Multispecific proteins and related methods

Abstract

Provided herein are multispecific proteins and compositions (e.g., pharmaceutical compositions) comprising the same; and methods of making the multispecific proteins and compositions. The multispecific proteins provided herein are useful in pharmaceutical compositions and methods, including, for example, methods of treating a disease (e.g., cancer), methods of activating T cells, and methods of inducing or enhancing an immune response.

Description

Related Applications

[0001] This application claims priority to U.S. Serial No. 63 / 413,765, filed October 6, 2022, the entire content of which is incorporated herein by reference. 1. Field

[0002] The present disclosure relates to multispecific proteins (and polypeptides thereof) and polynucleotides encoding the same. The present disclosure also relates to methods of preparing and utilizing such proteins. 2. Background Art

[0003] T cell engaging immunotherapies function by activating T cells (or subsets thereof, such as CD8+ T cells) to mediate, for example, tumor cell lysis. A subset of T cell engagers are multispecific and are designed to target activated T cells to tumor cells by simultaneously binding to a T cell antigen (such as CD3) and an antigen expressed on the surface of tumor cells (such as a tumor associated antigen). Exemplary T cell engagers include bispecific T cell engagers (BiTEs), bifunctional checkpoint inhibitory T cell engagers (CiTEs), simultaneously multiplexed interacting T cell engagers (SMITEs), trispecific killer engagers (TriKEs), and chimeric antigen receptor (CAR) T cells expressing BiTEs (CART.BiTE cells). 3. Summary

[0004] In particular, provided herein are multispecific proteins (and polypeptides thereof) and polynucleotides encoding the same; methods of making the same; pharmaceutical compositions; and methods of use, including, for example, methods of treating diseases (such as cancer) and methods of inducing an immune response.

[0005] In one aspect, the present disclosure provides multispecific proteins comprising: (a) a full-length antibody comprising: (i) a first light chain comprising, from the N-terminus to the C-terminus, a variable light chain (VL) region and a constant light chain (CL) region; (ii) a first heavy chain comprising, from the N-terminus to the C-terminus, a variable heavy chain (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second heavy chain comprising, from the N-terminus to the 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 the N-terminus to the C-terminus, a VL region and a CL region; wherein the first light chain associates with the first heavy chain to form a first antigen-binding domain; wherein the second light chain associates with the second heavy chain to form a second antigen-binding domain; and wherein the first heavy chain associates with the second heavy chain to form a dimer; (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, wherein the first scFv comprises, 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; and (c) a second scFv operably linked to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody, wherein the second scFv comprises, 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; wherein the first antigen-binding domain of the full-length antibody specifically binds to a first human tumor-associated antigen (hTAA); wherein the second antigen-binding domain of the full-length antibody specifically binds to a second hTAA; wherein the first scFv specifically binds to a human T cell costimulatory antigen (hTCSA) (e.g., hCD28, hCD2); wherein the second scFv specifically binds to human CD3 (hCD3); and wherein the CH3 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (e.g., a reference CH3 region, e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101), and wherein the CH3 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein 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; and wherein 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 contains one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not contain one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); the CH2 region of the second heavy chain of the full-length antibody contains one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not contain one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein one or more amino acid modifications in the CH2 region of the first heavy chain of 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 relative to a reference heavy chain that does not contain the one or more amino acid modifications (e.g., a heavy chain containing a wild-type CH2 region such as SEQ ID NO: 100): antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or binding affinity for 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))).;

[0006] In some embodiments, the first scFv is directly operably linked via a peptide bond 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 linked via a first peptide linker to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody. In some embodiments, the amino acid sequence of the first peptide linker contains glycine or glycine and serine amino acid residues or consists thereof. In some embodiments, the amino acid sequence of the first peptide linker contains 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, which contains 1, 2, or 3 amino acid substitutions or consists thereof.

[0008] In some embodiments, the second scFv is directly operably linked via a peptide bond 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 linked to the C-terminus of the CH3 domain of the second heavy chain of the full-length antibody via a second peptide linker. In some embodiments, the amino acid sequence of the second peptide linker comprises or consists of glycine or glycine and serine amino acid residues. In some embodiments, the amino acids of the second peptide linker comprise or consist of the following: (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, which comprises, consists of, or is composed of 1, 2, or 3 amino acid substitutions.

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

[0011] In some embodiments, the first hTAA and the second hTAA are expressed by the same tumor cell (e.g., expressed 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 comprise a cysteine amino acid residue, wherein the cysteine amino acid residue is capable of forming a disulfide bond. In some embodiments, 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, with amino acids numbered 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, with amino acids numbered according to 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, with amino acids numbered 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, with amino acids numbered 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 an 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, where the amino acid positions are numbered according to the Kabat EU index. 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 serine at amino acid position T366, alanine at amino acid position L368, and valine at amino acid position Y407, where the amino acid positions are numbered according to the Kabat EU index. 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, where the amino acid position is numbered according to the Kabat EU index. 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 cysteine at amino acid position Y349, where the amino acid position is numbered according to the Kabat EU index. 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, where the amino acid position is numbered according to the Kabat EU index. 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 tryptophan at amino acid position T366, where the amino acid position is numbered according to the Kabat EU index. 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, where the amino acid position is numbered according to the Kabat EU index. 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 cysteine at amino acid position S354, where the amino acid position is numbered according to the Kabat EU index.

[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, which amino acid positions are according to the EU index numbering 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 serine at amino acid position T366, alanine at amino acid position L368, and valine at amino acid position Y407, which amino acid positions are according to the EU index numbering 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, which amino acid position is according to the EU index numbering 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 cysteine at amino acid position Y349, which amino acid position is according to the EU index numbering 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, which amino acid position is according to the EU index numbering 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 tryptophan at amino acid position T366, which amino acid position is according to the EU index numbering 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, which amino acid position is according to the EU index numbering 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 cysteine at amino acid position S354, which amino acid position is according to the EU index numbering of Kabat.

[0016] In some embodiments, the multispecific protein substantially does not mediate ADCC, substantially does not mediate ADCP, substantially does not 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))).

[0017] In some embodiments, the full-length antibody is an IgG1 antibody, and the amino acid sequences of the first and second heavy chains each contain an amino acid substitution at amino acid position L234, and / or an amino acid substitution at amino acid position L235, where the amino acid positions are numbered according to Kabat's EU index. In some embodiments, the full-length antibody is an IgG4 antibody, and the amino acid sequences of the first and second heavy chains each contain alanine at amino acid position L234, and / or alanine at amino acid position L235, where the amino acid positions are numbered according to Kabat's EU index. In some embodiments, the full-length antibody is an IgG1 antibody, and the amino acid sequences of the first and second heavy chains each contain 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, where the amino acid positions are numbered according to Kabat's EU index.

[0018] In some embodiments, the full-length antibody is an IgG1 antibody, and the amino acid sequences of the first and second heavy chains each contain alanine at amino acid position L234, alanine at amino acid position L235, and / or alanine at amino acid position P329, where the amino acid positions are numbered according to Kabat's EU index. In some embodiments, the full-length antibody is an IgG1 antibody, and the amino acid sequences of the first and second heavy chains each contain alanine at amino acid position L234, alanine at amino acid position L235, and / or glycine at amino acid position P329, where the amino acid positions are numbered according to Kabat's EU index. In some embodiments, the full-length antibody is an IgG4 antibody, and the amino acid sequences of the first and second heavy chains each contain 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, where the amino acid positions are numbered according to Kabat's EU index. In some embodiments, the full-length antibody is an IgG4 antibody, and the amino acid sequences of the first and second heavy chains each contain proline at amino acid position S228, alanine at amino acid position F234, and / or alanine at amino acid position E235, where the amino acid positions are numbered according to Kabat's EU index.

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

[0020] In one aspect, the present disclosure provides multispecific proteins comprising: (a) a full-length antibody comprising: (i) a first light chain comprising a VL region and a CL region from the N-terminus to the C-terminus; (ii) a first heavy chain comprising a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; (iii) a second heavy chain comprising a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; (iv) a second light chain comprising a VL region and a CL region from the N-terminus to the C-terminus; wherein the first light chain associates with the first heavy chain to form a first antigen-binding domain; wherein the second light chain associates with the second heavy chain to form a second antigen-binding domain; and wherein the first heavy chain associates with the second heavy chain to form a dimer; (b) a first scFv operably linked to the N-terminus of the first heavy chain of the full-length antibody, wherein the first scFv comprises 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; and (c) a second scFv operably linked to the N-terminus of the second heavy chain of the full-length antibody, wherein the second scFv comprises 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; wherein the first antigen-binding domain of the full-length antibody specifically binds to a first hTAA; wherein the second antigen-binding domain of the full-length antibody specifically binds to a second hTAA; wherein the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); wherein the second scFv specifically binds to hCD3; and wherein the CH3 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (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 (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein 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; wherein 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; wherein the CH2 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100);The CH2 region of the second heavy chain of the full-length antibody contains one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not contain one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein one or more amino acid modifications in the CH2 region of the first heavy chain of 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 relative to a reference heavy chain that does not contain one or more amino acid modifications (e.g., a heavy chain containing a wild-type CH2 region such as SEQ ID NO: 100): ADCC, CDC, and / or binding affinity for 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))).;

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

[0022] In some embodiments, the first scFv is operably linked to the N-terminus of the VH region of the first heavy chain of the full-length antibody by a first peptide linker. In some embodiments, the amino acid sequence of the first peptide linker contains glycine or glycine and serine amino acid residues or consists thereof. In some embodiments, the amino acid sequence of the first peptide linker contains or consists of the following: (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, which contains 1, 2, or 3 amino acid substitutions or consists thereof.

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

[0024] In some embodiments, the second scFv is operably linked to the N-terminus of the VH region of the second heavy chain of the full-length antibody by a second peptide linker. In some embodiments, the amino acid sequence of the second peptide linker contains glycine or glycine and serine amino acid residues or consists thereof. In some embodiments, the second peptide linker amino acids contain or consist of the following: (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, which contains 1, 2, or 3 amino acid substitutions or consists thereof.

[0025] In some embodiments, the first scFv comprises, from the N-terminus to the C-terminus: a VL region, a peptide linker, and a VH region; wherein the C-terminus of the VH region of the scFv is operably linked 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 the N-terminus to the C-terminus: a VH region, a peptide linker, and a VL region; wherein the C-terminus of the VL region of the scFv is operably linked 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 the N-terminus to the C-terminus: a VL region, a peptide linker, and a VH region; and wherein the C-terminus of the VH region of the scFv is operably linked 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 the N-terminus to the C-terminus: a VH region, a peptide linker, and a VL region; and wherein the C-terminus of the VL region of the scFv is operably linked 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 the same tumor cell (e.g., expressed on its surface). 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; 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 comprise a cysteine amino acid residue, wherein the cysteine amino acid residue is capable of forming a disulfide bond. In some embodiments, 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, with amino acids numbered 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, with amino acids numbered according to 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, with amino acids numbered 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, with amino acids numbered 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 an hIgG1 or hIgG4 antibody.

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

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

[0031] In some embodiments, the multispecific protein substantially does not mediate ADCC, substantially does not mediate ADCP, substantially does not 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))).

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

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

[0034] In one aspect, the present disclosure provides multispecific proteins comprising: (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, wherein the first scFv comprises, 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, wherein the first Fc region comprises, from N-terminus to C-terminus, a CH2 region and a CH3 region; and (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, wherein the second scFv comprises, 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 Fc region operably linked to the C-terminus of the second scFv, wherein the second Fc region comprises, from N-terminus to C-terminus, a CH2 region and a CH3 region; and wherein the first Fab specifically binds to a first human hTAA; wherein the second Fab specifically binds to a second hTAA; wherein the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); wherein the second scFv specifically binds to hCD3; and wherein the CH3 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101), wherein the CH3 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein one or more amino acid modifications in the CH3 region of the first Fc region are different from one or more amino acid modifications in the CH3 region of the Fc region; wherein one or more amino acid modifications in the CH3 region of the first Fc region and one or more amino acid modifications in the CH3 region of the second Fc region promote heterodimerization of the first and second Fc regions; wherein the CH2 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100);wherein the CH2 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); 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 relative to a reference Fc region that does not comprise the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 domain such as SEQ ID NO: 100): ADCC, CDC, and / or binding affinity for 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))).;

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

[0036] In some embodiments, the first Fab is operably linked to the first scFv by a first peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises glycine or glycine and serine amino acid residues or consists thereof. 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, which comprises 1, 2, or 3 amino acid substitutions or consists thereof.

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

[0038] In some embodiments, the second Fab is operably linked to the second scFv by a second peptide linker. In some embodiments, the amino acid sequence of the second peptide linker comprises glycine or glycine and serine amino acid residues or consists thereof. In some embodiments, the amino acid 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, which comprises 1, 2, or 3 amino acid substitutions or consists thereof.

[0039] In some embodiments, the first scFv comprises, from the N-terminus to the C-terminus: a VL region, a peptide linker, and a VH region; and wherein the N-terminus of the VL region of the scFv is operably linked to the C-terminus of the CH1 region of the first Fab. In some embodiments, the first scFv comprises, from the N-terminus to the C-terminus: a VH region, a peptide linker, and a VL region; wherein the N-terminus of the VH region of the scFv is operably linked to the C-terminus of the CH1 region of the first Fab. In some embodiments, the second scFv comprises, from the N-terminus to the C-terminus: a VL region, a peptide linker, and a VH region; wherein the C-terminus of the VH region of the scFv is operably linked to the N-terminus of the CH1 region of the second Fab. In some embodiments, the second scFv comprises, from the N-terminus to the C-terminus: a VH region, a peptide linker, and a VL region; wherein the C-terminus of the VL region of the scFv is operably linked 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 the same tumor cell (e.g., expressed on its surface). 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; 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 comprise a cysteine amino acid residue, wherein the cysteine amino acid residue is capable of forming a disulfide bond. In some embodiments, 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 acids according to Kabat numbering; 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 acids according to Kabat numbering. 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 acids according to Kabat numbering; 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 acids according to Kabat numbering.

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

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

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

[0045] In some embodiments, the multispecific protein substantially does not mediate ADCC, substantially does not mediate ADCP, substantially does not 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))).

[0046] In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and wherein 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, said amino acid positions being according to the Kabat EU index numbering. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequences of the first Fc region and the second Fc region each comprise alanine at amino acid position L234, and / or alanine at amino acid position L235, said amino acid positions being according to the Kabat EU index numbering. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and wherein 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, said amino acid positions being according to the Kabat EU index numbering.

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

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

[0049] In one aspect, the present disclosure provides multispecific proteins comprising: (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 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 (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; wherein the C-terminus of the scFv is operably linked to the N-terminus of the Fab heavy chain of the first Fab; wherein the C-terminus of the Fab heavy chain is operably linked to the N-terminus of the first Fc region; wherein the C-terminus of the IgM CH2 mFab heavy chain is operably linked to the N-terminus of the second Fc region; wherein the scFv specifically binds to hTCSA (e.g., hCD28, hCD2); wherein the Fab specifically binds to hCD3; wherein the IgM CH2 mFab specifically binds to a human tumor-associated antigen (hTAA); wherein the CH3 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); wherein the CH3 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein one or more amino acid modifications in the CH3 region of the first Fc region are different from one or more amino acid modifications in the CH3 region of the second Fc region; wherein one or more amino acid modifications in the CH3 region of the first Fc region and one or more amino acid modifications in the CH3 region of the second Fc region promote heterodimerization of the first heavy chain and the second heavy chain; wherein the CH2 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein the CH2 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100);One or more amino acid modifications in the CH2 region of the Fc region and 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 relative to a reference Fc region that does not contain the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 domain such as SEQ ID NO: 100): ADCC, CDC, and / or binding affinity for 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))).;

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

[0051] In some embodiments, the scFv is operably linked to the N-terminus of the Fab heavy chain of the first Fab via a first peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises glycine or glycine and serine amino acid residues or consists thereof. 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, which comprises 1, 2, or 3 amino acid substitutions or consists thereof.

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

[0053] In some embodiments, the scFv comprises, from the N-terminus to the C-terminus: a VH region, a peptide linker, and a VL region; wherein the C-terminus of the VL region of the scFv is operably linked 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 comprise a cysteine amino acid residue, wherein the cysteine amino acid residue is capable of forming a disulfide bond. In some embodiments, 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, with amino acids numbered 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, with amino acids numbered according to 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, with amino acids numbered 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, with amino acids numbered 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 the IgG1 isotype, and wherein the amino acid sequence of the first Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407, the amino acid positions being numbered according to Kabat's EU index. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the first Fc region comprises serine at amino acid position T366, alanine at amino acid position L368, and valine at amino acid position Y407, the amino acid positions being numbered according to Kabat's EU index. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, the amino acid position being numbered according to Kabat's EU index. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the first Fc region comprises cysteine at amino acid position Y349, the amino acid position being numbered according to Kabat's EU index. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, the amino acid position being numbered according to Kabat's EU index. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises tryptophan at amino acid position T366, the amino acid position being numbered according to Kabat's EU index. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position S354, the amino acid position being numbered according to Kabat's EU index. In some embodiments, the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises cysteine at amino acid position S354, the amino acid position being numbered according to Kabat's EU index.

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

[0059] In some embodiments, the multispecific protein substantially does not mediate ADCC, substantially does not mediate ADCP, substantially does not 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))).

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

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

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

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

[0064] In one aspect, provided herein are polynucleotides encoding the multispecific proteins or one or more polypeptides thereof described herein. 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 are expression vectors comprising the polynucleotides described herein. In some embodiments, the expression vector is a viral vector or a plasmid.

[0066] In one aspect, provided herein are host cells comprising the multispecific proteins described herein, the polynucleotides described herein, or the expression vectors described herein.

[0067] In one aspect, provided herein are carriers comprising the multispecific proteins described herein, the polynucleotides described herein, or the expression vectors described herein.

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

[0069] In one aspect, provided herein are pharmaceutical compositions comprising the multispecific proteins described herein, the polynucleotides described herein, the expression vectors described herein, the host cells described herein, or the carriers described herein, and a pharmaceutically acceptable excipient.

[0070] In one aspect, provided herein are kits comprising the multispecific proteins described herein, the polynucleotides described herein, the expression vectors described herein, the host cells described herein, the carriers described herein, or the pharmaceutical compositions described herein.

[0071] In one aspect, provided herein are methods for preparing the multispecific proteins described herein, comprising: introducing the polynucleotides described herein or the vectors 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 are methods of delivering a multispecific protein, polynucleotide, expression vector, host cell, carrier, or pharmaceutical composition to a subject, the methods comprising administering the multispecific protein described herein, the polynucleotide described herein, the expression vector described herein, the host cell described herein, the carrier described herein, or the 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 are methods of inducing an immune response in a subject, the methods comprising administering to the subject the multispecific protein described herein, the polynucleotide described herein, the expression vector described herein, the host cell described herein, the carrier described herein, or the pharmaceutical composition described herein, thereby inducing an immune response in the subject.

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

[0075] In one aspect, provided herein are methods of preventing or treating cancer in a subject, the methods comprising administering to a subject in need thereof the multispecific protein described herein, the polynucleotide described herein, the expression vector described herein, the host cell described herein, the carrier described herein, or the pharmaceutical composition described herein, 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, thymic cancer, lung cancer, bronchial cancer, skin cancer, brain cancer, spinal cancer, head cancer, neck cancer, lip cancer, or oral cancer. 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1Illustration of a representative form of the BCA405 form of the multispecific protein described herein. In the specific embodiment shown, the multispecific protein comprises (i) a full-length antibody that specifically binds a first human tumor-associated antigen (hTAA) and a second hTAA; (ii) a first scFv operably linked to the C-terminus of the first heavy chain of the full-length antibody, wherein the first scFv specifically binds human CD3 (hCD3); and a second scFv operably linked to the C-terminus of the second heavy chain of the full-length antibody, wherein the second scFv specifically binds a human T cell co-stimulatory antigen (hTCSA) (e.g., human CD28 (hCD28), human CD2 (hCD2)). In some embodiments, the first and second hTAA are the same. In some embodiments, the first scFv is operably linked to the C-terminus of the first heavy chain of the full-length antibody via a peptide linker; and the second scFv is operably linked to the C-terminus of the second heavy chain of the full-length antibody via a peptide linker.

