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 may be a monospecific, bispecific, or trispecific binding protein having at least one antigen binding domain that binds to the CD38 polypeptide. The present disclosure also provides methods of making binding proteins that bind to CD38 polypeptides and uses of such binding proteins.

Description

[0001] This invention application is a divisional application based on a patent application with an application date of October 9, 2018, application number 201880079045.9, and invention name “Anti-CD38 antibodies and combinations with anti-CD3 and anti-CD28 antibodies”.

[0002] Cross-reference to related applications

[0003] 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.

[0004] Submit sequence listing as ASCII text file

[0005] The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of the Sequence Listing (File Name: 183952029941seqlist.TXT, Record Date: October 8, 2018, Size: 158KB). Technical Field

[0006] The present disclosure relates to binding proteins that bind 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 CD38 polypeptides, as well as polynucleotides, host cells, methods of production, and methods of use related thereto. Background Art

[0007] Monoclonal antibody-based biotherapeutics have become an important approach for new drug development. Monoclonal antibody technology provides specific targeting to specific cell populations, precise signal delivery and / or payload, and long-lasting biological effects through its Fc function. Antibody engineering efforts have allowed the development of multispecific antibodies that combine the specificities of multiple monoclonal antibodies for various biological applications, expanding the scope of antibody drug development.

[0008] 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, there is a need for therapeutic agents targeting CD38 with different modes of action and / or improved properties, including but not limited to high affinity binding to CD38, cross-reactivity between human and cynomolgus monkey CD38 polypeptides, binding to lymphoma cells (e.g., multiple myeloma large B-cell lymphoma cell lines), and the ability to induce apoptosis and / or antibody-dependent cell-mediated cytotoxicity (ADCC) and T cell-mediated anti-tumor activity. Summary of the Invention

[0009] Provided herein are binding proteins that bind to CD38 polypeptides (e.g., human and cynomolgus monkey 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 the granzyme / perforin mechanism, as seen with anti-CD38 antibodies such as (daratumumab) offers a different mode of action for anti-tumor activity. Furthermore, the ability to bind to both human and cynomolgus monkey CD38 polypeptides allows for easy testing of binding proteins in preclinical toxicology studies, for example, to evaluate their safety profile for subsequent clinical use.

[0010] 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 monkey 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 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, K D 1.5 nM or less, 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) expressed on the surface of a cell, as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell, 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 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; 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, 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 cell surface; and has one or more mutations (e.g., in the Fc region) that result in reduced binding to FcγRI and / or FcγRII compared to the same binding protein without the one or more mutations. For exemplary assays, see Examples 1, 3, and 4. In some embodiments, the KD is measured at 4°C or 25°C.

[0011] In some embodiments, provided herein are trispecific binding proteins that are bound to human CD38 polypeptides. In some embodiments, the trispecific binding protein binds (e.g., simultaneously) CD38 polypeptides (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 CD38 polypeptides. In some embodiments, the trispecific binding protein binds (e.g., simultaneously) CD38 polypeptides (e.g., expressed on the cell surface) and one or two target antigens expressed on the surface of T cells, 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 monkey CD38 polypeptides. In some embodiments, the trispecific binding protein binds to human isoform A and isoform E CD38 polypeptides. In some embodiments, the trispecific binding protein has one or more of the following 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 (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell, as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell, 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 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; 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, 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 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; induces proliferation of T cells (e.g., CD4+ and / or CD8+ T cells); induces Bcl-xL in T cells (e.g., CD4+ and / or CD8+ T cells) expression; inducing apoptosis of CD38+ cells; binding to CD38 expressed on the cell surface and one or more T cell target antigens expressed on the T cell surface; binding to CD38 expressed on the cell surface, CD28 expressed on the T cell surface, and CD3 expressed on the T cell surface; stimulating activation of the T cell receptor; inducing costimulation 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 PBMC; inducing cytokine release (e.g., IFN-γ and / or IL-6) by PBMC 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.

[0012] 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 the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and / or (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 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 CDR-L3 sequence of the amino acid sequence.In some embodiments, the antigen binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and / or (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the VH domain comprises 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 comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). 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.

[0013] In some embodiments, provided herein is a binding protein 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 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 monkey CD38 polypeptide. In some embodiments, the antigen binding site binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1. 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) of 0.05. D ) is 1.3 nM or less.

[0014] 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 comprising one or more mutations that reduce or eliminate Fc receptor binding and / or effector functions of the Fc region. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, according to the EU Index, the human IgG1 Fc region comprises amino acid substitutions 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, according to the EU Index, the human IgG1 Fc region comprises amino acid substitutions 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, 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 a deletion 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 label. In some embodiments, the binding protein is a bispecific binding protein comprising a first antigen binding site that binds to a CD38 polypeptide, and a second antigen binding site. In some embodiments, the binding protein is a trispecific binding protein comprising a first antigen binding site, a second antigen binding site and a third antigen binding site that bind 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.

[0015] 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 monkey 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 monkey 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 monkey 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 monkey CD38 polypeptide, an antigen binding site that binds to a human CD28 polypeptide, and an antigen binding site that binds to a human CD3 polypeptide. 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:

[0016] V L2 -L1-V L1 -L2-C L [I]

[0017] and the second polypeptide chain comprises a structure represented by the formula:

[0018] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0019] and the third polypeptide chain comprises a structure represented by the formula:

[0020] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0021] and the fourth polypeptide chain comprises a structure represented by the formula:

[0022] V L3 -C L [IV]

[0023] in:

[0024] V L1 is the first immunoglobulin light chain variable domain;

[0025] V L2 is the second immunoglobulin light chain variable domain;

[0026] V L3 is the third immunoglobulin light chain variable domain;

[0027] V H1 is the first immunoglobulin heavy chain variable domain;

[0028] V H2 is a second immunoglobulin heavy chain variable domain;

[0029] V H3 is the third immunoglobulin heavy chain variable domain;

[0030] C L is the immunoglobulin light chain constant domain;

[0031] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0032] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0033] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0034] The hinge is connected to the C H1 and C H2 domain of the immunoglobulin hinge region, and

[0035] L1, L2, L3, and L4 are amino acid linkers;

[0036] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair, and

[0037] Wherein: (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 a domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

[0038] (b) the 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

[0039] (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:

[0040] V L2 -L1-V L1 -L2-C L [I]

[0041] and the second polypeptide chain comprises a structure represented by the formula:

[0042] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0043] and the third polypeptide chain comprises a structure represented by the formula:

[0044] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0045] and the fourth polypeptide chain comprises a structure represented by the formula:

[0046] V L3 -C L [IV]

[0047] in:

[0048] V L1 is the first immunoglobulin light chain variable domain;

[0049] V L2 is the second immunoglobulin light chain variable domain;

[0050] V L3 is the third immunoglobulin light chain variable domain;

[0051] V H1 is the first immunoglobulin heavy chain variable domain;

[0052] V H2is a second immunoglobulin heavy chain variable domain;

[0053] V H3 is the third immunoglobulin heavy chain variable domain;

[0054] C L is the immunoglobulin light chain constant domain;

[0055] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0056] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0057] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0058] The hinge is connected to the C H1 and C H2 domain of the immunoglobulin hinge region, and

[0059] L1, L2, L3, and L4 are amino acid linkers;

[0060] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair, and

[0061] Wherein: (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 L1 a 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);

[0062] (b) the V H2The 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 a 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); or

[0063] (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 the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the V H1 The V 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 L1The 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); H1 The V domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and L1 The 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); H2 The V 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 the V L2 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); H2 The V domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and L2 The 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); H3The V 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 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 V domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and L3 The 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 V 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 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 H3The V domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and L3 The 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; 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; 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; H3 The domain comprises the amino acid sequence of SEQ ID NO: 23, and the V L3 The V domain comprises the amino acid sequence of SEQ ID NO: 18; or 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. 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:

[0064] V L2 -L1-V L1 -L2-C L [I]

[0065] and the second polypeptide chain comprises a structure represented by the formula:

[0066] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0067] and the third polypeptide chain comprises a structure represented by the formula:

[0068] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0069] and the fourth polypeptide chain comprises a structure represented by the formula:

[0070] V L3 -C L [IV]

[0071] in:

[0072] V L1 is the first immunoglobulin light chain variable domain;

[0073] V L2 is the second immunoglobulin light chain variable domain;

[0074] V L3 is the third immunoglobulin light chain variable domain;

[0075] V H1 is the first immunoglobulin heavy chain variable domain;

[0076] V H2 is a second immunoglobulin heavy chain variable domain;

[0077] V H3 is the third immunoglobulin heavy chain variable domain;

[0078] C L is the immunoglobulin light chain constant domain;

[0079] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0080] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0081] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0082] The hinge is connected to the C H1 and C H2 domain of the immunoglobulin hinge region, and

[0083] L1, L2, L3, and L4 are amino acid linkers;

[0084] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair, and

[0085] Wherein (a) V H1 The V 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 the V L1 a 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);

[0086] (b) the V H2 The V 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 the V L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46), or

[0087] (c) V H3 The V 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 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). In some embodiments, the V H3The V 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 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). In some embodiments, 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, 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; H2 The 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; 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; H2 The domain comprises the amino acid sequence of SEQ ID NO: 51, wherein 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 L1The domain comprises the amino acid sequence of SEQ ID NO: 54; 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; H2 The 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: 84, and the V L1 The domain comprises the amino acid sequence of SEQ ID NO: 85; H1 The domain comprises the amino acid sequence of SEQ ID NO: 51, 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 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: 84, and the V L1 The V 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 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: 13, and the V L3 The V domain comprises the amino acid sequence of SEQ ID NO: 14; or 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 VH2 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, at least one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments, L1, L2, L3, or L4 are each independently at least one amino acid in length. In some embodiments, (a) L1, L2, L3 and L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58) and GGSGSSGSGG (SEQ ID NO:59); or (b) L1, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58) and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO:58), L2 comprises the sequence TKGPS (SEQ ID NO:57), L3 comprises the sequence S, and L4 comprises the sequence RT; L1 comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO:55), the length of L3 is 0 amino acids, and the length of L4 is 0 amino acids; L1 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), the length of L3 is 0 amino acids, and the length of L4 is 0 amino acids; or L1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), the length of L2 is 0 amino acids, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), and the length of L4 is 0 amino acids. In some embodiments, the hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG4 hinge-C H2 -CH3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 The hinge-C domains of the second and third polypeptide chains 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. H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, and wherein according to EUIndex, the hinge-C H2 -C H3 The domains each comprise amino acid substitutions at positions 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 hinge-C H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, and wherein according to EUIndex, the hinge-C H2 -C H3 The hinge 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 hinge-C domains of the second and third polypeptide chains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1, wherein the amino acid substitutions are L234A, L235A, and P329A. 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, the hinge-C of the second polypeptide chain isH2 -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 the hinge-C of the third polypeptide chain is, according to the EU Index, H2 -C H3 The 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, the hinge-C of the second polypeptide chain is H2 -C H3 The 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; and wherein 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.

