Anti-CD38 antibodies and combinations with anti-CD3 and anti-CD28 antibodies

Abstract

The present disclosure provides binding proteins that bind to CD38 polypeptides (e.g., human and cynomolgus CD38 polypeptides). For example, the binding protein can be a monospecific, bispecific, or trispecific binding protein having at least one antigen binding domain that binds to a CD38 polypeptide. The present disclosure also provides methods for preparing binding proteins that bind to CD38 polypeptides and uses of such binding proteins.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 570,655 filed on October 10, 2017; U.S. Provisional Application Serial No. 62 / 570,660 filed on October 11, 2017; U.S. Provisional Application Serial No. 62 / 676,221 filed on May 24, 2018; and EP Application No. EP18187186.4 filed on August 3, 2018, all of which are incorporated herein by reference in their entirety.

[0003] Submit a sequence listing as an ASCII text file

[0004] The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of Sequence Listing (File Name: 183952029941seqlist.TXT, Record Date: October 8, 2018, Size: 158KB). Technical Field

[0005] 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 CD38 polypeptides, as well as polynucleotides, host cells, methods of production, and methods of use related thereto. Background Art

[0006] Biotherapeutics based on monoclonal antibodies have become an important approach for the development of new drugs. Monoclonal antibody technology provides specific targeting, precise signal delivery and / or payload to specific cell populations, and provides lasting biological effects through its Fc function. Antibody engineering efforts have allowed the development of multispecific antibodies that combine the specificity of multiple monoclonal antibodies for various biological applications, expanding the scope of antibody drug development.

[0007] CD38 is an attractive drug target because it is expressed on the cell surface of a variety of lymphoid tumor cells (see Stevenson, GT (2006) Mol. Med. 12:345-346). Daratumumab (daratumumab) is an anti-CD38 antibody approved for the treatment of multiple myeloma. However, therapeutic agents targeting CD38 are desired to have different modes of action and / or improved properties, including but not limited to high affinity binding to CD38, cross-reactivity between human and cynomolgus 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 anti-tumor activity. Summary of the invention

[0008] Provided herein are 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 site that binds to CD38 polypeptides. Advantageously, these binding proteins have the ability to recruit T cells to the vicinity of cancer cells, subsequently activate T cells, and promote the killing of adjacent cancer cells by activated T cells through a granzyme / perforin mechanism, from anti-CD38 antibodies such as (daratumumab) provides a different mode of action for anti-tumor activity. In addition, the ability to bind human and cynomolgus monkey CD38 polypeptides allows easy testing of binding proteins in preclinical toxicology studies, for example, to evaluate their safety profile for later clinical use.

[0009] In some embodiments, provided herein are monospecific binding proteins that bind to human CD38 polypeptides. In some embodiments, the binding protein cross-reacts with human and cynomolgus CD38 polypeptides. In some embodiments, the binding protein binds to human isoform A and isoform E CD38 polypeptides. In some embodiments, the binding protein has one or more of the following features (in any combination): binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising an amino acid sequence of SEQ ID NO: 1) as a purified protein, as determined by SPR; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising an amino acid sequence of SEQ ID NO: 1) as a purified protein, K D 1.5 nM or less, as determined by SPR; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), with an apparent K Dis 20 nM, 15 nM, 10 nM, 5 nM, 1 nM or less, as determined by flow cytometry; 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, as determined by SPR; 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, K D is 3.5 nM or less, as determined by SPR; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell, as determined by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell, with an apparent K D is 7.5 nM or less, as determined by flow cytometry; 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, as determined by ELISA; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) expressed on the surface of a cell, as determined by flow cytometry; induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells expressing CD38 on the surface of the cell; 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.

[0010] In some embodiments, provided herein are trispecific binding proteins that bind to human CD38 polypeptides. In some embodiments, the trispecific binding protein binds (e.g., simultaneously) a CD38 polypeptide (e.g., expressed on the cell surface) and one or more other target antigens expressed on the surface of a second cell, thereby recruiting a second cell close to a cell expressing a CD38 polypeptide. In some embodiments, the trispecific binding protein binds (e.g., simultaneously) a CD38 polypeptide (e.g., expressed on the cell surface) and one or two target antigens expressed on the surface of a T cell, thereby recruiting T cells close to cells expressing CD38 polypeptides. In some embodiments, the trispecific binding protein activates T cells and / or provides CD28-mediated co-stimulatory signals to T cells. 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 isotype A and isotype E CD38 polypeptides. In some embodiments, the trispecific binding protein has one or more of the following features (in any combination): binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, as determined by SPR; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, D is 1.5 nM or less, as determined by SPR; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), with an apparent K D is 20 nM, 15 nM, 10 nM, 5 nM, 1 nM or less, as determined by flow cytometry; 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, as determined by SPR; 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, K D is 3.5 nM or less, as determined by SPR; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell, as determined by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell, with an apparent K Dis 7.5 nM or less, as determined by flow cytometry; 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, as determined by ELISA; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) expressed on the surface of a cell, as determined by flow cytometry; induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells expressing CD38 on the surface of the cell; 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; induces proliferation of T cells (e.g., CD4+ and / or CD8+ T cells); induces T cell (e.g., CD4+ and / or CD8+ T cells) expression of Bcl-xL; induces apoptosis of CD38+ cells; binds to CD38 expressed on the surface of a cell and on the surface of a T cell One or more T cell target antigens expressed; binding to CD38 expressed on the cell surface, CD28 expressed on the T cell surface, and CD3 expressed on the T cell surface; stimulating the activation of the T cell receptor; inducing co-stimulation of T cell receptor signaling (e.g., mediated by CD28); and having one or more mutations (e.g., in the Fc region) compared to the same binding protein without one or more mutations, resulting in reduced induction of cytokine release (e.g., IFN-γ, IL-2, and / or TNF-α) by PBMCs; inducing cytokine release (e.g., IFN-γ and / or IL-6) by PBMCs in the presence of CD38+ target cells. For exemplary assays, see Examples 1, 3, and 4. In some embodiments, KD is measured at 4°C or 25°C.

[0011] In some embodiments, provided herein are binding proteins comprising an antigen binding site that binds a CD38 polypeptide, wherein the antigen binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising an amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising an amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising an 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 an amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising an amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 36). NO:40), and a CDR-L2 sequence containing 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-H3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 41). NO: 36) of the amino acid sequence of CDR-L3.In some embodiments, the antigen binding site comprises: (a) an antibody heavy chain variable (VH) domain, which 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 (b) an antibody light chain variable (VL) domain, which 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). In some embodiments, the VH domain comprises the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4 from N-terminus to C-terminus; wherein FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO: 87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); wherein FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); wherein FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94). NO:94); and wherein 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) an antibody heavy chain variable (VH) domain, which 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 (b) an antibody light chain variable (VL) domain, which 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). In some embodiments, the VH domain comprises the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4 from N-terminus to C-terminus; wherein FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO: 87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); wherein FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); wherein FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94). NO:94); and wherein 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.

[0012] 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 antigen binding site cross-reacts with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus 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. In some embodiments, the antigen binding site binds to the human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with an equilibrium dissociation constant (K D ) is 2.1 nM or less. 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 the cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30 with an equilibrium dissociation constant (K D ) is 1.3 nM or less.

[0013] 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 containing an Fc region. In some embodiments, the Fc region is a human Fc region, which comprises one or more mutations that reduce or eliminate Fc receptor binding and / or the effector function of the Fc region. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, according to EU Index, the human IgG1 Fc region comprises amino acid substitutions at positions corresponding to 234, 235, and 329 of human IgG1, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, according to EU Index, the human IgG1 Fc region comprises amino acid substitutions at positions corresponding to 298, 299, and 300 of human IgG1, wherein the amino acid substitutions are S298N, T299A, and Y300S. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, according to the EU Index, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4, wherein the amino acid substitutions are S228P and R409K. In some embodiments, according to the EU Index, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4, wherein the amino acid substitutions are F234A and L235A. In some embodiments, according to the EU Index, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 233-236 of human IgG4, wherein the amino acid substitutions are E233P, F234V, L235A and deletions at 236. In some embodiments, according to the EU Index, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 233-237 of human IgG4, wherein the sequence EFLGG is replaced by PVAG. In some embodiments, the binding protein comprises an antibody F(ab), F(ab')2, Fab'-SH, Fv or scFv fragment. In some embodiments, the binding protein is conjugated to a cytotoxic agent or a marker. 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, a second antigen binding site, and a third antigen binding site that binds to a CD38 polypeptide. In some embodiments, the first antigen binding site binds to the extracellular domain of a human CD38 polypeptide, and wherein 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 wherein (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.

[0014] In some embodiments, the present invention provides 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 CD38 polypeptide. In some embodiments, the binding protein cross-reacts with a human CD38 polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 105. In some embodiments, the binding protein cross-reacts with a cynomolgus CD38 polypeptide comprising an amino acid sequence of SEQ ID NO: 30. In some embodiments, the binding protein comprises an antigen binding site that cross-reacts with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus CD38 polypeptide, and two antigen binding sites that each bind to a T cell surface protein. In some embodiments, the binding protein comprises an antigen binding site that cross-reacts with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus 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. In some embodiments, the binding protein comprises four polypeptide chains forming the three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0015] V L2 -L 1 -V L1 -L 2 -C L [I]

[0016] and the second polypeptide chain comprises a structure represented by the formula:

[0017] V H1 -L 3 -V H2 -L 4 -C H1 -Hinge-C H2 -C H3 [II]

[0018] and the third polypeptide chain comprises a structure represented by the formula:

[0019] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0020] and the fourth polypeptide chain comprises a structure represented by the formula:

[0021] V L3 -C L [IV]

[0022] in:

[0023] V L1 is the first immunoglobulin light chain variable domain;

[0024] V L2 is a second immunoglobulin light chain variable domain;

[0025] V L3 is the third immunoglobulin light chain variable domain;

[0026] V H1 is the first immunoglobulin heavy chain variable domain;

[0027] V H2 is a second immunoglobulin heavy chain variable domain;

[0028] V H3 is the third immunoglobulin heavy chain variable domain;

[0029] C L is the immunoglobulin light chain constant domain;

[0030] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0031] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0032] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0033] The hinge is connected to the C H 1 and C H 2 domain immunoglobulin hinge region, and

[0034] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0035] wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair, and

[0036] Where: (a) 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); and the V 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);

[0037] (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); and the V 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

[0038] (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); and the VL3 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). In some embodiments, the binding protein comprises four polypeptide chains forming the three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0039] V L2 -L 1 -V L1 -L 2 -C L [I]

[0040] and the second polypeptide chain comprises a structure represented by the formula:

[0041] V H1 -L 3 -V H2 -L 4 -C H1 -Hinge-C H2 -C H3 [II]

[0042] and the third polypeptide chain comprises a structure represented by the formula:

[0043] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0044] and the fourth polypeptide chain comprises a structure represented by the formula:

[0045] V L3 -C L [IV]

[0046] in:

[0047] V L1 is the first immunoglobulin light chain variable domain;

[0048] V L2 is a second immunoglobulin light chain variable domain;

[0049] V L3 is the third immunoglobulin light chain variable domain;

[0050] V H1is the first immunoglobulin heavy chain variable domain;

[0051] V H2 is a second immunoglobulin heavy chain variable domain;

[0052] V H3 is the third immunoglobulin heavy chain variable domain;

[0053] C L is the immunoglobulin light chain constant domain;

[0054] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0055] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0056] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0057] The hinge is connected to the C H1 and C H2 The immunoglobulin hinge region of the domain, and

[0058] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0059] wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair, and

[0060] Where: (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); and the V L1a 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);

[0061] (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); and the V 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); or

[0062] (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); and the V L3 The domain comprises a CDR-L1 sequence comprising an amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising an amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising an amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the VH1 The domain comprises a CDR-H1 sequence comprising an amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising an amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising an amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and the 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); the V H1 The domain comprises a CDR-H1 sequence comprising an amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising an amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising an amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and the V L1 The V 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); the V H2 The domain comprises a CDR-H1 sequence comprising an amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising an amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising an amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and the V 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); the V H2The V domain comprises a CDR-H1 sequence comprising an amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising an amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising an amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and L2 The V 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); the V H3 The domain comprises a CDR-H1 sequence comprising an amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising an amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising an amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and the V L3 The V 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 H3 The domain comprises a CDR-H1 sequence comprising an amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising an amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising an amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and the V L The V3 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). In some embodiments, the V H3The V domain comprises a CDR-H1 sequence comprising an amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising an amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising an amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and L3 The V 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 H3 The domain comprises a CDR-H1 sequence comprising an amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising an amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising an amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and the V L3 The V 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). In some embodiments, the V H3 The domain comprises the amino acid sequence of SEQ ID NO: 5, and the V L3 The domain comprises the amino acid sequence of SEQ ID NO: 6; the V H3 The domain comprises the amino acid sequence of SEQ ID NO: 17, and the V L3 The domain comprises the amino acid sequence of SEQ ID NO: 18; the V H3 The domain comprises the amino acid sequence of SEQ ID NO: 21, and the V L3 The domain comprises the amino acid sequence of SEQ ID NO: 18; the V H3 The domain comprises the amino acid sequence of SEQ ID NO: 23, and the V L3 The domain comprises the amino acid sequence of SEQ ID NO: 18; or the V H3 The domain comprises the amino acid sequence of SEQ ID NO: 13, and the V L3The domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the binding protein comprises four polypeptide chains forming the three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0063] V L2 -L 1 -V L1 -L 2 -C L [I]

[0064] and the second polypeptide chain comprises a structure represented by the formula:

[0065] V H1 -L 3 -V H2 -L 4 -C H1 -Hinge-C H2 -C H3 [II]

[0066] and the third polypeptide chain comprises a structure represented by the formula:

[0067] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0068] and the fourth polypeptide chain comprises a structure represented by the formula:

[0069] V L3 -C L [IV]

[0070] in:

[0071] V L1 is the first immunoglobulin light chain variable domain;

[0072] V L2 is a second immunoglobulin light chain variable domain;

[0073] V L3 is the third immunoglobulin light chain variable domain;

[0074] V H1 is the first immunoglobulin heavy chain variable domain;

[0075] V H2 is a second immunoglobulin heavy chain variable domain;

[0076] V H3 is the third immunoglobulin heavy chain variable domain;

[0077] CL is the immunoglobulin light chain constant domain;

[0078] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0079] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0080] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0081] The hinge is connected to the C H1 and C H2 The immunoglobulin hinge region of the domain, and

[0082] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0083] wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair, and

[0084] Wherein (a) V H1 The domain comprises a CDR-H1 sequence comprising an amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising an amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising an amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and the V L1 The domain comprises a CDR-L1 sequence comprising an amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising an amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising an amino acid sequence of LQDYIYYPT (SEQ ID NO: 46);

[0085] (b) V H2 The V domain comprises a CDR-H1 sequence comprising an amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising an amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising an amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and the V L2The 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

[0086] (c) V H3 The V domain comprises a CDR-H1 sequence comprising an amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising an amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising an amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and the V L3 The V domain comprises a CDR-L1 sequence comprising an amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising an amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising an amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H3 The V domain comprises a CDR-H1 sequence comprising an amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising an amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising an amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and L3 The V domain comprises a CDR-L1 sequence comprising an amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising an amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising an amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H3 The domain comprises the amino acid sequence of SEQ ID NO: 9, and the V L3 The domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, wherein the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, wherein the V H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, and the V L2 The domain comprises the amino acid sequence of SEQ ID NO: 54; the V H2The domain comprises the amino acid sequence of SEQ ID NO: 49, wherein the V L2 The domain comprises the amino acid sequence of SEQ ID NO: 50, wherein the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 53, and the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 54; the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 51, wherein the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 52, wherein the V H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, and the V L2 The domain comprises the amino acid sequence of SEQ ID NO: 54; the V H 2 domain comprises the amino acid sequence of SEQ ID NO: 51, the V L2 The domain comprises the amino acid sequence of SEQ ID NO: 52, wherein the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 53, and the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 54; the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, wherein the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, wherein the V H2 The domain comprises the amino acid sequence of SEQ ID NO: 84, and the V L2 The domain comprises the amino acid sequence of SEQ ID NO: 85; the V H2 The domain comprises the amino acid sequence of SEQ ID NO: 49, the VL 2 The domain comprises the amino acid sequence of SEQ ID NO: 50, wherein the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 84, and the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 85; the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 51, wherein the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 52, wherein the V H2 The domain comprises the amino acid sequence of SEQ ID NO: 84, and the V L2 The V domain comprises the amino acid sequence of SEQ ID NO: 85; or H2 The domain comprises the amino acid sequence of SEQ ID NO: 51, wherein the VL2 The domain comprises the amino acid sequence of SEQ ID NO: 52, wherein the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 84, and the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, wherein the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, wherein the V H2 The VL2 domain comprises the amino acid sequence of SEQ ID NO: 54, the V H3 The domain comprises the amino acid sequence of SEQ ID NO: 13, and the V L3 The domain comprises the amino acid sequence of SEQ ID NO: 14; or the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, wherein the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, wherein the V H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, wherein the V L2 The domain comprises the amino acid sequence of SEQ ID NO: 54, wherein the V H3 The domain comprises the amino acid sequence of SEQ ID NO: 9, and the V L3 The domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, L 1 , L 2 , L 3 or L 4 The length of at least one of L is independently 0 amino acids. 1 , L 2 , L 3 or L 4 The length of each of L is independently at least one amino acid. 1 , L 2 , L 3 and L 4 Each is independently 0 amino acids in length or comprises 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 , L 2 , L 3 and L 4 Each independently comprises 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, L 1 Containing the sequence GQPKAAP (SEQ ID NO: 58), L 2 Containing the sequence TKGPS (SEQ ID NO: 57), L 3 Contains the sequence S, and L 4 Contains the sequence RT;L 1 Containing the sequence GGGGSGGGGS (SEQ ID NO: 55), L 2 Containing the sequence GGGGSGGGGS (SEQ ID NO: 55), L 3 is 0 amino acids in length, and L 4 The length of L is 0 amino acids; 1 Containing the sequence GGSGSSGSGG (SEQ ID NO: 59), L 2 Containing the sequence GGSGSSGSGG (SEQ ID NO: 59), L 3 is 0 amino acids in length, and L 4 is 0 amino acids in length; or L 1 Containing the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L 2 The length is 0 amino acids, L 3 comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), and L 4 The length of the hinge-C of the second and third polypeptide chains is 0 amino acids. H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 The domains each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG4, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the hinge-C H2 -C H3The domain is human IgG4 hinge-C H2 -C H3 domain, and wherein according to the EUIndex, the hinge-C H2 -C H3 The domains each comprise an amino acid substitution at a position corresponding to positions 233-236 of human IgG4, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-C H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, and wherein according to the EUIndex, the hinge-C H2 -C H3 The domains each comprise an amino acid substitution at positions corresponding to positions 228 and 409 of human IgG4, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the hinge-C H2 -C H3 The domain is human IgG1 hinge-C H2 -C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 The domains each comprise an amino acid substitution at positions corresponding to positions 234, 235, and 329 of human IgG1, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the hinge-C H2 -C H3 The domain is human IgG1 hinge-C H2 -C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 The domains each comprise an amino acid substitution at positions corresponding to positions 298, 299 and 300 of human IgG1, wherein the amino acid substitutions are S298N, T299A and Y300S. In some embodiments, according to the EU Index, the hinge-C H2 -C H3 The domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V; and wherein according to the EU Index, the hinge-C H2 -C H3The domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W. In some embodiments, according to the EU Index, the hinge-C H2 -C H3 The domain comprises an amino acid substitution at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W; and wherein according to the EU Index, the hinge-C H2 -C H3The domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are 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 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.

