amino acids (e.g., approximately 2 to 50 amino acids) as peptides and longer polymers of amino acids (e.g., more than approximately 50 amino acids) as polypeptides. However, the terms "peptide" and "polypeptide" are used interchangeably herein.

[0146] As used herein, the term "protein" refers to a polypeptide or a set of polypeptides (i.e., at least two). In embodiments where a protein comprises a set of polypeptides, the set of polypeptides associate to form a functional unit (i.e., a quaternary structure). In some embodiments, a polypeptide or a set of polypeptides is folded into its three-dimensional structure (i.e., a tertiary or quaternary structure). It should be understood that when a polypeptide or a set of polypeptides is contemplated herein, a protein comprising the polypeptide or set of polypeptides folded into its three-dimensional structure (i.e., a tertiary or quaternary structure) is also provided herein, and vice versa.

[0147] A "prophylactic" treatment is a treatment administered to a subject who shows no signs of disease, or who shows only early signs of disease, for the purpose of reducing the risk of developing the condition.

[0148] The terms "RNA" and "polyribonucleotide" are used interchangeably herein and refer to a polymer comprising multiple ribonucleotides polymerized through phosphodiester bonds. Ribonucleotides are nucleotides whose sugar is ribose. RNA may contain modified nucleotides; and may contain natural, non-natural, or modified internucleotide bonds, such as phosphoramidate or phosphorothioate bonds, instead of the phosphodiester bonds found between nucleotides in unmodified nucleic acid molecules.

[0149] The term "scFv" or "single-chain variable fragment" refers to an antibody comprising a VH region operably connected to a VL region via a peptide linker, wherein the VH and VL regions associate to specifically bind to an antigen (e.g., form an antigen-binding domain). In some embodiments, an scFv comprises, from N-terminus to C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, an scFv comprises, from N-terminus to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, an scFv further comprises one or more engineered disulfide bonds connecting the VH region to the VL region.

[0150] The term "(scFv)2" as used herein refers to an antibody comprising a first and a second scFv operably connected (e.g., via a peptide linker). The first scFv and the second scFv can specifically bind to the same or different antigens. In some embodiments, the first scFv and the second scFv are operably connected by a peptide linker.

[0151] The term "scFv-Fc," as used herein, refers to an antibody comprising an scFv operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, the scFv is operably connected to only the first Fc domain of a first and second Fc domain pair. In some embodiments, the first scFv is operably connected to the first Fc domain of a first and second Fc domain pair, and the second scFv is operably connected to the second Fc domain.

[0152] The term "(scFv)2-Fc," as used herein, refers to an (scFv)2 operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, the (scFv)2 is operably connected to only the first Fc domain of a first and second Fc domain pair. In some embodiments, the first (scFv)2 is operably connected to the first Fc domain of a first and second Fc domain pair, and the second (scFv)2 is operably connected to the second Fc domain.

[0153] As used herein, the term "single domain antibody" or "sdAb" refers to an antibody having a single monomeric variable antibody domain. An sdAb can specifically bind to a specific antigen. A VHH (as defined herein) is an example of an sdAb.

[0154] As used herein, the term "signal peptide" or "signal sequence" refers to a sequence (e.g., an amino acid sequence) that can direct the transport or localization of a protein to a particular organelle, cellular compartment, or export out of a cell. The term encompasses both signal sequence peptides and nucleic acid sequences that encode signal peptides. Thus, reference to a signal peptide in the context of a nucleic acid refers to a nucleic acid sequence that encodes the signal peptide.

[0155] As used herein, the term "specifically binds" refers to a preferential interaction, i.e., a significantly higher binding affinity, between a first moiety (e.g., a protein (e.g., a ligand)) and a second moiety (e.g., a protein (e.g., a cognate receptor for the ligand)) compared to other moieties (e.g., amino acid sequences). For example, when a first protein or polypeptide is said to "specifically bind" to a second protein or polypeptide, it is understood that the first protein or polypeptide specifically binds to an epitope of the second protein or polypeptide. The term "epitope" refers to the portion of the second protein or polypeptide that the first protein or polypeptide specifically recognizes. The term "specifically binds" includes molecules that are cross-reactive with the same epitope in different species. For example, an antibody that specifically binds to human CD28 may be cross-reactive with CD28 from another species (e.g., cynomolgus monkey, mouse, etc.), but still be considered herein to specifically bind to human CD28. A plurality of moieties (eg, proteins) can specifically bind to more than one moiety (eg, protein (eg, epitope)).

[0156] As used herein, the term "subject" includes any animal, e.g., a human or other animal. In some embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In some embodiments, the subject is a human. In some embodiments, the subject of the method is a non-human mammal. In some embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., a cow, a buffalo, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In some embodiments, the subject is a bird, e.g., a member of the avian taxonomic groups Galliformes (e.g., chickens, turkeys, pheasants, quails), Anseriformes (e.g., ducks, geese), Paleognatha (e.g., ostriches, emus), Columbiformes (e.g., pigeons, stilt pigeons), or Psittaciformes (e.g., parrots).

[0157] As used herein, the term "therapeutically effective amount" of an agent (e.g., a therapeutic agent) refers to any amount of an agent (e.g., a therapeutic agent) that, when used alone or in combination with another agent (e.g., a therapeutic agent), protects a subject from developing a disease or promotes disease regression as evidenced by a reduction in the severity of symptoms of an infectious disease, an increase in the frequency and duration of asymptomatic periods of a disease or infectious disease, or prevention of disability or incapacity due to contraction of a disease or infectious disease. The ability of an agent (e.g., a therapeutic agent) to promote disease regression can be assessed using a variety of methods known to physicians skilled in the art, such as by assaying the activity of the agent in human subjects in clinical trials, animal model systems predictive of efficacy in humans, or in vitro assays.

[0158] As used herein, the terms "treat," "treating," "treatment," and similar terms refer to reducing or ameliorating a disease and / or disease-related symptoms, or achieving a desired pharmacological and / or physiological effect. It is recognized, although not excluded, that treating a disease does not require the complete elimination of the disease or disease-related symptoms. In some embodiments, the effect is therapeutic, i.e., including but not limited to, the effect partially or completely reduces, impairs, abolishes, alleviates, relieves, reduces the intensity of, or cures the disease and / or adverse symptoms resulting from the disease. In some embodiments, the effect is prophylactic, i.e., the effect protects against or prevents the occurrence or recurrence of the disease. To this end, the methods described herein comprise administering a therapeutically effective amount of a composition described herein.

[0159] As used herein, the term "variable region" refers to a portion of an antibody, generally the light or heavy chain, that varies extensively in sequence and is used for binding and specificity of a particular antibody for its particular antigen; typically, approximately 110-120 or 110-125 amino acids at the amino terminus of a mature heavy chain and approximately 90-115 amino acids in a mature light chain. The variability in sequence is concentrated in those regions called complementarity-determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, the CDRs of the light and heavy chains are believed to be primarily responsible for the interaction and specificity of an antibody with an antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0160] The terms "VH" and "VH region" are used interchangeably and refer to an immunoglobulin heavy chain variable region. A VH region can be incorporated into an antibody, such as an scFv, Fab, or full-length antibody. For example, an scFv contains a VH region operably connected to a VL region via a peptide linker.

[0161] The terms "VL" and "VL region" are used interchangeably and refer to an immunoglobulin light chain variable region. The VL region can be incorporated into an antibody, such as an scFv, Fab, or full-length antibody. For example, an scFv contains a VL region operably connected to a VH region via a peptide linker.

[0162] The term "VHH" as used herein refers to a type of single domain antibody (sdAb) having a single monomeric heavy chain variable antibody domain (VH). Such antibodies can be found in or produced from camelids (e.g., camels, llamas) that naturally lack light chains, or can be produced synthetically.

[0163] 5.2 Multispecific proteins In one aspect, provided herein are multispecific proteins that specifically bind to a TAA (e.g., a human tumor-associated antigen (hTAA)), CD3 (e.g., human (hCD3)), and a TCSA (e.g., human TCSA (hTCSA) (e.g., hCD28, hCD2)). The components of the multispecific proteins described herein can be arranged in one of the following formats, as described in detail below: format BCA405 (see, e.g., Figure 1), format BCA406 (see, e.g., Figure 2), format BCA424 (see, e.g., Figure 3), or format BCA418 (see, e.g., Figure 4).

[0164] 5.2.1 Multispecific Protein Formats 5.2.1.1 Format BCA405 In one aspect, provided herein is a multispecific protein in format BCA405 (see, e.g., Figure 1 ). Generally, format BCA405 provides a multispecific protein comprising: (i) a full-length antibody that specifically binds to a first hTAA and a second hTAA; (ii) a first scFv operably linked to the C-terminus of a first heavy chain of the full-length antibody, wherein the first scFv specifically binds to hCD3; and a second scFv operably linked to the C-terminus of a second heavy chain of the full-length antibody, wherein the second scFv specifically binds to an hTCSA (e.g., hCD28, hCD2).

[0165] In some embodiments, the multispecific protein comprises: (a) a full-length antibody comprising: (i) a first light chain comprising, from N-terminus to C-terminus, a light chain variable region (VL) region and a light chain constant region (CL) region; (ii) a first heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable region (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iv) a second light chain comprising, from N-terminus to C-terminus, a VL region and a VH region, wherein the first light chain and the first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and the first heavy chain and the second heavy chain associate to form a dimer; and (b) a full-length antibody comprising the CH3 region of the first heavy chain of the full-length antibody. and (c) a second scFv operably connected to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody, the second scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; the first antigen-binding domain of the full-length antibody specifically binds to a first hTAA; the second antigen-binding domain of the full-length antibody specifically binds to a second hTAA; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); and the second scFv specifically binds to hCD3.

[0166] In some embodiments, the first scFv is operably connected directly to the C-terminus of the CH3 of the first heavy chain via a peptide bond. In some embodiments, the first scFv is operably connected indirectly to the C-terminus of the CH3 of the first heavy chain via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the second scFv is operably connected directly to the C-terminus of the CH3 of the second heavy chain via a peptide bond. In some embodiments, the second scFv is operably connected indirectly to the C-terminus of the CH3 of the second heavy chain via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the first scFv is operably connected directly to the C-terminus of the CH3 of the first heavy chain via a peptide bond; and the second scFv is operably connected directly to the C-terminus of the CH3 of the second heavy chain via a peptide bond. In some embodiments, the first scFv is operably connected to the C-terminus of the CH3 of the first heavy chain indirectly via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8); and the second scFv is operably connected to the C-terminus of the CH3 of the second heavy chain indirectly via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the amino acid sequence of the peptide linker operably connecting the first scFv to the C-terminus of the CH3 region of the first heavy chain and the amino acid sequence of the peptide linker operably connecting the second scFv to the C-terminus of the CH3 region of the second heavy chain are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, the amino acid sequence of the peptide linker operably connecting the first scFv to the C-terminus of the CH3 region of the first heavy chain and the amino acid sequence of the peptide linker operably connecting the second scFv to the C-terminus of the CH3 region of the second heavy chain are 100% identical.

[0167] In some embodiments, a first scFv is operably connected to the C-terminus of the CH3 region of a first heavy chain through the VH region of the first scFv (either directly or indirectly via a peptide linker). In some embodiments, a first scFv is operably connected to the C-terminus of the CH3 region of a first heavy chain through the VL region of the first scFv (either directly or indirectly via a peptide linker). In some embodiments, a second scFv is operably connected to the C-terminus of the CH3 region of a second heavy chain through the VH region of the second scFv (either directly or indirectly via a peptide linker). In some embodiments, a second scFv is operably connected to the C-terminus of the CH3 region of a second heavy chain through the VL region of the second scFv (either directly or indirectly via a peptide linker). In some embodiments, the first scFv is operably connected to the C-terminus of the CH3 region of the first heavy chain through the VH region of the first scFv (either directly or indirectly via a peptide linker); and the second scFv is operably connected to the C-terminus of the CH3 region of the second heavy chain through the VH region of the second scFv (either directly or indirectly via a peptide linker). In some embodiments, the first scFv is operably connected to the C-terminus of the CH3 region of the first heavy chain through the VL region of the first scFv (either directly or indirectly via a peptide linker); and the second scFv is operably connected to the C-terminus of the CH3 region of the second heavy chain through the VL region of the second scFv (either directly or indirectly via a peptide linker).

[0168] In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first hTAA and the second hTAA are different. In some embodiments, the first hTAA and the second hTAA are different but expressed by the same cancer cells. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds to the same hTAA as the second antigen-binding domain of the full-length antibody. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds to the same epitope as the second antigen-binding domain of the full-length antibody. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds to the same hTAA as the second antigen-binding domain of the full-length antibody, but specifically binds to a different epitope. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds to the same hTAA as the second antigen-binding domain of the full-length antibody, but specifically binds to a different, non-overlapping epitope (e.g., the full-length antibody is biparatopic). In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds to a different hTAA than the second antigen-binding domain of the full-length antibody.

[0169] In some embodiments, a multispecific protein comprises: (a) a first polypeptide comprising, from N-terminus to C-terminus, a first light chain comprising a VL region and a CL region; (b) a second polypeptide comprising, from N-terminus to C-terminus: (i) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (ii) an optional first peptide linker, and (iii) a first scFv comprising, from N-terminus to C-terminus, (iii(a)) a VL region, a peptide linker, and a VH region, or (iii(b)) a VH region, a peptide linker, and a VL region; and (c) from N-terminus to C-terminus: (i) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region, (ii) an optional second peptide linker; and (iii) a third polypeptide comprising, from N-terminus to C-terminus, (iii(a)) a VL region, a peptide linker, and a VH region, or (iii(b)) a VH region, a peptide linker, and a VL region; and (d) a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain comprising a VL region and a CL region; the VL of the first light chain and the VH of the first heavy chain associate to form a first antigen-binding domain that specifically binds to a first hTAA; the VH of the second heavy chain and the VL of the second light chain associate to form a second antigen-binding domain that specifically binds to a second hTAA; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); and the second scFv specifically binds to hCD3.