[0077] Figure 2 Illustration of a representative form of the BCA406 form of the multispecific protein described herein. In the specific embodiment shown, the multispecific protein comprises (i) a full-length antibody that specifically binds a first human hTAA and a second hTAA; (ii) a first scFv operably linked to the N-terminus of the first heavy chain of the full-length antibody, wherein the first scFv specifically binds hCD3; and a second scFv operably linked to the N-terminus of the second heavy chain of the full-length antibody, wherein the second scFv specifically binds hTCSA (e.g., hCD28, hCD2). In some embodiments, the first and 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.

[0078] Figure 3 Illustration of a representative form of the BCA424 form of the multispecific protein described herein. In the specific embodiment shown, the multispecific protein comprises a first Fab operably linked to a first scFv, the first scFv being operably linked to a first Fc region; and a second Fab operably linked to a second scFv, the second scFv being operably linked to a second Fc region; wherein the first Fab specifically binds a first hTAA, the second Fab specifically binds a second hTAA, the first scFv specifically binds hTCSA (e.g., hCD28, hCD2), and the second scFv specifically binds hCD3. In some embodiments, the first and second hTAA are the same. In some embodiments, the first Fab is operably linked to the first scFv via a peptide linker; and the second Fab is operably linked to the second scFv via a peptide linker.

[0079] Figure 4 This is a diagram of a representative form of the BCA418 form of the multispecific protein described herein. In the specific embodiment shown, the multispecific protein comprises a first scFv operably linked to the VH region of a first Fab, which first Fab is operably linked to a first Fc region; and a first IgM CH2 mFab operably linked to a second Fc region; wherein the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2), the first Fab specifically binds to hCD3, and the first IgM CH2 mFab specifically binds to hTAA. In some embodiments, the first Fab is operably linked to the VL region of the scFv. In some embodiments, the first Fab is operably linked to the VH region of the scFv. In some embodiments, the first Fab is operably linked to the first scFv via a peptide linker.

[0080] Figure 5A This is a graph showing the ELISA-based HER2 binding of the indicated multispecific protein or control at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows the EC50 values for n = 2 based on BCA401 and n = 3 for BCA405, BCA406, and BCA424. Figure 5B This is a graph showing the ELISA-based HER2 binding of the indicated multispecific protein or control at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows the EC50 values for n = 2 based on BCA418 and BCA401 and n = 3 for BCA418.

[0081] Figure 6 This is a graph showing the EGFR binding of the indicated multispecific proteins (BCA405.EG, BCA406.EG, BCA424.EG, which are in the same form as Figure 1 、 2 and 3, with anti-EGFR as the TAA targeting arm) or control (cetuximab, a positive control for EGFR binding) to A431 cells at the indicated concentrations. The table below the graph shows the EC50 (nM) values.

[0082] Figure 7A This is a graph showing the ELISA-based CD3 binding of the indicated multispecific protein or control at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows the EC50 values for n = 3 based on BCA405, BCA406, and BCA424. Figure 7BIt is a graph showing the ELISA-based CD3 binding of the indicated multispecific protein or control at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows the EC50 values for n = 2 based on BCA418.

[0083] Figure 8 It is a graph showing the CD3 binding of the indicated multispecific protein or control to CD28 knockout Jurkat cells at the indicated concentrations. The graph shows representative data from two independent experiments. The table below the graph shows the qualitative ranking based on the shift of the curve relative to BCA403 (anti-CD3 mAb). Qualitative ranking is based on the shift of the curve, where +, ++, +++ represent low, medium, and high binding compared to BCA403.

[0084] Figure 9A It is a graph showing the ELISA-based CD28 binding of the indicated multispecific protein or control at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows the EC50 values for n = 3 based on BCA405, BCA406, and BCA424. Figure 9B It is a graph showing the CD28 binding of the indicated multispecific protein or control at the indicated concentrations. The graph shows representative data from at least two independent experiments. The table below the graph shows the EC50 values for n = 2 based on BCA418.

[0085] Figure 10 It is a graph showing the CD28 binding of the indicated multispecific protein or control on CD3 knockout Jurkat cells at the indicated concentrations. The graph shows representative data from two independent experiments. The table below the graph was qualitatively ranked based on the shift of the curve relative to BCA402 (anti-CD28 mAb). Qualitative ranking is based on the shift of the curve, where +, ++, +++ represent low, medium, and high binding compared to BCA402.

[0086] Figure 11 It is a graph showing the percentage of cytotoxicity mediated by the indicated multispecific protein or control at the indicated concentrations in an SKBR3-PBMC co-culture assay. Target cells (tumor cells, T) were co-cultured with human PBMC (E) at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was evaluated using a luminescence readout (BioGlo). The formula used: Percentage of cytotoxicity = [100 - (RLU N / RLU 未处理的SKBR3-Luc ) X 100], where RLU N = RLU 所有组 - RLU 未刺激的PBMC。RLU = Relative Light Unit. Each point represents the mean ± SD of replicate values in the experiment. The figure shows representative data from at least two independent experiments using two PBMC donors. Human IgG isotype control antibody (hIgG) was used as a negative control. The table below the figure shows the EC50 (nM) values.

[0087] Figure 12 is a line graph showing the percentage of cytotoxicity mediated by the indicated multispecific protein or control at the indicated concentrations in the BxPC3-PBMC co-culture assay. Target cells were co-cultured with human PBMC at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was evaluated using a luminescence readout (BioGlo). The formula used: Percentage of cytotoxicity = [100 - (RLU N / RLU 未处理的SKBR3-Luc ) X 100], where RLU N = RLU 所有组 - RLU 未刺激的PBMC . RLU = Relative Light Unit. The percentage of cytotoxicity shown in the figure has been normalized to the hIgG control. Each point represents the mean ± SD of replicate values in one experiment and represents the results of two independent experiments. Figure 13A is a bar graph showing the levels of IL-2 and IFNγ released in SKBR3luc-PBMC co-cultures treated with the indicated multispecific protein or control at the indicated concentrations. Data are plotted as the mean ± SD of replicate wells in the experiment. Figure 13B is a bar graph showing the level of granzyme B released in SKBR3luc-PBMC co-cultures treated with the indicated multispecific protein or control at the indicated concentrations. Data are plotted as the mean ± SD of replicate wells in the experiment. Figure 13C is a bar graph showing the levels of IL-6 and TNFα released in SKBR3luc-PBMC co-cultures treated with the indicated multispecific protein or control at the indicated concentrations. Data are plotted as the mean ± SD of replicate wells in the experiment. Figure 13D is a bar graph showing the levels of IL-2 and IFNγ released in PBMC cultures treated with the indicated multispecific protein or control at the indicated concentrations. Figure 13E is a bar graph showing the level of granzyme B released in PBMC cultures treated with the indicated multispecific protein or control at the indicated concentrations. Data are plotted as the mean ± SD of replicate wells in the experiment. Figure 13F is a bar graph showing the levels of IL-6 and TNFα released in PBMC cultures treated with the indicated multispecific protein or control at the indicated concentrations.

[0089] Figure 14AIt is a bar graph showing the frequency of activated T cells (CD3+CD25+ cells) in PBMC cultures treated with the indicated multispecific protein or control at the indicated concentrations. The graph represents n = 3 experiments for BCA405, BCA406, BCA424, and BCA410 and n = 2 experiments for BCA418 in two PBMC donors. Figure 14B It is a bar graph showing the frequency of T cells expressing Bcl-xL (CD3+Bcl-xL+ cells) in PBMC cultures treated with the indicated multispecific protein or control at the indicated concentrations. The graph represents n = 3 experiments for BCA405, BCA406, BCA424, and BCA410 and n = 2 experiments for BCA418 in two PBMC donors. Figure 14C It is a bar graph showing the frequency of activated T cells (CD3+CD25+ cells) in SKBR3-PBMC co-cultures treated with the indicated multispecific protein or control at the indicated concentrations. The graph represents n = 3 for BCA405, BCA406, BCA424, and BCA410 and n = 2 for BCA418 in two PBMC donors. Figure 14D It is a bar graph showing the frequency of T cells expressing Bcl-xL (CD3+Bcl-xL+ cells) in SKBR3-PBMC co-cultures treated with the indicated multispecific protein or control at the indicated concentrations. The graph represents n = 3 for BCA405, BCA406, BCA424, and BCA410 and n = 2 for BCA418 in two PBMC donors.

[0090] Figure 15A It is a bar graph showing the frequency of T cells expressing Bcl-xL (CD3+Bcl-xL+ cells) in PBMC cultures treated with the indicated multispecific protein or control at the indicated concentrations. 15B is a bar graph showing the frequency of T cells expressing Bcl-xL (CD3+Bcl-xL+ cells) in FaDu-PBMC co-cultures treated with the indicated multispecific protein or control at the indicated concentrations.

[0091] Figure 16A It is a bar graph showing the frequency of T cells expressing Bcl-xL (CD3+Bcl-xL+ cells) in PBMC cultures treated with the indicated multispecific protein or control at the indicated concentrations. Figure 16B It is a bar graph showing the frequency of T cells expressing Bcl-xL (CD3+Bcl-xL+ cells) in SKBR3-PBMC co-cultures treated with the indicated multispecific protein or control at the indicated concentrations.

[0092] Figure 17AIs a line graph showing the percentage of cytotoxicity mediated by the indicated multispecific protein or control at the indicated concentrations in the SKBR3-PBMC co-culture assay. Target cells were co-cultured with human PBMC at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was evaluated using a luminescence readout (BioGlo). The formula used: Percentage cytotoxicity = [100 - (RLU N / RLU 未处理的SKBR3-Luc ) X 100], where RLU N = RLU 所有组 - RLU 未刺激的PBMC . RLU = relative light unit. Each point represents the mean ± standard deviation (SD) of duplicate values in the experiment. Figure 17B Is a line graph showing the percentage of cytotoxicity mediated by the indicated multispecific protein or control at the indicated concentrations in the SKBR3-PBMC co-culture assay. Target cells were co-cultured with human PBMC at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was evaluated using a luminescence readout (BioGlo). The formula used: Percentage cytotoxicity = [100 - (RLU N / RLU 未处理的SKBR3-Luc ) X 100], where RLU N = RLU 所有组 - RLU 未刺激的PBMC . RLU = relative light unit. Each point represents the mean ± standard deviation (SD) of duplicate values in the experiment. Figure 17C Is a line graph showing the percentage of cytotoxicity mediated by the indicated multispecific protein or control at the indicated concentrations in the SKBR3-PBMC co-culture assay. Target cells were co-cultured with human PBMC at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was evaluated using a luminescence readout (BioGlo). The formula used: Percentage cytotoxicity = [100 - (RLU N / RLU 未处理的SKBR3-Luc ) X 100], where RLU N = RLU 所有组 - RLU 未刺激的PBMC . RLU = relative light unit. Each point represents the mean ± standard deviation (SD) of duplicate values in the experiment. Figure 17D Is a line graph showing the percentage of cytotoxicity mediated by the indicated multispecific protein or control at the indicated concentrations in the SKBR3-PBMC co-culture assay. Target cells were co-cultured with human PBMC at a T:E ratio of 1:10 for 48 hours. Cytotoxicity was evaluated using a luminescence readout (BioGlo). The formula used: Percentage cytotoxicity = [100 - (RLU N / RLU 未处理的SKBR3-Luc ) X 100], where RLU N = RLU 所有组 - RLU未刺激的PBMC 。RLU = Relative Light Unit. Each point represents the mean ± standard deviation (SD) of the replicate values in the experiment.

[0093] Figure 18A It is a bar graph showing the percentage of activated CD4+ T cells (CD4+CD25+ cells) in FaDu-PBMC co-cultures treated with the indicated multi-specific protein or control at the indicated concentrations. Figure 18B It is a bar graph showing the percentage of activated CD8+ T cells (CD8+CD25+ cells) in FaDu-PBMC co-cultures treated with the indicated multi-specific protein or control at the indicated concentrations. Figure 18C It is a bar graph showing the percentage of activated CD4+ T cells (CD4+CD25+ cells) in PBMC cultures treated with the indicated multi-specific protein or control at the indicated concentrations. Figure 18D It is a bar graph showing the percentage of activated CD8+ T cells (CD8+CD25+ cells) in PBMC cultures treated with the indicated multi-specific protein or control at the indicated concentrations.

[0094] Figure 19A It is a line graph showing the percentage of activated CD4+CD25+ memory T cells (CD45RO and CCR7 (naïve CD45RO-CCR7+), (TEM CD45RO+CCR7-), (TCM CD45RO+CCR7+), (DN CD45RO-CCR7-)) in FaDu-PBMC co-cultures treated with the indicated control at the indicated concentrations. Figure 19B It is a line graph showing the percentage of activated CD8+CD25+ memory T cells (CD45RO and CCR7 (naïve CD45RO-CCR7+), (TEM CD45RO+CCR7-), (TCM CD45RO+CCR7+), (DN CD45RO-CCR7-)) in FaDu-PBMC co-cultures treated with the indicated control at the indicated concentrations. Figure 19C It is a line graph showing the percentage of activated CD4+CD25+ memory T cells (CD45RO and CCR7 (naïve CD45RO-CCR7+), (TEM CD45RO+CCR7-), (TCM CD45RO+CCR7+), (DN CD45RO-CCR7-)) in FaDu-PBMC co-cultures treated with the indicated multi-specific protein at the indicated concentrations. Figure 19DIt 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 co-cultures treated with the indicated multi-specific protein at the indicated concentration.

[0095] Figure 20A It is a line graph showing the level of IFNγ released in FaDu-PBMC co-cultures treated with the indicated multi-specific protein or control at the indicated concentration. Figure 20B It is a line graph showing the level of IL-2 released in FaDu-PBMC co-cultures treated with the indicated multi-specific protein or control at the indicated concentration. Figure 20C It is a line graph showing the level of TNF released in FaDu-PBMC co-cultures treated with the indicated multi-specific protein or control at the indicated concentration. Figure 20D It is a line graph showing the level of IL-6 released in FaDu-PBMC co-cultures treated with the indicated multi-specific protein or control at the indicated concentration. Figure 20E It is a line graph showing the level of IL-4 released in FaDu-PBMC co-cultures treated with the indicated multi-specific protein or control at the indicated concentration. Figure 20F It is a line graph showing the level of IL-10 released in FaDu-PBMC co-cultures treated with the indicated multi-specific protein or control at the indicated concentration. Figure 20G It is a line graph showing the level of IL-17A released in FaDu-PBMC co-cultures treated with the indicated multi-specific protein or control at the indicated concentration.

[0096] Figure 21A It is a schematic illustration of the structure of the multi-specific antibody BCA605. The structure of this multi-specific antibody is similar to the structure of BCA405 (as Figure 1 shown). 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 (as shown by the lines in the figure). Figure 21B It is a schematic illustration of the structure of the multi-specific antibody BCA606. The structure of the multi-specific antibody is similar to the structure of BCA406 (as Figure 2 shown). 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 (as shown by the lines in the figure). Figure 21CIt is a diagram of the structure of the multispecific antibody BCA624. The structure of the multispecific antibody is similar to the structure of BCA424 (as Figure 3 shown). The anti-TAA Fab of BCA605 specifically binds to MSLN. In some embodiments, the anti-CD3 and anti-hTCSA (such as CD28, CD2) scFv arms contain engineered disulfide bonds (as shown by the lines in the figure).

[0097] Figure 22A It is a graph showing the binding of the indicated multispecific proteins (BCA605 (MOR), BCA606 (MOR), BCA624 (MOR)) or control (BCA429 (MOR), hIgG) at the indicated concentrations to MSLN expressed on the surface of OVACAR-3 / CMV Luc cells. Figure 22B It is a graph showing the binding of the indicated multispecific proteins (BCA605 (FB6), BCA606 (FB6), BCA624 (FB6)) or control (hIgG) at the indicated concentrations to MSLN expressed on the surface of OVACAR-3 / CMV Luc cells.

[0098] Figure 23A It is a graph showing the binding of the indicated multispecific proteins (BCA605 (MOR), BCA606 (MOR), BCA624 (MOR)) or control (BCA402, BCA403, hIgG) at the indicated concentrations to CD28 expressed on the surface of CD3eKO Jurkat cells as measured by flow cytometry. Figure 23B It is a graph showing the binding of the indicated multispecific proteins (BCA605 (FB6), BCA606 (FB6), BCA624 (FB6)) or control (BCA402, BCA403, hIgG) at the indicated concentrations to CD28 expressed on the surface of CD3eKO Jurkat cells as measured by flow cytometry.

[0099] Figure 24A It is a graph showing the binding of the indicated multispecific proteins (BCA605 (MOR), BCA606 (MOR), BCA624 (MOR)) or control (BCA402, BCA403, hIgG) at the indicated concentrations to CD3 expressed on the surface of CD28eKO Jurkat cells as measured by flow cytometry. Figure 24B It is a graph showing the binding of the indicated multispecific proteins (BCA605 (FB6), BCA606 (FB6), BCA624 (FB6)) or control (BCA402, BCA403, hIgG) at the indicated concentrations to CD3 expressed on the surface of CD28eKO Jurkat cells as measured by flow cytometry. Figure 24CIt is a graph showing the binding of the indicated multispecific proteins (BCA605(FB6), BCA606(FB6), BCA624(FB6)) or control (BCA403) to CD3 at the indicated concentrations measured by ELISA.

[0100] Figure 25A It is a graph showing the percentage of cytotoxicity mediated by the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR), BCA605(FB6), BCA606(FB6), BCA624(FB6)) at the indicated concentrations in the OVCAR3-PBMC (1:10) co-culture assay. Figure 25B It is a graph showing the percentage of cytotoxicity mediated by the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR), BCA605(FB6), BCA606(FB6), BCA624(FB6)) at the indicated concentrations in the SCOV3-PBMC (1:10) co-culture assay.

[0101] Figure 26A It is a bar graph showing the release of IL-2 from the co-culture of tumor-PBMC with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or control (BCA429(MOR), hIgG, unstimulated co-culture (target + effector (T+E))) at the indicated concentrations. Figure 26B It is a bar graph showing the release of IL-6 from the co-culture of tumor-PBMC with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or control (BCA429(MOR), hIgG, unstimulated co-culture (target + effector (T+E))) at the indicated concentrations. Figure 26C It is a bar graph showing the release of IFNγ from the co-culture of tumor-PBMC with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or control (BCA429(MOR), hIgG, unstimulated co-culture (target + effector (T+E))) at the indicated concentrations. Figure 26D It is a bar graph showing the release of TNF from the co-culture of tumor-PBMC with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or control (BCA429(MOR), hIgG, unstimulated co-culture (target + effector (T+E))) at the indicated concentrations. Figure 26EBar graph showing IL-10 release from tumor-PBMC co-cultures with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or controls (BCA429(MOR), hIgG, unstimulated co-cultures (target + effector (T+E))) at the indicated concentrations. Figure 26F Bar graph showing IL-17 release from tumor-PBMC co-cultures with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or controls (BCA429(MOR), hIgG, unstimulated co-cultures (target + effector (T+E))) at the indicated concentrations. Figure 26G Bar graph showing IL-4 release from tumor-PBMC co-cultures with the indicated multispecific proteins (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or controls (BCA429(MOR), hIgG, unstimulated co-cultures (target + effector (T+E))) at the indicated concentrations.

[0102] Figure 27A Bar graph showing IFNγ release in PBMCs treated with the indicated multispecific antibodies (BCA605(MOR), BCA606(MOR), BCA624(MOR)) or controls (BCA403(MOR), hIgG) in soluble or tethered form. Data from two PBMC batches are shown. Figure 27B Bar graph showing IFNγ release in PBMCs treated with the indicated multispecific antibodies (BCA605(FB6), BCA606(FB6), BCA624(FB6)) or controls (BCA403(MOR), hIgG) in soluble or tethered form. Data from two PBMC batches are shown.

[0103] Figure 28 Graphical depiction of a control multispecific protein form referred to herein as BCA410.

[0104] Figure 29 Graphical representation of the overall structure of a naturally occurring full-length antibody. This structure is used for several control antibodies described herein, including, for example, BCA401 (anti-HER2 full-length antibody), BCA402 (anti-CD28 full-length antibody), BCA403 (anti-EGFR full-length antibody), BCA403 (αCD3ε full-length antibody), and cetuximab (anti-EGFR full-length antibody). 5. Detailed Description

[0105] T cell engager immunotherapies work by targeting T cells (or subsets thereof, such as CD8+ T cells) to tumors. For example, bispecific T cell engager single-chain antibodies (also known as BiTEs) are single-chain proteins that simultaneously bind an antigen on a tumor cell and an antigen expressed on the surface of a T cell, thereby activating the T cell and inducing tumor lysis. Although T cell engagers have shown promising efficacy in clinical assays, 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 (such as IL-6) triggered by T cell activation. T cell engager-mediated CRS particularly stems from peripheral toxicity - by activating peripheral T cells outside of the tumor microenvironment. In particular, the inventors have discovered novel multispecific protein forms that specifically bind hCD3, hTCSA (such as hCD28, hCD2), and hTAA and exhibit low to zero peripheral toxicity. Thus, the novel multispecific proteins described herein are good candidates for treating diseases (such as cancer). Accordingly, in particular, the present disclosure provides novel multispecific proteins for use in pharmaceutical compositions for treating diseases (such as cancer). 5.1 Definitions

[0106] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0107] 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 should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and do not limit any claimed subject matter.

