ANTI-CD38 ANTIBODIES AND METHODS OF USE

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application Serial No. 62/570,655, filed October 10, 2017; U.S. Provisional Application Serial No. 62/570,660, filed October 11, 2017; U.S. Provisional Application Serial No. 62/676,221, filed May 24, 2018; and EP Application No. EP18187186.4, filed August 3, 2018; all of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 183952029941seqlist.TXT, date recorded: October 8, 2018, size: 158 KB).

FIELD OF THE INVENTION

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

BACKGROUND

[0004] Monoclonal antibody based biotherapeutics have become an important avenue for new drug development. Monoclonal antibody technology offers specific targeting, precise signaling delivery and/or payload to specific cell population, and provides long lasting biological effect through its Fc functions. Efforts in antibody engineering have allowed developing multispecific antibodies combining the specificities of multiple monoclonal antibodies for various biological applications, expanding the scope of antibody drug development.

[0005] CD38 is an attractive drug target, since it is expressed on the cell surface of a variety of lymphoid tumor cells (see Stevenson, G.T. (2006) Mol. Med. 12:345-346).

DARZALEX® (daratumumab) is an anti-CD38 antibody approved for use in treating multiple myeloma. However, a need exists for therapeutics targeting CD38 with a different mode 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 activities.

BRIEF SUMMARY

[0006] Provided herein are binding proteins that bind CD38 polypeptides (e.g., human and cynomolgus monkey CD38 polypeptides), including monospecific, bispecific, or trispecific binding proteins with at least one antigen binding site that binds a CD38 polypeptide. Advantageously, these binding proteins have the ability to recruit T cells to the proximity of cancer cells, subsequently to activate T cells and promote the activated T cells killing of adjacent cancer cells through a Granzyme/Perforin mechanism, providing a different mode of action for anti-tumor activity from anti-CD38 antibodies such as

DARZALEX® (daratumumab). Moreover, the ability to bind both human and cynomolgus monkey CD38 polypeptides allows binding proteins to be readily tested in preclinical toxicological studies, e.g., to evaluate their safety profiles for later clinical use.

[0007] In some embodiments, provided herein are monospecific binding proteins that bind to a human CD38 polypeptide. In some embodiments, the binding proteins cross-react with human and cynomolgus monkey CD38 polypeptides. In some embodiments, the binding proteins bind to human isoform A and isoform E CD38 polypeptides. In some embodiments, the binding proteins possess one or more of the following features (in any combination): binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, as assayed by SPR; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein with a KD of 1.5nM or less, as assayed by SPR; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell, as assayed by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell with an apparent KD of 20nM, 15nM, lOnM, 5nM, InM, or less, as assayed by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30) as a purified protein, as

assayed by SPR; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30) as a purified protein with a KD of 3.5nM or less, as assayed by SPR; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30) expressed on the surface of a cell, as assayed by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell with an apparent KD of 7.5nM or less, as assayed by flow cytometry; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) as a purified protein, as assayed by ELISA; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) expressed on the surface of a cell, as assayed by flow cytometry; induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of a cell expressing CD38 on its cell surface; and has one or more mutations (e.g., in an Fc region) resulting in decreased binding to FcyRI and/or FcyRII, as compared to the same binding protein without the one or more mutations. For exemplary assays, see Examples 1, 3, and 4. In some embodiments, the KD is measured at 4°C or 25°C.

[0008] In some embodiments, provided herein are trispecific binding proteins that bind to a human CD38 polypeptide. In some embodiments, the trispecific binding proteins bind (e.g., simultaneously) to a CD38 polypeptide (e.g., expressed on the surface of a cell) and one or more other target antigens expressed on the surface of a second cell, thereby recruiting the second cell in proximity with the cell expressing the CD38 polypeptide. In some embodiments, the trispecific binding proteins bind (e.g., simultaneously) to a CD38 polypeptide (e.g., expressed on the surface of a cell) and one or two target antigens expressed on the surface of a T cell, thereby recruiting the T cell in proximity with the cell expressing the CD38 polypeptide. In some embodiments, the trispecific binding proteins activate the T cell and/or provide a CD28-mediated costimulatory signal to the T cell. In some embodiments, the trispecific binding proteins cross-react with human and

cynomolgus monkey CD38 polypeptides. In some embodiments, the trispecific binding proteins bind to human isoform A and isoform E CD38 polypeptides. In some

embodiments, the trispecific binding proteins possess one or more of the following features (in any combination): binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, as assayed by SPR; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein with a KD of 1.5nM or less, as assayed by SPR; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell, as assayed by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell with an apparent KD of 20nM, 15nM, lOnM, 5nM, InM, or less, as assayed by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30) as a purified protein, as assayed by SPR; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30) as a purified protein with a KD of 3.5nM or less, as assayed by SPR; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell, as assayed by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) expressed on the surface of a cell with an apparent KD of 7.5nM or less, as assayed by flow cytometry; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) as a purified protein, as assayed by ELISA; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) expressed on the surface of a cell, as assayed by flow cytometry; induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of a cell expressing CD38 on its cell surface; and has one or more mutations (e.g., in an Fc region) resulting in decreased binding to FcyRI and/or FcyRII, as compared to the same binding protein without the one or more mutations; induces T cell (e.g., CD4+ and/or CD8+ T cell) proliferation; induces T cell (e.g., CD4+ and/or CD8+ T cell) expression of Bcl-xL; induces apoptosis of CD38+ cells; binds to CD38 expressed on the surface of a cell and one or more T cell target antigen(s) expressed on the surface of a T cell; binds to CD38 expressed on the surface of a cell, CD28 expressed on the surface of a T cell, and CD3 expressed on the surface of a T cell; stimulates activation of the T cell receptor; induces costimulation of T cell receptor signaling (e.g., as mediated by CD28); and has one or more mutations (e.g., in an Fc region) resulting in decreased induction of cytokine release (e.g., IFN-γ, IL-2, and/or TNF-a) by PBMCs, as compared to the same binding protein without the one or more mutations;

