Anti-CD38 antibodies and methods of use

Abstract

To provide binding proteins that bind CD38 polypeptides, for example, human and cynomolgus monkey CD38 polypeptides.SOLUTION: For example, the binding proteins are monospecific, bispecific, or trispecific binding proteins with at least one antigen binding domain that binds a CD38 polypeptide. The disclosure also provides methods for making binding proteins that bind CD38 polypeptides and uses of such binding proteins.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 570,655, filed October 10, 2017, U.S. Provisional Application No. 62 / 570,660, filed October 11, 2017, U.S. Provisional Application No. 62 / 676,221, filed May 24, 2018, and European Patent Application No. EP18187186.4, filed August 3, 2018, all of which are incorporated herein by reference in their entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (Filename: 183952029941seqlist.TXT, Date Recorded: October 8, 2018, Size: 158KB).

[0003] The present disclosure relates to binding proteins that bind to CD38 polypeptides (e.g., human and cynomolgus CD38 polypeptides), including monospecific, bispecific, or trispecific binding proteins having at least one antigen-binding domain that binds to a CD38 polypeptide, as well as related polynucleotides, host cells, methods of production, and methods of use. [Background technology]

[0004] Monoclonal antibody-based biotherapeutics have become an important tool for new drug development. Monoclonal antibody technology offers specific targeting, precise signaling delivery and / or payload to specific cell populations, and long-lasting biological effects via its Fc function. Efforts in antibody engineering have enabled the development of multispecific antibodies that combine the specificities of multiple monoclonal antibodies for various biological applications, broadening the scope of antibody drug development.

[0005] CD38 is an attractive drug target because it is expressed on the cell surface of various lymphoid tumor cells (see Non-Patent Document 1). DARZALEX® (daratumumab) is an anti-CD38 antibody approved for use in the treatment of multiple myeloma. However, there is a need for CD38-targeted therapeutic agents with different modes of action and / or improved properties, including, but not limited to, high-affinity binding to CD38, cross-reactivity between human and cynomolgus monkey CD38 polypeptides, binding to lymphoma cells (e.g., multiple myeloma, large B-cell lymphoma cell lines), and the ability to induce apoptosis and / or antibody-dependent cell-mediated cytotoxicity (ADCC) and T-cell-mediated antitumor activity. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Stevenson, GT (2006) Mol.Med.12:345~346 Summary of the Invention [Means for solving the problem]

[0007] CD38 polypeptides (e.g., CD38 polypeptides), including monospecific, bispecific, or trispecific binding proteins having at least one antigen-binding site that binds to a CD38 polypeptide (e.g., Provided herein are binding proteins that bind to human and cynomolgus monkey CD38 polypeptides. Advantageously, these binding proteins have the ability to recruit T cells to the vicinity of cancer cells, subsequently activating the T cells and promoting killing of neighboring cancer cells by the activated T cells via a granzyme / perforin mechanism, providing a different mode of action than the anti-tumor activity derived from anti-CD38 antibodies such as DARZALEX® (daratumumab). Furthermore, the ability to bind to both human and cynomolgus monkey CD38 polypeptides allows the binding proteins to be readily tested in preclinical toxicology studies, for example, to assess their safety profile for subsequent clinical use.

[0008] In some embodiments, provided herein are monospecific binding proteins that bind to human CD38 polypeptides. In some embodiments, the binding proteins cross-react with human and cynomolgus monkey CD38 polypeptides. In some embodiments, the binding proteins bind to human isoform A and isoform E CD38 polypeptides. In some embodiments, the binding proteins have one or more of the following configurations (in any combination): as a purified protein, they bind to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) when assayed by SPR; a K of 1.5 nM or less when assayed by SPR; D binds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein; binds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the cell surface when assayed by flow cytometry; has an apparent K of 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, or less when assayed by flow cytometry. Dbinds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the cell surface; binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein when assayed by SPR; and has a K of 3.5 nM or less when assayed by SPR. D binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein; binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the cell surface when assayed by flow cytometry; and has an apparent K of 7.5 nM or less when assayed by flow cytometry. D The binding protein binds to the extracellular domain of a cell-surface-expressed cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) at a specific concentration; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) as a purified protein when assayed by ELISA; binds to the extracellular domain of a cell-surface-expressed human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) when assayed by flow cytometry; induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells expressing CD38 on their cell surface; and has one or more mutations (e.g., in the Fc region) that result in reduced binding to FcγRI and / or FcγRII compared to the same binding protein without the one or more mutations. For exemplary assays, see Examples 1, 3, and 4. In some embodiments, the KD is measured at 4°C or 25°C.

[0009] In some embodiments, provided herein is a trispecific binding protein that binds to a human CD38 polypeptide. In some embodiments, the trispecific binding protein binds (e.g., simultaneously) to a CD38 polypeptide (e.g., expressed on a cell surface) and one or more other target antigens expressed on the surface of a second cell, thereby recruiting the second cell to the proximity of the cell that expresses the CD38 polypeptide. In some embodiments, In some embodiments, the trispecific binding protein binds (e.g., simultaneously) to a CD38 polypeptide (e.g., expressed on a cell surface) and one or two target antigens expressed on the T cell surface, thereby recruiting the T cell to the proximity of cells expressing the CD38 polypeptide. In some embodiments, the trispecific binding protein activates the T cell and / or provides a CD28-mediated costimulatory signal to the T cell. In some embodiments, the trispecific binding protein cross-reacts with human and cynomolgus CD38 polypeptides. In some embodiments, the trispecific binding protein binds to human isoform A and isoform E CD38 polypeptides. In some embodiments, the trispecific binding protein has one or more of the following configurations (in any combination): as a purified protein, it binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1), when assayed by SPR; a K of 1.5 nM or less, when assayed by SPR; D binds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein; binds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the cell surface when assayed by flow cytometry; has an apparent K of 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, or less when assayed by flow cytometry. Dbinds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the cell surface; binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein when assayed by SPR; and has a K of 3.5 nM or less when assayed by SPR. D binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein; binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the cell surface when assayed by flow cytometry; and has an apparent K of 7.5 nM or less when assayed by flow cytometry. Dand binds to the extracellular domain of a cell surface-expressed cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30); as a purified protein, binds to the extracellular domain of a cell surface-expressed human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) when assayed by ELISA; as a purified protein, binds to the extracellular domain of a cell surface-expressed human isoform E when assayed by flow cytometry. binds to the extracellular domain of a CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105); induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells expressing CD38 on their cell surface; and has one or more mutations (e.g., in the Fc region) that result in decreased binding to FcγRI and / or FcγRII compared to the same binding protein without the one or more mutations; induces proliferation of T cells (e.g., CD4+ and / or CD8+ T cells); induces expression of Bcl-xL by T cells (e.g., CD4+ and / or CD8+ T cells); induces apoptosis of CD38+ cells; inhibits cell surface-expressed CD38 and T cell surface-expressed CD38. The binding protein binds to one or more T cell target antigens expressed on CD38, CD28, and CD3 expressed on the T cell surface; stimulates T cell receptor activation; induces costimulation of T cell receptor signaling (e.g., when mediated by CD28); and has one or more mutations (e.g., in the Fc region) that result in reduced induction of cytokine release (e.g., IFN-γ, IL-2, and / or TNF-α) by PBMCs compared to the same binding protein without the one or more mutations; induces cytokine release (e.g., IFN-γ and / or IL-6) by PBMCs in the presence of CD38+ target cells. See Examples 1, 3, and 4 for exemplary assays. In some embodiments, the KD is measured at 4°C or 25°C.

[0010] In some embodiments, a binding protein comprising an antigen binding site that binds to a CD38 polypeptide. Provided herein are binding proteins, wherein the antigen-binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and / or (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the antigen-binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and / or (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).In some embodiments, the antigen-binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and / or (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the VH domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO: 87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94); and FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96). In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 5 and / or the VL domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 17 and / or the VL domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 20.In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 21, and / or the VL domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 23, and / or the VL domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 24 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antigen-binding site comprises: (a) the amino acid sequence of GYTFTSYA (SEQ ID NO: 37). (b) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT, and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the VH domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO: 87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94); and FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96). In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 13 and / or the VL domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 16.

[0011]

[0010] In some embodiments, provided herein is a binding protein comprising an antigen-binding site that binds to a CD38 polypeptide, wherein the antigen-binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 9, and / or the VL domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody binding site cross-reacts with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide. In some embodiments, the antigen binding site binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 ... equilibrium dissociation constant (K D ) to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antigen-binding site binds to a human isoform E CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, the antigen-binding site binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antigen-binding site binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30 with an equilibrium dissociation constant (K D ) and binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30.

[0012] In some embodiments of any of the above embodiments, the binding protein is a chimeric or humanized antibody. In some embodiments, the binding protein is a human antibody. In some embodiments, the binding protein is a monoclonal antibody. In some embodiments, the binding protein comprises one or more full-length antibody heavy chains comprising an Fc region. In some embodiments, the Fc region is a human Fc region comprising one or more mutations that reduce or eliminate Fc receptor binding and / or effector function of the Fc region. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG1 The Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, with the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the human IgG1 Fc region comprises amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, with the amino acid substitutions being S298N, T299A, and Y300S. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, with the amino acid substitutions being S228P and R409K. In some embodiments, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, with the amino acid substitutions being F234A and L235A. In some embodiments, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index, where the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 233-237 of human IgG4 according to the EU index, where the sequence EFLGG is replaced by PVAG. In some embodiments, the binding protein comprises an antibody F(ab), F(ab'), Fab'-SH, Fv, or scFv fragment. In some embodiments, the binding protein is conjugated to a cytotoxic agent or label. In some embodiments, the binding protein is a bispecific binding protein comprising a first antigen-binding site that binds to a CD38 polypeptide and a second antigen-binding site. In some embodiments, the binding protein is a trispecific binding protein comprising a first antigen-binding site that binds to a CD38 polypeptide, a second antigen-binding site, and a third antigen-binding site. In some embodiments, the first antigen-binding site binds to the extracellular domain of a human CD38 polypeptide, and the second and third antigen-binding sites each bind to a T-cell surface protein.In some embodiments, the first antigen-binding site binds to the extracellular domain of a human CD38 polypeptide, and (a) the second antigen-binding site binds to a human CD28 polypeptide and the third antigen-binding site binds to a human CD3 polypeptide, or (b) the second antigen-binding site binds to a human CD3 polypeptide and the third antigen-binding site binds to a human CD28 polypeptide.

[0013] In some embodiments, provided herein are binding proteins comprising three antigen-binding sites, each binding to one or more target proteins, wherein at least one of the three antigen-binding sites cross-reacts with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide. In some embodiments, the binding protein cross-reacts with a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 105. In some embodiments, the binding protein cross-reacts with a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the binding protein comprises an antigen-binding site cross-reacting with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide, and two antigen-binding sites each binding to a T cell surface protein. In some embodiments, the binding protein comprises an antigen-binding site cross-reacting with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide, an antigen-binding site binding to a human CD28 polypeptide, and an antigen-binding site binding to a human CD3 polypeptide. In some embodiments, the binding protein comprises four polypeptide chains that form three antigen binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -CH3 [II] It includes a structure represented by The third polypeptide chain has the formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] It includes a structure represented by During the ceremony, V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 is an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; (a)V H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); (b)V H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNG (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or (c)V H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the binding protein comprises four polypeptide chains that form three antigen-binding sites, wherein the first polypeptide chain has the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by The third polypeptide chain has the formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] It includes a structure represented by During the ceremony, V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 is an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; (a)V H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); (b)V H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or (c)V H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence containing the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and L1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or V H3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or V H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and L3 The domain comprises a CDR-L1 sequence containing the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence containing the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the CDR-L3 sequence comprises a V H3 The domain comprises the amino acid sequence of SEQ ID NO: 5, L3 The domain comprises the amino acid sequence of SEQ ID NO: 6; H3 The domain comprises the amino acid sequence of SEQ ID NO: 17, L3 The domain comprises the amino acid sequence of SEQ ID NO: 18; H3 The domain comprises the amino acid sequence of SEQ ID NO: 21,L3 The domain comprises the amino acid sequence of SEQ ID NO: 18; H3 The domain comprises the amino acid sequence of SEQ ID NO: 23, L3 The domain comprises the amino acid sequence of SEQ ID NO: 18; or V H3 The domain comprises the amino acid sequence of SEQ ID NO: 13, L3 The domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the binding protein comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain has the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by The third polypeptide chain has the formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] It includes a structure represented by During the ceremony, V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 is an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; (a)V H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and V L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46); (b)V H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and V L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46); or (c)V H3 The domains are CDR-H1 sequence containing the amino acid sequence GFTFSSYG (SEQ ID NO: 41), CDR-H2 sequence containing the amino acid sequence IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and V L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H3 The domain comprises the amino acid sequence of SEQ ID NO: 9, L3 The V domain comprises the amino acid sequence of SEQ ID NO: 10. H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, L2 The domain comprises the amino acid sequence of SEQ ID NO: 54; H2 The domain comprises the amino acid sequence of SEQ ID NO: 49, L2 The domain comprises the amino acid sequence of SEQ ID NO: 50, H1 The domain comprises the amino acid sequence of SEQ ID NO: 53, L1 The domain comprises the amino acid sequence of SEQ ID NO: 54; H1 The domain comprises the amino acid sequence of SEQ ID NO: 51, L1The domain comprises the amino acid sequence of SEQ ID NO: 52, H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, L2 The domain comprises the amino acid sequence of SEQ ID NO: 54; H2 The domain comprises the amino acid sequence of SEQ ID NO: 51, L2 The domain comprises the amino acid sequence of SEQ ID NO: 52, H1 The domain comprises the amino acid sequence of SEQ ID NO: 53, L1 The domain comprises the amino acid sequence of SEQ ID NO: 54; H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, H2 The domain comprises the amino acid sequence of SEQ ID NO: 84, L2 The domain comprises the amino acid sequence of SEQ ID NO: 85; H2 The domain comprises the amino acid sequence of SEQ ID NO: 49, L2 The domain comprises the amino acid sequence of SEQ ID NO: 50, H1 The domain comprises the amino acid sequence of SEQ ID NO: 84, L1 The domain comprises the amino acid sequence of SEQ ID NO: 85; H1 The domain comprises the amino acid sequence of SEQ ID NO: 51, L1 The domain comprises the amino acid sequence of SEQ ID NO: 52, H2 The domain comprises the amino acid sequence of SEQ ID NO: 84, L2 The domain comprises the amino acid sequence of SEQ ID NO: 85; or V H2 The domain comprises the amino acid sequence of SEQ ID NO: 51, L2 The domain comprises the amino acid sequence of SEQ ID NO: 52, H1 The domain comprises the amino acid sequence of SEQ ID NO: 84, L1 The V domain comprises the amino acid sequence of SEQ ID NO: 85. H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, L2 The domain comprises the amino acid sequence of SEQ ID NO: 54,H3 The domain comprises the amino acid sequence of SEQ ID NO: 13, L3 The domain comprises the amino acid sequence of SEQ ID NO: 14; or V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, L2 The domain comprises the amino acid sequence of SEQ ID NO: 54, H3 The domain comprises the amino acid sequence of SEQ ID NO: 9, L3 The domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, at least one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments, L1, L2, L3, or L4 is each independently at least 1 amino acid in length. In some embodiments, (a) L1, L2, L3, and L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59); or (b) L1, L2, L3, and L4 are each independently GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPK AAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO:58), L2 comprises the sequence TKGPS (SEQ ID NO:57), L3 comprises the sequence S, and L4 comprises the sequence RT; L1 comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; L1 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; or L1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), and L4 is 0 amino acids in length. In some embodiments, the hinge-C of the second and third polypeptide chains H2 -C H3 The domain is the hinge-C of human IgG4. H2 -C H3 domain, hinge-C H2 -C H3 In some embodiments, the domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A. H2 -C H3 The domain is the hinge-C of human IgG4. H2 -C H3 domain, hinge-C H2 -C H3 Each domain comprises an amino acid substitution at a position corresponding to positions 233-236 of human IgG4 according to the EU index, the amino acid substitutions being E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-C region of the second and third polypeptide chains H2 -C H3 The domain is the hinge-C of human IgG4. H2 -C H3 domain, hinge-C H2 -CH3 In some embodiments, the domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. H2 -C H3 The domain is the hinge-C domain of human IgG1. H2 -C H3 domain, hinge-C H2 -C H3 The domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the hinge-C of the second and third polypeptide chains H2 -C H3 The domain is the hinge-C domain of human IgG1. H2 -C H3 domain, hinge-C H2 -C H3 The domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, the amino acid substitutions being S298N, T299A, and Y300S. In some embodiments, the hinge-C of the second polypeptide chain H2 -C H3 The domain contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; the hinge-C of the third polypeptide chain H2 -C H3 In some embodiments, the domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W. H2 -C H3The domain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; H2 -C H3 The domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 65, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 67, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the first The polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69.

[0014] In some embodiments, a binding protein comprises three antigen binding sites that each bind to one or more target proteins, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, and a first polypeptide chain has the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by The third polypeptide chain has the formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] It includes a structure represented by During the ceremony, V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 is an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair; (a) Hinge-C of the second and third polypeptide chains H2 -C H3 The domain is the hinge-C domain of human IgG1. H2 -C H3 domain, hinge-C H2 -C H3 The domain is (b) containing amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, the amino acid substitutions being S298N, T299A, and Y300S; or (b) containing amino acid substitutions at positions S298N, T299A, and Y300S in the hinge-C region of the second and third polypeptide chains, respectively. H2 -C H3 The domain is the hinge-C of human IgG4. H2 -C H3 domain, hinge-C H2 -C H3Provided herein are binding proteins, each of which contains an amino acid substitution at a position corresponding to positions 233-236 of human IgG4 according to the EU index, the amino acid substitutions being E233P, F234V, L235A, and a deletion at 236. In some embodiments, the domains include a deletion at position 236 of the hinge-C region of human IgG4. H2 -C H3 The domain contains amino acid substitutions at positions corresponding to positions 233-237 of human IgG4 according to the EU index, where the sequence EFLGG is replaced by PVAG. H1 and V L1 , V H2 and V L2 , and V H3 and V L3 At least one pair of V forms an antigen-binding site that binds to a CD38 polypeptide. H1 and V L1 , V H2 and V L2 , and V H3 and V L3One, two, or three pairs of these are: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD3 9, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi2 4, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6 , IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM,and XCR1. In some embodiments, V, H1 and V L1 , V H2 and V L2 , and V H3 and V L3 The first pair of V forms an antigen-binding site that binds to the human CD3 polypeptide, and V H1 and V L1 , V H2 and V L2 , and V H3 and V L3 a second pair of V forms an antigen-binding site that binds to the human CD28 polypeptide, and V H1 and V L1 , V H2 and V L2 , and V H3 and V L3 The third pair of antibodies is A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, and CD137. , CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R , IL2R beta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL 12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, S The antibody forms an antigen-binding site that binds to a human antigen target selected from the group consisting of LC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFα, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.

[0015] In some embodiments, a kit of polynucleotides includes: (a) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 72, a third polynucleotide comprising the sequence of SEQ ID NO: 74, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 75; (b) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 76, a third polynucleotide comprising the sequence of SEQ ID NO: 77, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 75; (c) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 78, a third polynucleotide comprising the sequence of SEQ ID NO: 79, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 75. (d) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:72, a third polynucleotide comprising the sequence of SEQ ID NO:80, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81; (e) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:76, a third polynucleotide comprising the sequence of SEQ ID NO:82, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81; or (f) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:78, a third polynucleotide comprising the sequence of SEQ ID NO:83, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81.

[0016] In some embodiments, provided herein is a polynucleotide comprising the binding protein of any one of the above embodiments. In some embodiments, provided herein is a vector comprising the polynucleotide comprising the binding protein of any one of the above embodiments.

[0017] In some embodiments, provided herein is a host cell comprising any one of the polynucleotide kits, polynucleotides, or vectors of the above embodiments. In some embodiments, provided herein is a method for producing a binding protein, comprising culturing any one of the host cells of the above embodiments so that the binding protein is produced. In some embodiments, the method further comprises recovering the binding protein from the host cell.

