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 divisional application of the present invention is based on the patent application with the application date of October 9, 2018, application number 201880079045.9, and invention title "Anti-CD38 Antibody and Combinations with Anti-CD3 and Anti-CD28 Antibodies".

[0002] Cross-reference to related applications

[0003] This application claims the priority of 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] Submission of a sequence listing in ASCII text file

[0005] The content of the following ASCII text file submitted is incorporated herein by reference in its entirety: computer-readable form (CRF) of the sequence listing (filename: 183952029941seqlist.TXT, date of record: October 8, 2018, size: 158 KB). Technical field

[0006] The present disclosure relates to 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 domain that binds to a CD38 polypeptide, as well as polynucleotides, host cells, production methods, and methods of use related thereto. Background art

[0007] Monoclonal antibody-based biotherapeutics have become an important approach in new drug development. Monoclonal antibody technology provides specific targeting, precise signal delivery, and / or payloads to specific cell populations and provides durable biological effects through their Fc functions. Efforts in antibody engineering 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, G.T. (2006) Mol. Med. 12:345 - 346). (Daratumumab) is an anti-CD38 antibody that has been approved for the treatment of multiple myeloma. However, there is a need for therapeutic agents that target CD38 and have different modes of action and / or improved properties, including but not limited to high affinity binding to CD38, cross-reactivity between human and cynomolgus 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 a CD38 polypeptide. Advantageously, these binding proteins have the ability to recruit T cells to the vicinity of cancer cells, subsequently activate the T cells, and promote the killing of adjacent cancer cells by the activated T cells through the granzyme / perforin mechanism, providing a different mode of anti-tumor activity from anti-CD38 antibodies such as (daratumumab). In addition, the ability to bind to both human and cynomolgus monkey CD38 polypeptides allows for easy testing of the binding proteins in preclinical toxicology studies, e.g., to evaluate their safety profiles 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 proteins cross-react with human and cynomolgus monkey CD38 polypeptides. In some embodiments, the binding proteins bind to human isotype A and isotype E CD38 polypeptides. In some embodiments, the binding proteins have one or more of the following characteristics (in any combination): binding to the extracellular domain of the human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purified protein, as determined by SPR; binding to the extracellular domain of the human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purified protein, with a K D of 1.5 nM or lower, as determined by SPR; binding to the extracellular domain of the human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) expressed on the cell surface, as determined by flow cytometry; binding to the extracellular domain of the human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) expressed on the cell surface, with an apparent K Dis 20 nM, 15 nM, 10 nM, 5 nM, 1 nM or lower, as determined by flow cytometry; binds to the extracellular domain of cynomolgus CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30) as a purified protein, as determined by SPR; binds to the extracellular domain of cynomolgus 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 lower, as determined by SPR; binds to the extracellular domain of cynomolgus CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:30), as determined by flow cytometry; binds to the extracellular domain of cynomolgus CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:30), its apparent K D is 7.5 nM or lower, as determined by flow cytometry; binds to the extracellular domain of human isotype 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 human isotype E CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:105), as determined by flow cytometry; induces apoptosis of cells expressing CD38 on the cell surface or antibody - dependent cellular cytotoxicity (ADCC); and has one or more mutations (e.g., in the Fc region) resulting in reduced binding to FcγRI and / or FcγRII compared to the same binding protein without one or more mutations. For exemplary assays, see Examples 1, 3, and 4. In some embodiments, KD is measured at 4 °C or 25 °C.

[0011] In some embodiments, trispecific binding proteins that bind to human CD38 polypeptides are provided herein. In some embodiments, the trispecific binding protein binds (e.g., simultaneously) to a CD38 polypeptide (e.g., expressed on the cell surface) and one or more other target antigens expressed on a second cell surface, thereby recruiting a second cell in proximity to the cell expressing the CD38 polypeptide. In some embodiments, the trispecific binding protein binds (e.g., simultaneously) to a CD38 polypeptide (e.g., expressed on the cell surface) and one or two target antigens expressed on the surface of T cells, thereby recruiting T cells in proximity to the cell expressing the CD38 polypeptide. In some embodiments, the trispecific binding protein activates T cells and / or provides CD28-mediated costimulatory 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 isotype A and isotype E CD38 polypeptides. In some embodiments, the trispecific binding protein has one or more of the following characteristics (in any combination): binds to the extracellular domain of the 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 the human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purified protein, K D is 1.5 nM or lower, as determined by SPR; binds to the extracellular domain of the human CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:1), as determined by flow cytometry; binds to the extracellular domain of the human CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:1), whose apparent K D is 20 nM, 15 nM, 10 nM, 5 nM, 1 nM or lower, 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 lower, as determined by SPR; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:30), as determined by flow cytometry; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:30), whose apparent K Dis 7.5 nM or less, as determined by flow cytometry; binds to the extracellular domain of the human isotype 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 the human isotype E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) expressed on the cell surface, as determined by flow cytometry; induces apoptosis of cells expressing CD38 on the cell surface or antibody-dependent cellular cytotoxicity (ADCC) of cells; and has one or more mutations (e.g., in the Fc region) resulting in reduced binding to FcγRI and / or FcγRII as compared to the same binding protein without one or more mutations; induces proliferation of T cells (e.g., CD4+ and / or CD8+ T cells); induces expression of Bcl-xL in T cells (e.g., CD4+ and / or CD8+ T cells); induces apoptosis of CD38+ cells; binds to CD38 expressed on the cell surface and one or more T cell target antigens expressed on the T cell surface; binds to CD38 expressed on the cell surface, CD28 expressed on the T cell surface, and CD3 expressed on the T cell surface; stimulates activation of the T cell receptor; induces co-stimulation of T cell receptor signaling (e.g., mediated by CD28); and has one or more mutations (e.g., in the Fc region) resulting in reduced induction of cytokine release (e.g., IFN-γ, IL-2, and / or TNF-α) by PBMC as compared to the same binding protein without one or more mutations; induces 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 antigen-binding sites that bind 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 / or (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). In some embodiments, 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 / or (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 QSVSSYGQG (SEQ ID NO: 132), 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).In some embodiments, the antigen-binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence having the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence having the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence having the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and / or (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence having the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence having the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence having the amino acid sequence 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 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). 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 having the amino acid sequence of SEQ ID NO:7 and an antibody light chain having 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 having the amino acid sequence of SEQ ID NO:19 and an antibody light chain having 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 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. 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 containing the amino acid sequence of SEQ ID NO: 24 and an antibody light chain containing 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 that comprises a CDR-H1 sequence containing the amino acid sequence GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence containing the amino acid sequence IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO: 33); and (b) an antibody light chain variable (VL) domain that comprises a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO: 40), and a CDR-L3 sequence containing the amino acid sequence 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 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).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 having the amino acid sequence of SEQ ID NO:15 and an antibody light chain having the amino acid sequence of SEQ ID NO:16.

[0013] In some embodiments, provided herein is a binding protein that comprises an antigen-binding site that binds to a CD38 polypeptide, wherein the antigen-binding site comprises: (a) an antibody heavy chain variable (VH) domain that comprises a CDR-H1 sequence having the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence having the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence having the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43); and (b) an antibody light chain variable (VL) domain that comprises a CDR-L1 sequence having the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence having the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence having 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 having the amino acid sequence of SEQ ID NO:11 and an antibody light chain having the amino acid sequence of SEQ ID NO:12. In some embodiments, the antigen-binding site cross-reacts with the extracellular domain of the human CD38 polypeptide and the extracellular domain of the 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, and the equilibrium dissociation constant (K D ) is 2.1 nM or less. In some embodiments, the antigen-binding site binds to a human isotype 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, and the equilibrium dissociation constant (K 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 that comprises 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 that comprises 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 that comprises a first antigen-binding site that binds to a CD38 polypeptide, a second antigen-binding site, and a third antigen-binding site. In some embodiments, the first antigen-binding site binds to the extracellular domain of a human CD38 polypeptide, and 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, provided herein is a binding protein that comprises 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 the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:105. In some embodiments, the binding protein cross-reacts with a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30. In some embodiments, the binding protein comprises an antigen-binding site 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 that form the three antigen-binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0016] V L2 -L 1 -V L1 -L 2 -C L [I]

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

[0018] V H1 -L 3 -V H2 -L 4 -C H1 -hinge-C H2 -C H3 [II]

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

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

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

[0022] V L3 -C L [IV]

[0023] wherein:

[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 the 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 is the immunoglobulin C H1 heavy chain constant domain;

[0032] C H2 is the immunoglobulin C H2 heavy chain constant domain;

[0033] C H3 is the immunoglobulin C H3 heavy chain constant domain;

[0034] The hinge is the immunoglobulin hinge region connecting the C H1 and C H2 domains, and

[0035] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

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

[0037] wherein: (a) the V H1The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L1 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36);

[0038] (b) The V H2 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36), or

[0039] (c) The V H3 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the VL3 The domain contains 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). In some embodiments, the binding protein comprises 4 polypeptide chains forming the 3 antigen-binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0040] V L2 -L 1 -V L1 -L 2 -C L [I]

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

[0042] V H1 -L 3 -V H2 -L 4 -C H1 -hinge-C H2 -C H3 [II]

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

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

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

[0046] V L3 -C L [IV]

[0047] wherein:

[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 H1is the first immunoglobulin heavy chain variable domain;

[0052] V H2 is the 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 is the immunoglobulin C H1 heavy chain constant domain;

[0056] C H2 is the immunoglobulin C H2 heavy chain constant domain;

[0057] C H3 is the immunoglobulin C H3 heavy chain constant domain;

[0058] The hinge is the immunoglobulin hinge region connecting the said C H1 and C H2 domains, and

[0059] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

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

[0061] wherein: (a) the said V H1 domain comprises a CDR - H1 sequence containing an amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR - H2 sequence containing an amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR - H3 sequence containing an amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and the said V L1The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36);

[0062] (b) The V H2 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36); or

[0063] (c) The V H3 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, the VH1 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L1 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36); the V H1 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L1 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36); the V H2 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36); the V H2The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36); the V H3 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36); or the V H3 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V H3The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36); or the V H3 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V H3 The domain contains the amino acid sequence of SEQ ID NO:5, and the V L3 The domain contains the amino acid sequence of SEQID NO:6; the V H3 The domain contains the amino acid sequence of SEQ ID NO:17, and the V L3 The domain contains the amino acid sequence of SEQ ID NO:18; the V H3 The domain contains the amino acid sequence of SEQ ID NO:21, and the V L3 The domain contains the amino acid sequence of SEQ ID NO:18; the V H3 The domain contains the amino acid sequence of SEQ ID NO:23, and the V L3 The domain contains the amino acid sequence of SEQ ID NO:18; or the V H3 The domain contains the amino acid sequence of SEQ ID NO:13, and the V L3The domain contains the amino acid sequence of SEQ ID NO:14. In some embodiments, the binding protein comprises 4 polypeptide chains that form the 3 antigen-binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0064] V L2 -L 1 -V L1 -L 2 -C L [I]

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

[0066] V H1 -L 3 -V H2 -L 4 -C H1 -hinge-C H2 -C H3 [II]

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

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

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

[0070] V L3 -C L [IV]

[0071] wherein:

[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 the second immunoglobulin heavy chain variable domain;

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

[0078] CL is the constant domain of an immunoglobulin light chain;

[0079] C H1 is immunoglobulin C H1 constant domain of the heavy chain;

[0080] C H2 is immunoglobulin C H2 constant domain of the heavy chain;

[0081] C H3 is immunoglobulin C H3 constant domain of the heavy chain;

[0082] The hinge is the immunoglobulin hinge region connecting the said C H1 and C H2 domains, and

[0083] L 1 、L 2 、L 3 and L 4 is the amino acid linker;

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

[0085] wherein (a) the said V H1 domain comprises a CDR - H1 sequence having an amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR - H2 sequence having an amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR - H3 sequence having an amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43); and the said V L1 domain comprises a CDR - L1 sequence having an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR - L2 sequence having an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR - L3 sequence having an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46);

[0086] (b) the said V H2 domain comprises a CDR - H1 sequence having an amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR - H2 sequence having an amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR - H3 sequence having an amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43); and the said V L2The domain contains a CDR-L1 sequence having an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence having an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence having an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46), or

[0087] (c) said V H3 The domain contains a CDR-H1 sequence having an amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence having an amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence having an amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43); and said V L3 The domain contains a CDR-L1 sequence having an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence having an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence having an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46). In some embodiments, said V H3 The domain contains a CDR-H1 sequence having an amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence having an amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence having an amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43); and said V L3 The domain contains a CDR-L1 sequence having an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence having an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence having an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46). In some embodiments, said V H3 The domain contains the amino acid sequence of SEQ ID NO:9, and said V L3 The domain contains the amino acid sequence of SEQ ID NO:10. In some embodiments, said V H1 The domain contains the amino acid sequence of SEQ ID NO:49, said V L1 The domain contains the amino acid sequence of SEQ ID NO:50, said V H2 The domain contains the amino acid sequence of SEQ ID NO:53, and said V L2 The domain contains the amino acid sequence of SEQ ID NO:54; said V H2The domain contains the amino acid sequence of SEQ ID NO: 49, and said V L2 The domain contains the amino acid sequence of SEQ ID NO: 50, and said V H1 The domain contains the amino acid sequence of SEQ ID NO: 53, and said V L1 The domain contains the amino acid sequence of SEQ ID NO: 54; and said V H1 The domain contains the amino acid sequence of SEQ ID NO: 51, and said V L1 The domain contains the amino acid sequence of SEQ ID NO: 52, and said V H2 The domain contains the amino acid sequence of SEQ ID NO: 53, and said V L2 The domain contains the amino acid sequence of SEQ ID NO: 54; and said V H2 The domain contains the amino acid sequence of SEQ ID NO: 51, and said V L2 The domain contains the amino acid sequence of SEQ ID NO: 52, and said V H1 The domain contains the amino acid sequence of SEQ ID NO: 53, and said V L1 The domain contains the amino acid sequence of SEQ ID NO: 54; and said V H1 The domain contains the amino acid sequence of SEQ ID NO: 49, and said V L1 The domain contains the amino acid sequence of SEQ ID NO: 50, and said V H2 The domain contains the amino acid sequence of SEQ ID NO: 84, and said V L2 The domain contains the amino acid sequence of SEQ ID NO: 85; and said V H2 The domain contains the amino acid sequence of SEQ ID NO: 49, and said V L2 The domain contains the amino acid sequence of SEQ ID NO: 50, and said V H1 The domain contains the amino acid sequence of SEQ ID NO: 84, and said V L1 The domain contains the amino acid sequence of SEQ ID NO: 85; and said V H1 The domain contains the amino acid sequence of SEQ ID NO: 51, and said V L1 The domain contains the amino acid sequence of SEQ ID NO: 52, and said V H2 The domain contains the amino acid sequence of SEQ ID NO: 84, and said V L2 The domain contains the amino acid sequence of SEQ ID NO: 85; or said V H2 The domain contains the amino acid sequence of SEQ ID NO: 51, and said VL2 The domain comprises the amino acid sequence of SEQ ID NO: 52, wherein the V H1 The domain comprises the amino acid sequence of SEQ ID NO: 84, and the V L1 The 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 V H2 The domain comprises the amino acid sequence of SEQ ID NO: 53, wherein the V L2 The domain comprises the amino acid sequence of SEQ ID NO: 54, wherein the V H3 The domain comprises the amino acid sequence of SEQ ID NO: 9, and the V L3 The domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, L 1 , L 2 , L 3 or L 4 The length of at least one of L is independently 0 amino acids. 1 , L 2 , L 3 or L 4 The length of each of L is independently at least one amino acid. 1 , L 2 , L 3 and L 4Each is independently 0 amino acids in length or comprises a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59); or (b) L 1 , L 2 , L 3 and L 4 Each independently comprises a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L 1 Containing the sequence GQPKAAP (SEQ ID NO: 58), L 2 Containing the sequence TKGPS (SEQ ID NO: 57), L 3 Contains the sequence S, and L 4 Contains the sequence RT;L 1 Containing the sequence GGGGSGGGGS (SEQ ID NO: 55), L 2 Containing the sequence GGGGSGGGGS (SEQ ID NO: 55), L 3 is 0 amino acids in length, and L 4 The length of L is 0 amino acids; 1 Containing the sequence GGSGSSGSGG (SEQ ID NO: 59), L 2 Containing the sequence GGSGSSGSGG (SEQ ID NO: 59), L 3 is 0 amino acids in length, and L 4 is 0 amino acids in length; or L 1 Containing the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L 2 The length is 0 amino acids, L 3 comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), and L 4 The length of the hinge-C of the second and third polypeptide chains is 0 amino acids. H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3domain, and wherein according to the EU Index, said hinge-C H2 -C H3 domains each contain amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4, wherein said amino acid substitutions are F234A and L235A. In some embodiments, the hinge-C H2 -C H3 domains of the second and third polypeptide chains are human IgG4 hinge-C H2 -C H3 domains, and wherein according to the EU Index, said hinge-C H2 -C H3 domains each contain amino acid substitutions at positions corresponding to positions 233-236 of human IgG4, wherein said amino acid substitutions are E233P, F234V, L235A and a deletion at 236. In some embodiments, the hinge-C H2 -C H3 domains of the second and third polypeptide chains are human IgG4 hinge-C H2 -C H3 domains, and wherein according to the EU Index, said hinge-C H2 -C H3 domains each contain amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4, wherein said amino acid substitutions are S228P and R409K. In some embodiments, the hinge-C H2 -C H3 domains of the second and third polypeptide chains are human IgG1 hinge-C H2 -C H3 domains, and wherein according to the EU Index, said hinge-C H2 -C H3 domains each contain amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG1, wherein said amino acid substitutions are L234A, L235A and P329A. In some embodiments, the hinge-C H2 -C H3 domains of the second and third polypeptide chains are human IgG1 hinge-C H2 -C H3 domains, and wherein according to the EU Index, said hinge-C H2 -C H3 domains each contain amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1, wherein said amino acid substitutions are S298N, T299A and Y300S. In some embodiments, according to the EU Index, the hinge-C H2-C H3 The -C domain contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein according to the EU Index, the hinge-C of the third polypeptide chain H2 -C H3 The -C domain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W. In some embodiments, according to the EU Index, the hinge-C of the second polypeptide chain H2 -C H3 The -C domain contains 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 according to the EU Index, the hinge-C of the third polypeptide chain H2 -C H3The domain contains 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 that comprises three antigen-binding sites each binding to one or more target proteins, wherein the binding protein comprises four polypeptide chains that form the three antigen-binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0089] V L2 -L 1 -V L1 -L2 -C L [I]