[0088] In some embodiments, provided herein is 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:

[0089] V L2 -L1-V L1 -L2-C L[I]

[0090] and the second polypeptide chain comprises a structure represented by the formula:

[0091] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0092] and the third polypeptide chain comprises a structure represented by the formula:

[0093] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0094] and the fourth polypeptide chain comprises a structure represented by the formula:

[0095] V L3 -C L [IV]

[0096] in:

[0097] V L1 is the first immunoglobulin light chain variable domain;

[0098] V L2 is the second immunoglobulin light chain variable domain;

[0099] V L3 is the third immunoglobulin light chain variable domain;

[0100] V H1 is the first immunoglobulin heavy chain variable domain;

[0101] V H2 is a second immunoglobulin heavy chain variable domain;

[0102] V H3 is the third immunoglobulin heavy chain variable domain;

[0103] C L is the immunoglobulin light chain constant domain;

[0104] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0105] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0106] C H3Immunoglobulin C H3 heavy chain constant domain;

[0107] The hinge is connected to the C H1 and C H2 domain of the immunoglobulin hinge region, and

[0108] L1, L2, L3, and L4 are amino acid linkers;

[0109] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair, and

[0110] 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 EUIndex, the hinge-C H2 -C H3 The hinge-C domains each comprise amino acid substitutions at positions 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 V L1 、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, and CLEC14. 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 forms an antigen binding site that binds to human CD3 polypeptide, 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.

[0111] 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: (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.

[0112] 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.

[0113] In some embodiments, a host cell is provided herein comprising a kit, polynucleotide, or vector of the polynucleotide class of any one of the above embodiments. In some embodiments, a method of producing a binding protein is provided herein, 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.

[0114] 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.

[0115] 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 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 surface. In some embodiments, cancer cells express CD38 and CD28. In some embodiments, cancer cells express CD38 but do not express CD28.

[0116] In some embodiments, provided herein is at least one binding protein of any one of the above embodiments or a pharmaceutical composition of any one of the above embodiments, for use in preventing and / or treating cancer in a patient (e.g., a patient in need thereof, such as a patient with cancer). In some embodiments, provided herein is at least one binding protein of any one of the above embodiments or a pharmaceutical composition of any one of the above embodiments, for use in the preparation of 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 comprising a first antigen binding site in conjunction with CD3, a second antigen binding site in conjunction with CD28, and a third antigen binding site in conjunction with the extracellular domain of a human CD38 polypeptide. In some embodiments, the at least one binding protein is co-administered with a chemotherapeutic agent. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma. In some embodiments, the patient is human. In some embodiments, the patient is selected for treatment because the cancer cells express human CD38 isoform E polypeptide (e.g., as 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.

[0117] 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.

[0118] The present invention comprises:

[0119] 1. A binding protein comprising an antigen binding site that binds to a CD38 polypeptide, wherein the antigen binding site comprises:

[0120] (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

[0121] (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0122] 2. The binding protein of claim 1 , wherein the antigen binding site comprises:

[0123] (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and

[0124] (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0125] 3. The binding protein of claim 1 or 2, wherein 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: NO:94); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO:96).

[0126] 4. The binding protein of item 1 or item 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 5, and the VL domain comprises the amino acid sequence of SEQ ID NO: 6.

[0127] 5. The binding protein of item 4, wherein 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.

[0128] 6. The binding protein of item 1 or item 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 17, and the VL domain comprises the amino acid sequence of SEQ ID NO: 18.

[0129] 7. The binding protein of item 6, wherein 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.

[0130] 8. The binding protein of item 1 or item 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 21, and the VL domain comprises the amino acid sequence of SEQ ID NO: 18.

[0131] 9. The binding protein of item 8, wherein 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.

[0132] 10. The binding protein of item 1 or item 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 23, and the VL domain comprises the amino acid sequence of SEQ ID NO: 18.

[0133] 11. The binding protein of claim 10, wherein 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.

[0134] 12. The binding protein of claim 1 , wherein the antigen binding site comprises:

[0135] (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and

[0136] (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0137] 13. The binding protein of claim 1 or 12, wherein 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: ID NO:94); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO:96).

[0138] 14. The binding protein of item 1 or item 12, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 13, and the VL domain comprises the amino acid sequence of SEQ ID NO: 14.

[0139] 15. The binding protein of claim 14, wherein 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.

[0140] 16. A binding protein comprising an antigen binding site that binds a CD38 polypeptide, wherein the antigen binding site comprises:

[0141] (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

[0142] (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).

[0143] 17. The binding protein of claim 16, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 9, and the VL domain comprises the amino acid sequence of SEQ ID NO: 10.

[0144] 18. The binding protein of item 16 or item 17, wherein 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.

[0145] 19. The binding protein of any one of items 1 to 18, wherein the antigen binding site cross-reacts with the extracellular domain of human CD38 polypeptide and the extracellular domain of cynomolgus monkey CD38 polypeptide.

[0146] 20. The binding protein of claim 19, wherein the antigen binding site binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0147] 21. The binding protein of item 20, wherein 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.

[0148] 22. The binding protein of claim 19, wherein the antigen binding site binds to a human isoform E CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 105.

[0149] 23. The binding protein of any one of items 19-22, wherein the antigen binding site binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30.

[0150] 24. The binding protein of claim 23, wherein 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.

[0151] 25. The binding protein of any one of items 1-24, wherein the binding protein is a chimeric or humanized antibody.

[0152] 26. The binding protein of any one of items 16-24, wherein the binding protein is a human antibody.

[0153] 27. The binding protein of any one of items 1 to 24, wherein the binding protein is a monoclonal antibody.

[0154] 28. The binding protein of any one of items 1-24, wherein the binding protein comprises one or more full-length antibody heavy chains containing an Fc region.

[0155] 29. The binding protein of claim 28, wherein the Fc region is a human Fc region comprising one or more mutations that reduce or eliminate Fc receptor binding and / or effector function of the Fc region.

[0156] 30. The binding protein of claim 28, wherein the Fc region is a human IgG1 Fc region.

[0157] 31. The binding protein of claim 30, wherein the human IgG1 Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A.

[0158] 32. The binding protein of claim 30, wherein the human IgG1 Fc region comprises amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1 according to the EU Index, wherein the amino acid substitutions are S298N, T299A and Y300S.

[0159] 33. The binding protein of item 28, wherein the Fc region is a human IgG4 Fc region.

[0160] 34. The binding protein of claim 33, wherein the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU Index, wherein the amino acid substitutions are S228P and R409K.

[0161] 35. The binding protein of item 33 or item 34, wherein the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU Index, wherein the amino acid substitutions are F234A and L235A.

[0162] 36. The binding protein of item 33 or item 34, wherein the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU Index, wherein the amino acid substitutions are E233P, F234V, L235A and a deletion at position 236.

[0163] 37. The binding protein of any one of items 1-24, wherein the binding protein comprises an antibody F(ab), F(ab')2, Fab'-SH, Fv or scFv fragment.

[0164] 38. The binding protein of any one of items 1 to 37, wherein the binding protein is conjugated to a cytotoxic agent or a label.

[0165] 39. The binding protein of any one of items 1-37, wherein 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.

[0166] 40. The binding protein of any one of items 1-37, wherein 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 bind to a CD38 polypeptide.

[0167] 41. The binding protein of claim 40, wherein the first antigen binding site binds to the extracellular domain of the human CD38 polypeptide, and wherein the second and third antigen binding sites each bind to a T cell surface protein.

[0168] 42. The binding protein of claim 41, wherein 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.

[0169] 43. A binding protein comprising three antigen binding sites, each of which binds to one or more target proteins, wherein at least one of the three antigen binding sites cross-reacts with the extracellular domain of human CD38 polypeptide and the extracellular domain of cynomolgus monkey CD38 polypeptide.

[0170] 44. The binding protein of claim 43, wherein the binding protein cross-reacts with a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 105.

[0171] 45. The binding protein of item 43 or item 44, wherein the binding protein cross-reacts with a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 30.

[0172] 46. ​​The binding protein of any one of items 43-45, wherein the binding protein comprises an antigen binding site that cross-reacts with the extracellular domain of human CD38 polypeptide and the extracellular domain of cynomolgus monkey CD38 polypeptide, and two antigen binding sites that each bind to a T cell surface protein.

[0173] 47. The binding protein of claim 46, wherein the binding protein comprises an antigen binding site that cross-reacts with the extracellular domain of human CD38 polypeptide and the extracellular domain of cynomolgus monkey CD38 polypeptide, an antigen binding site that binds to human CD28 polypeptide, and an antigen binding site that binds to human CD3 polypeptide.

[0174] 48. The binding protein of items 43-47, wherein 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:

[0175] V L2 -L1-V L1 -L2-C L [I]

[0176] and the second polypeptide chain comprises a structure represented by the formula:

[0177] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0178] and the third polypeptide chain comprises a structure represented by the formula:

[0179] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0180] and the fourth polypeptide chain comprises a structure represented by the formula:

[0181] V L3 -C L [IV]

[0182] in:

[0183] V L1 is the first immunoglobulin light chain variable domain;

[0184] V L2 is the second immunoglobulin light chain variable domain;

[0185] V L3 is the third immunoglobulin light chain variable domain;

[0186] V H1 is the first immunoglobulin heavy chain variable domain;

[0187] V H2 is a second immunoglobulin heavy chain variable domain;

[0188] VH3 is the third immunoglobulin heavy chain variable domain;

[0189] C L is the immunoglobulin light chain constant domain;

[0190] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0191] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0192] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0193] The hinge is connected to the C H1 and C H2 domain of the immunoglobulin hinge region, and

[0194] L1, L2, L3, and L4 are amino acid linkers;

[0195] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair, and

[0196] in:

[0197] (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 L1 a domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

[0198] (b) the V H2The 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 a domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or

[0199] (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 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).