[0018] In some embodiments, provided herein is a pharmaceutical composition comprising the binding protein of any one of the above embodiments and a pharmaceutically acceptable carrier.

[0019] In some embodiments, provided herein are methods for preventing and / or treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of at least one binding protein of any one of the above embodiments or a pharmaceutical composition of any one of the above embodiments. In some embodiments, the binding protein is a trispecific binding protein comprising a first antigen-binding site that binds to CD3, a second antigen-binding site that binds to CD28, and a third antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide. In some embodiments, the at least one binding protein is co-administered with a chemotherapeutic agent. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma. In some embodiments, the patient is human. In some embodiments, the patient is selected for treatment because the cancer cells express human CD38 isoform E polypeptide (e.g., as set forth in SEQ ID NO: 105) on their cell surface. In some embodiments, the cancer cells express CD38 and CD28. In some embodiments, the cancer cells express CD38 and do not express CD28.

[0020] In some embodiments, provided herein is at least one binding protein of any one of the above embodiments or a pharmaceutical composition of any one of the above embodiments for use in preventing and / or treating cancer in a patient (e.g., a patient in need thereof, such as a patient with cancer). In some embodiments, provided herein is at least one binding protein of any one of the above embodiments or a pharmaceutical composition of any one of the above embodiments for use in the manufacture of a medicament for preventing and / or treating cancer in a patient (e.g., a patient in need thereof, such as a patient with cancer). In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein comprising a first antigen-binding site that binds CD3, a second antigen-binding site that binds CD28, and a third antigen-binding site that binds the extracellular domain of human CD38 polypeptide. In some embodiments, the at least one binding protein is to be co-administered with a chemotherapeutic agent. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma. In some embodiments, the patient is human. In some embodiments, the patient is selected for treatment because the cancer cells express human CD38 isoform E polypeptide (e.g., as set forth in SEQ ID NO: 105) on their cell surface. In some embodiments, the cancer cells express CD38 and CD28. In some embodiments, the cancer cells express CD38 but do not express CD28.

[0021] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows.

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) are expected to be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0023] [Figure 1A-1] 1 is a graph showing binding of anti-CD38 antibody mAb1 (top) and isatuximab (bottom) to SU-DHL-8 human lymphoma cells or MOLP-8 human multiple myeloma cells using flow cytometry. [Figure 1A-2] Continued from Figure 1A-1. [Figure 1B] 1 is a graph showing the results of a flow cytometry binding assay investigating the binding of anti-CD38 antibody mAb1 or isatuximab (no binding observed) to cells expressing cynomolgus monkey CD38 on their surface. [Figure 2A-1] Figures 2A-2I are graphs showing the results of assays characterizing the binding of anti-CD38 antibodies to human and cynomolgus monkey CD38 polypeptides. Figure 2A is a graph showing the binding of humanized anti-CD38 antibody mAb2 to soluble human CD38 (top, "hCD38::Histag") or cynomolgus monkey CD38 (top, "cynoCD38::Histag") by ELISA, and the binding of mAb2 to the surface of cells expressing human CD38 (bottom, as indicated) or cynomolgus monkey CD38 (bottom, as indicated) by flow cytometry. [Figure 2A-2] Continued from Figure 2A-1. [Figure 2B] FIG. 2B is a graph showing the binding of mAb2 to human CD38 (top) or cynomolgus monkey CD38 (bottom) by surface plasmon resonance (SPR). [Figure 2C-1]Figure 2C is a graph showing the binding of humanized anti-CD38 antibody mAb3 to soluble human CD38 (top, "hCD38::Histag") or cynomolgus monkey CD38 (top, "cynoCD38::Histag") by ELISA, and the binding of mAb3 to the surface of cells expressing human CD38 (bottom, as indicated) or cynomolgus monkey CD38 (bottom, as indicated) by flow cytometry. [Figure 2C-2] Continued from Figure 2C-1. [Figure 2D] FIG. 2D is a graph showing the binding of mAb3 to human CD38 (top) or cynomolgus monkey CD38 (bottom) by SPR. [Figure 2E-1] Figure 2E is a graph showing the binding of humanized anti-CD38 antibody mAb5 to soluble human CD38 (top, "hCD38::Histag") or cynomolgus monkey CD38 (top, "cynoCD38::Histag") by ELISA, and the binding of mAb5 to the surface of cells expressing human CD38 (bottom, as indicated) or cynomolgus monkey CD38 (bottom, as indicated) by flow cytometry. [Figure 2E-2] Continued from Figure 2E-1. [Figure 2F] FIG. 2F is a graph showing the binding of mAb5 to human CD38 (top) or cynomolgus monkey CD38 (bottom) by SPR. [Figure 2G-1] Figure 2G is a graph showing the binding of the human anti-CD38 antibody hhy1370 to soluble human CD38 (top, "hCD38::Histag") or cynomolgus monkey CD38 (top, "cynoCD38::Histag") by ELISA, and the binding of hhy1370 to the surface of cells expressing human CD38 (bottom, as indicated) or cynomolgus monkey CD38 (bottom, as indicated) by flow cytometry. [Figure 2G-2] Continued from Figure 2G-1. [Figure 2H] FIG. 2H is a graph showing the binding of hhy1370 to human CD38 (top) or cynomolgus monkey CD38 (bottom) by SPR. [Figure 2I]Figure 2I is a table summarizing the binding data from ELISA, SPR, and FACS experiments, as well as the percentage identity of the VH ("H") or VL ("L") domain of each antibody to the sequence of the human V region, corresponding from top to bottom to mAb2, mAb3, mAb4, mAb5, and mAb6 (bottom row) (1195 HHKK-3 is not described by its VL / VH amino acid sequence in the following text). [Figure 2J] 1 is a graph showing the concentration-dependent induction of apoptosis in SU-DHL-8 cells by mAb7 and mAb1 after 72 hours of incubation at 37° C. [Figure 2K] 1 is a graph showing the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isatuximab and mAb1 against SU-DHL-8 cells in the presence of NK92 cells. [Figure 2L] Graph showing concentration-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isatuximab (right) and mAb1 (left) against SU-DHL-8 cells in the presence of NK92 cells after 4 hours at 37°C. [Figure 2M] Figure 2M shows the results of an apoptosis induction assay using the indicated anti-CD38 antibodies on SU-DHL-8 tumor cells. Apoptosis was quantified by measuring dual uptake of annexin V and propidium iodide by flow cytometry. Figure 2M shows the percentage of apoptotic cells induced by each antibody. [Figure 2N] Figures 2N-2Q show the dose-dependent induction of apoptosis in SU-DHL-8 lymphoma cells by anti-CD38 antibodies mAb2 (Figure 2N), mAb3 (Figure 2O), mAb4 (Figure 2P), and mAb5 (Figure 2Q), as well as the IC50 for each antibody. [Figure 2O] Figures 2N-2Q show the dose-dependent induction of apoptosis in SU-DHL-8 lymphoma cells by anti-CD38 antibodies mAb2 (Figure 2N), mAb3 (Figure 2O), mAb4 (Figure 2P), and mAb5 (Figure 2Q), as well as the IC50 for each antibody. [Figure 2P]Figures 2N-2Q show the dose-dependent induction of apoptosis in SU-DHL-8 lymphoma cells by anti-CD38 antibodies mAb2 (Figure 2N), mAb3 (Figure 2O), mAb4 (Figure 2P), and mAb5 (Figure 2Q), as well as the IC50 for each antibody. [Figure 2Q] Figures 2N-2Q show the dose-dependent induction of apoptosis in SU-DHL-8 lymphoma cells by anti-CD38 antibodies mAb2 (Figure 2N), mAb3 (Figure 2O), mAb4 (Figure 2P), and mAb5 (Figure 2Q), as well as the IC50 for each antibody. [Figure 3A] Figure 3A provides a schematic diagram of a trispecific binding protein containing four polypeptide chains that form three antigen-binding sites that bind to three target proteins: CD28, CD3, and CD38. The first pair of polypeptides has dual variable domains with crossover orientation (VH1-VH2 and VL2-VL1) that form two antigen-binding sites that recognize CD3 and CD28, and the second pair of polypeptides has single variable domains (VH3 and VL3) that form a single antigen-binding site that recognizes CD38. The trispecific binding protein shown in Figure 3A uses an IgG4 constant region with "knobs-into-holes" mutations, with the knobs on the second pair of polypeptides having a single variable domain. [Figure 3B] FIG. 1 provides a schematic of an SPR-based assay to investigate the ability of each antigen-binding domain of an anti-CD38 / anti-CD28 / anti-CD3 trispecific binding protein to bind to its cognate antigen. [Figure 3C] 1 is a graph showing the results of an SPR-based assay to investigate CD38 binding to an anti-CD38 / anti-CD28 / anti-CD3 trispecific binding protein. Binding of human CD38 to the trispecific binding protein was investigated alone (top left), after pre-binding with CD3 (top center), after pre-binding with CD28 (top right), after pre-binding to CD3 and then CD28 (bottom left), or after pre-binding to CD28 and then CD3 (bottom right). [Figure 4]1 is a graph showing sequential binding of human CD3, CD28, and CD38 polypeptides to an anti-CD38 / anti-CD28 / anti-CD3 trispecific binding protein as assayed by SPR. [Figure 5] 1 is a table summarizing the binding affinities of the indicated trispecific binding proteins to their cognate antigens (human CD3, CD28, and CD38) as measured by SPR. [Figure 6A-1] 3A is a graph comparing the apparent affinity of isatuximab antigen-binding domains in a human IgG1 format (second sheet) or in a trispecific binding protein format with isatuximab, anti-CD28, and anti-CD3 antigen-binding domains (FIG. 3A; format on first sheet) for binding to human (top) or cynomolgus monkey (bottom) CD38 polypeptides, as assayed by flow cytometry. [Figure 6A-2] Continuation of Figure 6A-1. [Figure 6B] Figures 6B-6D are graphs comparing the apparent affinity of the trispecific binding proteins CD38VH1xCD28supxCD3mid, CD38VH1xCD28cvnxCD3mid, or the monospecific anti-CD38 antibody mAb2 for binding to cells expressing human or cynomolgus CD38 polypeptides, as assayed by flow cytometry. Figure 6B shows binding of the trispecific binding protein CD38VH1xCD28supxCD3mid to cells expressing human (top) or cynomolgus CD38 polypeptides (bottom). [Figure 6C] FIG. 6C shows binding of the trispecific binding protein CD38VH1×CD28cvn×CD3mid to cells expressing human (top) or cynomolgus (bottom) CD38 polypeptides. [Figure 6D] FIG. 6D shows binding of the monospecific anti-CD38 antibody mAb2 to cells expressing human (top) or cynomolgus monkey (bottom) CD38 polypeptide. [Figure 6E-1]1 is a graph comparing the apparent affinity of the trispecific binding protein CD38HHY1370×CD28sup×CD3mid or the monospecific anti-CD38 antibody mAb6 for binding to cells expressing human (top) or cynomolgus monkey (bottom) CD38 polypeptides, as assayed by flow cytometry. [Figure 6E-2] Continued from Figure 6E-1. [Figure 6F] 1 is a table summarizing the binding affinities of the indicated anti-CD38×anti-CD28×anti-CD3 trispecific binding proteins to human CD38 as measured by SPR or flow cytometry (FACS). [Figure 6G] 1 is a graph showing the apparent affinity of the trispecific binding protein ΔCD38VH1×CD28sup×CD3mid, lacking the anti-CD38 antigen-binding domain, for binding to cells expressing human (top) or cynomolgus (bottom) CD38 polypeptide, as assayed by flow cytometry. [Figure 7A] 1 is a graph showing the results of an ELISA assay determining the binding affinity of various anti-CD38xCD28xCD3 IgG4 trispecific binding proteins, or control antibodies, to human and rhesus monkey CD3, CD28, and CD38 polypeptides. [Figure 7B] 1 is a graph showing the results of an ELISA assay determining the binding affinity of various anti-CD38xCD28xCD3 IgG4 trispecific binding proteins, or control antibodies, to human and rhesus monkey CD3, CD28, and CD38 polypeptides. [Figure 8A]Figures 8A-8D are graphs showing the results of antibody-mediated specific killing of CD38+ cells by PBMCs from three different human donors using the indicated anti-CD38xCD28xCD3 trispecific binding proteins and control antibodies. Representative results using the multiple myeloma cell lines RPMI8266 (Figure 8A), NCI-H929 (Figure 8B), KMS-26 (Figure 8C), and KMS-11 (Figure 8D) cell lines are shown, and EC50 values ​​are provided in Table N. EC50 values ​​obtained using NCI-H929, KMS-26, and KMS-11 cells are provided in Table OQ. [Figure 8B] Figures 8A-8D are graphs showing the results of antibody-mediated specific killing of CD38+ cells by PBMCs from three different human donors using the indicated anti-CD38xCD28xCD3 trispecific binding proteins and control antibodies. Representative results using the multiple myeloma cell lines RPMI8266 (Figure 8A), NCI-H929 (Figure 8B), KMS-26 (Figure 8C), and KMS-11 (Figure 8D) cell lines are shown, and EC50 values ​​are provided in Table N. EC50 values ​​obtained using NCI-H929, KMS-26, and KMS-11 cells are provided in Table OQ. [Figure 8C] Figures 8A-8D are graphs showing the results of antibody-mediated specific killing of CD38+ cells by PBMCs from three different human donors using the indicated anti-CD38xCD28xCD3 trispecific binding proteins and control antibodies. Representative results using the multiple myeloma cell lines RPMI8266 (Figure 8A), NCI-H929 (Figure 8B), KMS-26 (Figure 8C), and KMS-11 (Figure 8D) cell lines are shown, and EC50 values ​​are provided in Table N. EC50 values ​​obtained using NCI-H929, KMS-26, and KMS-11 cells are provided in Table OQ. [Figure 8D]Figures 8A-8D are graphs showing the results of antibody-mediated specific killing of CD38+ cells by PBMCs from three different human donors using the indicated anti-CD38xCD28xCD3 trispecific binding proteins and control antibodies. Representative results using the multiple myeloma cell lines RPMI8266 (Figure 8A), NCI-H929 (Figure 8B), KMS-26 (Figure 8C), and KMS-11 (Figure 8D) cell lines are shown, and EC50 values ​​are provided in Table N. EC50 values ​​obtained using NCI-H929, KMS-26, and KMS-11 cells are provided in Table OQ. [Figure 8E] 8A-8E are graphs showing the results of antibody-mediated specific killing of CD38+ cells by PBMCs from two different donors using the indicated anti-CD38xCD28xCD3 trispecific binding proteins with variant Fc regions and a control antibody. Representative results using the CD38+ KMS-11 (FIG. 8D) and U266 (FIG. 8E) cell lines are shown, and EC50 values ​​are provided in Tables Q2 and Q3. [Figure 8F] 8A-8E are graphs showing the results of antibody-mediated specific killing of CD38+ cells by PBMCs from two different donors using the indicated anti-CD38xCD28xCD3 trispecific binding proteins with variant Fc regions and a control antibody. Representative results using the CD38+ KMS-11 (FIG. 8D) and U266 (FIG. 8E) cell lines are shown, and EC50 values ​​are provided in Tables Q2 and Q3. [Figure 9] Figures 9A, 9B, and 10 are graphs showing the activation of human T cells (CD69+) treated with various anti-CD38xCD28xCD3 trispecific binding proteins or control antibodies for 24 hours. Figure 9A shows the activation of human CD3+ T cells (CD69+). Figure 9B shows the activation of human CD3+CD4+ T cells (CD69+). Figure 10 shows the activation of human CD3+CD8+ T cells (CD69+). [Figure 10]Figures 9A, 9B, and 10 are graphs showing the activation of human T cells (CD69+) treated with various anti-CD38xCD28xCD3 trispecific binding proteins or control antibodies for 24 hours. Figure 9A shows the activation of human CD3+ T cells (CD69+). Figure 9B shows the activation of human CD3+CD4+ T cells (CD69+). Figure 10 shows the activation of human CD3+CD8+ T cells (CD69+). [Figure 11A] Figure 11A shows the results of an in vitro cytokine release assessment of human PBMCs treated with the indicated anti-CD38xCD28xCD3 trispecific binding proteins or control antibodies, based on the dry plate method described in Stebbings, R. et al. (2007) J. Immunol. 179:3325-3331. Figure 11A shows results using 5 μg / mL of the indicated antibodies. Figure 11B shows results using 25 ng / mL of the indicated antibodies. [Figure 11B] Figure 11A shows the results of an in vitro cytokine release assessment of human PBMCs treated with the indicated anti-CD38xCD28xCD3 trispecific binding proteins or control antibodies, based on the dry plate method described in Stebbings, R. et al. (2007) J. Immunol. 179:3325-3331. Figure 11A shows results using 5 μg / mL of the indicated antibodies. Figure 11B shows results using 25 ng / mL of the indicated antibodies. [Figure 12A] Figures 12A-12E are graphs showing the in vivo activity of anti-CD38(VHI)xCD28(sup)xCD3(mid) trispecific binding protein in a CD34+ cord blood cell-humanized NSG mouse model engrafted with RPMI-8226 multiple myeloma cells. Figure 12A shows the change in tumor volume in mice treated with the indicated concentrations of anti-CD38(VHI)xCD28(sup)xCD3(mid) trispecific binding protein relative to mice treated with an anti-CD3 / CD38 bispecific antibody. [Figure 12B]Figure 12B shows the mean tumor volume at day 18 in mice treated with the indicated concentrations of anti-CD38(VHI) x CD28(sup) x CD3(mid) trispecific binding protein versus mice treated with the anti-CD3 / CD38 bispecific antibody. [Figure 12C] Figure 12C shows the mean terminal tumor weight in mice treated with the indicated concentrations of anti-CD38(VHI) x CD28(sup) x CD3(mid) trispecific binding protein versus mice treated with the anti-CD3 / CD38 bispecific antibody. [Figure 12D] Figure 12D shows the mean tumor growth curves over the experimental period in mice treated with the indicated concentrations of anti-CD38(VHI) x CD28(sup) x CD3(mid) trispecific binding protein versus mice treated with the anti-CD3 / CD38 bispecific antibody. [Figure 12E] Figure 12E shows the average weight change at multiple time points over the experimental period for mice treated with the indicated concentrations of anti-CD38(VHI)xCD28(sup)xCD3(mid) trispecific binding protein versus mice treated with the anti-CD3 / CD38 bispecific antibody. [Figure 13A] Figures 13A-13F are graphs showing the in vivo activity of anti-CD38(VHI)xCD28(sup)xCD3(mid) trispecific binding protein in a PBMC-humanized NSG mouse model engrafted with RPMI-8226 multiple myeloma cells. Figure 13A shows the change in tumor volume in mice treated with the indicated concentrations of anti-CD38(VHI)xCD28(sup)xCD3(mid) trispecific binding protein relative to mice treated with an anti-CD3 / CD38 bispecific antibody. [Figure 13B] Figure 13B shows tumor volumes at day 4 in mice treated with the indicated concentrations of anti-CD38(VHI)xCD28(sup)xCD3(mid) trispecific binding protein versus mice treated with anti-CD3 / CD38 bispecific antibody. [Figure 13C]Figure 13C shows tumor volumes at day 21 in mice treated with the indicated concentrations of anti-CD38(VHI)xCD28(sup)xCD3(mid) trispecific binding protein versus mice treated with anti-CD3 / CD38 bispecific antibody. [Figure 13D] Figure 13D shows the mean tumor volume at day 21 in mice treated with the indicated concentrations of anti-CD38(VHI)xCD28(sup)xCD3(mid) trispecific binding protein versus mice treated with anti-CD3 / CD38 bispecific antibody. [Figure 13E] Figure 13E shows the mean end-stage tumor weight in mice treated with the indicated concentrations of anti-CD38(VHI) x CD28(sup) x CD3(mid) trispecific binding protein versus mice treated with anti-CD3 / CD38 bispecific antibody. [Figure 13F] Figure 13F shows the mean tumor volumes at multiple time points over the course of the experiment in mice treated with the indicated concentrations of anti-CD38(VHI) x CD28(sup) x CD3(mid) trispecific binding protein versus mice treated with the anti-CD3 / CD38 bispecific antibody. [Figure 14-1]Figures 14A-14U are graphs showing the results of a dose-escalation study (0.5, 2.5, and 12.5 μg / kg) using anti-CD38(VHI)×CD28(sup)×CD3(mid), anti-CD38(VHI)×CD28(cvn)×CD3(mid), anti-CD38(hhy1370)×CD28(sup)×CD3(mid), and anti-CD38(hhy1370)×CD28(cvn)×CD3(mid) trispecific binding proteins in non-human primates. Figure 14A shows T cell activation (CD69+) of circulating CD3+ T cells after administration of various doses of anti-CD38(VHI)×CD28(sup)×CD3(mid) trispecific binding protein. Figure 14B shows T cell activation (CD69+) of circulating CD3+ T cells after administration of various doses of anti-CD38(VHI)×CD28(cvn)×CD3(mid) trispecific binding protein. Figure 14C shows T cell activation (CD69+) of circulating CD3+ T cells after administration of various doses of anti-CD38(hhy1370)×CD28(sup)×CD3(mid) trispecific binding protein. Figure 14D shows T cell activation (CD69+) of circulating CD3+ T cells after administration of various doses of anti-CD38(hhy1370)×CD28(cvn)×CD3(mid) trispecific binding protein. Figure 14E shows the change in the percentage of circulating CD4+ T cells after administration of the indicated doses of anti-CD38(VHI)×CD28(sup)×CD3(mid) trispecific binding protein. Figure 14F shows the change in the percentage of circulating CD8+ T cells after administration of the indicated doses of anti-CD38(VHI)xCD28(sup)xCD3(mid) trispecific binding protein. Figure 14G shows the change in the percentage of circulating CD4+ T cells after administration of the indicated doses of anti-CD38(VHI)xCD28(cvn)xCD3(mid) trispecific binding protein. Figure 14H shows the change in the percentage of circulating CD8+ T cells after administration of the indicated doses of anti-CD38(VHI)xCD28(cvn)xCD3(mid) trispecific binding protein. Figure 14I shows the change in the percentage of circulating CD4+ T cells after administration of the indicated doses of anti-CD38(hhy1370)xCD28(sup)xCD3(mid) trispecific binding protein.Figure 14J shows the change in the percentage of circulating CD8+ T cells after administration of the indicated doses of anti-CD38(hhy1370)xCD28(sup)xCD3(mid) trispecific binding protein. Figure 14K shows the change in the percentage of circulating CD4+ T cells after administration of the indicated doses of anti-CD38(hhy1370)xCD28(cvn)xCD3(mid) trispecific binding protein. Figure 14L shows the change in the percentage of circulating CD8+ T cells after administration of the indicated doses of anti-CD38(hhy1370)xCD28(cvn)xCD3(mid) trispecific binding protein. Figure 14M shows the change in the total number of CD4+ T cells 6, 24, and 48 hours after administration of 12.5 μg / kg of the indicated trispecific binding protein. Figure 14N shows the change in total NK cell numbers 6, 24, and 48 hours after administration of 12.5 μg / kg of the indicated trispecific binding proteins. Figure 14O shows the change in total CD8+ T cell numbers 6, 24, and 48 hours after administration of 12.5 μg / kg of the indicated trispecific binding proteins. Figure 14P shows the change in total B cell numbers 6, 24, and 48 hours after administration of 12.5 μg / kg of the indicated trispecific binding proteins. Figure 14Q shows the change in cytokine levels 6 hours after administration of three sequentially increasing doses (0.5, 2.5, 12.5 μg / kg) of anti-CD38(VH1)×CD28(sup)×CD3(mid) trispecific binding proteins (results from different test animals labeled "117065" and "117066"). Figure 14R shows the changes in cytokine levels 6 hours after administration of three successively increasing doses (0.5, 2.5, and 12.5 μg / kg) of anti-CD38(VH1)×CD28(cvn)×CD3(mid) trispecific binding protein (results from different test animals labeled "117067" and "117068"). Figure 14S shows the changes in cytokine levels 6 hours after administration of three successively increasing doses (0.5, 2.5, and 12.5 μg / kg) of anti-CD38(hhy1370)×CD28(sup)×CD3(mid) trispecific binding protein (results from different test animals labeled "117069" and "117070").Figure 14T shows the changes in cytokine levels 6 hours after administration of three successively increasing doses (0.5, 2.5, 12.5 μg / kg) of anti-CD38(hhy1370) x CD28(cvn) x CD3(mid) trispecific binding protein (results from different test animals labeled "117071" and "117072"). Figure 14U shows the changes in cytokine levels 24 hours after administration of three successively increasing doses (0.5, 2.5, 12.5 μg / kg) of the indicated trispecific binding protein (results shown from all test animals). [Figure 14-2] Continued from Figure 14-1. [Figure 14-3] Continued from Figure 14-2. [Figure 14-4] Continued from Figure 14-3. [Figure 14-5] Continued from Figure 14-4. [Figure 14-6] Continued from Figure 14-5. [Figure 14-7] Continued from Figure 14-6. [Figure 14-8] Continuation of Figure 14-7. [Figure 14-9] Continuation of Figure 14-8. [Figure 14-10] Figures 14V and 14W are graphs showing that anti-CD38(VHI) x CD28(sup) x CD3(mid) and anti-CD38(VHI) x CD28(cvn) x CD3(mid) trispecific binding proteins induced depletion of blood T cells in non-human primates at higher doses in vivo (6 hours post-administration). [Figure 14-11] Figures 14X and 14Y are graphs showing that anti-CD38(HHY1370)xCD28(sup)xCD3(mid) and anti-CD38(HHY1370)xCD28(cvn)xCD3(mid) trispecific binding proteins induced depletion of blood T cells in non-human primates at higher doses in vivo (6 hours post-administration). [Figure 14-12]Figures 14Z and 14AA are graphs showing blood T cell levels in non-human primates over time after administration of anti-CD38(VHI)xCD28(sup)xCD3(mid) or anti-CD38(VHI)xCD28(cvn)xCD3(mid) trispecific binding proteins. [Figure 14-13] Figures 14AB and 14AC are graphs showing blood T cell levels in non-human primates over time after administration of anti-CD38(HHY1370)xCD28(sup)xCD3(mid) or anti-CD38(HHY1370)xCD28(cvn)xCD3(mid) trispecific binding proteins. [Figure 14-14] Figures 14AD and 14AE are graphs showing the amount of CD4+ T cells bound to the trispecific binding protein after administration of a 100 μg / kg dose in non-human primates. [Figure 14-15] Figures 14AD and 14AE are graphs showing the amount of CD4+ T cells bound to the trispecific binding protein after administration of a 100 μg / kg dose in non-human primates. [Figure 14-16] Figures 14AF and 14AG are graphs showing the amount of CD8+ T cells bound to the trispecific binding protein after administration of a 100 μg / kg dose in non-human primates. [Figure 14-17] Figures 14AF and 14AG are graphs showing the amount of CD8+ T cells bound to the trispecific binding protein after administration of a 100 μg / kg dose in non-human primates. [Figure 15] Figures 15A-15C are graphs showing the binding or lack thereof of various Fc variants to the human Fc receptors FcγR I (Figure 15A), FcγR IIa (Figure 15B), and FcγR IIIb / c (Figure 15C). The variants tested were human IgG1, human IgG4, and human IgG4 with a FALA mutation. [Figure 16] 1 is a graph showing binding of human IgG4 with or without the FALA mutation to FcRn. [Figure 17]1 is a table summarizing the PK parameters of the indicated trispecific binding proteins (CD38VH1×CD28sup×CD3mid IgG4, CD38VH1×CD28sup×CD3mid IgG4 FALA, CD38VH1×CD28sup×CD3mid IgG1 LALA P329A, and CD38HHY1370×CD28sup×CD3mid IgG4 FALA) in NSG mice. [Figure 18-1] Figures 18A-18C are graphs showing Fc / FcR interaction-mediated (non-specific) release of INF-γ (Figure 18A), IL-2 (Figure 18B), or TNF-α (Figure 18C) by human PBMCs incubated with trispecific binding proteins having wild-type or FALA variant Fc regions. [Figure 18-2] Figures 18A-18C are graphs showing Fc / FcR interaction-mediated (non-specific) release of INF-γ (Figure 18A), IL-2 (Figure 18B), or TNF-α (Figure 18C) by human PBMCs incubated with trispecific binding proteins having wild-type or FALA variant Fc regions. [Figure 18-3] Figure 18D is a graph showing in vitro activation of human PBMCs by CD38VH1xCD28supxCD3mid and CD38HHY1370xCD28supxCD3mid trispecific binding proteins and their IgG1 and IgG4 Fc variants. [Figure 19-1] Figures 19A and 19B show that induction of Bcl-xL in CD4+ (Figure 19A) or CD8+ (Figure 19B) T cells by the trispecific binding protein CD38VH1xCD28supxCD3mid requires both the CD3 and CD28 antigen-binding domains. Bars = mean and standard deviation from three PBMC donors. *p=<0.009. [Figure 19-2]Figures 19C and 19D show that CD38VH1xCD28xCD3 with IgG4 FALA variant Fc upregulates Bcl-xL in CD4+ (Figure 19C) or CD8+ (Figure 19D) T cells to a greater extent than the benchmark bispecific antibody. Bars = mean and standard deviation from 3 PBMC donors. *p=<0.045 [Figure 19-3] Figure 19E is a graph showing that T cell activation by anti-CD38 x anti-CD28 x anti-CD3 trispecific binding proteins is dependent on the anti-CD3 antigen-binding domain, as assayed by IL-2 expression in a Jurkat T cell receptor line. [Figure 19-4] Figure 19F shows the release of cytokines TNF, IFNg, IL-2, IL-6, and IL-10 by the CD38VH1xCD28supxCD3mid trispecific binding protein compared to binding proteins with mutated anti-CD28, anti-CD38, or anti-CD28, anti-CD38, and anti-CD3, and benchmarks. [Figure 19-5] Figure 19G shows proliferation of T cells activated with an anti-CD38xanti-CD28xanti-CD3 trispecific binding protein with an IgG4 FALA variant Fc, a benchmark anti-CD38xanti-CD3 bispecific antibody, or an isotype control (trispecific binding protein with an IgG4 FALA variant Fc with a mutated binding domain). [Figure 20] FIG. 1 shows proliferation of T cells activated with an anti-CD38×anti-CD28×anti-CD3 trispecific binding protein with an IgG4 FALA variant Fc, an anti-CD38×anti-CD28×anti-CD3 trispecific binding protein with an IgG4 FALA variant Fc and mutations in the CD38, CD28, or CD3 antigen-binding domain, or an isotype control (trispecific binding protein with an IgG4 FALA variant Fc with three mutated binding domains). [Figure 21]1 is a graph showing the in vivo antitumor activity of the CD38VH1×CD28sup×CD3mid IgG4 FALA trispecific binding protein administered at the indicated doses in the NCI-H929-Luc disseminated tumor model in PBMC-humanized NSG mice. [Figure 22] 1 is a graph showing the in vivo antitumor activity of the CD38HHY1370×CD28sup×CD3mid IgG4 FALA trispecific binding protein administered at the indicated doses in the NCI-H929-Luc disseminated tumor model in PBMC-humanized NSG mice. [Figure 23-1] Figures 23A and 23B are graphs showing the potent in vitro tumor-killing activity of NCI-H929-Luc cells with CD38VH1xCD28supxCD3mid and CD38HHY1370xCD28supxCD3mid trispecific binding proteins and the benchmark anti-CD38xanti-CD3 bispecific antibody using human PBMCs from two donors in humanized NSG mice after 24 hours of incubation at an effector:PBMC ratio of 10:1. [Figure 23-2] Figure 23C is a graph showing the superior in vivo anti-tumor activity of CD38VH1xCD28supxCD3mid and CD38hhy1370xCD28supxCD3mid trispecific binding proteins administered at the indicated doses compared to the benchmark anti-CD38xanti-CD3 bispecific antibody in the NCI-H929-Luc disseminated tumor model in PBMC-humanized NSG mice. Binding proteins were administered by weekly intraperitoneal (IP) injection at 30 μg / kg. [Figure 24A] Graph showing the results of a luciferase reporter assay using GloResponse™ IL-2-luc2P Jurkat cells (Promega) after stimulation with 10 nM concentrations of CD38VH1 / CD28sup×CD3mid and its single binding site KO and triple KO mutants. [Figure 24B]