[0087] In some embodiments, the present invention provides a binding protein comprising three antigen binding sites each binding to one or more target proteins, wherein the binding protein comprises four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0088] V L2 -L 1 -V L1 -L2 -C L [I]

[0089] and the second polypeptide chain comprises a structure represented by the formula:

[0090] V H1 -L 3 -V H2 -L 4 -C H1 -Hinge-C H2 -C H3 [II]

[0091] and the third polypeptide chain comprises a structure represented by the formula:

[0092] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0093] and the fourth polypeptide chain comprises a structure represented by the formula:

[0094] V L3 -C L [IV]

[0095] in:

[0096] V L1 is the first immunoglobulin light chain variable domain;

[0097] V L2 is a second immunoglobulin light chain variable domain;

[0098] V L3 is the third immunoglobulin light chain variable domain;

[0099] V H1 is the first immunoglobulin heavy chain variable domain;

[0100] V H2 is a second immunoglobulin heavy chain variable domain;

[0101] V H3 is the third immunoglobulin heavy chain variable domain;

[0102] C L is the immunoglobulin light chain constant domain;

[0103] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0104] C H2 Immunoglobulin C H2heavy chain constant domain;

[0105] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0106] The hinge is connected to the C H1 and C H2 The immunoglobulin hinge region of the domain, and

[0107] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0108] wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair, and

[0109] wherein (a) the hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG1 hinge-C H2 -C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 The structural domains each comprise an amino acid substitution at positions corresponding to positions 298, 299 and 300 of human IgG1, wherein the amino acid substitutions are S298N, T299A and Y300S; or (b) the hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, and wherein according to the EUIndex, the hinge-C H2 -C H3 The hinge-C domains each comprise an amino acid substitution at a position corresponding to positions 233-236 of human IgG4, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the human IgG4 hinge-C H2 -C H3 The domain comprises an amino acid substitution at positions corresponding to positions 233-237 of human IgG4, wherein the sequence EFLGG is replaced by PVAG. H1 and V L1 、V H2 and V L2 and V H3 and V L3 In some embodiments, V H1 and VL1 、V H2 and V L2 and V H3 and V L31, 2 or 3 pairs of the antigen binding site that binds to an antigen target selected from the group consisting of A2AR, APRIL, ATP diphosphohydrolase, 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, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC9 1, 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, IL2Rβ, IL1, I L1A, 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, NTP diphosphohydrolase-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 H1 and V L1 、V H2 and V L2 and V H3 and V L3 The second pair forms an antigen binding site that binds to human CD28 polypeptide, and V H1 and V L1 、V H2 and V L2 and V H3 and V L3The third pair forms an antigen binding site that binds a human antigen target selected from the group consisting of A2AR, APRIL, ATP diphosphohydrolase, 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, C CL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, 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, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rβ, IL1, IL 1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, I L13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC Class II, NCR3LG1, NKG2D, NTP diphosphohydrolase-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.

[0110] In some embodiments, the present invention provides a kit of polynucleotides, comprising: (a) a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 72, a third polynucleotide comprising a sequence of SEQ ID NO: 74, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 75; (b) a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 76, a third polynucleotide comprising a sequence of SEQ ID NO: 77, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 75; (c) a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 78, a third polynucleotide comprising a sequence of SEQ ID NO: 79, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 75; (d) a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 72, a third polynucleotide comprising a sequence of SEQ ID NO: 80, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 81. (e) a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 76, a third polynucleotide comprising a sequence of SEQ ID NO: 82, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 81; or (f) a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 78, a third polynucleotide comprising a sequence of SEQ ID NO: 83, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 81.

[0111] 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 of any one of the above embodiments.

[0112] In some embodiments, a host cell is provided herein, comprising a kit, a polynucleotide or a vector of any one of the polynucleotides in the above embodiments. In some embodiments, a method for producing a binding protein is provided herein, the method comprising culturing the host cell of any one of the above embodiments to produce the binding protein. In some embodiments, the scheme further comprises recovering the binding protein from the host cell.

[0113] 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.

[0114] In some embodiments, the present invention provides a method 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, 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 a human. In some embodiments, the patient is selected for treatment because the cells of the cancer express human CD38 isoform E polypeptides (e.g., as shown in SEQ ID NO: 105) on their cell surfaces. In some embodiments, cancer cells express CD38 and CD28. In some embodiments, cancer cells express CD38 but not CD28.

[0115] 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, which is used to prevent and / or treat 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, which is used to prepare a drug 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, which comprises a first antigen binding site binding to CD3, a second antigen binding site binding to CD28, and a third antigen binding site binding 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 cells of the cancer express human CD38 isoform E polypeptide (e.g., as shown 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 not CD28.

[0116] It should be understood that one, some or all of the properties 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 become apparent to those skilled in the art. These and other embodiments of the present invention will be further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0118] Figure 1A Binding of the anti-CD38 antibody mAbl (upper panel) and isatuximab (lower panel) to SU-DHL-8 human lymphoma cells or MOLP-8 human multiple myeloma cells is shown using flow cytometry.

[0119] Figure 1B Shown are the results of a flow cytometric binding assay detecting binding of the anti-CD38 antibody mAbl or isatuximab (no binding observed) to cells expressing cynomolgus monkey CD38 on their surface.

[0120] Figure 2A-2I Shown are the results of assays characterizing the binding of anti-CD38 antibodies to human and cynomolgus CD38 polypeptides. Figure 2A Binding of the humanized anti-CD38 antibody mAb2 to soluble human CD38 (upper panel, "hCD38::Histag") or cynomolgus CD38 (upper panel, "cynoCD38::Histag") by ELISA is shown, as well as binding of mAb2 to the surface of cells expressing human CD83 (lower panel, as indicated) or cynomolgus CD38 (lower panel, as indicated) by flow cytometry. Figure 2B Binding of mAb2 to human CD38 (upper panel) or cynomolgus monkey CD38 (lower panel) is shown by surface plasmon resonance (SPR). Figure 2C Binding of the humanized anti-CD38 antibody mAb3 to soluble human CD38 (upper panel, "hCD38::Histag") or cynomolgus CD38 (upper panel, "cynoCD38::Histag") is shown by ELISA, and binding of mAb3 to the surface of cells expressing human CD83 (lower panel, as indicated) or cynomolgus CD38 (lower panel, as indicated) by flow cytometry. Figure 2D Binding of mAb3 to human CD38 (upper panel) or cynomolgus monkey CD38 (lower panel) by SPR is shown. Figure 2E Binding of the humanized anti-CD38 antibody mAb5 to soluble human CD38 (upper panel, "hCD38::Histag") or cynomolgus CD38 (upper panel, "cynoCD38::Histag") by ELISA is shown, as well as binding of mAb5 to the surface of cells expressing human CD83 (lower panel, as indicated) or cynomolgus CD38 (lower panel, as indicated) by flow cytometry. Figure 2F Binding of mAb5 to human CD38 (upper panel) or cynomolgus monkey CD38 (lower panel) by SPR is shown. Figure 2G Binding of the human anti-CD38 antibody hhy1370 to soluble human CD38 (upper panel, "hCD38::Histag") or cynomolgus CD38 (upper panel, "cynoCD38::Histag") by ELISA and binding of hhy1370 to the surface of cells expressing human CD83 (lower panel, as indicated) or cynomolgus CD38 (lower panel, as indicated) by flow cytometry are shown. Figure 2H Binding of hhy1370 to human CD38 (upper panel) or cynomolgus monkey CD38 (lower panel) is shown by SPR. Fig.2IBinding data from ELISA, SPR, and FACS experiments are summarized, along with the percent identity of each antibody VH ("H") or VL ("L") domain to the human V region sequence from top to bottom, corresponding to mAb2, mAb3, mAb4, mAb5, and mAb6 in the last row (1195HHKK-3 is not described below by its VL / VH amino acid sequence).

[0121] Figure 2J Shown is the concentration-dependent induction of apoptosis in SU-DHL-8 cells by mAb7 and mAbl after 72 h of incubation at 37°C.

[0122] Figure 2K The antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isatuximab and mAb1 against SU-DHL-8 cells in the presence of NK92 cells is shown.

[0123] Figure 2L Shown are the concentration-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) activities of isatuximab (right) and mAb1 (left) on SU-DHL-8 cells in the presence of NK92 cells after 4 hours at 37°C.

[0124] Figure 2M-2Q Results of apoptosis induction assays using the indicated anti-CD38 antibodies against SU-DHL-8 tumor cells are shown. Apoptosis was quantified by measuring double Annexin V and propidium iodide uptake via flow cytometry. Figure 2M The percentage of apoptotic cells induced by each antibody is shown. Figure 2N-2Q The anti-CD38 antibody mAb2 ( Figure 2N )、mAb3( Fig.2O )、mAb4( Figure 2P ) and mAb5( Figure 2Q ) Dose-dependent induction of apoptosis in SU-DHL-8 lymphoma cells, and the IC50 of each antibody.

[0125] Figure 3A A schematic diagram of a trispecific binding protein comprising four polypeptide chains forming three antigen binding sites that bind to the following three target proteins is provided: CD28, CD3 and CD38. The first pair of polypeptides has dual variable domains (VH1-VH2 and VL2-VL1) in a cross-orientation, forming two antigen binding sites that recognize CD3 and CD28, and the second pair of polypeptides has a single variable domain (VH3 and VL3), forming a single antigen binding site that recognizes CD38. Figure 3A The trispecific binding protein shown in uses an IgG4 constant region with a "knobs-into-holes" mutation, where the knobs are on a second pair of polypeptides with a single variable domain.

[0126] Figure 3B A schematic diagram of an SPR-based assay used to examine the ability of each antigen binding domain of an anti-CD38 / anti-CD28 / anti-CD3 trispecific binding protein to bind its cognate antigen is provided.

[0127] Figure 3C Results of an SPR-based assay for examining CD38 binding to an anti-CD38 / anti-CD28 / anti-CD3 trispecific binding protein are shown. Binding of human CD38 to the trispecific binding protein was examined alone (upper left), after pre-binding to CD3 (upper middle), after pre-binding to CD28 (upper right), after pre-binding to CD3 and then CD28 (lower left), or after pre-binding to CD28 and then CD3 (lower right).

[0128] Figure 4 Shown is the sequential binding of human CD3, CD28 and CD38 polypeptides to the anti-CD38 / anti-CD28 / anti-CD3 trispecific binding protein as determined by SPR.

[0129] Figure 5 Summarized are the binding affinities of the indicated trispecific binding proteins to their cognate antigens (human CD3, CD28 and CD38), as measured by SPR.

[0130] Fig. 6A The apparent affinity of the isatuximab antigen-binding domain in human IgG1 format (second sheet) or in trispecific binding protein format was compared with that of the isatuximab, anti-CD28, and anti-CD3 antigen-binding domains (according to Figure 3A 1st sheet) was used to bind human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide, as determined by flow cytometry.

[0131] Figure 6B-6D Comparison of the trispecific binding protein CD38 VH1 xCD28 sup xCd3 mid 、CD38 VH1 xCD28 cvn xCd3 mid Or the apparent affinity of binding of the monospecific anti-CD38 antibody mAb2 to cells expressing human or cynomolgus CD38 polypeptide as determined by flow cytometry. Figure 6B Display of trispecific binding protein CD38 VH1 xCD28 sup xCd3 mid Binding to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide. Figure 6C Display of trispecific binding protein CD38VH1 xCD28 cvn xCd3 mid Binding to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide. Fig.6D Binding of the monospecific anti-CD38 antibody mAb2 to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide is shown.

[0132] Fig. 6E Comparison of the trispecific binding protein CD38 HHY1370 xCD28 sup xCd3 mid Or the apparent affinity of the monospecific anti-CD38 antibody mAb6 binding to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide as determined by flow cytometry.

[0133] Fig. 6F Summarized are the binding affinities of the indicated anti-CD38xanti-CD28xanti-CD3 trispecific binding proteins to human CD38 as measured by SPR or flow cytometry (FACS).

[0134] Figure 6G A trispecific binding protein ΔCD38 lacking the anti-CD38 antigen binding domain is shown VH1 xCD28 sup xCd3 mid Apparent affinity for binding to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide as determined by flow cytometry.

[0135] Fig. 7A and 7B Shown are the results of ELISA assays determining the binding affinity of various anti-CD38xCD28xCD3IgG4 trispecific binding proteins or control antibodies to human and rhesus CD3, CD28 and CD38 polypeptides.

[0136] Figures 8A-8D Antibody-mediated CD38 in PBMCs from three different human donors using the indicated anti-CD38xCD28xCD3 trispecific binding proteins and control antibodies are shown. + Results of cell-specific killing were shown using the multiple myeloma cell line RPMI8266 ( Fig. 8A )、NCI-H929( Figure 8B )、KMS-26( Figure 8C ) and KMS-11 cell lines ( Fig.8D ) and the EC50 values ​​are provided in Table N. The EC50 values ​​obtained by using NCI-H929, KMS-26 and KMS-11 cells are provided in Table OQ.

[0137] Fig. 8E and 8F Antibody-mediated CD38 induction of PBMCs from two different donors using the indicated anti-CD38 x CD28 x CD3 trispecific binding proteins with variant Fc regions and a control antibody are shown. + Results of cell-specific killing were shown using CD38 + KMS-11( Fig.8D ) and U266( Fig. 8E ) cell lines, and EC50 values ​​are provided in Tables Q2 and Q3.

[0138] Fig.9A , 9B and 10 show activation of human T cells (CD69 + ). Fig.9A Display human CD3 + T cell activation (CD69 + ). Fig. 9B Display human CD3 + CD4 + T cell activation (CD69 + ). Fig.10 Display human CD3 + CD8 + T cell activation (CD69 + ).

[0139] Figures 11A-11B Shown are the results of 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 as described in Stebbings, R. et al. (2007) J. Immunol. 179:3325-3331. Fig.11A Results using 5 μg / mL of the indicated antibodies are shown. Fig. 11B Results using 25 ng / mL of the indicated antibodies are shown.

[0140] Figures 12A-12E Anti-CD38 in a CD34+ cord blood humanized NSG mouse model engrafted with RPMI-8226 multiple myeloma cells is shown (VHI) x CD28 (sup) x CD3 (mid) In vivo activity of trispecific binding proteins. Fig. 12A Anti-CD38 (VHI) x CD28 (sup) x CD3(mid) Changes in tumor volume in mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody. Fig. 12B CD38 (VHI) xCD28 (sup) x CD3 (mid) Mean tumor volume at day 18 in mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody. Fig. 12C Anti-CD38 (VHI )x CD28 (sup) x CD3 (mid) Mean terminal tumor weight of mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody. Fig.12D Anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) Mean tumor growth curves over the length of the experiment for mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody. Fig.12E Anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) Mean change in body weight of mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody at multiple time points over the length of the experiment.

[0141] Figures 13A-13F Anti-CD38 in a PBMC humanized NSG mouse model implanted with RPMI-8226 multiple myeloma cells is shown (VHI) x CD28 (sup) x CD3 (mid) In vivo activity of trispecific binding proteins. Fig.13A Anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) Changes in tumor volume in mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody. Fig. 13B Anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) Tumor volume at day 4 in mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody. Fig. 13C Anti-CD38 (VHI)x CD28 (sup) x CD3 (mid) Tumor volume at day 21 in mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody. Fig.13D Anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) Mean tumor volume at day 21 of mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody. Fig.13E Anti-CD38 (VHI) xCD28 (sup) x CD3 (mid) Mean terminal tumor weights of mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody. Fig.13F In the presence of anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) Mean tumor volume of mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody at multiple time points over the length of the experiment.