[0200] 49. The binding protein of item 48, wherein

[0201] (a) V H1 The V 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 the V L1the 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);

[0202] (b) the V H1 The V domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and L1 the domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

[0203] (c) V H2 The V 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 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);

[0204] (d) the V H2 The V domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and L2the domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

[0205] (e) V H3 The V 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 the V L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or

[0206] (h) V H3 The V domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0207] 50. The binding protein of item 49, wherein

[0208] (a) V H3 The V 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 the V L3The 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

[0209] (b) the V H3 The V domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0210] 51. The binding protein of item 50, wherein

[0211] (a) 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;

[0212] (b) 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;

[0213] (c) 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;

[0214] (d) 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

[0215] (e) 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.

[0216] 52. The binding protein of items 43-47, wherein 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:

[0217] V L2 -L1-V L1 -L2-C L [I]

[0218] and the second polypeptide chain comprises a structure represented by the formula:

[0219] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0220] and the third polypeptide chain comprises a structure represented by the formula:

[0221] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0222] and the fourth polypeptide chain comprises a structure represented by the formula:

[0223] V L3 -C L [IV]

[0224] in:

[0225] V L1 is the first immunoglobulin light chain variable domain;

[0226] V L2 is the second immunoglobulin light chain variable domain;

[0227] V L3 is the third immunoglobulin light chain variable domain;

[0228] V H1 is the first immunoglobulin heavy chain variable domain;

[0229] V H2 is a second immunoglobulin heavy chain variable domain;

[0230] V H3 is the third immunoglobulin heavy chain variable domain;

[0231] C Lis the immunoglobulin light chain constant domain;

[0232] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0233] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0234] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0235] The hinge is connected to the C H1 and C H2 domain of the immunoglobulin hinge region, and

[0236] L1, L2, L3, and L4 are amino acid linkers;

[0237] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair, and

[0238] in:

[0239] (a) V H1 The V 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 the V L1 a 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);

[0240] (b) the V H2 The V 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 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

[0241] (c) V H3 The V 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 the 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).

[0242] 53. The binding protein of item 52, wherein said V H3 The V 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 the 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).

[0243] 54. The binding protein of item 53, wherein said 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.

[0244] 55. The binding protein of items 48-54, wherein

[0245] (a) V H1 The domain comprises the amino acid sequence of SEQ ID NO: 49, wherein the V L1The 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;

[0246] (b) the V H2 The 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;

[0247] (c) 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;

[0248] (d) the V H2 The domain comprises the amino acid sequence of SEQ ID NO: 51, wherein 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;

[0249] (e) 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;

[0250] (f) V H2 The 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 H1The 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;

[0251] (g) 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 domain comprises the amino acid sequence of SEQ ID NO: 85; or

[0252] (h) V H2 The domain comprises the amino acid sequence of SEQ ID NO: 51, wherein 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: 84, and the V L1 The domain comprises the amino acid sequence of SEQ ID NO:85.

[0253] 56. The binding protein of item 55, wherein

[0254] (a) 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: 13, and the V L3 The domain comprises the amino acid sequence of SEQ ID NO: 14; or

[0255] (b) 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 L3The domain comprises the amino acid sequence of SEQ ID NO:10.

[0256] 57. The binding protein of any one of items 48-56, wherein at least one of L1, L2, L3 or L4 is independently 0 amino acids in length.

[0257] 58. The binding protein of any one of items 48-56, wherein the length of L1, L2, L3 or L4 is each independently at least one amino acid.

[0258] 59. The binding protein of any one of items 48-56, wherein (a) L1, L2, L3 and L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59); or (b) L1, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59).

[0259] 60. The binding protein of any one of items 48-56, wherein

[0260] (a) L1 comprises the sequence GQPKAAP (SEQ ID NO: 58), L2 comprises the sequence TKGPS (SEQ ID NO: 57), L3 comprises the sequence S, and L4 comprises the sequence RT;

[0261] (b) L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 55), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 55), L3 is 0 amino acids in length, and L4 is 0 amino acids in length;

[0262] (c) L1 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; or

[0263] (d) L1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), and L4 is 0 amino acids in length.

[0264] 61. The binding protein of any one of items 48-60, wherein the hinge-C 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 amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4, wherein the amino acid substitutions are F234A and L235A.

[0265] 62. The binding protein of any one of items 48-60, wherein 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 EU Index, the hinge-C H2 -C H3 The domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236.

[0266] 63. The binding protein of any one of items 48-60, wherein 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 EU Index, the hinge-C H2 -C H3 The domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4, wherein the amino acid substitutions are S228P and R409K.

[0267] 64. The binding protein of any one of items 48-60, wherein 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 EU Index, the hinge-CH2 -C H3 The domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG4, wherein the amino acid substitutions are L234A, L235A, and P329A.

[0268] 65. The binding protein of any one of items 48-60, wherein the hinge-C 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 amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG4, wherein the amino acid substitutions are S298N, T299A, and Y300S.

[0269] 66. The binding protein of any one of items 48-65, wherein 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 the hinge-C of the third polypeptide chain is, according to the EU Index. H2 -C H3 The 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.

[0270] 67. The binding protein of any one of items 48-65, wherein according to the EU Index, the hinge-C H2 -C H3 The 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; and wherein 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.

[0271] 68. The binding protein of item 48 or item 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:62, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63.

[0272] 69. The binding protein of item 48 or item 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:65, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63.

[0273] 70. The binding protein of item 48 or item 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63.

[0274] 71. The binding protein of item 48 or item 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69.

[0275] 72. The binding protein of item 48 or item 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:70, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69.

[0276] 73. The binding protein of item 48 or item 52, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69.

[0277] 74. 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 the three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0278] V L2 -L1-V L1 -L2-C L [I]

[0279] and the second polypeptide chain comprises a structure represented by the formula:

[0280] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0281] and the third polypeptide chain comprises a structure represented by the formula:

[0282] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0283] and the fourth polypeptide chain comprises a structure represented by the formula:

[0284] V L3 -C L [IV]

[0285] in:

[0286] V L1 is the first immunoglobulin light chain variable domain;

[0287] V L2 is the second immunoglobulin light chain variable domain;

[0288] V L3 is the third immunoglobulin light chain variable domain;

[0289] V H1 is the first immunoglobulin heavy chain variable domain;

[0290] V H2 is a second immunoglobulin heavy chain variable domain;

[0291] V H3 is the third immunoglobulin heavy chain variable domain;

[0292] C L is the immunoglobulin light chain constant domain;

[0293] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0294] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0295] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0296] The hinge is connected to the C H1 and C H2 The immunoglobulin hinge region of the domain; and

[0297] L1, L2, L3, and L4 are amino acid linkers;

[0298] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair; and

[0299] in:

[0300] (a) 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 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

[0301] (b) hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG4 C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 The domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236.

[0302] 75. The binding protein of item 74, wherein V H1 and V L1 、V H2 and V L2 and V H3 and V L3 At least one pair of them forms an antigen binding site that binds to the CD38 polypeptide.

[0303] 76. The binding protein of item 74, wherein V H1 and V L1 、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, and CLEC14. 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.

[0304] 77. The binding protein of item 74, wherein V H1 and V L1 、V H2 and V L2 and V H3 and V L3 The first pair forms an antigen binding site that binds to human CD3 polypeptide, 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.

[0305] 78. A polynucleotide kit comprising:

[0306] (a) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 72, a third polynucleotide comprising the sequence of SEQ ID NO: 74, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 75;

[0307] (b) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 76, a third polynucleotide comprising the sequence of SEQ ID NO: 77, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 75;

[0308] (c) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 78, a third polynucleotide comprising the sequence of SEQ ID NO: 79, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 75;

[0309] (d) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 72, a third polynucleotide comprising the sequence of SEQ ID NO: 80, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 81;

[0310] (e) a first polynucleotide comprising the sequence of SEQ ID NO: 73, a second polynucleotide comprising the sequence of SEQ ID NO: 76, a third polynucleotide comprising the sequence of SEQ ID NO: 82, and a fourth polynucleotide comprising the sequence of SEQ ID NO: 81; or

[0311] (f) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:78, a third polynucleotide comprising the sequence of SEQ ID NO:83, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81.

[0312] 79. A polynucleotide encoding the binding protein of any one of items 1-77.

[0313] 80. A vector comprising the polynucleotide of item 79.

[0314] 81. A host cell comprising a kit of polynucleotides of item 78, a polynucleotide of item 79 or a vector of item 80.

[0315] 82. A method for producing a binding protein, the method comprising culturing the host cell of item 81 to produce the binding protein.

[0316] 83. The method of claim 82, further comprising recovering the binding protein from the host cell.

[0317] 84. A pharmaceutical composition comprising the binding protein of any one of items 1-77 and a pharmaceutically acceptable carrier.

[0318] 85. 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 items 1-73 or the pharmaceutical composition of item 84.

[0319] 86. The method of claim 85, wherein the binding protein comprises one antigen binding site that binds to a T cell surface protein and another antigen binding site that binds to the extracellular domain of a human CD38 polypeptide.

[0320] 87. The method of claim 86, wherein the binding protein is a trispecific binding protein comprising a first antigen binding site that binds to CD3, a second antigen binding site that binds to CD28, and a third antigen binding site that binds to the extracellular domain of a human CD38 polypeptide.

[0321] 88. The method of any one of items 85-87, wherein at least one binding protein is co-administered with a chemotherapeutic agent.

[0322] 89. The method of any one of items 85-88, wherein the cancer is multiple myeloma.

[0323] 90. The method of any one of items 85-88, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma.

[0324] 91. The method of any one of items 85-90, wherein the patient is a human.