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

[0091] V H1 -L 3 -V H2 -L 4 -C H1 -hinge-C H2 -C H3 [II]

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

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

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

[0095] V L3 -C L [IV]

[0096] wherein:

[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 the 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 is the immunoglobulin C H1 heavy chain constant domain;

[0105] C H2 is the immunoglobulin C H2Heavy chain constant domain;

[0106] C H3 is immunoglobulin C H3 Heavy chain constant domain;

[0107] The hinge is the immunoglobulin hinge region that connects the said C H1 and C H2 domains, and

[0108] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

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

[0110] wherein (a) the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgG1 hinge-C H2 -C H3 domains, and wherein according to the EU Index, the hinge-C H2 -C H3 domains each contain 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; or (b) the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgG4 hinge-C H2 -C H3 domains, and wherein according to the EU Index, the hinge-C H2 -C H3 domains each contain 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, the human IgG4 hinge-C H2 -C H3 domains contain amino acid substitutions at positions corresponding to positions 233-237 of human IgG4, wherein the sequence EFLGG is replaced by PVAG. In some embodiments, V H1 and V L1 、V H2 and V L2 and V H3 and V L3 at least one pair of which form an antigen-binding site that binds to the CD38 polypeptide. In some embodiments, V H1 and VL1 , V H2 and V L2 as well as V H3 and V L31, 2, or 3 pairs thereof form antigen-binding sites that bind to antigen targets 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, 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, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, 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 of the first pair form an antigen-binding site that binds to the human CD3 polypeptide, V H1 and V L1 、V H2 and V L2 and V H3 and V L3 of the second pair form an antigen-binding site that binds to the human CD28 polypeptide, and V H1 and V L1 、V H2 and V L2 and V H3 and V L3The third pair forms antigen-binding sites that bind to human antigen targets 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, 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, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, 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 disclosure provides a kit of polynucleotides, comprising: (a) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:72, a third polynucleotide comprising the sequence of SEQ ID NO:74, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75; (b) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:76, a third polynucleotide comprising the sequence of SEQ ID NO:77, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75; (c) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:78, a third polynucleotide comprising the sequence of SEQ ID NO:79, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75; (d) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:72, a third polynucleotide comprising the sequence of SEQ ID NO:80, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81; (e) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:76, a third polynucleotide comprising the sequence of SEQ ID NO:82, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81; or (f) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:78, a third polynucleotide comprising the sequence of SEQ ID NO:83, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81.

[0112] In some embodiments, the present disclosure provides a polynucleotide comprising the binding protein of any one of the above embodiments. In some embodiments, the present disclosure provides a vector comprising the polynucleotide of any one of the above embodiments.

[0113] In some embodiments, the present disclosure provides a host cell comprising the kit of polynucleotides, polynucleotide or vector of any one of the above embodiments. In some embodiments, the present disclosure provides a method for producing a binding protein, the method comprising culturing the host cell of any one of the above embodiments to produce the binding protein. In some embodiments, the method 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, provided herein is a method of 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 CD3, a second antigen-binding site that binds CD28, and a third antigen-binding site that binds the extracellular domain of a human CD38 polypeptide. In some embodiments, at least one binding protein is co-administered with a chemotherapeutic agent. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B cell lymphoma. In some embodiments, the patient is a human. In some embodiments, the patient is selected for treatment because the cancer cells express the human CD38 isoform E polypeptide (e.g., as shown in SEQ ID NO: 105) on their cell surface. In some embodiments, the cancer cells express CD38 and CD28. In some embodiments, the cancer cells express CD38 but not CD28.

[0116] 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 preventing and / or treating cancer in a patient (e.g., a patient in need thereof, such as a patient suffering from 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 preparing a medicament for preventing and / or treating cancer in a patient (e.g., a patient in need thereof, such as a patient suffering from cancer). In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein comprising a first antigen-binding site that binds CD3, a second antigen-binding site that binds CD28, and a third antigen-binding site that binds the extracellular domain of the 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 a human. In some embodiments, the patient is selected for treatment because the cancer cells express the 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 are further described by the following detailed description.

[0118] The present invention includes:

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

[0120] (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

[0121] (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).

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

[0123] (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

[0124] (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).

[0125] 3. The binding protein of item 1 or item 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).

[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 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.

[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 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.

[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 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.

[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 item 10, wherein the binding protein comprises an antibody heavy chain containing the amino acid sequence of SEQ ID NO: 24 and an antibody light chain containing the amino acid sequence of SEQ ID NO: 20.

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

[0135] (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence containing the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence containing 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 containing the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence containing the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

[0137] 13. The binding protein of item 1 or item 12, 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).

[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 item 14, wherein the binding protein comprises an antibody heavy chain containing the amino acid sequence of SEQ ID NO:15 and an antibody light chain containing the amino acid sequence of SEQ ID NO:16.

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

[0141] (a) An antibody heavy chain variable (VH) domain that comprises a CDR-H1 sequence containing the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence containing the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence containing the amino acid sequence ARMFRGAFDY (SEQ ID NO:43); and

[0142] (b) An antibody light chain variable (VL) domain comprising a CDR-L1 sequence having an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence having an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence having an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46).

[0143] 17. The binding protein of item 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 having the amino acid sequence of SEQ ID NO:11 and an antibody light chain having the amino acid sequence of SEQ ID NO:12.

[0145] 19. The binding protein of any one of items 1-18, 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 item 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, and the equilibrium dissociation constant (K D ) is 2.1 nM or less.

[0148] 22. The binding protein of item 19, wherein the antigen-binding site binds to a human isotype 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 item 23, wherein the antigen-binding site binds to the cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30, and the 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 according to any one of items 16 - 24, wherein the binding protein is a human antibody.

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

[0154] 28. The binding protein according to 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 according to item 28, wherein the Fc region is a human Fc region, which comprises one or more mutations that reduce or eliminate Fc receptor binding and / or effector functions of the Fc region.

[0156] 30. The binding protein according to item 28, wherein the Fc region is a human IgG1 Fc region.

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

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

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

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

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

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

[0163] 37. The binding protein according to 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 according to any one of items 1-37, wherein the binding protein is conjugated to a cytotoxic agent or a label.

[0165] 39. The binding protein according to any one of items 1-37, wherein the binding protein is a bispecific binding protein that comprises a first antigen-binding site that binds to a CD38 polypeptide and a second antigen-binding site.

[0166] 40. The binding protein according to any one of items 1-37, wherein the binding protein is a trispecific binding protein that comprises a first antigen-binding site, a second antigen-binding site and a third antigen-binding site that each bind to a CD38 polypeptide.

[0167] 41. The binding protein according to item 40, wherein 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.

[0168] 42. The binding protein according to item 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 that comprises three antigen-binding sites that each bind 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.

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

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

[0172] 46. The binding protein according to any one of items 43-45, wherein 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.

[0173] 47. The binding protein of item 46, wherein 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.

[0174] 48. The binding protein of items 43-47, wherein the binding protein comprises 4 polypeptide chains that form the 3 antigen-binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0175] V L2 -L 1 -V L1 -L 2 -C L [I]

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

[0177] V H1 -L 3 -V H2 -L 4 -C H1 -hinge-C H2 -C H3 [II]

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

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

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

[0181] V L3 -C L [IV]

[0182] wherein:

[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 H2is the second immunoglobulin heavy chain variable domain;

[0188] V H3 is the third immunoglobulin heavy chain variable domain;

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

[0190] C H1 is immunoglobulin C H1 heavy chain constant domain;

[0191] C H2 is immunoglobulin C H2 heavy chain constant domain;

[0192] C H3 is immunoglobulin C H3 heavy chain constant domain;

[0193] The hinge is the immunoglobulin hinge region that connects the said C H1 and C H2 domains, and

[0194] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

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

[0196] wherein:

[0197] (a) the said V H1 domain contains a CDR - H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR - H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR - H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the said V L1 domain contains a CDR - L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR - L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR - L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36);

[0198] (b) The V H2 domain comprises 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 the V L2 domain comprises 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); or

[0199] (c) The V H3 domain comprises 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 the V L3 domain comprises 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).

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

[0201] (a) The V H1The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and said V L1 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36);

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

[0203] (c) Said V H2 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and said V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36);

[0204] (d) Said V H2The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and said V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36);

[0205] (e) said V H3 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and said V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36); or

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

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

[0208] (a) said V H3The domain contains a CDR-H1 sequence with the amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with the amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with the amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and the said V L3 The domain contains a CDR-L1 sequence with the amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with the amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with the amino acid sequence containing QQNKEDPWT (SEQ ID NO:36); or

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

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

[0211] (a) The said V H3 The domain contains the amino acid sequence of SEQ ID NO:5, and the said V L3 The domain contains the amino acid sequence of SEQ ID NO:6;

[0212] (b) The said V H3 The domain contains the amino acid sequence of SEQ ID NO:17, and the said V L3 The domain contains the amino acid sequence of SEQ ID NO:18;

[0213] (c) The said V H3 The domain contains the amino acid sequence of SEQ ID NO:21, and the said V L3 The domain contains the amino acid sequence of SEQ ID NO:18;

[0214] (d) The said V H3The domain contains the amino acid sequence of SEQ ID NO:23, and the V L3 The domain contains the amino acid sequence of SEQ ID NO:18; or

[0215] (e) The V H3 The domain contains the amino acid sequence of SEQ ID NO:13, and the V L3 The domain contains the amino acid sequence of SEQ ID NO:14.

[0216] 52. The binding protein of items 43 - 47, wherein the binding protein comprises 4 polypeptide chains forming the 3 antigen - binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0217] V L2 -L 1 -V L1 -L 2 -C L [I]

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

[0219] V H1 -L 3 -V H2 -L 4 -C H1 -hinge - C H2 -C H3 [II]

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

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

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

[0223] V L3 -C L [IV]

[0224] wherein:

[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 the second immunoglobulin heavy chain variable domain;

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

[0231] C L is the immunoglobulin light chain constant domain;

[0232] C H1 is the immunoglobulin C H1 heavy chain constant domain;

[0233] C H2 is the immunoglobulin C H2 heavy chain constant domain;

[0234] C H3 is the immunoglobulin C H3 heavy chain constant domain;

[0235] The hinge is the immunoglobulin hinge region that connects the said C H1 and C H2 domains, and

[0236] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

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

[0238] wherein:

[0239] (a) The said V H1 domain contains a CDR - H1 sequence with an amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR - H2 sequence with an amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR - H3 sequence with an amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43); and the said V L1 domain contains a CDR - L1 sequence with an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR - L2 sequence with an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR - L3 sequence with an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46);

[0240] (b) The said VH2 The domain contains a CDR-H1 sequence with the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence with the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence with the amino acid sequence ARMFRGAFDY (SEQ ID NO:43); and the V L2 The domain contains a CDR-L1 sequence with the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence with the amino acid sequence AAS (SEQ ID NO:45), and a CDR-L3 sequence with the amino acid sequence LQDYIYYPT (SEQ ID NO:46); or

[0241] (c) The V H3 The domain contains a CDR-H1 sequence with the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence with the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence with the amino acid sequence ARMFRGAFDY (SEQ ID NO:43); and the V L3 The domain contains a CDR-L1 sequence with the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence with the amino acid sequence AAS (SEQ ID NO:45), and a CDR-L3 sequence with the amino acid sequence LQDYIYYPT (SEQ ID NO:46).

[0242] 53. The binding protein of item 52, wherein the V H3 The domain contains a CDR-H1 sequence with the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence with the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence with the amino acid sequence ARMFRGAFDY (SEQ ID NO:43); and the V L3 The domain contains a CDR-L1 sequence with the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence with the amino acid sequence AAS (SEQ ID NO:45), and a CDR-L3 sequence with the amino acid sequence LQDYIYYPT (SEQ ID NO:46).

[0243] 54. The binding protein of item 53, wherein the V H3 The domain contains the amino acid sequence of SEQ ID NO:9, and the V L3The domain contains the amino acid sequence of SEQ ID NO:10.

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

[0245] (a) The V H1 domain contains the amino acid sequence of SEQ ID NO:49, the V L1 domain contains the amino acid sequence of SEQ ID NO:50, the V H2 domain contains the amino acid sequence of SEQ ID NO:53, and the V L2 domain contains the amino acid sequence of SEQ ID NO:54;

[0246] (b) The V H2 domain contains the amino acid sequence of SEQ ID NO:49, the V L2 domain contains the amino acid sequence of SEQ ID NO:50, the V H1 domain contains the amino acid sequence of SEQ ID NO:53, and the V L1 domain contains the amino acid sequence of SEQ ID NO:54;

[0247] (c) The V H1 domain contains the amino acid sequence of SEQ ID NO:51, the V L1 domain contains the amino acid sequence of SEQ ID NO:52, the V H2 domain contains the amino acid sequence of SEQ ID NO:53, and the V L2 domain contains the amino acid sequence of SEQ ID NO:54;

[0248] (d) The V H2 domain contains the amino acid sequence of SEQ ID NO:51, the V L2 domain contains the amino acid sequence of SEQ ID NO:52, the V H1 domain contains the amino acid sequence of SEQ ID NO:53, and the V L1 domain contains the amino acid sequence of SEQ ID NO:54;

[0249] (e) The V H1 domain contains the amino acid sequence of SEQ ID NO:49, the V L1 domain contains the amino acid sequence of SEQ ID NO:50, the V H2 domain contains the amino acid sequence of SEQ ID NO:84, and the V L2The domain contains the amino acid sequence of SEQ ID NO:85;

[0250] (f) The V H2 The domain contains the amino acid sequence of SEQ ID NO:49, and the V L2 The domain contains the amino acid sequence of SEQ IDNO:50, and the V H1 The domain contains the amino acid sequence of SEQ ID NO:84, and the V L1 The domain contains the amino acid sequence of SEQ ID NO:85;

[0251] (g) The V H1 The domain contains the amino acid sequence of SEQ ID NO:51, and the V L1 The domain contains the amino acid sequence of SEQ IDNO:52, and the V H2 The domain contains the amino acid sequence of SEQ ID NO:84, and the V L2 The domain contains the amino acid sequence of SEQ ID NO:85; or

[0252] (h) The V H2 The domain contains the amino acid sequence of SEQ ID NO:51, and the V L2 The domain contains the amino acid sequence of SEQ IDNO:52, and the V H1 The domain contains the amino acid sequence of SEQ ID NO:84, and the V L1 The domain contains the amino acid sequence of SEQ ID NO:85.