[0142] Figures 14A-14U Demonstrated use of anti-CD38 in nonhuman primates (VHI) x CD28 (sup) x CD3 (mid) , anti-CD38 (VHI) x CD28 (cvn) x CD3 (mid) , anti-CD38 (hhy1370) x CD28 (sup) x CD3 (mid) and anti-CD38 (hhy1370) xCD28 (cvn) x CD3 (mid) Results of a dose escalation study of a trispecific binding protein. Fig.14A It was shown that after administration of different doses of anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) Trispecific binding protein post-circulating CD3 + T cell activation (CD69 + ). Fig. 14B It was shown that after administration of different doses of anti-CD38 (VHI) x CD28 (cvn) x CD3 (mid) Trispecific binding protein post-circulating CD3 +T cell activation (CD69 + ). Fig. 14C Different doses of anti-CD38 (hhy1370) x CD28 (sup) xCd3 (mid) Trispecific binding protein post-circulating CD3 + T cell activation (CD69 + ). Fig.14D It was shown that after administration of different doses of anti-CD38 (hhy1370) x CD28 (cvn) x CD3 (mid) Trispecific binding protein post-circulating CD3 + T cell activation (CD69 + ). Fig.14E Anti-CD38 was administered at the indicated doses. (VHI) x CD28 (sup) x CD3 (mid) Trispecific binding protein post-circulating CD4 + Changes in T cell percentage. Fig.14F Anti-CD38 was administered at the indicated doses. (VHI) x CD28 (sup) x CD3 (mid) Trispecific binding protein post-circulating CD8 + Changes in the percentage of T cells. Figure 14G Anti-CD38 was used at the indicated doses. (VHI) xCD28 (cvn) x CD3 (mid) Trispecific binding protein post-circulating CD4 + Changes in the percentage of T cells. Fig.14H Anti-CD38 was administered at the indicated doses. (VHI) x CD28 (cvn) x CD3 (mid) Trispecific binding protein post-circulating CD8 + Changes in T cell percentage. Fig.14I Anti-CD38 was administered at the indicated doses. (hhy1370) x CD28 (sup) x CD3 (mid) Trispecific binding protein post-circulating CD4 + Changes in T cell percentage. Fig.14J Anti-CD38 was administered at the indicated doses. (hhy1370) x CD28 (sup) x CD3 (mid) Trispecific binding protein post-circulating CD8 + Changes in the percentage of T cells. Figure 14KAnti-CD38 was administered at the indicated doses. (hhyl370) x CD28 (cvn) x CD3 (mid) Trispecific binding protein post-circulating CD4 + Changes in the percentage of T cells. Figure 14L Anti-CD38 was administered at the indicated doses. (hhy1370) x CD28 (cvn) x CD3 (mid) Trispecific binding protein post-circulating CD8 + Changes in the percentage of T cells. Fig.14M Total CD4+ cells were shown 6, 24 and 48 hours after administration of 12.5 μg / kg of the indicated trispecific binding proteins. + Changes in T cells. Fig.14N Shown are changes in total NK cells 6, 24 and 48 hours after administration of 12.5 μg / kg of the indicated trispecific binding proteins. Fig.14O Total CD8 6, 24 and 48 hours after administration of 12.5 μg / kg of the indicated trispecific binding proteins are shown + Changes in T cells. Figure 14P Shown are changes in total B cells 6, 24 and 48 hours after administration of 12.5 μg / kg of the indicated trispecific binding proteins. Figure 14Q Three increasing doses (0.5, 2.5, 12.5 μg / kg) of anti-CD38 (VH1) x CD28 (sup) x CD3 (mid) Changes in cytokine levels 6 hours after trispecific binding protein (results from different test animals labeled "117065" and "117066"). Figure 14R Three increasing doses (0.5, 2.5, 12.5 μg / kg) of anti-CD38 (VH1) xCD28 (cvn) x CD3 (mid) Changes in cytokine levels 6 hours after trispecific binding protein (results from different experimental animals labeled "117067" and "117068"). Figure 14S Three increasing doses (0.5, 2.5, 12.5 μg / kg) of anti-CD38 (hhyl370) x CD28 (sup) x CD3 (mid) Changes in cytokine levels 6 hours after trispecific binding protein (results from different test animals labeled "117069" and "117070"). Figure 14T Three increasing doses (0.5, 2.5, 12.5 μg / kg) of anti-CD38 (hh y1370) x CD28 (cvn) x CD3 (mid) Changes in cytokine levels 6 hours after trispecific binding protein (results from different test animals labeled "117071" and "117072"). Figure 14U Shown are changes in cytokine levels 24 hours after administration of three increasing doses (0.5, 2.5, 12.5 μg / kg) of the indicated trispecific binding proteins (results shown for all experimental animals).

[0143] Figure 14V and 14W Display anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) and anti-CD38 (VHI) x CD28 (cvn) xCd3 (mid) The trispecific binding protein induced depletion of T cells in vivo in non-human primate blood at higher doses (6 hours after dosing).

[0144] Figure 14X and 14Y Display anti-CD38 (HHY1370) x CD28 (sup) x CD3 (mid) and anti-CD38 (HHY1370 x CD28 (cvn) xCd3 (mid) The trispecific binding protein induced depletion of T cells in vivo in non-human primate blood at higher doses (6 hours after dosing).

[0145] Figure 14Z and 14AA Anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) or anti-CD38 (VHI) x CD28 (cvn) xCd3 (mid) The amount of blood T cells in nonhuman primates changes over time after trispecific binding protein.

[0146] Fig.14AB and 14AC Anti-CD38 (HHY1370) x CD28 (sup) x CD3 (mid) or anti-CD38 (HHY1370) xCD28 (cvn) x CD3 (mid) The amount of blood T cells in nonhuman primates changes over time after trispecific binding protein.

[0147] Fig.14AD and 14AE Shown is the amount of CD4+ T cells with bound trispecific binding protein following administration of a 100 μg / kg dose in non-human primates.

[0148] Figure 14AF and 14AG Shown is the amount of CD8+ T cells with bound trispecific binding protein following administration of a 100 μg / kg dose in non-human primates.

[0149] Figures 15A-15C Multiple Fc variants were shown to bind to the human Fc receptor FcγR I ( Fig.15A )、FcγR IIa( Fig. 15B ) and FcγR IIIb / c( Fig. 15C ) binding or lack thereof. The variants tested were human IgG1, human IgG4, and human IgG4 with FALA mutations.

[0150] Fig.16 Binding of human IgG4 with or without FALA mutation to FcRn is shown.

[0151] Fig.17 A summary of the trispecific binding proteins shown in NSG mice (CD38 VH1 xCD28 sup xCd3 mid IgG4, CD38 VH1 xCD28 sup xCd3 mid IgG4 FALA, CD38 vH1 xCD28 sup xCd3 mid IgG1 LALA P329A and CD38 HHY1370 xCD28 sup xCd3 mid PK parameters of IgG4 FALA).

[0152] Figures 18A-18C Figure 3 shows Fc / FcR interaction-mediated (nonspecific) IFN-γ expression in human PBMCs incubated with trispecific heterobinding proteins bearing wild-type or FALA variant Fc regions. Fig.18A )、IL-2( Fig.18B ) or TNF-α( Fig. 18C )’s release.

[0153] Fig.18D Display CD38 VH1 xCD28 sup xCd3 mid and CD38HHY1370 xCD28 sup xCd3 mid In vitro activation of human PBMCs by the trispecific binding protein and its IgG1 and IgG4 Fc variants.

[0154] Fig.19A and 19B CD38 trispecific binding protein VH1 xCD28 sup xCd3 mid In CD4+( Fig.19A ) or CD8+( Fig.19B ) CD3 and CD28 antigen binding domains are required for Bcl-xL induction in T cells. Bar graph = mean and sd from 3 PBMC donors. *p = ≤ 0.009.

[0155] Fig.19C and 19D Display of CD38 with IgG4 FALA variant Fc VH1 xCD28xCD3 upregulated CD4+ ( Fig.19C ) or CD8+( Fig.19D ) Bcl-xL in T cells. Bar graph = mean and sd from 3 PBMC donors. *p = ≤ 0.045

[0156] Fig.19E It was shown that T cell activation of the anti-CD38xanti-CD28xanti-CD3 trispecific binding protein, as determined by IL-2 expression in a Jurkat T cell reporter cell line, is dependent on the anti-CD3 antigen binding domain.

[0157] Fig.19F CD38 was compared with anti-CD28, anti-CD38, or anti-CD28, anti-CD38, and anti-CD3 binding proteins with mutations, as well as benchmark binding proteins. VH1 xCD28 sup xCd3 mid The trispecific binding protein releases the cytokines TNF, IFNg, IL-2, IL-6, and IL-10.

[0158] Figure 19G Proliferation of T cells activated by anti-CD38xanti-CD28xanti-CD3 trispecific binding protein with IgG4 FALA variant Fc, benchmark anti-CD38xanti-CD3 bispecific antibody or isotype control (trispecific binding protein with IgG4 FALA variant Fc with mutant binding domains) is shown.

[0159] Fig. 20Proliferation of T cells activated by an anti-CD38xanti-CD28xanti-CD3 trispecific binding protein with an IgG4 FALA variant Fc, an anti-CD38xanti-CD28xanti-CD3 trispecific binding protein with an IgG4 FALA variant Fc and mutations in the CD38, CD28, or CD3 antigen binding domains, or an isotype control (an IgG4 FALA variant Fc trispecific binding protein with three mutant binding domains) is shown.

[0160] Fig.21 CD38 is shown in the NCI-H929-Luc disseminated tumor model in PBMC humanized NSG mice at the indicated doses. VH1 xCD28 sup xCd3 mid In vivo antitumor activity of IgG4 FALA trispecific binding protein.

[0161] Fig. 22 CD38 is shown in the NCI-H929-Luc disseminated tumor model in PBMC humanized NSG mice at the indicated doses. HHY1370 xCD28 sup xCd3 mid In vivo antitumor activity of IgG4 FALA trispecific binding protein.

[0162] Fig.23A and 23B The results show that human PBMCs used in in vivo studies have CD38 VH1 xCD28 sup xCd3 mid and CD38 HHY1370 xCD28 sup xCd3 mid Potent in vitro tumor killing activity of NCI-929-Luc cells of trispecific binding proteins and benchmark anti-CD38xanti-CD3 bispecific antibodies. Human PBMCs from 2 donor humanized NSG mice were used after 24 hours of incubation at an effector:PBMC ratio of 10:1.

[0163] Fig.23C Shown is the efficacy of CD38 administered at the indicated doses in the NCI-H929-Luc disseminated tumor model in PBMC humanized NSG mice compared to a benchmark anti-CD38xanti-CD3 bispecific antibody. VH1 xCD28 sup xCd3 mid and CD38 hhy1370 xCD28 sup xCd3 midExcellent in vivo antitumor activity of the trispecific binding protein. The binding protein was administered by weekly intraperitoneal (IP) injection at 30 μg / kg.

[0164] Fig.24A Display using GloResponse TM IL2-luc2P Jurkat cells (Promega) in CD38 VH1 / CD28 sup xCd3 mid Results of luciferase reporter assays after stimulation with single binding site KO and triple KO mutants at 10 nM concentration.

[0165] Fig. 24B Optimization of the anti-CD3xCD28 CODV-Fab antibody is shown. The optimal configuration of α-CD3 and α-CD28 in alternative positions of the CODV bispecific Fab was evaluated by an in vitro cytokine release assay using human PBMCs. Distal CD28x proximal CD3 was determined to be the optimal positioning based on the secretion of IFN-γ and IL-2 in the supernatant after 24 hours.

[0166] Fig.25 Display CD38 VH1 / CD28 sup xCd3 mid Induction of upregulation of the Bcl-2 family member Bcl-xL in primary T cells is CD28-dependent.

[0167] Fig.26 The anti-CD28 in the trispecific Ab was shown to provide the secondary signaling necessary to support the proliferation of primary T cells in vitro.

[0168] Fig. 27 The configuration of trispecific antibodies color-coded by the parent antibodies is shown (left). Dark shades (purple or green) represent heavy chain peptides; light shades represent light chain peptides. Also shown are the anti-CD38 VH1 Fab and CD28 sup / CD3 mid Crystal structure of CODVFab of CD38 VH1 / CD28 sup xCd3 mid Structural model of the trispecific antibody (right).

[0169] Fig.28A and 28B Shown to have high (RPMI-8226; Fig.28A ) and low (KMS-11; Fig.28B) CD38 surface-expressing multiple myeloma (MM) cells were efficiently lysed by human PBMC incubated with various concentrations of the trispecific Ab (E:T=10:1). The contribution of each binding site to the killing activity was demonstrated by binding site KO mutations, as shown.

[0170] Figures 29A-29C Anti-CD28 showing trispecific Ab sup KO mutants inhibit CD38 in vitro high 、CD38 mid and CD38 low MM cells showed significantly reduced antitumor activity. Fig.29A )、U266( Fig.29B ) or KMS-11( Fig.29C ) cell determination.

[0171] Fig.30 Display CD38 VH1 / CD28 sup xCd3 mid_ The reduction in tumor burden in the FALA trispecific antibody treatment group was dose-dependent and statistically significant in a disseminated human multiple myeloma cell line model using NSG mice reconstituted with in vitro expanded human primary T cells.

[0172] Fig.31A and 31B Figure 2 shows a key microscopic analysis of myeloma cell lysis in vitro by a CD38 trispecific Ab in the presence of primary human T cells. TM Human PBMCs incubated with RPMI-8226 myeloma cell line labeled with deep red dye were used as negative controls (triple KO triple specific; Fig.31A ) or CD38 / CD28xCD3 trispecific Ab ( Fig.31B ) Time-lapse microscopy images were performed. The images presented were collected after 24 h of incubation. Scale bar: 50 μm.

[0173] Fig.32 Shown are substitution mutations in the Fc region of IgG4 prepared for analysis in an Fc receptor binding assay. SEQ ID NOs: 111-116 are shown (from top to bottom, respectively).

[0174] Fig.33 Shown are the results of SPR assays used to measure the affinity of the indicated IgG4 Fc variants to the indicated human Fc receptors.

[0175] Fig.34CD38 trispecific binding proteins with minimal FcR binding were shown to reduce nonspecific cytokine release in human PBMCs in vitro. The proinflammatory effects of different FcR inactivating mutations (as indicated) on human IgG4 isotypes were analyzed. Human PBMCs were incubated in culture medium (unstimulated) or in the presence of myeloma cells RPMI-8226 (stimulated), and the bar graphs represent supernatant IFN-γ levels measured by ELISA.

[0176] Fig.35 CD38 trispecific binding proteins with minimal FcR binding are shown to lyse human multiple myeloma cells with varying levels of CD38 expression. Cytolysis of myeloma cells with IgG4 or the indicated Fc mutations was assessed in vitro using human PBMCs with the indicated tumor targets.

[0177] Fig.36 Shown is a comparison of the in vitro cytolytic activity of the trispecific anti-CD38 / CD28 / CD3 Ab and the anti-CD38 antibody daratumumab on the cell lines RPMI-8226, U266 and KMS-11 (E:T=10:1) measured using human PBMCs as effector cells.

[0178] Figures 37A-37D Characterization of in vitro T cell subset expansion in response to CD38 / CD3xCD28 is shown. Assessment of T cell subset expansion is performed by coating wells with 350ng / well CD38 tri-specific Ab in the absence of exogenous cytokines. T cell colonies are measured at the indicated time points. Triple mutant tri-specific Ab is used as a negative control. Flow cytometry is used to determine the central and effector memory CD4 T cells ( Fig.37A ), helper T cells ( Fig.37B ), central and effector memory CD8 T cells ( Fig.37C ) and CMV pp65-specific CD8 cells ( Fig.37D ). CMV-specific pp65 effector cells were analyzed by pentamer staining of PBMCs from HLA-A2CMV+ donors treated with CD38 trispecific or triple negative control.

[0179] Fig.38The contribution of CD28 expression on target cells to the susceptibility of cytolysis by CD38 / CD3xCD28 is shown. CD28 is knocked out in KMS-11 cells using CRISPR / Cas 9 gene targeting and used as a cytolysis target in vitro. Compared with parent KMS-11, CD38 expression is retained, while CD28 is eliminated, as confirmed by flow cytometry (upper figure, KMS-11 is relative to KMS-11 (CD28KO)). The cytolysis of CD28KO cells is checked with WT or CD28 invalid tri-specificity (lower figure; tri-specificity is relative to tri-specificity (CD28KO)).

[0180] Fig.39 CD38 / CD28xCD3 trispecific FALA mutant Abs were shown to be effective against the indicated CD38 + CD28 - Cytolytic activity of the CD38 + CD28 - Lineages include acute myeloid leukemia (AML (KG-1)), B-cell lymphoma (OCI-Ly19), acute T-lymphocytic leukemia (ALL (KOPN8)), and chronic lymphocytic lymphoma (CLL (Z-138)).

[0181] Fig.40 It was shown that in vitro activation of human PBMCs by an α-CD28 superagonist requires bivalency of the antibody. DETAILED DESCRIPTION

[0182] The present disclosure provides binding proteins comprising at least one antigen binding site that binds a CD38 polypeptide.

[0183] I. General Definitions

[0184] As used in accordance with the present disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated. Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and the like.

[0185] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of," aspects and embodiments.

[0186] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Such modifications include base modifications such as bromouridine; ribose modifications such as arabinoside and 2', 3'-dideoxyribose; and internucleotide bond modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, and phosphoroamidate. The term "polynucleotide" specifically includes DNA in single-stranded and double-stranded forms.

[0187] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, that: (1) is not associated with all or part of a polynucleotide with which the isolated polynucleotide is found in nature, (2) is associated with a polynucleotide with which it is not naturally associated, or (3) does not occur in nature as part of a larger sequence.

[0188] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides that are normally found, (2) is substantially free of other polypeptides from the same source (e.g., from the same species), (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other substances with which it is naturally associated, (5) is not associated (by covalent or non-covalent interactions) with a portion of the polypeptide with which the "isolated polypeptide" is naturally associated, (6) is operably associated (by covalent or non-covalent interactions) with a polypeptide that is not naturally associated, or (7) does not exist in nature. Such an isolated polypeptide may be encoded by genomic DNA, cDNA, mRNA or other RNA from a synthetic source, or any combination thereof. Preferably, the isolated polypeptide is substantially free of polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0189] Naturally occurring antibodies typically comprise tetramers. Each such tetramer is typically composed of two pairs of identical 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 to a target antigen. The amino terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids, which are typically responsible for antigen recognition. The carboxyl terminal portion of each chain typically defines the constant domains responsible for effector function. Thus, in naturally occurring antibodies, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (V H ) and three constant domains (C H1 , C H2 and C H3 ), where V H The domain is located between the amino terminus and the C H3 The domain is located at the carboxyl terminus. The full-length light chain immunoglobulin polypeptide includes the variable domain (V L ) and constant domain (C L ), where V L The domain is located between the amino terminus and the C L The domain is located at the carboxyl terminus.