[0170] In some embodiments, the second polypeptide comprises, from N-terminus to C-terminus: (i) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (ii) a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8); and (iii) a first scFv comprising, from N-terminus to C-terminus, (iii(a)) a VL region, a peptide linker, and a VH region, or (iii(b)) a VH region, a peptide linker, and a VL region. In some embodiments, the third polypeptide comprises, from N-terminus to C-terminus: (i) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (ii) a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8); and (iii) a second scFv comprising, from N-terminus to C-terminus, (iii(a)) a VL region, a peptide linker, and a VH region, or (iii(b)) a VH region, a peptide linker, and a VL region. In some embodiments, the second polypeptide comprises, from N-terminus to C-terminus: (i) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (ii) a first peptide linker (e.g., a linker described herein) (see, e.g., § 5.2.8); and (iii) a first scFv comprising, from N-terminus to C-terminus, (iii(a)) a VL region, a peptide linker, and a VH region, or (iii(b)) a VH region, a peptide linker, and a VL region. and the third polypeptide comprises, from N-terminus to C-terminus: (i) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region, and (ii) a second peptide linker (e.g., a linker described herein) (see, e.g., § 5.2.8); and (iii) a second scFv comprising, from N-terminus to C-terminus, (iii(a)) a VL region, a peptide linker, and a VH region, or (iii(b)) a VH region, a peptide linker, and a VL region.In some embodiments, the amino acid sequences of the first and second peptide linkers are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, hi some embodiments, the amino acid sequences of the first and second peptide linkers are 100% identical.

[0171] In some embodiments, the first scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the first scFv comprises, from N- to C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, the second scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the second scFv comprises, from N- to C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, the first scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region; and the second scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the first scFv comprises, from N-terminus to C-terminus, a VH region, a peptide linker, and a VL region; and the second scFv comprises, from N-terminus to C-terminus, a VH region, a peptide linker, and a VL region.

[0172] In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first hTAA and the second hTAA are different. In some embodiments, the first hTAA and the second hTAA are different but expressed by the same cancer cells. In some embodiments, the first antigen-binding domain specifically binds to the same hTAA as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds to the same epitope as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds to the same hTAA as the second antigen-binding domain, but to a different epitope. In some embodiments, the first antigen-binding domain specifically binds to the same hTAA as the second antigen-binding domain, but to a different, non-overlapping epitope. In some embodiments, the first antigen-binding domain specifically binds to a different hTAA than the second antigen-binding domain.

[0173] Generally, format BCA405 also includes (a) a first polypeptide comprising, from N-terminus to C-terminus, a first light chain comprising a VL region and a CL region; and (b) from N-terminus to C-terminus: (i) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (ii) an optional first peptide linker (e.g., a linker described herein) (see, e.g., § 5.2.8), and (iii) a second polypeptide comprising, from N-terminus to C-terminus: (iii(a)) a VL region, a peptide linker, and a VH region, or (iii(b)) a VH region, a peptide linker, and a VL region; and (c) a second polypeptide comprising, from N-terminus to C-terminus: (i) a second heavy chain comprising, from N-terminus to C-terminus: a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (ii) an optional second peptide linker (e.g., as described herein). and (iii) a third polypeptide comprising, from N-terminus to C-terminus, (iii(a)) a VL region, a peptide linker, and a VH region, or (iii(b)) a VH region, a peptide linker, and a VL region; and (d) a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain comprising a VL region and a CL region, wherein the VL of the first light chain and the VH of the first heavy chain associate to form a first antigen-binding domain that specifically binds to a first hTAA; the VH of the second heavy chain and the VL of the second light chain associate to form a second antigen-binding domain that specifically binds to a second hTAA; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); and the second scFv specifically binds to hCD3;and the CH3 region of the first heavy chain comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications, and the CH3 region of the second heavy chain comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications; and the one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody are different from the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody; the one or more amino acid modifications in the CH3 region of the heavy chain of the full-length antibody and the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody promote heterodimerization of the first and second heavy chains of the full-length antibody; the CH2 region of the first heavy chain comprises the one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); the CH2 region of the second heavy chain comprises the one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications;The one or more amino acid modifications in the CH2 region of the first heavy chain and the one or more amino acid modifications in the CH2 region of the second heavy chain may also be described as a multispecific protein, wherein the one or more amino acid modifications in the CH2 region of the first heavy chain reduce or eliminate one or more of the following heavy chain effector functions: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))) compared to a reference heavy chain that does not contain the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 domain, e.g., SEQ ID NO: 100). In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first hTAA and the second hTAA are different. In some embodiments, the first hTAA and the second hTAA are different but expressed by the same cancer cell;

[0174] 5.2.1.2 Format BCA406 In one aspect, provided herein is a multispecific protein in format BCA406 (see, e.g., Figure 2). Generally, format BCA406 provides a multispecific protein comprising: (i) a full-length antibody that specifically binds to a first hTAA and a second hTAA; (ii) a first scFv operably linked to the N-terminus of either a first heavy chain or a first light chain of the full-length antibody, wherein the first scFv specifically binds to hCD3; and a second scFv operably linked to the N-terminus of either a second heavy chain or a second light chain of the full-length antibody, wherein the second scFv specifically binds to an hTCSA (e.g., hCD28, hCD2).

[0175] In some embodiments, the multispecific protein comprises: (a) a full-length antibody comprising: (i) a first light chain comprising, from N-terminus to C-terminus, a VL region and a CL region; (ii) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iv) a second light chain comprising, from N-terminus to C-terminus, a VL region and a VH region, wherein the first light chain and the first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and the first heavy chain and the second heavy chain associate to form a dimer; and (b) a full-length antibody comprising: a second light chain operably connected to the N-terminus of the first heavy chain of the full-length antibody; and (c) a second scFv operably connected to the N-terminus of the second heavy chain of the full-length antibody, the second scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; the first antigen-binding domain of the full-length antibody specifically binds to a first hTAA; the second antigen-binding domain of the full-length antibody specifically binds to a second hTAA; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); and the second scFv specifically binds to hCD3.

[0176] In some embodiments, the first scFv is operably connected directly to the N-terminus of the first heavy chain of the full-length antibody via a peptide bond. In some embodiments, the first scFv is operably connected indirectly to the N-terminus of the first heavy chain of the full-length antibody via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the second scFv is operably connected directly to the N-terminus of the second heavy chain of the full-length antibody via a peptide bond. In some embodiments, the second scFv is operably connected indirectly to the N-terminus of the second heavy chain of the full-length antibody via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the first scFv is operably connected directly to the N-terminus of the first heavy chain of the full-length antibody via a peptide bond; and the second scFv is operably connected directly to the N-terminus of the second heavy chain of the full-length antibody via a peptide bond. In some embodiments, the first scFv is operably connected to the N-terminus of the first heavy chain of the full-length antibody indirectly via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8); and the second scFv is operably connected to the N-terminus of the second heavy chain of the full-length antibody indirectly via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the amino acid sequence of the peptide linker operably connecting the first scFv to the N-terminus of the first heavy chain of the full-length antibody and the amino acid sequence of the peptide linker operably connecting the second scFv to the N-terminus of the second heavy chain of the full-length antibody are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, the amino acid sequence of the peptide linker that operably connects the first scFv to the N-terminus of the first heavy chain of the full-length antibody and the amino acid sequence of the peptide linker that operably connects the second scFv to the N-terminus of the second heavy chain of the full-length antibody are 100% identical.

[0177] In some embodiments, a first scFv is operably connected to the N-terminus of a first heavy chain of a full-length antibody through the VH region of the first scFv (either directly or indirectly through a peptide linker). In some embodiments, a first scFv is operably connected to the N-terminus of a first heavy chain of a full-length antibody through the VL region of the first scFv (either directly or indirectly through a peptide linker). In some embodiments, a second scFv is operably connected to the N-terminus of a second heavy chain of a full-length antibody through the VH region of the second scFv (either directly or indirectly through a peptide linker). In some embodiments, a second scFv is operably connected to the N-terminus of a second heavy chain of a full-length antibody through the VL region of the second scFv (either directly or indirectly through a peptide linker). In some embodiments, the first scFv is operably connected to the N-terminus of the first heavy chain of the full-length antibody through the VH region of the first scFv (either directly or indirectly through a peptide linker); and the second scFv is operably connected to the N-terminus of the second heavy chain of the full-length antibody through the VH region of the second scFv (either directly or indirectly through a peptide linker). In some embodiments, the first scFv is operably connected to the N-terminus of the first heavy chain of the full-length antibody through the VL region of the first scFv (either directly or indirectly through a peptide linker); and the second scFv is operably connected to the N-terminus of the second heavy chain of the full-length antibody through the VL region of the second scFv (either directly or indirectly through a peptide linker).

[0178] In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first hTAA and the second hTAA are different. In some embodiments, the first hTAA and the second hTAA are different but expressed by the same cancer cells. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds to the same hTAA as the second antigen-binding domain of the full-length antibody. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds to the same epitope as the second antigen-binding domain of the full-length antibody. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds to the same hTAA as the second antigen-binding domain of the full-length antibody, but to a different epitope. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds to the same hTAA as the second antigen-binding domain of the full-length antibody, but to a different, non-overlapping epitope (e.g., the full-length antibodies are biparatopic). In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds to a different hTAA than the second antigen-binding domain of the full-length antibody.

[0179] In some embodiments, the multispecific protein comprises: (a) a first polypeptide comprising, from N-terminus to C-terminus, a first light chain comprising a VL region and a CL region; (b) a second polypeptide comprising, from N-terminus to C-terminus: (i) a first scFv comprising, from N-terminus to C-terminus: (i(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; (ii) an optional first peptide linker, and (iii) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (c) a second scFv comprising, from N-terminus to C-terminus: (i) a second scFv comprising, from N-terminus to C-terminus: (i(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; (ii) an optional second peptide linker; and (iii) a third polypeptide comprising, from N-terminus to C-terminus, a second heavy chain comprising a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (d) a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain comprising a VL region and a CL region; the VL region of the first light chain and the VH region of the first Ig heavy chain associate to form a first antigen-binding domain that specifically binds to a first hTAA; the VH region of the second heavy chain and the VL region of the second Ig light chain associate to form a second antigen-binding domain that specifically binds to a second hTAA; the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); and the second scFv specifically binds to hCD3.

[0180] In some embodiments, the second polypeptide comprises, from N-terminus to C-terminus: (i) a first scFv comprising, from N-terminus to C-terminus: (i(a)) a VL region, a peptide linker, and a VH region, or (i(b)) a VH region, a peptide linker, and a VL region; (ii) a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8); and (iii) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the third polypeptide comprises, from N-terminus to C-terminus: (i) a second scFv comprising, from N-terminus to C-terminus: (i(a)) a VL region, a peptide linker, and a VH region, or (i(b)) a VH region, a peptide linker, and a VL region; (ii) a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8); and (iii) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the second polypeptide comprises, from N-terminus to C-terminus: (i) a first scFv comprising, from N-terminus to C-terminus: (i(a)) a VL region, a peptide linker, and a VH region, or (i(b)) a VH region, a peptide linker, and a VL region; (ii) a first peptide linker (e.g., a linker described herein) (see, e.g., § 5.2.8); and (iii) a first heavy chain comprising, from N-terminus to C-terminus: a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. and the third polypeptide comprises, from N-terminus to C-terminus: (i) a second scFv comprising, from N-terminus to C-terminus: (i(a)) a VL region, a peptide linker, and a VH region, or (i(b)) a VH region, a peptide linker, and a VL region; (ii) a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8); and (iii) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.In some embodiments, the amino acid sequences of the first and second peptide linkers are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, hi some embodiments, the amino acid sequences of the first and second peptide linkers are 100% identical.

[0181] In some embodiments, the first scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the first scFv comprises, from N- to C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, the second scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the second scFv comprises, from N- to C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, the first scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region; and the second scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the first scFv comprises, from N-terminus to C-terminus, a VH region, a peptide linker, and a VL region; and the second scFv comprises, from N-terminus to C-terminus, a VH region, a peptide linker, and a VL region.

[0182] In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first hTAA and the second hTAA are different. In some embodiments, the first hTAA and the second hTAA are different but expressed by the same cancer cells. In some embodiments, the first antigen-binding domain specifically binds to the same hTAA as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds to the same epitope as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds to the same hTAA as the second antigen-binding domain, but to a different epitope. In some embodiments, the first antigen-binding domain specifically binds to the same hTAA as the second antigen-binding domain, but to a different, non-overlapping epitope. In some embodiments, the first antigen-binding domain specifically binds to a different hTAA than the second antigen-binding domain.

[0183] Generally, the BCA406 format also includes: (a) a first polypeptide comprising, from N-terminus to C-terminus, a first light chain comprising a VL region and a CL region; and (b) a first scFv comprising, from N-terminus to C-terminus: (i) a first scFv comprising, from N-terminus to C-terminus: (i(a)) a VL region, a peptide linker, and a VH region, or (i(b)) a VH region, a peptide linker, and a VL region; (ii) an optional first peptide linker (e.g., a peptide linker as described herein). linker) (see, e.g., § 5.2.8), and (iii) a second polypeptide comprising, from N-terminus to C-terminus, a first heavy chain comprising a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (c) a second scFv comprising, from N-terminus to C-terminus: (i) a second scFv comprising, from N-terminus to C-terminus: (i(a)) a VL region, a peptide linker, and a VH region, or (i(b)) a VH region, a peptide linker, and a VL region; (ii) any a multispecific protein comprising a selected second peptide linker (e.g., a linker described herein) (see, e.g., § 5.2.8), and (iii) a third polypeptide comprising, from N-terminus to C-terminus, a second heavy chain comprising a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (d) a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain comprising a VL region and a CL region, wherein the VL region of the first light chain and the VH region of the first Ig heavy chain associate to form a first antigen-binding domain that specifically binds to a first hTAA; the VH region of the second heavy chain and the VL region of a second Ig light chain associate to form a second antigen-binding domain that specifically binds to a second hTAA; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); and the second scFv specifically binds to hCD3;and the CH3 region of the first heavy chain comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications, and the CH3 region of the second heavy chain comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications; and the one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody are different from the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody; the one or more amino acid modifications in the CH3 region of the first heavy chain and the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody promote heterodimerization of the first and second heavy chains of the full-length antibody, wherein the CH2 region of the first heavy chain comprises the one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); and the CH2 region of the second heavy chain comprises the one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications;The one or more amino acid modifications in the CH2 region of the first heavy chain and the one or more amino acid modifications in the CH2 region of the second heavy chain may also be described as a multispecific protein, wherein the one or more amino acid modifications in the CH2 region of the first heavy chain reduce or eliminate one or more of the following heavy chain effector functions: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))) compared to a reference heavy chain that does not contain the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 domain, e.g., SEQ ID NO: 100). In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first hTAA and the second hTAA are different. In some embodiments, the first hTAA and the second hTAA are different but expressed by the same cancer cell;

[0184] 5.2.1.3 Format BCA424 In one aspect, provided herein is a multispecific protein according to format BCA424 (see, e.g., Figure 3). Generally, format BCA424 provides a multispecific protein comprising a first Fab operably connected (e.g., via a peptide linker) to a first scFv, which is operably connected (e.g., via a peptide linker) to a first Fc region; and a second Fab operably connected (e.g., via a peptide linker) to a second scFv operably connected (e.g., via a peptide linker) to a second Fc region, wherein the first Fab specifically binds to a first hTAA, the second Fab specifically binds to a second hTAA, the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2), and the second scFv specifically binds to hCD3.