[0325] 92. The method of any one of items 85-91, wherein the patient is selected for treatment because cells of the cancer express human CD38 isoform E polypeptide on their cell surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0326] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0327] Figure 1ABinding of the anti-CD38 antibody mAb1 (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.

[0328] 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.

[0329] Figure 2A-Figure 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") is shown by ELISA, and 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 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") is shown by ELISA, and 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 2GBinding of the human anti-CD38 antibody hhy1370 to soluble human CD38 (upper panel, "hCD38::Histag") or cynomolgus CD38 (upper panel, "cynoCD38::Histag") by ELISA is shown, as is 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. Figure 2H Binding of hhy1370 to human CD38 (upper panel) or cynomolgus monkey CD38 (lower panel) is shown by SPR. Figure 2I Binding 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 the last row of mAb6 (1195HHKK-3 is not described below by its VL / VH amino acid sequence).

[0330] Figure 2J Shown is the concentration-dependent induction of apoptosis in SU-DHL-8 cells by mAb7 and mAb1 after 72 h of incubation at 37°C.

[0331] Figure 2K Shown are the antibody-dependent cell-mediated cytotoxicity (ADCC) activities of isatuximab and mAb1 against SU-DHL-8 cells in the presence of NK92 cells.

[0332] 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.

[0333] Figure 2M-2Q Shown are the results of apoptosis induction assays using the indicated anti-CD38 antibodies against SU-DHL-8 tumor cells. Apoptosis was quantified by measuring dual Annexin V and propidium iodide uptake by flow cytometry. Figure 2M The percentage of apoptotic cells induced by each antibody is shown. Figure 2N-2Q Anti-CD38 antibody mAb2 ( Figure 2N )、mAb3( Figure 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.

[0334] Figure 3AA schematic diagram of a trispecific binding protein comprising four polypeptide chains that form three antigen-binding sites for 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 single variable domains (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.

[0335] 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.

[0336] Figure 3C Shown are the results of an SPR-based assay for examining CD38 binding to an anti-CD38 / anti-CD28 / anti-CD3 trispecific binding protein. 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).

[0337] Figure 4 Shown are sequential binding of human CD3, CD28, and CD38 polypeptides to the anti-CD38 / anti-CD28 / anti-CD3 trispecific binding protein as determined by SPR.

[0338] 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.

[0339] Figure 6A The apparent affinities of the isatuximab antigen-binding domain in human IgG1 format (second sheet) or trispecific binding protein format were compared with those of the isatuximab, anti-CD28, and anti-CD3 antigen-binding domains (according to Figure 3A 1st sheet) for binding of human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide, as determined by flow cytometry.

[0340] Figure 6B-6D Comparison of trispecific binding protein CD38 VH1 xCD28 sup xCD3mid 、CD38 VH1 xCD28 cvn xCD3 mid Or the apparent affinity of the monospecific anti-CD38 antibody mAb2 binding 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 CD38 VH1 xCD28 cvn xCD3 mid Binding to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide. Figure 6D Binding of the monospecific anti-CD38 antibody mAb2 to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide is shown.

[0341] Figure 6E Comparison of 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.

[0342] Figure 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).

[0343] 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.

[0344] Figure 7A and Figure 7B Shown are the results of ELISA assays determining the binding affinity of various anti-CD38 x CD28 x CD3 IgG4 trispecific binding proteins or control antibodies to human and rhesus CD3, CD28, and CD38 polypeptides.

[0345] Figures 8A-8DAntibody-mediated CD38 knockdown of PBMCs from three different human donors using the indicated anti-CD38 x CD28 x CD3 trispecific binding proteins and control antibodies are shown. + Results of cell-specific killing were shown using the multiple myeloma cell line RPMI8266 ( Figure 8A )、NCI-H929( Figure 8B )、KMS-26( Figure 8C ) and KMS-11 cell lines ( Figure 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.

[0346] Figure 8E and Figure 8F Antibody-mediated CD38 expression in 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 is shown. + Results of cell-specific killing were shown using CD38 + KMS-11( Figure 8D ) and U266( Figure 8E ) cell lines, and EC50 values ​​are provided in Tables Q2 and Q3.

[0347] Figure 9A 、 Figure 9B and Figure 10 Activation of human T cells (CD69 + ). Figure 9A Display human CD3 + T cell activation (CD69 + ). Figure 9B Display human CD3 + CD4 + T cell activation (CD69 + ). Figure 10 Display human CD3 + CD8 + T cell activation (CD69 + ).

[0348] Figures 11A-11BShown are the results of in vitro cytokine release assessment of human PBMCs treated with the indicated anti-CD38 x CD28 x CD3 trispecific binding proteins or a control antibody based on the dry plate method as described in Stebbings, R. et al. (2007) J. Immunol. 179: 3325-3331. Figure 11A Results using 5 μg / mL of the indicated antibodies are shown. Figure 11B Results using 25 ng / mL of the indicated antibodies are shown.

[0349] Figures 12A-12E Anti-CD38 expression in a humanized NSG mouse model of CD34+ umbilical cord blood cells engrafted with RPMI-8226 multiple myeloma cells was demonstrated. (VHI) x CD28 (sup) x CD3 (mid) In vivo activity of trispecific binding proteins. Figure 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. Figure 12B CD38 was used at the indicated concentrations. (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. Figure 12C Anti-CD38 (VHI) x CD28 (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. Figure 12D Anti-CD38 (VHI) xCD28 (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. Figure 12E The cells were treated with the indicated concentrations of 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.

[0350] Figures 13A-13F Anti-CD38 in a PBMC humanized NSG mouse model engrafted with RPMI-8226 multiple myeloma cells was shown. (VHI) x CD28 (sup) x CD3 (mid) In vivo activity of trispecific binding proteins. Figure 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. Figure 13B Anti-CD38 (VHI) x CD28 (sup) xCD3 (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. Figure 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. Figure 13D Anti-CD38 (VHI) x CD28 (sup) xCD3 (mid) Mean tumor volume at day 21 in mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody. Figure 13E Anti-CD38 (VHI) x CD28 (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. Figure 13F Anti-CD38 (VHI) x CD28 (sup) xCD3 (mid) Mean tumor volumes of mice treated with the trispecific binding protein relative to mice treated with the anti-CD3 / CD38 bispecific antibody at various time points over the length of the experiment.

[0351] Figures 14A-14U showed the use of anti-CD38 in nonhuman primates (VHI) x CD28 (sup) xCD3 (mid) , anti-CD38 (VHI) x CD28(cvn) x CD3 (mid) , anti-CD38 (hhy1370) x CD28 (sup) xCD3 (mid) and anti-CD38 (hhy1370) xCD28 (cvn) x CD3 (mid) Results of a dose-escalation study of a trispecific binding protein. Figure 14A The results showed that different doses of anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) trispecific binding protein post-circulating CD3 + T cell activation (CD69 + ). Figure 14B The results showed that different doses of anti-CD38 (VHI) x CD28 (cvn) x CD3 (mid) trispecific binding protein post-circulating CD3 + T cell activation (CD69 + ). Figure 14C Different doses of anti-CD38 (hhy1370) x CD28 (sup) xCD3 (mid) trispecific binding protein post-circulating CD3 + T cell activation (CD69 + ). Figure 14D The results showed that different doses of anti-CD38 (hhy1370) x CD28 (cvn) x CD3 (mid) trispecific binding protein post-circulating CD3 + T cell activation (CD69 + ). Figure 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 percentages. Figure 14F Anti-CD38 was administered at the indicated doses. (VHI) x CD28 (sup) xCD3 (mid) trispecific binding protein post-circulating CD8 + Changes in T cell percentages. Figure 14G Anti-CD38 was used at the indicated doses. (VHI) x CD28 (cvn) x CD3 (mid)trispecific binding protein post-circulating CD4 + Changes in T cell percentages. Figure 14H Anti-CD38 was administered at the indicated doses. (VHI) x CD28 (cvn) xCD3 (mid) trispecific binding protein post-circulating CD8 + Changes in T cell percentages. Figure 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 percentages. Figure 14J Anti-CD38 was administered at the indicated doses. (hhy1370) xCD28 (sup) x CD3 (mid) trispecific binding protein post-circulating CD8 + Changes in T cell percentages. Figure 14K Anti-CD38 was administered at the indicated doses. (hhy1370) x CD28 (cvn) x CD3 (mid) trispecific binding protein post-circulating CD4 + Changes in T cell percentages. 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 T cell percentages. Figure 14M Total CD4 T cells were expressed 6, 24, and 48 hours after administration of 12.5 μg / kg of the indicated trispecific binding proteins. + Changes in T cells. Figure 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. Figure 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) xCD28 (sup) xCD3 (mid)Changes in cytokine levels 6 hours after administration of the 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) x CD28 (cvn) x CD3 (mid) Changes in cytokine levels 6 hours after administration of the 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 (hhy1370) xCD28 (sup) x CD3 (mid) Changes in cytokine levels 6 hours after administration of the 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 (hhy1370) x CD28 (cvn) x CD3 (mid) Changes in cytokine levels 6 hours after administration of the 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).

[0352] Figure 14V and Figure 14W Anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) and anti-CD38 (VHI) xCD28 (cvn) xCD3 (mid) The trispecific binding protein induced T cell depletion in vivo (6 hours post-dose) in non-human primate blood at higher doses.

[0353] Figure 14X and Figure 14Y Anti-CD38 (HHY1370) x CD28 (sup) x CD3 (mid) and anti-CD38 (HHY1370 x CD28 (cvn) x CD3 (mid)The trispecific binding protein induced T cell depletion in vivo (6 hours post-dose) in non-human primate blood at higher doses.

[0354] Figure 14Z and Figure 14AA showed that administration of anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) or anti-CD38 (VHI) x CD28 (cvn) x CD3 (mid) The amount of blood T cells in nonhuman primates changes over time after the administration of trispecific binding proteins.

[0355] Figure 14AB and Figure 14AC showed that administration of 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 the administration of trispecific binding proteins.

[0356] Figure 14AD and Figure 14AE Shown are the amounts of CD4+ T cells with bound trispecific binding protein following a 100 μg / kg dose in non-human primates.

[0357] Figure 14AF and Figure 14AG Shown are the amounts of CD8+ T cells with bound trispecific binding protein following a 100 μg / kg dose in non-human primates.