induces cytokine release (e.g., IFN-γ and/or IL-6) by PBMCs in the presence of a CD38+ target cell. For exemplary assays, see Examples 1, 3, and 4. In some embodiments, the KD is measured at 4°C or 25°C.

[0009] In some embodiments, provided herein is a binding protein comprising an antigen binding site that binds a CD38 polypeptide, wherein the antigen binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and/or (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or

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

ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the antigen binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and/or (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence

comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the VH domain comprises the sequence, from N-terminus to C-terminus, FR1— CDR-H1— FR2— CDR-H2— FR3— CDR-H3— FR4; wherein FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86),

QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87), or

QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); wherein FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHW VKE APGQ GLEWIGY (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 comprising the amino acid sequence of SEQ ID NO:7 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 17, and/or the VL domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:21, and/or the VL domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:22 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:23, and/or the VL domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the antigen binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1

sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQ KEDPWT (SEQ ID NO:36). In some embodiments, the VH domain comprises the sequence, from N-terminus to C-terminus, FR1— CDR-H1— FR2— CDR-H2— FR3— CDR-H3— FR4; wherein FR1 comprises the sequence 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 MHW VKE APGQ GLEWIGY (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 comprising the amino acid sequence of SEQ ID NO: 15 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 16.

[0010] In some embodiments, provided herein is a binding protein comprising an antigen binding site that binds a CD38 polypeptide, wherein the antigen binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46). In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:9, and/or the VL domain comprises the amino acid sequence of SEQ ID NO: 10. In some

embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antigen binding site cross-reacts with an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide. In some embodiments, the antigen binding site binds a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antigen binding site binds the human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with an equilibrium dissociation constant (KD) of 2.1nM or less. In some embodiments, the antigen binding site binds a human isoform E CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, the antigen binding site binds a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30. In some embodiments, the antigen binding site binds the cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30 with an equilibrium dissociation constant (KD) of 1.3nM or less.

[0011] In some embodiments of any of the above embodiments, the binding protein is a chimeric or humanized antibody. In some embodiments, the binding protein is a human antibody. In some embodiments, the binding protein is a monoclonal antibody. In some embodiments, the binding protein comprises one or more full-length antibody heavy chains comprising an Fc region. In some embodiments, the Fc region is a human Fc region comprising one or more mutations that reduce or eliminate Fc receptor binding and/or effector function of the Fc region. In some embodiments, the Fc region is a human IgGl Fc region. In some embodiments, the human IgGl Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the human IgGl Fc region comprises amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, 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, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the human IgG4 Fc region comprises amino acid

substitutions at positions corresponding to positions 233-237 of human IgG4 according to EU Index, 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 label. In some embodiments, the binding protein is a bispecific binding protein comprising the first antigen binding site that binds the CD38 polypeptide and a second antigen binding site. In some embodiments, the binding protein is a trispecific binding protein comprising the first antigen binding site that binds the CD38 polypeptide, a second antigen binding site, and a third antigen binding site. In some embodiments, the first antigen binding site binds the extracellular domain of a human CD38 polypeptide, and wherein the second and third antigen binding sites each bind a T-cell surface protein. In some embodiments, the first antigen binding site binds the extracellular domain of a human CD38 polypeptide, and wherein (a) the second antigen binding site binds a human CD28 polypeptide, and the third antigen binding site binds a human CD3 polypeptide, or (b) the second antigen binding site binds a human CD3 polypeptide, and the third antigen binding site binds a human CD28 polypeptide.

[0012] In some embodiments, provided herein is a binding protein comprising three antigen binding sites that each bind one or more target proteins, wherein at least one of the three antigen binding sites cross-reacts with an extracellular domain of a human CD38 polypeptide and an 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 an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide and two antigen binding sites that each bind a T-cell surface protein. In some embodiments, the binding protein comprises an antigen binding site that cross-reacts with an extracellular domain of a human CD38 polypeptide and an

extracellular domain of a cynomolgus monkey CD38 polypeptide, an antigen binding site that binds a human CD28 polypeptide, and an antigen binding site that binds a human CD3 polypeptide. In some embodiments, the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-LI-VLI-L2-Cl [I]

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

VHi-L3-VH2-L4-CHi-hinge-CH2-CH3 [Π]

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

Vro-Cm-hinge-Cm-Cro [HI]

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

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHi heavy chain constant domain;

CH2 is an immunoglobulin Cm heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and

Li, L2, L3 and L4 are amino acid linkers;

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

wherein: (a) the VHi domain comprises a CDR-Hl sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VLi domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);

(b) the Vm domain comprises a CDR-Hl sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT

(SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of

ARTGGLRRAYFTY (SEQ ID NO:33), and the VL2 domain comprises a CDR-Ll 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 QQ KEDPWT (SEQ ID NO:36); or