[0185] In some embodiments, the multispecific protein comprises (a) a first Fab comprising (i) a first Fab heavy chain comprising, from N-terminus to C-terminus, a first VH region and a first CH1 region, and (ii) a first light chain comprising, from N-terminus to C-terminus, a first VL region and a first CL region; and (b) a peptide linker (e.g., a peptide linker, e.g., any one of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 31) operably connected to the C-terminus of the first CH1 region of the first Fab. (via 9) a first scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region; or (ii) a VL region, a peptide linker, and a VH region; (c) a first Fc region operably connected to the C-terminus of the first scFv, the first Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region; (d) a second Fab heavy chain comprising, from N-terminus to C-terminus, (i) a second VH region and a second CH1 region; and (ii) a second Fab comprising, from the N-terminus to the C-terminus, a second light chain comprising a second VL region and a first CL region; and (b) a second scFv operably connected to the C-terminus of the second CH1 region of the second Fab (e.g., via a peptide linker, e.g., any one of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319), comprising, from the N-terminus to the C-terminus, (i) a VH region, a peptide linker, and a VL region; or (ii) a VL region, a peptide linker and a second scFv comprising a VH region; (c) a second Fc region operably connected to the C-terminus of the second scFv, the second Fc region comprising a CH2 region and a CH3 region from the N-terminus to the C-terminus; the first Fab specifically binds to a first hTAA; the second Fab specifically binds to a second hTAA; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); and the second scFv specifically binds to hCD3.

[0186] In some embodiments, a first scFv is operably connected directly to the C-terminus of the first CH1 region of the first Fab via a peptide bond. In some embodiments, a first scFv is operably connected indirectly to the C-terminus of the first CH1 region of the first Fab via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any one of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319). In some embodiments, a second scFv is operably connected directly to the C-terminus of the second CH1 region of the second Fab via a peptide bond. In some embodiments, a second scFv is operably connected indirectly to the C-terminus of the second CH1 region of the second Fab via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any one of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319). In some embodiments, a first scFv is operably connected directly to the C-terminus of the first CH1 region of the first Fab via a peptide bond; and a second scFv is operably connected directly to the C-terminus of the second CH1 region of the second Fab via a peptide bond. In some embodiments, a first scFv is operably connected indirectly to the C-terminus of the first CH1 region of the first Fab via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any one of SEQ ID NOs:217-236 or 318-319, e.g., SEQ ID NO:319); and a second scFv is operably connected indirectly to the C-terminus of the second CH1 region of the second Fab via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any one of SEQ ID NOs:217-236 or 318-319, e.g., SEQ ID NO:319).In some embodiments, the amino acid sequence of a peptide linker operably connecting a first scFv to the C-terminus of the first CH1 region of the first Fab and the amino acid sequence of a peptide linker operably connecting a second scFv to the C-terminus of the second CH1 region of the second Fab are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, the amino acid sequence of a peptide linker operably connecting a first scFv to the C-terminus of the first CH1 region of the first Fab and the amino acid sequence of a peptide linker operably connecting a second scFv to the C-terminus of the second CH1 region of the second Fab are 100% identical.

[0187] In some embodiments, the first Fc region is operably connected directly to the C-terminus of the first scFv via a peptide bond. In some embodiments, the first Fc region is operably connected indirectly to the C-terminus of the first scFv via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the second Fc region is operably connected directly to the C-terminus of the second scFv via a peptide bond. In some embodiments, the second Fc region is operably connected indirectly to the C-terminus of the second scFv via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the first Fc region is operably connected directly to the C-terminus of the first scFv via a peptide bond; and the second Fc region is operably connected directly to the C-terminus of the second scFv via a peptide bond. In some embodiments, the first Fc region is operably connected to the C-terminus of the first scFv indirectly via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8); and the second Fc region is operably connected to the C-terminus of the second scFv indirectly via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the amino acid sequence of the peptide linker operably connecting the first Fc region to the C-terminus of the first scFv and the amino acid sequence of the peptide linker operably connecting the second Fc region to the C-terminus of the second scFv are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, the amino acid sequence of the peptide linker operably connecting the first Fc region to the C-terminus of the first scFv and the amino acid sequence of the peptide linker operably connecting the first Fc region to the C-terminus of the first scFv are 100% identical.

[0188] In some embodiments, the first Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the second Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region; and the second Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region comprises, from the N-terminus to the C-terminus, a partial hinge region, a CH2 region, and a CH3 region. In some embodiments, the second Fc region comprises, from the N-terminus to the C-terminus, a partial hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region comprises, from N-terminus to C-terminus, a partial hinge region, a CH2 region, and a CH3 region; and the second Fc region comprises, from N-terminus to C-terminus, a partial hinge region, a CH2 region, and a CH3 region.

[0189] In some embodiments, a first scFv is operably connected to the C-terminus of the first CH1 region of the first Fab through the VH region of the first scFv (either directly or indirectly through a peptide linker). In some embodiments, a first scFv is operably connected to the C-terminus of the first CH1 region of the first Fab through the VL region of the first scFv (either directly or indirectly through a peptide linker). In some embodiments, a second scFv is operably connected to the C-terminus of the second CH1 region of the second Fab through the VH region of the second scFv (either directly or indirectly through a peptide linker). In some embodiments, a second scFv is operably connected to the C-terminus of the second CH1 region of the second Fab through the VL region of the second scFv (either directly or indirectly through a peptide linker). In some embodiments, a first scFv is operably connected to the C-terminus of the first CH1 region of the first Fab through the VH region of the first scFv (either directly or indirectly through a peptide linker); and a second scFv is operably connected to the C-terminus of the second CH1 region of the second Fab through the VH region of the second scFv (either directly or indirectly through a peptide linker). In some embodiments, a first scFv is operably connected to the C-terminus of the first CH1 region of the first Fab through the VL region of the first scFv (either directly or indirectly through a peptide linker); and a second scFv is operably connected to the C-terminus of the second CH1 region of the second Fab through the VL region of the second scFv (either directly or indirectly through a peptide linker).

[0190] In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first hTAA and the second hTAA are different. In some embodiments, the first hTAA and the second hTAA are different but expressed by the same cancer cells. In some embodiments, the first Fab specifically binds to the same hTAA as the second Fab. In some embodiments, the first Fab specifically binds to the same epitope as the second Fab. In some embodiments, the first Fab specifically binds to the same hTAA as the second Fab, but to a different epitope. In some embodiments, the first Fab specifically binds to the same hTAA as the second Fab, but to a different, non-overlapping epitope. In some embodiments, the first Fab specifically binds to a different hTAA than the second Fab.

[0191] In some embodiments, the multispecific protein comprises: (a) a first polypeptide comprising, from N-terminus to C-terminus: (i) a first Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; operably connected thereto: (ii) a first scFv comprising, from N-terminus to C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; operably connected thereto: (b) a second polypeptide comprising, from N-terminus to C-terminus, a first light chain comprising, from N-terminus to C-terminus, a VL region and a CL region; and (c) a second Fab heavy chain comprising, from N-terminus to C-terminus: (i) a second Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; operably connected thereto: (ii) a first Fc region comprising, from N-terminus to C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region; and (ii(b)) a second scFv comprising an L region, a peptide linker, and a VH region, or (ii(b)) a second scFv comprising a VH region, a peptide linker, and a VL region; and (ii(c)) a third polypeptide operably connected thereto, the third polypeptide comprising a second Fc region from N-terminus to C-terminus, the second Fc region comprising a CH2 region and a CH3 region; and (ii(d)) a fourth polypeptide comprising a second light chain from N-terminus to C-terminus, the second light chain comprising a VL region and a CL region; the first Fab heavy chain and the first light chain associate to form a first antigen-binding domain that specifically binds to a first hTAA; the second Fab heavy chain and the second light chain associate to form a second antigen-binding domain that specifically binds to a second hTAA; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); and the second scFv specifically binds to hCD3.

[0192] In some embodiments, the first polypeptide comprises, from N-terminus to C-terminus: (i) a first Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; (ii) a first scFv comprising, from N-terminus to C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region, operably connected thereto via a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8); and a first Fc region operably connected thereto, comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region. In some embodiments, the third polypeptide comprises, from N-terminus to C-terminus: (i) a second Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; (ii) a second scFv comprising, from N-terminus to C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region, operably connected thereto via a second peptide linker (e.g., a linker described herein) (e.g., see §5.2.8, e.g., any one of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319); and (ii) a second Fc region operably connected thereto, comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region.In some embodiments, the first polypeptide comprises, from N-terminus to C-terminus: (i) a first Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; (ii) a first scFv comprising, from N-terminus to C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region, operably connected thereto via a first peptide linker (e.g., a linker described herein) (see, e.g., § 5.2.8); and (ii) a first Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region, operably connected thereto; and the third polypeptide comprises, from N-terminus to C-terminus, At the C-terminus: (i) a second Fab heavy chain comprising, from the N-terminus to the C-terminus, a VH region and a VL region; operably connected thereto via a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any one of SEQ ID NOS: 217-236 or 318-319, e.g., SEQ ID NO: 319); (ii) a second scFv comprising, from the N-terminus to the C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; operably connected thereto, a second Fc region comprising, from the N-terminus to the C-terminus, a CH2 region and a CH3 region. In some embodiments, the amino acid sequences of the first and second peptide linkers are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, hi some embodiments, the amino acid sequences of the first and second peptide linkers are 100% identical.

[0193] In some embodiments, the first polypeptide comprises, from N-terminus to C-terminus: (i) a first Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; operably connected thereto, (ii) a first scFv comprising, from N-terminus to C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; and a first Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region, operably connected thereto via a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the third polypeptide comprises, from N-terminus to C-terminus: (i) a second Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; and, operably connected thereto, (ii) a second scFv comprising, from N-terminus to C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; and, operably connected thereto via a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), a second Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region.In some embodiments, the first polypeptide comprises, from N-terminus to C-terminus: (i) a first Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; operably connected thereto, (ii) a first scFv comprising, from N-terminus to C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; and a first Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region, operably connected thereto via a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). and a third polypeptide comprising, from N-terminus to C-terminus: (i) a second Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; and, operably connected thereto, (ii) a second scFv comprising, from N-terminus to C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; and a second Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region, operably connected thereto via a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the amino acid sequences of the first and second peptide linkers are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, hi some embodiments, the amino acid sequences of the first and second peptide linkers are 100% identical.

[0194] In some embodiments, the first polypeptide comprises, from N-terminus to C-terminus: (i) a first Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; operably connected thereto via a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8; e.g., any one of SEQ ID NOs:217-236 or 318-319, e.g., SEQ ID NO:319); (ii) a first scFv comprising, from N-terminus to C-terminus: (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; and a first Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region, operably connected thereto via a third peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the third polypeptide comprises, from N-terminus to C-terminus: (i) a second Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; operably connected thereto via a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8; e.g., any one of SEQ ID NOs:217-236 or 318-319, e.g., SEQ ID NO:319); (ii) a second scFv comprising, from N-terminus to C-terminus: (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; and a second Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region, operably connected thereto via a fourth peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8).In some embodiments, the first polypeptide comprises, from N-terminus to C-terminus: (i) a first Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a VL region; (ii) a first scFv comprising, from N-terminus to C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region, operably connected thereto via a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8; e.g., any one of SEQ ID NOs:217-236 or 318-319, e.g., SEQ ID NO:319); and a first Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region, operably connected thereto via a third peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). and the third polypeptide comprises, from the N-terminus to the C-terminus: (i) a second Fab heavy chain comprising, from the N-terminus to the C-terminus, a VH region and a VL region; (ii) a second scFv comprising, from the N-terminus to the C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region, operably connected thereto via a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any one of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319); and a second Fc region comprising, from the N-terminus to the C-terminus, a CH2 region and a CH3 region, operably connected thereto via a fourth peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8). In some embodiments, the amino acid sequences of the third and fourth peptide linkers are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, hi some embodiments, the amino acid sequences of the third and fourth peptide linkers are 100% identical.

[0195] In some embodiments, the first Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the second Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region; and the second Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region comprises, from the N-terminus to the C-terminus, a partial hinge region, a CH2 region, and a CH3 region. In some embodiments, the second Fc region comprises, from the N-terminus to the C-terminus, a partial hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region comprises, from N-terminus to C-terminus, a partial hinge region, a CH2 region, and a CH3 region; and the second Fc region comprises, from N-terminus to C-terminus, a partial hinge region, a CH2 region, and a CH3 region.

[0196] In some embodiments, the first scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the first scFv comprises, from N- to C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, the second scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the second scFv comprises, from N- to C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, the first scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region; and the second scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the first scFv comprises, from N-terminus to C-terminus, a VH region, a peptide linker, and a VL region; and the second scFv comprises, from N-terminus to C-terminus, a VH region, a peptide linker, and a VL region.

[0197] In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first hTAA and the second hTAA are different. In some embodiments, the first hTAA and the second hTAA are different but expressed by the same cancer cells. In some embodiments, the first antigen-binding domain specifically binds to the same hTAA as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds to the same epitope as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds to the same hTAA as the second antigen-binding domain, but to a different epitope. In some embodiments, the first antigen-binding domain specifically binds to the same hTAA as the second antigen-binding domain, but to a different, non-overlapping epitope. In some embodiments, the first antigen-binding domain specifically binds to a different hTAA than the second antigen-binding domain.