[0108] In this application, unless otherwise expressly stated, the singular forms also include the plural forms. For example, in the specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" also include plural referents. Additionally, the use of the term "comprising" and other forms (such as "including," "containing," and "having") is not limiting.

[0109] It should be understood that when aspects are described herein using the term "comprising," similar aspects are also provided using the terms "consisting of" and "consisting essentially of."

[0110] The term "and / or" as used herein is to be understood as a specific disclosure of each of the two specified features or components (in the presence or absence of the other). Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" herein is intended to cover each of the following aspects: 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).

[0111] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range, and, where appropriate, fractions thereof (such as tenths and hundredths of an integer), unless otherwise specified.

[0112] The term "about" refers to a value or a composition that is within an acceptable error range determined by those skilled in the art for a particular value or composition, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measuring system. When a particular value or composition is provided in this application and the claims, the meaning of "about" should be assumed to be within the acceptable error range of that particular value or composition, unless otherwise stated.

[0113] Unless otherwise specified or the context clearly indicates, terms used herein similar to "first and second", "(a) and (b)", or "(i) and (ii)" do not denote order or direction, but are used to identify multiple components of a composition or method. When those skilled in the art can clearly understand from the context that these terms are intended to denote order or direction.

[0114] When a protein and / or polypeptide is described herein, it should be understood that polynucleotides encoding the protein or polypeptide (e.g., RNA (e.g., mRNA) or DNA polynucleotides) are also provided herein.

[0115] When a protein, polypeptide, polynucleotide, cell, expression vector, etc. is described herein, it should be understood that an isolated form of the protein, polypeptide, polynucleotide, cell, expression vector, etc. is also provided herein.

[0116] When a protein, polypeptide, polynucleotide, etc. is described herein, it should be understood that a recombinant form of the protein, polypeptide, polynucleotide, etc. is also provided herein.

[0117] When a polypeptide or group of polypeptides is described herein, it should be understood that a protein containing the polypeptide or group of polypeptides folded into its three-dimensional structure (i.e., tertiary or quaternary structure) is also provided herein, and vice versa.

[0118] 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 altered in a subject to produce a therapeutic agent in vitro) to a subject using a variety of methods and delivery systems known to those of skill in the art. Administration can be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0119] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to an immune mechanism that results in the lysis of target cells coated with an antibody (or a polypeptide or protein containing an Fc region) (e.g., an Ig Fc-containing fusion protein or polypeptide described herein) by immune effector cells (e.g., NK cells). As used herein, the terms "reduced ADCC" and the like refer to a decrease in the number of target cells lysed in the culture medium surrounding the target cells over a given time period at a given concentration of an antibody (or a polypeptide or protein containing an Fc region) (e.g., an Fc region-containing fusion protein or polypeptide described herein) by the mechanism of ADCC as defined above, and / or an increase in the concentration of an antibody (or a polypeptide or protein containing an Fc region) (e.g., an Fc region-containing fusion protein or polypeptide described herein) in the culture medium surrounding the target cells required to achieve lysis of a given number of target cells over a given time period by the mechanism of ADCC as defined above. Reduced ADCC is relative to ADCC mediated by the same antibody (or Fc region-containing polypeptide or protein) (e.g., an Fc region-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 not engineered (e.g., does not contain one or more amino acid modifications that mediate reduced ADCC, such as amino acid substitutions). For example, reduced ADCC mediated by an antibody (or Fc region-containing polypeptide or protein) (e.g., an Fc region-containing fusion protein or polypeptide described herein) that contains an amino acid substitution in its Fc region that reduces ADCC is relative to ADCC mediated by the same antibody (or Fc region-containing polypeptide or protein) (e.g., an Fc region-containing fusion protein or polypeptide described herein) that does not contain said amino acid substitution in its Fc region.

[0120] As used herein, the term "affinity" refers to the strength with which one protein (e.g., an antibody) binds to another protein (e.g., an antigen). The affinity of a protein is measured by the dissociation constant Kd, defined as [antibody] × [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 as 1 / Kd. Standard methods for measuring affinity are known to those of ordinary skill in the art. Exemplary methods for measuring affinity are described herein; see, for example, §5.2.5.

[0121] The term "antibody or antibodies" as used herein has the broadest meaning and encompasses various immunoglobulin (Ig) (e.g., human immunoglobulin (hIg)) structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific (e.g., bispecific, trispecific) antibodies, and antibody fragments (as long as they exhibit the desired antigen-binding activity) (i.e., antigen-binding fragments or variants). 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 scaffolds (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, intracellular antibodies, affibodies, diabodies, triabodies, 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-linked Fv (sdFv), Fc fusions (e.g., Fab-Fc, scFv-Fc, VHH-Fc, (scFv)2-Fc, (VHH)2-Fc) and antigen-binding fragments of any of the foregoing, as well as conjugates or fusion proteins comprising any of the foregoing. Antibodies can be of Ig isotypes (e.g., IgG, IgE, IgM, IgD, or IgA), any type of Ig (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ). In certain embodiments, the antibodies described herein are IgG antibodies, or a type (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 population of monoclonal or polyclonal antibodies. The antibodies described herein can be produced by any standard method known in the art, such as recombinantly in a host cell (see §5.3); or synthetically produced.

[0122] The term "antibody-like scaffold" as used herein refers to a non-Ig-based antigen-binding domain. Various antibody-like scaffolds are known in the art. Various antibody-like scaffolds are known in the art. For example, the 10th type III domain of fibronectin (e.g., ) and designed ankyrin repeat proteins (e.g., ) 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 scaffolds include, but are not limited to, lipocalins (see, for example, US7250297) (e.g., ), protein A-derived molecules such as the z-domain of protein A (see, for example, US5831012) (e.g., ), the A-domain of a membrane receptor stabilized by disulfide bonds and Ca2+ (see, for example, US7803907) (e.g., ), serum transferrin (see, for example, US2004023334) (e.g., ); designed ankyrin repeat proteins (see, for example, US7417130) (e.g. ), fibronectin (see, for example, US6818418) (e.g. ), C-type lectin domains (see, for example, US2004132094) (e.g. ); human γ-crystallin or ubiquitin (see, for example, US7838629) (e.g. ); the kunitz-type domain of human protease inhibitors (see, for example, US2004209243), C-type lectins (see, for example, US2004132094) (e.g. ), cysteine knots or knottins (see, for example, US7186524) (e.g. ), nucleic acid aptamers (see, for example, US5475096), thioredoxin A scaffolds (see, for example, US6004746) (peptide aptamers), the 10th type III domain of fibronectin (see, for example, US6818418) (e.g. ) and cystine dense peptides (see, e.g., WO2023023031). Other exemplary antibody-like scaffolds are known in the art, for example, as described 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 content of each of the foregoing references is incorporated herein by reference for all purposes. Antibody-like scaffolds include, for example, naturally occurring antigen-binding moieties, variants of naturally occurring antigen-binding moieties (e.g., functional variants), fragments of naturally occurring antigen-binding moieties (e.g., functional fragments), and synthetic antigen-binding moieties (i.e., non-naturally occurring antigen-binding moieties).

[0123] 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 an antigen. Exemplary antigen-binding domains include, but are not limited to, single-domain antibodies (e.g., VHH, (VHH)2), single-chain Fv (e.g., scFv; (scFv)2), Fab fragments (e.g., Fab, single-chain Fab (scFab), F(ab’)2), and disulfide-linked Fv (sdFv). An antigen-binding domain can be part of a larger polypeptide or protein (e.g., a full-length antibody, 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 is referred to using a target protein or polypeptide, the term "antigen" can be replaced with the name of the target protein or antigen. For example, an antigen-binding domain that specifically binds hCD3 can also be referred to herein as an "hCD3-binding domain".

[0124] The terms "cancer" and "tumor" are used interchangeably herein and refer to a large group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that can invade adjacent tissues and may also metastasize to distant parts of the body, such as through the lymphatic system or bloodstream.

[0125] As used herein, the term "CDR" or "complementary determining region" refers to the non - contiguous antigen - combining sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252, 6609 - 6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), the entire contents of which are incorporated herein by reference for all purposes. Unless otherwise specified, the term "CDR" is the CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609 - 6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991).

[0126] The terms "CH1" and "CH1 region" are used interchangeably herein and refer to the first constant region of an immunoglobulin heavy chain. An exemplary reference for the amino acid sequence of the hIgG1 CH1 region is shown in SEQ ID NO: 98; an exemplary reference for the amino acid sequence of the hIgG4 CH1 region is shown in SEQ ID NO: 111.

[0127] The terms "CH2" and "CH2 region" are used interchangeably herein and refer to the second constant region of an immunoglobulin heavy chain. An exemplary reference for the amino acid sequence of the hIgG1 CH2 region is shown in SEQ ID NO: 100; an exemplary reference for the amino acid sequence of the hIgG4 CH2 region is shown in SEQ ID NO: 113.

[0128] The terms "CH3" and "CH3 region" are used interchangeably herein and refer to the third constant region of an immunoglobulin heavy chain. An exemplary reference for the amino acid sequence of the hIgG1 CH3 region is shown in SEQ ID NO: 101; an exemplary reference for the amino acid sequence of the hIgG4 CH3 region is shown in SEQ ID NO: 113.

[0129] The terms "constant region" and "constant domain" are used interchangeably herein and refer to the carboxyl - terminal portion of the light and / or heavy chains of a full - length antibody that does not directly participate in antibody - antigen binding but can exhibit various effector functions, such as interaction with Ig Fc receptors (e.g., Fcγ receptors). The constant regions of immunoglobulin molecules generally have more conserved amino acid sequences than the immunoglobulin variable domains.

[0130] As used herein, the term "derived from", with respect to a polynucleotide, refers to a polynucleotide having at least 70% (e.g., at least 85%) sequence identity to a reference polynucleotide (e.g., a naturally occurring polynucleotide) or a fragment thereof. With respect to a polypeptide or protein, the term "derived from" refers to a polypeptide or protein comprising an amino acid sequence having at least 70% (e.g., at least 85%) sequence identity to the amino acid sequence of a reference polypeptide or protein (e.g., a naturally occurring polypeptide or protein). The term "derived from" as used herein does not refer to any particular process or method of obtaining the polynucleotide, polypeptide, or protein. For example, the polynucleotide, polypeptide, or protein may be recombinantly produced or chemically synthesized.

[0131] As used herein, the term "diagnosing" or "diagnosis" refers to determining the presence, absence, severity, or course of a disease (e.g., cancer). The term "diagnosis" encompasses the initial determination as well as subsequent determinations (e.g., monitoring) after the initial determination.

[0132] As used herein, the term "disease" refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, ailment, abnormality, pathology, condition, or syndrome in which physiological function is impaired, regardless of the nature of its cause.

[0133] The terms "hinge" or "hinge region" are used interchangeably herein and refer to the hinge region of an immunoglobulin heavy chain. An exemplary reference amino acid sequence of the hIgG1 hinge region is shown in SEQ ID NO: 99; an exemplary reference amino acid sequence of the hIgG4 hinge region is shown in SEQ ID NO: 112.

[0134] The terms "DNA" and "polydCxyribonucleotide" are used interchangeably herein and refer to a macromolecule comprising a plurality of deoxyribonucleotides polymerized by phosphodiester bonds. A deoxyribonucleotide refers to a nucleotide in which the sugar is deoxyribose.

[0135] When used in reference to an antibody, the term "effector function" refers to the biological activities attributed to the Fc region of the antibody, and thus these activities vary depending on the antibody isotype. Antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell 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.

[0136] As used herein, the term "EU numbering system" refers to the EU numbering convention for the constant regions of antibodies, as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78 - 85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th ed., 1991, the entire contents of which are incorporated herein by reference for all purposes.

[0137] The term "Fab" as used herein refers to an antigen - binding domain comprising a Fab heavy chain and a light chain, wherein the Fab heavy chain comprises a VH region and a CH1 region from the N - terminus to the C - terminus; the light chain comprises a VL region and a CL region from the N - terminus to the C - terminus; and wherein the Fab heavy chain and the light chain associate to form the antigen - binding domain.

[0138] 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.

[0139] The term "Fc region" as used herein refers to the C - terminal region of an hIg heavy chain, which comprises at least one CH2 region operably linked to a CH3 region from the N - terminus to the C - terminus. In some embodiments, the Fc region comprises an Ig hinge region or at least a portion of the Ig hinge region operably linked to the N - terminus of the CH2 region. In some embodiments, the Fc region is engineered relative to a reference Fc region (e.g., comprises one or more amino acid modifications), see, e.g., §5.2.7.1. More examples of proteins with engineered Fc regions can be found in Saunders 2019 (K.O. Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half - Life," 2019, Frontiers in Immunology, V.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 (e.g., C - terminal lysine; C - terminal glycine - lysine) relative to the wild - type CH3 region.

[0140] As used herein, the term "Fc-modified fusion protein or polypeptide" refers to a fusion polypeptide or protein comprising an Fc region, wherein the Fc region is modified relative to a reference Fc region (e.g., comprising one or more amino acid modifications (e.g., one or more amino acid substitutions, deletions, or additions)).

[0141] As used herein, the terms "first" and "second" with respect to Fc regions, etc. are for convenience in distinguishing cases where there is more than one of each type of moiety. Unless expressly stated, use of these terms is not intended to confer a particular order or orientation on the fusion protein.

[0142] 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 CDR region (e.g., using the Kabat definition of CDRs).

[0143] 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 comprises: (i) a first Ig light chain that, from the N-terminus to the C-terminus, comprises a variable light chain (VL) region and a constant light chain (CL) region; (ii) a first Ig heavy chain that, from the N-terminus to the C-terminus, comprises a variable heavy chain (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second Ig heavy chain that, from the N-terminus to the C-terminus, comprises a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iv) a second Ig light chain that, from the N-terminus to the C-terminus, comprises 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; wherein the second light chain and the second heavy chain associate to form a second antigen-binding domain; and wherein the first heavy chain and the second heavy chain associate to form a dimer. In some embodiments, the two heavy chains comprise substantially the same amino acid sequence; and the two light chains comprise substantially the same amino acid sequence. In some embodiments, the two heavy chains comprise substantially the same amino acid sequence, except for one or more amino acid modifications (e.g., as described herein) that facilitate proper heavy chain heterodimerization; and the two light chains comprise substantially the same amino acid sequence. The antibody chains may be substantially the same, but are not identical if they differ due to post-translational modifications (e.g., C-terminal cleavage of lysine residues, alternative glycosylation patterns, etc.).

[0144] As used herein, the term "functional variant" with respect to a polypeptide or protein refers to a polypeptide or protein that contains at least one but no more than 15%, no more than 12%, no more than 10%, no more than 8% amino acid variations (e.g., substitutions, deletions, additions) compared to the amino acid sequence of a reference polypeptide or protein, wherein the polypeptide or protein retains at least one specific function of the reference polypeptide or protein. A functional variant of a protein need not retain all functions of the reference polypeptide or protein (e.g., wild-type). In some cases, one or more functions are selectively reduced or eliminated. In some embodiments, the reference polypeptide or protein is a wild-type protein.

[0145] As used herein, the term "functional fragment" with respect 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 need not retain all functions of the reference polypeptide or protein. In some cases, one or more functions are selectively reduced or eliminated. In some embodiments, the reference polypeptide or protein is a wild-type protein.

[0146] As used herein, the term "fusion" and its grammatical equivalents refer to the operable linkage of at least a first polypeptide with a second polypeptide, wherein the first and second polypeptides are not naturally operably linked together. For example, the first and second polypeptides are derived from different proteins. The term "fusion" encompasses the direct linkage of at least two polypeptides through a peptide bond, as well as the indirect linkage through a linker (e.g., a peptide linker).

[0147] As used herein, the term "fusion protein" and its grammatical equivalents refer to a protein that contains at least one polypeptide operably linked to another polypeptide, wherein the first and second polypeptides are different and not operably linked together in nature. For example, the first and second polypeptides of a fusion protein are each derived from different proteins. At least two polypeptides of a fusion protein can be directly operably linked through a peptide bond; or can be indirectly operably linked through a linker (e.g., a peptide linker). Thus, for example, the term "fusion polypeptide" encompasses embodiments in which polypeptide A is directly operably linked 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).

[0148] As used herein, the term "heavy chain" refers to a portion of an immunoglobulin (e.g., a human immunoglobulin), typically comprising a heavy chain variable region (VH), a CH1 region, a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. The constant region of the heavy chain (i.e., the CH1 region, the hinge region, the CH2 region, and the CH3 region) can be any of the different isotypes, e.g., human alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence of the constant domain, giving rise to human antibodies of the hIgA, hIgD, IgE, hIgG, and hIgM classes, including the 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 of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence of the constant domain, giving rise to antibodies of the human IgA, IgD, IgE, IgG, and IgM classes, including the subclasses of human IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0149] As used herein, the term "half-life extending moiety" refers to a moiety (e.g., a small molecule, a polypeptide, a polynucleotide, a carbohydrate, a lipid, a synthetic polymer (e.g., a PEG polymer), etc.) that, when conjugated or otherwise operably linked (e.g., fused) to a polypeptide or protein (the polypeptide or protein of the present invention), increases the in vivo half-life of the polypeptide or protein of the present invention when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the polypeptide or protein can be evaluated using in vitro and in vivo models known in the art.

[0150] 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 (the polypeptide or protein of the present invention), increases the in vivo half-life of the polypeptide of the present invention when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the polypeptide or protein can be evaluated using in vitro and in vivo models known in the art.

[0151] As used herein, the term "heterologous" when used to describe a first element in relation to a second element means that the first and second elements do not exist in nature in the manner described. For example, a polypeptide comprising a "heterologous moiety" means a polypeptide that is linked to a moiety that is not linked to the polypeptide in nature (e.g., a small molecule, a polypeptide, a polynucleotide, a carbohydrate, a lipid, a synthetic polymer (e.g., a PEG polymer), etc.). For example, a non-limiting example of a heterologous moiety is a heterologous polypeptide (as defined herein).

[0152] As used herein, the term "heterologous signal peptide" refers to a signal peptide that is not operably linked to a target polypeptide or protein in nature. For example, with respect to a polypeptide comprising a signal peptide from human IL-2 (hIL-2) operably linked to hIL-12p40, the hIL-2 signal peptide constitutes a heterologous signal peptide.

[0153] As used herein, the term "homologous signal peptide" refers to a signal peptide that is operably linked to a target polypeptide or protein in nature. For example, with respect to a polypeptide comprising a human IL-2 signal peptide operably linked to hIL-2, the hIL-2 signal peptide constitutes a homologous signal peptide.

[0154] As used herein, the term "human T cell co-stimulatory 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 enhances T cell activation when bound to its cognate ligand. Exemplary hTCSAs are described herein, including, for example, hCD28, hCD2, and h41BB.

[0155] 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 co-stimulatory signals required for T cell activation and survival. An exemplary reference to the amino acid sequence of the mature hCD28 protein is shown as SEQ ID NO: 2.

[0156] 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 co-stimulatory signals. An exemplary reference to the amino acid sequence of the mature hCD2 protein is shown as SEQ ID NO: 38.

[0157] As used herein, the term "human CD3" or "hCD3" refers to the human CD3 protein complex. The human CD3 protein complex comprises four different proteins, namely hCD3ε, hCD3ζ, hCD3γ and hCD3δ. Accordingly, the term "CD3" as used herein encompasses hCD3ε, hCD3ζ, hCD3γ and hCD3δ. When referring to a specific hCD3 protein in the CD3 complex, that specific protein (hCD3ε, hCD3ζ, hCD3γ or hCD3δ) will be specifically indicated. For example, when referring to an "antigen-binding domain that specifically binds hCD3" and the like, it encompasses an antigen-binding domain that specifically binds any one of hCD3ε, hCD3ζ, hCD3γ or hCD3δ. An "antigen-binding domain that specifically binds hCD3ε" and the like refers to an antigen-binding domain that specifically binds hCD3ε but does not specifically bind hCD3ζ, hCD3γ or hCD3δ. An exemplary reference to the amino acid sequence of the mature hCD3ε protein is shown as SEQ ID NO: 22. An exemplary reference to the amino acid sequence of the mature hCD3δ protein is shown as SEQ ID NO: 28. An exemplary reference to the amino acid sequence of the mature hCD3γ protein is shown as SEQ ID NO: 26. An exemplary reference to the amino acid sequence of the mature hCD3ζ protein is shown as SEQ ID NO: 24.