(c) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of

ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or

QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

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

VHi-L3-VH2-L4-CHi-hinge-CH2-CH3 [Π]

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

VH3-CHi-hinge-CH2-CH3 [HI]

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

VL3-CL [IV]

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHI heavy chain constant domain;

CH2 is an immunoglobulin Cm heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and

Li, L2, L3 and L4 are amino acid linkers;

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

wherein: (a) the Vm domain comprises a CDR-Hl sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VLi domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);

(b) the VH2 domain comprises a CDR-Hl sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of

ARTGGLRRAYFTY (SEQ ID NO:33), and the VL2 domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or

QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or

(c) the VH3 domain comprises a CDR-Hl sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of

ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or

QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the Vm domain comprises a CDR-Hl sequence comprising the amino acid

sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VLi domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID

NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VLi domain comprises a CDR-L1 sequence comprising the amino acid sequence of

QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or the VH3 domain comprises a CDR-Hl sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of

GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of

QQNKEDPWT (SEQ ID NO:36). In some embodiments, the VH3 domain comprises the amino acid sequence of SEQ ID NO:5, and the VL3 domain comprises the amino acid sequence of SEQ ID NO:6; the VH3 domain comprises the amino acid sequence of SEQ ID NO: 17, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 18; the VH3 domain comprises the amino acid sequence of SEQ ID NO:21, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 18; the VH3 domain comprises the amino acid sequence of SEQ ID NO:23, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 18; or the VH3 domain comprises the amino acid sequence of SEQ ID NO: 13, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-LI-VLI-L2-Cl [I]

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

VHi-L3-VH2-L4-CHi-hinge-CH2-CH3 [Π]

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

Vro-Cm-hinge-Cm-Cro [HI] and a fourth polypeptide chain comprises a structure represented by the formula:

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHi heavy chain constant domain;

CH2 is an immunoglobulin Cm heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and

Li, L2, L3 and L4 are amino acid linkers;

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

wherein (a) the VHi domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGS K (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the VLI domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46); (b) the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2

sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46); or (c) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGS K (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46). In some embodiments, the VH3 domain comprises the amino acid sequence of SEQ ID NO:9, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the Vm domain comprises the amino acid sequence of SEQ ID NO:49, the VLI domain comprises the amino acid sequence of SEQ ID NO:50, the VH2 domain comprises the amino acid sequence of SEQ ID NO:53, and the VL2 domain comprises the amino acid sequence of SEQ ID NO:54; the Vm domain comprises the amino acid sequence of SEQ ID NO:49, the VL2 domain comprises the amino acid sequence of SEQ ID NO:50, the VHi domain comprises the amino acid sequence of SEQ ID NO:53, and the VLi domain comprises the amino acid sequence of SEQ ID NO:54; the Vm domain comprises the amino acid sequence of SEQ ID NO:51, the VLI domain comprises the amino acid sequence of SEQ ID NO:52, the Vm domain comprises the amino acid sequence of SEQ ID NO:53, and the VL2 domain comprises the amino acid sequence of SEQ ID NO:54; the Vm domain comprises the amino acid sequence of SEQ ID NO:51, the VL2 domain comprises the amino acid sequence of SEQ ID NO:52, the VHi domain comprises the amino acid sequence of SEQ ID NO:53, and the VLI domain comprises the amino acid sequence of SEQ ID NO:54 the Vm domain comprises the amino acid sequence of SEQ ID NO:49, the VLI domain comprises the amino acid sequence of SEQ ID NO:50, the Vm domain comprises the amino acid sequence of SEQ ID NO: 84, and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 85; the VH2 domain comprises the amino acid sequence of SEQ ID NO:49, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 50, the VHI domain comprises the amino acid sequence of SEQ ID NO: 84, and the VLI domain comprises the amino acid sequence of SEQ ID NO: 85; the VHi domain comprises the amino acid sequence of SEQ ID NO: 51 , the VLI domain comprises the amino acid sequence of SEQ ID NO: 52, the VH2 domain comprises the amino acid sequence of SEQ ID NO: 84, and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 85; or the VH2 domain comprises the amino acid sequence of SEQ ID NO: 51 , the VL2 domain comprises the amino acid sequence of SEQ ID NO: 52, the VHI domain comprises the amino acid sequence of SEQ ID NO: 84, and the VLi domain comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the VHI domain comprises the amino acid sequence of SEQ ID NO:49, the VLI domain comprises the amino acid sequence of SEQ ID NO: 50, the VH2 domain comprises the amino acid sequence of SEQ ID NO: 53, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 54, the VH3 domain comprises the amino acid sequence of SEQ ID NO: 13, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 14; or the VHi domain comprises the amino acid sequence of SEQ ID NO:49, the VLI domain comprises the amino acid sequence of SEQ ID NO: 50, the VH2 domain comprises the amino acid sequence of SEQ ID NO: 53, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 54, the VH3 domain comprises the amino acid sequence of SEQ ID NO: 9, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, at least one of Li, L2, L3 or L4 is independently 0 amino acids in length. In some embodiments, Li, L2, L3 or L4 are each independently at least one amino acid in length. In some embodiments, (a) Li, L2, L3 and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO : 58), and GGSGSSGSGG (SEQ ID NO: 59); or (b) Li, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO :55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO : 58), and

GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, Li comprises the sequence GQPKAAP (SEQ ID NO: 58), L2 comprises the sequence TKGPS (SEQ ID NO: 57), L3 comprises the sequence S, and L4 comprises the sequence RT; Li comprises the sequence GGGGSGGGGS (SEQ ID NO: 55), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 55), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; Li comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; or Li comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), and L4 is 0 amino acids in length. In some embodiments, the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgGl hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgGl hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S. In some embodiments, the hinge-Cm-Cm domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-Cm-Cmdomain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-Cm-Cm domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to

positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-Cm-Cm domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, 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.