[0198] Generally, the BCA424 format also includes (a) from N-terminus to C-terminus: (i) from N-terminus to C-terminus, a first Fab heavy chain comprising a VH region and a VL region; operably connected thereto (via an optional first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any one of SEQ ID NOS: 217-236 or 318-319, e.g., SEQ ID N0: 319)); (ii) from N-terminus to C-terminus (ii(a)) a VL region, a peptide linker, and a VH region, or or (ii(b)) a first polypeptide comprising, from the N-terminus to the C-terminus, a first Fc region comprising, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; (b) a second polypeptide comprising, from the N-terminus to the C-terminus, a first light chain comprising, from the N-terminus to the C-terminus, a VL region and a CL region; and (c) from the N-terminus to the C-terminus: (i) a second Fab heavy chain comprising, from the N-terminus to the C-terminus, a VH region and a VL region; (ii) a second scFv comprising, from the N-terminus to the C-terminus, (ii(a)) a VL region, a peptide linker, and a VH region, or (ii(b)) a VH region, a peptide linker, and a VL region; operably connected thereto; and (d) a third polypeptide comprising, from the N-terminus to the C-terminus, a second Fc region comprising, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; and a fourth polypeptide at its C-terminus comprising a second light chain comprising a VL region and a CL region, wherein the first Fab heavy chain and the first light chain associate to form a first antigen-binding domain that specifically binds to a first hTAA; the second Fab heavy chain and the second light chain associate to form a second antigen-binding domain that specifically binds to a second hTAA; the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); and the second scFv specifically binds to hCD3;the CH3 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications, and the CH3 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications; and the one or more amino acid modifications in the CH3 region of the first Fc region are different from the one or more amino acid modifications in the CH3 region of the Fc region; the one or more amino acid modifications in the CH3 region and the one or more amino acid modifications in the CH3 region of the second Fc region promote heterodimerization of the first and second heavy chains of the full-length antibody; the CH2 region of the first Fc region comprises the one or more amino acid modifications compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); the CH2 region of the second Fc region comprises the one or more amino acid modifications compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions);The one or more amino acid modifications in the CH2 region of the Fc region and the one or more amino acid modifications in the CH2 region of the second Fc region reduce or eliminate one or more of the following Fc region effector functions: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))) compared to a reference Fc region (e.g., a wild-type CH2 domain, e.g., a heavy chain comprising SEQ ID NO: 100) that does not contain the one or more amino acid modifications. In some embodiments, the first hTAA and the second hTAA are the same. In some embodiments, the first hTAA and the second hTAA are different. In some embodiments, the first hTAA and the second hTAA are different but expressed by the same cancer cell;

[0199] 5.2.1.4 Format BCA418 In one aspect, provided herein is a multispecific protein in format BCA418 (see, e.g., Figure 4). Generally, format BCA418 provides a multispecific protein comprising a first scFv operably connected (e.g., via a peptide linker) to a first Fab, which is operably connected (e.g., via a peptide linker) to a first Fc region; and a first IgM CH2 mFab operably connected (e.g., via a peptide linker) to a second Fc region, wherein the first scFv specifically binds to an hTCSA (e.g., hCD28, hCD2), the first Fab specifically binds to hCD3, and the first IgM CH2 mFab specifically binds to an hTAA.

[0200] In some embodiments, the multispecific protein comprises: (a) an scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; (b) a Fab comprising, from N-terminus to C-terminus, (i) a Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a CH1 region, and (ii) a light chain comprising, from N-terminus to C-terminus, a VL region and a CL region; (c) a first Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region; (d) a second Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region; and (e) an IgM CH2 mFab comprising, from N-terminus to C-terminus, (i) an IgM CH2 mFab heavy chain comprising, from N-terminus to C-terminus, a VH region and an IgM CH2 region, and (ii) an IgM CH2 mFab light chain comprising, from N-terminus to C-terminus, a VL region and an IgM CH2 region. the C-terminus of the scFv is operably connected to the N-terminus of the Fab heavy chain of the Fab; the C-terminus of the Fab heavy chain is operably connected to the N-terminus of the first Fc region; the C-terminus of the IgM CH2 mFab heavy chain is operably connected to the N-terminus of the second Fc region (e.g., via a peptide linker, e.g., any one of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 318); the IgM CH2 mFab specifically binds to hTAA; the scFv specifically binds to hTCSA (e.g., hCD28, hCD2); and the Fab specifically binds to hCD3.

[0201] In some embodiments, an scFv is operably linked to the N-terminus of the Fab heavy chain directly via a peptide bond, hi some embodiments, an scFv is operably linked to the N-terminus of the Fab heavy chain indirectly via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8).

[0202] In some embodiments, an scFv is operably connected to the N-terminus of the Fab heavy chain through the VH region of the scFv (either directly or indirectly through a peptide linker), hi some embodiments, an scFv is operably connected to the N-terminus of the Fab heavy chain through the VL region of the scFv (either directly or indirectly through a peptide linker).

[0203] In some embodiments, the C-terminus of the Fab heavy chain is operably connected to the N-terminus of the first Fc region; the C-terminus of the IgM CH2 mFab heavy chain is operably connected to the N-terminus of the second Fc region; and the IgM CH2 mFab specifically binds to an hTAA via a peptide linker, e.g., any one of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 318.

[0204] In some embodiments, the first Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the second Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region; and the second Fc region comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region comprises, from the N-terminus to the C-terminus, a partial hinge region, a CH2 region, and a CH3 region. In some embodiments, the second Fc region comprises, from the N-terminus to the C-terminus, a partial hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region comprises, from N-terminus to C-terminus, a partial hinge region, a CH2 region, and a CH3 region; and the second Fc region comprises, from N-terminus to C-terminus, a partial hinge region, a CH2 region, and a CH3 region.

[0205] In some embodiments, the multispecific protein comprises: (a) a first polypeptide comprising, from N-terminus to C-terminus, a first light chain comprising a VL region and a CL region; (b) a first scFv comprising, from N-terminus to C-terminus, (i) a first scFv comprising, from N-terminus to C-terminus, (i(a)) a VH region, a peptide linker, and a VL region, or (i(b)) a VL region, a peptide linker, and a VH region; (ii) a second polypeptide comprising, from N-terminus to C-terminus, a first heavy chain comprising, from N-terminus to C-terminus, a VH region, an IgG CH1 region, an IgG hinge region, an IgG CH2 region, and an IgG CH3 region; (c) a third polypeptide comprising, from N-terminus to C-terminus, a VH region, a first IgM CH2 region, and an Fc region comprising, from N-terminus to C-terminus, an IgG hinge region, an IgG CH2 region, and an IgG CH3 region; and (d) a third polypeptide comprising, from N-terminus to C-terminus, a VL region and a second IgM and a fourth polypeptide comprising a CH2 region; the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); the first light chain and the first heavy chain associate to form an antigen-binding domain that specifically binds to human CD3 (hCD3); and the third and fourth polypeptides associate to form an antigen-binding domain that specifically binds to hTAA.

[0206] In some embodiments, the second polypeptide comprises, from N-terminus to C-terminus: (i) a first scFv comprising, from N-terminus to C-terminus: (i(a)) a VH region, a peptide linker, and a VL region, or (i(b)) a VL region, a peptide linker, and a VH region; a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8); and (ii) a first heavy chain comprising, from N-terminus to C-terminus: a VH region, an IgG CH1 region, an IgG hinge region, an IgG CH2 region, and an IgG CH3 region.

[0207] In some embodiments, the first scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the first scFv comprises, from N- to C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, the second scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the second scFv comprises, from N- to C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, the first scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region; and the second scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the first scFv comprises, from N-terminus to C-terminus, a VH region, a peptide linker, and a VL region; and the second scFv comprises, from N-terminus to C-terminus, a VH region, a peptide linker, and a VL region.

[0208] Generally, the BCA418 format also includes (a) a first polypeptide comprising, from N-terminus to C-terminus, a first light chain comprising a VL region and a CL region; (b) a first scFv comprising, from N-terminus to C-terminus, (i) a first scFv comprising, from N-terminus to C-terminus, (i(a)) a VH region, a peptide linker, and a VL region, or (i(b)) a VL region, a peptide linker, and a VH region; and an optional first peptide linker (e.g., a linker described herein) (see, e.g., § 5.2.8); (iii) a second polypeptide comprising, from N-terminus to C-terminus, a VH region, an IgG CH1 region, an IgG hinge region, an IgG CH2 region, and an IgG CH3 region; and (c) a second polypeptide comprising, from N-terminus to C-terminus, a VH region, a first IgM CH2 region, and, from N-terminus to C-terminus, an IgG hinge region, an IgG CH2 region, and an IgG (d) a third polypeptide comprising, from the N-terminus to the C-terminus, a VL region and a second IgM and a fourth polypeptide comprising a CH2 region, wherein the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); the first light chain and the first heavy chain associate to form an antigen-binding domain that specifically binds to human CD3 (hCD3); the third and fourth polypeptides associate to form an antigen-binding domain that specifically binds to human hTAA; and the CH3 region of the first heavy chain comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions), and the CH3 region of the Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications; and the one or more amino acid modifications in the CH3 region of the first heavy chain are different from the one or more amino acid modifications in the CH3 region of the Fc region;the one or more amino acid modifications in the CH3 region of the first heavy chain and the one or more amino acid modifications in the CH3 region of the Fc region promote heterodimerization of the first heavy chain and the Fc region; the CH2 region of the first heavy chain comprises the one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); the CH2 region of the Fc region comprises the one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications; and the one or more amino acid modifications in the CH2 region of the heavy chain of SEQ ID NO: 100) reduce or eliminate one or more of the following Fc region effector functions: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))), compared to a reference Fc region that does not contain the one or more amino acid modifications (e.g., a wild-type Fc region, e.g., a heavy chain comprising SEQ ID NO: 100);

[0209] 5.2.1.5 Stabilized scFv The scFv component of any of the multispecific proteins described herein includes modified scFvs that promote stabilization of the scFv structure.Methods for stabilizing scFvs are known in the art, such as the introduction of cysteine ​​residues in the VH region of scFvs, where the cysteines can form disulfide bonds, and the introduction of cysteine ​​residues in the VL region of scFvs (see, for example, U.S. Pat. No. 6,147,203; Zhao, Jian-Xin et al. "Stabilization of the single-chain fragment variable by an interdomain disulfide bond and its effect on antibody affinity," International Journal of Molecular Sciences, Vol. 12, 11-11, 23 Dec. 2010, doi:10.3390 / ijms12010001; Duan, Ye et al. "A novel disulfide-stabilized single-chain variable antibody fragment against rabies virus G protein with enhanced in vivo neutralizing potency," Molecular Immunology, Vol. 51, 2(2012): 188-96). doi:10.1016 / j.molimm.2012.03.015; Eve E. Weatherill, et al., Towards a universal disulphide stabilized single-chain Fv format: importance of interchain disulphide bond location and VL-VH orientation, Protein Engineering, Design and Selection, Volume 25, Issue 7, July 2012, Pages 321-329, https: / / doi.org / 10.1093 / protein / gzs021); the entire contents of each of which are incorporated herein by reference for all purposes.

[0210] In some embodiments, the introduction of cysteines into the VH region and the VL region of an scFv does not substantially affect the binding affinity of the scFv to its cognate antigen. In some embodiments, cysteines are introduced into the framework regions of the VH region of an scFv; and cysteines are introduced into the framework regions of the VL region of an scFv.

[0211] In some embodiments, a cysteine ​​is introduced at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, numbered according to Kabat, in the VH region of the scFv; and a cysteine ​​is introduced at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, numbered according to Kabat, in the VL region of the scFv.

[0212] In some embodiments, a cysteine ​​is introduced into the framework region of the VH region of the scFv; and a cysteine ​​is introduced into the framework region of the VL region of the scFv. In some embodiments, a cysteine ​​is introduced at amino acid position 100 of the VH region of the scFv, numbered according to Kabat; and a cysteine ​​is introduced at amino acid position 44 or 45 of the VL region of the scFv, numbered according to Kabat. In some embodiments, a cysteine ​​is introduced into the framework region of the VH region of the scFv; and a cysteine ​​is introduced into the framework region of the VL region of the scFv ..., numbered according to Kabat. In some embodiments, a cysteine ​​is introduced into the framework region of the VH region of the scFv; and a cysteine ​​is introduced into the framework region of the VL region of the scFv. In some embodiments, a cysteine ​​is introduced at amino acid position 100, numbered according to Kabat, of the VH region of the scFv; and a cysteine ​​is introduced at amino acid position 45, numbered according to Kabat, of the VL region of the scFv.

[0213] 5.2.2 Human T-cell C-stimulatory antigen (hTCSA) binding domain The multispecific proteins described herein comprise an antigen-binding domain that specifically binds to a human T cell C-stimulatory antigen (hTCSA). As described above, hTCSA is a protein expressed on the surface of human T cells that is not part of the T cell receptor complex and enhances T cell activation when its cognate ligand binds. hTCSAs are known in the art. Exemplary hTCSAs include, but are not limited to, hCD28, hCD2, hCD137 (also known as 41BB), human CD27, human CD278 (also known as ICOS), human CD134 (also known as OX40), or human CD40. In some embodiments, the hTCSA is hCD28. In some embodiments, the hTCSA is hCD2. In some embodiments, the hTCSA is h41BB.

[0214] 5.2.2.1 CD28-binding domain CD28 is a costimulatory transmembrane protein expressed on the surface of T cells, including naive T cells. CD28 functions, for example, in T cell activation, inducing T cell proliferation, T cell cytokine production, and promoting T cell survival. CD28 is a receptor for CD80 and CD86 proteins and is a cell surface transmembrane protein expressed by antigen-presenting cells.

[0215] The amino acid sequences of the reference immature hCD28 polypeptide (with N-terminal signal peptide) and mature hCD28 polypeptide (without signal peptide) are set forth in SEQ ID NOs: 1 and 2, respectively. See Table 1 herein.

[0216] [Table 1]

[0217] In some embodiments, a multispecific protein described herein comprises an antigen-binding domain that specifically binds to hCD28, also referred to herein as an hCD28-binding domain or anti-hCD28 antibody (or functional fragment or variant thereof). In certain embodiments, the hCD28-binding domain is an scFv (see, e.g., §5.2.1). In certain embodiments, the multispecific protein or polypeptide is monovalent with respect to hCD28.

[0218] hCD28 binding domains that can be used in the multispecific proteins described herein are known in the art and are described, for example, in U.S. Pat. Nos. 7,585,960, 9,562,098, 7,723,482, 7,939,638, 9,908,937, 7,723,482, WO 2021155071, WO 20231, the entire contents of each of which are incorporated herein by reference for all purposes. 64510, WO 2023155845, WO 2023143547, WO 2023114701, WO 2017103003, WO 2011101791, WO 2022253867, WO 2022216915, and WO 2020127628, U.S. Patent Application Publication No. 20230265218, WO 2022094299, and U.S. Patent Application Publication No. 20220089766.

[0219] Exemplary hCD28 binding domains that can be used in the multispecific proteins described herein are described, eg, in §5.2.2, Table 2, see for example.

[0220] Exemplary hCD28 binding domains known in the art that can be used in the fusion proteins described herein also include TGN1412.