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

[0254] (a) The V H1 The domain contains the amino acid sequence of SEQ ID NO:49, and the V L1 The domain contains the amino acid sequence of SEQ IDNO:50, and the V H2 The domain contains the amino acid sequence of SEQ ID NO:53, and the V L2 The domain contains the amino acid sequence of SEQ ID NO:54, and the V H3 The domain contains the amino acid sequence of SEQ ID NO:13, and the V L3 The domain contains the amino acid sequence of SEQ ID NO:14; or

[0255] (b) The V H1 The domain contains the amino acid sequence of SEQ ID NO:49, and the V L1The domain contains the amino acid sequence of SEQ ID NO: 50, and said V H2 The domain contains the amino acid sequence of SEQ ID NO: 53, and said V L2 The domain contains the amino acid sequence of SEQ ID NO: 54, and said V H3 The domain contains the amino acid sequence of SEQ ID NO: 9, and said V L3 The domain contains the amino acid sequence of SEQ ID NO: 10.

[0256] 57. The binding protein according to any one of items 48 - 56, wherein L 1 、L 2 、L 3 or L 4 The length of at least one of them is independently 0 amino acids.

[0257] 58. The binding protein according to any one of items 48 - 56, wherein L 1 、L 2 、L 3 or L 4 The length of each of them is independently at least one amino acid.

[0258] 59. The binding protein according to any one of items 48 - 56, wherein (a) L 1 、L 2 、L 3 and L 4 Each independently has a length of 0 amino acids or contains 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) L 1 、L 2 、L 3 and L 4 Each independently contains 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 according to any one of items 48 - 56, wherein

[0260] (a) L 1Comprising the sequence GQPKAAP (SEQ ID NO:58), L 2 Comprising the sequence TKGPS (SEQ ID NO:57), L 3 Comprising the sequence S, and L 4 Comprising the sequence RT;

[0261] (b)L 1 Comprising the sequence GGGGSGGGGS (SEQ ID NO:55), L 2 Comprising the sequence GGGGSGGGGS (SEQ ID NO:55), L 3 has a length of 0 amino acids, and L 4 has a length of 0 amino acids;

[0262] (c)L 1 Comprising the sequence GGSGSSGSGG (SEQ ID NO:59), L 2 Comprising the sequence GGSGSSGSGG (SEQ ID NO:59), L 3 has a length of 0 amino acids, and L 4 has a length of 0 amino acids; or

[0263] (d)L 1 Comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L 2 has a length of 0 amino acids, L 3 Comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), and L 4 has a length of 0 amino acids.

[0264] 61. The binding protein according to any one of items 48 - 60, wherein the hinge - C of the second and the third polypeptide chains H2 -C H3 domain is a human IgG4 hinge - C H2 -C H3 domain, and wherein according to the EU Index, the hinge - C H2 -C H3 domains each contain 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 according to any one of items 48 - 60, wherein the hinge - C of the second and the third polypeptide chains H2 -C H3 domain is a human IgG4 hinge - C H2 -C H3domain, and wherein according to the EU Index, the hinge-C H2 -C H3 domains each contain 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.

[0266] 63. The binding protein of any one of items 48-60, wherein the hinge-C H2 -C H3 domain of the second and the third polypeptide chains is the human IgG4 hinge-C H2 -C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 domains each contain 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 H2 -C H3 domain of the second and the third polypeptide chains is the human IgG4 hinge-C H2 -C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 domains each contain 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 domain of the second and the third polypeptide chains is the human IgG4 hinge-C H2 -C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 domains each contain 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 H3The domain contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V, and wherein according to the EU Index, the hinge-C of the third polypeptide chain H2 -C H3 The domain contains 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 of the second polypeptide chain H2 -C H3 The domain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W; and wherein according to the EU Index, the hinge-C of the third polypeptide chain H2 -C H3 The domain contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4, 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 that comprises three antigen-binding sites each binding one or more target proteins, wherein the binding protein comprises four polypeptide chains that form the three antigen-binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0278] V L2 -L 1 -V L1 -L 2 -C L [I]

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

[0280] V H1 -L 3 -V H2 -L 4 -C H1 -hinge-C H2 -C H3 [II]

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

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

[0283] The fourth polypeptide chain contains a structure represented by the following formula:

[0284] V L3 -C L [IV]

[0285] Where:

[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 the 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 is the immunoglobulin C H1 heavy chain constant domain;

[0294] C H2 is the immunoglobulin C H2 heavy chain constant domain;

[0295] C H3 is the immunoglobulin C H3 heavy chain constant domain;

[0296] The hinge is the immunoglobulin hinge region connecting the C H1 and C H2 domains; and

[0297] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

[0298] Wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain - heavy chain pair; and

[0299] Where:

[0300] (a) The hinge - C of the second and the third polypeptide chainsH2 -C H3 The domain is the hinge-C of human IgG1 H2 -C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 domains each contain 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; or

[0301] (b) The hinge-C of the second and the third polypeptide chains H2 -C H3 domain is the C domain of human IgG4 H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 domains each contain 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.

[0302] 75. The binding protein of item 74, wherein V H1 and V L1 、V H2 and V L2 as well as V H3 and V L3 at least one pair of which form 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 as well as V H3 and V L31, 2 or 3 pairs thereof form antigen-binding sites that bind to antigen targets 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, 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, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, 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] The binding protein of item 74, wherein V H1 and V L1 、V H2 and V L2 as well as V H3 and V L3 of the first pair form an antigen-binding site that binds to the human CD3 polypeptide, V H1 and V L1 、V H2 and V L2 as well as V H3 and V L3 of the second pair form an antigen-binding site that binds to the human CD28 polypeptide, and V H1 and V L1 、V H2 and V L2 as well as V H3 and V L3The third pair forms an antigen-binding site that binds to 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, 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, 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, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, 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 kit of polynucleotides, 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 a binding protein according to any one of items 1-77.

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

[0314] 81. A host cell comprising the kit of polynucleotides of item 78, the polynucleotide of item 79, or the 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 item 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 item 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 item 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 the cancer cells express human CD38 isoform E polypeptide on their cell surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0326] This patent or application document contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fees.

[0327] Figure 1AShown is the binding 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 using flow cytometry.

[0328] Figure 1B Shown are the results of a flow cytometry binding assay detecting the binding of the anti-CD38 antibody mAb1 or isatuximab (no binding was observed) to cells expressing cynomolgus monkey CD38 on the surface.

[0329] Figures 2A - 2I Shown are the results of assays characterizing the binding of anti-CD38 antibodies to human and cynomolgus monkey CD38 polypeptides. Figure 2A Shown is the binding of the humanized anti-CD38 antibody mAb2 to soluble human CD38 (upper panel, “hCD38::Histag”) or cynomolgus monkey CD38 (upper panel, “cynoCD38::Histag”) by ELISA, and the binding of mAb2 to the cell surface expressing human CD83 (lower panel, as shown) or cynomolgus monkey CD38 (lower panel, as shown) by flow cytometry. Figure 2B Shown is the binding of mAb2 to human CD38 (upper panel) or cynomolgus monkey CD38 (lower panel) by surface plasmon resonance (SPR). Figure 2C Shown is the binding of the humanized anti-CD38 antibody mAb3 to soluble human CD38 (upper panel, “hCD38::Histag”) or cynomolgus monkey CD38 (upper panel, “cynoCD38::Histag”) by ELISA, and the binding of mAb3 to the cell surface expressing human CD83 (lower panel, as shown) or cynomolgus monkey CD38 (lower panel, as shown) by flow cytometry. Figure 2D Shown is the binding of mAb3 to human CD38 (upper panel) or cynomolgus monkey CD38 (lower panel) by SPR. Figure 2E Shown is the binding of the humanized anti-CD38 antibody mAb5 to soluble human CD38 (upper panel, “hCD38::Histag”) or cynomolgus monkey CD38 (upper panel, “cynoCD38::Histag”) by ELISA, and the binding of mAb5 to the cell surface expressing human CD83 (lower panel, as shown) or cynomolgus monkey CD38 (lower panel, as shown) by flow cytometry. Figure 2F Shown is the binding of mAb5 to human CD38 (upper panel) or cynomolgus monkey CD38 (lower panel) by SPR. Figure 2GShown is the binding of the human anti-CD38 antibody hhy1370 to soluble human CD38 (upper panel, “hCD38::Histag”) or cynomolgus monkey CD38 (upper panel, “cynoCD38::Histag”) by ELISA, and the binding of hhy1370 to the cell surface expressing human CD83 (lower panel, as indicated) or cynomolgus monkey CD38 (lower panel, as indicated) by flow cytometry. Figure 2H Shown is the binding of hhy1370 to human CD38 (upper panel) or cynomolgus monkey CD38 (lower panel) by SPR. Figure 2I Summarized are the binding data from ELISA, SPR, and FACS experiments, as well as the percentage identity of each antibody VH (“H”) or VL (“L”) domain with the human V region sequences from top to bottom, corresponding to the last row of mAb2, mAb3, mAb4, mAb5, and mAb6 (1195HHKK-3 is not described by its VL / VH amino acid sequence below).

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

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

[0332] Figure 2L Shown is the concentration-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isatuximab (right) and mAb1 (left) against SU-DHL-8 cells in the presence of NK92 cells after incubation at 37 °C for 4 hours.

[0333] Figures 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 annexin V and propidium iodide uptake via flow cytometry. Figure 2M Shown is the percentage of apoptotic cells induced by each antibody. Figures 2N - 2Q Shown is the anti-CD38 antibody mAb2 ( Figure 2N ), mAb3 ( Figure 2O ), mAb4 ( Figure 2P ), and mAb5 ( Figure 2Q ) dose-dependent induction of apoptosis of SU-DHL-8 lymphoma cells, and the IC50 of each antibody.

[0334] Figure 3ASchematic of a trispecific binding protein comprising four polypeptide chains forming three antigen-binding sites that bind the following three target proteins: CD28, CD3, and CD38. The first pair of polypeptides has cross-oriented dual variable domains (VH1-VH2 and VL2-VL1) that form two antigen-binding sites that recognize CD3 and CD28, and the second pair of polypeptides has single variable domains (VH3 and VL3) that form a single antigen-binding site that recognizes CD38. Figure 3A The trispecific binding protein shown in Figure 3A uses an IgG4 constant region with "knobs-into-holes" mutations, where the knobs are on the second pair of polypeptides with single variable domains.

[0335] Figure 3B Schematic 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.

[0336] Figure 3C Results of an SPR-based assay showing the binding of human CD38 to an anti-CD38 / anti-CD28 / anti-CD3 trispecific binding protein are shown. Binding of human CD38 to the trispecific binding protein alone (upper left) was examined after pre-binding CD3 (upper middle), after pre-binding CD28 (upper right), after pre-binding CD3 then CD28 (lower left), or after pre-binding CD28 then CD3 (lower right).

[0337] Figure 4 Sequential binding of human CD3, CD28, and CD38 polypeptides to an anti-CD38 / anti-CD28 / anti-CD3 trispecific binding protein is shown, as measured by SPR assay.

[0338] Figure 5 Binding affinities of the shown trispecific binding protein for its cognate antigens (human CD3, CD28, and CD38), as measured by SPR, are summarized.

[0339] Figure 6A Apparent affinities of the isatuximab antigen-binding domain in human IgG1 form (second panel) or trispecific binding protein form for isatuximab, anti-CD28, and anti-CD3 antigen-binding domains are compared (in the form of Figure 3A ; first panel) for binding to human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptides, as measured by flow cytometry.

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

[0341] Figure 6E Compared the trispecific binding protein CD38 HHY1370 xCD28 sup xCD3 mid or the apparent affinity of the monospecific anti-CD38 antibody mAb6 to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide, as determined by flow cytometry.

[0342] Figure 6F Summarized the binding affinities of the indicated anti-CD38 x anti-CD28 x anti-CD3 trispecific binding proteins to human CD38, as measured by SPR or flow cytometry (FACS).

[0343] Figure 6G Showing the trispecific binding protein ΔCD38 lacking the anti-CD38 antigen-binding domain VH1 xCD28 sup xCD3 mid for the apparent affinity to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide, as determined by flow cytometry.

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

[0345] Figures 8A - 8DResults of antibody-mediated CD38 cell-specific killing of PBMCs from three different human donors using the indicated anti-CD38 x CD28 x CD3 trispecific binding protein and control antibodies. + Results are shown using the multiple myeloma cell lines RPMI8266 ( Figure 8A ), NCI-H929 ( Figure 8B ), KMS-26 ( Figure 8C ), and KMS-11 cell lines ( Figure 8D ), and EC50 values are provided in Table N. EC50 values obtained using NCI-H929, KMS-26, and KMS-11 cells are provided in Tables O - Q.

[0346] Figure 8E and Figure 8F Results of antibody-mediated CD38 cell-specific killing of PBMCs from two different donors using the indicated anti-CD38 x CD28 x CD3 trispecific binding protein with variant Fc region and control antibodies. + Results are shown using + 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 Show activation (CD69 + ) of human T cells treated with various anti-CD38 x CD28 x CD3 trispecific binding proteins or control antibodies for 24 hours. Figure 9A Show activation of human CD3 + T cells (CD69 + ). Figure 9B Show activation of human CD3 + CD4 + T cells (CD69 + ). Figure 10 Show activation of human CD3 + CD8 + T cells (CD69 + ).

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

[0349] Figures 12A - 12E Shows the in vivo activity of an anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) trispecific binding protein in a humanized NSG mouse model implanted with RPMI-8226 multiple myeloma cells. Figure 12A Shows the change in tumor volume of mice treated with the indicated concentration of anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) trispecific binding protein relative to mice treated with an anti-CD3 / CD38 bispecific antibody. Figure 12B Shows the average tumor volume on day 18 of mice treated with the indicated concentration of CD38 (VHI) xCD28 (sup) x CD3 (mid) trispecific binding protein relative to mice treated with an anti-CD3 / CD38 bispecific antibody. Figure 12C Shows the average terminal tumor weight of mice treated with the indicated concentration of anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) trispecific binding protein relative to mice treated with an anti-CD3 / CD38 bispecific antibody. Figure 12D Shows the average tumor growth curve over the length of the experiment of mice treated with the indicated concentration of anti-CD38 (VHI) xCD28 (sup) x CD3 (mid) trispecific binding protein relative to mice treated with an anti-CD3 / CD38 bispecific antibody. Figure 12E Shows the average change in body weight at multiple time points over the length of the experiment of mice treated with the indicated concentration of anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) trispecific binding protein relative to mice treated with an anti-CD3 / CD38 bispecific antibody.

[0350] Figures 13A - 13F Shows the in vivo activity of an anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) trispecific binding protein in a humanized NSG mouse model implanted with PBMC and RPMI-8226 multiple myeloma cells.Figure 13A Shown with anti-CD38 at the indicated concentration (VHI) x CD28 (sup) x CD3 (mid) Change in tumor volume of mice treated with trispecific binding protein relative to mice treated with anti-CD3 / CD38 bispecific antibody. Figure 13B Shown with anti-CD38 at the indicated concentration (VHI) x CD28 (sup) xCD3 (mid) Tumor volume of mice treated with trispecific binding protein relative to mice treated with anti-CD3 / CD38 bispecific antibody on day 4. Figure 13C Shown with anti-CD38 at the indicated concentration (VHI) x CD28 (sup) x CD3 (mid) Tumor volume of mice treated with trispecific binding protein relative to mice treated with anti-CD3 / CD38 bispecific antibody on day 21. Figure 13D Shown with anti-CD38 at the indicated concentration (VHI) x CD28 (sup) xCD3 (mid) Average tumor volume of mice treated with trispecific binding protein relative to mice treated with anti-CD3 / CD38 bispecific antibody on day 21. Figure 13E Shown with anti-CD38 at the indicated concentration (VHI) x CD28 (sup) x CD3 (mid) Average terminal tumor weight of mice treated with trispecific binding protein relative to mice treated with anti-CD3 / CD38 bispecific antibody. Figure 13F Shown with anti-CD38 at the indicated concentration (VHI) x CD28 (sup) xCD3 (mid) Average tumor volume of mice treated with trispecific binding protein relative to mice treated with anti-CD3 / CD38 bispecific antibody at multiple time points during the length of the experiment.