[0013] In some embodiments, provided herein is a binding protein comprising three antigen binding sites that each bind one or more target proteins, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-LI-VLI-L2-Cl [I]

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

VHi-L3-VH2-L4-CHi-hinge-CH2-CH3 [Π]

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

Vro-Cm-hinge-Cm-Cro [HI]

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

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHI heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and

Li, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair; and

wherein (a) the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgGl hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S; or (b) the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the human IgG4 hinge-Cm-Cm domains comprise amino acid substitutions at positions corresponding to positions 233-237 of human IgG4 according to EU Index, wherein the sequence EFLGG is replaced by PVAG. In some embodiments, at least one pair of VHI and VLI, Vm and VL2, and Vm and VL3 forms an antigen binding site that binds a CD38 polypeptide. In some embodiments, one, two, or three pairs of Vm and VLI, VH2 and VL2, and VH3 and VL3 form an antigen binding site that binds an antigen target selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD 122, CD 137, 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, FCERIA, FCERl, FLAP, FOLHl, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGBl, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, ILIA, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhILlO, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, KG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TFM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1. In some embodiments, a first pair of Vm and VLI, VH2 and VL2, and VH3 and VL3 forms an antigen binding site that binds a human CD3 polypeptide, a second pair of Vm and VLI, VH2 and VL2, and VH3 and VL3 forms an antigen binding site that binds a human CD28 polypeptide, and a third pair of Vm and VLI, VH2 and VL2, and VH3 and VL3 forms an antigen binding site that binds a human antigen target selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, 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, FCERIA, FCERl, FLAP, FOLHl, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB l, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, ILIA, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhILlO, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II,

NCR3LG1, KG2D, NTPDase-1, OX40, OX40L, PD-IH, platelet receptor, PROMl, SI 52, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TTM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.

[0014] In some embodiments, provided herein is 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.

[0015] In some embodiments, provided herein is a polynucleotide comprising the binding protein of any one of the above embodiments. In some embodiments, provided herein is a vector comprising a polynucleotide comprising the binding protein of any one of the above embodiments.

[0016] In some embodiments, provided herein is a host cell comprising the kit of polynucleotides, polynucleotide, or vector of any one of the above embodiments. In some embodiments, provided herein is a method of producing a binding protein, the method comprising culturing the host cell of any one of the above embodiments such that the

binding protein is produced. In some embodiments, the method further comprises recovering the binding protein from the host cell.

[0017] 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.

[0018] 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 the 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, 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 a B cell lymphoma. In some embodiments, the patient is a human. In some embodiments, the patient is selected for treatment because cells of the cancer express a human CD38 isoform E polypeptide (e.g., as set forth in SEQ ID NO: 105) on their cell surface. In some embodiments, the cancer cells express CD38 and CD28. In some embodiments, the cancer cells express CD38 and do not express CD28.

[0019] In some embodiments, provided herein is at least one binding protein of any one of the above embodiments or the pharmaceutical composition of any one of the above embodiments for use in preventing and/or treating cancer in a patient (e.g., a patient in need thereof, such as a patient with cancer). In some embodiments, provided herein is at least one binding protein of any one of the above embodiments or the pharmaceutical composition of any one of the above embodiments for use in the manufacture of a medicament for preventing and/or treating cancer in a patient (e.g., a patient in need thereof, such as a patient with cancer). In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein comprising a first antigen binding site that binds CD3, a second antigen binding site that binds CD28, and a third antigen binding site that binds the extracellular domain of a human CD38 polypeptide. In some embodiments, the at least one binding protein is to be co-administered with a chemotherapeutic agent. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia

(ALL), chronic lymphocytic leukemia (CLL), or a B cell lymphoma. In some embodiments, the patient is a human. In some embodiments, the patient is selected for treatment because cells of the cancer express a human CD38 isoform E polypeptide (e.g., as set forth in SEQ ID NO: 105) on their cell surface. In some embodiments, the cancer cells express CD38 and CD28. In some embodiments, the cancer cells express CD38 and do not express CD28.

[0020] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] FIG. 1A shows the binding of anti-CD38 antibodies mAb l (top) and isatuximab (bottom) to SU-DHL-8 human lymphoma cells or MOLP-8 human multiple myeloma cells using flow cytometry.

[0023] FIG. IB shows the results of flow cytometry binding assays examining binding of anti-CD38 antibodies mAb l or isatuximab (no binding observed) to cells expressing cynomolgus monkey CD38 on their surface.