[0221] The amino acid sequences of exemplary hCD28 binding domains that can be utilized in the multispecific proteins described herein are provided in Table 2. The CDRs of the hCD28 binding domains in Table 2 are designated according to Kabat. One skilled in the art would be able to determine the CDRs as defined by other schemes, e.g., Chothia, IMGT, using routine methods known in the art.

[0222] [Table 2-1]

[0223] [Table 2-2]

[0224] In some embodiments, the hCD28 binding domain comprises an hCD28 binding domain provided in Table 2.

[0225] In some embodiments, the hCD28 binding domain comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3.

[0226] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence of the VH CDR1 of the VH listed in Table 2, or the amino acid sequence of the VH CDR1 of the VH listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence of the VH CDR2 of the VH listed in Table 2, or the amino acid sequence of the VH CDR2 of the VH listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence of the VH CDR3 of the VH listed in Table 2, or the amino acid sequence of the VH CDR3 of the VH listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0227] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence of the VH CDR1 of the VH listed in Table 2, or the amino acid sequence of the VH CDR1 of the VH listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence of the VH CDR2 of the VH listed in Table 2, or the amino acid sequence of the VH CDR2 of the VH listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence of the VH CDR3 of the VH listed in Table 2, or the amino acid sequence of the VH CDR3 of the VH listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0228] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence of the VH CDR1 set forth in Table 2, or the amino acid sequence of the VH CDR1 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence of the VH CDR2 set forth in Table 2, or the amino acid sequence of the VH CDR2 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence of the VH CDR3 set forth in Table 2, or the amino acid sequence of the VH CDR3 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0229] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence of the VH CDR1 set forth in Table 2, or the amino acid sequence of the VH CDR1 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence of the VH CDR2 set forth in Table 2, or the amino acid sequence of the VH CDR2 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence of the VH CDR3 set forth in Table 2, or the amino acid sequence of the VH CDR3 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0230] In some embodiments, the hCD28 binding domain comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3.

[0231] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 of a VL listed in Table 2, or the amino acid sequence of the VL CDR1 of a VL listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of a VL listed in Table 2, or the amino acid sequence of the VL CDR2 of a VL listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of a VL listed in Table 2, or the amino acid sequence of the VL CDR3 of a VL listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0232] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 of the VL of Table 2 listed in Table 2, or the amino acid sequence of the VL CDR1 of the VL of Table 2 listed in Table 2 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of the VL of Table 2 listed in Table 2, or the amino acid sequence of the VL CDR2 of the VL of Table 2 listed in Table 2 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of the VL of Table 2 listed in Table 2 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0233] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 set forth in Table 2, or the amino acid sequence of the VL CDR1 set forth in Table 2 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 set forth in Table 2, or the amino acid sequence of the VL CDR2 set forth in Table 2 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 set forth in Table 2, or the amino acid sequence of the VL CDR3 set forth in Table 2 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0234] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 set forth in Table 2, or the amino acid sequence of the VL CDR1 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 set forth in Table 2, or the amino acid sequence of the VL CDR2 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 set forth in Table 2, or the amino acid sequence of the VL CDR3 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0235] In some embodiments, the hCD28 binding domain comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3.

[0236] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence of the VH CDR1 of the VH listed in Table 2, or the amino acid sequence of the VH CDR1 of the VH listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence of the VH CDR2 of the VH listed in Table 2, or the amino acid sequence of the VH CDR2 of the VH listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence of the VH CDR3 of the VH listed in Table 2, or the amino acid sequence of the VH CDR3 of the VH listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR1 comprises or consists of the VL CDR1 of the VL listed in Table 2. the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of the VL of Table 2, or the amino acid sequence of the VL CDR2 of the VL of Table 2, comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of the VL of Table 2, or the amino acid sequence of the VL CDR3 of the VL of Table 2, comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0237] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 set forth in Table 2, or the amino acid sequence of VH CDR1 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 set forth in Table 2, or the amino acid sequence of VH CDR2 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 set forth in Table 2, or the amino acid sequence of VH CDR3 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of VL CDR1 set forth in Table 2. the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 set forth in Table 2, or the amino acid sequence of the VL CDR2 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 set forth in Table 2, or the amino acid sequence of the VL CDR3 set forth in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0238] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VH set forth in Table 2. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VL set forth in Table 2. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VH set forth in Table 2; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VL set forth in Table 2.

[0239] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, or the amino acid sequence set forth in SEQ ID NO: 3 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, or the amino acid sequence set forth in SEQ ID NO: 4 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, or the amino acid sequence set forth in SEQ ID NO: 5 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0240] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, or the amino acid sequence set forth in SEQ ID NO: 3 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, or the amino acid sequence set forth in SEQ ID NO: 4 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, or the amino acid sequence set forth in SEQ ID NO: 5 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0241] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, or the amino acid sequence set forth in SEQ ID NO: 6 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, or the amino acid sequence set forth in SEQ ID NO: 7 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8, or the amino acid sequence set forth in SEQ ID NO: 8 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0242] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, or the amino acid sequence set forth in SEQ ID NO: 6 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, or the amino acid sequence set forth in SEQ ID NO: 7 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8, or the amino acid sequence set forth in SEQ ID NO: 8 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0243] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:3, or the amino acid sequence set forth in SEQ ID NO:3 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:4, or the amino acid sequence set forth in SEQ ID NO:4 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, or the amino acid sequence set forth in SEQ ID NO:5 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, or the amino acid sequence set forth in SEQ ID NO:6 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); The amino acid sequence of CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:7, or the amino acid sequence set forth in SEQ ID NO:7 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8, or the amino acid sequence set forth in SEQ ID NO:8 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0244] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:10.

[0245] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11, or the amino acid sequence set forth in SEQ ID NO: 11 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, or the amino acid sequence set forth in SEQ ID NO: 12 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, or the amino acid sequence set forth in SEQ ID NO: 13 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0246] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11, or the amino acid sequence set forth in SEQ ID NO: 11 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, or the amino acid sequence set forth in SEQ ID NO: 12 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, or the amino acid sequence set forth in SEQ ID NO: 13 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0247] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14, or the amino acid sequence set forth in SEQ ID NO: 14 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, or the amino acid sequence set forth in SEQ ID NO: 15 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, or the amino acid sequence set forth in SEQ ID NO: 16 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0248] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14, or the amino acid sequence set forth in SEQ ID NO: 14 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, or the amino acid sequence set forth in SEQ ID NO: 15 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, or the amino acid sequence set forth in SEQ ID NO: 16 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0249] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11, or the amino acid sequence set forth in SEQ ID NO: 11 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, or the amino acid sequence set forth in SEQ ID NO: 12 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, or the amino acid sequence set forth in SEQ ID NO: 13 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14, or the amino acid sequence set forth in SEQ ID NO: 14 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); The amino acid sequence of CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, or the amino acid sequence set forth in SEQ ID NO: 15 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, or the amino acid sequence set forth in SEQ ID NO: 16 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0250] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 19.

[0251] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 20.

[0252] 5.2.2.2 CD2-binding domain CD2 is a costimulatory transmembrane protein expressed on the surface of T cells and NK cells.CD28 functions in the formation and construction of the immune synapse formed between T cells and antigen-presenting cells, for example, during cell-cell conjugation and related intracellular signal transduction.CD2 is the receptor for LFA3, a cell surface protein expressed by antigen-presenting cells.

[0253] The amino acid sequences of the reference immature hCD2 polypeptide (with N-terminal signal peptide) and mature hCD2 polypeptide (without signal peptide) are set forth in SEQ ID NOs: 37 and 38, respectively. See Table 16 herein.

[0254] [Table 3]

[0255] In some embodiments, a multispecific protein described herein comprises an antigen-binding domain that specifically binds to hCD2, also referred to herein as an hCD2-binding domain or anti-hCD2 antibody (or functional fragment or variant thereof). In certain embodiments, the hCD2-binding domain is an scFv (see, e.g., §5.2.1). In certain embodiments, the multispecific protein or polypeptide is monovalent with respect to hCD28.

[0256] Exemplary hCD2 binding domains that can be used in the multispecific proteins described herein are listed in §5.2.2, Table 17.

[0257] Exemplary hCD2 binding domains known in the art that can be used in the fusion proteins described herein also include, for example, siplizumab, LO-CD2b, and humanized versions of LO-CD2b (see, e.g., WO 1999003502).

[0258] hCD2 binding domains that can be used in the multispecific proteins described herein are known in the art and include, for example, those described in U.S. Pat. No. 5,656,438, EP 1,003,552, WO 2022067089, WO 2023126445, WO 2023126445, WO 2022157272, WO 2021259927, U.S. Patent Application Publication No. 20210260212, U.S. Pat. No. 7,678,582, U.S. Pat. No. 7,250,167, U.S. Pat. No. 7,332,157, U.S. Pat. No. 6,384,198, and WO 1999003502, the entire contents of each of which are incorporated herein by reference for all purposes.

[0259] The amino acid sequences of exemplary hCD2 binding domains that can be utilized in the multispecific proteins described herein are provided in Table 17. The CDRs of the hCD2 binding domains in Table 17 are set forth according to Kabat. One skilled in the art would be able to determine the CDRs as defined by other schemes, e.g., Chothia, IMGT, using routine methods known in the art.

[0260] [Table 4]

[0261] In some embodiments, the hCD2 binding domain comprises an hCD2 binding domain provided in Table 17.

[0262] In some embodiments, the hCD2 binding domain comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3.

[0263] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 of a VH listed in Table 17, or the amino acid sequence of VH CDR1 of a VH listed in Table 17 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 of a VH listed in Table 17, or the amino acid sequence of VH CDR2 of a VH listed in Table 17 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 of a VH listed in Table 17, or the amino acid sequence of VH CDR3 of a VH listed in Table 17 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0264] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence of the VH CDR1 of a VH listed in Table 17, or the amino acid sequence of the VH CDR1 of a VH listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence of the VH CDR2 of a VH listed in Table 17, or the amino acid sequence of the VH CDR2 of a VH listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence of the VH CDR3 of a VH listed in Table 17, or the amino acid sequence of the VH CDR3 of a VH listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0265] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 set forth in Table 17, or the amino acid sequence of VH CDR1 set forth in Table 17 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 set forth in Table 17, or the amino acid sequence of VH CDR2 set forth in Table 17 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 set forth in Table 17, or the amino acid sequence of VH CDR3 set forth in Table 17 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0266] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 set forth in Table 17, or the amino acid sequence of VH CDR1 set forth in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 set forth in Table 17, or the amino acid sequence of VH CDR2 set forth in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 set forth in Table 17, or the amino acid sequence of VH CDR3 set forth in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0267] In some embodiments, the hCD2 binding domain comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3.

[0268] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 of a VL listed in Table 17, or the amino acid sequence of the VL CDR1 of a VL listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of a VL listed in Table 17, or the amino acid sequence of the VL CDR2 of a VL listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of a VL listed in Table 17, or the amino acid sequence of the VL CDR3 of a VL listed in Table 2, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0269] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 of a VL listed in Table 2, or the amino acid sequence of the VL CDR1 of a VL listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of a VL listed in Table 17, or the amino acid sequence of the VL CDR2 of a VL listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of a VL listed in Table 17, or the amino acid sequence of the VL CDR3 of a VL listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0270] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 17, or the amino acid sequence of a VL CDR1 set forth in Table 17 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 17, or the amino acid sequence of a VL CDR2 set forth in Table 17 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 17, or the amino acid sequence of a VL CDR3 set forth in Table 17 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0271] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 set forth in Table 17, or the amino acid sequence of the VL CDR1 set forth in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 set forth in Table 17, or the amino acid sequence of the VL CDR2 set forth in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 set forth in Table 17, or the amino acid sequence of the VL CDR3 set forth in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0272] In some embodiments, the hCD2 binding domain comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3.

[0273] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 of a VH listed in Table 17, or the amino acid sequence of VH CDR1 of a VH listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 of a VH listed in Table 17, or the amino acid sequence of VH CDR2 of a VH listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 of a VH listed in Table 17, or the amino acid sequence of VH CDR3 of a VH listed in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of VL CDR1 of a VL listed in Table 17. the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of a VL listed in Table 17, or the amino acid sequence of the VL CDR2 of a VL listed in Table 17, comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of a VL listed in Table 17, or the amino acid sequence of the VL CDR3 of a VL listed in Table 17, comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0274] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 set forth in Table 17, or the amino acid sequence of VH CDR1 set forth in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 set forth in Table 17, or the amino acid sequence of VH CDR2 set forth in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 set forth in Table 17, or the amino acid sequence of VH CDR3 set forth in Table 17, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of VL CDR1 set forth in Table 17. the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 set forth in Table 17, or the amino acid sequence of the VL CDR2 set forth in Table 17, or the amino acid sequence of the VL CDR2 set forth in Table 17, or the amino acid sequence of the VL CDR2 set forth in Table 17, or the amino acid sequence of the VL CDR2 set forth in Table 17, or the amino acid sequence of the VL CDR2 set forth in Table 17, or the amino acid sequence of the VL CDR3 ...

[0275] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VH set forth in Table 17. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VL set forth in Table 17. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VH set forth in Table 17; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VL set forth in Table 17.

[0276] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60, or the amino acid sequence set forth in SEQ ID NO: 60 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 85, or the amino acid sequence set forth in SEQ ID NO: 85 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86, or the amino acid sequence set forth in SEQ ID NO: 86 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0277] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60, or the amino acid sequence set forth in SEQ ID NO: 60 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 85, or the amino acid sequence set forth in SEQ ID NO: 85 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86, or the amino acid sequence set forth in SEQ ID NO: 86 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0278] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 87, or the amino acid sequence set forth in SEQ ID NO: 87 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88, or the amino acid sequence set forth in SEQ ID NO: 88 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89, or the amino acid sequence set forth in SEQ ID NO: 89 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0279] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 87, or the amino acid sequence set forth in SEQ ID NO: 87 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88, or the amino acid sequence set forth in SEQ ID NO: 88 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89, or the amino acid sequence set forth in SEQ ID NO: 89 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0280] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60, or the amino acid sequence set forth in SEQ ID NO: 60 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 85, or the amino acid sequence set forth in SEQ ID NO: 85 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86, or the amino acid sequence set forth in SEQ ID NO: 86 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 87, or the amino acid sequence set forth in SEQ ID NO: 87 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); The amino acid sequence of CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88, or the amino acid sequence set forth in SEQ ID NO: 88 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89, or the amino acid sequence set forth in SEQ ID NO: 89 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0281] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:91. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 90; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 91.