[0023] Figure 1 shows the optimization of anti-CD3 x CD28 CODV-Fab antibodies. The optimal configuration of α-CD3 and α-CD28 in alternative positions of the CODV bispecific Fab was evaluated in vitro by cytokine release assay using human PBMCs. Based on the secretion of IFN-γ and IL-2 in the supernatant after 24 hours, distal CD28 x proximal CD3 was identified as the optimal configuration. [Figure 25] 1 is a graph showing that CD38VH1 / CD28sup×CD3mid-induced upregulation of the Bcl-2 family member Bcl-xL in primary T cells is CD28 dependent. [Figure 26] 1 is a graph showing that anti-CD28 in a trispecific Ab provided the secondary signaling necessary to support the proliferation of primary T cells in vitro. [Figure 27] Figure 1 shows the configuration of the trispecific antibody, color-coded by the parent antibody (left). Darker shades (purple or green) indicate heavy chain peptides; lighter shades indicate light chain peptides. A structural model of the CD38VH1 / CD28sup×CD3mid trispecific Ab, based on the crystal structures of anti-CD38 VH1 Fab and CD28sup / CD3mid CODV Fab, is also shown (right). [Figure 28] Figures 28A and 28B are graphs showing that multiple myeloma (MM) cells with high (RPMI-8226; Figure 28A) and low (KMS-11; Figure 28B) CD38 surface expression were efficiently lysed by human PBMCs (E:T = 10:1) incubated with various concentrations of the trispecific Ab. The contribution of each binding site to killing activity was demonstrated by knockout mutations of the binding sites, as indicated. [Figure 29] Figures 29A-29C are graphs showing that the KO mutants of the trispecific anti-CD28sup Ab exhibited significantly reduced antitumor activity against CD38high, CD38mid, and CD38low MM cells in vitro. Assays using RPMI-8226 (Figure 29A), U266 (Figure 29B), or KMS-11 (Figure 29C) cells are also shown. [Figure 30] 1 is a graph showing that the reduction in tumor burden in the CD38VH1 / CD28sup×CD3mid_FALA trispecific antibody treated group was dose-dependent and statistically different in a disseminated human multiple myeloma cell line model using NSG mice reconstituted with in vitro expanded human primary T cells. [Figure 31] Figures 31A and 31B are photographs showing intravital microscopy analysis of myeloma cell cytolysis by CD38 trispecific Ab in the presence of human primary T cells in vitro. Time-lapse microscopy images using a negative control (triplicate KO trispecific; Figure 31A) or a CD38 / CD28 x CD3 trispecific Ab (Figure 31B) were performed using human PBMCs incubated with CellTracker™ red dye-labeled RPMI-8226 myeloma cell line. Images shown were collected after 24 hours of incubation. Scale bar: 50 μm. [Figure 32] Figure 1 shows alternative mutations in the Fc region of IgG4 prepared for analysis in an Fc receptor binding assay. SEQ ID NOs: 111-116 (from top to bottom, respectively) are shown. [Figure 33] Graph showing the results of an SPR assay to measure the affinity of identified IgG4 Fc variants for the indicated human Fc receptors. [Figure 34]

[0023] Figure 1 is a graph showing that CD38 trispecific binding proteins with minimal FcR binding reduced nonspecific cytokine release by human PBMCs in vitro. Various FcR-inactivating mutations (as indicated) were analyzed for their pro-inflammatory effects on the human IgG4 isotype. Human PBMCs were incubated in medium (unstimulated) or in the presence of myeloma cells, RPMI-8226 (stimulated), and the bars indicate IFN-γ levels in the supernatants, measured by ELISA. [Figure 35]

[0023] Figure 1 is a graph showing that CD38 trispecific binding proteins with minimal FcR binding lysed human multiple myeloma cells with various CD38 expression levels. Cytolysis of myeloma cells bearing IgG4 or the indicated Fc mutations was assessed in vitro using human PBMCs containing the indicated tumor targets. [Figure 36] Graph showing a comparison of the in vitro cytolytic activity of trispecific anti-CD38 / CD28 / CD3 Ab and the anti-CD38 antibody daratumumab, measured using human PBMCs as effector cells against the cell lines RPMI-8226, U266, and KMS-11 (E:T=10:1). [Figure 37-1] Figures 37A-37D are graphs showing the characterization of in vitro T cell subset expansion in response to CD38 / CD3xCD28. Assessment of T cell subset expansion was performed by coating wells with 350 ng / well of CD38 trispecific Ab in the absence of exogenous cytokines. T cell populations were measured at the indicated time points. Triple mutant trispecific Ab was used as a negative control. Flow cytometry was used to determine central and effector memory CD4 T cells (Figure 37A), helper T cells (Figure 37B), central and effector memory CD8 T cells (Figure 37C), and CMV pp65-specific CD8 cells (Figure 37D), as described in Example 12. Analysis of CMV-specific pp65 effector cells was performed by pentamer staining of PBMCs from HLA-A2 CMV+ donors treated with CD38 trispecific or triple negative control. [Figure 37-2] Continuation of Figure 37-1. [Figure 37-3] Continued from Figure 37-2. [Figure 37-4] Continued from Figure 37-3. [Figure 38]Figure 1 shows the contribution of CD28 expression on target cells to susceptibility to cytolysis by CD38 / CD3 × CD28. CD28 was knocked out in KMS-11 cells using CRISPR / Cas 9 gene targeting and used as a cytolytic target in vitro. Compared to parental KMS-11, CD38 expression was preserved, while CD28 expression was eliminated, as demonstrated by flow cytometry (upper panel, KMS-11 vs. KMS-11(CD28KO)). Cytolysis of CD28 KO cells was investigated in the context of WT or CD28 trispecific deficiency (lower panel; trispecific vs. trispecific(CD28KO)). [Figure 39] 1 is a graph showing the cytolytic activity of CD38 / CD28×CD3 trispecific FALA mutant Abs against the indicated CD38+CD28− lines, including acute myeloid leukemia (AML(KG-1)), B-cell lymphoma (OCI-Ly19), acute T-lymphocytic leukemia (ALL(KOPN8)), and chronic lymphocytic lymphoma (CLL(Z-138)). [Figure 40] 1 is a graph showing that in vitro activation of human PBMCs by α-CD28 superagonists requires antibody bivalency. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present disclosure provides binding proteins comprising at least one antigen binding site that binds to a CD38 polypeptide.

[0025] I. General definition As utilized in accordance with this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. As used in this specification and the appended claims, the singular forms "a," "an," and "the" shall refer to the plural unless clearly defined otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0026] It will be understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0027] The term "polynucleotide" as used herein refers to a single-stranded or double-stranded nucleic acid polymer of at least 10 nucleotides in length.In certain embodiments, the nucleotides comprising polynucleotide are ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide.Such modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate and phosphoramidate.Specifically, the term "polynucleotide" includes single-stranded and double-stranded forms of DNA.

[0028] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, in which (1) the isolated polynucleotide is not associated with all or a portion of a polynucleotide found in nature, (2) it is linked to a polynucleotide with which it is not naturally linked, or (3) it does not occur in nature as part of a larger sequence.

[0029] An "isolated polypeptide" is one that (1) is free of at least some other polypeptides with which it is normally found; (2) is derived from the same source, e.g., is essentially free from other polypeptides from the same species; (3) is expressed by cells from a different species; (4) is separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) is not associated (by covalent or noncovalent interactions) with portions of polypeptides with which it is naturally associated; (6) is operably associated (by covalent or noncovalent interactions) with polypeptides with which it is not naturally associated; or (7) is not naturally occurring. Such isolated polypeptides may be encoded by genomic DNA, cDNA, mRNA, or other RNA, or may be of synthetic origin, or any combination thereof. Preferably, an isolated polypeptide is substantially free of polypeptides or other contaminants found in its natural environment that would be expected to interfere with its use (therapeutic, diagnostic, prophylactic, research, or other).