[0024] FIGS. 2A-2I show the results of assays characterizing the binding of anti-CD38 antibodies to human and cynomolgus monkey CD38 polypeptides. FIG. 2A shows the binding of humanized anti-CD38 antibody mAb2 to soluble human CD38 (top,

"hCD38: :Histag") or cynomolgus monkey CD38 (top, "cynoCD38: :Histag") by ELISA, as well as the binding of mAb2 to the surface of cells expressing human CD83 (bottom, as indicated) or cynomolgus monkey CD38 (bottom, as indicated) by flow cytometry. FIG. 2B shows the binding of mAb2 to human CD38 (top) or cynomolgus monkey CD38 (bottom) by surface plasmon resonance (SPR). FIG. 2C shows the binding of humanized anti-CD38 antibody mAb3 to soluble human CD38 (top, "hCD38: :Histag") or cynomolgus monkey CD38 (top, "cynoCD38: :Histag") by ELISA, as well as the binding of mAb3 to the surface of cells expressing human CD83 (bottom, as indicated) or cynomolgus monkey

CD38 (bottom, as indicated) by flow cytometry. FIG. 2D shows the binding of mAb3 to human CD38 (top) or cynomolgus monkey CD38 (bottom) by SPR. FIG. 2E shows the binding of humanized anti-CD38 antibody mAb5 to soluble human CD38 (top,

"hCD38: :Histag") or cynomolgus monkey CD38 (top, "cynoCD38: :Histag") by ELISA, as well as the binding of mAb5 to the surface of cells expressing human CD83 (bottom, as indicated) or cynomolgus monkey CD38 (bottom, as indicated) by flow cytometry. FIG. 2F shows the binding of mAb5 to human CD38 (top) or cynomolgus monkey CD38 (bottom) by SPR. FIG. 2G shows the binding of human anti-CD38 antibody hhyl370 to soluble human CD38 (top, "hCD38::Histag") or cynomolgus monkey CD38 (top,

"cynoCD38: :Histag") by ELISA, as well as the binding of hhyl370 to the surface of cells expressing human CD83 (bottom, as indicated) or cynomolgus monkey CD38 (bottom, as indicated) by flow cytometry. FIG. 2H shows the binding of hhyl370 to human CD38 (top) or cynomolgus monkey CD38 (bottom) by SPR. FIG. 21 summarizes the binding data from the ELISA, SPR, and FACS experiments, as well as the percentage identity of each antibody VH ("H") or VL ("L") domain to human V region sequence from top to bottom it corresponds to mAb2, mAb3, mAb 4, mAb 5, and mAb6 last line (1195 HHKK-3 has not been described by its VL/VH amino acid sequence in the text below).

[0025] FIG. 2J shows the concentration-dependent induction of SU-DHL-8 cells apoptosis by mAb7 and mAbl after incubation for 72 hours at 37°C.

[0026] FIG. 2K shows antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isatuximab and mAbl against SU-DHL-8 cells in the presence of K92 cells.

[0027] FIG. 2L shows concentration-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isatuximab (right) and mAbl (left) against SU-DHL-8 cells in the presence of K92 cells after 4 hours at 37°C.

[0028] FIGS. 2M-2Q showed the results of apoptosis induction assays using the indicated anti-CD38 antibodies against SU-DHL-8 tumor cells. Apoptosis was quantified by measuring dual Annexin V and Propidium iodide uptake via flow cytometry. FIG. 2M shows the percentage of apoptotic cells induced by each antibody. FIGS. 2N-2Q show dose-dependent induction of apoptosis in in SU-DHL-8 lymphoma cells by anti-CD38 antibodies mAb2 (FIG. 2N), mAb3 (FIG. 20), mAb4 (FIG. 2P), and mAb5 (FIG. 2Q), as well as the IC50 for each antibody.

[0029] FIG. 3A provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that binds three target proteins: CD28, CD3, and CD38. A first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites that recognize CD3 and CD28, and a second pair of polypeptides possess a single variable domain (VH3 and VL3) forming a single antigen binding site that recognizes CD38. The trispecific binding protein shown in FIG. 3A uses an IgG4 constant region with a "knobs-into-holes" mutation, where the knob is on the second pair of polypeptides with a single variable domain.

[0030] FIG. 3B provides a schematic representation of an SPR-based assay for examining the ability of each antigen binding domain of anti-CD38/anti-CD28/anti-CD3 trispecific binding proteins to bind its cognate antigen.

[0031] FIG. 3C shows the results of SPR-based assays for examining CD38 binding to anti-CD38/anti-CD28/anti-CD3 trispecific binding proteins. Binding of human CD38 to trispecific binding proteins was examined alone (top left), after pre-binding with CD3 (top center), after pre-binding with CD28 (top right), after pre-binding to CD3 then CD28 (bottom left), or after pre-binding to CD28 then CD3 (bottom right).

[0032] FIG. 4 shows the sequential binding of human CD3, CD28, and CD38 polypeptides to anti-CD38/anti-CD28/anti-CD3 trispecific binding proteins, as assayed by SPR.

[0033] FIG. 5 summarizes the binding affinities of indicated trispecific binding proteins against their cognate antigens (human CD3, CD28, and CD38) as measured by SPR.

[0034] FIG. 6A compares the apparent affinity of isatuximab antigen binding domain in human IgGl format (2nd sheet) or in a trispecific binding protein format with the isatuximab, anti-CD28, and anti-CD3 antigen binding domains (format according to FIG. 3A; 1st sheet) for binding human (top) or cynomolgus monkey (bottom) CD38

polypeptides, as assayed by flow cytometry.