[0158] As used herein, the term "human tumor-associated antigen" or "hTAA" refers to a protein expressed on the surface of human cancer cells that permits recruitment of the multispecific proteins described herein to human cancer cells. In some embodiments, the tumor-associated antigen is expressed by normal cells and cancer cells. In some embodiments, the tumor-associated antigen is overexpressed in cancer cells relative to normal cells, e.g., overexpressed 1-fold, 2-fold, 3-fold or more compared to normal cells. In some embodiments, the tumor-associated antigen is inappropriately synthesized in cancer cells, e.g., the protein contains amino acid modifications (e.g., amino acid deletions, additions and / or substitutions) compared to the protein expressed by normal cells. In some embodiments, the tumor-associated antigen is expressed only by cancer cells and is not detectable at a detectable level in normal cells. Methods for identifying and validating tumor-associated proteins are known to those of skill in the art and have been described in the literature (see, e.g., Bornstein, AAPS J. (2015), vol. 17(3), p. 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).

[0159] As used herein, the term "hIgM CH2 mFab" refers to a modified Fab comprising an hIgM CH2 mFab heavy chain and an 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.

[0160] As used herein, the term "hIgM CH2 mFab heavy chain" comprises a VH region and an hIgM CH2 region from the N-terminus to the C-terminus. The amino acid sequence of an exemplary hIgM CH2 region is shown in SEQ ID NO: 126.

[0161] As used herein, the term "hIgM CH2 mFab light chain" comprises a VL region and an hIgM CH2 region from the N-terminus to the C-terminus. The amino acid sequence of an exemplary hIgM CH2 region is shown in SEQ ID NO: 126.

[0162] As used herein, the term "isolated", with respect to a polypeptide, protein, polynucleotide, vector (and the like), refers to a polypeptide, protein, polynucleotide, vector (and the like) that is substantially free of other cellular components that are associated with it in its natural state.

[0163] As used herein, the term "light chain" refers to a portion of an immunoglobulin (e.g., a human immunoglobulin) that comprises a light chain variable region (VL) from the N-terminus to the C-terminus, which is operably linked to a light chain constant region (CL). The CL can be any different 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.

[0164] As used herein, the term "translatable RNA" refers to any RNA that encodes at least one peptide or protein and is capable of being 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.

[0165] As used herein, the term "modified", with respect to a polynucleotide, refers to a polynucleotide that contains at least one nucleotide substitution, alteration, inversion, addition, or deletion (e.g., one or more amino acid substitutions) compared to a reference polynucleotide. Modifications can include the insertion of non-naturally occurring nucleotide residues. As used herein, the term "modified", with respect to an amino acid sequence, refers to an amino acid sequence that contains at least one amino acid residue substitution, alteration, inversion, addition, or deletion compared to a reference amino acid sequence. Modifications can include the insertion of non-naturally occurring amino acid residues. Naturally occurring amino acid derivatives are not considered modified amino acids when determining the percent identity of two amino acid sequences. For example, the natural modification of a glutamic acid amino acid residue to a pyroglutamic acid amino acid residue is not considered an amino acid modification when determining the percent identity of two amino acid sequences. Additionally, for example, the naturally occurring modification of a glutamic acid amino acid residue to a pyroglutamic acid amino acid residue is not considered an amino acid "modification" as defined herein.

[0166] A "modification that promotes heterodimerization of a first Fc region and a second Fc region" (or similar phrase) is an operation on the peptide backbone or a post-translational modification of the Fc region that reduces or prevents the association of a polypeptide containing the Fc region with the same polypeptide to form a homodimer. Modifications that promote association as used herein particularly include modifications made to two Fc regions that are desired to associate (i.e., a first Fc region and a second Fc region), where the modifications are complementary to each other to promote the association of the two Fc regions. For example, a modification that promotes association can alter the structure or charge of one or both Fc regions such that their association is respectively spatially or electrostatically favorable. Thus, heterodimerization occurs between a polypeptide containing a first Fc region and a polypeptide containing a second Fc region, which may be different because the other components (e.g., antigen-binding domains) fused to each Fc region are different. In some embodiments, a modification that promotes association includes an amino acid mutation in the Fc region, specifically an amino acid substitution. In a particular embodiment, a modification that promotes association includes individual amino acid mutations in each of the first Fc region and the second Fc region, specifically one or more amino acid substitutions. See, e.g., §5.2.7.2.

[0167] The term "moiety" as used herein is generally used to describe any macromolecule or small molecule that can 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, synthetic polymers (e.g., PEG polymers).

[0168] As used herein, the term "multispecific" with respect to a protein or polypeptide (e.g., a protein or polypeptide described herein) refers to a protein or polypeptide that comprises at least two antigen-binding domains, wherein the at least two antigen-binding domains bind to different antigens. Thus, the term "multispecific" includes, for example, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and the like. In some embodiments, the multispecific antibody is trispecific. In some embodiments, the multispecific antibody is trispecific and tetravalent. In some embodiments, the multispecific antibody is trispecific and trivalent.

[0169] As used herein, the term "operably linked" refers to two components (e.g., two polypeptides or two polynucleotides) being linked in a functional relationship. For example, a polypeptide is operably linked to another polypeptide when they are linked in-frame (either directly or indirectly through a peptide linker) such that both polypeptides are functional (e.g., a fusion protein or polypeptide as described herein). Or, for example, a transcriptional regulatory polynucleotide (e.g., a promoter, enhancer, or other expression regulatory element) is operably linked to a polynucleotide encoding a protein when the transcriptional regulatory polynucleotide affects the transcription of the polynucleotide encoding the protein. The term "operably linked" can also refer to coupling a component to, for example, a polynucleotide or polypeptide (e.g., coupling a PEG polymer to a protein or polypeptide).

[0170] A mathematical algorithm can be used to determine the “percent identity” between two sequences, such as peptides or proteins (amino acid sequences) or polynucleotides (nucleic acid sequences). A specific, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87:2264-2268, which was modified in Karlin S & Altschul SF (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., score = 50, wordlength = 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, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used for iterative searches that detect distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the website ncbi.nlm.nih.gov of the National Center for Biotechnology Information (NCBI)). Another specific, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the 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, whether or not gaps are allowed. When calculating the percent identity, generally only exact matches are counted.

[0171] As used herein, the term "pharmaceutical composition" means a composition suitable for administration to an animal (e.g., a human subject) that comprises a therapeutic agent and a pharmaceutically acceptable carrier or diluent. "Pharmaceutically acceptable carrier or diluent" means a substance for contact with the tissues of humans and / or non-human animals that is not unduly toxic, irritating, allergic, or otherwise problematic or complication-causing, and that has a reasonable therapeutic benefit / risk ratio.

[0172] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of DNA or RNA. The nucleic acid molecule can be single-stranded or double-stranded; contain natural, unnatural, or modified nucleotides; and contain natural, unnatural, or modified internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, rather than the phosphodiester linkages between the nucleotides of an unmodified nucleic acid molecule. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules obtained by any available method in the art, including but not limited to recombinant methods, such as cloning nucleic acid molecules from recombinant libraries or cell genomes, using conventional cloning techniques and polymerase chain reaction, etc., and by synthetic methods. Those skilled in the art will understand that, unless otherwise stated, the nucleic acid sequences described in this application will refer to thymine (T) in the representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), thymine (T) will be replaced by uracil (U). Thus, any RNA polynucleotide encoded by a DNA identified by a specific sequence identifier may also contain the corresponding RNA (e.g., mRNA) sequence encoded by that DNA, where each thymine (T) of the DNA sequence is replaced by uracil (U).

[0173] As used herein, the term "polypeptide" refers to a polymer of at least 2 (e.g., at least 5) amino acids linked by peptide bonds. The term "polypeptide" does not denote a specific length of the amino acid polymer chain. In the art, shorter amino acid polymers (e.g., about 2 - 50 amino acids) are commonly referred to as peptides; longer amino acid polymers (e.g., about more than 50 amino acids) are referred to as polypeptides. However, the terms "peptide" and "polypeptide" are used interchangeably herein.

[0174] As used herein, the term "protein" refers to a polypeptide or a group (i.e., at least two) of polypeptides. In embodiments where the protein comprises a group of polypeptides, the group of polypeptides associates to form a functional unit (i.e., quaternary structure). In some embodiments, the polypeptide or group of polypeptides folds into its three-dimensional structure (i.e., tertiary or quaternary structure). When a polypeptide or group of polypeptides is covered herein, it should be understood that proteins comprising a polypeptide or group of polypeptides folded into its three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein, and vice versa.

[0175] "Prophylactic" treatment refers to treatment administered to a subject who does not exhibit signs of a disease or only exhibits early signs, with the aim of reducing the risk of developing a pathology.

[0176] The terms "RNA" and "polynucleotide" are used interchangeably herein and refer to a macromolecule comprising multiple ribonucleotides polymerized by phosphodiester bonds. A ribonucleotide is a nucleotide in which the sugar is ribose. RNA can contain modified nucleotides; and comprises natural, unnatural, or altered internucleotide linkages, such as phosphoramidate linkages or thiophosphate linkages, rather than the phosphodiester bonds between nucleotides of an unmodified nucleic acid molecule.

[0177] The term "scFv" or "single-chain variable fragment" refers to an antibody comprising a VH region operably linked to a VL region by a peptide linker, and wherein the VH region and the VL region associate to specifically bind an antigen (e.g., form an antigen-binding domain). In some embodiments, the scFv comprises, from the N-terminus to the C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, the scFv comprises, from the N-terminus to the C-terminus, a VL region, a peptide linker, and a VH region. In some embodiments, the scFv further comprises one or more engineered disulfide bonds that link the VH region and the VL region.

[0178] The term "(scFv)2" as used herein refers to an antibody comprising a first and a second scFv operably linked (e.g., by a peptide linker). The first and second scFvs can specifically bind the same or different antigens. In some embodiments, the first and second scFvs are operably linked by a peptide linker.

[0179] The term "scFv-Fc" as used herein refers to an antibody comprising a scFv operably linked (e.g., by a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, the scFv is operably linked only to the first Fc domain of a first and a second Fc domain pair. In some embodiments, a first scFv is operably linked to a first Fc domain, while a second scFv is operably linked to the second Fc domain of a first and a second Fc domain pair.

[0180] The term "(scFv)2-Fc" as used herein refers to a (scFv)2 operably linked (e.g., by a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, the (scFv)2 is operably linked only to the first Fc domain of a first and a second Fc domain pair. In some embodiments, a first (scFv)2 is operably linked to a first Fc domain, while a second (scFv)2 is operably linked to the second Fc domain of a first and a second Fc domain pair.

[0181] As used herein, the term "single-domain antibody" or "sdAb" refers to an antibody having a single monomeric variable antibody domain. An sdAb is capable of specifically binding to a particular antigen. A VHH (as defined herein) is an example of an sdAb.

[0182] As used herein, the term "signal peptide" or "signal sequence" refers to a sequence (e.g., an amino acid sequence) that is capable of directing the trafficking or localization of a protein to a particular organelle, cellular compartment, or extracellular export. The term encompasses signal peptides and the nucleic acid sequences encoding such signal peptides. Thus, in the context of nucleic acids, reference to a signal peptide refers to the nucleic acid sequence encoding such signal peptide.

[0183] As used herein, the term "specifically binds" refers to a preferential interaction between a first moiety (e.g., a protein (e.g., a ligand)) and a second moiety (e.g., a protein (e.g., the cognate receptor of the ligand)) relative to other moieties (e.g., amino acid sequences), i.e., a significantly higher binding affinity. For example, herein, when a first protein or polypeptide is described as "specifically binding" 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 is specifically recognized by the first protein or polypeptide. The term "specifically binds" includes molecules that cross-react with the same epitope from different species. For example, an antibody that specifically binds human CD28 may cross-react with CD28 from other species (e.g., cynomolgus monkey, mouse, etc.), but is still considered herein to specifically bind human CD28. A moiety (e.g., a protein) can specifically bind to multiple moieties (e.g., proteins (e.g., epitopes)).

[0184] As used herein, the term "subject" includes any animal, such as a human or other animal. In some embodiments, the subject is a vertebrate (e.g., a mammal, bird, fish, reptile, or 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, ape), ungulate (e.g., a cow, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horse, donkey), carnivore (e.g., a dog, cat), rodent (e.g., a rat, mouse), or lagomorph (e.g., a rabbit). In some embodiments, the subject is a bird, such as a member of an avian taxon: Galliformes (e.g., a chicken, turkey, pheasant, quail), Anseriformes (e.g., a duck, goose), Palaeognathae (e.g., an ostrich, emu), Columbiformes (e.g., a pigeon, dove), or Psittaciformes (e.g., a parrot).

[0185] The term "therapeutically effective amount" of an agent (e.g., a therapeutic agent) as used herein refers to any amount of an agent (e.g., a therapeutic agent) that, when used alone or in combination with other agents (e.g., therapeutic agents), protects a subject against the onset of a disease or promotes the regression of a disease, as evidenced by a decrease in the severity of the disease or infection symptoms, an increase in the frequency and duration of the asymptomatic period of the disease or infection, or the prevention of damage or disability caused by the disease or infection. The ability of an agent (e.g., a therapeutic agent) to promote the regression of a disease can be evaluated using a variety of methods known to a skilled practitioner, such as in human subjects during a clinical assay, in an animal model system predictive of human efficacy, or by assaying the activity of the agent in an in vitro assay.

[0186] The terms "treat", "treating", "treatment" and like terms as used herein refer to alleviating or ameliorating a disease and / or related symptoms, or obtaining a desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treating a disease does not necessarily require complete elimination of the disease or related symptoms. In some embodiments, the effect is therapeutic, i.e., but not limited to, the effect partially or completely alleviates, attenuates, eliminates, slows down, relieves, reduces the intensity of the disease and / or adverse symptoms caused by the disease or cures the disease and / or adverse symptoms caused by the disease. In some embodiments, the effect is prophylactic, i.e., the effect protects or prevents the occurrence or recurrence of a disease. To this end, the methods of the present disclosure include administering a therapeutically effective amount of the composition described herein.

[0187] The term "variable region" as used herein refers to a portion of an antibody, typically a portion of a light or heavy chain, typically about 110 to 120 amino acids or 110 to 125 amino acids at the amino terminus of a mature heavy chain, and about 90 to 115 amino acids in a mature light chain, which vary widely in sequence between antibodies and are used for the binding and specificity of a particular antibody to its particular antigen. The variability of the sequence is concentrated in those regions called complementarity determining regions (CDRs), while the more conserved regions in the variable domain are called framework regions (FRs). Without being bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains primarily determine the interaction and specificity of the antibody with the 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 a particular embodiment, 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 (e.g., non-human primate) framework regions (FRs).

[0188] The terms "VH" and "VH region" are used interchangeably and refer to the variable region of an immunoglobulin heavy chain. The VH region can be incorporated into antibodies such as scFv, Fab, full-length antibodies. For example, an scFv comprises a VH region operably linked via a peptide linker to a VL region.

[0189] The terms "VL" and "VL region" are used interchangeably and refer to the variable region of an immunoglobulin light chain. The VL region can be incorporated into antibodies such as scFv, Fab, full-length antibodies. For example, an scFv comprises a VL region operably linked via a peptide linker to a VH region.

[0190] As used herein, the term "VHH" refers to a type of single domain antibody (sdAb) having a single monomeric heavy chain variable antibody domain (VH). Such antibodies can be found or produced or synthetically generated in camelid mammals that are naturally lacking in light chains, such as camels, llamas. 5.2 Multispecific proteins

[0191] 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 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 forms: the BCA405 form (see, e.g., Figure 1 ), the BCA406 form (see, e.g., Figure 2 ), the BCA424 form (see, e.g., Figure 3 ), or the BCA418 form (see, e.g., Figure 4 ), as further described below. 5.2.1 Multispecific protein forms 5.2.1.1 BCA405 form

[0192] In one aspect, provided herein is a multispecific protein shown in the BCA405 form (see, e.g., Figure 1 ). Generally, the BCA405 form provides a multispecific protein that comprises: (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 the first heavy chain of the full-length antibody, wherein the first scFv specifically binds hCD3; and a second scFv operably linked to the C-terminus of the second heavy chain of the full-length antibody, wherein the second scFv specifically binds hTCSA (e.g., hCD28, hCD2).

[0193] In some embodiments, the multispecific protein comprises (a) a full-length antibody comprising: (i) a first light chain comprising, from the N-terminus to the C-terminus, a variable light chain (VL) region and a constant light chain (CL) region; (ii) a first heavy chain comprising, from the N-terminus to the C-terminus, a variable heavy chain (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second heavy chain comprising, from the N-terminus to the 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 the N-terminus to the 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; wherein the second light chain and the second heavy chain associate to form a second antigen-binding domain; and wherein the first heavy chain and the second heavy chain associate to form a dimer; (b) a first scFv operably linked to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody, wherein the first scFv comprises, 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; and (c) a second scFv operably linked to the C-terminus of the CH3 region of the second heavy chain of the full-length antibody, wherein the second scFv comprises, 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; wherein the first antigen-binding domain of the full-length antibody specifically binds to a first hTAA; wherein the second antigen-binding domain of the full-length antibody specifically binds to a second hTAA; wherein the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); and wherein the second scFv specifically binds to hCD3.

[0194] In some embodiments, the first scFv is operably linked directly by a peptide bond to the C-terminus of the CH3 of the first heavy chain. In some embodiments, the first scFv is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the C-terminus of the CH3 of the first heavy chain. In some embodiments, the second scFv is operably linked directly by a peptide bond to the C-terminus of the CH3 of the second heavy chain. In some embodiments, the second scFv is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the C-terminus of the CH3 of the second heavy chain. In some embodiments, the first scFv is operably linked directly by a peptide bond to the C-terminus of the CH3 of the first heavy chain; and the second scFv is operably linked directly by a peptide bond to the C-terminus of the CH3 of the second heavy chain. In some embodiments, the first scFv is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the C-terminus of the CH3 of the first heavy chain; and the second scFv is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the C-terminus of the CH3 of the second heavy chain. In some embodiments, the amino acid sequence of the peptide linker that operably links the first scFv to the C-terminus of the CH3 region of the first heavy chain 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 of the peptide linker that operably links the second scFv to the C-terminus of the CH3 region of the second heavy chain. In some embodiments, the amino acid sequence of the peptide linker that operably links the first scFv to the C-terminus of the CH3 region of the first heavy chain is 100% identical to the amino acid sequence of the peptide linker that operably links the second scFv to the C-terminus of the CH3 region of the second heavy chain.

[0195] In some embodiments, the first scFv is operably linked to the C-terminus of the CH3 domain of the first heavy chain (either directly or indirectly via a peptide linker) through the VH region of the first scFv. In some embodiments, the first scFv is operably linked to the C-terminus of the CH3 domain of the first heavy chain (either directly or indirectly via a peptide linker) through the VL region of the first scFv. In some embodiments, the second scFv is operably linked to the C-terminus of the CH3 domain of the second heavy chain (either directly or indirectly via a peptide linker) through the VH region of the second scFv. In some embodiments, the second scFv is operably linked to the C-terminus of the CH3 domain of the second heavy chain (either directly or indirectly via a peptide linker) through the VL region of the second scFv. In some embodiments, the first scFv is operably linked to the C-terminus of the CH3 domain of the first heavy chain (either directly or indirectly via a peptide linker) through the VH region of the first scFv; and the second scFv is operably linked to the C-terminus of the CH3 domain of the second heavy chain (either directly or indirectly via a peptide linker) through the VH region of the second scFv. In some embodiments, the first scFv is operably linked to the C-terminus of the CH3 domain of the first heavy chain (either directly or indirectly via a peptide linker) through the VL region of the first scFv; and the second scFv is operably linked to the C-terminus of the CH3 domain of the second heavy chain (either directly or indirectly via a peptide linker) through the VL region of the second scFv.