[0030] Naturally occurring antibodies typically comprise tetramers. Each such tetramer typically consists of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain," as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100 to 110 or more amino acids responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in naturally occurring antibodies, the full-length heavy chain immunoglobulin polypeptide contains a variable domain (V H ) and three constant domains (C H1 , C H2 , and C H3 ), including VH The domain is located at the amino terminus of the polypeptide and is C H3 The domains are located at the carboxyl terminus, and full-length light chain immunoglobulin polypeptides contain a variable domain (V L ) and constant domains (C L ), including V L The domain is located at the amino terminus of the polypeptide. Yes, C L The domain is at the carboxyl terminus.

[0031] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is similarly subdivided into subclasses, including but not limited to IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains are typically connected by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 more amino acids. See, for example, FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated herein by reference in its entirety for all purposes. The variable regions of each light chain / heavy chain pair typically form an antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, which enable binding to a specific epitope. Both light chain and heavy chain variable domains typically comprise, from the amino terminus to the carboxyl terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0032] The term "CDR set" refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined in various ways by different systems. The system described by Kabat (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries that define the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., 1989, Nature 342:877-83) have described the Kabat CDRs as It has been found that certain sub-portions within the CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These sub-portions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" represent the light chain and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs have been described by Padlan, 1995, FASEB J. 9:133-39; MacCallum, 1996, J. Mol. Biol. 262(5):732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27:55-77. Still other CDR boundary definitions may not strictly follow the system herein, but nevertheless adhere to the Kabat CDRs. Although they overlap the CDRs, they are shortened or extended in light of predictions or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly affect antigen binding.The methods used herein can utilize CDRs defined according to any of these systems, although certain embodiments use CDRs defined by Kabat or Chothia. Identification of predicted CDRs using amino acid sequences is described in Martin, A.C., "Protein sequence and structure," in Antibody Engineering, Vol. 2, Kontermann R., Dubel S., eds., Springer-Verlag, Berlin, pp. 33-51 (2010). Methods for determining CDR sequences are well known in the art, such as in "structure analysis of antibody variable domains." The amino acid sequences of the heavy and / or light chain variable domains are also examined to identify the sequences of the CDRs by other conventional methods, for example, by comparison with known amino acid sequences of other heavy and light chain variable regions, to determine regions of sequence hypervariability. Numbered sequences can be aligned visually or by using an alignment program such as one of the CLUSTAL suite of programs as described in Thompson, 1994, Nucleic Acids Res. 22:4673-80. Molecular models are conventionally used to accurately depict the framework and CDR regions and thus correct sequence-based assignments.

[0033] In some embodiments, the definition of the CDR / FR in an immunoglobulin light or heavy chain should be determined based on the IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

[0034] The term "Fc," as used herein, refers to a molecule, whether in monomeric or multimeric form, that is produced by digestion of an antibody or by other means and contains the sequence of a non-antigen-binding fragment containing the hinge region. The original immunoglobulin source of native Fc is preferably of human origin and is either an immunoglobulin. Fc molecules are composed of monomeric polypeptides linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from one to four, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG4). One example of an Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. The term "native Fc," as used herein, is inclusive of monomeric, dimeric, and multimeric forms.

[0035] F(ab) fragments typically contain one light chain and one heavy chain V H and C H1 domain, and the V of the F(ab) fragment H -C H1 The heavy chain portion cannot form disulfide bonds with another heavy chain polypeptide. As used herein, a F(ab) fragment also refers to one light chain containing two variable domains separated by an amino acid linker, and one light chain containing two variable domains and a C separated by an amino acid linker. H1 The antibody may comprise one heavy chain containing the domain.

[0036] F(ab') fragments typically contain one light chain and more of the constant region (C) such that interchain disulfide bonds can form between two heavy chains to form an F(ab')2 molecule. H1 and C H2 It comprises the portion of one heavy chain containing the nucleotides (between the nucleotide domains).

[0037] The term "binding protein," as used herein, refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen, e.g., a CD38 polypeptide of the present disclosure.

[0038] A "recombinant" molecule is one prepared, expressed, produced, or isolated by recombinant means.

[0039] One embodiment of the present disclosure provides binding proteins having biological and immunological specificity for between one and three target antigens. Another embodiment of the present disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Another embodiment of the present disclosure provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide chains that form such a binding protein. Yet another embodiment of the present disclosure provides a host cell that expresses such a binding protein (i.e., comprises a nucleic acid molecule or vector encoding the polypeptide chains that form such a binding protein).

[0040] The term "swapability," as used herein, refers to the interchangeability of variable domains within a binding protein format with retention of folding and ultimate binding affinity. "Full swappability" refers to the interchangeability of V in a polypeptide chain of Formula I or a polypeptide chain of Formula II while maintaining full functionality of the binding protein as evidenced by retention of binding affinity. H1 and V H2 Domain both order, therefore, V L1 and V L2 It refers to the ability to swap (i.e., reverse) the order of domains. H and V L refers only to the location of the domain on a particular protein chain in the final form. For example, V H1 and V H2 is the V in the parent antibody L1 and V L2domain and V in binding proteins H1 and V H2 Similarly, V L1 and V L2 is the V in the parent antibody H1 and V H2 domain and V in binding proteins H1 and V H2 It is located at position V. H and V L The nomenclature refers to the current location rather than the original location in the parent antibody. Thus, V H and V L Domains are "interchangeable."

[0041] The term "antigen" or "target antigen" or "antigen target," as used herein, refers to a molecule or portion of a molecule that can be bound by a binding protein and further used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein can compete with intact antibodies that recognize the target antigen.

[0042] "CD38" is a cluster of differentiation 38 polypeptide, a glycoprotein found on the surface of many immune cells. In some embodiments, the binding proteins of the present disclosure bind to the extracellular domain of one or more CD38 polypeptides. Exemplary CD38 extracellular domain polypeptide sequences include, but are not limited to, the extracellular domain of human CD38 (e.g., as set forth in SEQ ID NO: 1) and the extracellular domain of cynomolgus monkey CD38 (e.g., as set forth in SEQ ID NO: 30).

[0043] The term "T cell engager" refers to a binding protein that targets the host's immune system, more specifically the cytotoxic activity of T cells, as well as a tumor target protein.

[0044] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

[0045] The term "monovalent binding protein" refers to a binding protein that has one antigen-binding site.

[0046] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets. In some embodiments, a bispecific binding protein binds to two different antigens. In some embodiments, a bispecific binding protein binds to two different epitopes on the same antigen.

[0047] The term "bivalent binding protein" refers to a binding protein that has two binding sites.

[0048] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets. In some embodiments, a trispecific binding protein binds to three different antigens. In some embodiments, a trispecific binding protein binds to one, two, or three different epitopes on the same antigen.

[0049] The term "trivalent binding protein" refers to a binding protein having three binding sites. In certain embodiments, a trivalent binding protein can bind to one antigen target. In other embodiments, a trivalent binding protein can bind to two antigen targets. In other embodiments, a trivalent binding protein can bind to three antigen targets.

[0050] An "isolated" binding protein is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses of the binding protein and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein will be purified (1) to greater than 95% by weight, and most preferably greater than 99% by weight, of the antibody as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated binding protein includes the binding protein in situ within a recombinant cell, since at least one component of the binding protein's natural environment will not be present.

[0051] The terms "substantially pure" or "substantially purified," as used herein, refer to a compound or species that is the predominant species present (i.e., more abundant, on a molar basis, than any other individual species in a composition). In some embodiments, a substantially purified fraction is a composition in which a species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition comprises greater than about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In yet other embodiments, the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species.

[0052] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, have specific three-dimensional structural and / or charge characteristics. An epitope is a region of an antigen to which an antibody or binding protein binds. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, a binding protein has an equilibrium dissociation constant of ≦10 -8 M, more preferably the equilibrium dissociation constant is ≦10 -9 M, most preferably the dissociation constant is ≦10 -10 When the antibody is M, it is said to specifically bind to the antigen.

[0053] Dissociation constant (K D ) can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR) using a BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis also involves immobilizing the analyte (binding protein on a biosensor matrix). This can be achieved by activating the target antigen and presenting the ligand (target antigen). D " as used herein refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

[0054] The term "binds to," as used herein in reference to a binding protein, refers to binding to at least about 1 x 10 of the binding protein or antigen-binding fragment thereof. -6 M, 1 x 10 -7 M, 1 x 10 -8M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 x 10 -12 It refers to the ability to bind to an antigen containing the epitope with a Kd of M or greater, and / or the ability to bind to an epitope with an affinity at least two-fold greater than its affinity for a nonspecific antigen. In some embodiments, a binding protein of the disclosure binds to two or more antigens, e.g., human and cynomolgus CD38 polypeptides.

[0055] In some embodiments, the antigen binding domains and / or binding proteins of the present disclosure "cross-react" with human and cynomolgus monkey CD38 polypeptides, e.g., CD38 extracellular domains such as SEQ ID NO: 1 (human CD38 isoform A), SEQ ID NO: 105 (human CD38 isoform E), and SEQ ID NO: 30 (cynomolgus monkey CD38). Binding proteins that bind to antigen 1 (Ag1) are known to be associated with EC 50 is "cross-reactive" to antigen 2 (Ag2) if the affinity of Ag2 is within a similar range for both antigens. In the present application, a binding protein that binds to Ag1 is cross-reactive to Ag2 if the ratio of the affinity of Ag2 to the affinity of Ag1 is equal to or less than 10 (e.g., 5, 2, 1, or 0.5) (affinity measured in the same way for both antigens).

[0056] A binding protein that binds to Ag1 is "not significantly cross-reactive" with Ag2 if the affinities are significantly different for the two antigens. The affinity for Ag2 is not measurable if the binding response is too low. In this application, a binding protein that binds to Ag1 is not significantly cross-reactive with Ag2 if the binding response of the binding protein to Ag2 is less than 5% of the binding response of the same binding protein to Ag1 in the same experimental setting and at the same antibody concentration. In practice, the concentration of binding protein used is less than EC 50 or the concentration required to reach the saturation plateau obtained with Ag1.

[0057] The term "linker," as used herein, refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient flexibility for the light and heavy chain domains to fold into a crossover dual variable region immunoglobulin. At the sequence level, linkers are inserted at the transition between variable domains or between variable and constant domains, respectively. Because the approximate sizes of immunoglobulin domains are well understood, the transitions between domains can be identified. The precise location of the domain transitions can be determined by positioning peptide stretches that do not form secondary structure elements, such as beta sheets or alpha helices, as demonstrated by experimental data or can be predicted by modeling techniques or secondary structure prediction. The linkers described herein are intended to be used in conjunction with V L2 C-terminus and V L1 L1, located on the light chain between the N-terminus of the domain; and V L1 C-terminus and C L The heavy chain linker is designated L2, located on the light chain between the N-termini of the domains. H1 C-terminus and V H2 L3, located between the N-terminus of the domain; and V H2 C-terminus and C H1 Located between the N-terminus of the domain is known as L4.

[0058] The term "vector," as used herein, refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information into a host cell. The term "vector" includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. One type of vector is a circular double-stranded vector into which additional DNA segments can be inserted. A "plasmid" refers to a single-strand DNA molecule. Another type of vector is a viral vector, into which additional DNA segments can be inserted. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because plasmids are the most commonly used form of vector, the terms "plasmid" and "vector" can be used interchangeably herein. However, the present disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0059] The phrase "recombinant host cell" (or "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cell is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but such cells are still included within the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express binding proteins, including bacteria, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To recombinantly express a binding protein, a host cell is transformed or transfected with one or more recombinant expression vectors carrying a DNA fragment encoding the polypeptide chain of the binding protein, such that the polypeptide chain is expressed in the host cell and preferably secreted into the medium in which the host cell is cultured, from which the binding protein can be recovered.

[0060] The term "transformation," as used herein, refers to a change in the genetic characteristics of a cell; a cell is transformed when it has been modified to contain new DNA. For example, a cell that has been genetically modified from its native state is transformed. After transformation, the transforming DNA may be recombined with that of the cell by being physically integrated into the cell's chromosome, or may be transiently maintained as an episomal element without replication, or may replicate independently as a plasmid. A cell is considered stably transformed when the DNA replicates with cell division. The term "transfection," as used herein, refers to the uptake of foreign or exogenous DNA by a cell; a cell is "transfected" when exogenous DNA is introduced into the cell membrane. Several transfection techniques are well known in the art. Using such techniques, one or more exogenous DNA molecules can be introduced into a suitable host cell.

[0061] As used herein, when applied to an object, the term "naturally occurring" refers to the fact that the object is found in nature and has not been manipulated by humans.For example, naturally occurring polynucleotides or polypeptides exist in organisms (including viruses) that can be isolated from sources in nature and have not been intentionally modified by humans.Similarly, as used herein, "non-naturally occurring" refers to an object that is not found in nature or that has been structurally modified or synthesized by humans.

[0062] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids); unnatural amino acids and analogs such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids are also suitable components of the polypeptide chain of a binding protein. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.

[0063] Naturally occurring residues can be divided into classes based on common side chain properties: (1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro; (2) Polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro; (4) Aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro; (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (6) Acidic: Asp, Glu; (7) Basic: His, Lys, Arg; (8) Residues affecting chain orientation: Gly, Pro; (9) Aromatic: His, Trp, Tyr, Phe; and (10) Aromatic hydrophobic: Phe, Trp, Tyr.

[0064] Conservative amino acid substitutions involve exchanging a member of one of these classes for another member of the same class, while non-conservative substitutions involve exchanging a member of one of these classes for a member from another class.

[0065] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptide chain of binding protein.For example, those skilled in the art can identify suitable regions of the polypeptide chain that can be modified without destroying activity by targeting regions that are not considered important for activity.Alternatively, those skilled in the art can identify residues and molecular parts that are conserved between similar polypeptides.In addition, even regions that are important for biological activity or structure may be subjected to conservative amino acid substitution without destroying biological activity or adversely affecting polypeptide structure.

[0066] The term "patient", as used herein, includes human and animal subjects (eg, mammals such as dogs, pigs, horses, cats, cows, etc.).

[0067] The term "treatment" or "treating" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those with a disorder and those prone to have a disorder or those in whom a disorder is to be prevented. In certain embodiments, the binding protein can be used to treat a person with cancer or a person susceptible to cancer, or to ameliorate cancer in a human subject. The binding protein can also be used to prevent cancer in a human patient. In certain embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, Germinal center B-cell lymphoma or B-cell acute lymphoblastic leukemia.

[0068] The term "pharmaceutical composition" or "therapeutic composition," as used herein, refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0069] The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier," as used herein, refers to one or more formulation materials suitable for achieving or enhancing delivery of a binding protein.

[0070] The terms "effective amount" and "therapeutically effective amount," when used in connection with pharmaceutical compositions containing one or more binding proteins, refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of binding protein sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated for a period of time. The effective amount will vary depending on the particular binding protein being used and will also depend on various factors associated with the patient being treated and the severity of the condition or disorder. For example, if the binding protein is to be administered in vivo, factors such as the patient's age, weight, and health, as well as dose-response curves and toxicity data obtained in preclinical animal studies, among others, will be considered. Determining the effective or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of one of ordinary skill in the art.

[0071] One embodiment of the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

[0072] II. Anti-CD38 Binding Proteins Certain aspects of the present disclosure relate to binding proteins that comprise an antigen-binding site that binds to a CD38 polypeptide (e.g., a human or cynomolgus CD38 polypeptide). In some embodiments, the binding proteins are monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent.

[0073] Various features of exemplary monospecific, bispecific, or trispecific binding proteins are described herein. For example, in some embodiments, the binding protein or antigen-binding fragment thereof cross-reacts with human CD38 (e.g., human CD38 isoform A and / or isoform E polypeptide) and cynomolgus monkey CD38. In some embodiments, the binding protein induces apoptosis of CD38+ cells. In some embodiments, the binding protein recruits T cells to CD38+ cells and optionally activates the T cells (e.g., by TCR stimulation and / or costimulation).

[0074] In some embodiments, the binding protein comprises an antigen-binding site comprising: an antibody heavy chain variable (VH) domain comprising: a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); or an antibody light chain variable (VL) domain comprising: a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the binding protein comprises: a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the binding protein comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising a CDR-H2 sequence comprising the amino acid sequence of YPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36), as shown in Table G, H, or I. sup ×CD3 mid IgG4 FALA, mAb2×CD28 sup ×CD3 mid IgG1 LALA P329A, mAb2×CD28 sup ×CD3 mid IgG1 NNSA, mAb6×CD28 sup ×CD3 mid IgG4 FALA, mAb6×CD28 sup ×CD3 mid IgG1 LALA P329A, or mAb6×CD28 sup ×CD3 mid It comprises 1, 2, 3, 4, 5, or 6 CDRs derived from the antibody VH and / or VL domain sequences of IgG1 NNSA.

[0075] In some embodiments, the binding protein comprises an antigen-binding site comprising: an antibody heavy chain variable (VH) domain comprising: a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and an antibody light chain variable (VL) domain comprising: a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0076] In some embodiments, the binding protein comprises an antigen-binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the binding protein comprises an antigen-binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In other embodiments, the binding protein comprises an antigen-binding site comprising: an antibody heavy chain variable (VH) domain comprising: a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); or an antibody light chain variable (VL) domain comprising: a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the binding protein comprises: a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), IYPG The antibody heavy chain variable (VH) domain comprises an antibody heavy chain variable (VH) domain comprising a CDR-H2 sequence comprising the amino acid sequence of QGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0077] In some embodiments, the VH domain comprises, from N-terminus to C-terminus, the sequence FR1--CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO: 87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94); and FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96). In some embodiments, the VL domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4; FR1 comprises the sequence DIVLTQSPATLSLSPGERATISCRAS (SEQ ID NO: 97); FR2 comprises the sequence MHWYQQKPGQPPRLLIY (SEQ ID NO: 99); FR3 comprises the sequence SRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYC (SEQ ID NO: 101); and FR4 comprises the sequence FGGGTKLEIK (SEQ ID NO: 103).

[0078] In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5; and / or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17; and / or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18.In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21; and / or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 100% identical to the amino acid sequence of SEQ ID NO: 18. It comprises an amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the present invention. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23; and / or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13; and / or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0079] In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18.In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain , comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0080] In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:5; and the VL domain comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:17; and the VL domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:21; and the VL domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:23; and the VL domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:13; and the VL domain comprises the amino acid sequence of SEQ ID NO:14.

[0081] In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:7 and / or an antibody light chain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:19 and / or an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:22 and / or an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and / or an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:15 and / or an antibody light chain comprising the amino acid sequence of SEQ ID NO:16.

[0082] In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:7 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:19 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:22 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:15 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:16.

[0083] In some embodiments, the binding protein has the amino acid sequence of: GFTFSSYG (SEQ ID NO: 41). an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK, and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, the binding protein comprises an antigen-binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

[0084] In some embodiments, the VH domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; FR1 comprises the sequence QVQLVESGGGVVQPGRSLRLSCAAS (SEQ ID NO: 89); FR2 comprises the sequence MHWVRQAPGKGLEWVAV (SEQ ID NO: 92); FR3 comprises the sequence YYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 95); and FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96). In some embodiments, the VL domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4; FR1 comprises the sequence AIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 98); FR2 comprises the sequence GWYQQKPGKAPKLLIY (SEQ ID NO: 100); FR3 comprises the sequence SLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYC (SEQ ID NO: 102); and FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 104).

[0085] In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9; and / or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:10.

[0086] In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:9. the VL domain comprises the amino acid sequence of SEQ ID NO:10.

[0087] In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 11 or an antibody light chain comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0088] In some embodiments, a binding protein of the disclosure comprises one, two, three, four, five, or six CDR sequences of an antibody sequence shown in Table G. In some embodiments, a binding protein of the disclosure comprises one, two, three, four, five, or six CDR sequences, VH domain sequences, and / or VL domain sequences of an antibody sequence shown in Table H. In some embodiments, a binding protein of the disclosure comprises one, two, three, four, five, or six CDR sequences, VH domain sequences, and / or VL domain sequences of an antibody sequence shown in Table I. In some embodiments, a binding protein of the disclosure comprises one, two, three, or four polypeptide sequences shown in Table I.