[0351] Figures 14A - 14U Shown using anti-CD38 in non-human 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 dose escalation study of trispecific binding protein. Figure 14A Showing circulating CD3 (VHI) x CD28 (sup) x CD3 (mid) T cell activation (CD69 + of circulating CD3 T cells after administration of trispecific binding protein. + ) Figure 14B Showing circulating CD3 (VHI) x CD28 (cvn) x CD3 (mid) T cell activation (CD69 + of circulating CD3 T cells after administration of trispecific binding protein. + ) Figure 14C Showing administration of different doses of anti-CD38 (hhy1370) x CD28 (sup) xCD3 (mid) T cell activation (CD69 + of circulating CD3 T cells after administration of trispecific binding protein. + ) Figure 14D Showing circulating CD3 (hhy1370) x CD28 (cvn) x CD3 (mid) T cell activation (CD69 + of circulating CD3 T cells after administration of trispecific binding protein. + ) Figure 14E Showing administration of the indicated dose of anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) Change in percentage of circulating CD4 + T cells after administration of trispecific binding protein. Figure 14F Showing administration of the indicated dose of anti-CD38 (VHI) x CD28 (sup) xCD3 (mid) Change in percentage of circulating CD8 + T cells after administration of trispecific binding protein. Figure 14G Showing use of the indicated dose of anti-CD38 (VHI) xCD28 (cvn) x CD3 (mid) Change in percentage of circulating CD4 + T cells after administration of trispecific binding protein. Figure 14H Showing administration of the indicated dose of anti-CD38 (VHI) x CD28 (cvn) xCD3 (mid) Change in percentage of circulating CD8 T cells after administration of trispecific binding protein.+ Change in percentage of T cells. Figure 14I Showing the administration of the indicated dose of anti-CD38 (hhy1370) x CD28 (sup) x CD3 (mid) tri-specific binding protein on the change in the percentage of circulating CD4 + T cells. Figure 14J Showing the administration of the indicated dose of anti-CD38 (hhy1370) xCD28 (sup) x CD3 (mid) tri-specific binding protein on the change in the percentage of circulating CD8 + T cells. Figure 14K Showing the administration of the indicated dose of anti-CD38 (hhy1370) xCD28 (cvn) x CD3 (mid) tri-specific binding protein on the change in the percentage of circulating CD4 + T cells. Figure 14L Showing the administration of the indicated dose of anti-CD38 (hhy1370) x CD28 (cvn) x CD3 (mid) tri-specific binding protein on the change in the percentage of circulating CD8 + T cells. Figure 14M Showing the change in total CD4 + T cells at 6, 24, and 48 hours after administration of 12.5 μg / kg of the indicated tri-specific binding protein. Figure 14N Showing the change in total NK cells at 6, 24, and 48 hours after administration of 12.5 μg / kg of the indicated tri-specific binding protein. Figure 14O Showing the change in total CD8 + T cells at 6, 24, and 48 hours after administration of 12.5 μg / kg of the indicated tri-specific binding protein. Figure 14P Showing the change in total B cells at 6, 24, and 48 hours after administration of 12.5 μg / kg of the indicated tri-specific binding protein. Figure 14Q Showing the administration of three increasing doses (0.5, 2.5, 12.5 μg / kg) of anti-CD38 (VH1) x CD28 (sup) xCD3 (mid) tri-specific binding protein on the change in cytokine levels at 6 hours (results from different test animals labeled "117065" and "117066"). Figure 14R Showing the administration of 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 the trispecific binding protein (results from different test animals labeled "117067" and "117068"). Figure 14S Showing administration of 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 the trispecific binding protein (results from different test animals labeled "117069" and "117070"). Figure 14T Showing administration of 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 the trispecific binding protein (results from different test animals labeled "117071" and "117072"). Figure 14U Showing 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 protein (results shown for all test animals).

[0352] Figure 14V and Figure 14W Showing anti-CD38 (VHI) x CD28 (sup) x CD3 (mid) and anti-CD38 (VHI) xCD28 (cvn) xCD3 (mid) The trispecific binding protein induces in vivo depletion of T cells in the blood of non-human primates at higher doses (6 hours after administration).

[0353] Figure 14X and Figure 14Y Showing anti-CD38 (HHY1370) x CD28 (sup) x CD3 (mid) and anti-CD38 (HHY1370 x CD28 (cvn) x CD3 (mid) The trispecific binding protein induces in vivo depletion of T cells in the blood of non-human primates at higher doses (6 hours after administration).

[0354] Figure 14Z and Figure 14AA Showing 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 over time in non - human primates after a trispecific binding protein.

[0355] Figure 14AB and Figure 14AC Showing 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 over time in non - human primates after a trispecific binding protein.

[0356] Figure 14AD and Figure 14AE Showing the amount of CD4+ T cells with bound trispecific binding protein in non - human primates after administration of a 100 μg / kg dose.

[0357] Figure 14AF and Figure 14AG Showing the amount of CD8+ T cells with bound trispecific binding protein in non - human primates after administration of a 100 μg / kg dose.

[0358] Figures 15A - 15C Showing the binding or lack thereof of various Fc variants to human Fc receptors FcγR I ( Figure 15A ), FcγR IIa ( Figure 15B ), and FcγR IIIb / c ( Figure 15C ). The variants tested were human IgG1, human IgG4, and human IgG4 with the FALA mutation.

[0359] Figure 16 Showing the binding of human IgG4 with or without the FALA mutation to FcRn.

[0360] Figure 17 Summarizes the trispecific binding proteins shown in NSG mice (CD38 VH1 xCD28 sup xCD3 mid IgG4, CD38 VH1 xCD28 sup xCD3 mid IgG4 FALA, CD38 VH1 xCD28 sup xCD3 mid IgG1 LALA P329A and CD38 HHY1370 xCD28 sup xCD3 midPK parameters of IgG4 FALA).

[0361] Figures 18A - 18C Fc / FcR interaction-mediated (non-specific) IFN-γ ( Figure 18A ) and IL-2 ( Figure 18B ) or TNF-α ( Figure 18C ) release in human PBMCs incubated with trispecific heterobivalent proteins with wild-type or FALA variant Fc regions.

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

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

[0364] Figure 19C and Figure 19D Show that CD38 with IgG4 FALA variant Fc VH1 xCD28xCD3 upregulates Bcl-xL in CD4+ ( Figure 19C ) or CD8+ ( Figure 19D ) T cells more than the reference bispecific antibody. Bar graphs = mean and s.d. from 3 PBMC donors. *p = ≤0.045

[0365] Figure 19E Show that T cell activation of anti-CD38 x anti-CD28 x anti-CD3 trispecific binding protein measured by IL-2 expression in Jurkat T cell reporter cell line depends on the anti-CD3 antigen-binding domain.

[0366] Figure 19F Show that CD38 compared with binding proteins with mutated anti-CD28, anti-CD38 or anti-CD28, anti-CD38 and anti-CD3 and the referenceVH1 xCD28 sup xCD3 mid The trispecific binding protein releases cytokines TNF, IFNg, IL-2, IL-6, and IL-10.

[0367] Figure 19G Shows the proliferation of T cells activated by an anti-CD38xanti-CD28xanti-CD3 trispecific binding protein with an IgG4 FALA variant Fc, a benchmark anti-CD38xanti-CD3 bispecific antibody, or an isotype control (trispecific binding protein with an IgG4 FALA variant Fc with a mutated binding domain).

[0368] Figure 20 Shows 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 a mutation in the CD38, CD28, or CD3 antigen-binding domain, or an isotype control (trispecific binding protein with an IgG4 FALA variant Fc with three mutated binding domains).

[0369] Figure 21 Shows CD38 administered at the indicated doses in the NCI-H929-Luc disseminated tumor model in PBMC-humanized NSG mice VH1 xCD28 sup xCD3 mid The in vivo antitumor activity of the IgG4 FALA trispecific binding protein.

[0370] Figure 22 Shows CD38 administered at the indicated doses in the NCI-H929-Luc disseminated tumor model in PBMC-humanized NSG mice HHY1370 xCD28 sup xCD3 mid The in vivo antitumor activity of the IgG4 FALA trispecific binding protein.

[0371] Figure 23A and Figure 23B Shows the use of human PBMCs with CD38 used in in vivo studies VH1 xCD28 sup xCD3 mid and CD38 HHY1370 xCD28 sup xCD3 midThe effective in vitro tumor killing activity of trispecific binding proteins and a benchmark anti-CD38xanti-CD3 bispecific antibody against NCI-929-Luc cells. Human PBMCs from 2 donor humanized NSG mice were used after incubation for 24 hours at an effector:PBMC ratio of 10:1.

[0372] Figure 23C Shows CD38 administered at the indicated doses in a disseminated tumor model of NCI-H929-Luc 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 The excellent in vivo anti-tumor activity of the trispecific binding protein. The binding protein was administered by intraperitoneal (IP) injection at 30 μg / kg weekly.

[0373] Figure 24A Shows the results of a luciferase reporter gene assay using GloResponse TM IL2-luc2P Jurkat cells (Promega) stimulated with CD38 VH1 / CD28 sup xCD3 mid and its single binding site KO and triple KO mutants at a concentration of 10 nM.

[0374] Figure 24B Shows the optimization of the anti-CD3xCD28 CODV-Fab antibody. The optimal configuration of α-CD3 and α-CD28 in alternative positions of the CODV bispecific Fab was evaluated by a cytokine release assay using human PBMCs in vitro. Based on the secretion of IFN-γ and IL-2 in the supernatant after 24 hours, distal CD28 x proximal CD3 was determined to be the optimal orientation.

[0375] Figure 25 Shows that CD38 VH1 / CD28 sup xCD3 mid Induces upregulation of the Bcl-2 family member Bcl-xL in primary T cells in a CD28-dependent manner.

[0376] Figure 26 Shows that the anti-CD28 in the trispecific Ab provides the secondary signaling necessary to support primary T cell proliferation in vitro.

[0377] Figure 27Shows the configuration of the trispecific antibody color-coded by the parental antibody (left). Dark tones (purple or green) represent heavy chain peptides; light tones represent light chain peptides. Also shown is based on anti-CD38 VH1Fab and CD28 sup / CD3 mid COD VFab crystal structure of CD38 VH1 / CD28 sup xCD3 mid Structural model of the trispecific antibody (right).

[0378] Figure 28A and Figure 28B Show that multiple myeloma (MM) cells with high (RPMI-8226; Figure 28A ) and low (KMS-11; Figure 28B ) CD38 surface expression are effectively lysed by human PBMC incubated with various concentrations of the trispecific Ab (E:T = 10:1). The contribution of each binding site to the killing activity was demonstrated by binding site KO mutations, as shown.

[0379] Figures 29A - 29C Show that the anti-CD28 sup KO mutant of the trispecific Ab shows significantly reduced anti-tumor activity against CD38 high , CD38 mid and CD38 low MM cells. Assays using RPMI-8226 ( Figure 29A ), U266 ( Figure 29B ) or KMS-11 ( Figure 29C ) cells are shown.

[0380] Figure 30 Show that the reduction of tumor burden in the CD38 VH1 / CD28 sup xCD3 mid _FALA trispecific antibody treatment group is dose-dependent and statistically different in a disseminated human multiple myeloma cell line model of NSG mice reconstituted with in vitro expanded human primary T cells.

[0381] Figure 31A and Figure 31B Show important microscopic analysis of myeloma cell lysis by CD38 trispecific Ab in vitro in the presence of primary human T cells. Using human PBMC incubated with the RPMI-8226 myeloma cell line labeled with the CellTracker TM dark red dye, using a negative control (triple KO trispecific; Figure 31A ) or CD38 / CD28xCD3 trispecific Ab (Figure 31B )Performing time-lapse photography of microscopic images. The presented images were collected after 24 hours of incubation. Scale bar: 50 μm.

[0382] Figure 32 Showing substitution mutations in the Fc region of IgG4 prepared for analysis in the Fc receptor binding assay. SEQ ID NO:111 - 116 are shown (from top to bottom respectively).

[0383] Figure 33 Showing the results of the SPR assay for measuring the affinity of the specified IgG4 Fc variants for the indicated human Fc receptors.

[0384] Figure 34 Showing that the CD38 trispecific binding protein with minimal FcR binding reduces non-specific cytokine release in human PBMCs in vitro. Different FcR inactivating mutations (as shown) were analyzed for their pro-inflammatory effects on the human IgG4 isotype. Human PBMCs were incubated in medium (unstimulated) or in the presence of myeloma cells RPMI - 8226 (stimulated), and the bar graph represents the supernatant IFN-γ levels measured by ELISA.

[0385] Figure 35 Showing that the CD38 trispecific binding protein with minimal FcR binding lyses human multiple myeloma cells with different CD38 expression levels. The cytolysis of myeloma cells with IgG4 or the indicated Fc mutations was evaluated in vitro using human PBMCs with the indicated tumor targets.

[0386] Figure 36 Showing a comparison of the in vitro cytolytic activities 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.

[0387] Figures 37A - 37D Showing the characterization of in vitro T cell subset expansion in response to CD38 / CD3xCD28. The evaluation of T cell subset expansion was performed by coating wells with 350 ng / well of the CD38 trispecific Ab in the absence of exogenous cytokines. T cell populations were measured at the indicated time points. The triple mutant trispecific ab was used as a negative control. Flow cytometry was used to determine central and effector memory CD4 T cells ( Figure 37A ), helper T cells ( Figure 37B ), central and effector memory CD8 T cells ( Figure 37C ), and CMV pp65-specific CD8 cells ( Figure 37D)。CMV-specific pp65 effector cells were analyzed by pentamer staining of PBMCs from HLA-A2 CMV+ donors treated with CD38 trispecific or trispecific negative controls.

[0388] Figure 38 Show the contribution of CD28 expression on target cells to the susceptibility of lysis by CD38 / CD3xCD28. CD28 was knocked out in KMS-11 cells using CRISPR / Cas 9 gene targeting and used as a lysis target in vitro. CD38 expression was retained while CD28 was eliminated compared to parental KMS-11, as confirmed by flow cytometry (upper panel, KMS-11 vs. KMS-11 (CD28KO)). The lysis of CD28 KO cells was examined with WT or CD28-null trispecifics (lower panel; trispecific vs. trispecific (CD28KO)).

[0389] Figure 39 Show the cytolytic activity of the CD38 / CD28xCD3 trispecific FALA mutant Ab against the indicated CD38 + CD28 - lines, where the CD38 + CD28 - lines include acute myeloid leukemia (AML (KG-1)), B cell lymphoma (OCI-Ly19), acute T lymphoblastic leukemia (ALL (KOPN8)), and chronic lymphocytic lymphoma (CLL (Z-138)).

[0390] Figure 40 Show that the in vitro activation of human PBMCs by an α-CD28 superagonist requires the bivalency of the antibody. DETAILED DESCRIPTION

[0391] The present disclosure provides a binding protein comprising at least one antigen-binding site that binds a CD38 polypeptide.

[0392] I. GENERAL DEFINITIONS

[0393] As used in accordance with the present disclosure, unless otherwise specified, the following terms shall be understood to have the following meanings. Unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, etc.

[0394] It should be understood that the aspects and embodiments of the present disclosure described herein include "comprising" aspects and embodiments, "consisting of" (the aspects and embodiments), and "consisting essentially of" (the aspects and embodiments).

[0395] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer that is at least 10 nucleotides in length. 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 arabinose and 2',3'-dideoxyribose; and internucleotide bond modifications, such as phosphorothioate, phosphorodithioate, phosphororoselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphororanilidate, and phosphoramidate. The term "polynucleotide" specifically includes DNA in both single-stranded and double-stranded forms.

[0396] "Isolated polynucleotide" is a polynucleotide or certain combinations thereof from genomic, cDNA, or synthetic sources that: (1) is not associated with all or part of the polynucleotide with which it is found in nature, (2) is associated with a polynucleotide to which it is not naturally associated, or (3) does not occur in nature as part of a larger sequence.

[0397] "Isolated polypeptide" is one that: (1) does not contain at least some of the other polypeptides that would normally be 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 bound (by covalent or non-covalent interactions) to polypeptide moieties that are not "isolated polypeptides" naturally associated with it, (6) is operably bound (by covalent or non-covalent interactions) to a polypeptide to which it is not naturally associated, or (7) does not exist in nature. Such isolated polypeptides can be encoded by genomic DNA, cDNA, mRNA, or other RNA from synthetic sources, 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).