[0190] Human light chains are usually classified as kappa and lambda light chains, and human heavy chains are usually classified as mu, delta, gamma, alpha or epsilon, and the isotype of the antibody is defined 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 the full-length light and heavy chains, the variable region and constant domain are usually connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, for example, F UNDAMENTAL I MMUNOLOGY(Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable region of each light / heavy chain pair generally forms an antigen binding site. The variable domains of naturally occurring antibodies generally exhibit the same general structure of relatively conservative framework regions (FRs) connected by three hypervariable regions, also referred to as complementary determining regions or CDRs. The CDRs from the two chains of each pair are generally aligned by the framework regions, which can enable binding to a specific epitope. From the amino terminus to the carboxyl terminus, the light and heavy chain variable domains generally comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0191] The term "CDR set" refers to a group of three CDRs that occur in a single variable region that is capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., S EQUENCES OF P ROTEINS oF MMUNOLOGICAL I NTEREST(National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17; Chothia et al., 1989, Nature 342: 877-83) found that despite great diversity at the amino acid sequence level, certain subsections of the Kabat CDRs adopt nearly identical peptide backbone conformations. These subsections are designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" represent light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with Kaba CDRs are 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. Other CDR boundary definitions may not strictly follow one of the systems described herein, but will still overlap with Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results for specific residues or groups of residues, or even entire CDRs, which does not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs. The use of amino acid sequences to identify predicted CDRs is well known in the art (such as in Martin, AC "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2. Kontermann R., Dübel S., eds. Springer-Verlag, Berlin, p. 33-51 (2010)). The amino acid sequence of the heavy and / or light chain variable domains can also be examined to identify the sequence of the CDR by other conventional methods (e.g., by comparing the known amino acid sequences of other heavy and light chain variable regions to determine the sequence hypervariable regions).Numbered sequences can be aligned by eye or by using an alignment program (such as one of the CLUSTAL program suite) as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular modeling is often used to correctly delineate framework and CDR regions and thus correct sequence-based assignments.

[0192] In some embodiments, the CDR / FR definitions in an immunoglobulin light or heavy chain are determined based on the IMGT definitions (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

[0193] As used herein, the term "Fc" refers to a molecule comprising a sequence of a non-antigen binding fragment produced by antibody digestion or otherwise produced in a monomeric or multimeric form, and may include a hinge region. The original immunoglobulin source of native Fc is preferably human-derived, and may be any immunoglobulin. The Fc molecule is composed of a monomeric polypeptide, which can be connected into a dimer or multimeric form by covalent (i.e., disulfide bond) and non-covalent bonding. The number of intermolecular disulfide bonds between the monomer subunits of a native Fc molecule ranges from 1 to 4, depending on class (e.g., IgG, IgA and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2 and IgG4). An example of Fc is a disulfide bonded dimer produced by papain digestion of IgG. As used herein, the term "native Fc" is universal for monomeric, dimer and multimeric forms.

[0194] The F(ab) fragment usually consists of a light chain and a heavy chain V H and C H1 domain, in which the V H -C H1 The heavy chain portion cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab) fragment may also include a light chain containing two variable domains separated by an amino acid linker and a light chain containing two variable domains separated by an amino acid linker and a C H1 domain of the heavy chain.

[0195] The F(ab') fragment usually includes a light chain and a portion of a heavy chain, which contains more constant regions (in the C H1 and C H2 domains), thereby allowing for the formation of an interchain disulfide bond between the two heavy chains to form the F(ab′) 2 molecular.

[0196] As used herein, the term "binding protein" refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen, for example, a CD38 polypeptide of the present disclosure.

[0197] A "recombinant" molecule is one that is prepared, expressed, generated or isolated by recombinant methods.

[0198] One embodiment of the present disclosure provides a binding protein with biological and immunological specificity for 1 to 3 target antigens. Another embodiment of the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Another embodiment of the present disclosure provides an expression vector comprising a nucleic acid molecule containing a polypeptide chain encoding such a binding protein. Yet another embodiment of the present disclosure provides a host cell expressing such a binding protein (i.e., a nucleic acid molecule or vector comprising a polypeptide chain encoding such a binding protein).

[0199] As used herein, the term "interchangeability" refers to the interchangeability of variable domains within a binding protein form with preservation of folding and ultimate binding affinity. "Complete interchangeability" refers to the ability to exchange V in a polypeptide chain of Formula I or a polypeptide chain of Formula II. H1 and V H2 The order of the domains and thus the exchange of V L1 and V L2 The order of the domains can be altered (i.e., inverted) while maintaining the ability to bind the protein to full function as evidenced by retention of binding affinity. H and V L Refers only to the position of the domain on a particular protein chain in its final form. For example, V H1 and V H2 Can be derived from V in the parent antibody L1 and V L2 domain, and is placed in the V H1 and V H2 Position. Similarly, V L1 and V L2 Can be derived from V in the parent antibody H1 and V H2 domain, and is placed in the V H1 and V H2 position. Therefore, V n and V L The names refer to the current position rather than the original position in the parent antibody. H and V L The domains are "exchangeable".

[0200] As used herein, the term "antigen" or "target antigen" or "antigenic target" refers to a molecule or a portion of a molecule that can be bound by a binding protein and that can additionally be used in an animal to produce antibodies that can bind to an epitope of the antigen. A target antigen may have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein is able to compete with an intact antibody that recognizes the target antigen.

[0201] "CD38" is a cluster of differentiation 38 polypeptide and is 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 shown in SEQ ID NO: 1) and the extracellular domain of cynomolgus monkey CD38 (e.g., as shown in SEQ ID NO: 30).

[0202] The term "T cell engager" refers to binding proteins directed against the host immune system, more specifically the cytotoxic activity of T cells, as well as binding proteins directed against tumor target proteins.

[0203] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigenic target.

[0204] The term "monovalent binding protein" refers to a binding protein with one antigen binding site.

[0205] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets. In some embodiments, the bispecific binding protein binds to two different antigens. In some embodiments, the bispecific binding protein binds to two different epitopes on the same antigen.

[0206] The term "bivalent binding protein" refers to a binding protein having two binding sites.

[0207] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets. In some embodiments, the trispecific binding protein binds to three different antigens. In some embodiments, the trispecific binding protein binds to one, two, or three different epitopes on the same antigen.

[0208] The term "trivalent binding protein" refers to a binding protein with three binding sites. In certain embodiments, the trivalent binding protein can bind to one antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets.

[0209] An "isolated" binding protein is a protein that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that interfere with the diagnostic or therapeutic use of the binding protein, and may include enzymes, hormones, and other proteins or non-protein solutes. In some embodiments, the binding protein is purified: (1) to greater than 95% by weight of the antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) by using a spinning cup sequencer to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) by SDS-PAGE to homogeneity under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. An isolated binding protein includes an in situ binding protein within a recombinant cell, since at least one component of the natural environment of the binding protein is absent.

[0210] As used herein, the term "substantially pure" or "substantially purified" refers to a compound or substance that is the predominant species present (i.e., it is more abundant on a molar basis than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise greater than about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, a substance is purified to substantial homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

[0211] The term "epitope" includes any determinant, preferably a polypeptide determinant, that is capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen to which an antigen 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, when the equilibrium dissociation constant is ≤10 -8 M, more preferably when the equilibrium dissociation constant is ≤10 -9 M, and most preferably when the dissociation constant is ≤10 -10 When M, the binding protein is said to specifically bind to the antigen.

[0212] The dissociation constant (K) of the binding protein D) can be determined, for example, by surface plasmon resonance. Typically, surface plasmon resonance analysis uses a BIAcore system (Pharmacia Biosensor; Piscataway, NJ) to measure the real-time binding interaction between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). As used herein, the term " D ” refers to the dissociation constant of the interaction between a specific binding protein and the target antigen.

[0213] The term "binding" as used herein in reference to a binding protein refers to the ability of a binding protein or antigen-binding fragment thereof to bind to an antigen containing an epitope with a Kd of at least about 1 x 10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 - 11 M, 1x10 -12 M or higher, and / or binds to the epitope with an affinity at least two times greater than its affinity for a nonspecific antigen. In some embodiments, the binding proteins of the present disclosure bind to two or more antigens, such as human and cynomolgus monkey CD38 polypeptides.

[0214] In some embodiments, the antigen binding domains and / or binding proteins of the present disclosure "cross-react" with human and cynomolgus CD38 polypeptides (such as 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 CD38)). A binding protein that binds to antigen 1 (Ag1) "cross-reacts" with antigen 2 (Ag2) when the EC50 is in a similar range for the two antigens. In the present application, a binding protein that binds to Ag1 cross-reacts with Ag2 when 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), and the affinity is detected by the same method for the two antigens.

[0215] A binding protein that binds to Ag1 "does not significantly cross-react" with Ag2 when the affinities of the two antigens are very different. If the binding reaction is too low, the affinity of Ag2 may not be measurable. In the present application, a binding protein that binds to Ag1 does not significantly cross-react with Ag2 when the binding reaction of the binding protein to Ag2 is less than 5% of the binding reaction of the same binding protein to Ag1 in the same experimental setting and at the same antibody concentration. In practice, the concentration of the binding protein used can be the EC50 or the concentration required to reach a saturation plateau obtained with Ag1.

[0216] The term "linker" as used herein refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light chain and the heavy chain to fold into a cross-linked dual variable region immunoglobulin. At the sequence level, linkers are inserted between variable domains or at transitions between variable domains and constant domains, respectively. Transitions between domains can be identified because the approximate sizes of immunoglobulin domains are well understood. The precise location of domain transitions can be determined by locating peptides that do not form secondary structural elements (such as β-sheets or α-helices), as demonstrated by experimental data, or can be assumed by modeling techniques or secondary structure predictions. The linker described herein is referred to as L 1 , which is located at V L2 The C-terminus and V L1 on the light chain between the N-termini of the domain; and L 2 , which is located at V L1 The C-terminus and C L The heavy chain linker is called the L 3 , which is located at V H1 The C-terminus and V H2 between the N-termini of the domain; and L 4 , which is located at V H2 The C-terminus and C H1 between the N-termini of the domain.

[0217] As used herein, the term "vector" refers to any molecule (for example, nucleic acid, plasmid or virus) used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule that can transport another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule that can insert an additional DNA segment. Another type of vector is a viral vector, in which an additional DNA segment can be inserted into the viral genome. Some vectors can replicate autonomously in the host cell in which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the expression of the genes to which they are operably connected. Such a vector is referred to as a "recombinant expression vector" (or simply "expression vector") in this article. In general, an expression vector useful in recombinant DNA technology is usually in the form of a plasmid. The terms "plasmid" and "vector" are used interchangeably herein because plasmids are the most commonly used vector forms. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0218] As used herein, the phrase "recombinant host cell" (or "host cell") refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cells or host cells refer not only to a specific subject cell, but also to the progeny of such cells. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may actually be different from the parent cell, but such cells are still included in the scope of the term "host cell" used herein. A 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. In order to recombinantly express the binding protein, the host cell is transformed or transfected with one or more recombinant expression vectors carrying a DNA fragment encoding the binding protein polypeptide chain, so that the polypeptide chain is expressed in the host cell and preferably secreted into the culture medium of the host cell, from which the binding protein can be recovered.

[0219] The term "conversion" as used herein refers to the change of the genetic characteristics of the cell, and has been transformed when the cell is modified to contain new DNA. For example, the cell is transformed, wherein genetic modification is performed from its native state. After transformation, the transforming DNA can be physically integrated into the chromosome of the cell and recombined with the DNA of the cell, or can be maintained instantaneously as an additional element without being replicated, or can be replicated independently as a plasmid. When DNA replicates with cell division, it is considered that the cell has been stably transformed. The term "transfection" as used herein refers to the cell taking in foreign or exogenous DNA, and when exogenous DNA has been introduced into the cell membrane, the cell has been "transfected". Many transfection techniques are well known in the art. Such technology can be used to introduce one or more exogenous DNA molecules into suitable host cells.

[0220] The term "naturally occurring" as used herein and applied to an object refers to the fact that an object can be found in nature and has not been manipulated by humans. For example, polynucleotides or polypeptides present in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified by humans are naturally occurring. Similarly, as used herein, "non-naturally occurring" refers to an object that is not found in nature or has been structurally modified or synthesized by humans.

[0221] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty conventional 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 may also be suitable components of binding protein polypeptide chains. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, gN, 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 symbols used herein, according to standard usage and convention, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction.

[0222] Naturally occurring residues can be divided into the following categories based on common side chain properties:

[0223] (1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

[0224] (2) Polar hydrophilicity: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

[0225] (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro;

[0226] (4) Aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro;

[0227] (5) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0228] (6) Acidic: Asp, Glu;

[0229] (7) Basic: His, Lys, Arg;

[0230] (8) Residues that affect chain orientation: Gly, Pro;

[0231] (9) Aromatic: His, Trp, Tyr, Phe; and

[0232] (10) Aromatic hydrophobicity: Phe, Trp, Tyr.

[0233] Conservative amino acid substitutions may involve exchanging a member of one of these classes for another member of the same class. Non-conservative substitutions may involve exchanging a member of one of these classes for a member of another class.

[0234] Those skilled in the art will be able to determine suitable variants of the polypeptide chain of the binding protein using well-known techniques. For example, those skilled in the art can identify suitable regions of the polypeptide chain that can be altered without destroying the activity by targeting regions that are not considered important for activity. Alternatively, those skilled in the art can identify residues and portions of the molecule that are conserved among similar polypeptides. In addition, even regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying the biological activity or adversely affecting the polypeptide structure.

[0235] As used herein, the term "patient" includes both human and animal subjects (eg, mammals such as dogs, pigs, horses, cats, cows, etc.).

[0236] As used herein, the term "treatment" or "treatment" refers to therapeutic treatment and prophylactic or preventive measures. Patients in need of treatment include patients with a disorder and patients susceptible to a disorder or patients to prevent a disorder. In a specific embodiment, the binding protein can be used to treat a person with cancer, or a person susceptible to cancer, or to improve cancer in a human subject. The binding protein can also be used to prevent cancer in a human patient. In a specific embodiment, 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.

[0237] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0238] As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing the delivery of a binding protein.

[0239] When used to refer to a pharmaceutical composition comprising one or more binding proteins, the terms "effective amount" and "therapeutically effective amount" refer to an amount or dosage sufficient to produce a desired therapeutic outcome. More specifically, a therapeutically effective amount is an amount of binding protein sufficient to suppress one or more clinically defined pathological processes associated with the treated condition over a period of time. The effective amount can vary depending on the specific binding protein used, and also depends on the severity of the various factors and conditions and illnesses associated with the treated patient. 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 work will be one of the factors considered. It is entirely within the capabilities of those skilled in the art to determine the effective amount or therapeutically effective amount of a given pharmaceutical composition.

[0240] One embodiment of the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

[0241] II. Anti-CD38 Binding Protein

[0242] Certain aspects of the present disclosure relate to binding proteins comprising an antigen binding site that binds a CD38 polypeptide (e.g., human and cynomolgus CD38 polypeptides). In some embodiments, the binding protein is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent.

[0243] Various features of exemplary monospecific, bispecific or trispecific binding proteins are described herein. For example, in some embodiments, the binding protein or its antigen binding fragment cross-reacts with human CD38 (e.g., human CD38 isoform A and / or isoform E polypeptides) and cynomolgus 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 T cells (e.g., by TCR stimulation and / or co-stimulation).

[0244] 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-H3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 41). NO: 36) of the amino acid sequence of CDR-L3. 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 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-H3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 41). NO: 36) of the amino acid sequence of CDR-L3.In some embodiments, the binding protein comprises 1, 2, 3, 4, 5 or 6 CDRs from an antibody VH and / or VL domain sequence of mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb2xCD28supxCD3midIgG4 FALA, mAb2xCD28supxCD3mid IgG1LALA P329A, mAb2xCD28supxCD3mid IgG1NNSA, mAb6xCD28supxCD3mid IgG4 FALA, mAb6xCD28supxCD3mid IgG1LALA P329A, or mAb6xCD28supxCD3mid IgG1NNSA as shown in Tables G, H or I.

[0245] 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-H3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 41). NO: 36) of the amino acid sequence of CDR-L3.

[0246] In some embodiments, the binding protein comprises an antigen binding site, which comprises: an antibody heavy chain variable (VH) domain, which 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); or an antibody light chain variable (VL) domain, which comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQID 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, which antigen binding site comprises: an antibody heavy chain variable (VH) domain, which 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 an antibody light chain variable (VL) domain, which 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). 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 an antigen binding site, which antigen binding site comprises: an antibody heavy chain variable (VH) domain, which 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 an antibody light chain variable (VL) domain, which 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).

[0247] In some embodiments, the VH domain comprises the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4 from N-terminus to C-terminus; wherein FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO: 87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); wherein FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); wherein FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94). NO: 94); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96). In some embodiments, the VL domain comprises the sequence FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4 from N-terminus to C-terminus; wherein FR1 comprises the sequence DIVLTQSPATLSLSPGERATISCRAS (SEQ ID NO: 97); wherein FR2 comprises the sequence MHWYQQKPGQPPRLLIY (SEQ ID NO: 99); wherein FR3 comprises the sequence SRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYC (SEQ ID NO: 101); and wherein FR4 comprises the sequence FGGGTKLEIK (SEQ ID NO: 103).

[0248] 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 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: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.

[0249] 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 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 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.

[0250] 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.

[0251] In some embodiments, the binding proteins of the present disclosure comprise 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, the binding proteins of the present disclosure comprise 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, the binding proteins of the present disclosure comprise 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, the binding proteins of the present disclosure comprise 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, the binding proteins of the present disclosure comprise 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.

[0252] In some embodiments, the 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, the 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, the 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, the 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, the 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.

[0253] 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); 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).

[0254] In some embodiments, the VH domain comprises the sequence, from N-terminus to C-terminus, FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; wherein FR1 comprises the sequence QVQLVESGGGVVQPGRSLRLSCAAS (SEQ ID NO: 89); wherein FR2 comprises the sequence MHWVRQAPGKGLEWVAV (SEQ ID NO: 92); wherein FR3 comprises the sequence YYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 95); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96). In some embodiments, the VL domain comprises the sequence, from N-terminus to C-terminus, FR1-CDR-L1-FR2-CDR-L2--FR3-CDR-L3-FR4; wherein FR1 comprises the sequence AIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 98); wherein FR2 comprises the sequence GWYQQKPGKAPKLLIY (SEQ ID NO: 100); wherein FR3 comprises the sequence SLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYC (SEQ ID NO: 102); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 104).

[0255] 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.

[0256] 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; and the VL domain comprises the amino acid sequence of SEQ ID NO: 10.

[0257] In some embodiments, the 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, the 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.