[0089] [Table 1]

[0090] Table 2 Table 3

[0091] Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13

[0092] Table 14 Table 15 Table 16 [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26]

[0093] CD38 polypeptide In some embodiments, the binding protein of the present disclosure comprises an antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide. Exemplary assays for determining whether an antigen-binding site binds to an antigen are described herein and are known in the art. In some embodiments, binding is determined by an ELISA assay, for example, as described below. In some embodiments, binding is determined by an SPR assay, for example, as described below. In some embodiments, binding is determined by a flow cytometry assay using cells that express a CD38 polypeptide on their cell surface, for example, as described below. See, e.g., Examples 1, 3, and 4.

[0094] In some embodiments, a binding protein of the disclosure binds to a purified polypeptide or fragment thereof comprising the amino acid sequence of SEQ ID NO: 1 and / or 30 (e.g., as measured by ELISA or SPR). In some embodiments, a binding protein of the disclosure binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and / or 30 when expressed on the surface of a cell (e.g., as measured by flow cytometry).

[0095] In some embodiments, a binding protein of the disclosure binds to a CD38 isoform A polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1). In some embodiments, a binding protein of the disclosure binds to a CD38 isoform A polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105 but not SEQ ID NO: 1). In some embodiments, the binding proteins of the present disclosure bind to CD38 isoform E polypeptides (e.g., comprising the amino acid sequence of SEQ ID NO: 1) and CD38 isoform E polypeptides (e.g., comprising the amino acid sequence of SEQ ID NO: 1 but not including the entire amino acid sequence of SEQ ID NO: 1, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 105). Without wishing to be bound by theory, binding to CD38 isoform E polypeptides is believed to be advantageous, for example, in targeting binding proteins of the present disclosure to cells expressing CD38 isoform E polypeptides.

[0096] Human CD38 isoform A extracellular domain polypeptide sequence RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI (SEQ ID NO: 1) Human CD38 isoform E polypeptide sequence RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRHFWECGSP (SEQ ID NO: 105)

[0097] In some embodiments, the extracellular domain of a human CD38 polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the extracellular domain of a cynomolgus monkey CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:30. Cynomolgus monkey CD38 polypeptide sequence RWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQSVWDAFKGAFISKYPCNITEEDYQPLVKLGTQTVPCNKTLLWSRIKDLAHQFTQVQRDMFTLEDMLLGYLADDLTWCGEFNTFEINYQSCPDWRKDCSNNPVSVFWKTVSRRFAETACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQALEAWVIHGGREDSRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKFLQCVKNPEDSSCLSGI (SEQ ID NO: 30)

[0098] Multispecific (e.g., bispecific, trispecific, or multispecific) binding proteins that bind to CD38 polypeptides In some embodiments, the binding proteins of the disclosure are bispecific binding proteins comprising an antigen-binding site that binds one or more CD38 polypeptides and a second antigen-binding site that binds a different target antigen, hi some embodiments, the binding proteins of the disclosure are bispecific binding proteins comprising an antigen-binding site that binds one or more CD38 polypeptides and a second antigen-binding site that binds one or more CD38 polypeptides.

[0099] In some embodiments, the binding proteins of the disclosure bind to one or more CD38 polypeptides. For example, in some embodiments, the antigen-binding site is a trispecific binding protein comprising an antigen-binding site that binds a polypeptide, a second antigen-binding site, and a third antigen-binding site. One of the antigen-binding sites binds to one or more CD38 polypeptides (e.g., the extracellular domain of a human and / or cynomolgus monkey CD38 polypeptide), and one or two of the antigen-binding sites bind to a T cell surface protein. In some embodiments, one of the antigen-binding sites binds to one or more CD38 polypeptides (e.g., the extracellular domain of a human and / or cynomolgus monkey CD38 polypeptide), one of the antigen-binding sites binds to a human CD3 polypeptide, and one of the antigen-binding sites binds to a human CD28 polypeptide. Human CD3 and CD28 polypeptides are known in the art. Exemplary, non-limiting, amino acid sequences of antibody variable domains that bind to human CD3 and CD28 polypeptides are provided herein.

[0100] In some embodiments, provided herein are binding proteins comprising three antigen-binding sites, each of which binds to one or more target proteins. In some embodiments, at least one of the three antigen-binding sites binds to the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide. In some embodiments, the human CD38 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, and / or the cynomolgus monkey CD38 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the binding protein comprises an antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide, and two antigen-binding sites that bind to a T cell surface protein (e.g., a human CD28 polypeptide and / or a human CD3 polypeptide), respectively.

[0101] In some embodiments, the binding proteins of the present disclosure bind to one or more target proteins (e.g., one or more CD38 polypeptides) and one or more T cell target proteins. In some embodiments, the binding proteins are capable of binding to one tumor target protein (e.g., one or more CD38 polypeptides) and two different epitopes on a single T cell target protein. In some embodiments, the binding proteins are capable of binding to one tumor target protein (e.g., one or more CD38 polypeptides) and two different T cell target proteins (e.g., CD28 and CD3). In some embodiments, the binding proteins are capable of binding to one T cell target protein and two different epitopes on a single tumor target protein (e.g., one or more CD38 polypeptides). In some embodiments, the binding proteins are capable of binding to one T cell target protein and two different tumor target proteins (e.g., one or more CD38 polypeptides and another tumor target protein). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen-binding sites that target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen-binding site that binds to one or more CD38 polypeptides. In some embodiments, the first and second polypeptide chains of the binding protein form two antigen-binding sites that target two tumor target proteins (e.g., one or more CD38 polypeptides and another tumor target protein), and the third and fourth polypeptide chains of the binding protein form an antigen-binding site that binds to a T cell target protein. In some embodiments, the one or more T cell target proteins are one or more of CD3 and CD28.

[0102] In some embodiments, the binding proteins specifically bind to one or more target proteins on T cells, including one or more CD38 polypeptides and the T cell receptor complex. These T cell engager binding proteins can transiently recruit T cells to target cells and simultaneously activate the cytolytic activity of the T cells. Examples of target proteins on T cells include, but are not limited to, CD3 and CD28, among others. Further examples of such antigen targets or target proteins are provided above. In some embodiments, trispecific binding proteins can be generated by combining the antigen-binding domains of two or more monospecific antibodies (parent antibodies) into one antibody.

[0103] Bispecific binding protein format In some embodiments, the binding proteins of the present disclosure are bispecific and / or bivalent binding proteins comprising four polypeptide chains (e.g., having a structure described in International Publication No. WO 2012 / 135345) that form four antigen-binding sites that bind to one or more (e.g., two) different antigen targets or target proteins. In some embodiments, the binding proteins are bivalent and / or bispecific. In some embodiments, the binding proteins are tetravalent and / or tetraspecific. In some embodiments, the binding proteins are tetravalent and / or bispecific. In some embodiments, at least one of the antigen-binding sites binds to a CD38 polypeptide (e.g., the extracellular domain of a human and / or cynomolgus CD38 polypeptide).

[0104] In some embodiments, the binding protein has the formula: V L1 -L1-V L2 -L2-C L [I] It contains two polypeptide chains having the structure The two polypeptide chains have the formula: V H2 -L3-V H1 -L4-CH1 -Fc [II] It has a structure represented by During the ceremony: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; Fc is an immunoglobulin hinge region and C H2 , C H3 Contains an immunoglobulin heavy chain constant domain; L1, L2, L3, and L4 are amino acid linkers; The polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair. H1 and V L1 forms the antigen-binding domain that binds to the CD38 polypeptide, and V H2 and V L2 In some embodiments, V forms an antigen binding domain that binds to another antigen target. H2 and V L2 forms the antigen-binding domain that binds to the CD38 polypeptide, and V H1 and V L1 forms an antigen-binding domain that binds to another antigen target.

[0105] In some embodiments, the binding protein comprises two polypeptide chains that form two antigen binding sites, the first polypeptide chain comprising: V L1 -L1-V L2 -L2-C L -Fc Including, The second polypeptide chain is V H2 -L3-V HI -L4-C H1 -Fc Including, During the ceremony: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; Fc is an immunoglobulin hinge region and C H2 , C H3 Contains an immunoglobulin heavy chain constant domain; L1, L2, L3, and L4 are amino acid linkers; The first and second polypeptides form a crossover light chain-heavy chain pair. H1 and V L1 forms the antigen-binding domain that binds to the CD38 polypeptide, and V H2 and V L2 In some embodiments, V forms an antigen binding domain that binds to another antigen target. H2 and V L2 forms the antigen-binding domain that binds to the CD38 polypeptide, and V H1 and V L1 forms an antigen-binding domain that binds to another antigen target.

[0106] In some embodiments, the binding protein comprises three polypeptide chains that form two antigen binding sites, the first polypeptide chain comprising: V L1 -L1-V L2 -L2-C L Including, The second polypeptide chain is V H2 -L3-V HI -L4-C H1 -Fc Including, the third polypeptide chain comprises an antibody Fc region; During the ceremony: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; Fc is an immunoglobulin hinge region and C H2 , C H3 Contains an immunoglobulin heavy chain constant domain; L1, L2, L3, and L4 are amino acid linkers; The first and second polypeptides form a crossover light chain-heavy chain pair. H1 and V L1 forms the antigen-binding domain that binds to the CD38 polypeptide, and V H2and V L2 In some embodiments, V forms an antigen binding domain that binds to another antigen target. H2 and V L2 forms the antigen-binding domain that binds to the CD38 polypeptide, and V H1 and V L1 forms an antigen-binding domain that binds to another antigen target.

[0107] In some embodiments, the binding protein has the formula: V L1 -L1-V L2 -L2-C L [I] and a first polypeptide chain comprising a structure represented by the formula: V H2 -L3-V H1 -L4-C H1 [II] a second polypeptide chain comprising a structure represented by During the ceremony: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair. H1 and V L1 forms the antigen-binding domain that binds to the CD38 polypeptide, and V H2 and V L2In some embodiments, V forms an antigen binding domain that binds to another antigen target. H2 and V L2 is a CD38 polypeptide forms the antigen-binding domain that binds to V H1 and V L1 forms an antigen-binding domain that binds to another antigen target.

[0108] In any of the bispecific proteins described above, the target antigen other than CD38 is any of the following exemplary antigen targets: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (MCP-1), CCL8 (MCP-1), CCL9 (MCP-1), CCL10 (MCP-1), CCL11 (MCP-1), CCL12 (MCP-1), CCL13 (MCP-1), CCL14 (MCP-1), CCL15 (MCP-1), CCL16 (MCP-1), CCL17 (MCP-1), CCL18 (MCP-1), CCL19 (MCP-1), CCL20 (MCP-1), CCL21 (MCP-1), CCL22 (MCP-1), CCL23 (MCP-1), CCL24 (MCP-1), CCL25 (MCP-1), CCL26 (MCP-1), CCL27 (MCP-1), CCL28 (MCP-1), CCL29 (MCP-1), CCL29 (MCP-1), CCL21 (MCP-1), CCL21 (MCP-1), CCL21 (MCP-1), CCL22 (MCP-1), CCL23 (MCP-1), CCL24 (MCP-1), CCL25 (MCP-1), CCL26 (MCP-1), CCL27 (MCP-1), CCL28 (MCP-1), CCL2 P-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (reactive protein for IgE) CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (IC OS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1,Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2R beta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin) , ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the coreceptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, , Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments, one or more of the antigen targets is a human antigen target.

[0109] Any of the bispecific binding proteins described above may use any linker or combination of linkers described herein. For example, in some embodiments, at least one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments, L1, L2, L3, or L4 is independently at least 1 amino acid in length. In some embodiments, L1, L2, L3, and L4 are independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO:58), L2 comprises the sequence TKGPS (SEQ ID NO:57), L3 comprises the sequence S, and L4 comprises the sequence RT. In some embodiments, L1 comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence GGSGSSGSGG (SEQ ID NO: 59), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO: 59), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence GGGGSGGGSGGGGGS (SEQ ID NO: 56), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGSGGGGGS (SEQ ID NO: 56), and L4 is 0 amino acids in length.

[0110] Trispecific binding protein that binds to CD38 polypeptide In some embodiments, the binding proteins of the present disclosure are trispecific and / or trivalent binding proteins comprising four polypeptide chains that form three antigen-binding sites that bind to one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one of the antigen-binding sites binds to a CD38 polypeptide (e.g., the extracellular domain of a human and / or cynomolgus CD38 polypeptide). In some embodiments, the first polypeptide chain has the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by The third polypeptide chain has the formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] It includes a structure represented by During the ceremony: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 is an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair.

[0111] In some embodiments, the binding proteins of the present disclosure are trispecific and / or trivalent binding proteins comprising four polypeptide chains that form three antigen-binding sites that bind to one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one of the antigen-binding sites binds to a CD38 polypeptide (e.g., the extracellular domain of a human and / or cynomolgus CD38 polypeptide). In some embodiments, the first polypeptide chain has the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 [II] It includes a structure represented by The third polypeptide chain has the formula: V H3 -C H1 [III] It includes a structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] It includes a structure represented by During the ceremony, V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair. In some embodiments, the second and third polypeptide chains are C H1 and further comprising an Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 Contains an immunoglobulin heavy chain constant domain.

[0112] In some embodiments, the first polypeptide chain and the second polypeptide chain have a crossover orientation forming two separate antigen-binding sites. In some embodiments, VH1 and VL1 form a binding pair to form a first antigen-binding site. In some embodiments, VH2 and VL2 form a binding pair to form a second antigen-binding site. In some embodiments, the first antigen-binding site binds to a CD3 polypeptide (e.g., human CD3) and the second antigen-binding site binds to a CD28 polypeptide (e.g., human CD28). In some embodiments, the second antigen-binding site binds to a CD3 polypeptide (e.g., human CD3). ), and the first antigen-binding site binds a CD28 polypeptide (e.g., human CD28). In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen-binding site. In some embodiments, VH3 and VL3 form a binding pair to form a third antigen-binding site. In some embodiments, the third antigen-binding site binds a CD38 polypeptide (e.g., human and optionally cynomolgus CD38). Exemplary binding protein formats with crossover orientations contemplated for use in the present invention are also described in U.S. Patent Application No. 15 / 487,243 and International Application No. PCT / US2017 / 027488.

[0113] In some embodiments of any of the bispecific, trispecific, or multispecific binding proteins described herein, the antigen-binding site binds to a CD38 polypeptide (e.g., human and optionally cynomolgus CD38). In some embodiments, the other antigen-binding site (e.g., not binding to CD38) of any of the bispecific, trispecific, or multispecific binding proteins described herein binds to CD28 or CD3. In some embodiments, the V H1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H2 The domain contains a CDR-H1 sequence containing the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), and the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38). a CDR-H2 sequence, and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L3 The domain includes a CDR-L1 sequence having the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence having the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence having the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0114] In some embodiments, V H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and / or V L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and / or V L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and / or V L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

[0115] In some embodiments, V H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and V L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and V L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H3 The domains are CDR-H1 sequences containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), IWYDGSNK (SEQ ID NO: 42), 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), L3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

[0116] In some embodiments, V H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and / or L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and / or L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and / or L2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and / or L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and / or L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and / or L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0117] In some embodiments, V H1 The domain comprises a CDR-H1 sequence containing the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence containing the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence containing the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33). CDR-H3 sequence containing V L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H1 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and L2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0118] In some embodiments, V H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and / or L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and / or L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0119] In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 5, or L3 The V domain comprises the amino acid sequence of SEQ ID NO: 6. H3 The domain comprises the amino acid sequence of SEQ ID NO: 17, or L3 The V domain comprises the amino acid sequence of SEQ ID NO: 18. H3 The domain comprises the amino acid sequence of SEQ ID NO: 21, or L3 The V domain comprises the amino acid sequence of SEQ ID NO: 18. H3 The domain comprises the amino acid sequence of SEQ ID NO: 23, or L3 The V domain comprises the amino acid sequence of SEQ ID NO: 18. H3 The domain comprises the amino acid sequence of SEQ ID NO: 13, or L3 The domain comprises the amino acid sequence of SEQ ID NO:14.

[0120] In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 5, and / or V L3 The V domain comprises the amino acid sequence of SEQ ID NO: 6. H3 The domain comprises the amino acid sequence of SEQ ID NO: 17, L3 The V domain comprises the amino acid sequence of SEQ ID NO: 18. H3The domain comprises the amino acid sequence of SEQ ID NO: 21, L3 The V domain comprises the amino acid sequence of SEQ ID NO: 18. H3 The domain comprises the amino acid sequence of SEQ ID NO: 23, L3 The V domain comprises the amino acid sequence of SEQ ID NO: 18. H3 The domain comprises the amino acid sequence of SEQ ID NO: 13, L3 The domain comprises the amino acid sequence of SEQ ID NO:14.

[0121] In some embodiments, V H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and / or V L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

[0122] In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 9, and / or L3 The domain comprises the amino acid sequence of SEQ ID NO:10.

[0123] In some embodiments of any of the trispecific binding proteins of the present disclosure, one antigen-binding domain binds to a CD3 polypeptide (e.g., human CD3) and one antigen-binding domain binds to a CD28 polypeptide (e.g., human CD28). H1 The domain comprises three CDRs from SEQ ID NO: 49 or 51, as shown in Table H, and L1 The V domain comprises three CDRs from SEQ ID NO: 50 or 52, as shown in Table H. In some embodiments, the V domain H2The domain comprises three CDRs from SEQ ID NO: 49 or 51, as shown in Table H, and L2 The V domain comprises three CDRs from SEQ ID NO: 50 or 52, as shown in Table H. In some embodiments, the V domain H1 The domain comprises three CDRs from SEQ ID NO: 53 or 84, as shown in Table H, and L1 The V domain comprises three CDRs from SEQ ID NO: 54 or 85, as shown in Table H. In some embodiments, the V H2 The domain comprises three CDRs from SEQ ID NO: 53 or 84, as shown in Table H, and L2 The domain comprises three CDRs from SEQ ID NO: 54 or 85 as shown in Table H.

[0124] In some embodiments, V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, L2 The V domain comprises the amino acid sequence of SEQ ID NO: 54. H2 The domain comprises the amino acid sequence of SEQ ID NO: 49, L2 The domain is comprising the amino acid sequence of SEQ ID NO: 50, H1 The domain comprises the amino acid sequence of SEQ ID NO: 53, L1 The V domain comprises the amino acid sequence of SEQ ID NO: 54. H1 The domain comprises the amino acid sequence of SEQ ID NO: 51, L1 The domain comprises the amino acid sequence of SEQ ID NO: 52, H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, L2 The V domain comprises the amino acid sequence of SEQ ID NO: 54. H2 The domain comprises the amino acid sequence of SEQ ID NO: 51, L2 The domain comprises the amino acid sequence of SEQ ID NO: 52, H1The domain comprises the amino acid sequence of SEQ ID NO: 53, L1 The domain comprises the amino acid sequence of SEQ ID NO:54.

[0125] In some embodiments, V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, L2 The domain comprises the amino acid sequence of SEQ ID NO: 54, H3 The domain comprises the amino acid sequence of SEQ ID NO: 13, L3 The V domain comprises the amino acid sequence of SEQ ID NO: 14. H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, L2 The domain comprises the amino acid sequence of SEQ ID NO: 54, H3 The domain comprises the amino acid sequence of SEQ ID NO: 9, L3 The domain comprises the amino acid sequence of SEQ ID NO:10.

[0126] In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 65, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69.In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 70, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, and the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 66. the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain comprises a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO:69.

[0127] In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 65, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 67, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69.

[0128] In any of the trispecific binding proteins described above, the target antigen other than CD38 is any of the following exemplary antigen targets: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 ( CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as IgE) CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 ( ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH 1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2R beta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1 , leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFα, TNFRSF7, Tp55, TREM1, TSLP (also known as the coreceptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments, one or more of the above antigen targets is a human antigen target.

[0129] In some embodiments, the binding protein of the present disclosure is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody.

[0130] The binding proteins of the present disclosure can be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.

[0131] Linker In some embodiments, linkers L1, L2, L3, and L4 range from no amino acids (length=0) to about 100 amino acids in length, or less than 100, 50, 40, 30, 20, or 15 amino acids or less. Linkers may also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in length. L1, L2, L3, and L4 in a single binding protein may all have the same amino acid sequence or all have different amino acid sequences.