[0398] Naturally occurring antibodies typically comprise a tetramer. Each such tetramer typically consists of two pairs of identical polypeptide chains, each pair having one full-length "light chain" (usually having a molecular weight of about 25 kDa) and one full-length "heavy chain" (usually having a molecular weight of about 50 - 70 kDa). As used herein, the terms "heavy chain" and "light chain" refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically comprises a variable domain of about 100 to 110 or more amino acids, which is generally responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines the constant domain responsible for effector function. Thus, in a naturally occurring antibody, the full-length heavy chain immunoglobulin polypeptide comprises a variable domain (V H ) and three constant domains (C H1 , C H2 and C H3 ), where the V H domain is located at the amino terminus of the polypeptide and the C H3 domain is located at the carboxyl terminus, and the full-length light chain immunoglobulin polypeptide comprises a variable domain (V L ) and a constant domain (C L ), where the V L domain is located at the amino terminus of the polypeptide and the C L domain is located at the carboxyl terminus.

[0399] Human light chains are generally classified as kappa and lambda light chains, and human heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon, and the isotypes of antibodies are 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 and constant domains are typically joined 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, e.g., FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair typically form the antigen-binding site. The variable domains of naturally occurring antibodies generally exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also known as complementarity-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 a specific epitope. From the amino terminus to the carboxyl terminus, the light and heavy chain variable domains typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0400] The term "CDR set" refers to a set of three CDRs that occur in a single variable region capable of binding an antigen. Depending on the system, the exact boundaries of these CDRs have been defined differently. 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 a clear residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries that define the three CDRs. These CDRs can be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., 1989, Nature 342:877-83) found that despite great diversity at the amino acid sequence level, certain subparts of the Kabat CDRs adopt almost identical peptide backbone conformations. These subparts are designated as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" denote the light chain and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs overlapping with the 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 the Kabat CDRs, although they may be shortened or lengthened based on the prediction or experimental results of 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 some embodiments use Kabat- or Chothia-defined CDRs. Identifying predicted CDRs using amino acid sequences is well known in the art (such as in Martin, A.C. "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2. Kontermann R., Dübel S., eds. Springer-Verlag, Berlin, p. 33–51 (2010)).The amino acid sequences of the heavy and / or light chain variable domains can also be examined to identify the sequences of the CDRs by other conventional methods (e.g., determining sequence hypervariable regions by comparison to known amino acid sequences of other heavy and light chain variable regions). The numbered sequences can be aligned by eye or by using an alignment program such as one of the CLUSTAL program suites, as described in Thompson, 1994, Nucleic Acids Res. 22:4673-80. Molecular models are typically used to correctly depict the framework and CDR regions and thus correct sequence-based assignments.

[0401] In some embodiments, the CDR / FR definitions in the immunoglobulin light or heavy chains are determined based on the IMGT definitions (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

[0402] As used herein, the term "Fc" refers to a molecule that contains, in monomeric or multimeric form, a sequence of non-antigen-binding fragments produced by digestion of an antibody or otherwise produced, and may include a hinge region. The original immunoglobulin source of native Fc is preferably of human origin and can be any immunoglobulin. The Fc molecule consists of monomeric polypeptides that can be joined into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG4). An example of Fc is the disulfide-bonded dimer produced by papain digestion of IgG. The term "native Fc" as used herein is generic to monomeric, dimeric, and multimeric forms.

[0403] The F(ab) fragment typically includes the V H and C H1 domains of one light chain and one heavy chain, wherein the V H -C H1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, the F(ab) fragment may also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a C H1 domain.

[0404] The F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more constant regions (at C H1 and C H2between domains), so that interchain disulfide bonds can be formed between the two heavy chains to form F(ab') 2 molecule.

[0405] 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, e.g., a CD38 polypeptide of the present disclosure.

[0406] A "recombinant" molecule is a molecule prepared, expressed, produced, or isolated by recombinant methods.

[0407] One embodiment of the present disclosure provides a binding protein that is biologically and immunologically specific 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 containing a nucleotide sequence encoding a polypeptide chain that forms such a binding protein. Yet another embodiment of the present disclosure provides a host cell that expresses such a binding protein (i.e., comprising a nucleic acid molecule or vector encoding a polypeptide chain that forms such a binding protein).

[0408] As used herein, the term "exchangeability" refers to the interchangeability of variable domains within a binding protein form and having retention of folding and ultimate binding affinity. "Complete exchangeability" refers to the ability to exchange the order of the V H1 and V H2 domains of the polypeptide chain of Form I or the polypeptide chain of Form II, and thus exchange the order of the V L1 and V L2 domains (i.e., reverse the order) while maintaining the complete function of the binding protein as evidenced by retention of binding affinity. In addition, it should be noted that the designations V H and V L refer only to the domain positions on a particular protein chain in the final form. For example, V H1 and V H2 can be derived from the V L1 and V L2 domains in a parental antibody and placed in the V H1 and V H2 positions in the binding protein. Similarly, V L1 and V L2 can be derived from the V H1 and V H2 domains in a parental antibody and placed in the V H1 and V H2 positions in the binding protein. Thus, the V H and V L designations refer to the current positions rather than the original positions in the parental antibody. Thus, V H and VL The domain is "exchangeable".

[0409] As used herein, the terms "antigen", "target antigen", or "antigen target" refer to a molecule or a portion of a molecule that can be bound by a binding protein and that can additionally be used in an animal to generate an antibody that can bind to an epitope of the antigen. The target antigen can have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein is capable of competing with a complete antibody that recognizes the target antigen.

[0410] "CD38" is the cluster of differentiation 38 polypeptide and is a glycoprotein found on the surface of many immune cells. In some embodiments, the binding proteins of the present disclosure bind to the extracellular domain of one or more CD38 polypeptides. Exemplary CD38 extracellular domain polypeptide sequences include, but are not limited to, the extracellular domain of human CD38 (e.g., as shown in SEQ ID NO:1) and the extracellular domain of cynomolgus monkey CD38 (e.g., as shown in SEQ ID NO:30).

[0411] The term "T cell conjugate" refers to a binding protein that targets the cytotoxic activity of the host immune system, more specifically T cells, and a binding protein that targets a tumor target protein.

[0412] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

[0413] The term "monovalent binding protein" refers to a binding protein that has one antigen binding site.

[0414] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets. In some embodiments, the bispecific binding protein binds two different antigens. In some embodiments, the bispecific binding protein binds to two different epitopes on the same antigen.

[0415] The term "bivalent binding protein" refers to a binding protein that has two binding sites.

[0416] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets. In some embodiments, the trispecific binding protein binds three different antigens. In some embodiments, the trispecific binding protein binds to one, two, or three different epitopes on the same antigen.

[0417] The term "trivalent binding protein" refers to a binding protein that has three binding sites. In certain embodiments, the trivalent binding protein can bind to one antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets.

[0418] A "separated" binding protein is a protein that has been identified, separated and / or recovered from the components of its natural environment. The 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 protein or non-protein solutes. In some embodiments, the binding protein will be purified: (1) to greater than 95% by weight antibody as determined by the Lowry method, and most preferably greater than 99% by weight; (2) to at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Separated binding proteins include in situ binding proteins within recombinant cells because at least one component of the natural environment of the binding protein is absent.

[0419] As used herein, the terms "substantially pure" or "substantially purified" refer to a compound or substance that is the major species present (i.e., it is more abundant, on a molar basis, than any other individual substance in the composition). In some embodiments, a substantially purified fraction is a composition in which the substance comprises at least about 50% (on a molar basis) of all macromolecular substances present. In other embodiments, a substantially pure composition will comprise greater than about 80%, 85%, 90%, 95% or 99% of all macromolecular substances present in the composition. In other embodiments, the substance is purified to substantial homogeneity (no contaminating substances can be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular substance.

[0420] The term "epitope" includes any determinant capable of specifically binding to an immunoglobulin or T cell receptor, preferably a polypeptide determinant. In certain embodiments, epitope determinants include the chemically reactive surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge characteristics. An epitope is the antigenic region 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 ≤ 10 -8 M, more preferably when the equilibrium dissociation constant ≤ 10 -9 M, and most preferably when the dissociation constant ≤ 10 -10 M, the binding protein is said to specifically bind the antigen.

[0421] The dissociation constant (K D) It can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis uses a BIAcore system (Pharmacia Biosensor; Piscataway, NJ) to measure the real-time binding interaction between a ligand (the target antigen on the biosensor matrix) and an analyte (the binding protein in solution) through surface plasmon resonance (SPR). Surface plasmon analysis can also be performed by immobilizing the analyte (the binding protein on the biosensor matrix) and presenting the ligand (the target antigen). As used herein, the term "K D " refers to the dissociation constant of the interaction between a specific binding protein and a target antigen.

[0422] As used herein when referring to a binding protein, the term "binds" refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope, with a Kd of at least about 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 x 10 -12 M or higher, and / or binds to the epitope with an affinity that is at least two-fold greater than its affinity for a non-specific antigen. In some embodiments, the binding proteins of the present disclosure bind two or more antigens, such as human and cynomolgus monkey CD38 polypeptides.

[0423] In some embodiments, the antigen-binding domain and / or binding protein of the present disclosure "cross-reacts" with human and cynomolgus monkey CD38 polypeptides (such as the CD38 extracellular domain, such as SEQ ID NO:1 (human CD38 isoform A), SEQ ID NO:105 (human CD38 isoform E), and SEQ ID NO:30 (cynomolgus monkey CD38)). A binding protein that binds to antigen 1 (Ag1) "cross-reacts" with antigen 2 (Ag2) when the EC50 is within a similar range for the two antigens. In the present application, a binding protein that binds to Ag1 cross-reacts with Ag2 when the ratio of the affinity for Ag2 to the affinity for Ag1 is equal to or less than 10 (e.g., 5, 2, 1, or 0.5), and the affinity is detected using the same method for both antigens.

[0424] When the affinities of two antigens are very different, the 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, when the binding reaction of the binding protein with Ag2 is less than 5% of the binding reaction of the same binding protein with Ag1 in the same experimental setting and the same antibody concentration, the binding protein that binds to Ag1 does not significantly cross-react with Ag2. In practice, the binding protein concentration used can be the EC50 or the concentration required to reach the saturated steady state obtained with Ag1.

[0425] 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-bivalent immunoglobulin. At the sequence level, linkers are inserted at the junctions between variable domains or at the junctions between variable and constant domains. The junctions between domains can be identified because the approximate sizes of immunoglobulin domains are well understood. The precise location of the domain transition can be determined by locating peptide segments that do not form secondary structure elements such as beta-sheets or alpha-helices, as demonstrated by experimental data, or can be hypothesized by modeling techniques or secondary structure prediction. The linkers described herein are designated L 1 , which is located on the light chain between the C-terminus of V L2 and the N-terminus of V L1 domain; and L 2 , which is located on the light chain between the C-terminus of V L1 and the N-terminus of C L domain. The heavy chain linker is designated L 3 , which is located between the C-terminus of V H1 and the N-terminus of V H2 domain; and L 4 , which is located between the C-terminus of V H2 and the N-terminus of C H1 domain.

[0426] As used herein, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer encoded information to a host cell. The term "vector" includes nucleic acid molecules capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, into which additional DNA segments can be inserted into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell after being introduced into the host cell and, thereby, replicate with the host genome. Additionally, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are usually in the form of plasmids. The terms "plasmid" and "vector" are used interchangeably herein because plasmids are the most commonly used form of vectors. However, the present disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions.

[0427] As used herein, the phrase "recombinant host cell" (or "host cell") refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell refers not only to a particular subject cell but also to the progeny of such a cell. Since certain modifications may occur in the progeny due to mutation or environmental influences, such progeny may actually be different from the parental 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 a binding protein, 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. To recombinantly express a binding protein, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chain of the binding protein so that the polypeptide chain is expressed in the host cell and preferably secreted into the culture medium of the host cell, from which the binding protein can be recovered.

[0428] As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell, and a cell has been transformed when it has been modified to contain new DNA. For example, a cell is transformed where it is genetically modified from its natural state. After transformation, the transforming DNA can recombine with the cell's DNA by physical integration into the cell's chromosomes, or can be transiently maintained as an episomal element without replication, or can replicate independently as a plasmid. A cell is considered to have been stably transformed when the DNA replicates with cell division. As used herein, the term "transfection" refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced within the cell membrane. Many transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into a suitable host cell.

[0429] As used herein and applied to an object, the term "naturally occurring" refers to the fact that the object can be found in nature and has not been manipulated by a human. For example, a polynucleotide or polypeptide that exists in a living organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by a human 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 a human.

[0430] 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); unnatural amino acids and analogs such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids can also be suitable components of a polypeptide chain of a binding protein. Examples of non-conventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, 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 notations 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.

[0431] Naturally occurring residues can be divided into the following classes based on common side-chain properties:

[0432] (1) Hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

[0433] (2) Polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

[0434] (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro;

[0435] (4) Aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro;

[0436] (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0437] (6) Acidic: Asp, Glu;

[0438] (7) Basic: His, Lys, Arg;

[0439] (8) Residues affecting chain orientation: Gly, Pro;

[0440] (9) Aromatic: His, Trp, Tyr, Phe; and

[0441] (10) Aromatic hydrophobic: Phe, Trp, Tyr.

[0442] Conservative amino acid substitutions may involve exchanging a member of one of these classes with 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.

[0443] 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 may identify suitable regions of the polypeptide chain that can be altered without disrupting activity by targeting regions not considered important for activity. Alternatively, those skilled in the art can identify residues and portions of molecules that are conserved in similar polypeptides. Additionally, even regions that are important for biological activity or structure may be subject to conservative amino acid substitutions without disrupting biological activity or having an adverse effect on the polypeptide structure.

[0444] As used herein, the term "patient" includes human and animal subjects (e.g., mammals such as dogs, pigs, horses, cats, cows, etc.).

[0445] As used herein, the terms "treatment" or "treating" refer to therapeutic treatment and prophylactic or preventive measures. Patients in need of treatment include those suffering from a disorder as well as those predisposed to a disorder or to be protected from a disorder. In specific embodiments, the binding protein can be used to treat a human suffering from cancer, or predisposed to cancer, or to ameliorate cancer in a human subject. The binding protein can also be used to prevent cancer in a human patient. In 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.

[0446] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition that is capable of inducing a desired therapeutic effect when appropriately administered to a patient.

[0447] 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.

[0448] When used in reference to a pharmaceutical composition comprising one or more binding proteins, the terms "effective amount" and "therapeutically effective amount" refer to an amount or dose sufficient to produce a desired therapeutic outcome. More specifically, a therapeutically effective amount is an amount of a binding protein sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated over a period of time. The effective amount can vary depending on the specific binding protein being used and also depends on a variety of factors related to the patient being treated, the condition, and the severity of the disorder. For example, if a binding protein is to be administered in vivo, factors obtained in preclinical animal work such as the patient's age, weight, and health, as well as the dose-response curve and toxicity data, would be among 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.

[0449] One embodiment of the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

[0450] II. Anti-CD38 Binding Proteins

[0451] Certain aspects of the present disclosure relate to binding proteins that comprise antigen-binding sites that bind to CD38 polypeptides (e.g., human and cynomolgus monkey CD38 polypeptides). In some embodiments, the binding protein is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent.

[0452] Multiple characteristics of exemplary monospecific, bispecific, or trispecific binding proteins are described herein. For example, in some embodiments, the binding protein or an antigen-binding fragment thereof 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 the T cells (e.g., via TCR stimulation and / or co-stimulation).

[0453] In some embodiments, the binding protein comprises an antigen-binding site, the antigen-binding site comprising: 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); or 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). In some embodiments, the binding protein comprises an antigen-binding site, the antigen-binding site comprising: 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); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence having an amino acid sequence containing ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), 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).In some embodiments, the binding protein comprises 1, 2, 3, 4, 5, or 6 CDRs of the antibody VH and / or VL domain sequences from 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.

[0454] In some embodiments, the binding protein comprises an antigen-binding site that comprises: an antibody heavy chain variable (VH) domain that comprises 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 an antibody light chain variable (VL) domain that comprises 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).