[0258] In some embodiments, the binding proteins of the present disclosure comprise 1, 2, 3, 4, 5, or 6 CDR sequences of the antibody sequences shown in Table G. In some embodiments, the binding proteins of the present disclosure comprise 1, 2, 3, 4, 5, or 6 CDR sequences, VH domain sequences, and / or VL domain sequences of the antibody sequences shown in Table H. In some embodiments, the binding proteins of the present invention comprise 1, 2, 3, 4, 5, or 6 CDR sequences, VH domain sequences, and / or VL domain sequences of the antibody sequences shown in Table I. In some embodiments, the binding proteins of the present invention comprise 1, 2, 3, or 4 polypeptide sequences shown in Table I.

[0259]

[0260] Table H. Variable domain sequences of anti-CD38 (mAb1-7) and other binding proteins.

[0261]

[0262]

[0263]

[0264] Note: CDR sequences are bold and underlined in the amino acid sequence above.

[0265] Table I. Full length sequences of binding proteins.

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282] Table J. Full-length polynucleotide sequences of binding proteins.

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304] CD38 peptide

[0305] In some embodiments, the binding proteins of the present disclosure comprise an antigen binding site that binds to the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus 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 flow cytometry using cells expressing CD38 polypeptides on their cell surfaces, for example, as described below. See, for example, Examples 1, 3, and 4.

[0306] In some embodiments, the binding proteins of the present disclosure bind to purified polypeptides or fragments thereof comprising the amino acid sequence of SEQ ID NO: 1 and / or 30 (e.g., measured by ELISA or SPR). In some embodiments, the binding proteins of the present disclosure bind to polypeptides or fragments thereof comprising the amino acid sequence of SEQ ID NO: 1 and / or 30 when expressed on the surface of a cell (e.g., measured by flow cytometry).

[0307] In some embodiments, the binding proteins of the present disclosure bind to CD38 isoform A polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1). In some embodiments, the binding proteins of the present disclosure bind to CD38 isoform E polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105, and does not comprise the complete amino acid sequence of SEQ ID NO: 1, consists of the amino acid sequence of SEQ ID NO: 105, or consists essentially of the amino acid sequence of SEQ ID NO: 105). In some embodiments, the binding proteins of the present disclosure bind to CD38 isoform A polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) and CD38 isoform E polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105, and does not comprise the complete amino acid sequence of SEQ ID NO: 1, consists of the amino acid sequence of SEQ ID NO: 105, or consists essentially of the amino acid sequence of SEQ ID NO: 105). Without wishing to be bound by theory, it is believed that binding to CD38 isoform E polypeptide may be advantageous, for example, to target the binding proteins of the present disclosure to cells expressing CD38 isoform E polypeptide.

[0308] Human CD38 isoform A extracellular domain polypeptide sequence

[0309] Human CD38 isoform E polypeptide sequence

[0310] In some embodiments, the extracellular domain of the human CD38 polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the extracellular domain of the cynomolgus monkey CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:30.

[0311] Cynomolgus monkey CD38 polypeptide sequence

[0312] Multispecific (e.g., bispecific, trispecific, or multispecific) binding proteins that bind to CD38 polypeptides

[0313] In some embodiments, the binding proteins of the present 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. In some embodiments, the binding proteins of the present 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.

[0314] In some embodiments, the binding protein of the present disclosure is a trispecific binding protein, which comprises an antigen binding site, a second antigen binding site, and a third antigen binding site that binds one or more CD38 polypeptides. For example, in some embodiments, one of the antigen binding sites binds to one or more CD38 polypeptides (e.g., the extracellular domain of human and / or cynomolgus CD38 polypeptides), and one or two antigen binding sites bind to T cell surface proteins. In some embodiments, one of the antigen binding sites binds to one or more CD38 polypeptides (e.g., the extracellular domain of human and / or cynomolgus CD38 polypeptides), one antigen binding site binds to human CD3 polypeptides, and one antigen binding site binds to human CD28 polypeptides. Human CD3 and CD28 polypeptides are known in the art. Provided herein are exemplary and non-limiting amino acid sequences of antibody variable domains that bind to human CD3 and CD28 polypeptides.

[0315] In some embodiments, provided herein is a binding protein comprising three antigen binding sites, each antigen binding site binding 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 CD38 polypeptide. In some embodiments, the human CD38 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, and / or the cynomolgus 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 CD38 polypeptide and two antigen binding sites that each bind to a T cell surface protein (e.g., a human CD28 polypeptide and / or a human CD3 polypeptide).

[0316] In some embodiments, the binding protein of the present disclosure binds to one or more tumor target proteins (e.g., one or more CD38 polypeptides) and one or more T cell target proteins. In some embodiments, the binding protein is capable of binding to two different epitopes on a tumor target protein (e.g., one or more CD38 polypeptides) and a single T cell target protein. In some embodiments, the binding protein is capable of binding to a 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 protein is capable of binding to two different epitopes on a T cell target protein and a single tumor target protein (e.g., one or more CD38 polypeptides). In some embodiments, the binding protein is capable of binding to a 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 targeting two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form antigen binding sites that bind 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.

[0317] In some embodiments, the binding protein specifically binds to one or more CD38 polypeptides and one or more target proteins on T cells, including T cell receptor complexes. These T cell engagers binding proteins can transiently recruit T cells into target cells while activating the cytolytic activity of T cells. Examples of target proteins on T cells include, but are not limited to, CD3 and CD28, etc. Other examples of such antigen targets or target proteins are provided above. In some embodiments, a trispecific binding protein can be produced by combining the antigen binding domains of two or more monospecific antibodies (parent antibodies) into one antibody.

[0318] Bispecific Binding Protein Format

[0319] In some embodiments, the binding protein of the present disclosure is a bispecific and / or bivalent binding protein comprising four polypeptide chains that form four antigen binding sites that bind to one or more (e.g., two) different antigen targets or target proteins (e.g., having a structure described in International Publication No. WO2012 / 135345). In some embodiments, the binding protein is bivalent and / or bispecific. In some embodiments, the binding protein is tetravalent and / or tetraspecific. In some embodiments, the binding protein is tetravalent and / or bispecific. In some embodiments, at least one antigen binding site binds to a CD38 polypeptide (e.g., an extracellular domain of a human and / or cynomolgus CD38 polypeptide).

[0320] In some embodiments, the binding protein comprises two polypeptide chains having a structure represented by the following formula:

[0321] V L1 -L 1 -V L2 -L 2 -C L [I]

[0322] The two polypeptide chains have the structure represented by the following formula:

[0323] V H2 -L 3 -V H1 -L 4 -C H1 -Fc [II]

[0324] in:

[0325] V L1 is the first immunoglobulin light chain variable domain;

[0326] V L2 is a second immunoglobulin light chain variable domain;

[0327] V H1 is the first immunoglobulin heavy chain variable domain;

[0328] V H2 is a second immunoglobulin heavy chain variable domain;

[0329] C L is the immunoglobulin light chain constant domain;

[0330] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0331] Fc contains the immunoglobulin hinge region and C H2 , C H3Immunoglobulin heavy chain constant region;

[0332] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0333] And wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair. In some embodiments, V H1 and V L1 Form an antigen binding domain that binds to the CD38 polypeptide, and V H2 and V L2 In some embodiments, V H2 and V L2 Form an antigen binding domain that binds to the CD38 polypeptide, and V H1 and V L1 An antigen binding domain is formed that binds another antigen target.

[0334] In some embodiments, the binding protein comprises two polypeptide chains that form two antigen binding sites, wherein the first polypeptide chain comprises

[0335] V L1 -L 1 -V L2 -L 2 -CL-Fc

[0336] and the second polypeptide chain comprises

[0337] V H2 -L 3 -V HI -L 4 -C H1 -Fc

[0338] in:

[0339] V L1 is the first immunoglobulin light chain variable domain;

[0340] V L2 is a second immunoglobulin light chain variable domain;

[0341] V H1 is the first immunoglobulin heavy chain variable domain;

[0342] V H2 is a second immunoglobulin heavy chain variable domain;

[0343] CL is the immunoglobulin light chain constant domain;

[0344] C H1Immunoglobulin C H1 heavy chain constant domain;

[0345] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0346] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0347] Fc contains the immunoglobulin hinge region and C H2 , C H3 an immunoglobulin heavy chain constant region; and

[0348] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0349] Wherein the first and second polypeptides form a crossover light chain-heavy chain pair. In some embodiments, V H1 and V L1 Form an antigen binding domain that binds to the CD38 polypeptide, and V H2 and V L2 In some embodiments, V H2 and V L2 Form an antigen binding domain that binds to the CD38 polypeptide, and V H1 and V L1 An antigen binding domain is formed that binds another antigen target.

[0350] In some embodiments, the binding protein comprises three polypeptide chains forming two antigen binding sites, wherein the first polypeptide chain comprises

[0351] V L1 -L 1 -V L2 -L 2 -CL

[0352] The second polypeptide chain comprises

[0353] V H2 -L 3 -V HI -L 4 -C H1 -Fc

[0354] The third polypeptide chain comprises the antibody Fc region

[0355] in:

[0356] V L1is the first immunoglobulin light chain variable domain;

[0357] V L2 is a second immunoglobulin light chain variable domain;

[0358] V H1 is the first immunoglobulin heavy chain variable domain;

[0359] V H2 is a second immunoglobulin heavy chain variable domain;

[0360] CL is the immunoglobulin light chain constant domain;

[0361] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0362] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0363] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0364] Fc contains the immunoglobulin hinge region and C H2 , C H3 an immunoglobulin heavy chain constant region; and

[0365] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0366] Wherein the first and second polypeptides form a crossover light chain-heavy chain pair. In some embodiments, V H1 and V L1 Form an antigen binding domain that binds to the CD38 polypeptide, and V H2 and V L2 In some embodiments, V H2 and V L2 Form an antigen binding domain that binds to the CD38 polypeptide, and V H1 and V L1 An antigen binding domain is formed that binds another antigen target.

[0367] In some embodiments, the binding protein comprises a first polypeptide chain comprising a structure represented by the following formula:

[0368] V L1 -L 1 -V L2 -L2 -C L [I]

[0369] and a second polypeptide chain comprising a structure represented by the formula:

[0370] V H2 -L 3 -V H1 -L 4 -C H1 [II]

[0371] in:

[0372] V L1 is the first immunoglobulin light chain variable domain;

[0373] V L2 is a second immunoglobulin light chain variable domain;

[0374] V H1 is the first immunoglobulin heavy chain variable domain;

[0375] V H2 is a second immunoglobulin heavy chain variable domain;

[0376] C L is the immunoglobulin light chain constant domain;

[0377] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0378] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0379] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair. H1 and V L1 Form an antigen binding domain that binds to the CD38 polypeptide, and V H2 and V L2 In some embodiments, V H2 and V L2 Form an antigen binding domain that binds to the CD38 polypeptide, and V H1 and V L1 An antigen binding domain is formed that binds another antigen target.

[0380] In any of the above bispecific binding proteins, the target antigen other than CD38 can be any of the following exemplary antigen targets: A2AR, APRIL, ATP diphosphohydrolase, 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 MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2,an IgE receptor), 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), 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、IL2Rβ、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, NTP diphosphohydrolase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as IL33 receptor), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments,One or more of the above antigenic targets are human antigenic targets.

[0381] In any of the bispecific binding proteins described above, any linker or linker combination described herein can be used. For example, in some embodiments, L 1 , L 2 , L 3 or L 4 In some embodiments, at least one of L is independently 0 amino acids in length. 1 , L 2 , L 3 or L 4 The length of each of L is independently at least 1 amino acid. 1 , L 2 , L 3 and L 4 Each is independently 0 amino acids in length or comprises 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, L 1 , L 2 , L 3 and L 4 Each independently comprises 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, L 1 Containing the sequence GQPKAAP (SEQ ID NO: 58), L 2 Containing the sequence TKGPS (SEQ ID NO: 57), L 3 Contains the sequence S, L 4 In some embodiments, L 1 Containing the sequence GGGGSGGGGS (SEQ ID NO: 55), L 2 Containing the sequence GGGGSGGGGS (SEQ ID NO: 55), L 3 The length is 0 amino acids, L 4 The length of L is 0 amino acids. 1Containing the sequence GGSGSSGSGG (SEQ ID NO: 59), L 2 Containing the sequence GGSGSSGSGG (SEQ ID NO: 59), L 3 The length is 0 amino acids, L 4 The length of L is 0 amino acids. 1 Containing the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L 2 The length is 0 amino acids, L 3 Containing the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L 4 The length of is 0 amino acids.

[0382] Trispecific binding protein that binds to CD38 polypeptide

[0383] In some embodiments, the binding protein of the present disclosure is a trispecific and / or trivalent binding protein 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 antigen binding site binds to a CD38 polypeptide (e.g., an extracellular domain of a human and / or cynomolgus CD38 polypeptide). In some embodiments, the first polypeptide chain comprises a structure represented by the following formula:

[0384] V L2 -L 1 -V L1 -L 2 -C L [I]

[0385] and the second polypeptide chain comprises a structure represented by the formula:

[0386] V H1 -L 3 -V H2 -L 4 -C H1 -Hinge-C H2 -C H3 [II]

[0387] and the third polypeptide chain comprises a structure represented by the formula:

[0388] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0389] and the fourth polypeptide chain comprises a structure represented by the formula:

[0390] V L3 -CL [IV]

[0391] in:

[0392] V L1 is the first immunoglobulin light chain variable domain;

[0393] V L2 is a second immunoglobulin light chain variable domain;

[0394] V L3 is the third immunoglobulin light chain variable domain;

[0395] V H1 is the first immunoglobulin heavy chain variable domain;

[0396] V H2 is a second immunoglobulin heavy chain variable domain;

[0397] V H3 is the third immunoglobulin heavy chain variable domain;

[0398] C L is the immunoglobulin light chain constant domain;

[0399] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0400] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0401] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0402] The hinge is connected to C H1 and C H2 The immunoglobulin hinge region of the domain, and

[0403] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0404] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair.

[0405] In some embodiments, the binding protein of the present disclosure is a trispecific and / or trivalent binding protein 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 antigen binding site binds to a CD38 polypeptide (e.g., an extracellular domain of a human and / or cynomolgus CD38 polypeptide). In some embodiments, the first polypeptide chain comprises a structure represented by the following formula:

[0406] V L2 -L 1 -V L1 -L 2 -C L [I]

[0407] and the second polypeptide chain comprises a structure represented by the formula:

[0408] V H1 -L 3 -V H2 -L 4 -C H1 [II]

[0409] and the third polypeptide chain comprises a structure represented by the formula:

[0410] V H3 -C H1 [III]

[0411] and the fourth polypeptide chain comprises a structure represented by the formula:

[0412] V L3 -C L [IV]

[0413] in:

[0414] V L1 is the first immunoglobulin light chain variable domain;

[0415] V L2 is a second immunoglobulin light chain variable domain;

[0416] V L3 is the third immunoglobulin light chain variable domain;

[0417] V H1 is the first immunoglobulin heavy chain variable domain;

[0418] V H2 is a second immunoglobulin heavy chain variable domain;

[0419] V H3 is the third immunoglobulin heavy chain variable domain;

[0420] C L is the immunoglobulin light chain constant domain;

[0421] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0422] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0423] and wherein 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 further comprise a C H1 The Fc region contains the immunoglobulin hinge region and the C H2 and C H3 Immunoglobulin heavy chain constant domain.

[0424] In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross orientation, which forms two different antigen binding sites. In some embodiments, VH1 and VL1 form a binding pair and form a first antigen binding site. In some embodiments, VH2 and VL2 form a binding pair and 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 to 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 and form a third antigen binding site. In some embodiments, the third antigen binding site binds to a CD38 polypeptide (e.g., human and optional cynomolgus monkey CD38). Exemplary binding protein formats having cross-orientations contemplated for use herein are also described in U.S. Patent Application No. 15 / 487,243 and International Application No. PCT / US2017 / 027488.

[0425] In some embodiments of any 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 (e.g., non-CD38) antigen binding site of any bispecific, trispecific, or multispecific binding proteins described herein binds to CD28 or CD3. In some embodiments, 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), and V 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). In some embodiments, 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), and V 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). In some embodiments, 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), and V 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, 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), V 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). In some embodiments, 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), V 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). In some embodiments, V 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), V L3 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).

[0426] In some embodiments, V H1 The structural 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 a V L1 The V domain comprises a CDR-L1 sequence comprising an amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising an amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising an amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H2 The structural 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 a V L2 The V domain comprises a CDR-L1 sequence comprising an amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising an amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising an amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H3The structural 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 a 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).

[0427] In some embodiments, V H1 The structural domain comprises a CDR-H1 sequence comprising an amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising an amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising an amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and V L1 The V domain comprises a CDR-L1 sequence comprising an amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising an amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising an amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H2 The structural domain comprises a CDR-H1 sequence comprising an amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising an amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising an amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and V L2 The V domain comprises a CDR-L1 sequence comprising an amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising an amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising an amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). H3The structural domain comprises a CDR-H1 sequence comprising an amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising an amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising an 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).

[0428] 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 a 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). In some embodiments, 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), and / or a V L1 The V 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). H2The 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 a V 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). In some embodiments, 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), and / or a V L2 The V 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 a 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). In some embodiments, 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 / or a V 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).

[0429] 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 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). In some embodiments, 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), and V L1 The V 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). H2The 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 V 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). In some embodiments, 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), and V L2 The V 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 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). In some embodiments, 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 V 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).

[0430] 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 a 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). In some embodiments, 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 / or a V 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).

[0431] In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 5, or V L3The domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 17, or V L3 The domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 21, or V L3 The domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, V H The V domain comprises the amino acid sequence of SEQ ID NO: 23, or V L3 The domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 13, or V L3 The domain comprises the amino acid sequence of SEQ ID NO:14.

[0432] In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 5, and / or V L3 The domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 17, and V L3 The domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 21, and V L3 The domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 23, and V L3 The domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 13, and V L3 The domain comprises the amino acid sequence of SEQ ID NO:14.

[0433] In some embodiments, V H3 The structural 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 a VL3 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).

[0434] In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 9, and / or V L3 The domain comprises the amino acid sequence of SEQ ID NO:10.