[0196] 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 are expressed by the same cancer cell. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds 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 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 the same hTAA as the second antigen-binding domain of the full-length antibody, but binds different epitopes. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds the same hTAA as the second antigen-binding domain of the full-length antibody, but binds different and non-overlapping epitopes (e.g., the full-length antibody is bispecific). In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds a different hTAA from the second antigen-binding domain of the full-length antibody.

[0197] In some embodiments, the multispecific protein comprises (a) a first polypeptide comprising a first light chain that, from N-terminus to C-terminus, comprises a VL region and a CL region; (b) a second polypeptide that, from N-terminus to C-terminus, comprises: (i) a first heavy chain that, from N-terminus to C-terminus, comprises 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 that, from N-terminus to C-terminus, comprises (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; (c) a third polypeptide that, from N-terminus to C-terminus, comprises: (i) a second heavy chain that, from N-terminus to C-terminus, comprises 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 second scFv that, from N-terminus to C-terminus, comprises (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 a second light chain that, from N-terminus to C-terminus, comprises a VL region and a CL region; wherein the VL of the first light chain associates with the VH of the first heavy chain to form a first antigen-binding domain that specifically binds a first hTAA; wherein the VH of the second heavy chain associates with the VL of the second light chain to form a second antigen-binding domain that specifically binds a second hTAA; wherein the first scFv specifically binds hTCSA (e.g., hCD28, hCD2); and wherein the second scFv specifically binds hCD3.

[0198] In some embodiments, the second polypeptide comprises, from the N-terminus to the C-terminus: (i) a first heavy chain that comprises, from the N-terminus to the 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 that comprises, from the N-terminus to the 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 the N-terminus to the C-terminus: (i) a second heavy chain that comprises, from the N-terminus to the 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 that comprises, from the N-terminus to the 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 the N-terminus to the C-terminus: (i) a first heavy chain that comprises, from the N-terminus to the 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 that comprises, from the N-terminus to the 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 the N-terminus to the C-terminus: (i) a second heavy chain that comprises, from the N-terminus to the 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 that comprises, from the N-terminus to the 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 sequence of the first peptide linker 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 of the second peptide linker. In some embodiments, the amino acid sequence of the first peptide linker is 100% identical to the amino acid sequence of the second peptide linker.

[0199] In some embodiments, the first scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus. In some embodiments, the second scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the second scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus; and the second scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus; and the second scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus.

[0200] 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 are expressed by the same cancer cell. In some embodiments, the first antigen-binding domain specifically binds the same hTAA as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds the same epitope as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds the same hTAA as the second antigen-binding domain but binds different epitopes. In some embodiments, the first antigen-binding domain specifically binds the same hTAA as the second antigen-binding domain but binds different and non-overlapping epitopes. In some embodiments, the first antigen-binding domain specifically binds a different hTAA from the second antigen-binding domain.

[0201] Generally, the BCA405 format can also be described as a multispecific protein comprising: (a) a first polypeptide comprising a first light chain that, from the N-terminus to the C-terminus, comprises a VL region and a CL region; (b) a second polypeptide that, from the N-terminus to the C-terminus, comprises: (i) a first heavy chain that, from the N-terminus to the C-terminus, comprises a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (ii) an optional first peptide linker (e.g., the linkers described herein) (see, e.g., §5.2.8); and (iii) a first scFv that, from the N-terminus to the C-terminus, comprises (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; (c) a third polypeptide that, from the N-terminus to the C-terminus, comprises: (i) a second heavy chain that, from the N-terminus to the C-terminus, comprises a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region, (ii) an optional second peptide linker (e.g., the linkers described herein) (see, e.g., §5.2.8); and (iii) a second scFv that, from the N-terminus to the C-terminus, comprises (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 a second light chain that, from the N-terminus to the C-terminus, comprises 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 a first hTAA; wherein the VH of the second heavy chain associates with the VL of the second light chain to form a second antigen-binding domain that specifically binds a second hTAA; wherein the first scFv specifically binds hTCSA (e.g., hCD28, hCD2); wherein the second scFv specifically binds hCD3; and wherein the CH3 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (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 (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein 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; wherein 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;wherein the CH2 region of the first heavy chain comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein the CH2 region of the second heavy chain comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein 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 reduce or eliminate one or more of the following heavy chain effector functions relative to a reference heavy chain that does not comprise the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 domain, e.g., SEQ ID NO: 100): ADCC, CDC, and / or binding affinity for 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))). 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 are expressed by the same cancer cell. 5.2.1.2 BCA406 form

[0202] In one aspect, provided herein is a multispecific protein in the BCA406 form (see, e.g., Figure 2 ). Generally, the BCA406 form provides a multispecific protein that comprises: (i) a full-length antibody that specifically binds a first hTAA and a second hTAA; (ii) a first scFv operably linked to the N-terminus of the first heavy chain or the first light chain of the full-length antibody, wherein the first scFv specifically binds hCD3; and a second scFv operably linked to the N-terminus of the second heavy chain or the second light chain of the full-length antibody, wherein the second scFv specifically binds hTCSA (e.g., hCD28, hCD2).

[0203] In some embodiments, the multispecific protein comprises (a) a full-length antibody comprising: (i) a first light chain comprising a VL region and a CL region from the N-terminus to the C-terminus; (ii) a first heavy chain comprising a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; (iii) a second heavy chain comprising a VH region, a CH1 region, a hinge region, a CH2 region, and an sCH3 region from the N-terminus to the C-terminus; (iv) a second light chain comprising a VL region and a VH region from the N-terminus to the C-terminus; wherein the first light chain and the first heavy chain associate to form a first antigen-binding domain; wherein the second light chain and the second heavy chain associate to form a second antigen-binding domain; and wherein the first heavy chain and the second heavy chain associate to form a dimer; (b) a first scFv operably linked to the N-terminus of the first heavy chain of the full-length antibody, wherein the first scFv comprises, 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; and (c) a second scFv operably linked to the N-terminus of the second heavy chain of the full-length antibody, wherein the second scFv comprises, 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; wherein the first antigen-binding domain of the full-length antibody specifically binds a first hTAA; wherein the second antigen-binding domain of the full-length antibody specifically binds a second hTAA; wherein the first scFv specifically binds hTCSA (e.g., hCD28, hCD2); and wherein the second scFv specifically binds hCD3.

[0204] In some embodiments, the first scFv is operably linked directly by a peptide bond to the N-terminus of the first heavy chain of the full-length antibody. In some embodiments, the first scFv is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the N-terminus of the first heavy chain of the full-length antibody. In some embodiments, the second scFv is operably linked directly by a peptide bond to the N-terminus of the second heavy chain of the full-length antibody. In some embodiments, the second scFv is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the N-terminus of the second heavy chain of the full-length antibody. In some embodiments, the first scFv is operably linked directly by a peptide bond to the N-terminus of the first heavy chain of the full-length antibody; and the second scFv is operably linked directly by a peptide bond to the N-terminus of the second heavy chain of the full-length antibody. In some embodiments, the first scFv is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the N-terminus of the first heavy chain of the full-length antibody; and the second scFv is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the N-terminus of the second heavy chain of the full-length antibody. In some embodiments, the amino acid sequence of the peptide linker that operably links the first scFv to the N-terminus of the first heavy chain of the full-length antibody 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 of the peptide linker that operably links the second scFv to the N-terminus of the second heavy chain of the full-length antibody. In some embodiments, the amino acid sequence of the peptide linker that operably links the first scFv to the N-terminus of the first heavy chain of the full-length antibody is 100% identical to the amino acid sequence of the peptide linker that operably links the second scFv to the N-terminus of the second heavy chain of the full-length antibody.

[0205] In some embodiments, the first scFv is operably linked to the N-terminus of the first heavy chain of the full-length antibody via the VH region of the first scFv (either directly or indirectly via a peptide linker). In some embodiments, the first scFv is operably linked to the N-terminus of the first heavy chain of the full-length antibody via the VL region of the first scFv (either directly or indirectly via a peptide linker). In some embodiments, the second scFv is operably linked to the N-terminus of the second heavy chain of the full-length antibody via the VH region of the second scFv (either directly or indirectly via a peptide linker). In some embodiments, the second scFv is operably linked to the N-terminus of the second heavy chain of the full-length antibody via the VL region of the second scFv (either directly or indirectly via a peptide linker). In some embodiments, the first scFv is operably linked to the N-terminus of the first heavy chain of the full-length antibody via the VH region of the first scFv (either directly or indirectly 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 the VH region of the second scFv (either directly or indirectly via a peptide linker). In some embodiments, the first scFv is operably linked to the N-terminus of the first heavy chain of the full-length antibody via the VL region of the first scFv (either directly or indirectly 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 the VL region of the second scFv (either directly or indirectly via a peptide linker).

[0206] 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 are expressed by the same cancer cell. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds 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 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 the same hTAA as the second antigen-binding domain of the full-length antibody, but binds a different epitope. In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds the same hTAA as the second antigen-binding domain of the full-length antibody, but binds different and non-overlapping epitopes (e.g., the full-length antibody is bispecific). In some embodiments, the first antigen-binding domain of the full-length antibody specifically binds a different hTAA than the second antigen-binding domain of the full-length antibody.

[0207] In some embodiments, the multispecific protein comprises (a) a first polypeptide comprising a first light chain that comprises a VL region and a CL region from the N-terminus to the C-terminus; (b) a second polypeptide that comprises, from the N-terminus to the C-terminus: (i) a first scFv that comprises, from the N-terminus to the 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 that comprises a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; (c) a third polypeptide that comprises, from the N-terminus to the C-terminus: (i) a second scFv that comprises, from the N-terminus to the 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 second heavy chain that comprises a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; and (d) a fourth polypeptide comprising a second light chain that comprises a VL region and a CL region from the N-terminus to the C-terminus; wherein the VL region of the first light chain associates with the VH region of the first Ig heavy chain to form a first antigen-binding domain that specifically binds a first hTAA; wherein the VH region of the second heavy chain associates with the VL region of the second Ig light chain to form a second antigen-binding domain that specifically binds a second hTAA; wherein the first scFv specifically binds hTCSA (e.g., hCD28, hCD2); and wherein the second scFv specifically binds hCD3.

[0208] In some embodiments, the second polypeptide comprises, from the N-terminus to the C-terminus: (i) a first scFv that comprises, from the N-terminus to the 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) a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), and (iii) a first heavy chain that comprises, from the N-terminus to the 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 the N-terminus to the C-terminus: (i) a second scFv that comprises, from the N-terminus to the 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) a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), and (iii) a second heavy chain that comprises, from the N-terminus to the 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 the N-terminus to the C-terminus: (i) a first scFv that comprises, from the N-terminus to the 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) a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), and (iii) a first heavy chain that comprises, from the N-terminus to the C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and the third polypeptide comprises, from the N-terminus to the C-terminus: (i) a second scFv that comprises, from the N-terminus to the 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) a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), and (iii) a second heavy chain that comprises, from the N-terminus to the C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the amino acid sequence of the first peptide linker 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 of the second peptide linker. In some embodiments, the amino acid sequence of the first peptide linker is 100% identical to the amino acid sequence of the second peptide linker.

[0209] In some embodiments, the first scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus. In some embodiments, the second scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the second scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus; and the second scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus; the second scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus.

[0210] 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 are expressed by the same cancer cell. In some embodiments, the first antigen-binding domain specifically binds the same hTAA as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds the same epitope as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds the same hTAA as the second antigen-binding domain, but binds a different epitope. In some embodiments, the first antigen-binding domain specifically binds the same hTAA as the second antigen-binding domain, but binds different and non-overlapping epitopes. In some embodiments, the first antigen-binding domain specifically binds a different hTAA from the second antigen-binding domain.

[0211] Generally, the BCA406 form can also be described as a multispecific protein, comprising: (a) a first polypeptide comprising a first light chain that, from the N-terminus to the C-terminus, comprises a VL region and a CL region; (b) a second polypeptide that, from the N-terminus to the C-terminus, comprises: (i) a first scFv that, from the N-terminus to the C-terminus, comprises (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 (e.g., the linker described herein) (see, e.g., §5.2.8), and (iii) a first heavy chain that, from the N-terminus to the C-terminus, comprises a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (c) a third polypeptide that, from the N-terminus to the C-terminus, comprises: (i) a second scFv that, from the N-terminus to the C-terminus, comprises (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 (e.g., the linker described herein) (see, e.g., §5.2.8), and (iii) a second heavy chain that, from the N-terminus to the C-terminus, comprises a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (d) a fourth polypeptide comprising a second light chain that, from the N-terminus to the C-terminus, comprises 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 a first hTAA; wherein the VH region of the second heavy chain associates with the VL region of a second Ig light chain to form a second antigen-binding domain that specifically binds a second hTAA; wherein the first scFv specifically binds hTCSA (e.g., hCD28, hCD2); wherein the second scFv specifically binds hCD3; and wherein the CH3 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (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 (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein 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; wherein 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 comprises one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); the CH2 region of the second heavy chain comprises one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); 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 reduce or eliminate one or more of the following heavy chain effector functions relative to a reference heavy chain that does not comprise the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 domain such as SEQ ID NO: 100): ADCC, CDC, and / or binding affinity for 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))). 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 are expressed by the same cancer cell. 5.2.1.3 BCA424 format

[0212] In one aspect, provided herein is a multispecific protein in the BCA424 format (see, e.g., Figure 3 ). Generally, the BCA424 format provides a multispecific protein comprising: a first Fab operably linked (e.g., via a peptide linker) to a first scFv, the first scFv operably linked (e.g., via a peptide linker) to a first Fc region; and a second Fab operably linked (e.g., via a peptide linker) to a second scFv, the second scFv operably linked (e.g., via a peptide linker) to a second Fc region; wherein the first Fab specifically binds a first hTAA, the second Fab specifically binds a second hTAA, the first scFv specifically binds hTCSA (e.g., hCD28, hCD2), and the second scFv specifically binds hCD3.

[0213] 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; (b) a first scFv operably linked to the C-terminus of the first CH1 region of the first Fab (e.g., via a peptide linker such as any one of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319), wherein the first scFv comprises, 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, wherein the first Fc region comprises, from N-terminus to C-terminus, a CH2 region and a CH3 region; and (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 region of the second Fab (e.g., via a peptide linker such as any one of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319), wherein the second scFv comprises, 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 Fc region operably linked to the C-terminus of the second scFv, wherein the second Fc region comprises, from N-terminus to C-terminus, a CH2 region and a CH3 region; and wherein the first Fab specifically binds to a first hTAA; wherein the second Fab specifically binds to a second hTAA; wherein the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); wherein the second scFv specifically binds to hCD3.

[0214] In some embodiments, the first scFv is operably linked directly by a peptide bond to the C-terminus of the first CH1 domain of the first Fab. In some embodiments, the first scFv is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319) to the C-terminus of the first CH1 domain of the first Fab. In some embodiments, the second scFv is operably linked directly by a peptide bond to the C-terminus of the second CH1 domain of the second Fab. In some embodiments, the second scFv is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319) to the C-terminus of the second CH1 domain of the second Fab. In some embodiments, the first scFv is operably linked directly by a peptide bond to the C-terminus of the first CH1 domain of the first Fab; and the second scFv is operably linked directly by a peptide bond to the C-terminus of the second CH1 domain of the second Fab. In some embodiments, the first scFv is operably linked indirectly via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319) to the C-terminus of the first CH1 domain of the first Fab; and the second scFv is operably linked indirectly via a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, e.g., any of SEQ ID NOs: 217-236 or 318-319, e.g., SEQ ID NO: 319) to the C-terminus of the second CH1 domain of the second Fab. In some embodiments, the amino acid sequence of the peptide linker that operably links the first scFv to the C-terminus of the first CH1 domain of the first Fab 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 of the peptide linker that operably links the second scFv to the C-terminus of the second CH1 domain of the second Fab. In some embodiments, the amino acid sequence of the peptide linker that operably links the first scFv to the C-terminus of the first CH1 domain of the first Fab is 100% identical to the amino acid sequence of the peptide linker that operably links the second scFv to the C-terminus of the second CH1 domain of the second Fab.

[0215] In some embodiments, the first Fc region is operably linked directly by a peptide bond to the C-terminus of the first scFv. In some embodiments, the first Fc region is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the C-terminus of the first scFv. In some embodiments, the second Fc region is operably linked directly by a peptide bond to the C-terminus of the second scFv. In some embodiments, the second Fc region is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the C-terminus of the second scFv. In some embodiments, the first Fc region is operably linked directly by a peptide bond to the C-terminus of the first scFv; and the second Fc region is operably linked directly by a peptide bond to the C-terminus of the second scFv. In some embodiments, the first Fc region is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the C-terminus of the first scFv; and the second Fc region is operably linked indirectly by a peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8) to the C-terminus of the second scFv. In some embodiments, the amino acid sequence of the peptide linker operably linking the first Fc region, which is operably linked to the C-terminus of the first scFv, 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 of the peptide linker operably linking the second Fc region, which is operably linked to the C-terminus of the second scFv. In some embodiments, the amino acid sequence of the peptide linker operably linking the first Fc region, which is operably linked to the C-terminus of the first scFv, is 100% identical to the amino acid sequence of the peptide linker operably linking the second Fc region, which is operably linked to the C-terminus of the second scFv.

[0216] In some embodiments, the first Fc region comprises a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. In some embodiments, the second Fc region comprises a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. In some embodiments, the first Fc region comprises a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; and the second Fc region comprises a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. In some embodiments, the first Fc region comprises a partial hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. In some embodiments, the second Fc region comprises a partial hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. In some embodiments, the first Fc region comprises a partial hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; and the second Fc region comprises a partial hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus.

[0217] In some embodiments, the first scFv is operably linked (either directly or indirectly through a peptide linker) to the C-terminus of the first CH1 domain of the first Fab through the VH domain of the first scFv. In some embodiments, the first scFv is operably linked (either directly or indirectly through a peptide linker) to the C-terminus of the first CH1 domain of the first Fab through the VL domain of the first scFv. In some embodiments, the second scFv is operably linked (either directly or indirectly through a peptide linker) to the C-terminus of the second CH1 domain of the second Fab through the VH domain of the second scFv. In some embodiments, the second scFv is operably linked (either directly or indirectly through a peptide linker) to the C-terminus of the second CH1 domain of the second Fab through the VL domain of the second scFv. In some embodiments, the first scFv is operably linked (either directly or indirectly through a peptide linker) to the C-terminus of the first CH1 domain of the first Fab through the VH domain of the first scFv; and the second scFv is operably linked (either directly or indirectly through a peptide linker) to the C-terminus of the second CH1 domain of the second Fab through the VH domain of the second scFv. In some embodiments, the first scFv is operably linked (either directly or indirectly through a peptide linker) to the C-terminus of the first CH1 domain of the first Fab through the VL domain of the first scFv; and the second scFv is operably linked (either directly or indirectly through a peptide linker) to the C-terminus of the second CH1 domain of the second Fab through the VL domain of the second scFv.

[0218] 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 are expressed by the same cancer cell. In some embodiments, the first Fab specifically binds the same hTAA as the second Fab. In some embodiments, the first Fab specifically binds the same epitope as the second Fab. In some embodiments, the first Fab specifically binds the same hTAA as the second Fab, but binds to different epitopes. In some embodiments, the first Fab specifically binds the same hTAA as the second Fab, but binds to different and non-overlapping epitopes. In some embodiments, the first Fab specifically binds a different hTAA than the second Fab.

[0219] In some embodiments, the multispecific protein comprises (a) a first polypeptide that, from N-terminus to C-terminus, comprises: (i) a first Fab heavy chain that, from N-terminus to C-terminus, comprises a VH region and a VL region; operably linked to (ii) a first scFv that, from N-terminus to C-terminus, comprises (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 linked to a first Fc region that, from N-terminus to C-terminus, comprises a CH2 region and a CH3 region; (b) a second polypeptide that comprises a first light chain that, from N-terminus to C-terminus, comprises a VL region and a CL region; (c) a third polypeptide that, from N-terminus to C-terminus, comprises: (i) a second Fab heavy chain that, from N-terminus to C-terminus, comprises a VH region and a VL region; operably linked to (ii) a second scFv that, from N-terminus to C-terminus, comprises (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 linked to a second Fc region that, from N-terminus to C-terminus, comprises a CH2 region and a CH3 region; (d) a fourth polypeptide that comprises a second light chain that, from N-terminus to C-terminus, comprises 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 a first hTAA; wherein the second Fab heavy chain and the second light chain associate to form a second antigen-binding domain that specifically binds a second hTAA; wherein the first scFv specifically binds hTCSA (e.g., hCD28, hCD2); and wherein the second scFv specifically binds hCD3.