[0035] FIGS. 6B-6D compare the apparent affinities of trispecific binding protein CD38vHixCD28supxCD3mid, CD38vHixCD28cvnxCD3mid, or monospecific anti-CD38 antibody mAb2 for binding to cells expressing human or cynomolgus monkey CD38 polypeptides, as assayed by flow cytometry. FIG. 6B shows the binding of trispecific binding protein CD38vHixCD28SUpxCD3mid to cells expressing human (top) or cynomolgus monkey (bottom) CD38 polypeptides. FIG. 6C shows the binding of trispecific binding protein CD38vHixCD28cvnxCD3mid to cells expressing human (top) or cynomolgus monkey (bottom) CD38 polypeptides. FIG. 6D shows the binding of monospecific anti-CD38

antibody mAb2 to cells expressing human (top) or cynomolgus monkey (bottom) CD38 polypeptides.

[0036] FIG. 6E compares the apparent affinities of trispecific binding protein

CD38HHYi37oxCD28supxCD3mid or monospecific anti-CD38 antibody mAb6 for binding to cells expressing human (top) or cynomolgus monkey (bottom) CD38 polypeptides, as assayed by flow cytometry.

[0037] FIG. 6F summarizes the binding affinity of the indicated anti-CD38xanti-CD28xanti-CD3 trispecific binding proteins for human CD38, as measured by SPR or flow cytometry (FACS).

[0038] FIG. 6G shows the apparent affinity of trispecific binding protein

ACD38vHixCD28supxCD3mid lacking the anti-CD38 antigen binding domain for binding to cells expressing human (top) or cynomolgus monkey (bottom) CD38 polypeptides, as assayed by flow cytometry.

[0039] FIGS. 7A & 7B show the results of an ELISA assay determining the binding affinities of various anti-CD38 x CD28 x CD3 IgG4 trispecific binding proteins, or control antibodies, to human and rhesus monkey CD3, CD28 and CD38 polypeptides.

[0040] FIGS. 8A-8D show the results of antibody-mediated specific killing of CD38+ cells by PBMCs from three different human donors using the indicated anti-CD38 x CD28 x CD3 trispecific binding proteins and control antibodies. Representative results using multiple myeloma cell lines RPMI8266 (FIG. 8A), NCI-H929 (FIG. 8B), KMS-26 (FIG. 8C), and KMS-11 cell lines (FIG. 8D) are shown, and EC50 values are provided in Table N. EC50 values obtained by using NCI-H929, KMS-26, and KMS-11 cells are provided in Tables O-Q.

[0041] FIGS. 8E & 8F show the results of antibody-mediated specific killing of CD38+ cells by PBMCs from two different donors using the indicated anti-CD38 x CD28 x CD3 trispecific binding proteins with variant Fc regions and control antibodies. Representative results using CD38+ KMS-11 (FIG. 8D) and U266 (FIG. 8E) cell lines are shown, and EC50 values are provided in Tables Q2 and Q3.

[0042] FIGS. 9 A, 9B, & 10 show the activation (CD69+) of human T cells treated with various anti-CD38 x CD28 x CD3 trispecific binding proteins or control antibodies for 24 hours. FIG. 9A shows the activation (CD69+) of human CD3+ T cells. FIG. 9B shows the activation (CD69+) of human CD3+ CD4+ T cells. FIG. 10 shows the activation (CD69+) of human CD3+ CD8+ T cells.

[0043] FIGS. 11A-11B show the results of in vitro cytokine release assessments of human PBMCs treated with the indicated anti-CD38 x CD28 x CD3 trispecific binding proteins or control antibodies based on dried plate method as described in Stebbings, R. et al. (2007) J. Immunol. 179:3325-3331. FIG. 11A shows the results using 5μg/mL of the indicated antibodies. FIG. 11B shows the results using 25ng/mL of the indicated antibodies.

[0044] FIGS. 12A-12E show the in vivo activity of the anti-CD38(VHi) x CD28(sup) x CD3(mid) trispecific binding protein in the CD34+ umbilical cord blood cells humanized NSG mouse model implanted with RPMI-8226 multiple myeloma cells. FIG. 12A shows the change in tumor volume in mice treated with the indicated concentrations of the anti-CD38(VHI) x CD28(Sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG. 12B shows the average tumor volume at day 18 in mice treated with the indicated concentrations of the anti-CD38(vHi) x CD28(sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG. 12C shows the average terminal tumor weight in mice treated with the indicated concentrations of the anti-CD38(VHi) x CD28(Sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG. 12D shows the average tumor growth curve over the length of the experiment in mice treated with the indicated concentrations of the anti-CD38(vHi) x CD28(sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG. 12E shows the average change in body weight at multiple time points over the length of the experiment of mice treated with the indicated concentrations of the anti- CD38(VHI) X CD28(sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody.

[0045] FIGS. 13A-13F show the in vivo activity of the anti-CD38(VHi) x CD28(sup) x CD3(mid) trispecific binding protein in the PBMCs humanized NSG mouse model implanted with RPMI-8226 multiple myeloma cells. FIG. 13A shows the change in tumor volume in mice treated with the indicated concentrations of the anti-CD38(vHi) x CD28(Sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG. 13B shows the tumor volume at day 4 in mice treated with the indicated

concentrations of the anti-CD38(VHi) x CD28(sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG. 13C shows the tumor volume at day 21 in mice treated with the indicated concentrations of the anti-CD38(VHi) x CD28(Sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG. 13D shows the average tumor volume at day 21 in mice treated with the indicated concentrations of the anti-CD38(vHi) x CD28(sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG. 13E shows the average terminal tumor weight in mice treated with the indicated concentrations of the anti-CD38(VHi) CD28(Sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody. FIG. 13F shows the average tumor volume at multiple time points over the length of the experiment in mice treated with the indicated concentrations of the anti-CD38(vHi) x CD28(sup) x CD3(mid) trispecific binding protein vs. mice treated with an anti-CD3/CD38 bispecific antibody.