[0282] 5.2.3 CD3-binding domain The CD3 complex is a set of polypeptides that function with the T cell receptor (TCR) to activate T cells. The CD3 complex contains two CD3ε polypeptides, two CD3ζ polypeptides, a CD3δ polypeptide, and a CD3γ polypeptide. The intracellular tail of each of the CD3 chains (CD3ε, CD3ζ, CD3γ, and CD3δ) contains at least one single conserved motif, also known as an immunoreceptor tyrosine-based activation motif (ITAM), which is essential for the signaling ability of the TCR.

[0283] The amino acid sequences of the reference immature hCD3ε polypeptide and mature hCD3ε polypeptide are set forth in SEQ ID NOs: 21 and 22, respectively. The amino acid sequences of the reference immature hCD3ζ polypeptide and mature hCD3ζ polypeptide are set forth in SEQ ID NOs: 23 and 24, respectively. The amino acid sequences of the reference immature hCD3γ polypeptide and mature hCD3γ polypeptide are set forth in SEQ ID NOs: 25 and 26, respectively. The amino acid sequences of the reference immature hCD3δ polypeptide and mature hCD3δ polypeptide are set forth in SEQ ID NOs: 27 and 28, respectively. See Table 3 herein.

[0284] [Table 5]

[0285] The multispecific proteins described herein comprise an antigen-binding domain that specifically binds to hCD3, also referred to herein as an hCD3-binding domain or an anti-hCD3 antibody (or functional fragment or variant thereof). In certain embodiments, the hCD3-binding domain is an scFv (see, e.g., §5.2.1). In certain embodiments, the hCD3-binding domain is a Fab (see, e.g., §5.2.1). In certain embodiments, the multispecific protein is monovalent with respect to hCD3.

[0286] In some embodiments, the hCD3 binding domain specifically binds to hCD3ε. In some embodiments, the hCD3 binding domain specifically binds to hCD3δ. In some embodiments, the hCD3 binding domain specifically binds to hCD3γ. In some embodiments, the hCD3 binding domain specifically binds to hCD3ζ.

[0287] Exemplary hCD3 binding domains that can be used in the multispecific proteins described herein are listed in §5.2.3, Table 4.

[0288] hCD3 binding domains that can be used in the multispecific proteins described herein are known in the art and are described, for example, in International Publication Nos. WO 2023044402, WO 2023014809, WO 2020247929, U.S. Pat. No. 8,530,629, U.S. Pat. App. Pub. No. 20220041721, U.S. Pat. No. 7,820,166, U.S. Pat. No. 8,101,722, WO 2021121215, U.S. Pat. App. Pub. No. 2021121215, U.S. Pat ... Patent Publication No. 20220010015, International Publication No. 2021240388, International Publication No. 2021141996, International Publication No. 2021063330, U.S. Patent Application Publication No. 20230212289, U.S. Patent Application Publication No. 20210054077, International Publication No. 2020204708, International Publication No. 2023044402, International Publication No. 2020157210, U.S. Patent Application Publication No. 20210317213, International Publication No. 2023014809, International Publication No. 2023273914, International Publication No. 2022170740, U.S. Publication No. 20210269525, Publication No. 20210277118, Publication No. 20200048349, Publication No. 20190382486, U.S. Patent No. 11203646, Publication No. 20210155694, Publication No. 20200339686, Publication No. 20200317779, Publication No. 20200299384, Publication No. 20200157217 ... Patent Application Publication No. 20200377594, U.S. Patent Application Publication No. 20200115449, International Publication No. 2017053856, U.S. Patent Application Publication No. 20180273622, U.S. Patent Application Publication No. 20190284278, U.S. Patent No. 9914777, U.S. Patent No. 11007267, U.S. Patent Application Publication No. 20210054087, U.S. Patent No. 10066015, U.S. Patent No. 10669337, U.S. Patent No. 9611325, U.S. Patent Application Publication No. 20230002487,Including those described in U.S. Pat. Nos. 11,603,405, 11,639,388, 10,407,501, 10,640,572, 10,968,276, 10,000,567, 10,202,452, 10,150,812, 7,728,114, U.S. Patent Application Publication No. 20190359712, U.S. Patent Application Publication No. 20200048,348, and U.S. Patent No. 10,066,016.

[0289] Exemplary hCD3-binding domains known in the art that can be used in the fusion proteins and polypeptides described herein also include OKT3 (also known as muromonab), SP34, otelixizumab, teplizumab, visilizumab, catumaxomab, blinatumomab, and foralarumab; and chimeric or humanized versions of any of the foregoing, e.g., humanized versions of OKT3 and SP34. The anti-hCD3 antibody referred to herein as OKT3 (see, e.g., Table 4) specifically binds to hCD3ε. The anti-hCD3 antibody referred to herein as SP34 (see, e.g., Table 4) specifically binds to hCD3ε.

[0290] The amino acid sequences of exemplary hCD3 binding domains that can be utilized in the multispecific proteins described herein are provided in Table 4. The CDRs of the hCD3 binding domains in Table 4 are designated according to Kabat. One skilled in the art would be able to determine the CDRs as defined by other schemes, e.g., Chothia, IMGT, using routine methods known in the art.

[0291] [Table 6-1]

[0292] [Table 6-2]

[0293] In some embodiments, the hCD3 binding domain comprises an hCD3 binding domain provided in Table 4.

[0294] In some embodiments, the hCD3 binding domain comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3.

[0295] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence of the VH CDR1 of the VH listed in Table 4, or the amino acid sequence of the VH CDR1 of the VH listed in Table 4, which comprises or consists of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence of the VH CDR2 of the VH listed in Table 4, or the amino acid sequence of the VH CDR2 of the VH listed in Table 4, which comprises or consists of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence of the VH CDR3 of the VH listed in Table 4, or the amino acid sequence of the VH CDR3 of the VH listed in Table 4, which comprises or consists of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0296] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence of the VH CDR1 of the VH listed in Table 4, or the amino acid sequence of the VH CDR1 of the VH listed in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence of the VH CDR2 of the VH listed in Table 4, or the amino acid sequence of the VH CDR2 of the VH listed in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence of the VH CDR3 of the VH listed in Table 4, or the amino acid sequence of the VH CDR3 of the VH listed in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0297] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence of the VH CDR1 set forth in Table 4, or the amino acid sequence of the VH CDR1 set forth in Table 4 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence of the VH CDR2 set forth in Table 4, or the amino acid sequence of the VH CDR2 set forth in Table 4 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence of the VH CDR3 set forth in Table 4, or the amino acid sequence of the VH CDR3 set forth in Table 4 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0298] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence of the VH CDR1 set forth in Table 4, or the amino acid sequence of the VH CDR1 set forth in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence of the VH CDR2 set forth in Table 4, or the amino acid sequence of the VH CDR2 set forth in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence of the VH CDR3 set forth in Table 4, or the amino acid sequence of the VH CDR3 set forth in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0299] In some embodiments, the hCD3 binding domain comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3.

[0300] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 of a VL listed in Table 4, or the amino acid sequence of the VL CDR1 of a VL listed in Table 4 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of a VL listed in Table 4, or the amino acid sequence of the VL CDR2 of a VL listed in Table 4 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of a VL listed in Table 4, or the amino acid sequence of the VL CDR3 of a VL listed in Table 4 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0301] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 of a VL listed in Table 4, or the amino acid sequence of the VL CDR1 of a VL listed in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of a VL listed in Table 4, or the amino acid sequence of the VL CDR2 of a VL listed in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of a VL listed in Table 4, or the amino acid sequence of the VL CDR3 of a VL listed in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0302] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 set forth in Table 4, or the amino acid sequence of the VL CDR1 set forth in Table 4 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 set forth in Table 4, or the amino acid sequence of the VL CDR2 set forth in Table 4 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 set forth in Table 4, or the amino acid sequence of the VL CDR3 set forth in Table 4 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0303] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 set forth in Table 4, or the amino acid sequence of the VL CDR1 set forth in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 set forth in Table 4, or the amino acid sequence of the VL CDR2 set forth in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 set forth in Table 4, or the amino acid sequence of the VL CDR3 set forth in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0304] In some embodiments, the hCD3 binding domain comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3.

[0305] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 of a VH listed in Table 4, or the amino acid sequence of VH CDR1 of a VH listed in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 of a VH listed in Table 4, or the amino acid sequence of VH CDR2 of a VH listed in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 of a VH listed in Table 4, or the amino acid sequence of VH CDR3 of a VH listed in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of VL CDR1 of a VL listed in Table 4. the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of the VL of Table 4, or the amino acid sequence of the VL CDR2 of the VL of Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of the VL of Table 4, or the amino acid sequence of the VL CDR3 of the VL of Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0306] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 set forth in Table 4, or the amino acid sequence of VH CDR1 set forth in Table 4 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 set forth in Table 4, or the amino acid sequence of VH CDR2 set forth in Table 4 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 set forth in Table 4, or the amino acid sequence of VH CDR3 set forth in Table 4 comprising or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of VL CDR1 set forth in Table 4. the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 set forth in Table 4, or the amino acid sequence of the VL CDR2 set forth in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 set forth in Table 4, or the amino acid sequence of the VL CDR3 set forth in Table 4, which comprises or consists of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0307] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VH set forth in Table 4. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VL set forth in Table 4. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VH set forth in Table 4; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VL set forth in Table 4.

[0308] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29, or the amino acid sequence set forth in SEQ ID NO: 29 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, or the amino acid sequence set forth in SEQ ID NO: 30 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, or the amino acid sequence set forth in SEQ ID NO: 31 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0309] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29, or the amino acid sequence set forth in SEQ ID NO: 29 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, or the amino acid sequence set forth in SEQ ID NO: 30 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, or the amino acid sequence set forth in SEQ ID NO: 31 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0310] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32, or the amino acid sequence set forth in SEQ ID NO: 32 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33, or the amino acid sequence set forth in SEQ ID NO: 33 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34, or the amino acid sequence set forth in SEQ ID NO: 34 with on...

Claims

1. (a)(i) a first light chain comprising, from N-terminus to C-terminus, a light chain variable region (VL) region and a light chain constant region (CL) region; (ii) a first heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable region (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iv) a second light chain comprising, from N-terminus to C-terminus, a VL region and a CL region. A full-length antibody comprising: the first light chain and the first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and a full-length antibody, wherein the first heavy chain and the second heavy chain associate to form a dimer; (b) a first single-chain variable fragment (scFv) operably connected to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody, the first single-chain variable fragment (scFv) comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; (c) a second scFv operably linked to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody, the second scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; a multispecific protein comprising the first antigen-binding domain of the full-length antibody specifically binds to a first human tumor-associated antigen (hTAA); the second antigen-binding domain of the full-length antibody specifically binds to a second hTAA; the first scFv specifically binds to a human T cell costimulatory antigen (hTCSA) (e.g., hCD28, hCD2); the second scFv specifically binds to human CD3 (hCD3); and the CH3 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a reference CH3 region, e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions); the CH3 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions); and the one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody are different from the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody; the one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody and the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody promote heterodimerization of the first and second heavy chains of the full-length antibody; the CH2 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications; the CH2 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); the one or more amino acid modifications in the CH2 region of the first heavy chain of the full length antibody and the one or more amino acid modifications in the CH2 region of the second heavy chain of the full length antibody, compared to a reference heavy chain that does not contain the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 region, e.g., SEQ ID NO: 100), enhance the following heavy chain effector functions: antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or binding affinity to one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa)) reduce or eliminate one or more of Multispecific proteins.

2. The multispecific protein of claim 1 , wherein the first scFv is operably linked to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody directly through a peptide bond.

3. 2. The multispecific protein of claim 1, wherein the first scFv is operably connected indirectly to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody through a first peptide linker.

4. The multispecific protein of claim 3 , wherein the amino acid sequence of the first peptide linker comprises or consists of glycine amino acid residues or glycine and serine amino acid residues.

5. 5. The multispecific protein of claim 3, wherein the amino acid sequence of the first peptide linker comprises or consists of: (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319 comprising or consisting of one, two, or three amino acid substitutions.

6. 10. The multispecific protein of any one of the preceding claims, wherein the second scFv is operably linked via a peptide bond directly to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody.

7. 6. The multispecific protein of any one of claims 1 to 5, wherein the second scFv is operably connected to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody via a second peptide linker.

8. The multispecific protein of claim 7 , wherein the amino acid sequence of the second peptide linker comprises or consists of glycine amino acid residues or glycine and serine amino acid residues.

9. the amino acid of the second peptide linker is (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319, comprising or consisting of one, two, or three amino acid substitutions.

9. The multispecific protein of claim 7, comprising or consisting of:

10. 10. The multispecific protein of any one of the preceding claims, wherein the first scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; and the N-terminus of the VL region of the scFv is operably connected to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody.

11. 10. The multispecific protein of any one of the preceding claims, wherein the first scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; and the N-terminus of the VH region of the scFv is operably connected to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody.

12. 10. The multispecific protein of any one of the preceding claims, wherein the second scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; and the N-terminus of the VL region of the scFv is operably connected to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody.

13. 10. The multispecific protein of any one of the preceding claims, wherein the second scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; and the N-terminus of the VH region of the scFv is operably connected to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody.

14. (a) (i) a first light chain comprising, from N-terminus to C-terminus, a VL region and a CL region; (ii) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iv) a second light chain comprising, from N-terminus to C-terminus, a VL region and a CL region. A full-length antibody comprising: the first light chain and first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and The first heavy chain and the second heavy chain associate to form a dimer. a full-length antibody; (b) a first scFv operably linked to the N-terminus of the first heavy chain of the full-length antibody, the first scFv comprising, from N-terminus to C-terminus, (i) a VH domain, a peptide linker, and a VL domain, or (ii) a VL domain, a peptide linker, and a VH domain; (c) a second scFv operably linked to the N-terminus of the second heavy chain of the full-length antibody, the second scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; 1. A multispecific protein comprising: the first antigen-binding domain of the full-length antibody specifically binds to a first hTAA; the second antigen-binding domain of the full-length antibody specifically binds to a second hTAA; the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); the second scFv specifically binds to hCD3; and the CH3 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications; the CH3 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions); and the one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody are different from the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody; the one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody and the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody promote heterodimerization of the first and second heavy chains of the full-length antibody; the CH2 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications; the CH2 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications (e.g., substitutions); the one or more amino acid modifications in the CH2 region of the first heavy chain of the full length antibody and the one or more amino acid modifications in the CH2 region of the second heavy chain of the full length antibody, compared to a reference heavy chain that does not contain the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 region, e.g., SEQ ID NO: 100), enhance the following heavy chain effector functions: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa)) reduce or eliminate one or more of Multispecific proteins.