[0132] Examples of suitable linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues; a peptide with five glycine residues; a peptide with six glycine residues; a peptide with seven glycine residues; and a peptide with eight glycine residues. Other combinations of amino acid residues may also be used, such as the peptide GGGGSGGGGS (SEQ ID NO: 55), the peptide GGGGSGGGGSGGGGGS (SEQ ID NO: 56), the peptide TKGPS (SEQ ID NO: 57), the peptide GQPKAAP (SEQ ID NO: 58), and the peptide GGSGSSGSGG (SEQ ID NO: 59). The examples listed above are in no way intended to limit the scope of the present disclosure, and linkers containing randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable for binding proteins. For further description of linker sequences, see, e.g., WO2012135345 and International Application No. PCT / US2017 / 027488.

[0133] The identity and sequence of the amino acid residues in the linker are determined based on the sequence required to achieve The amino acid residues vary depending on the type of secondary structural element. For example, glycine, serine, and alanine are best for linkers with maximum flexibility. If a stronger, extended linker is needed, some combination of glycine, proline, threonine, and serine is useful. Depending on the desired properties, any amino acid residue can be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as needed.

[0134] In some embodiments, at least one of L1, L2, L3, or L4 is independently 0 amino acid long. In some embodiments, L1, L2, L3, or L4 is each independently at least 1 amino acid long. In some embodiments, L1 is at least twice as long as L3. In some embodiments, L2 is at least twice as long as L4. In some embodiments, L1 is at least twice as long as L3, and L2 is at least twice as long as L4. In some embodiments, L1 is 3 to 12 amino acid residues long, L2 is 3 to 14 amino acid residues long, L3 is 1 to 8 amino acid residues long, and L4 is 1 to 3 amino acid residues long. In some embodiments, L1 is 5 to 10 amino acid residues long, L2 is 5 to 8 amino acid residues long, L3 is 1 to 5 amino acid residues long, and L4 is 1 to 2 amino acid residues long. In some embodiments, L1 is 7 amino acid residues in length, L2 is 5 amino acid residues in length, L3 is 1 amino acid residue in length, and L4 is 2 amino acid residues in length. In some embodiments, L1 is 10 amino acid residues in length, L2 is 10 amino acid residues in length, L3 is 0 amino acid residues in length, and L4 is 0 amino acid residues in length. In some embodiments, L1, L2, L3, and L4 each have a length independently selected from 0 to 15 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids), and at least two of the linkers have a length of 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). In some embodiments, L1, L2, L3, and L4 are each 0 amino acids in length.

[0135] In some embodiments, L1, L2, L3, and / or L4 comprise a sequence derived from a naturally occurring sequence at the junction between an antibody variable domain and an antibody constant domain (e.g., as described in WO2012 / 135345). For example, in some embodiments, the linker is a sequence derived from a naturally occurring sequence at the junction between an endogenous V H and C H1 Between domains, or endogenous V L and C L In some embodiments, the linker comprises a sequence found at the transition between domains (e.g., kappa or lambda). H and C H1 Between domains, or endogenous human V L and C L Includes sequences found at the transitions between domains (eg, human kappa or lambda).

[0136] In some embodiments, L1, L2, L3, and L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L1, L2, L3, and L4 are each independently GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59).

[0137] In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 58), L2 comprises the sequence TKGPS (SEQ ID NO: 57), L3 comprises the sequence S, and L4 comprises the sequence RT. In some embodiments, L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 55), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence GGGGGSGGGGSGGGGS (SEQ ID NO:56), L2 is 0 amino acids in length, L3 comprises the sequence GGGGGSGGGGSGGGGS (SEQ ID NO:56), and L4 is 0 amino acids in length.

[0138] Fc region and constant domains In some embodiments, the binding proteins of the present disclosure comprise a full-length antibody heavy chain or polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region comprises the hinge, C, or C regions of an antibody. H1 , C H2 , C H3 , sometimes C H4 In some embodiments, the Fc region comprises a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region comprises one or more of the mutations described above.

[0139] In some embodiments, the binding proteins of the present disclosure comprise one or two Fc variants. The term "Fc variant," as used herein, refers to a molecule or sequence that has been modified from a native Fc but still contains a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term "Fc variant" can include molecules or sequences that have been humanized from a non-human native Fc. Additionally, native Fcs contain regions that can be removed to provide structural features or biological activities not required for the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues or in which one or more Fc sites or residues have been altered that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0140] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and / or an effector function of the Fc region (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC)).

[0141] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0142] In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and / or FcγII binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and / or FcγII binding without affecting FcRn ... according to the EU index. The Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to positions 28 and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is S228P and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to positions 234 and / or 235 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is F234A and / or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to positions 228, 234, 235, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is S228P, F234A, L235A, and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to positions 233 to 236 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and / or R409K.

[0143] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to improve purification, for example, by modulating affinity for purification reagents. For example, it is known that heterodimeric binding proteins can be selectively purified away from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains a mutation that reduces or eliminates binding to Protein A, because the heterodimeric form has intermediate affinity for Protein A-based purification than either homodimeric form and can be selectively eluted from Protein A, for example, by using a different pH (see, e.g., Smith, EJ et al. (2015) Sci. Rep. 5:17943). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, with the amino acid substitutions being H435R and Y436F. In some embodiments, the binding protein comprises a C H1 a second polypeptide chain further comprising a first Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 a second polypeptide chain comprising an immunoglobulin heavy chain constant domain; and H1 a third polypeptide chain further comprising a second Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 and a third polypeptide chain comprising an immunoglobulin heavy chain constant domain; and only one of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being H435R and Y436F. In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and one or more mutations to improve purification. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region.

[0144] To improve the yield of some binding proteins (e.g., bispecific or trispecific binding proteins), C cleavage can be achieved by, for example, the "knob-into-hole" technique, which is described in detail with some examples in International Publication No. WO 96 / 027011; Ridgway et al., 1996, Protein Eng. 9:617-21; and Merchant et al., 1998, Nat. Biotechnol. 16:677-81. H3 You can change the domain. H3 The interaction surface of the domain is between these two C H3 The two C domains are altered to increase heterodimerization of both heavy chains. H3 Each of the domains (of the two heavy chains) may be a "knob" while the other is a "hole." The introduction of disulfide bridges further stabilizes the heterodimer. (Merchant et al., 1998; Atwell et al., 1997, J. Mol. Biol. 270:26-35), increasing yield. In certain embodiments, the knobs are on the second pair of polypeptides having a single variable domain. In other embodiments, the knobs are on the first pair of polypeptides having a crossover orientation. In still other embodiments, the C H3 The domain does not include knobs-in-holes.

[0145] In some embodiments, a binding protein (e.g., a trispecific binding protein) of the disclosure comprises a "knob" mutation on the second polypeptide chain and a "hole" mutation on the third polypeptide chain. In some embodiments, a binding protein of the disclosure comprises a "knob" mutation on the third polypeptide chain and a "hole" mutation on the second polypeptide chain. In some embodiments, the "knob" mutation comprises a substitution at a position corresponding to positions 354 and / or 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the "knob" mutation comprises a substitution at a position corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is S354C and T366W. In some embodiments, the "hole" mutation comprises a substitution at a position corresponding to position 407, and optionally positions 349, 366, and / or 368, of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is Y407V or Y407T, and optionally Y349C, T366S, and / or L368A. In some embodiments, the "hole" mutation comprises a substitution at a position corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

[0146] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises an amino acid substitution at a position corresponding to position 366, and optionally position 354, of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W or T366Y, and optionally S354C; and the third polypeptide chain and a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises an amino acid substitution at a position corresponding to position 407, and optionally positions 349, 366, and / or 368 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is Y407V or Y407T, and optionally Y349C, T366S, and / or L368A.

[0147] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and the first Fc region comprises an amino acid substitution at a position corresponding to position 407, and optionally positions 349, 366, and / or 368 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is Y407V or Y407T, optionally Y349C, and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising an amino acid substitution at a position corresponding to position 366, and optionally position 354, of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W or T366Y, and optionally S354C.

[0148] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3. the first Fc region comprises an immunoglobulin heavy chain constant domain, the first Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W; the third polypeptide chain further comprises a second Fc region linked to the CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 366, 368, and / or 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366S, L368A, and / or Y407V.

[0149] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises an amino acid substitution at a position corresponding to position 366, 368, and / or 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366S, L368A, and / or Y407V; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W.

[0150] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, with the amino acid substitutions being Y349C, T366S, L368A, and Y407V; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, with the amino acid substitutions being S354C and T366W. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region.

[0151] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains. and a CH3 immunoglobulin heavy chain constant domain, and the second Fc region contains amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains. The second Fc region contains amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, S354C, T366W, and R409K.

[0152] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, S354C, and T366W; and the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains. The second Fc region contains amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, with the amino acid substitutions being F234A, L235A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V; and the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains. The second Fc region contains amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, S354C, and T366W.

[0153] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; and the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K. It contains amino acid substitutions at positions corresponding to 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4, the amino acid substitutions being S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; and the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains. The second Fc region contains amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, with the amino acid substitutions being S228P, F234A, L235A, S354C, T366W, and R409K.

[0154] In some embodiments, the binding proteins of the disclosure comprise one or more mutations to improve serum half-life (see, e.g., Hinton, PR et al. (2006) J. Immunol. 176(1):346-56). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, with the amino acid substitutions being M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first and / or second Fc region comprises amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being M428L and N434S. In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and one or more mutations to improve serum half-life. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region.

[0155] In some embodiments, the binding proteins of the disclosure comprise one or more mutations, for example, in the hinge region and / or dimer interface of IgG4 to improve stability (see, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. In some embodiments, the binding proteins comprise one or more mutations, for example, in the hinge region and / or dimer interface of IgG4 to improve stability (see, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. H1a second polypeptide chain further comprising a first Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 a second polypeptide chain comprising an immunoglobulin heavy chain constant domain; and H1 a third polypeptide chain further comprising a second Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 and a third polypeptide chain comprising an immunoglobulin heavy chain constant domain; the first and second Fc regions are human IgG4 Fc regions; and the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, respectively, the amino acid substitutions being S228P and R409K. In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and stability In some embodiments, the first and / or second Fc region is a human IgG4 Fc region.

[0156] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to improve purification, for example, by modulating affinity for purification reagents. For example, it is known that heterodimeric binding proteins can be selectively purified away from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains a mutation that reduces or eliminates binding to Protein A, because the heterodimeric form has intermediate affinity for Protein A-based purification than either homodimeric form and can be selectively eluted from Protein A, for example, by using a different pH (see, e.g., Smith, EJ et al. (2015) Sci. Rep. 5:17943). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, with the amino acid substitutions being H435R and Y436F. In some embodiments, the binding protein comprises a C H1a second polypeptide chain further comprising a first Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 a second polypeptide chain comprising an immunoglobulin heavy chain constant domain; and H1 a third polypeptide chain further comprising a second Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 and a third polypeptide chain comprising an immunoglobulin heavy chain constant domain; and only one of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being H435R and Y436F. In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and one or more mutations to improve purification. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region.

[0157] In some embodiments, the binding proteins of the disclosure comprise one or more mutations to improve serum half-life (see, e.g., Hinton, PR et al. (2006) J. Immunol. 176(1):346-56). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, with the amino acid substitutions being M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first and / or second Fc region comprises amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being M428L and N434S. In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and one or more mutations to improve serum half-life. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region.

[0158] In some embodiments, the binding proteins of the disclosure comprise one or more mutations to reduce effector function, e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain comprises a C H1 and a first Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 the third polypeptide chain comprises an immunoglobulin heavy chain constant domain; H1and a second Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 the first and second Fc regions are human IgG1 Fc regions; and the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, the amino acid substitutions being L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, the amino acid substitutions being L234A and L235A. In some embodiments, the second polypeptide chain comprises a C H1 and a first Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 the third polypeptide chain comprises an immunoglobulin heavy chain constant domain; H1 and a second Fc region linked to an immunoglobulin hinge region and a C H2 and C H3the first and second Fc regions are human IgG1 Fc regions, each comprising amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, with the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, each comprising amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, with the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG4 Fc regions, each comprising amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, with the amino acid substitutions being L234A and L235A. In some embodiments, the binding protein is C H1 a second polypeptide chain further comprising a first Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 a second polypeptide chain comprising an immunoglobulin heavy chain constant domain; and H1 a third polypeptide chain further comprising a second Fc region linked to an immunoglobulin hinge region and a C H2 and C H3 and a third polypeptide chain comprising an immunoglobulin heavy chain constant domain; the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4, according to the EU index, respectively, the amino acid substitutions being F234A and L235A.

[0159] In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and one or more mutations to reduce effector function. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region. For further description of Fc mutations at position 329, see, for example, Shields, RL et al. (2001) J. Biol. Chem. 276:6591-6604 and WO1999051642.

[0160] In some embodiments, the types of mutations described above can be combined in any order or combination. For example, a binding protein of the disclosure includes two or more "knob" and "hole" mutations, one or more mutations to improve serum half-life, one or more mutations to improve IgG4 stability, one or more mutations to improve purification, and / or one or more mutations to reduce effector function as described above.

[0161] In some embodiments, binding proteins of the present disclosure comprise antibody fragments, including but not limited to, F(ab), F(ab')2, Fab'-SH, Fv, or scFv fragments.

[0162] Assay The present disclosure provides antigen binding proteins that bind to human and / or cynomolgus monkey CD38 polypeptides and induce proliferation of T cells (e.g., CD4+ and / or CD8+ T cells) and / or induce apoptosis of CD38+ cells. Exemplary assays for measuring these parameters and identifying such binding proteins are provided herein. For example, in some embodiments, the binding affinity between a binding protein or its antigen-binding fragment and a purified CD38 polypeptide is measured by SPR (e.g., as described above), and the binding affinity between a binding protein or its antigen-binding fragment and a CD38 polypeptide expressed on the cell surface is measured by flow cytometry (e.g., as described above).

[0163] In some embodiments, the antigen-binding site of a binding protein of the disclosure has an equilibrium dissociation constant (K) of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, or 0.8 nM or less, when measured by a flow cytometry assay using cells expressing a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1 on their cell surface, e.g., as described above. D ) that binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antigen-binding site has an equilibrium dissociation constant (K ) of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, or 0.75 nM or less, as measured by a flow cytometry assay using cells expressing a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30 on their cell surface, e.g., as described above. D) binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antigen-binding site has an equilibrium dissociation constant (K) of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, or 0.83 nM or less, as measured by an SPR assay using a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1, e.g., as described above. D ) that binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antigen-binding site has an equilibrium dissociation constant (K) of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 3.5 nM or less, 1.5 nM or less, or 1.0 nM or less, as measured by an SPR assay using a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30, e.g., as described above. D ) to a cynomolgus CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30. As demonstrated herein, in some embodiments, the binding proteins of the disclosure have one or more of the exemplary binding characteristics described herein. In some embodiments, the KD is measured at 4°C or 25°C.

[0164] In some embodiments, a monospecific binding protein of the disclosure has one or more of the following configurations: binds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein when assayed by SPR or ELISA; has a K of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1.5 nM or less when assayed by SPR or ELISA. Dand binds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein; binds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the cell surface when assayed by flow cytometry; has an apparent K of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less when assayed by flow cytometry. D and binds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the cell surface; binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein when assayed by SPR or ELISA; and has a K of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less when assayed by SPR or ELISA. D binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein; binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the cell surface when assayed by flow cytometry; has an apparent K of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less when assayed by flow cytometry. Dand binds to the extracellular domain of a cell-surface-expressed cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30); binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) as a purified protein when assayed by SPR or ELISA; binds to the extracellular domain of a cell-surface-expressed human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) when assayed by flow cytometry; induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells that express CD38 on their cell surface; and has one or more mutations (e.g., in the Fc region) that result in decreased binding to FcγRI and / or FcγRII compared to the same binding protein without the one or more mutations. In some embodiments, the binding proteins of the present disclosure bind to CD38 polypeptides (e.g., humans or cynomolgus monkeys) expressed on the cell surface with an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less when assayed by flow cytometry. In some embodiments, the binding proteins of the present disclosure bind to CD38 polypeptides (e.g., humans or cynomolgus monkeys) as purified proteins with an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less when assayed by ELISA. In some embodiments, the KD is measured at 4°C or 25°C.

[0165] In some embodiments, the trispecific binding proteins of the disclosure have one or more of the following properties: induce proliferation of T cells (e.g., CD4+ and / or CD8+ T cells); induce expression of Bcl-xL by T cells (e.g., CD4+ and / or CD8+ T cells); induce apoptosis of CD38+ cells (e.g., as measured by Annexin V staining and / or propidium iodide uptake); bind to cell surface-expressed CD38 and one or more T cell target antigens expressed on the surface of T cells; bind to cell surface-expressed CD38, T cell surface-expressed CD28, and T cell surface-expressed CD3; stimulate T cell receptor activation (e.g., as measured by CD69 expression); induce costimulation of T cell receptor signaling (e.g., as mediated by CD28); decrease cytokine release by PBMCs (e.g., as measured by CD69 expression) compared to the same binding protein without the one or more mutations. have one or more mutations (e.g., in the Fc region) that result in reduced induction of cytokines (e.g., IFN-γ, IL-2, and / or TNF-α); induce cytokine release (e.g., IFN-γ and / or IL-6) by PBMCs in the presence of CD38+ target cells (e.g., as measured by immunoassay); bind to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein when assayed by SPR or ELISA; have a K of 1.5 nM or less when assayed by SPR or ELISA D binds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein; binds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the cell surface when assayed by flow cytometry; and has an apparent K of 12 nM or less when assayed by flow cytometry. Dbinds to the extracellular domain of human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the cell surface; binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein when assayed by SPR or ELISA; and has a K of 3.5 nM or less when assayed by SPR or ELISA. D binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein; binds to the extracellular domain of cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the cell surface when assayed by flow cytometry; and has an apparent K of 7.5 nM or less when assayed by flow cytometry. Dand binds to the extracellular domain of a cell-surface-expressed cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30); binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) as a purified protein when assayed by SPR or ELISA; binds to the extracellular domain of a cell-surface-expressed human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) when assayed by flow cytometry; induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells that express CD38 on their cell surface; and has one or more mutations (e.g., in the Fc region) that result in decreased binding to FcγRI and / or FcγRII compared to the same binding protein without the one or more mutations. In some embodiments, a binding protein of the disclosure binds to a CD38 polypeptide (e.g., a human or cynomolgus monkey) expressed on the cell surface with an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less when assayed by flow cytometry. In some embodiments, a binding protein of the disclosure binds to a CD38 polypeptide (e.g., a human or cynomolgus monkey) as a purified protein with an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less when assayed by ELISA.

[0166] nucleic acid Standard recombinant DNA methods are used to construct polynucleotides encoding polypeptides that form binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See, e.g., Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. Unless a specific definition is provided, the terms "antibody" and "antibody" are used in analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry described herein. The nomenclature utilized in connection with, and the laboratory procedures and techniques of, biological and pharmaceutical chemistry are well known and commonly used in the art. Similarly, conventional techniques are used for chemical synthesis, chemical analysis, pharmaceutical manufacture, formulation, delivery, and treatment of patients.

[0167] Other aspects of the present disclosure relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein. In some embodiments, the isolated nucleic acid molecule is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 60-83. and / or comprising the sequences shown in Table J.

[0168] Certain aspects of the present disclosure relate to kits of polynucleotides. In some embodiments, one or more of the polynucleotides is a vector (e.g., an expression vector). The kit may find use, among other things, in producing one or more of the binding proteins described herein, e.g., the trispecific binding proteins of the present disclosure. In some embodiments, the kit includes one, two, three, or four polynucleotides shown in Table J (e.g., mAb2xCD28supxCD3mid IgG4 FALA, mAb2xCD28supxCD3mid IgG1LALA P329A, mAb2xCD28supxCD3mid IgG1 NNSA, mAb6xCD28supxCD3mid IgG4 FALA, mAb6xCD28supxCD3mid IgG1LALA P329A, or mAb6xCD28supxCD3mid IgG1 NNSA's). In some embodiments, the polynucleotide kit comprises: a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:72, a third polynucleotide comprising the sequence of SEQ ID NO:74, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75. In some embodiments, the polynucleotide kit comprises: a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:76, a third polynucleotide comprising the sequence of SEQ ID NO:77, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75. In some embodiments, the polynucleotide kit comprises: a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:78, a third polynucleotide comprising the sequence of SEQ ID NO:79, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75. In some embodiments, the polynucleotide kit comprises: a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:72, a third polynucleotide comprising the sequence of SEQ ID NO:80, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81. In some embodiments, the kit of polynucleotides comprises: a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 76, a third polynucleotide comprising the sequence of SEQ ID NO: 82, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 81. In some embodiments, the kit of polynucleotides comprises: a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 78, a third polynucleotide comprising the sequence of SEQ ID NO: 83, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 81.