[0358] Figures 15A-15C Multiple Fc variants were shown to bind to the human Fc receptor FcγR I ( Figure 15A )、FcγR IIa( Figure 15B ) and FcγR IIIb / c( Figure 15C The variants tested were human IgG1, human IgG4, and human IgG4 with FALA mutations.

[0359] Figure 16 Binding of human IgG4 with or without FALA mutation to FcRn is shown.

[0360] Figure 17 A summary of the trispecific binding proteins shown in NSG mice (CD38 VH1 xCD28 sup xCD3 mid IgG4, CD38 VH1xCD28 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).

[0361] Figures 18A-18C Figure 3 shows (nonspecific) IFN-γ mediated by Fc / FcR interactions of human PBMC incubated with trispecific heterobinding proteins bearing wild-type or FALA variant Fc regions. Figure 18A ), IL-2( Figure 18B ) or TNF-α( Figure 18C )’s release.

[0362] Figure 18D Display CD38 VH1 xCD28 sup xCD3 mid and CD38 HHY1370 xCD28 sup xCD3 mid In vitro activation of human PBMCs by the trispecific binding protein and its IgG1 and IgG4 Fc variants.

[0363] Figure 19A and Figure 19B trispecific binding protein CD38 VH1 xCD28 sup xCD3 mid In CD4+( Figure 19A ) or CD8+( Figure 19B ) Induction of Bcl-xL in T cells requires both CD3 and CD28 antigen binding domains. Bar graph = mean and sd from 3 PBMC donors. *p= < 0.009.

[0364] Figure 19C and Figure 19D CD38 with IgG4 FALA variant Fc was shown VH1 xCD28xCD3 upregulated CD4+ ( Figure 19C ) or CD8+( Figure 19D )Bcl-xL in T cells.

[0365] Bar graph = mean and sd from 3 PBMC donors. *p= < 0.045

[0366] Figure 19E T cell activation of the anti-CD38xanti-CD28xanti-CD3 trispecific binding protein, as measured by IL-2 expression in a Jurkat T cell reporter cell line, was shown to be dependent on the anti-CD3 antigen binding domain.

[0367] Figure 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.

[0368] Figure 19G Shown are the proliferation of T cells activated by the anti-CD38xanti-CD28xanti-CD3 trispecific binding protein with IgG4 FALA variant Fc, the benchmark anti-CD38xanti-CD3 bispecific antibody, or an isotype control (an IgG4 FALA variant Fc trispecific binding protein with mutant binding domains).

[0369] Figure 20 Shown are the proliferation 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).

[0370] Figure 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.

[0371] Figure 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.

[0372] Figure 23A and Figure 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 with the trispecific binding protein and a benchmark anti-CD38xanti-CD3 bispecific antibody Human PBMCs from two donor humanized NSG mice were used after 24 hours of incubation at an effector:PBMC ratio of 10:1.

[0373] Figure 23C Shown is the effect 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 mid Excellent in vivo antitumor activity of the trispecific binding protein. The binding protein was administered by weekly intraperitoneal (IP) injection at 30 μg / kg.

[0374] Figure 24A Display using GloResponse TM IL2-luc2P Jurkat cells (Promega) in CD38 VH1 / CD28 sup xCD3 mid Results of luciferase reporter gene assays after stimulation with single binding site KO and triple KO mutants at 10 nM concentration.

[0375] Figure 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 in an in vitro cytokine release assay using human PBMCs. The distal CD28 x proximal CD3 positioning was determined to be optimal based on IFN-γ and IL-2 secretion in the supernatant after 24 hours.

[0376] Figure 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.

[0377] Figure 26 We show that the anti-CD28 component of the trispecific Ab provides the secondary signaling necessary to support primary T cell proliferation in vitro.

[0378] Figure 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).

[0379] Figure 28A and Figure 28B Shown to have high (RPMI-8226; Figure 28A ) and low (KMS-11; Figure 28B ) CD38 surface-expressing multiple myeloma (MM) cells were efficiently lysed by human PBMC incubated with various concentrations of 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.

[0380] Figures 29A-29C Anti-CD28 displaying trispecific Ab sup KO mutants inhibit CD38 in vitro high 、CD38 mid and CD38 low MM cells showed significantly reduced antitumor activity. Figure 29A )、U266( Figure 29B ) or KMS-11( Figure 29C ) cell determination.

[0381] Figure 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.

[0382] Figure 31A and Figure 31BFigure 3 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 were incubated with RPMI-8226 myeloma cell line labeled with deep red dye, and negative controls (triple KO trispecific; Figure 31A ) or CD38 / CD28xCD3 trispecific Ab ( Figure 31B ) Time-lapse microscopy images were taken. Images presented were collected after 24 hours of incubation. Scale bar: 50 μm.

[0383] Figure 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).

[0384] Figure 33 Shown are the results of SPR assays measuring the affinity of the indicated IgG4 Fc variants to the indicated human Fc receptors.

[0385] Figure 34 The CD38 trispecific binding protein with minimal FcR binding was shown to reduce nonspecific cytokine release in human PBMCs in vitro. The proinflammatory effects of different FcR inactivating mutations (as shown) 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 graph represents the supernatant IFN-γ levels measured by ELISA.

[0386] Figure 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 harboring IgG4 or the indicated Fc mutations was assessed in vitro using human PBMCs harboring the indicated tumor targets.

[0387] Figure 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 against the cell lines RPMI-8226, U266, and KMS-11 (E:T=10:1) measured using human PBMCs as effector cells.

[0388] Figures 37A-37DCharacterization of in vitro T cell subset expansion in response to CD38 / CD3xCD28 was shown. Assessment of T cell subset expansion was performed by coating wells with 350ng / well of CD38 trispecific Ab in the absence of exogenous cytokines. T cell colonies were measured at the indicated time points. Triple mutant trispecific Ab was used as a negative control. Flow cytometry was used to determine the central and effector memory CD4 T cells ( Figure 37A ), helper T cells ( Figure 37B ), central and effector memory CD8 T cells ( Figure 37C ) and CMV pp65-specific CD8 cells ( Figure 37D CMV-specific pp65 effector cells were analyzed by pentamer staining of PBMCs from HLA-A2 CMV+ donors treated with CD38 trispecific or triple-negative control.

[0389] Figure 38 Show that CD28 expression on target cells is to the contribution of the susceptibility of cytolysis by CD38 / CD3xCD28.Knock out CD28 in KMS-11 cells using CRISPR / Cas 9 gene targeting, and use it as cytolysis target in vitro.Compared with parent KMS-11, CD38 expression is retained, and CD28 is eliminated, as confirmed by flow cytometry (upper figure, KMS-11 is relative to KMS-11 (CD28KO)).With WT or CD28 invalid three specificity check CD28KO cell cytolysis (lower figure; Three specificity is relative to three specificity (CD28KO)).

[0390] Figure 39 CD38 / CD28xCD3 trispecific FALA mutant Abs are shown for the indicated CD38 + CD28 - The cytolytic activity of the CD38 + CD28 - Lines 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)).

[0391] Figure 40 It was shown that in vitro activation of human PBMCs by an α-CD28 superagonist requires bivalency of the antibody. DETAILED DESCRIPTION

[0392] The present disclosure provides binding proteins comprising at least one antigen binding site that binds a CD38 polypeptide.

[0393] I. General Definition

[0394] As used in accordance with this 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 so forth.

[0395] 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.

[0396] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer having a length of at least 10 nucleotides. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or modified forms 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, selenophosphate, diselenophosphate, phosphoroanilothioate, phosphoranilidate, and phosphoramidate. The term "polynucleotide" specifically includes DNA in single-stranded and double-stranded forms.

[0397] 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.

[0398] 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 with which it is not naturally associated, or (7) does not exist in nature. Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA, or other RNA of a synthetic origin, 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).

[0399] 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 the 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 is 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, the full-length heavy chain immunoglobulin polypeptide includes variable domains (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.

[0400] Human light chains are generally classified as kappa and lambda light chains, and human heavy chains are generally classified as μ, δ, γ, α or ε, 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 generally 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, FUNDAMENTAL IMMUNOLOGY (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 typically 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 typically aligned by the framework regions, which can enable binding to specific epitopes. From amino terminus to carboxyl terminus, the light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0401] The term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides 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 certain subportions of the Kabat CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These subportions are designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" denote the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs. CDRs that have boundaries that overlap with Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat 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 CDRs defined by Kabat or Chothia. Identifying predicted CDRs using amino acid sequences is well established 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)) is well known.The amino acid sequence of the heavy chain and / or light chain variable domain can also be inspected 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 region). The numbered sequences can be aligned by eye or by employing an alignment program (such as one of the CLUSTAL program suites), as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular modeling is typically used to correctly depict framework and CDR regions, and therefore sequence-based allocation is corrected.

[0402] 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).

[0403] As used herein, term " Fc " refers to a molecule comprising a sequence of non-antigen binding fragments produced by antibody digestion or produced by other means in 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 monomeric polypeptides, which can be connected into dimer or multimeric form by covalent (i.e., disulfide bond) and non-covalent binding. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from 1 to 4, depending on classification (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, term " native Fc " is general for monomeric, dimer, and multimeric form.

[0404] F(ab) fragments usually contain a light chain and a heavy chain V H and C H1 domain, wherein the V H -C H1 The heavy chain portion cannot form disulfide bonds 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.

[0405] The F(ab') fragment usually consists of a light chain and a portion of a heavy chain, which contains more of the constant region (in the C H1 and C H2domains), thereby allowing for the formation of an interchain disulfide bond between the two heavy chains to form a F(ab')2 molecule.

[0406] 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.

[0407] A "recombinant" molecule is one that is prepared, expressed, produced, or isolated by recombinant means.

[0408] 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 that forms such a binding protein. Another embodiment of the present disclosure provides an expression vector comprising a nucleic acid molecule encoding a polypeptide chain that forms such a binding protein. Yet another embodiment of the present disclosure provides a host cell expressing such a binding protein (i.e., comprising a nucleic acid molecule or vector encoding a polypeptide chain that forms such a binding protein).