15. 15. The multispecific protein of claim 14, wherein the first scFv is operably linked via a peptide bond directly to the N-terminus of the VH region of the first heavy chain of the full-length antibody.

16. 15. The multispecific protein of claim 14, wherein the first scFv is operably connected to the N-terminus of the VH region of the first heavy chain of the full-length antibody via a first peptide linker.

17. 17. The multispecific protein of claim 16, wherein the amino acid sequence of the first peptide linker comprises or consists of glycine amino acid residues or glycine and serine amino acid residues.

18. the amino acid sequence of the first peptide linker is (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319, comprising or consisting of one, two, or three amino acid substitutions.

17. The multispecific protein of claim 15 or 16, comprising or consisting of:

19. 19. The multispecific protein of any one of claims 14 to 18, wherein the second scFv is operably linked via a peptide bond directly to the N-terminus of the VH region of the second heavy chain of the full-length antibody.

20. 19. The multispecific protein of any one of claims 14 to 18, wherein the second scFv is operably connected to the N-terminus of the VH region of the second heavy chain of the full-length antibody via a second peptide linker.

21. 21. The multispecific protein of claim 20, wherein the amino acid sequence of the second peptide linker comprises or consists of glycine amino acid residues or glycine and serine amino acid residues.

22. the amino acid of the second peptide linker is (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319, comprising or consisting of one, two, or three amino acid substitutions.

22. The multispecific protein of claim 20, comprising or consisting of:

23. 23. The multispecific protein of any one of claims 14 to 22, wherein the first scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; and the C-terminus of the VH region of the scFv is operably connected to the N-terminus of the VH region of the first heavy chain of the full-length antibody.

24. 24. The multispecific protein of any one of claims 14 to 23, wherein the first scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; and the C-terminus of the VL region of the scFv is operably connected to the N-terminus of the VH region of the first heavy chain of the full-length antibody.

25. 25. The multispecific protein of any one of claims 14 to 24, wherein the second scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; and the C-terminus of the VH region of the scFv is operably connected to the N-terminus of the VH region of the second heavy chain of the full-length antibody.

26. 26. The multispecific protein of any one of claims 14 to 25, wherein the second scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; and the C-terminus of the VL region of the scFv is operably connected to the N-terminus of the VH region of the second heavy chain of the full-length antibody.

27. 10. The multispecific protein of any one of the preceding claims, wherein the first hTAA and the second hTAA are expressed by (e.g., on the surface of) the same tumor cell.

28. 10. The multispecific protein of claim 1, wherein the first hTAA and the second hTAA are the same.

29. 10. The multispecific protein of any one of the preceding claims, wherein the first scFv and the second scFv specifically bind to the same epitope of the same hTAA.

30. 10. The multispecific protein of any one of the preceding claims, wherein the first scFv and the second scFv specifically bind to different epitopes of the same hTAA.

31. 10. The multispecific protein of any one of the preceding claims, wherein the first tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2.

32. 10. The multispecific protein of claim 9, wherein the amino acid sequence of the VH region of the first scFv and the amino acid sequence of the VL region of the first scFv each comprise a cysteine ​​amino acid residue, wherein the cysteine ​​amino acid residue is capable of forming a disulfide bond.

33. 33. The multispecific protein of claim 32, wherein the amino acid sequence of the VH region of the first scFv comprises a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, amino acid numbering according to Kabat; and the amino acid sequence of the VL region of the first scFv comprises a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, amino acid numbering according to Kabat.

34. 34. The multispecific protein of claim 32 or 33, wherein the amino acid sequence of the VH region of the second scFv comprises a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, amino acid numbering according to Kabat; and the amino acid sequence of the VL region of the second scFv comprises a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, amino acid numbering according to Kabat.

35. 10. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is a human IgG (hIgG) antibody.

36. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is a hIgG1 or hIgG4 antibody.

37. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the first heavy chain of the full length antibody comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbering according to the EU index of Kabat.

38. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the first heavy chain of the full length antibody comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbering according to the EU index of Kabat.

39. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the first heavy chain of the full length antibody comprises an amino acid substitution at amino acid position Y349, numbering according to the EU index of Kabat.

40. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the first heavy chain of the full length antibody comprises a cysteine ​​at amino acid position Y349, numbering according to the EU index of Kabat.

41. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the second heavy chain of said full length antibody comprises an amino acid substitution at amino acid position T366, numbering according to the EU index of Kabat.

42. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the second heavy chain of the full length antibody comprises tryptophan at amino acid position T366, numbering according to the EU index of Kabat.

43. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the second heavy chain of the full length antibody comprises an amino acid substitution at amino acid position S354, numbering according to the EU index of Kabat.

44. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the second heavy chain of the full length antibody comprises a cysteine ​​at amino acid position S354, numbering according to the EU index of Kabat.

45. 37. The multispecific protein of any one of claims 1 to 36, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the second heavy chain of the full length antibody comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbering according to the EU index of Kabat.

46. 46. ​​The multispecific protein of any one of claims 1 to 36 or 45, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the second heavy chain of said full length antibody comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbering according to the EU index of Kabat.

47. 47. The multispecific protein of any one of claims 1 to 36 or 45 to 46, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the second heavy chain of said full length antibody comprises an amino acid substitution at amino acid position Y349, numbering according to the EU index of Kabat.

48. 48. The multispecific protein of any one of claims 1 to 36 or 45 to 47, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the second heavy chain of the full length antibody comprises a cysteine ​​at amino acid position Y349, numbering according to the EU index of Kabat.

49. 49. The multispecific protein of any one of claims 1 to 36 or 45 to 48, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the first heavy chain of said full length antibody comprises an amino acid substitution at amino acid position T366, numbering according to the EU index of Kabat.

50. 50. The multispecific protein of any one of claims 1 to 36 or 45 to 49, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the first heavy chain of the full length antibody comprises tryptophan at amino acid position T366, numbering according to the EU index of Kabat.

51. 51. The multispecific protein of any one of claims 1 to 36 or 45 to 50, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the first heavy chain of the full length antibody comprises an amino acid substitution at amino acid position S354, numbering according to the EU index of Kabat.

52. 52. The multispecific protein of any one of claims 1 to 36 or 45 to 51, wherein the full length antibody is an IgG1 antibody and the amino acid sequence of the first heavy chain of the full length antibody comprises a cysteine ​​at amino acid position S354, numbering according to the EU index of Kabat.

53. 10. The multispecific protein of any one of the preceding claims, wherein the multispecific protein does not substantially mediate ADCC, does not substantially mediate ADCP, does not substantially mediate CDC, and / or does not bind to one or more Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

54. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an amino acid substitution at amino acid position L234 and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat.

55. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbering according to the EU index of Kabat.

56. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an amino acid substitution at amino acid position L234, an amino acid substitution at amino acid position L235, and / or an amino acid substitution at amino acid position P329, numbering according to the EU index of Kabat.

57. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG1 antibody and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or an alanine at amino acid position P329, numbering according to the EU index of Kabat.

58. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG4 antibody and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or a glycine at amino acid position P329, numbering according to the EU index of Kabat.

59. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG4 antibody and the amino acid sequences of the first heavy chain and the second heavy chain each comprise an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position E235, numbering according to the EU index of Kabat.

60. 10. The multispecific protein of any one of the preceding claims, wherein the full length antibody is an IgG4 antibody and the amino acid sequences of the first heavy chain and the second heavy chain each comprise a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position E235, numbering according to the EU index of Kabat.

61. (a) a first Fab comprising (i) a first Fab heavy chain comprising, from N-terminus to C-terminus, a first VH region and a first CH1 region, and (ii) a first light chain comprising, from N-terminus to C-terminus, a first VL region and a first CL region; (b) a first scFv operably linked to the C-terminus of the first CH1 region of the first Fab, the first scFv comprising, from N-terminus to C-terminus: (i) a VH region, a peptide linker, and a VL region; or (ii) a VL region, a peptide linker, and a VH region; (c) a first Fc region operably linked to the C-terminus of the first scFv, the first Fc region comprising, from N-terminus to C-terminus, a CH2 region and a CH3 region; (d) a second Fab comprising (i) a second Fab heavy chain comprising, from N-terminus to C-terminus, a second VH region and a second CH1 region, and (ii) a second light chain comprising, from N-terminus to C-terminus, a second VL region and a first CL region; (b) a second scFv operably linked to the C-terminus of the second CH1 of the second Fab, the second scFv comprising, from N-terminus to C-terminus, (i) a VH domain, a peptide linker, and a VL domain; or (ii) a VL domain, a peptide linker, and a VH domain; (c) a second Fc region operably linked to the C-terminus of the second scFv, the second Fc region comprising, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; 1. A multispecific protein comprising: the first Fab specifically binds to a first human hTAA; the second Fab specifically binds to a second hTAA; the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); the second scFv specifically binds to hCD3; and the CH3 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications; the CH3 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions); and the one or more amino acid modifications in the CH3 region of the first Fc region are different from the one or more amino acid modifications in the CH3 region of the Fc region; the one or more amino acid modifications in the CH3 region of the first Fc region and the one or more amino acid modifications in the CH3 region of the second Fc region promote heterodimerization of the first and second Fc regions; the CH2 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications; the CH2 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications; wherein said one or more amino acid modifications in the CH2 region of said Fc region and said one or more amino acid modifications in the CH2 region of said second Fc region enhance the following Fc region effector function, as compared to a reference Fc region (e.g., a heavy chain comprising a wild-type CH2 domain, e.g., SEQ ID NO: 100) that does not contain said one or more amino acid modifications: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa)) A multispecific protein that reduces or eliminates one or more of:

62. 62. The multispecific protein of claim 61 , wherein the first Fab is operably linked to the first scFv directly through a peptide bond.

63. 62. The multispecific protein of claim 61 , wherein the first Fab is operably connected to the first scFv through a first peptide linker.

64. 64. The multispecific protein of claim 63, wherein the amino acid sequence of the first peptide linker comprises or consists of glycine amino acid residues or glycine and serine amino acid residues.

65. 65. The multispecific protein of any one of claims 63 or 64, wherein the amino acid sequence of the first peptide linker comprises or consists of: (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319 comprising or consisting of one, two, or three amino acid substitutions.

66. 66. The multispecific protein of any one of claims 61 to 65, wherein the second Fab is operably linked to the second scFv directly through a peptide bond.

67. 66. The multispecific protein of any one of claims 61 to 65, wherein the second Fab is operably connected to the second scFv through a second peptide linker.

68. 68. The multispecific protein of claim 67, wherein the amino acid sequence of the second peptide linker comprises or consists of glycine amino acid residues or glycine and serine amino acid residues.

69. the amino acid of the second peptide linker is (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319, comprising or consisting of one, two, or three amino acid substitutions.

69. The multispecific protein of any one of claims 67 or 68, comprising or consisting of:

70. 70. The multispecific protein of any one of claims 61 to 69, wherein the first scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; and the N-terminus of the VL region of the scFv is operably connected to the C-terminus of the CH1 region of the first Fab.

71. 71. The multispecific protein of any one of claims 61 to 70, wherein the first scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; and the N-terminus of the VH region of the scFv is operably connected to the C-terminus of the CH1 region of the first Fab.

72. 72. The multispecific protein of any one of claims 61 to 71, wherein the second scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; and the C-terminus of the VH region of the scFv is operably connected to the N-terminus of the CH1 region of the second Fab.

73. 73. The multispecific protein of any one of claims 61 to 72, wherein the second scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; and the C-terminus of the VL region of the scFv is operably connected to the N-terminus of the CH1 region of the second Fab.

74. 74. The multispecific protein of any one of claims 61 to 73, wherein the first hTAA and the second hTAA are expressed by (e.g., on the surface of) the same tumor cell.

75. 75. The multispecific protein of any one of claims 61 to 74, wherein the first hTAA and the second hTAA are the same.

76. 76. The multispecific protein of any one of claims 61 to 75, wherein the first scFv and the second scFv specifically bind to the same epitope of the same hTAA.

77. 77. The multispecific protein of any one of claims 61 to 76, wherein the first scFv and the second scFv specifically bind to different epitopes of the same hTAA.

78. 78. The multispecific protein of any one of claims 61 to 77, wherein the first tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2.

79. 79. The multispecific protein of any one of claims 61 to 78, wherein the amino acid sequence of the VH region of the first scFv and the amino acid sequence of the VL region of the first scFv each comprise a cysteine ​​amino acid residue, wherein the cysteine ​​amino acid residue is capable of forming a disulfide bond.

80. 80. The multispecific protein of claim 79, wherein the amino acid sequence of the VH region of the first scFv comprises a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, amino acid numbering according to Kabat; and the amino acid sequence of the VL region of the first scFv comprises a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, amino acid numbering according to Kabat.

81. 81. The multispecific protein of claim 79 or 80, wherein the amino acid sequence of the VH region of the second scFv comprises a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, amino acid numbering according to Kabat; and the amino acid sequence of the VL region of the second scFv comprises a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, amino acid numbering according to Kabat.

82. (a) an scFv comprising, from N-terminus to C-terminus, (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; (b) a Fab comprising (i) a Fab heavy chain comprising, from N-terminus to C-terminus, a VH region and a CH1 region, and (ii) a light chain comprising, from N-terminus to C-terminus, a VL region and a CL region; (c) a first Fc region comprising, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; (d) a second Fc region comprising, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; (e) an IgM CH2 mFab comprising (i) an IgM CH2 mFab heavy chain comprising, from N-terminus to C-terminus, a VH region and an IgM CH2 region, and (ii) an IgM CH2 mFab light chain comprising, from N-terminus to C-terminus, a VL region and an IgM CH2 region; 1. A multispecific protein comprising: the C-terminus of the scFv is operably connected to the N-terminus of the Fab heavy chain of the first Fab; the C-terminus of the Fab heavy chain is operably connected to the N-terminus of the first Fc region; the C-terminus of the IgM CH2 mFab heavy chain is operably connected to the N-terminus of the second Fc region; The scFv specifically binds to hTCSA (e.g., hCD28, hCD2); the Fab specifically binds to hCD3; the IgM CH2 mFab specifically binds to a human tumor-associated antigen (hTAA); the CH3 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications; the CH3 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101) that does not contain the one or more amino acid modifications (e.g., substitutions); and the one or more amino acid modifications in the CH3 region of the first Fc region are different from the one or more amino acid modifications in the CH3 region of the Fc region; the one or more amino acid modifications in the CH3 region of the first Fc region and the one or more amino acid modifications in the CH3 region of the second Fc region promote heterodimerization of the first and second heavy chains; the CH2 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications; the CH2 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) compared to the amino acid sequence of a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not contain the one or more amino acid modifications; wherein said one or more amino acid modifications in the CH2 region of said Fc region and said one or more amino acid modifications in the CH2 region of said second Fc region enhance the following Fc region effector function, as compared to a reference Fc region (e.g., a heavy chain comprising a wild-type CH2 domain, e.g., SEQ ID NO: 100) that does not contain said one or more amino acid modifications: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa)) A multispecific protein that reduces or eliminates one or more of:

83. 83. The multispecific protein of claim 82, wherein the scFv is operably linked to the N-terminus of the Fab heavy chain of the first Fab directly through a peptide bond.