[0220] In some embodiments, the first polypeptide comprises, from the N-terminus to the C-terminus: (i) a first Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked, via a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), to (ii) a first scFv that comprises, 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 linked to a first Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region. In some embodiments, the third polypeptide comprises, from the N-terminus to the C-terminus: (i) a second Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked, via a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, such as any one of SEQ ID NOs: 217-236 or 318-319, such as SEQ ID NO: 319), to (ii) a second scFv that comprises, 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 linked to a second Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region. In some embodiments, the first polypeptide comprises, from the N-terminus to the C-terminus: (i) a first Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked, via a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), to (ii) a first scFv that comprises, 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 linked to a first Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; and the third polypeptide comprises, from the N-terminus to the C-terminus: (i) a second Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked, via a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8, such as, for example, any one of SEQ ID NOs: 217-236 or 318-319, such as, for example, SEQ ID NO: 319), to (ii) a second scFv that comprises, 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 linked to a second Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region. In some embodiments, the amino acid sequence of the first peptide linker 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 of the second peptide linker. In some embodiments, the amino acid sequence of the first peptide linker is 100% identical to the amino acid sequence of the second peptide linker.

[0221] In some embodiments, the first polypeptide comprises, from the N-terminus to the C-terminus: (i) a first Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; the Fab heavy chain is operably linked to (ii) a first scFv that comprises, 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; and is operably linked, via a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), to a first Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region. In some embodiments, the third polypeptide comprises, from the N-terminus to the C-terminus: (i) a second Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked to (ii) a second scFv that comprises, 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; and is operably linked, via a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), to a second Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region. In some embodiments, the first polypeptide comprises, from the N-terminus to the C-terminus: (i) a first Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked to (ii) a first scFv that comprises, 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; and is operably linked, via a first peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), to a first Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; and the third polypeptide comprises, from the N-terminus to the C-terminus: (i) a second Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked to (ii) a second scFv that comprises, 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; and is operably linked, via a second peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), to a second Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region. In some embodiments, the amino acid sequence of the first peptide linker and the amino acid sequence of the second peptide linker 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 first peptide linker and the amino acid sequence of the second peptide linker are 100% identical.

[0222] In some embodiments, the first polypeptide comprises, from the N-terminus to the C-terminus: (i) a first Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked, 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), to (ii) a first scFv that comprises, 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 linked, via a third peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), to a first Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region. In some embodiments, the third polypeptide comprises, from the N-terminus to the C-terminus: (i) a second Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked, 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), to (ii) a second scFv that comprises, 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 linked, via a fourth peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), to a second Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region.In some embodiments, the first polypeptide comprises, from the N-terminus to the C-terminus: (i) a first Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked, 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), to (ii) a first scFv that comprises, 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 linked, via a third peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), to a first Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; and the third polypeptide comprises, from the N-terminus to the C-terminus: (i) a second Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a VL region; operably linked, 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), to (ii) a second scFv that comprises, 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 linked, via a fourth peptide linker (e.g., a linker described herein) (see, e.g., §5.2.8), to a second Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region. In some embodiments, the amino acid sequence of the third peptide linker 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 of the fourth peptide linker. In some embodiments, the amino acid sequence of the third peptide linker is 100% identical to the amino acid sequence of the fourth peptide linker.

[0223] 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 the N-terminus to the C-terminus, a partial hinge region, a CH2 region, and a CH3 region; and the second Fc region comprises, from the N-terminus to the C-terminus, a partial hinge region, a CH2 region, and a CH3 region.

[0224] In some embodiments, the first scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus. In some embodiments, the second scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the second scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus; and the second scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus; the second scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus.

[0225] 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 are expressed by the same cancer cells. In some embodiments, the first antigen-binding domain specifically binds the same hTAA as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds the same epitope as the second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds the same hTAA as the second antigen-binding domain but binds to different epitopes. In some embodiments, the first antigen-binding domain specifically binds the same hTAA as the second antigen-binding domain but binds to different and non-overlapping epitopes. In some embodiments, the first antigen-binding domain specifically binds different hTAA from the second antigen-binding domain.

[0226] Generally, the BCA424 format can also be described as a multispecific protein that comprises: (a) a first polypeptide that, from the N-terminus to the C-terminus, comprises: (i) a first Fab heavy chain that, from the N-terminus to the C-terminus, comprises a VH region and a VL region; operably linked (by 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 NO: 319)) to (ii) a first scFv that, from the N-terminus to the C-terminus, comprises (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 linked to a first Fc region that, from the N-terminus to the C-terminus, comprises a CH2 region and a CH3 region; (b) a second polypeptide that comprises a first light chain that, from the N-terminus to the C-terminus, comprises a VL region and a CL region; (c) a third polypeptide that, from the N-terminus to the C-terminus, comprises: (i) a second Fab heavy chain that, from the N-terminus to the C-terminus, comprises a VH region and a VL region; operably linked (by an optional 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)) to (ii) a second scFv that, from the N-terminus to the C-terminus, comprises (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 linked to a second Fc region that, from the N-terminus to the C-terminus, comprises a CH2 region and a CH3 region; (d) a fourth polypeptide that comprises a second light chain that, from the N-terminus to the C-terminus, comprises a VL region and a CL region; wherein the first Fab heavy chain associates with the first light chain to form a first antigen-binding domain that specifically binds a first hTAA; wherein the second Fab heavy chain associates with the second light chain to form a second antigen-binding domain that specifically binds a second hTAA; wherein the first scFv specifically binds hTCSA (e.g., hCD28, hCD2); wherein the second scFv specifically binds hCD3; wherein the CH3 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101), and wherein the CH3 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein 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 second Fc region;One or more amino acid modifications in the CH3 region of the first Fc region and 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; wherein the CH2 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein the CH2 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein the one or more amino acid modifications in the CH2 region of the first 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 relative to a reference Fc region that does not comprise the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 domain such as SEQ ID NO: 100): ADCC, CDC, and / or binding affinity for 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))). 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 are expressed by the same cancer cell. 5.2.1.4 BCA418 form

[0227] In one aspect, the present disclosure provides a multispecific protein in the BCA418 form (see, e.g., Figure 4 ). Generally, the BCA418 form provides a multispecific protein that comprises: a first scFv operably linked (e.g., via a peptide linker) to a first Fab, the first Fab operably linked (e.g., via a peptide linker) to a first Fc region; and a first IgM CH2 mFab operably linked (e.g., via a peptide linker) to a second Fc region; wherein the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2), the first Fab specifically binds to hCD3, and the first IgM CH2 mFab specifically binds to hTAA.

[0228] In some embodiments, the multispecific protein comprises (a) an scFv that, from the N-terminus to the C-terminus, comprises (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 that comprises (i) a Fab heavy chain that, from the N-terminus to the C-terminus, comprises a VH region and a CH1 region, and (ii) a light chain that, from the N-terminus to the C-terminus, comprises a VL region and a CL region; (c) a first Fc region that, from the N-terminus to the C-terminus, comprises a CH2 region and a CH3 region; (d) a second Fc region that, from the N-terminus to the C-terminus, comprises a CH2 region and a CH3 region; (e) an IgM CH2 mFab that comprises (i) an IgM CH2 mFab heavy chain that, from the N-terminus to the C-terminus, comprises a VH region and an IgM CH2 region, and (ii) an IgM CH2 mFab light chain that, from the N-terminus to the C-terminus, comprises a VL region and an IgM CH2 region; wherein the C-terminus of the scFv is operably linked to the N-terminus of the Fab heavy chain of the Fab; wherein the C-terminus of the Fab heavy chain is operably linked to the N-terminus of the first Fc region; wherein the C-terminus of the IgM CH2 mFab heavy chain is operably linked (e.g., via a peptide linker, such as any one of SEQ ID NOs: 217 - 236 or 318 - 319, such as SEQ ID NO: 318) to the N-terminus of the second Fc region; wherein the IgM CH2 mFab specifically binds to an hTAA; wherein the scFv specifically binds to an hTCSA (e.g., hCD28, hCD2); wherein the Fab specifically binds to hCD3.

[0229] In some embodiments, the scFv is directly operably linked to the N-terminus of the Fab heavy chain via a peptide bond. In some embodiments, the scFv is indirectly operably linked to the N-terminus of the Fab heavy chain via a peptide linker (e.g., the linkers described herein) (see, e.g., §5.2.8).

[0230] In some embodiments, the scFv is operably linked to the N-terminus of the Fab heavy chain via the VH region of the scFv (either directly or indirectly via a peptide linker). In some embodiments, the scFv is operably linked to the N-terminus of the Fab heavy chain via the VL region of the scFv (either directly or indirectly via a peptide linker).

[0231] In some embodiments, the C-terminus of the Fab heavy chain is operably linked to the N-terminus of the first Fc region; wherein the C-terminus of the IgM CH2 mFab heavy chain is operably linked to the N-terminus of the second Fc region; wherein the IgM CH2 mFab specifically binds to an hTAA via a peptide linker (such as any one of SEQ ID NOs: 217 - 236 or 318 - 319, such as SEQ ID NO: 318).

[0232] In some embodiments, the first Fc region comprises a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. In some embodiments, the second Fc region comprises a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. In some embodiments, the first Fc region comprises a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; and the second Fc region comprises a hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. In some embodiments, the first Fc region comprises a partial hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. In some embodiments, the second Fc region comprises a partial hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus. In some embodiments, the first Fc region comprises a partial hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus; and the second Fc region comprises a partial hinge region, a CH2 region, and a CH3 region from the N-terminus to the C-terminus.

[0233] In some embodiments, the multispecific protein comprises (a) a first polypeptide comprising a first light chain that comprises a VL region and a CL region from the N-terminus to the C-terminus; (b) a second polypeptide that comprises, from the N-terminus to the C-terminus, (i) a first scFv that comprises, from the N-terminus to the 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 first heavy chain that comprises a VH region, an IgG CH1 region, an IgG hinge region, an IgG CH2 region, and an IgG CH3 region; (c) a third polypeptide that comprises a VH region, a first IgM CH2 region, and an Fc region, the Fc region comprising an IgG hinge region, an IgG CH2 region, and an IgG CH3 region from the N-terminus to the C-terminus; and (d) a fourth polypeptide that comprises a VL region and a second IgM CH2 region from the N-terminus to the C-terminus; wherein the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); wherein the first light chain and the first heavy chain associate to form an antigen-binding domain that specifically binds to human CD3 (hCD3); and wherein the third and fourth polypeptides associate to form an antigen-binding domain that specifically binds to hTAA.

[0234] In some embodiments, the second polypeptide comprises, from the N-terminus to the C-terminus: (i) a first scFv that comprises, from the N-terminus to the 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., the linker described herein) (see, e.g., §5.2.8); (ii) a first heavy chain that comprises a VH region, an IgG CH1 region, an IgG hinge region, an IgG CH2 region, and an IgG CH3 region.

[0235] In some embodiments, the first scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus. In some embodiments, the second scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the second scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus; and the second scFv comprises a VL region, a peptide linker, and a VH region from the N-terminus to the C-terminus. In some embodiments, the first scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus; and the second scFv comprises a VH region, a peptide linker, and a VL region from the N-terminus to the C-terminus.

[0236] Generally, the BCA418 form can also be described as a multispecific protein comprising: (a) a first polypeptide comprising a first light chain that from the N-terminus to the C-terminus comprises a VL region and a CL region; (b) a second polypeptide that from the N-terminus to the C-terminus comprises (i) a first scFv that from the N-terminus to the C-terminus comprises (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; an optional first peptide linker (e.g., the linker described herein) (see, e.g., §5.2.8); (iii) a first heavy chain that from the N-terminus to the C-terminus comprises a VH region, an IgG CH1 region, an IgG hinge region, an IgG CH2 region, and an IgG CH3 region; (c) a third polypeptide that from the N-terminus to the C-terminus comprises a VH region, a first IgM CH2 region, and an Fc region that from the N-terminus to the C-terminus comprises an IgG hinge region, an IgG CH2 region, and an IgG CH3 region; and (d) a fourth polypeptide that from the N-terminus to the C-terminus comprises a VL region and a second IgM CH2 region; wherein the first scFv is specific for hTCSA (e.g., hCD28, hCD2); wherein the first light chain and the first heavy chain associate to form an antigen-binding domain that specifically binds human CD3 (hCD3); wherein the third and fourth polypeptides associate to form an antigen-binding domain that specifically binds human hTAA; and wherein the CH3 region of the first heavy chain comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101), wherein the CH3 region of the Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein 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; wherein 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; wherein the CH2 region of the first heavy chain comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein the CH2 region of the Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100);One or more amino acid modifications in the CH2 region of the first heavy chain and one or more amino acid modifications in the CH2 region of the Fc region reduce or eliminate one or more of the following Fc region effector functions relative to a reference Fc region that does not contain the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type Fc region such as SEQ ID NO: 100): ADCC, CDC, and / or binding affinity for 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))).; 5.2.1.5 Stabilized scFv

[0237] The scFv component of any multispecific protein described herein includes a modified scFv that promotes the stability of the scFv structure. Methods for stabilizing scFv are known in the art, for example, introducing a cysteine residue in the VH region of the scFv and introducing a cysteine residue in the VL region of the scFv, wherein the cysteines are capable of forming a disulfide bond (see, e.g., US6147203; 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 destabilised single chain Fv format: importance of interchain disulphide bond location and VL-VH orientation, Protein Engineering, Design and Selection, vol. 25, no. 7, July 2012, pp. 321-329, https: / / doi.org / 10.1093 / protein / gzs021); the entire contents of which are incorporated herein by reference for all purposes).

[0238] In some embodiments, introducing cysteine into the VH region and the VL region of the scFv does not significantly affect the binding affinity of the scFv for its cognate antigen. In some embodiments, cysteine is introduced into the framework region of the VH region of the scFv; and cysteine is introduced into the framework region of the VL region of the scFv.

[0239] In some embodiments, cysteine is introduced at amino acid positions 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106 in the VH region of the scFv, with the amino acids numbered according to Kabat; and cysteine is introduced at amino acid positions 40, 41, 42, 43, 44, 45, 46, or 47 in the VL region of the scFv, with the amino acids numbered according to Kabat.

[0240] In some embodiments, cysteine is introduced into the framework region of the VH region of the scFv; and cysteine is introduced into the framework region of the VL region of the scFv. In some embodiments, cysteine is introduced at amino acid position 100 in the VH region of the scFv, with the amino acids numbered according to Kabat; and cysteine is introduced at amino acid position 44 or 45 in the VL region of the scFv, with the amino acids numbered according to Kabat. In some embodiments, cysteine is introduced into the framework region of the VH region of the scFv; and cysteine is introduced into the framework region of the VL region of the scFv. In some embodiments, cysteine is introduced at amino acid position 100 in the VH region of the scFv, with the amino acids numbered according to Kabat; and cysteine is introduced at amino acid position 44 in the VL region of the scFv, with the amino acids numbered according to Kabat. In some embodiments, cysteine is introduced into the framework region of the VH region of the scFv; and cysteine is introduced into the framework region of the VL region of the scFv. In some embodiments, cysteine is introduced at amino acid position 100 in the VH region of the scFv, with the amino acids numbered according to Kabat; and cysteine is introduced at amino acid position 45 in the VL region of the scFv, with the amino acids numbered according to Kabat. 5.2.2 Human T cell co-stimulatory antigen (hTCSA) binding domain

[0241] The multispecific proteins described herein comprise antigen-binding domains that specifically bind to human T cell co-stimulatory antigens (hTCSA). As described above, hTCSA are proteins expressed on the surface of human T cells that are not part of the T cell receptor complex and enhance T cell activation when bound to their cognate ligands. hTCSA are known in the art. Exemplary hTCSA 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. 5.2.2.1 CD28 Binding Domain

[0242] CD28 is a co-stimulatory transmembrane protein expressed on the surface of T cells, including naive T cells. Functions of CD28 include, for example, activating T cells, inducing T cell proliferation, producing T cell cytokines, and promoting T cell survival. CD28 is a receptor for the CD80 and CD86 proteins, which are cell surface transmembrane proteins expressed by antigen-presenting cells.

[0243] The amino acid sequences of the reference immature hCD28 polypeptide (with a signal peptide at the N-terminus) and the mature hCD28 polypeptide (without a signal peptide) are shown as SEQ ID NO: 1 and 2, respectively. See Table 1 herein. Table 1. Amino Acid Sequences of Reference hCD28 Polypeptides

[0244] In some embodiments, the multispecific proteins described herein comprise antigen-binding domains that specifically bind to hCD28, also referred to herein as hCD28 binding domains or anti-hCD28 antibodies (or functional fragments or variants thereof). In a specific embodiment, the hCD28 binding domain is an scFv (see, for example, §5.2.1). In a specific embodiment, the multispecific protein or polypeptide is monovalent for hCD28.

[0245] hCD28 binding domains useful for the multispecific proteins described herein are known in the art and include, for example, those described in, e.g., US7585960, US9562098, US7723482, US7939638B2, US9908937, US7723482B2, WO2021155071, WO2023164510A1, WO2023155845A1, WO2023143547A1, WO2023114701A2, WO2017103003A1, WO2011101791A1, WO2022253867A1, WO2022216915A1, and WO2020127628A1, US20230265218A1, WO2022094299, US20220089766A1, the entire contents of which are incorporated herein by reference for all purposes.

[0246] Exemplary hCD28 binding domains useful for the multispecific proteins described herein are described, for example, in Table 2 of §5.2.2.

[0247] Exemplary hCD28 binding domains known in the art useful for the fusion proteins described herein also include TGN1412.

[0248] The amino acid sequences of exemplary hCD28 binding domains useful for the multispecific proteins described herein are provided in Table 2. The CDRs of the hCD28 binding domains in Table 2 are represented according to Kabat. Those of ordinary skill in the art can determine the CDRs defined by other schemes (e.g., Chothia, IMGT) using conventional methods known in the art. Table 2. Amino Acid Sequences of Exemplary hCD28 Binding Domains

[0249] In some embodiments, the hCD28 binding domain comprises the hCD28 binding domain provided in Table 2.

[0250] In some embodiments, the hCD28 binding domain comprises a VH that comprises: VH CDR1, VH CDR2, and VH CDR3.

[0251] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 of VH shown in Table 2 or comprises the amino acid sequence of VH CDR1 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of. In some embodiments, the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 of VH shown in Table 2 or comprises the amino acid sequence of VH CDR2 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of. In some embodiments, the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 of VH shown in Table 2 or comprises the amino acid sequence of VH CDR3 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of.

[0252] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 of VH shown in Table 2 or comprises the amino acid sequence of VH CDR1 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of; the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 of VH shown in Table 2 or comprises the amino acid sequence of VH CDR2 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of; and the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 of VH shown in Table 2 or comprises the amino acid sequence of VH CDR3 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 of VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of.

[0253] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 shown in Table 2 or comprises the amino acid sequence of VH CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of. In some embodiments, the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 shown in Table 2 or comprises the amino acid sequence of VH CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of. In some embodiments, the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 shown in Table 2 or comprises the amino acid sequence of VH CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of.

[0254] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 shown in Table 2 or comprises or consists of the amino acid sequence of VH CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 shown in Table 2 or comprises or consists of the amino acid sequence of VH CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 shown in Table 2 or comprises or consists of the amino acid sequence of VH CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0255] In some embodiments, the hCD28 binding domain comprises a VL, which comprises: VL CDR1, VL CDR2, and VL CDR3.

[0256] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 of VL shown in Table 2 or comprises or consists of the amino acid sequence of VL CDR1 of VL shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 of VL shown in Table 2 or comprises or consists of the amino acid sequence of VL CDR2 of VL shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 of VL shown in Table 2 or comprises or consists of the amino acid sequence of VL CDR3 of VL shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0257] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 shown in Table 2 or comprises the amino acid sequence of VL CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 shown in Table 2 or comprises the amino acid sequence of VL CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; and the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 shown in Table 2 or comprises the amino acid sequence of VL CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof.

[0258] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 shown in Table 2 or comprises the amino acid sequence of VL CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof. In some embodiments, the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 shown in Table 2 or comprises the amino acid sequence of VL CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof. In some embodiments, the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 shown in Table 2 or comprises the amino acid sequence of VL CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof.

[0259] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 shown in Table 2 or comprises the amino acid sequence of VL CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 shown in Table 2 or comprises the amino acid sequence of VL CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; and the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 shown in Table 2 or comprises the amino acid sequence of VL CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof.