[0409] 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. "Fully interchangeable" refers to the ability to exchange V domains 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 ability to modify the order of the domains (i.e., reverse the order) while maintaining the full functionality of the binding protein 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 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 parent antibody H1 and V H2 domain, and is placed in the V H1 and V H2 Position. Therefore, V H and V L The designation refers to the current position rather than the original position in the parent antibody. H and V LThe domains are "exchangeable".

[0410] As used herein, the term "antigen" or "target antigen" or "antigen target" refers to a molecule or portion of a molecule that can be bound by a binding protein and that can be used in an animal to produce antibodies that can bind to an epitope of the antigen. A target antigen can have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein can compete with an intact antibody that recognizes the target antigen.

[0411] "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 set forth in SEQ ID NO: 1) and the extracellular domain of cynomolgus monkey CD38 (e.g., as set forth in SEQ ID NO: 30).

[0412] 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.

[0413] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigenic target.

[0414] The term "monovalent binding protein" refers to a binding protein with one antigen binding site.

[0415] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets. In some embodiments, a bispecific binding protein binds to two different antigens. In some embodiments, a bispecific binding protein binds to two different epitopes on the same antigen.

[0416] The term "bivalent binding protein" refers to a binding protein with two binding sites.

[0417] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigenic 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.

[0418] The term "trivalent binding protein" refers to a binding protein with three binding sites. In certain embodiments, a trivalent binding protein can bind to one antigenic target. In other embodiments, a trivalent binding protein can bind to two antigenic targets. In other embodiments, a trivalent binding protein can bind to three antigenic targets.

[0419] An "isolated" binding protein is one 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 would interfere with the diagnostic or therapeutic use of the binding protein and may include enzymes, hormones, and other proteinaceous or non-proteinaceous 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 use of a spinning cup sequencer to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated binding protein includes the binding protein in situ within recombinant cells because at least one component of the binding protein's natural environment is absent.

[0420] As used herein, the terms "substantially pure" or "substantially purified" refer 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, the species 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.

[0421] 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.

[0422] 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 "K D ” refers to the dissociation constant of the interaction between a specific binding protein and its target antigen.

[0423] 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, 1x 10 -7 M, 1x10 -8 M, 1x 10 -9 M, 1x 10 -10 M, 1x10 -11 M, 1x 10 -12 M or higher, and / or binds to the epitope with an affinity at least two-fold 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.

[0424] 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. For purposes of this application, a binding protein that binds to Ag1 cross-reacts with Ag2 when the ratio of the affinity for Ag2 to the affinity for Ag1 is equal to or less than 10 (e.g., 5, 2, 1, or 0.5), the affinity being determined using the same method for both antigens.

[0425] When the affinities of the two antigens are very different, a binding protein that binds to Ag1 "does not significantly cross-react" with Ag2. 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 saturated plateau obtained with Ag1.

[0426] As used herein, the term "linker" refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into a cross-linked dual variable region immunoglobulin. At the sequence level, linkers are inserted between variable domains or at the transition between variable and constant domains, respectively. The 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 hypothesized by modeling techniques or secondary structure predictions. The linker described herein is called L1, which is located between V L2 The C-terminus and V L1 on the light chain between the N-termini of the V L1 The C-terminus and C L The heavy chain linker is called L3 and is located between the N-termini of the V H1 The C-terminus and V H2 between the N-termini of the V H2 The C-terminus and C H1 between the N-termini of the domains.

[0427] As used herein, the term "vector" refers to any molecule (for example, nucleic acid, plasmid or virus) for transferring coding information to a host cell. The term "vector" includes a nucleic acid molecule capable of transporting 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 gene to which they are operably connected. Such a vector is referred to as a "recombinant expression vector" (or simply "expression vector") in this article. Generally speaking, an expression vector useful in recombinant DNA technology is typically in the form of a plasmid. The terms "plasmid" and "vector" are used interchangeably herein because plasmid is the most commonly used vector form. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0428] 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 cell or host cell refers not only to a specific subject cell, but also to the progeny of such a cell. 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 within the scope of the term "host cell" as used herein. A variety of host cell expression systems can be used to express binding proteins, including bacterial, 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 in which the host cells are cultured, from which the binding protein can be recovered.

[0429] As used herein, the term "conversion" 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 carried out 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 believed that the cell has been stably transformed. As used herein, the term "transfection" 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 for introducing one or more exogenous DNA molecules into suitable host cells.

[0430] As used herein and as applied to an object, the term "naturally occurring" refers to the fact that an object can be found in nature and has not been manipulated by humans. For example, a polynucleotide or polypeptide present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by humans is 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.

[0431] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids); non-natural 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 the binding protein polypeptide chain. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide 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.

[0432] Naturally occurring residues can be divided into the following categories based on common side chain properties:

[0433] (1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

[0434] (2) Polar hydrophilicity: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

[0435] (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro;

[0436] (4) Aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro;

[0437] (5) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0438] (6) Acidic: Asp, Glu;

[0439] (7) Basic: His, Lys, Arg;

[0440] (8) Residues that affect chain orientation: Gly, Pro;

[0441] (9) Aromatic: His, Trp, Tyr, Phe; and

[0442] (10) Aromatic hydrophobicity: Phe, Trp, Tyr.

[0443] 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.

[0444] 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 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. Additionally, it may even be possible to make conservative amino acid substitutions in regions that are important for biological activity or structure without destroying the biological activity or adversely affecting the polypeptide structure.

[0445] As used herein, the term "patient" includes both human and animal subjects (eg, mammals such as dogs, pigs, horses, cats, cows, etc.).

[0446] As used herein, the terms "treat" or "treating" refer to both therapeutic treatment and prophylactic or preventative measures. Patients in need of treatment include patients with a condition as well as patients susceptible to a condition or patients for whom a condition is to be prevented. In specific embodiments, the binding proteins can be used to treat a person suffering from cancer, or a person susceptible to cancer, or to ameliorate cancer in a human subject. The binding proteins can also be used to prevent cancer in a human patient. In specific embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, germinal center B cell lymphoma, or B cell acute lymphoblastic leukemia.

[0447] 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.

[0448] 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.

[0449] 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 the 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 a variety of factors associated with the patient being treated and the severity of the condition and illness. 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. Determining the effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of those skilled in the art.

[0450] One embodiment of the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

[0451] II. Anti-CD38 Binding Protein

[0452] 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 polypeptide). In some embodiments, the binding protein is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent.

[0453] 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 monkey CD38. In some embodiments, the binding protein induces apoptosis of CD38+ cells. In some embodiments, the binding protein recruits T cells to CD38+ cells and optionally activates T cells (e.g., by TCR stimulation and / or co-stimulation).

[0454] 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 CDR-L3 sequence of the amino acid sequence. 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 CDR-L3 sequence of the amino acid sequence.In some embodiments, the binding protein comprises 1, 2, 3, 4, 5, or 6 CDRs from the antibody VH and / or VL domain sequence of mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb2xCD28supxCD3midIgG4 FALA, mAb2xCD28supxCD3mid IgG1LALA P329A, mAb2xCD28supxCD3mid IgG1 NNSA, mAb6xCD28supxCD3mid IgG4 FALA, mAb6xCD28supxCD3mid IgG1LALA P329A, or mAb6xCD28supxCD3mid IgG1 NNSA as shown in Tables G, H, or I.

[0455] 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 CDR-L3 sequence of the amino acid sequence.

[0456] In some embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In other embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).In some embodiments, the binding protein comprises 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); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0457] In some embodiments, the VH domain comprises, from N-terminus to C-terminus, the sequence FR1—CDR-H1—FR2—CDR-H2—FR3—CDR-H3—FR4; 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).

[0458] 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.

[0459] 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.

[0460] 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.

[0461] 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.

[0462] 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.

[0463] 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).

[0464] 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).

[0465] 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.

[0466] 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.

[0467] 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.

[0468] 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 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 I. In some embodiments, the binding proteins of the present disclosure comprise 1, 2, 3, 4, 5, or 6 polypeptide sequences shown in Table I.

[0469]

[0470] Table H. Variable domain sequences of anti-CD38 (mAbs 1-7) and other binding proteins.

[0471]

[0472]

[0473]

[0474] Note: CDR sequences are bolded and underlined in the above amino acid sequence.

[0475] Table I. Full-length sequences of binding proteins.

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491] Table J. Full-length polynucleotide sequences of binding proteins.

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512] CD38 polypeptide

[0513] In some embodiments, the binding proteins of the present disclosure comprise an antigen binding site that binds to the extracellular domain of the human CD38 polypeptide and the extracellular domain of the cynomolgus monkey CD38 polypeptide. Exemplary assays for determining whether the antigen binding site binds to the 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 the CD38 polypeptide on their cell surface, for example, as described below. See, for example, Examples 1, 3, and 4.

[0514] 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., as 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., as measured by flow cytometry).

[0515] 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 entire 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 entire 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 polypeptides may be advantageous, for example, to target the binding proteins of the present disclosure to cells expressing CD38 isoform E polypeptides.

[0516] Human CD38 isoform A extracellular domain polypeptide sequence

[0517] RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCP DWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI(SEQ ID NO:1)

[0518] Human CD38 isoform E polypeptide sequence

[0519] RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRHFWECGSP(SEQ ID NO:105)

[0520] 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.

[0521] Cynomolgus monkey CD38 polypeptide sequence

[0522] RWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQSVWDAFKGAFISKYPCNITEEDYQPLVKLGTQTVPCNKTLLWSRIKDLAHQFTQVQRDMFTLEDMLLGYLADDLTWCGEFNTFEINYQSCP DWRKDCSNNPVSVFWKTVSRRFAETACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQALEAWVIHGGREDSRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKFLQCVKNPEDSSCLSGI(SEQ ID NO:30)

[0523] Multispecific (e.g., bispecific, trispecific, or multispecific) binding proteins that bind to CD38 polypeptides

[0524] 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.

[0525] In some embodiments, the binding protein of the present disclosure is a trispecific binding protein comprising an antigen binding site, a second antigen binding site, and a third antigen binding site that binds to 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 monkey 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 monkey 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 amino acid sequences of exemplary and non-limiting antibody variable domains that bind to human CD3 and CD28 polypeptides.