[0455] In some embodiments, the binding protein comprises an antigen-binding site, and the antigen-binding site comprises: 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); or 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). In some embodiments, the binding protein comprises an antigen-binding site, and the antigen-binding site comprises: 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 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). In other embodiments, the binding protein comprises an antigen-binding site, and the antigen-binding site comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence having an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence having an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence having an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence having an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence having an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence having an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36).In some embodiments, the binding protein comprises an antigen-binding site, the antigen-binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence having an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence having an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence having an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence having an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence having an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence having an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36).

[0456] In some embodiments, 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). In some embodiments, the VL domain comprises the sequence FR1—CDR-L1—FR2—CDR-L2—FR3—CDR-L3—FR4 from the N-terminus to the 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); wherein FR4 comprises the sequence FGGGTKLEIK (SEQ ID NO:103).

[0457] In some embodiments, the VH domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO:5; and / or the VL domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO:17; and / or the VL domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO:21; and / or the VL domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO:18.In some embodiments, the VH domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO: 23; and / or the VL domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO: 13; and / or the VL domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO: 14.

[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 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 having 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% identity to the amino acid sequence of SEQ ID NO: 23; and the VL domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO: 13; and the VL domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO: 14.

[0459] 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.

[0460] In some embodiments, the binding protein of the present disclosure comprises an antibody heavy chain containing the amino acid sequence of SEQ ID NO:7 and / or an antibody light chain containing the amino acid sequence of SEQ ID NO:8. In some embodiments, the binding protein of the present disclosure comprises an antibody heavy chain containing the amino acid sequence of SEQ ID NO:19 and / or an antibody light chain containing the amino acid sequence of SEQ ID NO:20. In some embodiments, the binding protein of the present disclosure comprises an antibody heavy chain containing the amino acid sequence of SEQ ID NO:22 and / or an antibody light chain containing the amino acid sequence of SEQ ID NO:20. In some embodiments, the binding protein of the present disclosure comprises an antibody heavy chain containing the amino acid sequence of SEQ ID NO:24 and / or an antibody light chain containing the amino acid sequence of SEQ ID NO:20. In some embodiments, the binding protein of the present disclosure comprises an antibody heavy chain containing the amino acid sequence of SEQ ID NO:15 and / or an antibody light chain containing the amino acid sequence of SEQ ID NO:16.

[0461] In some embodiments, the binding protein of the present disclosure 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. In some embodiments, the binding protein of the present disclosure 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. In some embodiments, the binding protein of the present disclosure 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. In some embodiments, the binding protein of the present disclosure comprises an antibody heavy chain containing the amino acid sequence of SEQ ID NO:24 and an antibody light chain containing the amino acid sequence of SEQ ID NO:20. In some embodiments, the binding protein of the present disclosure comprises an antibody heavy chain containing the amino acid sequence of SEQ ID NO:15 and an antibody light chain containing the amino acid sequence of SEQ ID NO:16.

[0462] In some embodiments, the binding protein comprises an antigen-binding site, the antigen-binding site comprising: a variable heavy (VH) domain of an antibody, which comprises a CDR-H1 sequence having an amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence having an amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence having an amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43); or a variable light (VL) domain of an antibody, which comprises a CDR-L1 sequence having an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence having an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence having an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46). In some embodiments, the binding protein comprises an antigen-binding site, the antigen-binding site comprising: a variable heavy (VH) domain of an antibody, which comprises a CDR-H1 sequence having an amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence having an amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence having an amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43); and a variable light (VL) domain of an antibody, which comprises a CDR-L1 sequence having an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence having an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence having an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46).

[0463] In some embodiments, 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 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, FR1—CDR-L1—FR2—CDR-L2—FR3—CDR-L3—FR4 from the N-terminus to the C-terminus; 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).

[0464] In some embodiments, the VH domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO:9; and / or the VL domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO:10.

[0465] In some embodiments, the VH domain comprises an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO:9; and the VL domain comprises an amino acid sequence having 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% identity 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.

[0466] 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.

[0467] In some embodiments, the binding protein of the present disclosure comprises 1, 2, 3, 4, 5 or 6 CDR sequences of the antibody sequences shown in Table G. In some embodiments, the binding protein of the present disclosure comprises 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 protein of the present invention comprises 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 protein of the present invention comprises 1, 2, 3 or 4 polypeptide sequences shown in Table I.

[0468]

[0469] Table H. Variable domain sequences of anti-CD38 (mAb1-7) and other binding proteins.

[0470]

[0471]

[0472]

[0473] Note: The CDR sequences are bolded and underlined in the above amino acid sequences.

[0474] Table I. Full-length sequences of the binding proteins.

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490] Table J. Full-length polynucleotide sequences of the binding proteins.

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511] CD38 polypeptide

[0512] In some embodiments, the binding proteins of the present disclosure comprise antigen-binding sites that bind to the extracellular domain of human CD38 polypeptide and the extracellular domain of cynomolgus monkey CD38 polypeptide. Exemplary assays for determining whether an antigen-binding site binds an antigen are described herein and are known in the art. In some embodiments, binding is determined by ELISA assay, for example, as described below. In some embodiments, binding is determined by SPR assay, for example, as described below. In some embodiments, binding is determined by flow cytometry assay using cells expressing CD38 polypeptide on their cell surface, for example, as described below. See, for example, Examples 1, 3, and 4.

[0513] In some embodiments, the binding proteins of the present disclosure bind to a purified polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and / or 30 or a fragment thereof (e.g., as measured by ELISA or SPR). In some embodiments, when expressed on the cell surface (e.g., as measured by flow cytometry), the binding proteins of the present disclosure bind to a polypeptide or to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and / or 30.

[0514] In some embodiments, the binding proteins of the present disclosure bind to CD38 isoform A polypeptides (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 polypeptides (e.g., comprising the amino acid sequence of SEQ ID NO:105, and not comprising the complete amino acid sequence of SEQ ID NO:1, consisting of the amino acid sequence of SEQ ID NO:105, or consisting 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 polypeptides (e.g., comprising the amino acid sequence of SEQ ID NO:1) and CD38 isoform E polypeptides (e.g., comprising the amino acid sequence of SEQ ID NO:105, and not comprising the complete amino acid sequence of SEQ ID NO:1, consisting of the amino acid sequence of SEQ ID NO:105, or consisting 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, e.g., for targeting the binding proteins of the present disclosure to cells that express CD38 isoform E polypeptides.

[0515] Amino acid sequence of the extracellular domain of human CD38 isotype A

[0516] RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI(SEQ ID NO:1)

[0517] Amino acid sequence of human CD38 isotype E

[0518] RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRHFWECGSP(SEQ ID NO:105)

[0519] 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.

[0520] Amino acid sequence of cynomolgus monkey CD38

[0521] RWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQSVWDAFKGAFISKYPCNITEEDYQPLVKLGTQTVPCNKTLLWSRIKDLAHQFTQVQRDMFTLEDMLLGYLADDLTWCGEFNTFEINYQSCPDWRKDCSNNPVSVFWKTVSRRFAETACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQALEAWVIHGGREDSRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKFLQCVKNPEDSSCLSGI(SEQ ID NO:30)

[0522] Multispecific (e.g., bispecific, trispecific, or multispecific) binding protein that binds to a CD38 polypeptide

[0523] In some embodiments, the binding protein of the present disclosure is a bispecific binding protein that comprises an antigen-binding site that binds to one or more CD38 polypeptides and a second antigen-binding site that binds to a different target antigen. In some embodiments, the binding protein of the present disclosure is a bispecific binding protein that comprises an antigen-binding site that binds to one or more CD38 polypeptides and a second antigen-binding site that binds to one or more CD38 polypeptides.

[0524] In some embodiments, the binding protein of the present disclosure is a trispecific binding protein that comprises an antigen-binding site, a second antigen-binding site, and a third antigen-binding site that each bind 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 a human and / or cynomolgus monkey CD38 polypeptide), and one or two of the antigen-binding sites bind to a T cell surface protein. In some embodiments, one of the antigen-binding sites binds to one or more CD38 polypeptides (e.g., the extracellular domain of a human and / or cynomolgus monkey CD38 polypeptide), one antigen-binding site binds to a human CD3 polypeptide, and one antigen-binding site binds to a human CD28 polypeptide. Human CD3 and CD28 polypeptides are known in the art. Amino acid sequences of exemplary and non-limiting antibody variable domains that bind to human CD3 and CD28 polypeptides are provided herein.

[0525] 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 the human CD38 polypeptide and the extracellular domain of the cynomolgus monkey CD38 polypeptide. In some embodiments, the human CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:1, and / or the cynomolgus monkey CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:30. In some embodiments, the binding protein comprises an antigen-binding site that binds to the extracellular domain of the human CD38 polypeptide and the extracellular domain of the cynomolgus monkey CD38 polypeptide and two antigen-binding sites that each bind to a T cell surface protein (e.g., the human CD28 polypeptide and / or the human CD3 polypeptide).

[0526] 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 protein is capable of binding to two different epitopes on one tumor target protein (e.g., one or more CD38 polypeptides) and a single T cell target protein. In some embodiments, the binding protein is capable of binding to one tumor target protein (e.g., one or more CD38 polypeptides) and two different T cell target proteins (e.g., CD28 and CD3). In some embodiments, the binding protein is capable of binding to one T cell target protein and two different epitopes on a single tumor target protein (e.g., one or more CD38 polypeptides). In some embodiments, the binding protein is capable of binding to one T cell target protein and two different tumor target proteins (e.g., one or more CD38 polypeptides and another tumor target protein). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen-binding sites that target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen-binding site that binds to one or more CD38 polypeptides. In some embodiments, the first and second polypeptide chains of the binding protein form two antigen-binding sites that target two tumor target proteins (e.g., one or more CD38 polypeptides and another tumor target protein), and the third and fourth polypeptide chains of the binding protein form an antigen-binding site that binds to a T cell target protein. In some embodiments, one or more of the T cell target proteins are one or more of CD3 and CD28.

[0527] In some embodiments, the binding protein specifically binds to one or more CD38 polypeptides and one or more target proteins on T cells, including the T cell receptor complex. These T cell engager binding proteins are capable of transiently recruiting T cells into target cells while activating the lytic activity of the T cells. Examples of target proteins on T cells include, but are not limited to, CD3 and CD28, among others. Additional examples of such antigen targets or target proteins are provided above. In some embodiments, a trispecific binding protein can be generated by combining the antigen-binding domains of two or more monospecific antibodies (parent antibodies) into one antibody.

[0528] Bispecific binding protein format

[0529] In some embodiments, the binding proteins of the present disclosure are bispecific and / or bivalent binding proteins that comprise four polypeptide chains that form four antigen-binding sites that bind to one or more (e.g., two) different antigen targets or target proteins (e.g., having the structure described in International Publication No. WO2012 / 135345). In some embodiments, the binding protein is bivalent and / or bispecific. In some embodiments, the binding protein is tetravalent and / or tetraspecific. In some embodiments, the binding protein is tetravalent and / or bispecific. In some embodiments, at least one antigen-binding site binds to a CD38 polypeptide (e.g., the extracellular domain of a human and / or cynomolgus monkey CD38 polypeptide).

[0530] In some embodiments, the binding protein comprises two polypeptide chains having a structure represented by the following formula:

[0531] V L1 -L 1 -V L2 -L 2 -C L [I]

[0532] and two polypeptide chains have a structure represented by the following formula:

[0533] V H2 -L 3 -V H1 -L 4 -C H1 -Fc [II]

[0534] Wherein:

[0535] V L1 is the first immunoglobulin light chain variable domain;

[0536] V L2 is the second immunoglobulin light chain variable domain;

[0537] V H1is the first immunoglobulin heavy chain variable domain;

[0538] V H2 is the second immunoglobulin heavy chain variable domain;

[0539] C L is the immunoglobulin light chain constant domain;

[0540] C H1 is immunoglobulin C H1 heavy chain constant domain;

[0541] Fc contains the immunoglobulin hinge region and C H2 、C H3 immunoglobulin heavy chain constant region;

[0542] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

[0543] and wherein the polypeptide of Formula I and the polypeptide of Formula II form cross light chain - heavy chain pairs. 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 form an antigen - binding domain that binds to another antigen target. 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 form an antigen - binding domain that binds to another antigen target.

[0544] In some embodiments, the binding protein comprises two polypeptide chains that form two antigen - binding sites, wherein the first polypeptide chain comprises

[0545] V L1 -L 1 -V L2 -L 2 -CL - Fc

[0546] and the second polypeptide chain comprises

[0547] V H2 -L 3 -V HI -L 4 -C H1 -Fc

[0548] wherein:

[0549] V L1is the first immunoglobulin light chain variable domain;

[0550] V L2 is the second immunoglobulin light chain variable domain;

[0551] V H1 is the first immunoglobulin heavy chain variable domain;

[0552] V H2 is the second immunoglobulin heavy chain variable domain;

[0553] CL is the immunoglobulin light chain constant domain;

[0554] C H1 is the immunoglobulin C H1 heavy chain constant domain;

[0555] C H2 is the immunoglobulin C H2 heavy chain constant domain;

[0556] C H3 is the immunoglobulin C H3 heavy chain constant domain;

[0557] Fc contains the immunoglobulin hinge region and C H2 、C H3 immunoglobulin heavy chain constant regions; and

[0558] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

[0559] wherein the first and second polypeptides form a cross 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 form an antigen - binding domain that binds to another antigen target. 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 form an antigen - binding domain that binds to another antigen target.

[0560] In some embodiments, the binding protein comprises three polypeptide chains that form two antigen - binding sites, wherein the first polypeptide chain comprises

[0561] V L1 -L 1 -V L2 -L2 -CL

[0562] The second polypeptide chain comprises

[0563] V H2 -L 3 -V HI -L 4 -C H1 -Fc

[0564] The third polypeptide chain comprises the antibody Fc region

[0565] Wherein:

[0566] V L1 is the variable domain of the first immunoglobulin light chain;

[0567] V L2 is the variable domain of the second immunoglobulin light chain;

[0568] V H1 is the variable domain of the first immunoglobulin heavy chain;

[0569] V H2 is the variable domain of the second immunoglobulin heavy chain;

[0570] CL is the constant domain of the immunoglobulin light chain;

[0571] C H1 is the immunoglobulin C H1 constant domain of the heavy chain;

[0572] C H2 is the immunoglobulin C H2 constant domain of the heavy chain;

[0573] C H3 is the immunoglobulin C H3 constant domain of the heavy chain;

[0574] Fc comprises the immunoglobulin hinge region and C H2 、C H3 constant domains of the immunoglobulin heavy chain; and

[0575] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

[0576] Wherein the first and second polypeptides form a cross 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 L2Form an antigen-binding domain that binds to another antigen target. 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 form an antigen-binding domain that binds to another antigen target.

[0577] In some embodiments, the binding protein comprises a first polypeptide chain comprising a structure represented by the following formula:

[0578] V L1 -L 1 -V L2 -L 2 -C L [I]

[0579] and a second polypeptide chain comprising a structure represented by the following formula:

[0580] V H2 -L 3 -V H1 -L 4 -C H1 [II]

[0581] Wherein:

[0582] V L1 is the first immunoglobulin light chain variable domain;

[0583] V L2 is the second immunoglobulin light chain variable domain;

[0584] V H1 is the first immunoglobulin heavy chain variable domain;

[0585] V H2 is the second immunoglobulin heavy chain variable domain;

[0586] C L is the immunoglobulin light chain constant domain;

[0587] C H1 is the immunoglobulin C H1 heavy chain constant domain; and

[0588] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

[0589] Wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain-heavy chain pair. In some embodiments, V H1 and V L1Form an antigen-binding domain that binds to a CD38 polypeptide, and V H2 and V L2 Form an antigen-binding domain that binds to another antigen target. In some embodiments, V H2 and V L2 Form an antigen-binding domain that binds to a CD38 polypeptide, and V H1 and V L1 Form an antigen-binding domain that binds to another antigen target.

[0590] 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 eosinophil chemotactic factor), 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 / eosinophil chemotactic factor-2), CCL25 (also known as TECK), CCL26 (also known as eosinophil chemotactic factor-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 the IL33 receptor), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the common 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 antigen targets are human antigen targets.