[0435] In some embodiments of any of the trispecific binding proteins disclosed herein, one antigen binding domain binds a CD3 polypeptide (e.g., human CD3), and one antigen binding domain binds 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 V L1 The domain comprises three CDRs from SEQ ID NO: 50 or 52, as shown in Table H. In some embodiments, V H2 The domain comprises three CDRs from SEQ ID NO: 49 or 51, as shown in Table H, and V L2 The domain comprises three CDRs from SEQ ID NO: 50 or 52, as shown in Table H. In some embodiments, V H1 The domain comprises three CDRs from SEQ ID NO: 53 or 84, as shown in Table H, and V L1 The domain comprises three CDRs from SEQ ID NO: 54 or 85, as shown in Table H. In some embodiments, V H2 The domain comprises three CDRs from SEQ ID NO: 53 or 84, as shown in Table H, and V L2 The domain comprises three CDRs from SEQ ID NO: 54 or 85, as shown in Table H.

[0436] In some embodiments, V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, V L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, V H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, and V L2 The domain comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, VH2 The domain comprises the amino acid sequence of SEQ ID NO: 49, V L2 The domain comprises the amino acid sequence of SEQ ID NO: 50, V H1 The domain comprises the amino acid sequence of SEQ ID NO: 53, and V L1 The domain comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, V H1 The domain comprises the amino acid sequence of SEQ ID NO: 51, V L1 The domain comprises the amino acid sequence of SEQ ID NO: 52, V H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, and V L2 The domain comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, V H2 The domain comprises the amino acid sequence of SEQ ID NO: 51, V L2 The domain comprises the amino acid sequence of SEQ ID NO: 52, V H1 The domain comprises the amino acid sequence of SEQ ID NO: 53, and V L1 The domain comprises the amino acid sequence of SEQ ID NO:54.

[0437] In some embodiments, V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, V L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, V H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, V L2 The domain comprises the amino acid sequence of SEQ ID NO: 54, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 13, and V L3 The domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, V L1 The domain comprises the amino acid sequence of SEQ ID NO: 50, V H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, V L2 The domain comprises the amino acid sequence of SEQ ID NO: 54, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 9, and V L3 The domain comprises the amino acid sequence of SEQ ID NO:10.

[0438] In certain embodiments, the first polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 62, and the fourth polypeptide chain comprises a polypeptide sequence 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 at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 65, and the fourth polypeptide chain comprises a polypeptide sequence 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 at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and the fourth polypeptide chain comprises a polypeptide sequence 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 at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 68, and the fourth polypeptide chain comprises a polypeptide sequence 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 at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 70, and the fourth polypeptide chain comprises a polypeptide sequence 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 at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 71, and the fourth polypeptide chain comprises a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 69.

[0439] 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.

[0440] In any of the trispecific binding proteins described above, the target antigen other than CD38 can be any of the following exemplary antigen targets: A2AR, APRIL, ATP diphosphohydrolase, 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-1). 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 (also known as FCER2,an IgE receptor), 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), 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、IL2Rβ、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, NTP diphosphohydrolase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as IL33 receptor), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments,One or more of the above antigenic targets are human antigenic targets.

[0441] 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 antibody, a humanized antibody, or a human antibody.

[0442] The binding proteins of the 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).

[0443] Connectors

[0444] In some embodiments, the linker L 1 , L 2 , L 3 and L 4 The range of length is from no amino acids (length = 0) to about 100 amino acids long, or less than 100, 50, 40, 30, 20, or 15 amino acids or less. The linker can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long. 1 , L 2 , L 3 and L 4 They may all have the same amino acid sequence, or they may all have different amino acid sequences.

[0445] 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 be used, such as the peptide GGGGSGGGGS (SEQ ID NO: 55), the peptide GGGGSGGGGSGGGGS (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 not intended to limit the scope of the present disclosure in any way, and show that linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline are suitable for binding proteins. For further description of linker sequences, see, for example, WO2012135345 and International Application No. PCT / US2017 / 027488.

[0446] The identity and sequence of amino acid residues in the joint can vary according to the type of secondary structure elements required to be realized in the joint. For example, glycine, serine and alanine are most suitable for joints with maximum flexibility. If a more rigid and extended joint is desired, some combination of glycine, proline, threonine and serine is useful. Depending on the desired properties, any amino acid residue can be considered as a joint combined with other amino acid residues to build a required larger peptide joint.

[0447] In some embodiments, L 1 , L 2 , L 3 or L 4 In some embodiments, at least one of L is independently 0 amino acids in length. 1 , L 2 , L 3 or L 4 The length of each independently is at least 1 amino acid. 1 The length is L 3 In some embodiments, L 2 The length is L 4 In some embodiments, L 1 The length is L 3 at least twice the length of L 2 The length is L 4 In some embodiments, L 1 The length of L is 3 to 12 amino acid residues. 2 The length of L is 3 to 14 amino acid residues. 3 The length of L is 1 to 8 amino acid residues. 4 The length of L is 1 to 3 amino acid residues. 1 The length of L is 5 to 10 amino acid residues. 2 The length of L is 5 to 8 amino acid residues. 3 The length of L is 1 to 5 amino acid residues. 4 The length of L is 1 to 2 amino acid residues. 1 The length of L is 7 amino acid residues. 2 The length of L is 5 amino acid residues. 3 The length of L is 1 amino acid residue. 4 The length of L is 2 amino acid residues. 1 The length of L is 10 amino acid residues. 2 The length of L is 10 amino acid residues. 3 The length is 0 amino acid residues, L4 The length of L is 0 amino acid residues. 1 , L 2 , L 3 and L 4 Each has an independently selected length of 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), wherein at least two linkers have a length of 1 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). In some embodiments, L 1 , L 2 , L 3 and L 4 Each is 0 amino acids in length.

[0448] In some embodiments, L 1 , L 2 , L 3 and / or L 4 The linker comprises 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 comprises an endogenous V H and C H1 between domains or in endogenous V L and C L In some embodiments, the linker comprises a sequence found at a transition between endogenous human V H and C H1 domains or in endogenous human V L and C L Sequences found at transitions between domains (e.g., human kappa or lambda).

[0449] In some embodiments, L 1 , L 2 , L 3 and L 4 Each is independently 0 amino acids in length or comprises 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, L 1 , L 2 , L 3 and L 4Each independently comprises 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).

[0450] In some embodiments, L 1 Containing the sequence GQPKAAP (SEQ ID NO: 58), L 2 Containing the sequence TKGPS (SEQ ID NO: 57), L 3 Contains the sequence S, and L 4 In some embodiments, L 1 Containing the sequence GGGGSGGGGS (SEQ ID NO: 55), L 2 Containing the sequence GGGGSGGGGS (SEQ ID NO: 55), L 3 is 0 amino acids in length, and L 4 The length of L is 0 amino acids. 1 Containing the sequence GGSGSSGSGG (SEQ ID NO: 59), L 2 Containing the sequence GGSGSSGSGG (SEQ ID NO: 59), L 3 is 0 amino acids in length, and L 4 The length of L is 0 amino acids. 1 Containing the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L 2 The length is 0 amino acids, L 3 comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), and L 4 The length of is 0 amino acids.

[0451] Fc region and constant domains

[0452] In some embodiments, the binding proteins of the present disclosure comprise a full-length antibody heavy chain or a polypeptide chain containing 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 includes an antibody hinge, C H1 , C H2 , C H3 and optionally C H4In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region includes one or more mutations described below.

[0453] In some embodiments, the binding proteins of the present disclosure include one or two Fc variants. As used herein, the term "Fc variant" refers to a molecule or sequence modified from a native Fc but still comprising a binding site for a salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with salvage receptors are known in the art. Therefore, the term "Fc variant" may include a molecule or sequence of a humanized non-human native Fc. In addition, the native Fc includes regions that can be removed because they provide structural features or biological activities that are not required by the antibody-like binding proteins of the present invention. Therefore, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues, or wherein one or more Fc sites or residues are modified, affecting or participating in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed 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).

[0454] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and / or 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)).

[0455] In some embodiments, the Fc region is a human IgG1 Fc region, which, according to the EU Index, comprises one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1. 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, which, according to the EU Index, comprises amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0456] 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 but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region, which comprises amino acid substitutions at positions corresponding to 228 and / or 409 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitutions are S228P and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region, which comprises amino acid substitutions at positions corresponding to 234 and / or 235 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitutions are F234A and / or L235A. In some embodiments, the Fc region is a human IgG4 Fc region, which, according to the EU Index, comprises an amino acid substitution at a position corresponding to 228, 234, 235, and / or 409 of human IgG4. 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, which, according to the EU Index, comprises an amino acid substitution at a position corresponding to 233-236 of human IgG4. In some embodiments, the amino acid substitution is E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region, which, according to the EU Index, comprises an amino acid mutation at a substitution corresponding to 228, 233-236, and / or 409 of human IgG4. In some embodiments, the amino acid mutation is S228P; E233P, F234V, L235A, and deletion at 236; and / or R409K.

[0457] In some embodiments, the binding protein of the present disclosure comprises one or more mutations to improve purification, for example, by adjusting the affinity for the purification reagent. For example, it is well known that if one of the two Fc regions of the heterodimeric form contains a mutation that reduces or eliminates binding to protein A, the heterodimeric binding protein can be selectively purified from its homodimeric form because the heterodimeric form will have an intermediate affinity for purification based on protein A compared to any of the homodimeric forms, and can be selectively eluted from protein A, for example, by using different pH (see, e.g., Smith, EJ et al. (2015) Sci. Rep. 5: 17943). In some embodiments, the mutation comprises a substitution at a position corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain, which further comprises a C H1 The first Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 The immunoglobulin heavy chain constant region, and the third polypeptide chain further comprises H1 The second Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 Immunoglobulin heavy chain constant region; and wherein only one of the first and second Fc regions comprises an amino acid substitution at positions 435 and 436 corresponding to human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitution is H435R and Y436F. In some embodiments, the binding proteins of the present disclosure comprise a knob-in-hole mutation 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.

[0458] To increase the yield of some binding proteins (e.g., bispecific or trispecific binding proteins), the CH3 domain can be altered by the "knob-in-hole" technique, which is described in detail in several examples, such as 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. Specifically, the two CH3 domains are altered. H3 domains to increase the interaction surface containing these two C H3 Heterodimerization of the two heavy chains of the two C H3Each of the domains (two heavy chains) can be a "knob" and the other a "hole". The introduction of disulfide bridges further stabilizes the heterodimers (Merchant et al., 1998; Atwell et al., 1997, J. Mol. Biol. 270: 26-35) and improves yield. In certain embodiments, the knob is located on the second pair of polypeptides with a single variable domain. In other embodiments, the knob is located on the first pair of polypeptides with a cross-shaped orientation. In other embodiments, C H3 The domain does not include the knob in the mortar.

[0459] In some embodiments, the binding proteins of the present disclosure (e.g., trispecific binding proteins) comprise a "knob" mutation on the second polypeptide chain and a "hole" mutation on the third polypeptide chain. In some embodiments, the binding proteins of the present disclosure comprise 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 position 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 position 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 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 position 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU Index. In some embodiments, the amino acid substitution is Y349C, T366S, L368A and Y407V.

[0460] 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 regions, wherein according to the EU Index, the first Fc region comprises an amino acid substitution at a position corresponding to 366 and optionally 354 of human IgG1 or IgG4, wherein the amino acid substitution is T366W or T366Y, and optionally S354C; and wherein 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 regions, wherein according to the EU Index, the second Fc region comprises an amino acid substitution at a position corresponding to 407 and optionally 349, 366 and / or 368 of human IgG1 or IgG4, wherein the amino acid substitution is Y407V or Y407T, and optionally Y349C, T366S and / or L368A.

[0461] 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 regions, wherein according to the EU Index, the first Fc region comprises an amino acid substitution at a position corresponding to position 407 and optionally 349, 366 and / or 368 of human IgG1 or IgG4, wherein the amino acid substitution is Y407V or Y407T, and optionally Y349C, T366S and / or L368A; and wherein 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 regions, wherein according to the EU Index, the second Fc region comprises an amino acid substitution at a position corresponding to position 366 and optionally 354 of human IgG1 or IgG4, wherein the amino acid substitution is T366W or T366Y, and optionally S354C.

[0462] In some embodiments, the second polypeptide chain further comprises a first Fc region connected to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein according to the EU Index, the first Fc region comprises an amino acid substitution at a position corresponding to 366 of human IgG1 or IgG4, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region connected to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein according to the EU Index, the second Fc region comprises an amino acid substitution at a position corresponding to 366, 368 and / or 407 of human IgG1 or IgG4, wherein the amino acid substitution is T366S, L368A and / or Y407V.

[0463] In some embodiments, the second polypeptide chain further comprises a first Fc region connected to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein according to the EU Index, the first Fc region comprises an amino acid substitution at a position corresponding to 366, 368 and / or 407 of human IgG1 or IgG4, wherein the amino acid substitution is T366S, L368A and / or Y407V; and wherein the third polypeptide chain further comprises a second Fc region connected to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein according to the EU Index, the second Fc region comprises an amino acid substitution at a position corresponding to 366 of human IgG1 or IgG4, wherein the amino acid substitution is T366W.

[0464] In some embodiments, the second polypeptide chain further comprises a first Fc region connected to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W; wherein the third polypeptide chain further comprises a second Fc region connected to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to 349, 366, 368 and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region connected to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to 349, 366, 368 and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V; wherein the third polypeptide chain further comprises a second Fc region connected to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are 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.

[0465] 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 according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and wherein 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, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to 228, 349, 366, 368, 407 and 409 of human IgG4, wherein the amino acid substitutions are 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 according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein 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, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to 228, 354, 366 and 409 of human IgG4, wherein the amino acid substitutions are S228P, S354C, T366W and R409K.

[0466] 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 according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W; and wherein 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, wherein according to the EU Index Index, the second Fc region comprises amino acid substitutions at positions corresponding to 234, 235, 349, 366, 368 and 407 of human IgG4, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region connected 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 according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to 234, 235, 349, 366, 368 and 407 of human IgG4, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A and Y407V; and wherein the third polypeptide chain further comprises a second Fc region connected 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, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354 and 366 of human IgG4, wherein the amino acid substitutions are F234A, L235A, S354C and T366W.

[0467] 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 according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to 228, 234, 235, 354, 366, and 409 of human IgG4, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; and wherein 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, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to 228, 234, 235, 349, 366, 368, 407 and 409 of human IgG4, wherein the amino acid substitutions are 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, wherein according to the EU Index, 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, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V and R409K; and wherein 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, wherein according to the EU Index, second Fc region, comprising amino acid substitutions at positions corresponding to 228, 234, 235, 354, 366 and 409 of human IgG4, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W and R409K.

[0468] In some embodiments, the binding protein of the present disclosure comprises 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, according to the EU Index, the mutation comprises a substitution at a position corresponding to 428 and 434 of human IgG1 or IgG4, wherein the amino acid substitution is M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain, which further comprises a first Fc region connected to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and the third polypeptide chain further comprises a second Fc region connected to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the first and / or second Fc region comprises an amino acid substitution at a position corresponding to 428 and 434 of human IgG1, wherein the amino acid substitution is 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.

[0469] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to improve the stability of the hinge region and / or dimer interface of, for example, IgG4 (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, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein comprises a second polypeptide chain, which further comprises a C H 1 connected to the first Fc region, the first Fc region includes the immunoglobulin hinge region and C H2 and C H3 The immunoglobulin heavy chain constant domain, and the third polypeptide chain further comprises a C H1 The second Fc region comprises an immunoglobulin hinge region and a C H2 and C H3Immunoglobulin heavy chain constant domain; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein according to the EU Index, the first and second Fc regions each comprise an amino acid substitution at positions corresponding to positions 228 and 409 of human IgG4, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and one or more mutations to improve stability. In some embodiments, the first and / or second Fc regions are human IgG4 Fc regions.

[0470] In some embodiments, the binding protein of the present disclosure comprises one or more mutations to improve purification, for example, by adjusting affinity for purification reagents. For example, it is known that if one of the two Fc regions of the heterodimeric form contains a mutation that reduces or eliminates binding to protein A, the heterodimeric binding protein can be selectively purified from its homodimeric form because the heterodimeric form will have an intermediate affinity for purification based on protein A compared to either homodimeric form, and can be selectively eluted from protein A, for example, by using different pH (see, e.g., Smith, EJ et al. (2015) Sci. Rep. 5: 17943). In some embodiments, according to the EU Index, the mutation comprises a substitution at a position corresponding to positions 435 and 436 of human IgG1 or IgG4, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain, which further comprises a C H1 The first Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 The immunoglobulin heavy chain constant domain, and the third polypeptide chain further comprises a C H1 The second Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 Immunoglobulin heavy chain constant domain; and wherein according to the EU Index, only one of the first and second Fc regions comprises an amino acid substitution at a position corresponding to positions 435 and 436 of human IgG1 or IgG4, wherein the amino acid substitution is 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.

[0471] In some embodiments, the binding protein of the present disclosure comprises 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, according to the EU Index, the mutation comprises a substitution at a position corresponding to 428 and 434 of human IgG1 or IgG4, wherein the amino acid substitution is M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain, which further comprises a first Fc region connected to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and the third polypeptide chain further comprises a second Fc region connected to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the first and / or second Fc region comprises an amino acid substitution at a position corresponding to 428 and 434 of human IgG1, wherein the amino acid substitution is 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.

[0472] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to reduce effector functions, such as 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 further comprises a C H1 The first Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 Immunoglobulin heavy chain constant domain; wherein the third polypeptide chain further comprises a C H1 The second Fc region comprises an immunoglobulin hinge region and a C H2 and C H3Immunoglobulin heavy chain constant domain; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein according to the EU Index, the first and second Fc regions each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG1, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and wherein according to the EU Index, the Fc regions each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG1, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the second polypeptide chain further comprises a C H1 The first Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 Immunoglobulin heavy chain constant domain; wherein the third polypeptide chain further comprises a C H1 The second Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 Immunoglobulin heavy chain constant domain; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein according to the EU Index, the first and second Fc regions each comprise an amino acid substitution at positions corresponding to positions 234, 235, and 329 of human IgG1, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and wherein according to the EU Index, the Fc regions each comprise an amino acid substitution at positions corresponding to positions 234, 235, and 329 of human IgG1, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG4 Fc regions, and according to the EU Index, the Fc regions each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG4, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the binding protein comprises a second polypeptide chain, which further comprises a C H1 The first Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 The immunoglobulin heavy chain constant domain, and the third polypeptide chain further comprises a C H1 The second Fc region comprises an immunoglobulin hinge region and a C H2 and C H3An immunoglobulin heavy chain constant domain; and wherein the first and second Fc regions each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG4 according to the EU Index, wherein the amino acid substitutions are F234A and L235A.