[0046] FIGS. 14A-14U show the results of a dose escalation study (0.5, 2.5, 12.5 μg/kg) using the anti-CD38(VHi) x CD28(Sup) x CD3(mid) , the anti-CD38(VHi) x CD28(cvn) x CD3 (mid), the anti-CD38(hhyi370) x CD28(Sup) x CD3(mid) , and the anti-CD38(hhyi370) x

CD28(Cvn) x CD3(mid) trispecific binding proteins in non-human primates. FIG. 14A shows T cell activation (CD69+) of circulating CD3+ T cells after administration of different doses of the anti-CD38(VHi) x CD28(Sup) x CD3(imd) trispecific binding protein. FIG. 14B shows T cell activation (CD69+) of circulating CD3+ T cells after administration of different doses of the anti-CD38(VHi) x CD28(cvn) x CD3(mid) trispecific binding protein. FIG. 14C shows T cell activation (CD69+) of circulating CD3+ T cells after administration of different doses of the anti-CD38(hhyi370) x CD28(Sup) x CD3(imd) trispecific binding protein. FIG. 14D shows T cell activation (CD69+) of circulating CD3+ T cells after administration of different doses of the anti-CD38(hiiyi370) x CD28(cvn) x CD3(mid) trispecific binding protein. FIG. 14E shows the changes in percentage of circulating CD4+ T cells after administration of the indicated doses of the anti-CD38(vHi) x CD28(Sup) x CD3(imd) trispecific binding protein. FIG. 14F shows the changes in percentage of circulating CD8+ T cells after administration of the indicated doses of the anti-CD38(VHi) x CD28(sup) x CD3(mid) trispecific binding protein. FIG. 14G shows the changes in percentage of circulating CD4+ T cells after administration of the indicated doses of the anti-CD38(vHi) x CD28(cvn) x CD3(imd) trispecific binding protein. FIG. 14H shows the changes in percentage of circulating CD8+ T cells after administration of the indicated doses of the anti-CD38(vHi) x CD28(cvn) x CD3(mid) trispecific binding protein. FIG. 141 shows the changes in percentage of circulating CD4+ T cells after administration of the indicated doses of the anti-CDSS^nvo) x CD28(sup) x CD3(mid) trispecific binding protein. FIG. 14J shows the changes in percentage of circulating CD8+ T cells after administration of the indicated doses of the anti-CD38(hhyi370) x CD28(Sup) x CD3(mid) trispecific binding protein. FIG. 14K shows the changes in percentage of circulating CD4+ T cells after administration of the indicated doses of the anti-CD38(hhyi370) x CD28(CVN) x CD3(mid) trispecific binding protein. FIG. 14L shows the changes in percentage of circulating CD8+ T cells after administration of the indicated doses of the anti-CD38(hhyi370) x CD28(CVN) x CD3(mid) trispecific binding protein. FIG. 14M shows the changes in total CD4+ T cells 6, 24, and 48 hours after administration of 12.5 μg/kg of the indicated trispecific binding proteins. FIG. 14N shows the changes in total K cells 6, 24, and 48 hours after administration of 12.5 μg/kg of the indicated trispecific binding proteins. FIG. 140 shows the changes in total CD8+ T cells 6, 24, and 48 hours after administration of 12.5 μg/kg of the indicated trispecific binding proteins. FIG. 14P shows the changes in total B cells 6, 24, and 48 hours after administration of 12.5 μg/kg of the indicated trispecific binding proteins. FIG. 14Q shows the changes in cytokine levels 6 hours after administration of the three ascending doses (0.5, 2.5, 12.5 μg/kg) of the anti-CD38(vHi) x CD28(SUP) x CD3(mid) trispecific binding protein (results from different test animals labeled as " 1 17065" and " 1 17066"). FIG. 14R shows the changes in cytokine levels 6 hours after administration of the three ascending doses (0.5, 2.5, 12.5 μg/kg) of the anti-CD38(vHi) x CD28(CVN) x CD3(mid) trispecific binding protein (results from different test animals labeled as " 1 17067" and " 1 17068"). FIG. 14S shows the changes in cytokine levels 6 hours after administration of the three ascending doses (0.5, 2.5, 12.5 μg/kg) of the anti-CD38(hhyi370) x CD28(SUP) x CD3(mid) trispecific binding protein (results from different test animals labeled as " 1 17069" and " 1 17070"). FIG. 14T shows the changes in cytokine levels 6 hours after administration of the three ascending doses (0.5, 2.5, 12.5 μg/kg) of the anti-CD38(hhyi370) x CD28(CVN) x CD3(mid) trispecific binding protein (results from different test animals labeled as " 1 17071" and " 1 17072"). FIG. 14U shows the changes in cytokine levels 24 hours after administration of the three ascending doses (0.5, 2.5, 12.5 μg/kg) of the indicated trispecific binding proteins (results shown from all test animals).

[0047] FIGS. 14V & 14W show that anti-CD38(VHi) x CD28(SUP) x CD3(mid) and anti- CD38(VHI) x CD28(CVN) x CD3(mid) trispecific binding proteins induced depletion of T cells in vivo in non-human primate blood at higher doses (6 hours post-dose).