[0591] In any of the bispecific binding proteins described above, any linker or combination of linkers described herein can be used. For example, in some embodiments, L 1 , L 2 , L 3 or L 4 The length of at least one of them is independently 0 amino acids. In some embodiments, L 1 , L 2 , L 3 or L 4 The lengths are each independently at least 1 amino acid. In some embodiments, L 1 , L 2 , L 3 and L 4 Each independently has a length of 0 amino acids or contains a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L 1 , L 2 , L 3 and L 4 Each independently contains a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L 1 contains the sequence GQPKAAP (SEQ ID NO:58), L 2 contains the sequence TKGPS (SEQ ID NO:57), L 3 contains the sequence S, L 4 contains the sequence RT. In some embodiments, L 1 contains the sequence GGGGSGGGGS (SEQ ID NO:55), L 2 contains the sequence GGGGSGGGGS (SEQ ID NO:55), L 3 has a length of 0 amino acids, L 4 has a length of 0 amino acids. In some embodiments, L 1Comprising the sequence GGSGSSGSGG (SEQ ID NO:59), L 2 Comprising the sequence GGSGSSGSGG (SEQ ID NO:59), L 3 has a length of 0 amino acids, L 4 has a length of 0 amino acids. In some embodiments, L 1 Comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L 2 has a length of 0 amino acids, L 3 Comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L 4 has a length of 0 amino acids.

[0592] Trispecific binding protein that binds to a CD38 polypeptide

[0593] In some embodiments, the binding proteins of the present disclosure are trispecific and / or trivalent binding proteins that comprise four polypeptide chains that form three antigen-binding sites that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one antigen-binding site binds to a CD38 polypeptide (e.g., the extracellular domain of a human and / or cynomolgus monkey CD38 polypeptide). In some embodiments,

[0594] The first polypeptide chain comprises a structure represented by the following formula:

[0595] V L2 -L 1 -V L1 -L 2 -C L [I]

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

[0597] V H1 -L 3 -V H2 -L 4 -C H1 -hinge-C H2 -C H3 [II]

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

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

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

[0601] V L3 -C L [IV]

[0602] Wherein:

[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 the 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 is the immunoglobulin C H1 heavy chain constant domain;

[0611] C H2 is the immunoglobulin C H2 heavy chain constant domain;

[0612] C H3 is the immunoglobulin C H3 heavy chain constant domain;

[0613] The hinge is the immunoglobulin hinge region that connects the C H1 and C H2 domains, and

[0614] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

[0615] Wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain - heavy chain pair.

[0616] In some embodiments, the binding proteins of the present disclosure are trispecific and / or trivalent binding proteins that comprise four polypeptide chains that form three antigen-binding sites that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one antigen-binding site binds a CD38 polypeptide (e.g., the extracellular domain of a human and / or cynomolgus monkey CD38 polypeptide). In some embodiments, the first polypeptide chain comprises a structure represented by the formula:

[0617] V L2 -L 1 -V L1 -L 2 -C L [I]

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

[0619] V H1 -L 3 -V H2 -L 4 -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 -C L [IV]

[0624] wherein:

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

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

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

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

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

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

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

[0632] C H1 is the immunoglobulin C H1 constant domain of the heavy chain; and

[0633] L 1 、L 2 、L 3 and L 4 are amino acid linkers;

[0634] and wherein the polypeptide of Formula I and the polypeptide of Formula II form a cross light chain - heavy chain pair. In some embodiments, the second and third polypeptide chains further comprise an Fc region linked to C H1 wherein the Fc region comprises an immunoglobulin hinge region and C H2 and C H3 constant domains of the immunoglobulin heavy chain.

[0635] In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross - orientation which forms two different antigen - binding sites. In some embodiments, VH1 and VL1 form a binding pair and form a first antigen - binding site. In some embodiments, VH2 and VL2 form a binding pair and form a second antigen - binding site. In some embodiments, the first antigen - binding site binds a CD3 polypeptide (e.g., human CD3), and the second antigen - binding site binds a CD28 polypeptide (e.g., human CD28). In some embodiments, the second antigen - binding site binds a CD3 polypeptide (e.g., human CD3), and the first antigen - binding site binds a CD28 polypeptide (e.g., human CD28). In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen - binding site. In some embodiments, VH3 and VL3 form a binding pair and form a third antigen - binding site. In some embodiments, the third antigen - binding site binds a CD38 polypeptide (e.g., human and optionally cynomolgus monkey CD38). Exemplary binding protein forms with cross - orientation 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 of the bispecific, trispecific or multispecific binding proteins described herein, the antigen - binding site binds a CD38 polypeptide (e.g., human and optionally cynomolgus monkey CD38). In some embodiments, other (e.g., not binding CD38) antigen - binding sites of any of the bispecific, trispecific or multispecific binding proteins described herein bind CD28 or CD3. In some embodiments, V H1The domain contains 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 V L1 The domain contains 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). In some embodiments, V H2 The domain contains 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 V L2 The domain contains 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). In some embodiments, V H3 The domain contains 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 V L3The domain contains 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 contains a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), V L1 The domain contains 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 contains 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 contains a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H3The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36).

[0637] In some embodiments, V H1 The domain contains a CDR-H1 sequence with an amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence with an amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence with an amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43), and / or V L1 The domain contains a CDR-L1 sequence with an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence with an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence with an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46). In some embodiments, V H2 The domain contains a CDR-H1 sequence with an amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence with an amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence with an amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43), and / or V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence with an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence with an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46). In some embodiments, V H3The domain contains a CDR-H1 sequence with the amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence with the amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence with the amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43), and / or V L3 The domain contains a CDR-L1 sequence with the amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence with the amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence with the amino acid sequence containing LQDYIYYPT (SEQ ID NO:46).

[0638] In some embodiments, V H1 The domain contains a CDR-H1 sequence with the amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence with the amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence with the amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43), and V L1 The domain contains a CDR-L1 sequence with the amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence with the amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence with the amino acid sequence containing LQDYIYYPT (SEQ ID NO:46). In some embodiments, V H2 The domain contains a CDR-H1 sequence with the amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence with the amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence with the amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43), and V L2 The domain contains a CDR-L1 sequence with the amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence with the amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence with the amino acid sequence containing LQDYIYYPT (SEQ ID NO:46). In some embodiments, V H3The domain contains a CDR-H1 sequence with an amino acid sequence containing GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence with an amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence with an amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43), and V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence with an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence with an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46).

[0639] In some embodiments, V H1 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and / or V L1 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H1 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and / or V L1 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H2The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and / or V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H2 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and / or V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H3 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and / or V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ IDNO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H3The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and / or V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36).

[0640] In some embodiments, V H1 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and V L1 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H1 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and V L1 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H2The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H2 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and V L2 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H3 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ IDNO:33), and V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H3The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36).

[0641] In some embodiments, V H3 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence with an amino acid sequence containing IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ ID NO:33), and / or V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence with an amino acid sequence containing LAS (SEQ ID NO:35), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36). In some embodiments, V H3 The domain contains a CDR-H1 sequence with an amino acid sequence containing GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence with an amino acid sequence containing IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence with an amino acid sequence containing ARTGGLRRAYFTY (SEQ IDNO:33), and / or V L3 The domain contains a CDR-L1 sequence with an amino acid sequence containing QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence with an amino acid sequence containing GAS (SEQ ID NO:40), and a CDR-L3 sequence with an amino acid sequence containing QQNKEDPWT (SEQ ID NO:36).

[0642] In some embodiments, V H3 The domain contains the amino acid sequence of SEQ ID NO:5, or V L3The domain contains the amino acid sequence of SEQ ID NO:6. In some embodiments, the V H3 domain contains the amino acid sequence of SEQ ID NO:17, or the V L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V H3 domain contains the amino acid sequence of SEQID NO:21, or the V L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V H3 domain contains the amino acid sequence of SEQ ID NO:23, or the V L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V H3 domain contains the amino acid sequence of SEQ ID NO:13, or the V L3 domain contains the amino acid sequence of SEQ IDNO:14.

[0643] In some embodiments, the V H3 domain contains the amino acid sequence of SEQ ID NO:5, and / or the V L3 domain contains the amino acid sequence of SEQ ID NO:6. In some embodiments, the V H3 domain contains the amino acid sequence of SEQ ID NO:17, and the V L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V H3 domain contains the amino acid sequence of SEQID NO:21, and the V L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V H3 domain contains the amino acid sequence of SEQ ID NO:23, and the V L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V H3 domain contains the amino acid sequence of SEQ ID NO:13, and the V L3 domain contains the amino acid sequence of SEQ IDNO:14.

[0644] In some embodiments, the V H3 domain contains the CDR-H1 sequence with the amino acid sequence containing GFTFSSYG (SEQ ID NO:41), the CDR-H2 sequence with the amino acid sequence containing IWYDGSNK (SEQ ID NO:42), and the CDR-H3 sequence with the amino acid sequence containing ARMFRGAFDY (SEQ ID NO:43), and / or the VL3 The domain contains a CDR-L1 sequence with an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence with an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence with an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46).

[0645] In some embodiments, V H3 The domain contains the amino acid sequence of SEQ ID NO:9, and / or V L3 The domain contains the amino acid sequence of SEQ ID NO:10.

[0646] In some embodiments of any trispecific binding protein of the present disclosure, one antigen-binding domain binds to a CD3 polypeptide (e.g., human CD3), and one antigen-binding domain binds to a CD28 polypeptide (e.g., human CD28). In some embodiments, V H1 The domain contains three CDRs from SEQ ID NO:49 or 51, as shown in Table H, and V L1 The domain contains three CDRs from SEQ ID NO:50 or 52, as shown in Table H. In some embodiments, V H2 The domain contains three CDRs from SEQ ID NO:49 or 51, as shown in Table H, and V L2 The domain contains three CDRs from SEQ ID NO:50 or 52, as shown in Table H. In some embodiments, V H1 The domain contains three CDRs from SEQ ID NO:53 or 84, as shown in Table H, and V L1 The domain contains three CDRs from SEQ ID NO:54 or 85, as shown in Table H. In some embodiments, V H2 The domain contains three CDRs from SEQ ID NO:53 or 84, as shown in Table H, and V L2 The domain contains three CDRs from SEQ ID NO:54 or 85, as shown in Table H.

[0647] In some embodiments, V H1 The domain contains the amino acid sequence of SEQ ID NO:49, V L1 The domain contains the amino acid sequence of SEQ ID NO:50, V H2 The domain contains the amino acid sequence of SEQ ID NO:53, and V L2 The domain contains the amino acid sequence of SEQ ID NO:54. In some embodiments, VH2 The domain contains the amino acid sequence of SEQ ID NO:49, V L2 The domain contains the amino acid sequence of SEQ ID NO:50, V H1 The domain contains the amino acid sequence of SEQ ID NO:53, and V L1 The domain contains the amino acid sequence of SEQ ID NO:54. In some embodiments, V H1 The domain contains the amino acid sequence of SEQ ID NO:51, V L1 The domain contains the amino acid sequence of SEQ ID NO:52, V H2 The domain contains the amino acid sequence of SEQ ID NO:53, and V L2 The domain contains the amino acid sequence of SEQ ID NO:54. In some embodiments, V H2 The domain contains the amino acid sequence of SEQ ID NO:51, V L2 The domain contains the amino acid sequence of SEQ ID NO:52, V H1 The domain contains the amino acid sequence of SEQ ID NO:53, and V L1 The domain contains the amino acid sequence of SEQ ID NO:54.

[0648] In some embodiments, V H1 The domain contains the amino acid sequence of SEQ ID NO:49, V L1 The domain contains the amino acid sequence of SEQ ID NO:50, V H2 The domain contains the amino acid sequence of SEQ ID NO:53, V L2 The domain contains the amino acid sequence of SEQ ID NO:54, V H3 The domain contains the amino acid sequence of SEQ ID NO:13, and V L3 The domain contains the amino acid sequence of SEQ ID NO:14. In some embodiments, V H1 The domain contains the amino acid sequence of SEQ ID NO:49, V L1 The domain contains the amino acid sequence of SEQ ID NO:50, V H2 The domain contains the amino acid sequence of SEQ ID NO:53, V L2 The domain contains the amino acid sequence of SEQ ID NO:54, V H3 The domain contains the amino acid sequence of SEQ ID NO:9, and V L3 The domain contains the amino acid sequence of SEQ ID NO:10.

[0649] In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 65, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 64, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 70, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 66, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 71, and the fourth polypeptide chain comprises a polypeptide sequence that is 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-3), CCL8 (also known as mcp-2), CCL11 (also known as eosinophil chemotactic factor), 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 / eosinophil chemotactic factor-2), CCL25 (also known as TECK), CCL26 (also known as eosinophil chemotactic factor-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 the IL33 receptor), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the common 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 antigen targets are human antigen targets.

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

[0653] The binding proteins of the present disclosure can be prepared using domains or sequences obtained or derived from any human or non-human antibody (including, for example, human, murine or humanized antibodies).

[0654] Linker

[0655] In some embodiments, linker L 1 、L 2 、L 3 and L 4 ranges from no amino acids (length = 0) to about 100 amino acids in length, or less than 100, 50, 40, 30, 20 or 15 amino acids or fewer. The linker can also be 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid in length. The L 1 、L 2 、L 3 and L 4 in one 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 having four glycine residues; a peptide having five glycine residues; a peptide having six glycine residues; a peptide having seven glycine residues; and a peptide having eight glycine residues. Other combinations of amino acid residues can 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 it is shown that linkers containing randomly selected amino acids from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine and proline are suitable for binding proteins. For other descriptions of linker sequences, see, for example, WO2012135345 and International Application No. PCT / US2017 / 027488.

[0657] The identity and sequence of amino acid residues in the linker can vary depending on the type of secondary structure element desired in the linker. For example, glycine, serine, and alanine are most suitable for linkers with maximum flexibility. If a more rigid and extended linker is needed, certain combinations of glycine, proline, threonine, and serine are useful. Depending on the desired properties, any amino acid residue can be considered as part of a linker in combination with other amino acid residues to construct a larger peptide linker of the desired size.

[0658] In some embodiments, the length of at least one of L 1 , L 2 , L 3 or L 4 is independently 0 amino acids. In some embodiments, the length of each of L 1 , L 2 , L 3 or L 4 is independently at least 1 amino acid. In some embodiments, the length of L 1 is at least twice the length of L 3 . In some embodiments, the length of L 2 is at least twice the length of L 4 . In some embodiments, the length of L 1 is at least twice the length of L 3 , and the length of L 2 is at least twice the length of L 4 . In some embodiments, the length of L 1 is 3 to 12 amino acid residues, the length of L 2 is 3 to 14 amino acid residues, the length of L 3 is 1 to 8 amino acid residues, and the length of L 4 is 1 to 3 amino acid residues. In some embodiments, the length of L 1 is 5 to 10 amino acid residues, the length of L 2 is 5 to 8 amino acid residues, the length of L 3 is 1 to 5 amino acid residues, and the length of L 4 is 1 to 2 amino acid residues. In some embodiments, the length of L 1 is 7 amino acid residues, the length of L 2 is 5 amino acid residues, the length of L 3 is 1 amino acid residue, and the length of L 4 is 2 amino acid residues. In some embodiments, the length of L 1 is 10 amino acid residues, the length of L 2 is 10 amino acid residues, the length of L 3 is 0 amino acid residues, L4 has a length of 0 amino acid residues. In some embodiments, L 1 , L 2 , L 3 and L 4 each independently have a length of from 0 to 15 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids), wherein at least two of the linkers have a length of from 1 to 15 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). In some embodiments, L 1 , L 2 , L 3 and L 4 each have a length of 0 amino acids.

[0659] In some embodiments, L 1 , L 2 , L 3 and / or L 4 comprises a sequence derived from a naturally occurring sequence at the junction between an antibody variable domain and an antibody constant domain (e.g., as described in WO2012 / 135345). For example, in some embodiments, the linker comprises a sequence found at the transition between an endogenous V H and C H1 domain or between an endogenous V L and C L domain (e.g., κ or λ). In some embodiments, the linker comprises a sequence found at the transition between an endogenous human V H and C H1 domain or between an endogenous human V L and C L domain (e.g., human κ or λ).

[0660] In some embodiments, L 1 , L 2 , L 3 and L 4 each independently is a length of 0 amino acids or comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L 1 , L 2 , L 3 and L 4Each independently comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58), and GGSGSSGSGG (SEQ ID NO:59).