[0473] 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, e.g., Shields, RL et al. (2001) J. Biol. Chem. 276: 6591-6604 and WO1999051642.

[0474] In some embodiments, the types of mutations described above can be combined in any order or combination. For example, a binding protein of the present disclosure may comprise 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.

[0475] In some embodiments, the binding proteins of the present disclosure comprise antibody fragments, including but not limited to antibody F(ab), F(ab')2, Fab'SH, Fv or scFv fragments.

[0476] Determination

[0477] The present disclosure provides antigen binding proteins that bind to human and / or cynomolgus CD38 polypeptides, induce T cell (e.g., CD4+ and / or CD8+ T cells) proliferation, and / or induce CD38+ cell apoptosis. Exemplary assays for measuring these parameters and identifying such binding proteins are provided herein. For example, in some embodiments, the binding affinity between the binding protein or its antigen binding fragment and the purified CD38 polypeptide is measured by SPR (e.g., as described below), and the binding affinity between the binding protein or its antigen binding fragment and the CD38 polypeptide expressed on the cell surface is measured by flow cytometry (e.g., as described below).

[0478] In some embodiments, the antigen binding site of the binding protein of the present disclosure binds to the human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with an equilibrium dissociation constant (K D) is 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, as 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 below. 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 ) is 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 below. In some embodiments, the antigen binding site binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with an equilibrium dissociation constant (K D ) is 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 below. 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 ) is 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 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 below. As demonstrated herein, in some embodiments, the binding proteins of the present disclosure may have one or more of the exemplary binding properties described herein. In some embodiments, the KD is measured at 4°C or 5°C.

[0479] In some embodiments, the monospecific binding proteins of the present disclosure have one or more of the following features: bind to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, as determined by SPR or ELISA; bind to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, and its K Dis 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, as determined by SPR or ELISA; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), with an apparent K D is 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, as determined by flow cytometry; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein, as determined by SPR or ELISA; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein, whose K D is 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, as determined by SPR or ELISA; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell, as determined by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell, with an apparent K Dis 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, as determined by flow cytometry; 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, as determined by SPR or ELISA; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) expressed on the surface of a cell, as determined 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. In some embodiments, the binding proteins of the present disclosure bind to CD38 polypeptides expressed on the surface of cells (e.g., humans or cynomolgus monkeys) 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, as determined 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, as determined by ELISA. In some embodiments, the KD is measured at 4°C or 25°C.

[0480] In some embodiments, the trispecific binding proteins of the present disclosure have one or more of the following characteristics: induce T cell (e.g., CD4+ and / or CD8+ T cell) proliferation; induce T cell (e.g., CD4+ and / or CD8+ T cell) expression of Bcl-xL; induce apoptosis of CD38+ cells (e.g., as measured by annexin V staining and / or propidium iodide uptake); bind to CD38 expressed on the surface of a cell and one or more T cell target antigens expressed on the surface of a T cell; bind to CD38 expressed on the surface of a cell, CD28 expressed on the surface of a T cell, and CD3 expressed on the surface of a T cell; stimulate activation of a T cell receptor (e.g., as measured by CD69 expression); inducing co-stimulation of T cell receptor signaling (e.g., as mediated by CD28); having 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; inducing cytokine release (e.g., IFN-γ and / or IL-6) by PBMCs in the presence of CD38+ target cells (e.g., as measured by an immunoassay); binding to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, as determined by SPR or ELISA; binding to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, its K D is 1.5 nM or less, as determined by SPR or ELISA; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), with an apparent K D is 12 nM or less, as determined by flow cytometry; 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, as determined by SPR or ELISA; 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, its K D is 3.5 nM or less, as determined by SPR or ELISA; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell, as determined by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell, with an apparent K Dis 7.5 nM or less, as determined by flow cytometry; 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, as determined by SPR or ELISA; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) expressed on the surface of a cell, as determined by flow cytometry; induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells expressing CD38 on their cell surface; 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. In some embodiments, the binding proteins of the present disclosure bind to CD38 polypeptides expressed on the surface of cells (e.g., human or cynomolgus monkey) 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, as determined by flow cytometry. In some embodiments, the binding proteins of the present disclosure bind to CD38 polypeptides (e.g., human or cynomolgus monkey) 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, as determined by ELISA.

[0481] Nucleic Acids

[0482] 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, for example, Sambrook et al., 2001, M. OLECULAR C LONING :AL ABORATORY M ANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques may be performed according to the manufacturer's instructions, as commonly accomplished in the art, or as described herein. Unless specific definitions are provided, the nomenclature and laboratory procedures and techniques used in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Similarly, conventional techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0483] 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 molecules comprise a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% identical to SEQ ID NOs: 60-83. 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 and / or the sequence shown in Table J.

[0484] Certain aspects of the present disclosure relate to a kit of polynucleotides. In some embodiments, one or more polynucleotides are vectors (e.g., expression vectors). The kit is particularly useful for producing one or more binding proteins as described herein, such as tri-specific binding proteins of the present disclosure. In some embodiments, the kit is included in one, two, three or four nucleotides of (e.g., mAb2xCD28supxCD3mid IgG4 FALA, mAb2xCD28supxCD3mid IgG1LALA P329A, mAb2xCD28supxCD3mid IgG1 NNSA, mAb6xCD28supxCD3mid IgG4 FALA, mAb6xCD28supxCD3mid IgG1LALA P329A or mAb6xCD28supxCD3mid IgG1 NNSA) shown in Table J. In some embodiments, the polynucleotide kit comprises: a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 72, a third polynucleotide comprising a sequence of SEQ ID NO: 74, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 75. In some embodiments, the polynucleotide kit comprises: a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 76, a third polynucleotide comprising a sequence of SEQ ID NO: 77, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 75. In some embodiments, the polynucleotide kit comprises: a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 78, a third polynucleotide comprising a sequence of SEQ ID NO: 79, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 75. In some embodiments, the polynucleotide kit comprises: a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 72, a third polynucleotide comprising a sequence of SEQ ID NO: 80, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 81. In some embodiments, the polynucleotide kit comprises: a first polynucleotide comprising a sequence of SEQ ID NO: 73, a second polynucleotide comprising a sequence of SEQ ID NO: 76, a third polynucleotide comprising a sequence of SEQ ID NO: 82, and a fourth polynucleotide comprising a sequence of SEQ ID NO: 81.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:83, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81.

[0485] In some embodiments, the isolated nucleic acid is operably connected to a heterologous promoter to direct the transcription of a binding protein encoding nucleic acid sequence. A promoter may refer to a nucleic acid control sequence that directs the transcription of a nucleic acid. When a first nucleic acid sequence is in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably connected to the second nucleic acid sequence. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operably connected to the coding sequence of the binding protein. Examples of promoters may include, but are not limited to, promoters obtained from the genome of viruses (such as polyoma virus, fowl pox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40) and the like), promoters from heterologous eukaryotic promoters (such as actin promoter, immunoglobulin promoter, from heat shock promoters and the like), CAG promoter (Niwa et al., Gene 108(2):193-9, 1991), phosphoglycerate kinase (PGK)-promoter, tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005), lac system, trp system, tac system, trc system, major operator and promoter region of bacteriophage λ, promoter of 3-phosphoglycerate kinase, promoter of yeast acid phosphatase, and promoter of yeast α-mating factor. The polynucleotides encoding the binding proteins of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or other suitable promoters readily identified by those skilled in the art.

[0486] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. The expression vector may include one or more regulatory sequences operably connected to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globulin, elastase, albumin, α-fetoprotein, insulin, etc.), and enhancer sequences from eukaryotic cell viruses (such as SV40 enhancers on the rear side of the replication origin (bp100-270), cytomegalovirus early promoter enhancers, polyoma enhancers on the rear side of the replication origin, adenovirus enhancers, etc.). Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons and viral vectors (e.g., adenovirus, vaccinia virus, Sindbis virus (Sindbis-viral), measles, herpes virus, lentivirus, retrovirus, adeno-associated virus vectors, etc.). Expression vectors can be used to transfect host cells, such as, for example, bacterial cells, yeast cells, insect cells and mammalian cells. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art and can be used to transfect any cell of interest.

[0487] Other aspects of the present disclosure relate to a vector system comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any binding protein 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. The one or more vectors of the vector system can be any vector described herein. In some embodiments, one or more vectors are expression vectors.

[0488] Isolated host cells

[0489] Other aspects of the present disclosure relate to isolated host cells comprising one or more isolated polynucleotides, polynucleotide kits, vectors and / or vector systems 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., a Saccharomyces cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells may include, for example, a Drosophila cell (e.g., an S2 cell), a Trichoplusia ni cell (e.g., a High Five antagonist cell), a Drosophila cerevisiae ... TM 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 can include, for example, human embryonic kidney cells (e.g., 293 or 293 cells subcloned for growth in suspension culture), Expi293TM 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, HB8065), mouse breast tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (e.g., HepG2), and myeloma cells (e.g., NS0 and Sp2 / 0 cells).

[0490] Other aspects of the present disclosure relate to methods for producing any binding protein described herein. In some embodiments, the method includes a) culturing a host cell (e.g., any host cell described herein) containing an isolated nucleic acid, a vector and / or a vector system (e.g., any isolated nucleic acid, a vector and / or a vector system described herein) under conditions where the host cell expresses the binding protein; b) isolating the binding protein from the host cell. Methods for culturing host cells under conditions where the protein is expressed are well known to those of ordinary skill in the art. Methods for isolating proteins from cultured host cells are well known to those of ordinary skill in the art, including, for example, affinity chromatography (e.g., two-step affinity chromatography including protein A affinity chromatography followed by size exclusion chromatography).

[0491] In some embodiments, the binding proteins of the present 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), and optionally 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 present 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), and optionally 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 comprising a C H3 domain, and according to the EU Index, only one C H3 The domain comprises an amino acid substitution at a position corresponding to positions 435 and 436 of human IgG1 or IgG4, wherein the amino acid substitution is H435R and Y436F. In some embodiments, the binding protein of the present disclosure is purified sequentially by protein A affinity chromatography, then κ light chain affinity chromatography (e.g., using KappaSelect resin according to the manufacturer's instructions; GE Healthcare), and then optionally λ light chain affinity chromatography (e.g., using LambdaFabSelect resin according to the manufacturer's instructions; GE Healthcare). In some embodiments, the binding protein of the present disclosure is purified sequentially by protein A affinity chromatography, then λ light chain affinity chromatography (e.g., using LambdaFabSelect resin according to the manufacturer's instructions; GE Healthcare), and then optionally κ 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, in a suitable for converting the binding protein from a C containing 0 or 2 amino acids substituted with H435R and Y436F H3 The binding proteins containing the λC domain are eluted from protein A under conditions suitable for separating the binding proteins from the λC domain alone, contacted with a κ light chain affinity matrix (e.g., a resin as used in KappaSelect; GE Healthcare), and stirred under conditions suitable for separating the binding proteins from the λC domain alone. LThe binding protein of the present invention is eluted from the κ light chain affinity medium under conditions suitable for separating the binding protein of the κ CL domain (e.g., according to the manufacturer's instructions). Suitable conditions for protein A elution are known in the art, including but not limited to a step-wise elution gradient of pH 4.5-2.8. In some embodiments, protein A or protein A variants that can be used for protein purification are used. In some embodiments, protein A is attached to a substrate or resin, for example, as part of a chromatographic medium. In some embodiments, after elution from the κ light chain affinity medium, the binding protein is contacted with a λ light chain affinity medium (e.g., as used in LambdaFabSelect resin; GE Healthcare) and eluted from the λ light chain affinity medium under conditions suitable for separating the binding protein from the binding protein containing only the κ CL domain (e.g., according to the manufacturer's instructions). In some embodiments, HIC chromatography is used to detect the binding protein of the present invention. In some embodiments, the binding protein comprises: a first polypeptide chain, which comprises a λC L Domain; C of the second polypeptide chain H3 A domain 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; C of the third polypeptide chain H3 A domain comprising 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; and a fourth polypeptide chain comprising κ C L In some embodiments, the binding protein is produced by a host cell. In some embodiments, the binding protein is purified from a cell culture medium or a host cell extract. In some embodiments, the binding protein is secreted by a host cell or produced and extracted from a host cell (e.g., before contacting with protein A). In some embodiments, when contacted with protein A, the binding protein is in a cell culture medium or a host cell extract. In some embodiments, the binding protein is purified from other binding proteins, polypeptides, and / or other cellular components.

[0492] III. Trispecific Binding Proteins

[0493] In some embodiments, the binding protein of the present disclosure is a trispecific and / or trivalent binding protein comprising four polypeptide chains forming three antigen binding sites that bind to one or more (e.g., three) different antigen targets or target proteins. In some embodiments, the first polypeptide chain comprises a structure represented by the following formula:

[0494] V L2 -L 1-V L1 -L 2 -C L [I]

[0495] and the second polypeptide chain comprises a structure represented by the formula:

[0496] V H1 -L 3 -V H2 -L 4 -C H1 -Hinge-C H2 -C H3 [II]

[0497] and the third polypeptide chain comprises a structure represented by the formula:

[0498] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0499] and the fourth polypeptide chain comprises a structure represented by the formula:

[0500] V L3 -C L [IV]

[0501] in:

[0502] V L1 is the first immunoglobulin light chain variable domain;

[0503] V L2 is a second immunoglobulin light chain variable domain;

[0504] V L3 is the third immunoglobulin light chain variable domain;

[0505] V H1 is the first immunoglobulin heavy chain variable domain;

[0506] V H2 is a second immunoglobulin heavy chain variable domain;

[0507] V H3 is the third immunoglobulin heavy chain variable domain;

[0508] C L is the immunoglobulin light chain constant domain;

[0509] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0510] C H2Immunoglobulin C H2 heavy chain constant domain;

[0511] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0512] The hinge is connected to C H1 and C H2 The immunoglobulin hinge region of the domain; and

[0513] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0514] Wherein the polypeptide of Formula I and the polypeptide of Formula II form a cross light chain-heavy chain pair. In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross orientation, which forms two different antigen binding sites. As described above, the second and third polypeptide chains include an Fc region (e.g., comprising a hinge-C H2 -C H3 Domain). In some embodiments, one or both Fc regions are human IgG4 Fc regions, which, according to the EU Index, comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and deletions at 236. In some embodiments, the Fc region is a human IgG4 Fc region, which, according to the EU Index, comprises amino acid mutations at substitutions corresponding to positions 228, 233-236, and / or 409 of human IgG4. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and deletions at 236; and / or R409K. In some embodiments, one or both Fc regions are human IgG1 Fc regions, which, according to the EU Index, comprise one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1. In some embodiments, the amino acid substitution is L234A, L235A and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region, which comprises an amino acid substitution at a position corresponding to position 298, 299 and / or 300 of human IgG1 according to the EU Index. In some embodiments, the amino acid substitution is S298N, T299A and / or Y300S.

[0515] In some embodiments, the binding protein of the present disclosure is a trispecific and / or trivalent binding protein comprising four polypeptide chains forming 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 antigen binding site binds to a CD38 polypeptide (e.g., an extracellular domain of a human and / or cynomolgus CD38 polypeptide). In some embodiments, the first polypeptide chain comprises a structure represented by the following formula:

[0516] V L2 -L 1 -V L1 -L 2 -C L [I]

[0517] and the second polypeptide chain comprises a structure represented by the formula:

[0518] V H1 -L 3 -V H2 -L 4 -C H1 [II]

[0519] and the third polypeptide chain comprises a structure represented by the formula:

[0520] V H3 -C H1 [III]

[0521] and the fourth polypeptide chain comprises a structure represented by the formula:

[0522] V L3 -C L [IV]

[0523] in:

[0524] V L1 is the first immunoglobulin light chain variable domain;

[0525] V L2 is a second immunoglobulin light chain variable domain;

[0526] V L3 is the third immunoglobulin light chain variable domain;

[0527] V H1 is the first immunoglobulin heavy chain variable domain;

[0528] V H2 is a second immunoglobulin heavy chain variable domain;

[0529] V H3 is the third immunoglobulin heavy chain variable domain;

[0530] C L is the immunoglobulin light chain constant domain;

[0531] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0532] L 1 , L 2 , L 3 and L 4 is an amino acid linker;

[0533] and wherein 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 further comprise a C H1 The Fc region contains the immunoglobulin hinge region and the C H2 and C H3 Immunoglobulin heavy chain constant domain. In some embodiments, one or two Fc regions are human IgG4 Fc regions, which, according to the EU Index, comprise amino acid substitutions at positions corresponding to 233-236 of human IgG4. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and deletions at 236. In some embodiments, the Fc region is a human IgG4 Fc region, which, according to the EU Index, comprises amino acid mutations at substitutions corresponding to 228, 233-236, and / or 409 of human IgG4. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and deletions at 236; and / or R409K. In some embodiments, one or two Fc regions are human IgG1 Fc regions, which, according to the EU Index, comprise one or more amino acid substitutions at positions corresponding to 234, 235, and / or 329 of human IgG1. In some embodiments, the amino acid substitution is L234A, L235A and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region, which comprises an amino acid substitution at a position corresponding to position 298, 299 and / or 300 of human IgG1 according to the EU Index. In some embodiments, the amino acid substitution is S298N, T299A and / or Y300S.

[0534] In some embodiments, VH1 and VL1 form a binding pair and form a first antigen binding site. In some embodiments, VH2 and VL2 form a binding pair and form a second antigen binding site. In some embodiments, VH3 and VL3 form a binding pair and form a third antigen binding site. The binding protein can also be used for cell activation, tumor targeting, neutralization of cytokine activity, neutralization of viral infection, combination of multiple signaling events to treat cancer, arthritis and / or inflammatory disorders. For example, in some embodiments, the binding protein specifically binds to one, two, or three antigenic targets selected from the group consisting of A2AR, APRIL, ATP diphosphohydrolase, 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 MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2,an IgE receptor), 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), 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、IL2Rβ、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, NTP diphosphohydrolase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as IL33 receptor), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments,One or more of the above antigenic targets are human antigenic targets.