[0260] In some embodiments, the hCD28 binding domain comprises a VH that comprises: VH CDR1, VH CDR2, and VH CDR3; and a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[0261] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 of the VH shown in Table 2 or comprises or consists of the amino acid sequence of VH CDR1 of the VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 of the VH shown in Table 2 or comprises or consists of the amino acid sequence of VH CDR2 of the VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 of the VH shown in Table 2 or comprises or consists of the amino acid sequence of VH CDR3 of the VH shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 of the VL shown in Table 2 or comprises or consists of the amino acid sequence of VL CDR1 of the VL shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 of the VL shown in Table 2 or comprises or consists of the amino acid sequence of VL CDR2 of the VL shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 of the VL shown in Table 2 or comprises or consists of the amino acid sequence of VL CDR3 of the VL shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0262] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 shown in Table 2 or comprises the amino acid sequence of VH CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 shown in Table 2 or comprises the amino acid sequence of VH CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 shown in Table 2 or comprises the amino acid sequence of VH CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 shown in Table 2 or comprises the amino acid sequence of VL CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR1 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 shown in Table 2 or comprises the amino acid sequence of VL CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR2 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; and the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 shown in Table 2 or comprises the amino acid sequence of VL CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR3 shown in Table 2 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof.

[0263] In some embodiments, the amino acid sequence of 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 of VH shown in Table 2. In some embodiments, the amino acid sequence of 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 of VL shown in Table 2. In some embodiments, the amino acid sequence of 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 of VH shown in Table 2; and the amino acid sequence of 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 of VL shown in Table 2.

[0264] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3 with 1, 2 or 3 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 shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4 with 1, 2 or 3 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 shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5 with 1, 2 or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0265] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence shown in SEQ ID NO: 3 that contains 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence shown in SEQ ID NO: 4 that contains 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; and the amino acid sequence of VH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence shown in SEQ ID NO: 5 that contains 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0266] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence shown in SEQ ID NO: 6 that contains 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same. In some embodiments, the amino acid sequence of VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence shown in SEQ ID NO: 7 that contains 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same. In some embodiments, the amino acid sequence of VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence shown in SEQ ID NO: 8 that contains 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0267] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence shown in SEQ ID NO: 6 that contains 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence shown in SEQ ID NO: 7 that contains 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; and the amino acid sequence of VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence shown in SEQ ID NO: 8 that contains 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0268] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence shown in SEQ ID NO: 3 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence shown in SEQ ID NO: 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence shown in SEQ ID NO: 5 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VL CDR1 comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence shown in SEQ ID NO: 6 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence shown in SEQ ID NO: 7 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; and the amino acid sequence of VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence shown in SEQ ID NO: 8 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0269] In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 9. In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 10. In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 9; and the amino acid sequence of 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 shown in SEQ ID NO: 10.

[0270] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence shown in SEQ ID NO: 11 with 1, 2 or 3 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 shown in SEQ ID NO: 12 or an amino acid sequence shown in SEQ ID NO: 12 with 1, 2 or 3 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 shown in SEQ ID NO: 13 or an amino acid sequence shown in SEQ ID NO: 13 with 1, 2 or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0271] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence shown in SEQ ID NO: 11 having 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence shown in SEQ ID NO: 12 having 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; and the amino acid sequence of VH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence shown in SEQ ID NO: 13 having 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0272] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence shown in SEQ ID NO: 14 having 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same. In some embodiments, the amino acid sequence of VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence shown in SEQ ID NO: 15 having 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same. In some embodiments, the amino acid sequence of VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence shown in SEQ ID NO: 16 having 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0273] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence shown in SEQ ID NO: 14 having 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence shown in SEQ ID NO: 15 having 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; and the amino acid sequence of VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence shown in SEQ ID NO: 16 having 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0274] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence set forth in SEQ ID NO: 11 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence set forth in SEQ ID NO: 12 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence set forth in SEQ ID NO: 13 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence set forth in SEQ ID NO: 14 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence set forth in SEQ ID NO: 15 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; and the amino acid sequence of VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence set forth in SEQ ID NO: 16 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0275] In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 17. In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 19. In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 17; and the amino acid sequence of 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 shown in SEQ ID NO: 19.

[0276] In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 18. In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 20. In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 18; and the amino acid sequence of 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 shown in SEQ ID NO: 20. 5.2.2.2 CD2 Binding Domain

[0277] CD2 is a co-stimulatory transmembrane protein that is expressed on the surface of T cells and NK cells. CD28 functions in the formation and organization of the immunological synapse that forms between T cells and antigen-presenting cells, for example, after intercellular coupling and associated intracellular signaling. CD2 is a receptor for LFA3, a cell surface protein expressed by antigen-presenting cells.

[0278] The amino acid sequences of the reference immature hCD2 polypeptide (with a signal peptide at the N-terminus) and the mature hCD2 polypeptide (without a signal peptide) are shown in SEQ ID NO: 37 and 38, respectively. See Table 16 herein. Table 16. Amino Acid Sequences of Reference hCD2 Polypeptides

[0279] In some embodiments, the multispecific proteins described herein comprise an antigen-binding domain that specifically binds hCD2, also referred to herein as an hCD2 binding domain or an anti-hCD2 antibody (or a functional fragment or variant thereof). In a specific embodiment, the hCD2 binding domain is a scFv (see, for example, §5.2.1). In a specific embodiment, the multispecific protein or polypeptide is monovalent for hCD28.

[0280] Exemplary hCD2 binding domains useful for the multispecific proteins described herein are described in Table 17 of §5.2.2.

[0281] Exemplary hCD2 binding domains known in the art and useful for the fusion proteins described herein also include, for example, siltuximab, LO-CD2b, and the humanized form of LO-CD2b (see, for example, WO1999003502).

[0282] hCD2 binding domains useful for the multispecific proteins described herein are known in the art and include, for example, those described in US5656438A, EP1003552B1, WO2022067089, WO2023126445A1, WO2023126445A1, WO2022157272A1, WO2021259927A2, US20210260212A1, US7678582B2, US7250167B2, US7332157B2, US6384198B1, and WO1999003502, the entire contents of which are incorporated herein by reference for all purposes.

[0283] Table 17 provides the amino acid sequences of exemplary hCD2-binding domains that can be used for the multispecific proteins described herein. The CDRs of the hCD2-binding domains in Table 17 are according to the Kabat representation. Those skilled in the art can determine the CDRs defined by other schemes (such as Chothia, IMGT) using conventional methods known in the art. Table 17. Amino acid sequences of exemplary hCD2-binding domains

[0284] In some embodiments, the hCD2-binding domain comprises the hCD2-binding domain provided in Table 17.

[0285] In some embodiments, the hCD2-binding domain comprises a VH that comprises: VH CDR1, VH CDR2, and VH CDR3.

[0286] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 of the VH shown in Table 17 or comprises or consists of the amino acid sequence of VH CDR1 of the VH shown in Table 17 with 1, 2, or 3 amino acid modifications (such as substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 of the VH shown in Table 17 or comprises or consists of the amino acid sequence of VH CDR2 of the VH shown in Table 17 with 1, 2, or 3 amino acid modifications (such as substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 of the VH shown in Table 17 or comprises or consists of the amino acid sequence of VH CDR3 of the VH shown in Table 17 with 1, 2, or 3 amino acid modifications (such as substitutions, deletions, additions, etc.).

[0287] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 shown in Table 17 or comprises, consists of, or consists of the amino acid sequence of VH CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 shown in Table 17 or comprises, consists of, or consists of the amino acid sequence of VH CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 shown in Table 17 or comprises, consists of, or consists of the amino acid sequence of VH CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0288] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 shown in Table 17 or comprises, consists of, or consists of the amino acid sequence of VH CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 shown in Table 17 or comprises, consists of, or consists of the amino acid sequence of VH CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 shown in Table 17 or comprises, consists of, or consists of the amino acid sequence of VH CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0289] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 shown in Table 17 or comprises, consists of, or consists of the amino acid sequence of VH CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 shown in Table 17 or comprises, consists of, or consists of the amino acid sequence of VH CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 shown in Table 17 or comprises, consists of, or consists of the amino acid sequence of VH CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0290] In some embodiments, the hCD2 binding domain comprises a VL, which VL comprises: VL CDR1, VL CDR2, and VL CDR3.

[0291] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 of the VL shown in Table 17 or comprises the amino acid sequence of VL CDR1 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR1 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR1 of the VL shown in Table 17. In some embodiments, the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 of the VL shown in Table 17 or comprises the amino acid sequence of VL CDR2 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR2 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR2 of the VL shown in Table 17. In some embodiments, the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 of the VL shown in Table 17 or comprises the amino acid sequence of VL CDR3 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR3 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR3 of the VL shown in Table 17.

[0292] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 of the VL shown in Table 2 or comprises the amino acid sequence of VL CDR1 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR1 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR1 of the VL shown in Table 17; the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 of the VL shown in Table 17 or comprises the amino acid sequence of VL CDR2 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR2 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR2 of the VL shown in Table 17; and the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 of the VL shown in Table 17 or comprises the amino acid sequence of VL CDR3 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR3 of the VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR3 of the VL shown in Table 17.

[0293] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 shown in Table 17 or comprises the amino acid sequence of VL CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of or consists of the amino acid sequence of VL CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 shown in Table 17 or comprises the amino acid sequence of VL CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of or consists of the amino acid sequence of VL CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 shown in Table 17 or comprises the amino acid sequence of VL CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of or consists of the amino acid sequence of VL CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0294] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 shown in Table 17 or comprises the amino acid sequence of VL CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of or consists of the amino acid sequence of VL CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 shown in Table 17 or comprises the amino acid sequence of VL CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of or consists of the amino acid sequence of VL CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 shown in Table 17 or comprises the amino acid sequence of VL CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of or consists of the amino acid sequence of VL CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0295] In some embodiments, the hCD2 binding domain comprises VH, which comprises: VH CDR1, VH CDR2, and VH CDR3; and VL, which comprises: VL CDR1, VL CDR2, and VL CDR3.

[0296] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 of VH shown in Table 17 or comprises or consists of the amino acid sequence of VH CDR1 of VH shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 of VH shown in Table 17 or comprises or consists of the amino acid sequence of VH CDR2 of VH shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 of VH shown in Table 17 or comprises or consists of the amino acid sequence of VH CDR3 of VH shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 of VL shown in Table 17 or comprises or consists of the amino acid sequence of VL CDR1 of VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 of VL shown in Table 17 or comprises or consists of the amino acid sequence of VL CDR2 of VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 of VL shown in Table 17 or comprises or consists of the amino acid sequence of VL CDR3 of VL shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0297] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 shown in Table 17 or comprises the amino acid sequence of VH CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 shown in Table 17 or comprises the amino acid sequence of VH CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 shown in Table 17 or comprises the amino acid sequence of VH CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 shown in Table 17 or comprises the amino acid sequence of VL CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR1 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; the amino acid sequence of VL CDR2 comprises the amino acid sequence of VL CDR2 shown in Table 17 or comprises the amino acid sequence of VL CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR2 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof; and the amino acid sequence of VL CDR3 comprises the amino acid sequence of VL CDR3 shown in Table 17 or comprises the amino acid sequence of VL CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR3 shown in Table 17 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists thereof.

[0298] In some embodiments, the amino acid sequence of 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 of VH shown in Table 17. In some embodiments, the amino acid sequence of 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 of VL shown in Table 17. In some embodiments, the amino acid sequence of 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 of VH shown in Table 17; and the amino acid sequence of 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 of VL shown in Table 17.

[0299] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence shown in SEQ ID NO: 60 that contains 1, 2 or 3 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 shown in SEQ ID NO: 85 or an amino acid sequence shown in SEQ ID NO: 85 that contains 1, 2 or 3 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 shown in SEQ ID NO: 86 or an amino acid sequence shown in SEQ ID NO: 86 that contains 1, 2 or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0300] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence shown in SEQ ID NO: 60 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 85 or an amino acid sequence shown in SEQ ID NO: 85 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; and the amino acid sequence of VH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 86 or an amino acid sequence shown in SEQ ID NO: 86 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0301] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence shown in SEQ ID NO: 87 or an amino acid sequence shown in SEQ ID NO: 87 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same. In some embodiments, the amino acid sequence of VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 88 or an amino acid sequence shown in SEQ ID NO: 88 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same. In some embodiments, the amino acid sequence of VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 89 or an amino acid sequence shown in SEQ ID NO: 89 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0302] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence shown in SEQ ID NO: 87 or an amino acid sequence shown in SEQ ID NO: 87 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 88 or an amino acid sequence shown in SEQ ID NO: 88 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; and the amino acid sequence of VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 89 or an amino acid sequence shown in SEQ ID NO: 89 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0303] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence shown in SEQ ID NO: 60 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 85 or an amino acid sequence shown in SEQ ID NO: 85 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 86 or an amino acid sequence shown in SEQ ID NO: 86 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VLCDR1 comprises the amino acid sequence shown in SEQ ID NO: 87 or an amino acid sequence shown in SEQ ID NO: 87 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; the amino acid sequence of VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 88 or an amino acid sequence shown in SEQ ID NO: 88 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same; and the amino acid sequence of VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 89 or an amino acid sequence shown in SEQ ID NO: 89 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the same.

[0304] In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 90. In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 91. In some embodiments, the amino acid sequence of 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 shown in SEQ ID NO: 90; and the amino acid sequence of 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 shown in SEQ ID NO: 91. 5.2.3 CD3 Binding Domain

[0305] The CD3 complex is a group of polypeptides that act together with the T cell receptor (TCR) to activate T cells. The CD3 complex consists of two CD3ε polypeptides, two CD3ζ polypeptides, one CD3δ polypeptide, and one CD3γ polypeptide. The intracellular tail of each CD3 chain (CD3ε, CD3ζ, CD3γ, and CD3δ) contains at least one conserved motif called the immunoreceptor tyrosine-based activation motif (ITAM), which is crucial for TCR signal transduction.

[0306] The amino acid sequences of the reference immature hCD3ε polypeptide and mature hCD3ε polypeptide are shown in SEQ ID NO: 21 and 22, respectively. The amino acid sequences of the reference immature hCD3ζ polypeptide and mature hCD3ζ polypeptide are shown in SEQ ID NO: 23 and 24, respectively. The amino acid sequences of the reference immature hCD3γ polypeptide and mature hCD3γ polypeptide are shown in SEQ ID NO: 25 and 26, respectively. The amino acid sequences of the reference immature hCD3δ polypeptide and mature hCD3δ polypeptide are shown in SEQ ID NO: 27 and 28, respectively. See Table 3 herein. Table 3. Amino Acid Sequences of Reference hCD3 Polypeptides

[0307] The multispecific proteins described herein comprise an antigen-binding domain that specifically binds hCD3, also referred to herein as the hCD3-binding domain or anti-hCD3 antibody (or functional fragment or variant thereof). In certain embodiments, the hCD3-binding domain is a 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 for hCD3.

[0308] In some embodiments, the hCD3-binding domain specifically binds hCD3ε. In some embodiments, the hCD3-binding domain specifically binds hCD3δ. In some embodiments, the hCD3-binding domain specifically binds hCD3γ. In some embodiments, the hCD3-binding domain specifically binds hCD3ζ.

[0309] Exemplary hCD3-binding domains useful for the multispecific proteins described herein are described in Table 4 of §5.2.3.

[0310] hCD3 binding domains that can be used for the multispecific proteins described herein are known in the art, including those described in, for example, WO2023044402A1, WO2023014809A2, WO2020247929, US8530629, US20220041721A1, US7820166B2, US8101722, WO2021121215A1, US20220010015A1, WO2021240388A1, WO2021141996A1, WO2021063330A1, US20230212289A1, US20210054077A1, WO2020204708A1, WO2023044402A1, WO2020157210A1, US20210317213A1, WO2023014809A2, WO2023273914A1, WO2022170740A1, US20210269525A1, US20210277118A1, US20200048349A1, US20190382486A1, US11203646B2, US20210155694A1, US20200339686A1, US20200317779A1, US20200299384A1, US20200157217A1, US20200377594A1, US20200115449, WO2017053856A1, US20180273622A1, US20190284278A1, US9914777B2, US11007267B2, US20210054087A1, US10066015B2, US10669337B2, US9611325, US20230002487A1, US11603405B2, US11639388B2, US10407501B2, US10640572B2, US10968276B2, US10000567B2, US10202452B2, US10150812B2, US7728114B2, US20190359712A1, US20200048348A1, US10066016, the entire contents of each of which are incorporated herein by reference for all purposes.

[0311] Exemplary hCD3 binding domains known in the art and useful for the fusion proteins and polypeptides described herein also include OKT3 (also known as muromonab), SP34, otelixizumab, teplizumab, visilizumab, catumaxomab, blinatumomab, and foralumab; and chimeric or humanized versions of any of the foregoing, such as the humanized form of OKT3, the humanized form of SP34. The anti-hCD3 antibody referred to herein as OKT3 (see, e.g., Table 4) specifically binds hCD3ε. The anti-hCD3 antibody referred to herein as SP34 (see, e.g., Table 4) specifically binds hCD3ε.

[0312] The amino acid sequences of exemplary hCD3 binding domains useful for the multispecific proteins described herein are shown in Table 4. The CDRs of the hCD3 binding domains in Table 4 are represented according to Kabat. Those skilled in the art can use conventional methods known in the art to determine the CDRs defined by other schemes (e.g., Chothia, IMGT). Table 4. Amino Acid Sequences of Exemplary hCD3 Binding Domains

[0313] In some embodiments, the hCD3 binding domain comprises the hCD3 binding domain provided in Table 4.

[0314] In some embodiments, the hCD3 binding domain comprises a VH that comprises: VH CDR1, VH CDR2, and VH CDR3.

[0315] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 of VH shown in Table 4 or comprises the amino acid sequence of VH CDR1 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 of VH shown in Table 4 or comprises the amino acid sequence of VH CDR2 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 of VH shown in Table 4 or comprises the amino acid sequence of VH CDR3 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0316] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 of VH shown in Table 4 or comprises the amino acid sequence of VH CDR1 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 of VH shown in Table 4 or comprises the amino acid sequence of VH CDR2 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 of VH shown in Table 4 or comprises the amino acid sequence of VH CDR3 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 of VH shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0317] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 shown in Table 4 or comprises the amino acid sequence of VH CDR1 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 shown in Table 4 or comprises the amino acid sequence of VH CDR2 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 shown in Table 4 or comprises the amino acid sequence of VH CDR3 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0318] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of VH CDR1 shown in Table 4 or comprises the amino acid sequence of VH CDR1 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR1 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of; the amino acid sequence of VH CDR2 comprises the amino acid sequence of VH CDR2 shown in Table 4 or comprises the amino acid sequence of VH CDR2 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR2 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of; and the amino acid sequence of VH CDR3 comprises the amino acid sequence of VH CDR3 shown in Table 4 or comprises the amino acid sequence of VH CDR3 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VH CDR3 shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of.

[0319] In some embodiments, the hCD3 binding domain comprises a VL, which comprises: VL CDR1, VL CDR2, and VL CDR3.

[0320] In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of VL CDR1 of VL shown in Table 4 or comprises the amino acid sequence of VL CDR1 of VL shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions, deletions, additions, etc.) or consists of the amino acid sequence of VL CDR1 of VL shown in Table 4 with 1, 2, or 3 amino acid modifications (e.g., substitutions...

Claims

1. A multispecific protein, comprising: (a) A full-length antibody, said full-length antibody comprising: (i) A first light chain, which from the N-terminus to the C-terminus comprises a variable light chain (VL) region and a constant light chain (CL) region; (ii) A first heavy chain, which from the N-terminus to the C-terminus comprises a variable heavy chain (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) A second heavy chain, which from the N-terminus to the C-terminus comprises a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iv) A second light chain, which from the N-terminus to the C-terminus comprises a VL region and a CL region; wherein said first light chain and said first heavy chain associate to form a first antigen-binding domain; wherein said second light chain and said second heavy chain associate to form a second antigen-binding domain; and wherein said first heavy chain and said second heavy chain associate to form a dimer; (b) A first single-chain variable fragment (scFv) operably linked to the C-terminus of the CH3 region of said first heavy chain of said full-length antibody, wherein said first scFv from the N-terminus to the C-terminus comprises (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 said second heavy chain of said full-length antibody, wherein said second scFv from the N-terminus to the C-terminus comprises (i) a VH region, a peptide linker, and a VL region, or (ii) a VL region, a peptide linker, and a VH region; wherein said first antigen-binding domain of said full-length antibody specifically binds to a first human tumor-associated antigen (hTAA); wherein said second antigen-binding domain of said full-length antibody specifically binds to a second hTAA; wherein said first scFv specifically binds to a human T cell co-stimulatory antigen (hTCSA) (e.g., hCD28, hCD2); wherein said second scFv specifically binds to human CD3 (hCD3); and wherein the CH3 region of said first heavy chain of said full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise said one or more amino acid modifications (e.g., a reference CH3 region, e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); wherein the CH3 region of said second heavy chain of said full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise said one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein said one or more amino acid modifications in the CH3 region of said first heavy chain of said full-length antibody are different from said one or more amino acid modifications in the CH3 region of said second heavy chain of said 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 (e.g., substitutions) relative to a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not comprise 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) relative to a reference CH2 region (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100) that does not comprise the one or more amino acid modifications; One or more amino acid modifications in the CH2 region of the first heavy chain of 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 relative to a reference heavy chain that does not comprise the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 region such as SEQ ID NO: 100): antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or binding affinity for 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))); 2. The multispecific protein of claim 1, wherein the first scFv is directly operably linked via a peptide bond to the C-terminus of the CH3 region of the first heavy chain of the full-length antibody.