[0169] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct the transcription of the nucleic acid sequence encoding the binding protein. A promoter refers to a nucleic acid control sequence that directs the transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is positioned in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence of a binding protein when the promoter affects the transcription or expression of the coding sequence. Examples of promoters include: Examples of such promoters include, but are not limited to, promoters derived from the genomes of viruses (such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.), promoters derived from heterologous eukaryotic promoters (such as actin promoters, immunoglobulin promoters, heat shock promoters, etc.), the CAG promoter (Niwa et al., Gene 108(2):193-9, 1991), the phosphoglycerate kinase (PGK) promoter, tetracycline-inducible promoters (Masui et al., Nucleic Acids Res. 33:e43, 2005), the lac system, the trp system, the tac system, the trc system, the major operator and promoter region of phage lambda, the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, and the promoter of yeast alpha mating factor. A polynucleotide encoding a binding protein of the present disclosure is under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or readily recognized by one of skill in the art.

[0170] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. An expression vector comprises one or more regulatory sequences operably linked to a polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers include, but are not limited to, enhancer sequences from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, etc.) and enhancer sequences from eukaryotic viruses (such as the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, etc.). Examples of suitable vectors include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus, vaccinia virus, Sindbis virus, measles, herpes virus, lentivirus, retrovirus, adeno-associated virus vectors, etc.). Expression vectors can be used to transfect host cells, such as bacterial cells, yeast cells, insect cells, and mammalian cells. Biologically functional viral and plasmid DNA vectors capable of expression and replication in hosts are known in the art and can be used to transfect any cell of interest.

[0171] Another aspect of the present disclosure relates to a vector system comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. One or more vectors of the system are any of the vectors described herein. In some embodiments, one or more vectors are expression vectors.

[0172] Isolated host cells Other aspects of the present disclosure relate to isolated host cells, polynucleotide kits, vectors, and / or vector systems comprising one or more isolated polynucleotides described herein. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g., an S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells include, for example, human embryonic kidney cells (e.g., 293 or 293 cells subcloned for growth in suspension culture), Expi293™ cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical cancer cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB 8065), mouse breast tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (e.g., Hep G2), and myeloma cells (e.g., NS0 and Sp2 / 0 cells).

[0173] Another aspect of the present disclosure relates to a method for producing any of the binding proteins described herein. In some embodiments, the method includes: a) culturing a host cell (e.g., any of the host cells described herein) containing an isolated nucleic acid, vector, and / or vector system (e.g., any of the isolated nucleic acids, vectors, and / or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods for culturing host cells under conditions for protein expression are well known to those of skill in the art. For example, methods for isolating proteins from cultured host cells are well known to those of skill in the art, including affinity chromatography (e.g., two-step affinity chromatography including Protein A affinity chromatography followed by size exclusion chromatography).

[0174] In some embodiments, the binding proteins of the disclosure are purified by Protein A affinity chromatography, kappa light chain affinity chromatography (e.g., using KappaSelect resin according to the manufacturer's instructions; GE Healthcare), optionally by lambda light chain affinity chromatography (e.g., using LambdaFabSelect resin according to the manufacturer's instructions; GE Healthcare). In some embodiments, the binding proteins of the disclosure are purified by Protein A affinity chromatography, lambda light chain affinity chromatography (e.g., using LambdaFabSelect resin according to the manufacturer's instructions; GE Healthcare), optionally by kappa light chain affinity chromatography (e.g., using KappaSelect resin according to the manufacturer's instructions; GE Healthcare). In some embodiments, the binding proteins comprise two Fc regions, each of which is a C H3 domain, C H3Only one of the domains contains amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being H435R and Y436F. In some embodiments, binding proteins of the disclosure are purified, in order, by Protein A affinity chromatography, followed by kappa light chain affinity chromatography (e.g., using KappaSelect resin according to the manufacturer's instructions; GE Healthcare), and then optionally by lambda light chain affinity chromatography (e.g., using LambdaFabSelect resin according to the manufacturer's instructions; GE Healthcare). In some embodiments, binding proteins of the disclosure are purified, in order, by Protein A affinity chromatography, followed by lambda light chain affinity chromatography (e.g., using LambdaFabSelect resin according to the manufacturer's instructions; GE Healthcare), and then optionally by kappa light chain affinity chromatography (e.g., using KappaSelect resin according to the manufacturer's instructions; GE Healthcare). For example, in some embodiments, the binding protein is contacted with Protein A and a C 100 containing zero or two amino acid substitutions that are H435R and Y436F. H3 The binding protein is eluted from Protein A under conditions suitable for isolating the binding protein away from the binding protein containing the domain, contacted with a kappa light chain affinity medium (e.g., as used in KappaSelect resin; GE Healthcare), and isolated from the lambda C LThe binding protein is eluted from the kappa light chain affinity medium under conditions suitable for isolating the binding protein away from binding proteins containing only the kappa light chain domain (e.g., according to the manufacturer's instructions). Suitable conditions for Protein A elution are known in the art and include, but are not limited to, a stepwise elution gradient from pH 4.5 to 2.8. In some embodiments, Protein A or a variant of Protein A useful for protein purification is used. In some embodiments, Protein A is attached to a substrate or resin, for example, as part of a chromatography medium. In some embodiments, after elution from the kappa light chain affinity medium, the binding protein is contacted with a lambda light chain affinity medium (e.g., as used in LambdaFabSelect resin; GE Healthcare) to isolate the kappa light chain. L The binding protein is eluted from the lambda light chain affinity medium under conditions suitable for isolating the binding protein away from binding proteins containing only the lambda light chain domain (e.g., according to the manufacturer's instructions). In some embodiments, the binding protein of the present disclosure is detected using HIC chromatography. In some embodiments, the binding protein is: lambda C L a first polypeptide chain comprising a C domain; a second polypeptide chain comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W. H3 Domain: C of the third polypeptide chain containing amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F. H3 domain; and kappa C LIn some embodiments, the binding protein comprises a fourth polypeptide chain comprising a domain. In some embodiments, the binding protein is produced by a host cell. In some embodiments, the binding protein is purified from cell culture medium or a host cell extract. In some embodiments, the binding protein is secreted by or produced and extracted from a host cell (e.g., before contacting with Protein A). In some embodiments, the binding protein is in the cell culture medium or a host cell extract when contacted with Protein A. In some embodiments, the binding protein is purified away from other binding proteins, polypeptides, and / or other cellular components.

[0175] III. Trispecific Binding Proteins In some embodiments, the binding proteins of the disclosure are trispecific and / or trivalent binding proteins comprising four polypeptide chains that form three antigen-binding sites that bind to one or more (e.g., three) different antigen targets or target proteins. In embodiments, the first polypeptide chain has the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by The third polypeptide chain has the formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] It includes a structure represented by During the ceremony: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 is an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair. In some embodiments, the first polypeptide chain and the second polypeptide chain have a crossover orientation that forms two separate antigen binding sites. As described above, the second and third polypeptide chains are connected by a F-C region (e.g., a hinge-C region). H2 -C H3In some embodiments, one or both of the Fc regions are human IgG4 Fc regions comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc regions are human IgG4 Fc regions comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and / or R409K. In some embodiments, one or both Fc regions are human IgG1 Fc regions comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0176] In some embodiments, the binding proteins of the present disclosure are trispecific and / or trivalent binding proteins comprising four polypeptide chains that form three antigen-binding sites that bind to one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one of the antigen-binding sites binds to a CD38 polypeptide (e.g., the extracellular domain of a human and / or cynomolgus CD38 polypeptide). In some embodiments, the first polypeptide chain has the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by The second polypeptide chain has the formula: VH1 -L3-V H2 -L4-C H1 [II] It includes a structure represented by The third polypeptide chain has the formula: V H3 -C H1 [III] It includes a structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] It includes a structure represented by During the ceremony, V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair. In some embodiments, the second and third polypeptide chains are C H1 and further comprising an Fc region linked to an immunoglobulin hinge region and a C H2 and C H3They comprise an immunoglobulin heavy chain constant domain. In some embodiments, one or both Fc regions are human IgG4 Fc regions containing amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region containing amino acid mutations at positions corresponding to positions 228, 233-236, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and / or R409K. In some embodiments, one or both Fc regions are human IgG1 Fc regions containing one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0177] In some embodiments, VH1 and VL1 form a binding pair to form a first antigen-binding site. In some embodiments, VH2 and VL2 form a binding pair to form a second antigen-binding site. In some embodiments, VH3 and VL3 form a binding pair to form a third antigen-binding site. Binding proteins can also be used for cell activation, tumor targeting, neutralization of cytokine activity, neutralization of viral infection, and combination of multiple signaling events to treat cancer, arthritis, and / or inflammatory disorders. For example, in some embodiments, the binding proteins specifically target A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MIP-IF- 2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, the receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L) ), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL1 2 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2R beta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10,rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, The antibodies bind to one, two, or three antigen targets selected from SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFα, TNFRSF7, Tp55, TREM1, TSLP (also known as the coreceptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments, one or more of the antigen targets are human antigen targets.

[0178] In some embodiments, one of the three antigen-binding sites binds to a CD3 polypeptide (e.g., a human CD3 polypeptide), one of the three antigen-binding sites binds to a CD28 polypeptide (e.g., a human CD28 polypeptide), and one of the three antigen-binding sites binds to a third polypeptide. In some embodiments, the antigen binding site that specifically binds to an antigen target other than CD3 or CD28 is selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL26 (also known as MIP-1e), CCL27 (also known as MIP-1f), CCL28 (also known as MIP-1f), CCL29 (also known as MIP-1f), CCL30 (also known as MIP-1f), CCL31 (also known as MIP-1f), CCL32 (also known as MIP-1f), CCL33 (also known as MIP-1f), CCL34 (also known as MIP-1f), CCL35 (also known as MIP-1f), CCL36 (also known as MIP-1f), CCL37 (also known as MIP-1f), CCL38 (also known as MIP-1f), CCL39 (also known as MIP-1f), CCL39 (also known as MIP-1f), CCL39 (also known as MIP-1f), CCL310 (also known as MIP-1f), CCL311 (also known as MIP-1f), CCL312 (also known CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD19, CD20, CD23 (also known as FCER2, the receptor for IgE), CD24, CD27, CD38, CD39, CD40, CD70, CD80 (also known as B7-1 ), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRT H2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R , IL2R beta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II,The antigenic target binds to an antigenic target selected from NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFα, TNFRSF7, Tp55, TREM1, TSLP (also known as the coreceptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments, one or more of the antigenic targets is a human antigenic target.

[0179] Any of the trispecific binding proteins described above can use any linker or combination of linkers described herein. For example, in some embodiments, at least one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments, L1, L2, L3, or L4 is each independently at least 1 amino acid in length. In some embodiments, L1, L2, L3, and L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 58), L2 comprises the sequence TKGPS (SEQ ID NO: 57), L3 comprises the sequence S, and L4 comprises the sequence RT. In some embodiments, L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 59). 55), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 55), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence GGSGSSGSGG (SEQ ID NO: 59), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO: 59), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence GGGGGSGGGGSGGGGS (SEQ ID NO: 56), L2 is 0 amino acids in length, L3 comprises the sequence GGGGGSGGGGSGGGGS (SEQ ID NO: 56), and L4 is 0 amino acids in length.

[0180] IV. USES FOR BINDING PROTEINS The binding proteins can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, for the detection and quantification of one or more target antigens. The binding proteins bind to one or more target antigens with an affinity that is appropriate for the assay method being used.

[0181] For diagnostic applications, in certain embodiments, the binding protein can be labeled with a detectable moiety. The detectable moiety is any that is capable of producing a detectable signal, either directly or indirectly. For example, the detectable moiety can be: 3 H, 14 C. 32 P, 35 S, 125 I, 99 Tc, 111 In, or 67 a radioisotope such as Ga; a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0182] Binding protein is also useful for in vivo imaging.The binding protein labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed.The binding protein can be labeled with any moiety that can be detected in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

[0183] For clinical or research applications, in certain embodiments, binding proteins can be conjugated to cytotoxic drugs.Various antibodies linked to cytotoxic drugs (i.e., antibody-drug conjugates) have been used to target cytotoxic payloads to specific tumor cells.Cytotoxic drugs and linkers that conjugate them to antibodies are known in the art; for example, see Parslow, AC et al. (2016) Biomedicines 4:14 and Kalim, M. et al. (2017) Drug Des.Devel.Ther. 11:2265-2276.

[0184] The present disclosure also relates to kits containing binding proteins and other reagents useful for detecting target antigen levels in biological samples. Such reagents include a detectable label, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kits include compositions containing any of the binding proteins, polynucleotides, vectors, vector systems, and / or host cells described herein. In some embodiments, the kits include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers are formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective, by itself or in combination with another composition, to treat, prevent, and / or diagnose a condition and has a sterile access port (e.g., the container is an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). In some embodiments, the label or package insert indicates a composition that is effective for preventing, diagnosing, and / or treating a selected condition. or indicates that the composition is used to treat. Alternatively, or in addition, the article of manufacture or kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0185] In some embodiments, the binding proteins of the present disclosure are administered to a patient in need thereof for the treatment or prevention of cancer. In some embodiments, the disclosure relates to methods for preventing and / or treating a proliferative disease or disorder (e.g., cancer). In some embodiments, the method comprises administering to a patient a therapeutically effective amount of at least one of the binding proteins described herein, or pharmaceutical compositions related thereto. In some embodiments, the disclosure relates to the use of at least one of the binding proteins described herein, or pharmaceutical compositions related thereto, for preventing and / or treating a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the disclosure relates to at least one of the binding proteins described herein, or pharmaceutical compositions related thereto, for use in the manufacture of a medicament for preventing and / or treating a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the patient is a human. In some embodiments, the binding protein comprises one antigen-binding site that binds to a protein on the surface of a T cell and another antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide, e.g., as described in Section II above. In some embodiments, the binding protein comprises an antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide, an antigen-binding site that binds to a human CD28 polypeptide, and an antigen-binding site that binds to a human CD3 polypeptide.

[0186] In some embodiments, the cancer cells express a human CD38 isoform A polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) on their cell surface. In some embodiments, the cancer cells express a human CD38 isoform E polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) on their cell surface. In some embodiments, the patient is selected for treatment because the cancer cells express a human CD38 isoform E polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) on their cell surface. In some embodiments, the cancer cells express CD38 and CD28. In some embodiments, the cancer cells express CD38 but do not express CD28.

[0187] In some embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, germinal center B-cell lymphoma, or B-cell acute lymphoblastic leukemia. In certain embodiments, the cancer is multiple myeloma. In certain embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma.

[0188] In certain embodiments, the cancer is multiple myeloma. Anti-CD38 antibodies, such as daratumumab and isatuximab, have been tested for the treatment of multiple myeloma. However, while multiple myeloma is considered treatable, relapse is inevitable in almost all patients, leading to the development of treatment-refractory disease. In some embodiments, the cancer is relapsed or refractory multiple myeloma. In some embodiments, the patient has been treated with a previous multiple myeloma treatment. In some embodiments, the binding proteins of the present disclosure are administered to the patient as a first-line, second-line, or third-line treatment for multiple myeloma. Without wishing to be bound by theory, the anti-CD38×anti-CD28×anti-CD3 binding proteins of the present disclosure may be administered, for example, as an anti-CD38 (or anti-CD28 / anti-CD3) binding protein. CD28 is thought to be useful in treating multiple myeloma by recruiting T cells to tumor cells via anti-CD3 / anti-CD28, activating T cells engaged by anti-CD3 / anti-CD28, and / or killing tumor cells via a perforin / granzyme-based mechanism. CD28 has been reported as a novel cancer marker for multiple myeloma. See Nair, JR et al. (2011) J. Immunol. 187:1243-1253.

[0189] In some embodiments, at least one binding protein is administered (or should be administered) in combination with one or more anti-cancer therapies (e.g., any anti-cancer therapies known in the art, such as chemotherapeutic agents or treatments). In some embodiments, at least one binding protein is administered (or should be administered) prior to one or more anti-cancer therapies. In some embodiments, at least one binding protein is administered (or should be administered) simultaneously with one or more anti-cancer therapies. In some embodiments, at least one binding protein is administered (or should be administered) after one or more anti-retroviral therapies.

[0190] V. Binding Protein Therapeutic Compositions and Their Administration Therapeutic or pharmaceutical compositions comprising the binding proteins are within the scope of this disclosure. Such therapeutic or pharmaceutical compositions may include a therapeutically effective amount of the binding protein, or binding protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation selected for compatibility with the mode of administration.

[0191] Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.

[0192] Pharmaceutical compositions can contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition.Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borate, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), bulking agents, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives ( benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancers (such as sucrose or sorbitol), osmolality enhancers (such as alkali metal halides—preferably sodium or potassium chloride—or mannitol sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants (e.g., see REMINGTON'S Pharmaceuticals, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2 PHARMACEUTICAL SCIENCES (18th ed., edited by A.R. Gennaro, Mack Publishing Company. 1990) and subsequent editions of the same).

[0193] The optimal pharmaceutical composition will be determined by the skilled artisan depending, for example, on the intended route of administration, delivery format, and desired dosage. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.

[0194] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, suitable vehicles or carriers for injection are water, saline, or artificial cerebrospinal fluid, possibly supplemented with other ingredients common in parenteral compositions. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute. In one embodiment of the present disclosure, binding protein compositions can be prepared for storage by mixing a selected composition having the desired purity with optional formulations in the form of a lyophilized cake or aqueous solution. Additionally, binding proteins can be formulated as lyophilizates using appropriate excipients, such as sucrose.

[0195] The pharmaceutical compositions of the present disclosure can be selected for parenteral or subcutaneous delivery. Alternatively, the compositions can be selected for inhalation or delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of those in the art.

[0196] Formulation components are present in concentrations that are acceptable for the site of administration. For example, buffers are used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0197] When parenteral administration is intended, the therapeutic composition for use is in the form of a pyrogen-free, parenterally acceptable aqueous solution, which contains the desired binding protein in a pharmaceutically acceptable vehicle.A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the binding protein is formulated as a sterile isotonic solution, which is appropriately preserved.Yet another preparation involves formulating the desired molecule with agents such as injectable microspheres, biodegradable particles, polymer compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can produce a slow or sustained release of the product and then be delivered by depot injection.Hyaluronic acid can also be used, which has the effect of promoting sustained duration in circulation.Other suitable means for introducing the desired molecule include implantable drug delivery devices.

[0198] In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the binding protein can be formulated as a dry powder for inhalation. The binding protein inhalation solution can also be formulated using a propellant for aerosol delivery. In yet another embodiment, the solution can be nebulized.

[0199] It is also contemplated that certain formulations can be administered orally. In one embodiment of the present disclosure, binding proteins administered in this manner can be formulated with or without carriers customarily used in the formulation of solid dosage forms such as tablets and capsules. For example, capsules can be designed to release the active portion of the formulation at the time in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to promote the absorption of the binding protein. Diluents, flavoring agents, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be used.

[0200] Another pharmaceutical composition may involve an effective amount of binding protein in a mixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving tablets in sterile water or another suitable vehicle, a solut...

Claims

1. 1. A binding protein comprising an antigen-binding site that binds to a CD38 polypeptide, the antigen-binding site comprising: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); The binding protein comprising:

2. The antigen binding site is (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); The binding protein of claim 1 , comprising:

3. The VH domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); and FR2 comprises the sequence MHWVKEAPGQRLE 3. The binding protein of claim 1 or 2, wherein FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94); and FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96).

4. 3. The binding protein of claim 1 or 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 5 and the VL domain comprises the amino acid sequence of SEQ ID NO:

6.

5. 5. The binding protein of claim 4, comprising an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:7 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:

8.

6. 3. The binding protein of claim 1 or 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 17 and the VL domain comprises the amino acid sequence of SEQ ID NO:

18.

7. 7. The binding protein of claim 6, comprising an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:

20.