84. 83. The multispecific protein of claim 82, wherein the scFv is operably connected to the N-terminus of the Fab heavy chain of the first Fab through a first peptide linker.

85. 85. The multispecific protein of claim 84, wherein the amino acid sequence of the first peptide linker comprises or consists of glycine amino acid residues or glycine and serine amino acid residues.

86. the amino acid sequence of the first peptide linker is (a) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319; or (b) the amino acid sequence of any one of SEQ ID NOs: 217-236 or 318-319, comprising or consisting of one, two, or three amino acid substitutions.

86. The multispecific protein of any one of claims 84 or 85, comprising or consisting of:

87. 87. The multispecific protein of any one of claims 82-86, wherein the scFv comprises, from N-terminus to C-terminus: a VL region, a peptide linker, and a VH region; and the C-terminus of the VH region of the scFv is operably connected to the N-terminus of the Fab heavy chain of the first Fab.

88. 88. The multispecific protein of any one of claims 82-87, wherein the scFv comprises, from N-terminus to C-terminus: a VH region, a peptide linker, and a VL region; and the C-terminus of the VL region of the scFv is operably connected to the N-terminus of the Fab heavy chain of the first Fab.

89. 89. The multispecific protein of any one of claims 82 to 88, wherein the tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2.

90. 90. The multispecific protein of any one of claims 82 to 89, wherein the amino acid sequence of the VH region of the first scFv and the amino acid sequence of the VL region of the first scFv each contain a cysteine ​​amino acid residue, wherein the cysteine ​​amino acid residue is capable of forming a disulfide bond.

91. 91. The multispecific protein of claim 90, wherein the amino acid sequence of the VH region of the first scFv comprises a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, amino acid numbering according to Kabat; and the amino acid sequence of the VL region of the first scFv comprises a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, amino acid numbering according to Kabat.

92. 92. The multispecific protein of claim 90 or 91, wherein the amino acid sequence of the VH region of the second scFv comprises a cysteine ​​at amino acid position 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, amino acid numbering according to Kabat; and the amino acid sequence of the VL region of the second scFv comprises a cysteine ​​at amino acid position 40, 41, 42, 43, 44, 45, 46, or 47, amino acid numbering according to Kabat.

93. 93. The multispecific protein of any one of claims 61 to 92, wherein the first Fc region and the second Fc region are of the human IgG (hIgG) isotype.

94. 94. The multispecific protein of any one of claims 61 to 93, wherein the first Fc region and the second Fc region are of the hIgG1 or hIgG4 isotype.

95. 95. The multispecific protein of any one of claims 61 to 94, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the first Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407, EU numbering according to the EU index of Kabat.

96. 96. The multispecific protein of any one of claims 61 to 95, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the first Fc region comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbering according to the EU index of Kabat.

97. 97. The multispecific protein of any one of claims 61 to 96, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, numbering according to the EU index of Kabat.

98. 98. The multispecific protein of any one of claims 61 to 97, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the first Fc region comprises a cysteine ​​at amino acid position Y349, numbering according to the EU index of Kabat.

99. 99. The multispecific protein of any one of claims 61 to 98, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, numbering according to the EU index of Kabat.

100. 100. The multispecific protein of any one of claims 61 to 99, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the second Fc region comprises tryptophan at amino acid position T366, numbering according to the EU index of Kabat.

101. 101. The multispecific protein of any one of claims 61 to 100, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position S354, numbering according to the EU index of Kabat.

102. 102. The multispecific protein of any one of claims 61 to 101, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the second Fc region comprises a cysteine ​​at amino acid position S354, numbering according to the EU index of Kabat.

103. 103. The multispecific protein of any one of claims 61 to 102, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the second Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407, numbering according to the EU index of Kabat.

104. 104. The multispecific protein of any one of claims 61 to 94 or 103, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the second Fc region comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbered according to the EU index of Kabat.

105. 105. The multispecific protein of any one of claims 61 to 94 or 103 to 104, wherein the first Fc region and the second Fc region are of the IgG1 isotype and the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position Y349, numbering according to the EU index of Kabat.

106. 106. The multispecific protein of any one of claims 61 to 94 or 103 to 105, wherein the first Fc region and the second Fc region are of the IgG1 isotype and the amino acid sequence of the second Fc region comprises a cysteine ​​at amino acid position Y349, numbering according to the EU index of Kabat.

107. 107. The multispecific protein of any one of claims 61 to 94 or 103 to 106, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position T366, numbering according to the EU index of Kabat.

108. 108. The multispecific protein of any one of claims 61 to 94 or 103 to 107, wherein the first Fc region and the second Fc region are of the IgG1 isotype and the amino acid sequence of the first Fc region comprises tryptophan at amino acid position T366, numbering according to the EU index of Kabat.

109. 109. The multispecific protein of any one of claims 61 to 94 or 103 to 108, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position S354, numbering according to the EU index of Kabat.

110. 110. The multispecific protein of any one of claims 61 to 94 or 103 to 109, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequence of the first Fc region comprises a cysteine ​​at amino acid position S354, numbering according to the EU index of Kabat.

111. 111. The multispecific protein of any one of claims 61 to 110, wherein the multispecific protein does not substantially mediate ADCC, does not substantially mediate ADCP, does not substantially mediate CDC, and / or does not bind to one or more Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

112. 112. The multispecific protein of any one of claims 61 to 111, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position L234 and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat.

113. 113. The multispecific protein of any one of claims 61 to 112, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbering according to the EU index of Kabat.

114. 114. The multispecific protein of any one of claims 61 to 113, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position L234, an amino acid substitution at amino acid position L235, and / or an amino acid substitution at amino acid position P329, numbering according to the EU index of Kabat.

115. 115. The multispecific protein of any one of claims 61 to 114, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or an alanine at amino acid position P329, numbering according to the EU index of Kabat.

116. 116. The multispecific protein of any one of claims 61 to 115, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234, an alanine at amino acid position L235, and / or a glycine at amino acid position P329, numbering according to the EU index of Kabat.

117. 117. The multispecific protein of any one of claims 61 to 116, wherein the first Fc region and the second Fc region are of the IgG4 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position E235, numbering according to the EU index of Kabat.

118. 118. The multispecific protein of any one of claims 61 to 117, wherein the first Fc region and the second Fc region are of the IgG4 isotype, and the amino acid sequences of the first Fc region and the second Fc region each comprise a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position E235, numbering according to the EU index of Kabat.

119. 119. The multispecific protein of any one of claims 61 to 118, wherein the tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA, or hHER2.

120. 10. The multispecific protein of any one of the preceding claims, wherein the hTCSA is hCD28, hCD2, hCD137, hCD27, hCD278, hCD134, or hCD40.

121. 10. The multispecific protein of any one of the preceding claims, wherein the hTCSA is hCD28.

122. 10. The multispecific protein of any one of the preceding claims, wherein the hTCSA is hCD2.

123. 10. A polynucleotide encoding a multispecific protein or one or more polypeptides thereof according to any one of the preceding claims.

124. 124. The polynucleotide of claim 123, wherein the polynucleotide is RNA (e.g., mRNA) or DNA.

125. 125. The polynucleotide of claim 123 or 124, which is codon-optimized.

126. An expression vector comprising the polynucleotide of any one of claims 123 to 125.

127. 127. The expression vector of claim 126, which is a viral vector or a plasmid.

128. 128. A host cell comprising a multispecific protein according to any one of claims 1 to 122, a polynucleotide according to any one of claims 123 to 125, or an expression vector according to any one of claims 126 to 127.

129. A carrier comprising a multispecific protein according to any one of claims 1 to 122, a polynucleotide according to any one of claims 123 to 125, or an expression vector according to any one of claims 126 to 127.

130. 130. The carrier of claim 129, wherein the carrier is a lipid nanoparticle, a liposome, a lipoplex, or a nanoliposome.

131. 130. A pharmaceutical composition comprising a multispecific protein according to any one of claims 1 to 122, a polynucleotide according to any one of claims 123 to 125, an expression vector according to any one of claims 126 to 127, a host cell according to claim 128, or a carrier according to any one of claims 129 to 130, and a pharmaceutically acceptable excipient.

132. 132. A kit comprising a multispecific protein according to any one of claims 1 to 122, a polynucleotide according to any one of claims 123 to 125, an expression vector according to any one of claims 126 to 127, a host cell according to claim 128, a carrier according to any one of claims 129 to 130, or a pharmaceutical composition according to claim 131.

133. A method for producing a multispecific protein according to any one of claims 1 to 122, comprising introducing into an in vitro or ex vivo cell population a polynucleotide according to any one of claims 123 to 125 or a vector according to any one of claims 126 to 127; culturing the cell population under conditions sufficient for the cell population to express the multispecific protein; and Optionally isolating and / or purifying said multispecific protein. A method comprising:

134. 131. A method of delivering a multispecific protein, polynucleotide, expression vector, host cell, carrier, or pharmaceutical composition to a subject, the method comprising administering to the subject a multispecific protein of any one of claims 1 to 122, a polynucleotide of any one of claims 123 to 125, an expression vector of any one of claims 126 to 127, a host cell of claim 128, a carrier of any one of claims 129 to 130, or the pharmaceutical composition of claim 131, thereby delivering the multispecific protein, polynucleotide, expression vector, host cell, carrier, or pharmaceutical composition to the subject.

135. 132. A method of inducing an immune response in a subject, the method comprising administering to the subject a multispecific protein according to any one of claims 1 to 122, a polynucleotide according to any one of claims 123 to 125, an expression vector according to any one of claims 126 to 127, a host cell according to claim 128, a carrier according to any one of claims 129 to 130, or a pharmaceutical composition according to claim 131, thereby inducing an immune response in the subject.

136. 132. A method of activating a T cell or population of T cells in a subject, the method comprising administering to the subject a multispecific protein of any one of claims 1 to 122, a polynucleotide of any one of claims 123 to 125, an expression vector of any one of claims 126 to 127, a host cell of claim 128, a carrier of any one of claims 129 to 130, or a pharmaceutical composition of claim 131, thereby activating a T cell or population of T cells in the subject.

137. 132. A method of preventing or treating cancer in a subject, comprising administering to said subject in need thereof a multispecific protein of any one of claims 1 to 122, a polynucleotide of any one of claims 123 to 125, an expression vector of any one of claims 126 to 127, a host cell of claim 128, a carrier of any one of claims 129 to 130, or a pharmaceutical composition of claim 131, thereby preventing or treating cancer in said subject.

138. 138. The method of claim 137, wherein the cancer is a solid tumor.

139. 139. The method of claim 137 or 138, wherein the cancer is breast cancer, ovarian cancer, endometrial cancer, uterine cancer, cervical cancer, anal cancer, prostate cancer, rectal cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, pancreatic cancer, thyroid cancer, thymus cancer, lung cancer, bronchial cancer, skin cancer, brain cancer, spinal cancer, head cancer, neck cancer, lip cancer, or oral cavity cancer.

140. 132. The multispecific protein of any one of claims 1 to 122, the polynucleotide of any one of claims 123 to 125, the expression vector of any one of claims 126 to 127, the host cell of claim 128, the carrier of any one of claims 129 to 130, or the pharmaceutical composition of claim 131 for use in a method of preventing or treating cancer in a subject, comprising administering to the subject the multispecific protein, the polynucleotide, the expression vector, the host cell, the carrier, or the pharmaceutical composition, thereby preventing or treating cancer in the subject.

141. 132. Use of a multispecific protein according to any one of claims 1 to 122, a polynucleotide according to any one of claims 123 to 125, an expression vector according to any one of claims 126 to 127, a host cell according to claim 128, a carrier according to any one of claims 129 to 130, or a pharmaceutical composition according to claim 131 for the manufacture of a medicament for preventing or treating cancer in a subject, comprising administering to the subject the multispecific protein, the polynucleotide, the expression vector, the host cell, the carrier, or the pharmaceutical composition, thereby preventing or treating cancer in the subject.

142. 132. The multispecific protein of any one of claims 1 to 122, the polynucleotide of any one of claims 123 to 125, the expression vector of any one of claims 126 to 127, the host cell of claim 128, the carrier of any one of claims 129 to 130, or the pharmaceutical composition of claim 131 for use in a method of inducing an immune response in a subject, comprising administering to the subject the multispecific protein, the polynucleotide, the expression vector, the host cell, the carrier, or the pharmaceutical composition, thereby inducing an immune response in the subject.

143. 132. Use of a multispecific protein according to any one of claims 1 to 122, a polynucleotide according to any one of claims 123 to 125, an expression vector according to any one of claims 126 to 127, a host cell according to claim 128, a carrier according to any one of claims 129 to 130, or a pharmaceutical composition according to claim 131 for the manufacture of a medicament for inducing an immune response in a subject, comprising administering to a subject the multispecific protein, the polynucleotide, the expression vector, the host cell, the carrier, or the pharmaceutical composition, thereby inducing an immune response in the subject.

144. 132. The multispecific protein of any one of claims 1 to 122, the polynucleotide of any one of claims 123 to 125, the expression vector of any one of claims 126 to 127, the host cell of claim 128, the carrier of any one of claims 129 to 130, or the pharmaceutical composition of claim 131 for use in a method of activating a T cell or a population of T cells in a subject, comprising administering to the subject the multispecific protein, the polynucleotide, the expression vector, the host cell, the carrier, or the pharmaceutical composition, thereby activating the T cell or population of T cells in the subject.

145. 132. Use of a multispecific protein according to any one of claims 1 to 122, a polynucleotide according to any one of claims 123 to 125, an expression vector according to any one of claims 126 to 127, a host cell according to claim 128, a carrier according to any one of claims 129 to 130, or a pharmaceutical composition according to claim 131 for the manufacture of a medicament for activating a T cell or a population of T cells in a subject, comprising administering to a subject the multispecific protein, the polynucleotide, the expression vector, the host cell, the carrier, or the pharmaceutical composition, thereby activating the T cell or population of T cells in the subject.

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