[0526] In some embodiments, provided herein are binding proteins 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 human CD38 polypeptides and the extracellular domain of cynomolgus monkey CD38 polypeptides. In some embodiments, the human CD38 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, and / or the cynomolgus monkey CD38 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the binding protein comprises an antigen binding site that binds to the extracellular domain of human CD38 polypeptides and the extracellular domain of cynomolgus monkey CD38 polypeptides and two antigen binding sites that each bind to a T cell surface protein (e.g., human CD28 polypeptide and / or human CD3 polypeptide).

[0527] In some embodiments, the binding proteins of the present disclosure bind 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 proteins are 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 proteins are 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 proteins are 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 proteins are 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 proteins form two antigen binding sites targeting two T cell target proteins, and the third and fourth polypeptide chains of the binding proteins 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.

[0528] 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 engager 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. Other examples of such antigen targets or target proteins are provided above. In some embodiments, trispecific binding proteins can be produced by combining the antigen binding domains of two or more monospecific antibodies (parent antibodies) into one antibody.

[0529] Bispecific binding protein formats

[0530] In some embodiments, the binding proteins of the present disclosure are bispecific and / or bivalent binding proteins comprising four polypeptide chains that form four antigen binding sites that bind to one or more (e.g., two) different antigenic targets or target proteins (e.g., having the structure described in International Publication No. WO2012 / 135345). In some embodiments, the binding proteins are bivalent and / or bispecific. In some embodiments, the binding proteins are tetravalent and / or tetraspecific. In some embodiments, the binding proteins are tetravalent and / or bispecific. In some embodiments, at least one antigen binding site binds to a CD38 polypeptide (e.g., the extracellular domain of a human and / or cynomolgus monkey CD38 polypeptide).

[0531] In some embodiments, the binding protein comprises two polypeptide chains having a structure represented by the following formula:

[0532] V L1 -L1-V L2 -L2-C L [I]

[0533] The two polypeptide chains have the structure represented by the following formula:

[0534] V H2 -L3-V H1 -L4-C H1 -Fc [II]

[0535] in:

[0536] V L1 is the first immunoglobulin light chain variable domain;

[0537] V L2 is the second immunoglobulin light chain variable domain;

[0538] V H1 is the first immunoglobulin heavy chain variable domain;

[0539] VH2 is a second immunoglobulin heavy chain variable domain;

[0540] C L is the immunoglobulin light chain constant domain;

[0541] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0542] Fc contains the immunoglobulin hinge region and C H2 、C H3 immunoglobulin heavy chain constant region;

[0543] L1, L2, L3, and L4 are amino acid linkers;

[0544] 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.

[0545] In some embodiments, the binding protein comprises two polypeptide chains that form two antigen binding sites, wherein the first polypeptide chain comprises

[0546] V L1 -L1-V L2 -L2-CL-Fc

[0547] and the second polypeptide chain comprises

[0548] V H2 -L3-V HI -L4-C H1 -Fc

[0549] in:

[0550] V L1 is the first immunoglobulin light chain variable domain;

[0551] V L2 is the second immunoglobulin light chain variable domain;

[0552] V H1 is the first immunoglobulin heavy chain variable domain;

[0553] V H2 is a second immunoglobulin heavy chain variable domain;

[0554] CL is the immunoglobulin light chain constant domain;

[0555] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0556] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0557] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0558] Fc contains the immunoglobulin hinge region and C H2 、C H3 an immunoglobulin heavy chain constant region; and

[0559] L1, L2, L3, and L4 are amino acid linkers;

[0560] wherein the first and second polypeptides 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.

[0561] In some embodiments, the binding protein comprises three polypeptide chains that form two antigen binding sites, wherein the first polypeptide chain comprises

[0562] V L1 -L1-V L2 -L2-CL

[0563] The second polypeptide chain comprises

[0564] V H2 -L3-V HI -L4-C H1 -Fc

[0565] The third polypeptide chain comprises the antibody Fc region

[0566] in:

[0567] V L1 is the first immunoglobulin light chain variable domain;

[0568] V L2 is the second immunoglobulin light chain variable domain;

[0569] V H1 is the first immunoglobulin heavy chain variable domain;

[0570] V H2 is a second immunoglobulin heavy chain variable domain;

[0571] CL is the immunoglobulin light chain constant domain;

[0572] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0573] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0574] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0575] Fc contains the immunoglobulin hinge region and C H2 、C H3 an immunoglobulin heavy chain constant region; and

[0576] L1, L2, L3, and L4 are amino acid linkers;

[0577] wherein the first and second polypeptides 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.

[0578] In some embodiments, the binding protein comprises a first polypeptide chain comprising a structure represented by the following formula:

[0579] V L1 -L1-V L2 -L2-C L [I]

[0580] and a second polypeptide chain comprising a structure represented by the formula:

[0581] V H2 -L3-V H1 -L4-C H1 [II]

[0582] in:

[0583] V L1 is the first immunoglobulin light chain variable domain;

[0584] V L2 is the second immunoglobulin light chain variable domain;

[0585] V H1 is the first immunoglobulin heavy chain variable domain;

[0586] V H2 is a second immunoglobulin heavy chain variable domain;

[0587] C L is the immunoglobulin light chain constant domain;

[0588] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0589] L1, L2, L3, and L4 are amino acid linkers;

[0590] 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.

[0591] 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.

[0592] In any of the bispecific binding proteins described above, any linker or linker combination described herein can be used. For example, in some embodiments, at least one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments, L1, L2, L3, or L4 is each independently at least 1 amino acid in length. In some embodiments, L1, L2, L3, and L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 58), L2 comprises the sequence TKGPS (SEQ ID NO: 57), L3 comprises the sequence S, and L4 comprises the sequence RT. In some embodiments, L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 55), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 55), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence GGSGSSGSGG (SEQ ID NO: 59), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO: 59), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), and L4 is 0 amino acids in length.

[0593] Trispecific binding protein that binds to CD38 polypeptide

[0594] In some embodiments, the binding proteins of the present disclosure are trispecific and / or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that bind to one or more (e.g., three) different antigenic 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:

[0595] V L2 -L1-V L1 -L2-C L [I]

[0596] and the second polypeptide chain comprises a structure represented by the formula:

[0597] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0598] and the third polypeptide chain comprises a structure represented by the formula:

[0599] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0600] and the fourth polypeptide chain comprises a structure represented by the formula:

[0601] V L3 -C L [IV]

[0602] in:

[0603] V L1 is the first immunoglobulin light chain variable domain;

[0604] V L2 is the second immunoglobulin light chain variable domain;

[0605] V L3 is the third immunoglobulin light chain variable domain;

[0606] V H1 is the first immunoglobulin heavy chain variable domain;

[0607] V H2 is a second immunoglobulin heavy chain variable domain;

[0608] V H3 is the third immunoglobulin heavy chain variable domain;

[0609] C L is the immunoglobulin light chain constant domain;

[0610] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0611] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0612] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0613] The hinge is connected to C H1 and C H2 domain of the immunoglobulin hinge region, and

[0614] L1, L2, L3, and L4 are amino acid linkers;

[0615] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair.

[0616] In some embodiments, the binding proteins of the present disclosure are trispecific and / or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that bind to one or more (e.g., three) different antigenic 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:

[0617] V L2 -L1-V L1 -L2-C L [I]

[0618] and the second polypeptide chain comprises a structure represented by the formula:

[0619] V H1 -L3-V H2 -L4-C H1 [II]

[0620] and the third polypeptide chain comprises a structure represented by the formula:

[0621] V H3 -C H1 [III]

[0622] and the fourth polypeptide chain comprises a structure represented by the formula:

[0623] V L3 -CL [IV]

[0624] in:

[0625] V L1 is the first immunoglobulin light chain variable domain;

[0626] V L2 is the second immunoglobulin light chain variable domain;

[0627] V L3 is the third immunoglobulin light chain variable domain;

[0628] V H1 is the first immunoglobulin heavy chain variable domain;

[0629] V H2 is a second immunoglobulin heavy chain variable domain;

[0630] V H3 is the third immunoglobulin heavy chain variable domain;

[0631] C L is the immunoglobulin light chain constant domain;

[0632] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0633] L1, L2, L3, and L4 are amino acid linkers;

[0634] 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.

[0635] In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross-orientation that 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.

[0636] 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 monkey 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 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 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 H2The 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 L3 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 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 L1The 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 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), 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).

[0637] In some embodiments, V H1The 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 V L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, V 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 V L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). 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 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).

[0638] In some embodiments, V H1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and V L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, V H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and V L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, V H3 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and 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).

[0639] In some embodiments, V H1The 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 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 V L1 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, V H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and / or 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 H2The 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 V L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). 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 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 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).

[0640] In some embodiments, V H1The 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 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, V H2 The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), 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 H2The 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 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, 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 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 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).

[0641] In some embodiments, V H3The 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 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 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).

[0642] In some embodiments, V H3 The domain comprises the amino acid sequence of SEQ ID NO: 5, 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, 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 H3 The 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 L3The domain comprises the amino acid sequence of SEQ ID NO:14.

[0643] 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.

[0644] 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 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).

[0645] 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.

[0646] 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.

[0647] 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, V H2 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 VL2 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.

[0648] 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.

[0649] 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.

[0650] 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.

[0651] 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.

[0652] In some embodiments, the binding proteins of the present disclosure are antibodies. In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, the antibodies are chimeric antibodies, humanized antibodies, or human antibodies.

[0653] 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.

[0654] connector

[0655] In some embodiments, linkers L1, L2, L3, and L4 range 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. Linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long. L1, L2, L3, and L4 in a binding protein can all have the same amino acid sequence, or can all have different amino acid sequences.

[0656] 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 use in binding proteins. For further description of linker sequences, see, for example, WO2012135345 and International Application No. PCT / US2017 / 027488.

[0657] In the joint, the identity and sequence of amino acid residues can be changed according to the type of realizing required secondary structure elements in the joint. For example, glycine, serine and alanine are most suitable for joints with maximum flexibility. If more rigid and extended joints are needed, some combination of glycine, proline, threonine and serine is useful. According to the desired property, any amino acid residue can be considered as a joint in combination with other amino acid residues to build required larger peptide joints.