8. 3. The binding protein of claim 1 or 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 21 and the VL domain comprises the amino acid sequence of SEQ ID NO:

18.

9. 9. The binding protein of claim 8, comprising an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:22 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:

20.

10. 3. The binding protein of claim 1 or 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 23 and the VL domain comprises the amino acid sequence of SEQ ID NO:

18.

11. 11. The binding protein of claim 10, comprising an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:

20.

12. The antigen binding site is: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); The binding protein of claim 1 , comprising:

13. The VH domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); and FR2 comprises the sequence MHWVKEAPGQRLE 13. The binding protein of claim 1 or 12, wherein FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94); and FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96).

14. 13. The binding protein of claim 1 or 12, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 13 and the VL domain comprises the amino acid sequence of SEQ ID NO:

14.

15. 15. The binding protein of claim 14, comprising an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:

16.

16. 1. A binding protein comprising an antigen-binding site that binds to a CD38 polypeptide, wherein the antigen-binding site is: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46); The binding protein comprising:

17. 17. The binding protein of claim 16, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 9 and the VL domain comprises the amino acid sequence of SEQ ID NO:

10.

18. 18. The binding protein of claim 16 or 17, comprising an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:

12.

19. 19. The binding protein of any one of claims 1 to 18, wherein the antibody binding site cross-reacts with the extracellular domain of human CD38 polypeptide and the extracellular domain of cynomolgus monkey CD38 polypeptide.

20. 20. The binding protein of claim 19, wherein the antigen-binding site binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:

1.

21. The antigen-binding site has an equilibrium dissociation constant (K Ｄ 21. The binding protein of claim 20, which binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 at the C3 domain.

22. 20. The binding protein of claim 19, wherein the antigen-binding site binds to a human isoform E CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:

105.

23. 23. The binding protein of any one of claims 19 to 22, wherein the antigen-binding site binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:

30.

24. The antigen-binding site has an equilibrium dissociation constant (K Ｄ 24. The binding protein of claim 23, which binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:

30.

25. 25. The binding protein of any one of claims 1 to 24, which is a chimeric or humanized antibody.

26. The binding protein of any one of claims 16 to 24, which is a human antibody.

27. 25. The binding protein of any one of claims 1 to 24, which is a monoclonal antibody.

28. 25. The binding protein of any one of claims 1 to 24, comprising one or more full-length antibody heavy chains comprising an Fc region.

29. 29. The binding protein of claim 28, wherein the Fc region is a human Fc region comprising one or more mutations that reduce or eliminate Fc receptor binding and / or effector function of the Fc region.

30. 29. The binding protein of claim 28, wherein the Fc region is a human IgGl Fc region.

31. 31. The binding protein of claim 30, wherein the human IgGl Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to the EU index, the amino acid substitutions being L234A, L235A, and P329A.

32. The human IgG1 Fc region is 298, 299 of human IgG1 according to the EU index.

31. The binding protein of claim 30, comprising amino acid substitutions at positions corresponding to positions S298N, T299A, and Y300S.

33. 29. The binding protein of claim 28, wherein the Fc region is a human IgG4 Fc region.

34. 34. The binding protein of claim 33, wherein the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K.

35. 35. The binding protein of claim 33 or 34, wherein the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A.

36. 35. The binding protein of claim 33 or 34, wherein the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index, the amino acid substitutions being E233P, F234V, L235A, and a deletion at 236.

37. 25. The binding protein of any one of claims 1 to 24, comprising an antibody F(ab), F(ab')2, Fab'-SH, Fv, or scFv fragment.

38. 38. The binding protein of any one of claims 1 to 37, conjugated to a cytotoxic agent or label.

39. 38. The binding protein of any one of claims 1 to 37, which is a bispecific binding protein comprising a first antigen-binding site that binds to a CD38 polypeptide and a second antigen-binding site.

40. 38. The binding protein of any one of claims 1 to 37, which is a trispecific binding protein comprising a first antigen-binding site that binds to a CD38 polypeptide, a second antigen-binding site, and a third antigen-binding site.

41. 41. The binding protein of claim 40, wherein the first antigen-binding site binds to the extracellular domain of a human CD38 polypeptide, and the second and third antigen-binding sites each bind to a T-cell surface protein.

42. 42. The binding protein of claim 41 , wherein the first antigen-binding site binds to the extracellular domain of a human CD38 polypeptide, and (a) the second antigen-binding site binds to a human CD28 polypeptide and the third antigen-binding site binds to a human CD3 polypeptide, or (b) the second antigen-binding site binds to a human CD3 polypeptide and the third antigen-binding site binds to a human CD28 polypeptide.

43. A binding protein comprising three antigen-binding sites, each of which binds to one or more target proteins, wherein at least one of the three antigen-binding sites cross-reacts with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide.

44. 44. The binding protein of claim 43, which cross-reacts with a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

105.

45. 45. The binding protein of claim 43 or 44, which cross-reacts with a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:

30.

46. 46. ​​The binding protein of any one of claims 43 to 45, comprising an antigen binding site that cross-reacts with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide, and two antigen binding sites that each bind to a T-cell surface protein.

47. 47. The binding protein of claim 46, comprising an antigen-binding site that cross-reacts with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide, an antigen-binding site that binds to a human CD28 polypeptide, and an antigen-binding site that binds to a human CD3 polypeptide.

48. and four polypeptide chains that form three antigen-binding sites, the first polypeptide chain having the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] It includes a structure represented by The second polypeptide chain has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] It includes a structure represented by The third polypeptide chain has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] It includes a structure represented by The fourth polypeptide chain has the formula: V Ｌ３ -C Ｌ [IV] It includes a structure represented by During the ceremony, V Ｌ１ is a first immunoglobulin light chain variable domain; V Ｌ２ is a second immunoglobulin light chain variable domain; V Ｌ３ is a third immunoglobulin light chain variable domain; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; The hinge is C Ｈ１ and C Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; (a) V Ｈ１ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), Ｌ１ The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); (b) V Ｈ２ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), Ｌ２ the domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or (c) V Ｈ３ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), Ｌ３ 48. The binding protein of any one of claims 43 to 47, wherein the domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

49. (a) V Ｈ１ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), Ｌ１ The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); (b) V Ｈ１ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), Ｌ１ The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); (c) V Ｈ２ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), Ｌ２ The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); (d) V Ｈ２ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), Ｌ２ The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); (e) V Ｈ３ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33). -H3 sequence, V Ｌ３ the domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or (h) V Ｈ３ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), Ｌ３ 49. The binding protein of claim 48, wherein the domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

50. (a) V Ｈ３ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), Ｌ３ the domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or (b) V Ｈ３ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), Ｌ３ 50. The binding protein of claim 49, wherein the domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

51. (a) V Ｈ３ The domain comprises the amino acid sequence of SEQ ID NO: 5, Ｌ３ The domain comprises the amino acid sequence of SEQ ID NO:6; (b) V Ｈ３ The domain comprises the amino acid sequence of SEQ ID NO: 17, Ｌ３ The domain comprises the amino acid sequence of SEQ ID NO: 18; (c) V Ｈ３ The domain comprises the amino acid sequence of SEQ ID NO: 21, Ｌ３ The domain comprises the amino acid sequence of SEQ ID NO: 18; (d) V Ｈ３ The domain comprises the amino acid sequence of SEQ ID NO: 23, Ｌ３ The domain comprises the amino acid sequence of SEQ ID NO: 18; or (e) V Ｈ３ The domain comprises the amino acid sequence of SEQ ID NO: 13, Ｌ３ 51. The binding protein of claim 50, wherein the domain comprises the amino acid sequence of SEQ ID NO:

14.

52. and four polypeptide chains that form three antigen-binding sites, the first polypeptide chain having the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] It includes a structure represented by The second polypeptide chain has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] It includes a structure represented by The third polypeptide chain has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] It includes a structure represented by The fourth polypeptide chain has the formula: V Ｌ３ -C Ｌ [IV] It includes a structure represented by During the ceremony: V Ｌ１ is a first immunoglobulin light chain variable domain; V Ｌ２ is a second immunoglobulin light chain variable domain; V Ｌ３ is a third immunoglobulin light chain variable domain; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; The hinge is C Ｈ１ and C Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; (a) V Ｈ１ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), Ｌ１ The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46); (b) V Ｈ２ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), Ｌ２ the domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46); or (c) V Ｈ３ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), Ｌ３ 48. The binding protein of any one of claims 43 to 47, wherein the domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

53. V Ｈ３ The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), Ｌ３ 53. The binding protein of claim 52, wherein the domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

54. V Ｈ３ The domain comprises the amino acid sequence of SEQ ID NO: 9, Ｌ３ 54. The binding protein of claim 53, wherein the domain comprises the amino acid sequence of SEQ ID NO:

10.

55. (a) V Ｈ１ The domain comprises the amino acid sequence of SEQ ID NO: 49, Ｌ１ The domain comprises the amino acid sequence of SEQ ID NO: 50, Ｈ２ The domain comprises the amino acid sequence of SEQ ID NO:

53. Mi, V Ｌ２ The domain comprises the amino acid sequence of SEQ ID NO:54; (b) V Ｈ２ The domain comprises the amino acid sequence of SEQ ID NO: 49, Ｌ２ The domain comprises the amino acid sequence of SEQ ID NO: 50, Ｈ１ The domain comprises the amino acid sequence of SEQ ID NO: 53, Ｌ１ The domain comprises the amino acid sequence of SEQ ID NO:54; (c) V Ｈ１ The domain comprises the amino acid sequence of SEQ ID NO: 51, Ｌ１ The domain comprises the amino acid sequence of SEQ ID NO: 52, Ｈ２ The domain comprises the amino acid sequence of SEQ ID NO: 53, Ｌ２ The domain comprises the amino acid sequence of SEQ ID NO:54; (d) V Ｈ２ The domain comprises the amino acid sequence of SEQ ID NO: 51, Ｌ２ The domain comprises the amino acid sequence of SEQ ID NO: 52, Ｈ１ The domain comprises the amino acid sequence of SEQ ID NO: 53, Ｌ１ The domain comprises the amino acid sequence of SEQ ID NO: 54 (e) V Ｈ１ The domain comprises the amino acid sequence of SEQ ID NO: 49, Ｌ１ The domain comprises the amino acid sequence of SEQ ID NO: 50, Ｈ２ The domain comprises the amino acid sequence of SEQ ID NO: 84, Ｌ２ The domain comprises the amino acid sequence of SEQ ID NO: 85; (f) V Ｈ２ The domain comprises the amino acid sequence of SEQ ID NO: 49, Ｌ２ The domain comprises the amino acid sequence of SEQ ID NO: 50, Ｈ１ The domain comprises the amino acid sequence of SEQ ID NO: 84, Ｌ１ The domain comprises the amino acid sequence of SEQ ID NO: 85; (g) V Ｈ１ The domain comprises the amino acid sequence of SEQ ID NO: 51, Ｌ１ The domain comprises the amino acid sequence of SEQ ID NO: 52, Ｈ２ The domain comprises the amino acid sequence of SEQ ID NO: 84, Ｌ２ The domain comprises the amino acid sequence of SEQ ID NO: 85; or (h) V Ｈ２ The domain comprises the amino acid sequence of SEQ ID NO: 51, Ｌ２ The domain comprises the amino acid sequence of SEQ ID NO: 52, Ｈ１ The domain comprises the amino acid sequence of SEQ ID NO: 84, Ｌ１ 55. The binding protein of any one of claims 48 to 54, wherein the domain comprises the amino acid sequence of SEQ ID NO:

85.

56. (a) V Ｈ１ The domain comprises the amino acid sequence of SEQ ID NO: 49, Ｌ１ The domain comprises the amino acid sequence of SEQ ID NO: 50, Ｈ２ The domain comprises the amino acid sequence of SEQ ID NO: 53, Ｌ２ The domain comprises the amino acid sequence of SEQ ID NO: 54, Ｈ３ The domain comprises the amino acid sequence of SEQ ID NO: 13, Ｌ３ The domain comprises the amino acid sequence of SEQ ID NO: 14; or (b) V Ｈ１ The domain comprises the amino acid sequence of SEQ ID NO: 49, Ｌ１ The domain comprises the amino acid sequence of SEQ ID NO: 50, Ｈ２ The domain comprises the amino acid sequence of SEQ ID NO: 53, Ｌ２ The domain comprises the amino acid sequence of SEQ ID NO: 54, Ｈ３ The domain comprises the amino acid sequence of SEQ ID NO: 9, Ｌ３ 56. The binding protein of claim 55, wherein the domain comprises the amino acid sequence of SEQ ID NO:

10.

57. L １ , L ２ , L ３ or L ４ 57. The binding protein of any one of claims 48-56, wherein at least one of: is independently 0 amino acids in length.

58. L １ , L ２ , L ３ or L ４ is each independently at least one amino acid in length.

59. (a) L １ , L ２ , L ３ and L ４ are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGGS (SEQ ID NO:56), S,RT,TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59); or (b) L １ , L ２ , L ３ and L ４ each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGGS (SEQ ID NO:56), S,RT,TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59).

60. (a) L １ contains the sequence GQPKAAP (SEQ ID NO: 58), and L ２ contains the sequence TKGPS (SEQ ID NO: 57), and L ３ contains a sequence S, and L ４ contains the sequence RT; (b) L １ contains the sequence GGGGSGGGGS (SEQ ID NO: 55), and L ２ contains the sequence GGGGSGGGGS (SEQ ID NO: 55), and L ３ is 0 amino acids long, and L ４ is 0 amino acids in length; (c) L １ contains the sequence GGSGSSGSGG (SEQ ID NO: 59), and L ２ contains the sequence GGSGSSGSGG (SEQ ID NO: 59), and L ３ is 0 amino acids long, and L ４ is 0 amino acids in length; or (d) L １ contains the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 56), and L ２ is 0 amino acids long, and L ３ contains the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 56), and L ４ The binding protein of any one of claims 48 to 56, wherein is 0 amino acids in length.

61. Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is the hinge C of human IgG4. Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ 61. The binding protein of any one of claims 48 to 60, wherein each domain comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A.

62. Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is the hinge C of human IgG4. Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ 61. The binding protein of any one of claims 48 to 60, wherein each domain comprises amino acid substitutions at positions corresponding to positions 233 to 236 of human IgG4 according to the EU index, the amino acid substitutions being E233P, F234V, L235A, and a deletion at 236.

63. Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is the hinge C of human IgG4. Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ 63. The binding protein of any one of claims 48 to 62, wherein each domain comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K.

64. Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is the hinge C of human IgG1. Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ 61. The binding protein of any one of claims 48 to 60, wherein the domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A.

65. Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is the hinge C of human IgG1. Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ 61. The binding protein of any one of claims 48 to 60, wherein the domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, the amino acid substitutions being S298N, T299A, and Y300S.

66. Hinge-C of the second polypeptide chain Ｈ２ -C Ｈ３ The domain contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; Ｈ２ -C Ｈ３ The domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W.

66. The binding protein of any one of items 48 to 65.

67. Hinge-C of the second polypeptide chain Ｈ２ -C Ｈ３ The domain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; Ｈ２ -C Ｈ３ 66. The binding protein of any one of claims 48 to 65, wherein the domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V.

68. 53. The binding protein of claim 48 or 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

63.

69. 53. The binding protein of claim 48 or 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 65, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

63.

70. 53. The binding protein of claim 48 or 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 67, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

63.

71. 53. The binding protein of claim 48 or 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

69.

72. 53. The binding protein of claim 48 or 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

69.

73. 53. The binding protein of claim 48 or 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

69.

74. 1. A binding protein comprising three antigen-binding sites that each bind to one or more target proteins, the binding protein comprising four polypeptide chains that form the three antigen-binding sites, the first polypeptide chain having the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] It includes a structure represented by The second polypeptide chain has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] It includes a structure represented by The third polypeptide chain has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] It includes a structure represented by The fourth polypeptide chain has the formula: V Ｌ３ -C Ｌ [IV] It includes a structure represented by During the ceremony, V Ｌ１ is a first immunoglobulin light chain variable domain; V Ｌ２ is a second immunoglobulin light chain variable domain; V Ｌ３ is a third immunoglobulin light chain variable domain; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; The hinge is C Ｈ１ and C Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair; (a) Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is the hinge C of human IgG1. Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ the domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, the amino acid substitutions being S298N, T299A, and Y300S; or (b) Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is C of human IgG4 Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ The binding protein, wherein each domain contains amino acid substitutions at positions corresponding to positions 233 to 236 of human IgG4 according to the EU index, and the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.

75. V Ｈ１ and V Ｌ１ , V Ｈ２ and V Ｌ２ , and V Ｈ３ and V Ｌ３ 75. The binding protein of claim 74, wherein at least one pair of said amino acids forms an antigen-binding site that binds to a CD38 polypeptide.

76. V Ｈ１ and V Ｌ１ , V Ｈ２ and V Ｌ２ , and V Ｈ３ and V Ｌ３ One, two, or three pairs of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL1 5, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD2 4, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD1 60, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1 , EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1 , HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, I L5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, P 75. The binding protein of claim 74, which forms an antigen binding site that binds to an antigen target selected from the group consisting of ROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.

77. V Ｈ１ and V Ｌ１ , V Ｈ２ and V Ｌ２ , and V Ｈ３ and V Ｌ３ a first pair of V forms an antigen-binding site that binds to the human CD3 polypeptide, and V Ｈ１ and V Ｌ１ , V Ｈ２ and V Ｌ２ , and V Ｈ３ and V Ｌ３ a second pair of V forms an antigen-binding site that binds to the human CD28 polypeptide, and V Ｈ１ and V Ｌ１ , V Ｈ２ and V Ｌ２ , and V Ｈ３ and V Ｌ３ The third pair of proteins is A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, C D40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, I GF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, I L7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, I L15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, K IR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM,75. The binding protein of claim 74, which forms an antigen-binding site that binds to a human antigen target selected from the group consisting of: XCR1, XCR2, and XCR3.

78. 1. A kit of polynucleotides comprising: (a) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:72, a third polynucleotide comprising the sequence of SEQ ID NO:74, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75; (b) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 76, a third polynucleotide comprising the sequence of SEQ ID NO: 77, and a a fourth polynucleotide comprising the sequence of sequence number 75; (c) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:78, a third polynucleotide comprising the sequence of SEQ ID NO:79, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75; (d) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:72, a third polynucleotide comprising the sequence of SEQ ID NO:80, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81; (e) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 76, a third polynucleotide comprising the sequence of SEQ ID NO: 82, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 81; or (f) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 78, a third polynucleotide comprising the sequence of SEQ ID NO: 83, and a fourth polynucleotide comprising the sequence of SEQ ID NO:

81. The kit comprising:

79. A polynucleotide encoding the binding protein of any one of claims 1 to 77.

80. 80. A vector comprising the polynucleotide of claim 79.

81. 81. A host cell comprising the kit of polynucleotides of claim 78, the polynucleotide of claim 79, or the vector of claim 80.

82. 82. A method of producing a binding protein, comprising culturing the host cell of claim 81 so that the binding protein is produced.

83. 83. The method of claim 82, further comprising recovering the binding protein from the host cell.

84. 78. A pharmaceutical composition comprising the binding protein of any one of claims 1 to 77 and a pharmaceutically acceptable carrier.

85. 85. A method for preventing and / or treating cancer in a patient, said method comprising administering to the patient a therapeutically effective amount of at least one binding protein of any one of claims 1 to 73 or the pharmaceutical composition of claim 84.

86. 86. The method of claim 85, wherein the binding protein comprises one antigen binding site that binds to a T cell surface protein and another antigen binding site that binds to the extracellular domain of a human CD38 polypeptide.

87. 87. The method of claim 86, wherein the binding protein is a trispecific binding protein comprising a first antigen-binding site that binds to CD3, a second antigen-binding site that binds to CD28, and a third antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide.

88. 88. The method of any one of claims 85 to 87, wherein the at least one binding protein is co-administered with a chemotherapeutic agent.

89. The method of any one of claims 85 to 88, wherein the cancer is multiple myeloma.

90. Any one of claims 85 to 88, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma. The method described in paragraph .

91. The method of any one of claims 85 to 90, wherein the patient is a human.

92. 92. The method of any one of claims 85 to 91, wherein the patient is selected for treatment because the cancer cells express human CD38 isoform E polypeptide on their cell surface.

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