[0535] In some embodiments, one of the three antigen binding sites binds a CD3 polypeptide (e.g., a human CD3 polypeptide), one of the three antigen binding sites binds a CD28 polypeptide (e.g., a human CD28 polypeptide), and one of the three antigen binding sites binds a third polypeptide. In some embodiments, the antigen binding site that specifically binds to an antigen target other than CD3 or CD28 binds an antigen target selected from the group consisting of A2AR, APRIL, ATP diphosphohydrolase, 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 M CP-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 (also known as FCER2,an IgE receptor), 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), 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、IL2Rβ、IL1、IL1A、IL1B、IL1F10、IL2、IL4、IL4Ra、IL5、 IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ral, 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, NTP diphosphohydrolase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as IL33 receptor), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments,One or more of the above antigenic targets are human antigenic targets.

[0536] In any of the trispecific binding proteins described above, any linker or combination of linkers described herein may be used. For example, in some embodiments, L 1 , L 2 , L 3 or L 4 In some embodiments, at least one of L is independently 0 amino acids in length. 1 , L 2 , L 3 or L 4 The length of each of L is independently at least 1 amino acid. 1 , L 2 , L 3 and L 4 Each is independently 0 amino acids in length or comprises 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, L 1 , L 2 , L 3 and L 4 Each independently comprises 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, L 1 Containing the sequence GQPKAAP (SEQ ID NO: 58), L 2 Containing the sequence TKGPS (SEQ ID NO: 57), L 3 Contains the sequence S, L 4 In some embodiments, L 1 Containing the sequence GGGGSGGGGS (SEQ ID NO: 55), L 2 Containing the sequence GGGGSGGGGS (SEQ ID NO: 55), L 3 The length is 0 amino acids, L 4 The length of L is 0 amino acids. 1Containing the sequence GGSGSSGSGG (SEQ ID NO: 59), L 2 Containing the sequence GGSGSSGSGG (SEQ ID NO: 59), L 3 The length is 0 amino acids, L 4 The length of L is 0 amino acids. 1 Containing the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L 2 The length is 0 amino acids, L 3 Containing the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L 4 The length of is 0 amino acids.

[0537] IV. Uses of Binding Proteins

[0538] The binding protein can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for detecting and quantifying one or more target antigens. The binding protein will bind to one or more target antigens with an affinity suitable for the assay method used.

[0539] For diagnostic applications, in certain embodiments, the binding protein may be labeled with a detectable moiety. The detectable moiety may be any moiety that is capable of directly or indirectly producing a detectable signal. For example, the detectable moiety may be a radioactive isotope, such as 3 H. 14 C. 32 P. 35 S. 125 I. 99 Tc, 111 In or 67 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.

[0540] The binding protein can also be used for in vivo imaging. The binding protein labeled with a detectable moiety can be administered to an animal, preferably administered to the bloodstream, and the presence and location of the labeled antibody in the host is determined. The binding protein can be used in an animal by any moiety label that is detectable by nuclear magnetic resonance, radiology, or other detection means known in the art.

[0541] For clinical or research applications, in certain embodiments, the binding protein can be conjugated to a cytotoxic agent. A variety of antibodies conjugated to cytotoxic agents (i.e., antibody-drug conjugates) have been used to target cytotoxic payloads to specific tumor cells. Cytotoxic agents and linkers for conjugating agents to antibodies are known in the art; see, e.g., Parslow, AC et al. (2016) Biomedicines 4: 14 and Kalim, M. et al. (2017) Drug Des. Devel. Ther. 11: 2265-2276.

[0542] The present disclosure also relates to a kit comprising binding proteins and other reagents for detecting target antigen levels in biological samples. Such reagents may include detectable markers, blocking serum, positive and negative control samples and detection reagents. In some embodiments, the kit comprises a composition comprising any binding proteins, polynucleotides, vectors, vector systems and / or host cells described herein. In some embodiments, the kit comprises 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, etc. The container may be formed of a variety of materials, such as glass or plastic. The container holds the composition itself, which is itself or in combination with another effective treatment, prevention and / or diagnosis of the patient's condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). In some embodiments, a label or package insert indicates that the composition is used for prevention, diagnosis and / or treatment of the selected patient's condition. Alternatively, or in addition, the article of manufacture or kit may further comprise a second (or third) container comprising 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 perspective, including other buffers, diluents, filters, needles and syringes.

[0543] In some embodiments, the binding protein of the present disclosure is applied to a patient in need to treat or prevent cancer. In some embodiments, the present disclosure relates to a method for preventing and / or treating a proliferative disease or condition (e.g., cancer). In some embodiments, the method includes administering a therapeutically effective amount of at least one binding protein as described herein or a pharmaceutical composition associated therewith to a patient. In some embodiments, the present disclosure relates to at least one binding protein as described herein or a pharmaceutical composition associated therewith for the prevention and / or treatment of a proliferative disease or condition (e.g., cancer) in a patient in need. In some embodiments, the present disclosure relates to at least one binding protein as described herein or a pharmaceutical composition associated therewith for the preparation of a drug for the prevention and / or treatment of a proliferative disease or condition (e.g., cancer) in a patient in need. In some embodiments, the patient is a human. In some embodiments, the binding protein comprises an antigen binding site in conjunction with a T cell surface protein and another antigen binding site in conjunction with the extracellular domain of a human CD38 polypeptide, for example, as described in Part 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.

[0544] In some embodiments, the cancer cells express a human CD38 isoform A polypeptide (e.g., comprising an 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 an amino acid sequence of SEQ ID NO: 105) on their cell surface. In some embodiments, the patient is selected for treatment based on the cancer cells expressing a human CD38 isoform E polypeptide (e.g., comprising an 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 not CD28.

[0545] In some embodiments, cancer is multiple myeloma, acute lymphocytic 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, cancer is multiple myeloma. In certain embodiments, cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) or B cell lymphoma.

[0546] In certain embodiments, cancer is multiple myeloma. Anti-CD38 antibodies have been tested for the treatment of multiple myeloma, such as daratumumab and isatuximab. However, although multiple myeloma is considered to be treatable, almost all patients inevitably relapse, leading to the development of refractory diseases. In some embodiments, cancer is recurrent or refractory multiple myeloma. In some embodiments, the patient has been treated with previous multiple myeloma treatment. In some embodiments, the binding protein of the present disclosure is administered to the patient as the first, second or third line treatment of multiple myeloma. Without wishing to be bound by theory, it is believed that the anti-CD38x anti-CD28x anti-CD3 binding protein of the present disclosure can be used, for example, by recruiting T cells to tumor cells via anti-CD38 (or anti-CD28 / anti-CD38), activating T cells involved via anti-CD3 / anti-CD28, and / or killing tumor cells by a mechanism based on perforin / granzyme to treat multiple myeloma. CD28 has been reported as a novel cancer marker for multiple myeloma. See Nair, JR et al. (2011) J. Immunol. 187: 1243-1253.

[0547] In some embodiments, at least one binding protein is administered (or is to be administered) in combination with one or more anti-cancer therapies (e.g., any anti-cancer therapy known in the art, such as a chemotherapeutic agent or chemotherapy). In some embodiments, at least one binding protein is administered (or is to be administered) prior to one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered (or is to be administered) simultaneously with the one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered (or is to be administered) after one or more antiretroviral therapies.

[0548] V. Binding Protein Therapeutic Compositions and Administration

[0549] Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the present disclosure. Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of a binding protein or binding protein-drug conjugate mixed with a pharmaceutically or physiologically acceptable formulation selected for the mode of administration.

[0550] Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed.

[0551] The pharmaceutical composition may contain formulation materials used to alter, maintain or preserve, for example, the pH, osmolarity, 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 borates, bicarbonates, Tris-HCl, citrates, phosphates or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), bulking agents, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrin), proteins (such as serum albumin, gelatin or immunoglobulins), colorants, flavorings and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions, The composition may be a pharmaceutical composition comprising: a preservative (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide), a solvent (such as glycerol, propylene glycol or polyethylene glycol), a sugar alcohol (such as mannitol or sorbitol), a suspending agent, a surfactant or wetting agent (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), a stability enhancer (such as sucrose or sorbitol), a tonicity enhancer (such as an alkali metal halide - preferably sodium chloride or potassium chloride - or mannitol sorbitol), a delivery vehicle, a diluent, an excipient and / or a pharmaceutical adjuvant (see, e.g., R EMINGTON′ S PHARMACEUTICAL SCIENCES (18th Ed., AR Gennaro, ed., Mack Publishing Company 1990), and subsequent editions, which are incorporated herein by reference for any purpose).

[0552] The optimal pharmaceutical composition will be determined by the skilled artisan based on, for example, the intended route of administration, delivery form, and desired dosage. Such a composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0553] The main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous. For example, the suitable vehicle or carrier for injection can be water, physiological saline solution or artificial cerebrospinal fluid, which may be supplemented with other materials common in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or suitable substitutes. In one embodiment of the present disclosure, by mixing the selected composition with the desired purity with the optional preparaton in the form of lyophilized cake or aqueous solution, the bound protein composition can be prepared for storage. In addition, suitable excipients, such as sucrose, can be used to formulate the bound protein as a lyophilisate.

[0554] The pharmaceutical composition of the present disclosure can be selected for parenteral delivery or subcutaneous delivery. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract, such as oral administration. The preparation of such pharmaceutically acceptable compositions is within the technical scope of the art.

[0555] The formulation components are present in concentrations acceptable to the site of administration. For example, buffering agents are used to maintain the composition at physiological pH or slightly lower pH, generally within the pH range of about 5 to about 8.

[0556] When considering parenteral administration, the therapeutic composition used can be in the form of pyrogen-free, parenterally acceptable, aqueous solution, which comprises the associated protein of expectation in a pharmaceutically acceptable vehicle. The particularly suitable vehicle for parenteral injection is sterile distilled water, wherein the associated protein is formulated as a sterile isotonic solution of suitable preservation. Another preparation can be related to the desired molecule prepared with a medicament, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), pearls or liposomes, which provide controlled or sustained release of a product, and the product can then be sent by depot injection (depot injection). Hyaluronic acid can also be used, which can have the effect of promoting the duration of circulation. Other suitable methods for introducing the desired molecule include implantable drug delivery devices.

[0557] 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 with a propellant for aerosol delivery. In another embodiment, the solution can be atomized.

[0558] It is also contemplated that certain preparations can be administered orally. In one embodiment of the present disclosure, the bound protein administered in this manner may be formulated with or without those carriers that are commonly used to formulate solid dosage forms such as tablets and capsules. For example, when bioavailability is maximized and pre-systemic degradation is minimized, capsules may be designed to release the active portion of the preparation at points in the gastrointestinal tract. Additional agents may be included to promote the absorption of the bound protein. Diluents, flavoring agents, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and adhesives may also be used.

[0559] Another pharmaceutical composition may comprise an effective amount of the binding protein in a mixture with a non-toxic excipient suitable for preparing tablets. Solutions may be prepared in unit dosage form by dissolving the tablets in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or acacia; or lubricants such as magnesium stearate, stearic acid or talc.

[0560] Other pharmaceutical compositions of the present disclosure will be apparent to those skilled in the art, including preparations related to protein-bound preparations that are continuously or controlled for delivery. The technology used to prepare a variety of other continuous or controlled delivery methods, such as liposome carriers, bioerodible microparticles or porous beads and reservoir injections, is also well known to those skilled in the art. Other examples of sustained-release formulations include semipermeable polymer matrices in the form of shaped articles, for example, films or microcapsules. Sustained-release matrix may include copolymers of polyester, hydrogel, polylactide, L-glutamic acid and L-glutamic acid-γ-ethyl ester, poly-(2-hydroxyethyl-methacrylate), ethylene-vinyl acetate or poly-D (-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which may be prepared by any of several methods known in the art.

[0561] Pharmaceutical compositions for in vivo administration must generally be sterile. This can be accomplished by filtration through a sterile filtration membrane. In the case of a lyophilized composition, sterilization using this method can be performed before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution form. In addition, parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle.

[0562] Once the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid or as a dehydrated or lyophilized powder. Such a preparation can be stored in a ready-to-use form or in a form that requires reconstitution (e.g., lyophilization) before administration.

[0563] The present disclosure also encompasses kits for producing single-dose administration units. The kits may each comprise a first container with a dried protein and a second container with an aqueous formulation. Also included within the scope of the present disclosure are kits comprising single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes).

[0564] The effective amount of the protein-binding pharmaceutical composition used in treatment will depend on, for example, the treatment context and purpose. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will therefore depend in part on the molecule delivered, the indication for the use of the protein-binding agent, the route of administration, and the size (body weight, body surface or organ size) and condition (age and general health) of the patient. Therefore, clinicians can titrate the dosage and change the route of administration to obtain the best therapeutic effect.

[0565] The frequency of administration will depend on the pharmacokinetic parameters of the protein-binding agent in the formulation used. Generally speaking, the clinician will use the composition until the dosage for achieving the desired effect is reached. Therefore, the composition can be used as a single dose, as two or more dosages (which may or may not contain the same amount of the desired molecule) are used over time, or as a continuous infusion via an implant or catheter. Further improvements to appropriate dosages are routinely performed by those of ordinary skill in the art, and within the scope of their routinely performed tasks. Appropriate dosage can be determined by using appropriate dose-response data.

[0566] The administration route of the pharmaceutical composition is consistent with known methods, such as oral administration; injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional routes; by sustained release system; or by implant device. If necessary, the composition can be administered continuously by bolus injection or by infusion or by implant device.

[0567] The composition can also be administered locally via an implanted membrane, sponge or other appropriate material into which the desired molecule has been absorbed or encapsulated. Where an implant device is used, the device can be implanted in any suitable tissue or organ and the desired molecule can be delivered via diffusion, timed release bolus or continuous administration.

[0568] Example

[0569] The following examples illustrate specific embodiments of the present disclosure and their various uses. They are presented for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0570] The following terms may be used interchangeably in the examples and figures herein to refer to specific anti-CD38 antigen binding domains or antibodies:

[0571] Anti-CD38_C2-CD38-1: mAb1

[0572] Anti-CD38_C2-CD38-1_VH1-VL1 or CD38 VH1 :mAb2

[0573] Anti-CD38_C2-CD38-1_VH3-VL3: mAb3

[0574] Anti-CD38_C2-CD38-1_VH5-VL3: mAb4

[0575] Anti-CD38_C2-CD38-1_VH6-VL3: mAb5

[0576] Anti-CD38_1370 or CD38 HHY1370 :mAb6

[0577] Anti-CD38_SB19 or isatuximab: mAb7.

[0578] Example 1: Generation and characterization of monoclonal anti-CD38 antibodies

[0579] The following examples describe the generation and characterization of monoclonal anti-CD38 antibodies. Advantageously, the antibodies provided herein cross-react with human and monkey CD38 proteins, thereby providing molecules that can be used for safety and clinical studies. These antibodies are also able to kill CD38+ cells by apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0580] This example describes an efficient workflow for generating CD38-specific and cross-reactive monoclonal antibodies from single murine B cells.

[0581] Materials and methods

[0582] Monoclonal antibody generation ...

Claims

1. A monospecific binding protein that binds to a CD38 polypeptide, wherein the monospecific binding protein comprises: (a) an antibody heavy chain comprising an antibody heavy chain variable (VH) domain, wherein the antibody heavy chain variable (VH) domain comprises a CDR-H1 sequence consisting of the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence consisting of the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence consisting of the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and (b) an antibody light chain comprising an antibody light chain variable (VL) domain, wherein the antibody light chain variable (VL) domain comprises a CDR-L1 sequence consisting of the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence consisting of the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence consisting of the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

2. The monospecific binding protein of claim 1 , wherein the VH domain comprises, from N-terminus to C-terminus, the sequence FR1—CDR-H1—FR2—CDR-H2—FR3—CDR-H3—FR4; wherein FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 86); wherein FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90); wherein FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96).

3. The monospecific binding protein of claim 1, 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. 4 . The monospecific binding protein of claim 3 , wherein the antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 15 and the antibody light chain comprises the amino acid sequence of SEQ ID NO:

16.

5. The monospecific binding protein of claim 1, wherein the monospecific binding protein cross-reacts with the extracellular domain of human CD38 polypeptide and the extracellular domain of cynomolgus monkey CD38 polypeptide.

6. The monospecific binding protein of claim 5, wherein the monospecific binding protein binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:

1.

7. The monospecific binding protein of claim 6, wherein the monospecific binding protein binds to the human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with an equilibrium dissociation constant (K D ) is 2.1 nM or less.

8. The monospecific binding protein of claim 5, wherein the monospecific binding protein binds to a human isoform E CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:

105.

9. The monospecific binding protein of claim 5, wherein the monospecific binding protein binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:

30.

10. The monospecific binding protein of claim 9, wherein the monospecific binding protein binds to the cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30 with an equilibrium dissociation constant (K D ) is 1.3 nM or less.

11. The monospecific binding protein of claim 1, wherein the monospecific binding protein is a humanized antibody.

12. The monospecific binding protein of claim 1, wherein the monospecific binding protein is a monoclonal antibody.

13. The monospecific binding protein of claim 1, wherein the monospecific binding protein comprises two full-length antibody heavy chains containing an Fc region.

14. The monospecific binding protein of claim 13, wherein the Fc region is a human IgG1 Fc region.

15. The monospecific binding protein of claim 1, wherein the monospecific binding protein is conjugated to a label selected from the group consisting of a radioisotope, a fluorescent compound, a chemiluminescent compound, and an enzyme.

16. A polynucleotide encoding the monospecific binding protein of any one of claims 1-15.

17. A vector comprising the polynucleotide of claim 16.

18. A host cell comprising the polynucleotide of claim 16.

19. A method of producing a binding protein, the method comprising culturing the host cell of claim 18 so as to produce the monospecific binding protein.

20. The method of claim 19, further comprising recovering the monospecific binding protein from the host cell.

21. A host cell comprising the vector of claim 17.

22. A method of producing a binding protein, the method comprising culturing the host cell of claim 21 so as to produce the monospecific binding protein.

23. The method of claim 22, further comprising recovering the monospecific binding protein from the host cell.

24. A pharmaceutical composition comprising the monospecific binding protein of any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

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