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

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

5. The multispecific protein according to any one of claims 3 or 4, wherein the amino acid sequence of the first peptide linker comprises or consists of the following: (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, which comprises 1, 2, or 3 amino acid substitutions or consists thereof.

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

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

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

9. The multispecific protein of any one of claims 7 or 8, wherein the amino acids of the second peptide linker comprise or consist of the following: (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, which comprises 1, 2 or 3 amino acid substitutions or consists thereof.

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

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

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

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

14. A multispecific protein comprising: (a) A full-length antibody, said full-length antibody comprising: (i) A first light chain, which comprises a VL region and a CL region from the N-terminus to the C-terminus; (ii) A first heavy chain, which comprises a VH region, a CH1 region, a hinge region, a CH2 region and a CH3 region from the N-terminus to the C-terminus; (iii) A second heavy chain, which comprises a VH region, a CH1 region, a hinge region, a CH2 region and a CH3 region from the N-terminus to the C-terminus; (iv) A second light chain, which comprises a VL region and a CL region from the N-terminus to the C-terminus; wherein the first light chain associates with the first heavy chain to form a first antigen-binding domain; wherein the second light chain associates with the second heavy chain to form a second antigen-binding domain; and wherein the first heavy chain associates with the second heavy chain to form a dimer; (b) A first scFv operably linked to the N-terminus of the first heavy chain of the full-length antibody, wherein the first scFv comprises, 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; and (c) A second scFv operably linked to the N-terminus of the second heavy chain of said full-length antibody, wherein said second scFv comprises, 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; wherein the first antigen-binding domain of the full-length antibody specifically binds to a first hTAA; wherein the second antigen-binding domain of the full-length antibody specifically binds to a second hTAA; wherein the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); wherein the second scFv specifically binds to hCD3; and wherein the CH3 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (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 (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein 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; wherein 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; wherein the CH2 region of the first heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein the CH2 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein 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 reduce or eliminate one or more of the following heavy-chain effector functions relative to a reference heavy chain that does not comprise the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 region such as SEQ ID NO: 100): ADCC, CDC, and / or binding affinity for 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))).

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

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

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

18. The multispecific protein of any one of claims 15 or 16, wherein the amino acid sequence of the first peptide linker comprises or consists of the following: (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, which comprises 1, 2, or 3 amino acid substitutions or consists thereof.

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

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

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

22. The multispecific protein of any one of claims 20 or 21, wherein the amino acids of the second peptide linker comprise or consist of the following: (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, which comprises 1, 2, or 3 amino acid substitutions or consists thereof.

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

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

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

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

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

28. A multispecific protein according to any one of the preceding claims, wherein the first hTAA and the second hTAA are the same.

29. A multispecific protein according to 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. A multispecific protein according to any one of the preceding claims, wherein the first scFv and the second scFv specifically bind to different epitopes of the same hTAA.

31. A multispecific protein according to 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. A multispecific protein according to any one of the preceding claims, 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. 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 acids numbered 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 acids numbered according to Kabat.

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 acids numbered 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 acids numbered according to Kabat.

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

36. A multispecific protein according to any one of the preceding claims, wherein the full-length antibody is an hIgG1 or hIgG4 antibody. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein 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, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein the amino acid sequence of the first heavy chain of the full-length antibody comprises serine at amino acid position T366, alanine at amino acid position L368, and valine at amino acid position Y407, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein the amino acid sequence of the first heavy chain of the full-length antibody comprises an amino acid substitution at amino acid position Y349, the amino acid position being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein the amino acid sequence of the first heavy chain of the full-length antibody comprises cysteine at amino acid position Y349, the amino acid position being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein the amino acid sequence of the second heavy chain of the full-length antibody comprises an amino acid substitution at amino acid position T366, the amino acid position being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein the amino acid sequence of the second heavy chain of the full-length antibody comprises tryptophan at amino acid position T366, the amino acid position being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein the amino acid sequence of the second heavy chain of the full-length antibody comprises an amino acid substitution at amino acid position S354, the amino acid position being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein the amino acid sequence of the second heavy chain of the full-length antibody comprises cysteine at amino acid position S354, the amino acid position being numbered according to the EU index of Kabat. The multispecific protein of any one of claims 1-36, wherein the full-length antibody is an IgG1 antibody, and wherein 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, the amino acid positions being numbered according to the EU index of Kabat.

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

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

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

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

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

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

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

53. A multispecific protein according to any of the preceding claims, wherein the multispecific protein substantially does not mediate ADCC, substantially does not mediate ADCP, substantially does not 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))). The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein 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, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein the amino acid sequences of the first heavy chain and the second heavy chain each comprise alanine at amino acid position L234, and / or alanine at amino acid position L235, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein 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, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG1 antibody, and wherein the amino acid sequences of the first heavy chain and the second heavy chain each comprise alanine at amino acid position L234, alanine at amino acid position L235, and / or alanine at amino acid position P329, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG4 antibody, and wherein the amino acid sequences of the first heavy chain and the second heavy chain each comprise alanine at amino acid position L234, alanine at amino acid position L235, and / or glycine at amino acid position P329, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG4 antibody, and wherein 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, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein of any one of the preceding claims, wherein the full-length antibody is an IgG4 antibody, and wherein the amino acid sequences of the first heavy chain and the second heavy chain each comprise proline at amino acid position S228, alanine at amino acid position F234, and / or alanine at amino acid position E235, the amino acid positions being numbered according to the EU index of Kabat. A multispecific protein comprising: (a) A first Fab, comprising: (i) a first Fab heavy chain that, from the N-terminus to the C-terminus, comprises a first VH region and a first CH1 region; and (ii) a first light chain that, from the N-terminus to the C-terminus, comprises 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, wherein the first scFv, from the N-terminus to the C-terminus, comprises (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, wherein the first Fc region, from the N-terminus to the C-terminus, comprises a CH2 region and a CH3 region; and (d) A second Fab, comprising: (i) a second Fab heavy chain that, from the N-terminus to the C-terminus, comprises a second VH region and a second CH1 region; and (ii) a second light chain that, from the N-terminus to the C-terminus, comprises a second VL region and a first CL region; (b) A second scFv operably linked to the C-terminus of the second CH1 region of the second Fab, wherein the second scFv, from the N-terminus to the C-terminus, comprises (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 Fc region operably linked to the C-terminus of the second scFv, wherein the second Fc region, from the N-terminus to the C-terminus, comprises a CH2 region and a CH3 region; and wherein the first Fab specifically binds to a first human hTAA; wherein the second Fab specifically binds to a second hTAA; wherein the first scFv specifically binds to hTCSA (e.g., hCD28, hCD2); wherein the second scFv specifically binds to hCD3; and wherein the CH3 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); wherein the CH3 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein 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; wherein 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; wherein the CH2 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); wherein the CH2 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); 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 relative to a reference Fc region that does not comprise the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 domain such as SEQ ID NO: 100): ADCC, CDC, and / or binding affinity for 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))); 62. The multispecific protein of claim 61, wherein the first Fab is operably linked directly to the first scFv by a peptide bond.

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

64. The multispecific protein of claim 63, wherein the amino acid sequence of the first peptide linker comprises glycine or glycine and serine amino acid residues or consists thereof. 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 the following: (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, which comprises 1, 2, or 3 amino acid substitutions or consists thereof.

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

67. The multispecific protein of any one of claims 61-65, wherein the second Fab is operably linked to the second scFv by a second peptide linker.

68. The multispecific protein of claim 67, wherein the amino acid sequence of the second peptide linker comprises glycine or glycine and serine amino acid residues or consists thereof. The multispecific protein of any one of claims 67 or 68, wherein the amino acids of the second peptide linker comprise or consist of the following: (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, which comprises 1, 2, or 3 amino acid substitutions or consists thereof.

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

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

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

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

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

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

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

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

78. A multispecific protein according to any one of claims 61-77, 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. A multispecific protein according to any one of claims 61-78, 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. The multispecific protein of claim 79, wherein the amino acid sequence of the VH region of the first scFv comprises cysteine at amino acid positions 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106, amino acids according to Kabat numbering; and the amino acid sequence of the VL region of the first scFv comprises cysteine at amino acid positions 40, 41, 42, 43, 44, 45, 46 or 47, amino acids according to Kabat numbering.

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, with amino acids numbered 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, with amino acids numbered according to Kabat.

82. A multispecific protein comprising: (a) an scFv that comprises, 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; (b) a Fab that comprises (i) a Fab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and a CH1 region, and (ii) a light chain that comprises, from the N-terminus to the C-terminus, a VL region and a CL region; (c) a first Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; (d) a second Fc region that comprises, from the N-terminus to the C-terminus, a CH2 region and a CH3 region; (e) an IgM CH2 mFab that comprises (i) an IgM CH2 mFab heavy chain that comprises, from the N-terminus to the C-terminus, a VH region and an IgM CH2 region; and (ii) an IgM CH2 mFab light chain that comprises, from the N-terminus to the C-terminus, a VL region and an IgM CH2 region; wherein the C-terminus of the scFv is operably linked to the N-terminus of the Fab heavy chain of the first Fab; wherein the C-terminus of the Fab heavy chain is operably linked to the N-terminus of the first Fc region; wherein the C-terminus of the IgM CH2 mFab heavy chain is operably linked to the N-terminus of the second Fc region; wherein the scFv specifically binds to hTCSA (e.g., hCD28, hCD2); wherein the Fab specifically binds to hCD3; wherein the IgM CH2 mFab specifically binds to a human tumor-associated antigen (hTAA); wherein the CH3 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); wherein the CH3 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region that does not comprise one or more amino acid modifications (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 101); and wherein 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; One or more amino acid modifications in the CH3 region of the first Fc region and one or more amino acid modifications in the CH3 region of the second Fc region promote heterodimerization of the first heavy chain and the second heavy chain; The CH2 region of the first Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); The CH2 region of the second Fc region comprises one or more amino acid modifications (e.g., substitutions) relative to a reference CH2 region that does not comprise the one or more amino acid modifications (e.g., a wild-type CH2 region, e.g., SEQ ID NO: 100); One or more amino acid modifications in the CH2 region of the first Fc region and 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 relative to a reference Fc region that does not comprise the one or more amino acid modifications (e.g., a heavy chain comprising a wild-type CH2 domain such as SEQ ID NO: 100): ADCC, CDC, and / or binding affinity for 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))); 83. The multispecific protein of claim 82, wherein the scFv is operably linked directly via a peptide bond to the N-terminus of the Fab heavy chain of the first Fab.

84. 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 via a first peptide linker.

85. The multispecific protein of claim 84, wherein the amino acid sequence of the first peptide linker comprises glycine or glycine and serine amino acid residues or consists thereof. The multispecific protein of any one of claims 84 or 85, wherein the amino acid sequence of the first peptide linker comprises or consists of the following: (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, which comprises 1, 2, or 3 amino acid substitutions or consists thereof.

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

88. The multispecific protein of any one of claims 82-87, wherein the scFv comprises, from the N-terminus to the C-terminus: a VH region, a peptide linker, and a VL region; and wherein the C-terminus of the VL region of the scFv is operably linked to the N-terminus of the Fab heavy chain of the first Fab. The multispecific protein of any one of claims 82-88, wherein the tumor-associated antigen is hEGFR, hMSLN, hPSMA or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA or hHER2. The multispecific protein of any one of claims 82-89, 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. The multispecific protein of claim 90, wherein the amino acid sequence of the VH region of the first scFv contains cysteine at amino acid positions 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106, with the amino acids numbered according to Kabat; and the amino acid sequence of the VL region of the first scFv contains cysteine at amino acid positions 40, 41, 42, 43, 44, 45, 46 or 47, with the amino acids numbered according to Kabat. The multispecific protein of claim 90 or 91, wherein the amino acid sequence of the VH region of the second scFv contains cysteine at amino acid positions 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106, with the amino acids numbered according to Kabat; and the amino acid sequence of the VL region of the second scFv contains cysteine at amino acid positions 40, 41, 42, 43, 44, 45, 46 or 47, with the amino acids numbered according to Kabat. The multispecific protein of any one of claims 61-92, wherein the first Fc region and the second Fc region are of human IgG (hIgG) isotype. The multispecific protein of any one of claims 61-93, wherein the first Fc region and the second Fc region are of hIgG1 or hIgG4 isotype. The multispecific protein of any one of claims 61-94, wherein the first Fc region and the second Fc region are of IgG1 isotype, and wherein the amino acid sequence of the first Fc region contains amino acid substitutions at amino acid positions T366, L368 and Y407, with the amino acid positions numbered according to the EU index of Kabat. The multispecific protein of any one of claims 61-95, wherein the first Fc region and the second Fc region are of IgG1 isotype, and wherein the amino acid sequence of the first Fc region contains serine at amino acid position T366, alanine at amino acid position L368 and valine at amino acid position Y407, with the amino acid positions numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-96, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, the amino acid position being numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-97, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the first Fc region comprises a cysteine at amino acid position Y349, the amino acid position being numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-98, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, the amino acid position being numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-99, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises a tryptophan at amino acid position T366, the amino acid position being numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-100, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position S354, the amino acid position being numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-101, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises a cysteine at amino acid position S354, the amino acid position being numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-102, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises amino acid substitutions at amino acid positions T366, L368 and Y407, the amino acid positions being numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-94 or 103, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein 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, the amino acid positions being numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-94 or 103-104, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position Y349, the amino acid position being according to the EU index numbering of Kabat. A multispecific protein according to any one of claims 61-94 or 103-105, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the second Fc region comprises a cysteine at amino acid position Y349, the amino acid position being according to the EU index numbering of Kabat. A multispecific protein according to any one of claims 61-94 or 103-106, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position T366, the amino acid position being according to the EU index numbering of Kabat. A multispecific protein according to any one of claims 61-94 or 103-107, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the first Fc region comprises a tryptophan at amino acid position T366, the amino acid position being according to the EU index numbering of Kabat. A multispecific protein according to any one of claims 61-94 or 103-108, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position S354, the amino acid position being according to the EU index numbering of Kabat. A multispecific protein according to any one of claims 61-94 or 103-109, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequence of the first Fc region comprises a cysteine at amino acid position S354, the amino acid position being according to the EU index numbering of Kabat. A multispecific protein according to any one of claims 61-110, wherein the multispecific protein substantially does not mediate ADCC, substantially does not mediate ADCP, substantially does not 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))). The multispecific protein according to any one of claims 61-111, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein 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, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein according to any one of claims 61-112, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequences of the first Fc region and the second Fc region each comprise alanine at amino acid position L234 and / or alanine at amino acid position L235, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein according to any one of claims 61-113, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein 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, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein according to any one of claims 61-114, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequences of the first Fc region and the second Fc region each comprise alanine at amino acid position L234, alanine at amino acid position L235 and / or alanine at amino acid position P329, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein according to any one of claims 61-115, wherein the first Fc region and the second Fc region are of the IgG1 isotype, and wherein the amino acid sequences of the first Fc region and the second Fc region each comprise alanine at amino acid position L234, alanine at amino acid position L235 and / or glycine at amino acid position P329, the amino acid positions being numbered according to the EU index of Kabat. The multispecific protein according to any one of claims 61-116, wherein the first Fc region and the second Fc region are of the IgG4 isotype, and wherein 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, the amino acid positions being numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-117, wherein the first Fc region and the second Fc region are of the IgG4 isotype, and wherein 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, the amino acid positions being numbered according to the EU index of Kabat. A multispecific protein according to any one of claims 61-118, wherein the tumor-associated antigen is hEGFR, hMSLN, hPSMA or hHER2; and the second tumor-associated antigen is hEGFR, hMSLN, hPSMA or hHER2. A multispecific protein according to any one of the preceding claims, wherein the hTCSA is hCD28, hCD2, hCD137, hCD27, hCD278, hCD134 or hCD40. A multispecific protein according to any one of the preceding claims, wherein the hTCSA is hCD28. A multispecific protein according to any one of the preceding claims, wherein the hTCSA is hCD2. A polynucleotide encoding a multispecific protein according to any one of the preceding claims or one or more polypeptides thereof. The polynucleotide of claim 123, wherein the polynucleotide is RNA (e.g., mRNA) or DNA. The polynucleotide of claim 123 or 124, wherein the polynucleotide is codon-optimized. An expression vector comprising the polynucleotide of any one of claims 123-125. The expression vector of claim 126, wherein the expression vector is a viral vector or a plasmid. A host cell comprising the multispecific protein of any one of claims 1-122, the polynucleotide of any one of claims 123-125, or the expression vector of any one of claims 126-127. A carrier comprising the multispecific protein of any one of claims 1-122, the polynucleotide of any one of claims 123-125, or the expression vector of any one of claims 126-127. The carrier of claim 129, wherein the carrier is a lipid nanoparticle, a liposome, a lipid complex or a nanoliposome. A pharmaceutical composition comprising the multispecific protein of any one of claims 1-122, the polynucleotide of any one of claims 123-125, the expression vector of any one of claims 126-127, the host cell of claim 128, or the carrier of any one of claims 129-130, and a pharmaceutically acceptable excipient.

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

133. A method for preparing a multispecific protein according to any one of claims 1-122, comprising: introducing a polynucleotide according to any one of claims 123-125 or a vector according to any one of claims 126-127 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.

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

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

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

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

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

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, thymic cancer, lung cancer, bronchial cancer, skin cancer, brain cancer, spinal cord cancer, head cancer, neck cancer, lip cancer or oral cancer.

140. A multispecific protein according to any one of claims 1-122, a polynucleotide according to any one of claims 123-125, an expression vector according to any one of claims 126-127, a host cell according to claim 128, a carrier according to any one of claims 129-130 or a pharmaceutical composition according to claim 131 for use in a method of preventing or treating cancer in a subject, the method 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. Use of a multispecific protein according to any one of claims 1-122, a polynucleotide according to any one of claims 123-125, an expression vector according to any one of claims 126-127, a host cell according to claim 128, a carrier according to any one of claims 129-130 or a pharmaceutical composition according to claim 131 in 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. A multispecific protein according to any one of claims 1-122, a polynucleotide according to any one of claims 123-125, an expression vector according to any one of claims 126-127, a host cell according to claim 128, a carrier according to any one of claims 129-130 or a pharmaceutical composition according to claim 131 for use in a method of inducing an immune response in a subject, the method 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. Use of a multispecific protein according to any one of claims 1-122, a polynucleotide according to any one of claims 123-125, an expression vector according to any one of claims 126-127, a host cell according to claim 128, a carrier according to any one of claims 129-130 or a pharmaceutical composition according to claim 131 in the manufacture of a medicament for 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. A multispecific protein according to any one of claims 1-122, a polynucleotide according to any one of claims 123-125, an expression vector according to any one of claims 126-127, a host cell according to claim 128, a carrier according to any one of claims 129-130, or a pharmaceutical composition according to claim 131, for use in a method of activating T cells or a population of T cells in a subject, the method comprising administering to the subject the multispecific protein, the polynucleotide, the expression vector, the host cell, the carrier or the pharmaceutical composition, thereby activating T cells or a population of T cells in the subject. Use of a multispecific protein according to any one of claims 1-122, a polynucleotide according to any one of claims 123-125, an expression vector according to any one of claims 126-127, a host cell according to claim 128, a carrier according to any one of claims 129-130, or a pharmaceutical composition according to claim 131, in the manufacture of a medicament for activating T cells 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 T cells or a population of T cells in the subject.

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