[0661] In some embodiments, L 1 comprises the sequence GQPKAAP (SEQ ID NO:58), L 2 comprises the sequence TKGPS (SEQID NO:57), L 3 comprises the sequence S, and L 4 comprises the sequence RT. In some embodiments, L 1 comprises the sequence GGGGSGGGGS(SEQID NO:55), L 2 comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L 3 has a length of 0 amino acids, and L 4 has a length of 0 amino acids. In some embodiments, L 1 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L 2 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L 3 has a length of 0 amino acids, and L 4 has a length of 0 amino acids. In some embodiments, L 1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L 2 has a length of 0 amino acids, L 3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), and L 4 has a length of 0 amino acids.

[0662] Fc region and constant domain

[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 comprises an antibody hinge, C H1 、C H2 、C H3 and optionally C H4Domain. 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 comprise one or two Fc variants. As used herein, the term "Fc variant" refers to a molecule or sequence modified from native Fc but still containing the binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term "Fc variant" can encompass humanized non-human native Fc molecules or sequences. Additionally, native Fc contains regions that can be removed because they provide structural features or biological activities not required for the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" encompasses molecules or sequences lacking one or more native Fc sites or residues, or in which one or more Fc sites or residues are modified and affect or participate in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, 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 functions of the Fc region (e.g., Fc receptor-mediated antibody-dependent cell 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 that contains 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 that contains 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 that comprises 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 that comprises one or more mutations that reduce or eliminate FcγI and / or FcγII binding without affecting FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region that comprises amino acid substitutions at positions corresponding to positions 228 and / or 409 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitutions are S228P and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region that comprises amino acid substitutions at positions corresponding to positions 234 and / or 235 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitutions are F234A and / or L235A. In some embodiments, the Fc region is a human IgG4 Fc region that comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, and / or 409 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A, and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region that comprises amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region that comprises amino acid mutations at substitutions corresponding to positions 228, 233-236, and / or 409 of human IgG4 according to the EU Index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and / or R409K.

[0668] In some embodiments, the binding proteins of the present disclosure include one or more mutations to improve purification, for example, by modulating the affinity for purification reagents. For example, it is well known that if one of the two Fc regions in the heterodimeric form contains a mutation that reduces or eliminates binding to Protein A, the heterodimeric binding protein can be selectively purified from its homodimeric form because the heterodimeric form will have an intermediate affinity for Protein A-based purification compared to either homodimeric form and can be selectively eluted from Protein A, for example, by using different pHs (see, for example, Smith, E.J. et al. (2015) Sci. Rep. 5:17943). In some embodiments, the mutation comprises 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 protein comprises a second polypeptide chain that further comprises a first Fc region linked to C H1 The first Fc region comprises an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant regions, and the third polypeptide chain further comprises a second Fc region linked to C H1 The second Fc region comprises an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant regions; and wherein only one of the first and second Fc regions contains 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 include a "stapling" 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 increase the yield of some binding proteins (e.g., bispecific or trispecific binding proteins), the CH3 domain can be altered by the "stapling" 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 interaction surface of the two C H3 domains is altered to increase the heterodimerization of the two heavy chains containing these two C H3 domains. The two C H3Each of the domains (two heavy chains) can be a "pestle", and the other is a "mortar". The introduction of disulfide bridges further stabilizes the heterodimer (Merchant et al., 1998; Atwell et al., 1997, J. Mol. Biol. 270:26-35) and increases the yield. In certain embodiments, the pestle is located on the second pair of polypeptides having a single variable domain. In other embodiments, the pestle is located on the first pair of polypeptides with a cross-orientation. In other embodiments, the C H3 domain does not include the pestle in the mortar.

[0670] In some embodiments, the binding proteins of the present disclosure (e.g., trispecific binding proteins) comprise a "pestle" mutation on the second polypeptide chain and a "mortar" mutation on the third polypeptide chain. In some embodiments, the binding proteins of the present disclosure comprise a "pestle" mutation on the third polypeptide chain and a "mortar" mutation on the second polypeptide chain. In some embodiments, the "pestle" mutation comprises a substitution at positions corresponding to positions 354 and / or 366 of human IgG1 or IgG4 according to the EU Index. In some embodiments, the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the "pestle" mutation comprises a substitution at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU Index. In some embodiments, the amino acid substitution is S354C and T366W. In some embodiments, the "mortar" mutation comprises a substitution at positions corresponding to position 407 and optionally positions 349, 366 and / or 368 of human IgG1 or IgG4 according to the EU Index. In some embodiments, the amino acid substitutions are Y407V or Y407T, and optionally Y349C, T366S and / or L368A. In some embodiments, the "mortar" mutation comprises a substitution at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU Index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A and Y407V.

[0671] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4, wherein the amino acid substitutions are T366W or T366Y, and optionally S354C; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to positions 407 and optionally 349, 366 and / or 368 of human IgG1 or IgG4, wherein the amino acid substitutions are 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 according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 407 and optionally 349, 366 and / or 368 of human IgG1 or IgG4, wherein the amino acid substitutions are Y407V or Y407T, and optionally Y349C, T366S and / or L368A; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4, wherein the amino acid substitutions are 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 according to the EU Index, the first Fc region comprises an amino acid substitution at the position corresponding to position 366 of human IgG1 or IgG4, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to positions 366, 368 and / or 407 of human IgG1 or IgG4, wherein the amino acid substitutions are 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 according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 366, 368 and / or 407 of human IgG1 or IgG4, wherein the amino acid substitutions are 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 according to the EU Index, the second Fc region comprises an amino acid substitution at the position corresponding to position 366 of human IgG1 or IgG4, 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 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 349, 366, 368 and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V; wherein the third polypeptide chain further comprises a second Fc region 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 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region.

[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 according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to 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 CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to 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 CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, 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 CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region 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 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 CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366 and 409 of human IgG4, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the second Fc region comprises amino acid substitutions at positions corresponding to 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 CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407 and 409 of human IgG4, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein according to the EU Index, the second Fc region, comprises 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 include one or more mutations to improve serum half-life (see, e.g., Hinton, P.R. et al. (2006) J. Immunol. 176(1):346-56). In some embodiments, according to the EU Index, the mutations include substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein includes a second polypeptide chain that further includes a first Fc region linked to CH1, the first Fc region including an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further includes a second Fc region linked to CH1, the second Fc region including 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 regions include amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding proteins of the present disclosure include a staphylococcal nuclease and a mutant and one or more mutations to improve serum half-life. In some embodiments, the first and / or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and / or second Fc regions are human IgG4 Fc regions.

[0680] In some embodiments, the binding proteins of the present disclosure include one or more mutations to improve the stability of, for example, the hinge region and / or dimer interface of IgG4 (see, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, according to the EU Index, the mutations include substitutions at positions corresponding to positions 228 and 409 of human IgG4, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein includes a second polypeptide chain that further includes a first Fc region linked to C H1 linked to the first Fc region including an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further includes a second Fc region linked to C H1 linked to the second Fc region including an immunoglobulin hinge region and C H2 and C H3Immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein according to the EU Index, the first and second Fc regions each contain 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, the binding proteins of the present disclosure contain a staphylococcal protein A 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 proteins of the present disclosure contain one or more mutations to improve purification, for example, by modulating the affinity for purification reagents. For example, it is known that if one of the two Fc regions in the heterodimeric form contains a mutation that reduces or eliminates binding to protein A, the heterodimeric binding protein can be selectively purified from its homodimeric form because the heterodimeric form will have an intermediate affinity for protein A-based purification compared to either homodimeric form and can be selectively eluted from protein A, for example, by using different pHs (see, for example, Smith, E.J. et al. (2015) Sci. Rep. 5:17943). In some embodiments, according to the EU Index, the mutations contain substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein contains a second polypeptide chain, which further contains a first Fc region linked to C H1 linked first Fc region, the first Fc region containing an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further contains a second Fc region linked to C H1 linked second Fc region, the second Fc region containing an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; and wherein according to the EU Index, only one of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding proteins of the present disclosure contain a staphylococcal protein A and one or more mutations to improve purification. In some embodiments, the first and / or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and / or second Fc regions are human IgG4 Fc regions.

[0682] In some embodiments, the binding proteins of the present disclosure contain one or more mutations to improve serum half-life (see, e.g., Hinton, P.R. et al. (2006) J. Immunol. 176(1):346 - 56). In some embodiments, according to the EU Index, the mutations contain substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein contains a second polypeptide chain that further contains a first Fc region linked to CH1, the first Fc region containing an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further contains a second Fc region linked to CH1, the second Fc region containing 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 regions contain amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding proteins of the present disclosure contain a staphylococcal protein A and one or more mutations to improve serum half-life. In some embodiments, the first and / or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and / or second Fc regions are human IgG4 Fc regions.

[0683] In some embodiments, the binding proteins of the present disclosure contain one or more mutations to reduce effector functions, such as Fc receptor-mediated antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain further contains a first Fc region linked to C H1 The first Fc region contains an immunoglobulin hinge region and C H2 and C H3 Immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further contains a second Fc region linked to C H1 The second Fc region contains an immunoglobulin hinge region and 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 EUIndex, the first and second Fc regions each contain amino acid substitutions 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 contain amino acid substitutions 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 first Fc region linked to C H1 linked first Fc region, the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to C H1 linked second Fc region, the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein according to the EUIndex, the first and second Fc regions each contain 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, 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 contain 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, the Fc regions of the second and third polypeptide chains are human IgG4 Fc regions, and according to the EU Index, the Fc regions each contain 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, the binding protein comprises a second polypeptide chain, which further comprises a first Fc region linked to C H1 linked first Fc region, the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and the third polypeptide chain further comprises a second Fc region linked to C H1 linked second Fc region, the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3An immunoglobulin heavy chain constant domain; and wherein according to the EU Index, the first and second Fc regions each contain amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4, wherein the amino acid substitutions are F234A and L235A.

[0684] In some embodiments, the binding proteins of the present disclosure comprise a "stalk" and "socket" mutation 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 the Fc mutations at position 329, see, e.g., Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591-6604 and WO1999051642.

[0685] In some embodiments, the above-described types of mutations can be combined in any order or combination. For example, the binding proteins of the present disclosure can comprise two or more "stalk" and "socket" 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] Assay

[0688] The present disclosure provides antigen-binding proteins that bind to human and / or cynomolgus monkey CD38 polypeptides, induce proliferation of T cells (e.g., CD4+ and / or CD8+ T cells), and / or induce apoptosis of CD38+ cells. Exemplary assays for measuring these parameters and identifying such binding proteins are provided herein. For example, in some embodiments, the binding affinity between the binding protein or its antigen-binding fragment and a 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 a 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 proteins of the present disclosure binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1, the 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 flow cytometry using cells expressing a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1 on their cell surface, for example, as described hereinbelow. In some embodiments, the antigen-binding site binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30, the 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 flow cytometry using cells expressing a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30 on their cell surface, for example, as described hereinbelow. In some embodiments, the antigen-binding site binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1, the 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 SPR using a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1, for example, as described hereinbelow. In some embodiments, the antigen-binding site binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30, the 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 SPR using a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30, for example, as described hereinbelow. 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, 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 characteristics: binding to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purified protein, as measured by SPR or ELISA; binding to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purified protein, the 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 the human CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:1), as determined by flow cytometry; binds to the extracellular domain of the human CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:1), the 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, the 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 the cynomolgus monkey CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:30), as determined by flow cytometry; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:30), the 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 measured by flow cytometry; binds to the extracellular domain of a human isotype E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) as a purified protein, as measured by SPR or ELISA; binds to the extracellular domain of a human isotype E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) expressed on the cell surface, as measured by flow cytometry; induces apoptosis of cells expressing CD38 or antibody-dependent cellular cytotoxicity (ADCC) of antibody-dependent cells on its cell surface; and has one or more mutations (e.g., in the Fc region) resulting in reduced binding to FcγRI and / or FcγRII as compared to the same binding protein without one or more mutations. In some embodiments, the binding protein of the present disclosure binds to a CD38 polypeptide (e.g., human or cynomolgus monkey) expressed on the cell surface, and its EC50 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 measured by flow cytometry. In some embodiments, the binding protein of the present disclosure binds to a CD38 polypeptide (e.g., human or cynomolgus monkey) as a purified protein, and its EC50 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 measured 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: inducing the proliferation of T cells (e.g., CD4+ and / or CD8+ T cells); inducing the expression of Bcl-xL in T cells (e.g., CD4+ and / or CD8+ T cells); inducing apoptosis of CD38+ cells (e.g., as measured by annexin V staining and / or propidium iodide uptake); binding to CD38 expressed on the cell surface and one or more T cell target antigens expressed on the surface of T cells; binding to CD38 expressed on the cell surface, CD28 expressed on the surface of T cells, and CD3 expressed on the surface of T cells; stimulating the 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) resulting in a reduced induction of cytokine release (e.g., IFN-γ, IL-2, and / or TNF-α) by PBMCs compared to the same binding protein without 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 immunoassay); binding to the extracellular domain of the human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purified protein, as determined by SPR or ELISA; binding to the extracellular domain of the human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purified protein, the K D is 1.5 nM or less, as determined by SPR or ELISA; binding to the extracellular domain of the human CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:1), as determined by flow cytometry; binding to the extracellular domain of the human CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:1), the apparent K D is 12 nM or less, as determined by flow cytometry; binding 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; binding 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, the K D is 3.5 nM or less, as determined by SPR or ELISA; binding to the extracellular domain of the cynomolgus monkey CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:30), as determined by flow cytometry; binding to the extracellular domain of the cynomolgus monkey CD38 polypeptide expressed on the cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:30), the apparent K Dis 7.5 nM or less, as determined by flow cytometry; binds to the extracellular domain of a human isotype 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 isotype E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) expressed on the cell surface, as determined by flow cytometry; induces apoptosis of cells expressing CD38 on their cell surface or antibody-dependent cellular cytotoxicity (ADCC); has one or more mutations (e.g., in the Fc region) resulting in reduced binding to FcγRI and / or FcγRII as compared to the same binding protein without one or more mutations. In some embodiments, the binding protein of the present disclosure binds to a CD38 polypeptide (e.g., human or cynomolgus monkey) expressed on the cell surface, and the EC50 is 20 nM or less, 15 nM or less, ...

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. 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 the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence having the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence having the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43); and (b) An antibody light chain variable (VL) domain comprising a CDR-L1 sequence having an amino acid sequence containing QGIRND (SEQ ID NO:44), a CDR-L2 sequence having an amino acid sequence containing AAS (SEQ ID NO:45), and a CDR-L3 sequence having an amino acid sequence containing LQDYIYYPT (SEQ ID NO:46).

9. 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 the human CD38 polypeptide and the extracellular domain of the cynomolgus monkey CD38 polypeptide.

10. A binding protein comprising three antigen-binding sites that each bind to one or more target proteins, wherein the binding protein comprises four polypeptide chains that form the three antigen-binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula: V L2 -L 1 -V L1 -L 2 -C L [I] and the second polypeptide chain comprises a structure represented by the following formula: V H1 -L 3 -V H2 -L 4 -C H1 -Hinge - C H2 -C H3 [II] and the third polypeptide chain comprises a structure represented by the following formula: V H3 -C H1 -Hinge - C H2 -C H3 [III] and the fourth polypeptide chain comprises a structure represented by the following formula: V L3 -C L [IV] wherein ： V L1 is the first immunoglobulin light chain variable domain; V L2 is the second immunoglobulin light chain variable domain; V L3 is the third immunoglobulin light chain variable domain; V H1 is the first immunoglobulin heavy chain variable domain; V H2 is the second immunoglobulin heavy chain variable domain; V H3 is the third immunoglobulin heavy chain variable domain; C L is the constant domain of the immunoglobulin light chain; C H1 is immunoglobulin C H1 constant heavy chain domain; C H2 is the constant domain of the immunoglobulin C heavy chain; H2 constant domain of the heavy chain; C H3 is immunoglobulin C H3 constant heavy chain domain; The hinge is the immunoglobulin hinge region that connects the said C H1 and C H2 domains; and L 1 、L 2 、L 3 and L 4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain-heavy chain pair; and wherein: (a) The hinge-C of the second and the third polypeptide chains H2 -C H3 domain is a human IgG1 hinge-C H2 -C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 domains each contain 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; or (b) The hinge-C of the second and the third polypeptide chains H2 -C H3 domain is a human IgG4 C H3 domain, and wherein according to the EU Index, the hinge-C H2 -C H3 domains each contain 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.

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