[0658] In some embodiments, at least one of L1, L2, L3, or L4 is independently 0 amino acids long. In some embodiments, L1, L2, L3, or L4 is each independently at least 1 amino acid long. In some embodiments, the length of L1 is at least twice the length of L3. In some embodiments, the length of L2 is at least twice the length of L4. In some embodiments, the length of L1 is at least twice the length of L3, and the length of L2 is at least twice the length of L4. In some embodiments, the length of L1 is 3 to 12 amino acid residues, the length of L2 is 3 to 14 amino acid residues, the length of L3 is 1 to 8 amino acid residues, and the length of L4 is 1 to 3 amino acid residues. In some embodiments, the length of L1 is 5 to 10 amino acid residues, the length of L2 is 5 to 8 amino acid residues, the length of L3 is 1 to 5 amino acid residues, and the length of L4 is 1 to 2 amino acid residues. In some embodiments, the length of L1 is 7 amino acid residues, the length of L2 is 5 amino acid residues, the length of L3 is 1 amino acid residue, and the length of L4 is 2 amino acid residues. In some embodiments, the length of L1 is 10 amino acid residues, the length of L2 is 10 amino acid residues, the length of L3 is 0 amino acid residues, and the length of L4 is 0 amino acid residues. In some embodiments, L1, L2, L3 and L4 each have 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, the length of L1, L2, L3 and L4 each is 0 amino acid.

[0659] In some embodiments, L1, L2, L3, and / or L4 comprise sequences derived from naturally occurring sequences 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 Land C L In some embodiments, the linker comprises a sequence found at a transition between endogenous human V H and C H1 between domains or in endogenous human V L and C L Sequences found at transitions between domains (e.g., human kappa or lambda).

[0660] In some embodiments, L1, L2, L3, and L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L1, L2, L3, and L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59).

[0661] In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 58), L2 comprises the sequence TKGPS (SEQ ID NO: 57), L3 comprises the sequence S, and L4 comprises the sequence RT. In some embodiments, L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 55), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 55), the length of L3 is 0 amino acids, and the length of L4 is 0 amino acids. In some embodiments, L1 comprises the sequence GGSGSSGSGG (SEQ ID NO: 59), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO: 59), the length of L3 is 0 amino acids, and the length of L4 is 0 amino acids. In some embodiments, L1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), the length of L2 is 0 amino acids, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), and the length of L4 is 0 amino acids.

[0662] Fc region and constant domains

[0663] 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 the antibody hinge, C H1 、C H2 、C H3 and optionally C H4 In 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 comprises one or more mutations described below.

[0664] 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 that is modified from a native Fc but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with salvage receptors are known in the art. Thus, the term "Fc variant" may include a molecule or sequence of a humanized non-human native Fc. In addition, the native Fc contains 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. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues, or in which one or more Fc sites or residues are modified to affect or participate 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).

[0665] 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)).

[0666] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU Index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU Index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0667] 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 comprising an amino acid substitution at a position corresponding to position 228 and / or 409 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitution is S228P and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to position 234 and / or 235 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitution is F234A and / or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to position 228, 234, 235, and / or 409 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitution is S228P, F234A, L235A, and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to position 233-236 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitution is E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid mutation at a substitution corresponding to position 228, 233-236, and / or 409 of human IgG4 according to the EU Index. In some embodiments, the amino acid mutation is S228P; E233P, F234V, L235A, and deletion at 236; and / or R409K.

[0668] 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 purification reagents. 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 has an intermediate affinity for purification based on protein A compared to any homodimeric form and can be selectively eluted from protein A, for example, by using different pH values ​​(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 further comprising a C H1 The first Fc region of the immunoglobulin comprises the hinge region and the C H2 and C H3 immunoglobulin heavy chain constant region, and the third polypeptide chain further comprises a C H1 The second Fc region comprises the immunoglobulin hinge region and the 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 substitutions are 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.

[0669] To improve 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 C H3 The interaction surface of the domains is increased to contain 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 heterodimer (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 having a single variable domain. In other embodiments, the knob is located on the first pair of polypeptides having a cross-shaped orientation. In other embodiments, C H3 The domain does not include the knob in the mortar.

[0670] 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.

[0671] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein the first Fc region comprises an amino acid substitution at a position corresponding to position 366 and optionally 354 of human IgG1 or IgG4 according to the EU Index, 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 CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein the second 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 according to the EU Index, wherein the amino acid substitution is Y407V or Y407T, and optionally Y349C, T366S and / or L368A.

[0672] 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 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 according to the EU Index, 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 the second Fc region comprises an amino acid substitution at a position corresponding to position 366 and optionally 354 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitution is T366W or T366Y, and optionally S354C.

[0673] 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 the first Fc region comprises an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitution is T366W; 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 the second Fc region comprises an amino acid substitution at a position corresponding to position 366, 368 and / or 407 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitution is T366S, L368A and / or Y407V.

[0674] 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 the first Fc region comprises an amino acid substitution at a position corresponding to position 366, 368 and / or 407 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitution is T366S, L368A and / or Y407V; and 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 the second Fc region comprises an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitution is T366W.

[0675] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the first Fc region 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; 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 domains, wherein according to the EU Index, the second Fc region 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 second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU Index, 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.

[0676] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and 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 positions 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 CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU Index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, 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 228, 354, 366, and 409 of human IgG4, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K.

[0677] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU Index, 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 CHI, 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, 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 linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU Index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, 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.

[0678] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, 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 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 CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, 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 positions 228, 234, 235, 354, 366, and 409 of human IgG4, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K.

[0679] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to improve serum half-life (see, e.g., Hinton, PR et al. (2006) J. Immunol. 176(1):346-56). In some embodiments, the mutation comprises a substitution at a position corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitution is M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising 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, the third polypeptide chain further comprising 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 positions 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.

[0680] 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 further comprising a C H1 The first Fc region of the immunoglobulin comprises the hinge region and the 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 the immunoglobulin hinge region and the C H2 and C H3Immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and second Fc regions each comprise an amino acid substitution 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 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.

[0681] 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 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 has 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 values ​​(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 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 further comprising a C H1 The first Fc region of the immunoglobulin comprises the hinge region and the 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 the immunoglobulin hinge region and the C H2 and C H3 Immunoglobulin heavy chain constant domain; and wherein only one of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitutions are 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.

[0682] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to improve serum half-life (see, e.g., Hinton, PR et al. (2006) J. Immunol. 176(1):346-56). In some embodiments, the mutation comprises a substitution at a position corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitution is M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising 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 a third polypeptide chain further comprising 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 positions 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.

[0683] 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 of the immunoglobulin comprises the hinge region and the 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 the immunoglobulin hinge region and the 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 of the immunoglobulin comprises the hinge region and the 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 the immunoglobulin hinge region and the C H2 and C H3 Immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and second Fc regions each comprise an amino acid substitution at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU Index, 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 the Fc regions each comprise an amino acid substitution at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU Index, 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 wherein the 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. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a C H1 The first Fc region of the immunoglobulin comprises the hinge region and the 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 the immunoglobulin hinge region and the 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.

[0684] 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, R. Let al. (2001) J. Biol. Chem. 276:6591-6604 and WO1999051642.

[0685] 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.

[0686] 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.

[0687] Determination

[0688] The present disclosure provides antigen binding proteins that bind to human and / or cynomolgus monkey CD38 polypeptides, induce T cell (e.g., CD4+ and / or CD8+ T cell) 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).

[0689] 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 can have one or more of the exemplary binding properties described herein. In some embodiments, the KD is measured at 4°C or 25°C.

[0690] In some embodiments, the monospecific binding proteins of the present disclosure have one or more of the following features: 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 or ELISA; 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, 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 (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell, as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell, 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., 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 protein with an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less, as determined by ELISA. In some embodiments, the KD is measured at 4°C or 25°C.

[0691] In some embodiments, the trispecific binding proteins of the present disclosure have one or more of the following characteristics: induction of T cell (e.g., CD4+ and / or CD8+ T cell) proliferation; induction of Bcl-xL-expressing T cells (e.g., CD4+ and / or CD8+ inducing apoptosis of CD38+ cells (e.g., as measured by Annexin V staining and / or propidium iodide uptake); binding 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; binding 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; stimulating activation of the 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 SEQ ID NO:1 amino acid sequence) as a purified protein, as determined by SPR or ELISA; the extracellular domain of the human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, which D is 1.5 nM or less, as determined by SPR or ELISA; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell, as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell, with an apparent K D is 12 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 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) in cells that express 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 polypeptide (e.g., human or cynomolgus monkey) expressed on the surface of a cell 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 polypeptide (e.g., human or cynomolgus monkey) as a purified protein with an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less, as determined by ELISA.

[0692] Nucleic Acids

[0693] Standard recombinant DNA methods are used to construct the polynucleotides encoding the protein-binding polypeptide, these polynucleotides are incorporated into recombinant expression vectors, and such vectors are imported into host cells.See, for example, Sambrook et al., 2001, MOLECULAR CLONING:A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reaction and purification techniques can be carried out according to the manufacturer's instructions, as generally achieved in this area, or as described herein. Unless specific definition is provided, the nomenclature and laboratory procedures and techniques used in combination with analytical chemistry as herein described, synthetic organic chemistry and medicine and medicinal chemistry are those well known in the art and commonly used. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, preparation, delivery and patient treatment.

[0694] 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 SEQ ID NOs: 60-83 and / or the sequences shown in Table J.

[0695] Some aspects of the present disclosure relate to a test kit of polynucleotides. In some embodiments, one or more polynucleotides are vectors (e.g., expression vectors). The test kit is particularl...

Claims

1. 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 having an amino acid sequence containing GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence having an amino acid sequence containing IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence having an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence having an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence having an amino acid sequence containing LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence having an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36).

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

3. The binding protein of claim 1 or claim 2, wherein the VH domain comprises the sequence FR1—CDR-H1—FR2—CDR-H2—FR3—CDR-H3—FR4 from the N-terminus to the 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); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO:96).

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

6.

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

8.

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

18.

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

20.

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

18.

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

20.

10. The binding protein of claim 1 or claim 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO:23, and the VL domain comprises the amino acid sequence of SEQ ID NO:18.

Images