[0048] FIGS. 14X & 14Y show that anti-CD38(HHYi370) x CD28(SUP) x CD3(MID) and anti- CD38(HHYI37O x CD28(CVN) x CD3(mid) trispecific binding proteins induced depletion of T cells in vivo in non-human primate blood at higher doses (6 hours post-dose).

[0049] FIGS. 14Z & 14AA show the amount of blood T cells in non-human primates over time after administration of anti-CD38(vHi) x CD28(SUP) x CD3(mid) or anti-CD38(vHi) x

CD28(CVN) x CD3 (mid) trispecific binding proteins.

[0050] FIGS. 14AB & 14AC show the amount of blood T cells in non-human primates over time after administration of anti-CD38(HHYi370) x CD28(SUP) x CD3(mid) or anti-CD38(HHYI370) x CD28(CVN) x CD3(mid)trispecific binding proteins.

[0051] FIGS. 14AD & 14AE show the amount of CD4+ T cells with trispecific binding protein bound after administration of 10C^g/kg dose in non-human primates.

[0052] FIGS. 14AF & 14AG show the amount of CD8+ T cells with trispecific binding protein bound after administration of 10C^g/kg dose in non-human primates.

[0053] FIGS. 15A-15C show binding or lack thereof of various Fc variants to human Fc receptors FcyR I (FIG. 15A), FcyR Ila (FIG. 15B), and FcyR Illb/c (FIG. 15C).

Variants tested were human IgGl, human IgG4, and human IgG4 with FALA mutations.

[0054] FIG. 16 shows binding of human IgG4, with or without FALA mutations, to FcRn.

[0055] FIG. 17 summarizes PK parameters of the indicated trispecific binding proteins (CD38vHixCD28supxCD3mid IgG4, CD38vHixCD28supxCD3mid IgG4 FALA,

CD38vHixCD28supxCD3mid IgGl LALA P329A, and CD38HHYi37oxCD28SUpxCD3mid IgG4 FALA) in NSG mice.

[0056] FIGS. 18A-18C show Fc/FcR interaction-mediated (non-specific) release of IFN-γ (FIG. 18A), IL-2 (FIG. 18B), or TNF-a (FIG. 18C) by human PBMCs incubated with trispecific binding proteins having wild-type or FALA variant Fc regions.

[0057] FIG. 18D shows in vitro activation of human PBMCs by

CD38VHixCD28SUpxCD3mid and CD38HHYi37oxCD28supxCD3mid trispecific binding proteins, as well as IgGl and IgG4 Fc variants thereof.

[0058] FIGS. 19A&19B show that induction of Bcl-xL in CD4+ (FIG. 19A) or CD8+ (FIG. 19B) T cells by trispecific binding protein CD38vHixCD28supxCD3mid requires both CD3 and CD28 antigen binding domains. Bar graph=mean and s.d. from 3 PBMC donors. *p=<0.009.

[0059] FIGS. 19C&19D show that CD38vmxCD28xCD3 with IgG4 FALA variant Fc upregulates Bcl-xL in CD4+ (FIG. 19C) or CD8+ (FIG. 19D) T cells greater than a benchmark bispecific antibody. Bar graph=mean and s.d. from 3 PBMC donors.

*p=<0.045

[0060] FIG. 19E shows that T cell activation by anti-CD38xanti-CD28xanti-CD3 trispecific binding proteins, as assayed by IL-2 expression in a Jurkat T cell reporter line, is dependent upon the anti-CD3 antigen binding domain.

[0061] FIG. 19F shows the release of cytokines TNF, IFNg, IL-2, JL-6, and IL-10 by CD38vHixCD28supxCD3mid trispecific binding proteins, as compared to binding proteins with mutated anti-CD28, anti-CD38, or anti-CD28, anti-CD38, and anti-CD3, as well as the benchmark.

[0062] FIG. 19G shows proliferation of T cells activated by anti-CD38xanti-CD28xanti-CD3 trispecific binding protein with IgG4 FALA variant Fc, benchmark anti-CD38xanti-CD3 bispecific antibody, or isotype control (trispecific binding protein with IgG4 FALA variant Fc having mutated binding domains).

[0063] FIG. 20 shows proliferation of T cells activated by anti-CD38xanti-CD28xanti-CD3 trispecific binding protein with IgG4 FALA variant Fc, anti-CD38xanti-CD28xanti-CD3 trispecific binding proteins with IgG4 FALA variant Fc and a mutation in the CD38, CD28, or CD3 antigen binding domain, or isotype control (trispecific binding protein with IgG4 FALA variant Fc having three mutated binding domains).

[0064] FIG. 21 shows in vivo anti -tumor activity of CD38vHixCD28supxCD3mid IgG4 FALA trispecific binding protein administered at the indicated doses in an NCI-H929-Luc disseminated tumor model in PBMC humanized NSG mice.

[0065] FIG. 22 shows in vivo anti -tumor activity of CD38HHYi37oxCD28supxCD3mid IgG4 FALA trispecific binding protein administered at the indicated doses in an NCI-H929-Luc disseminated tumor model in PBMC humanized NSG mice.

[0066] FIGS. 23 A & 23B show potent in vitro tumor killing activity of NCI-929-Luc cells with CD38vHixCD28supxCD3mid and CD38HHYi37oxCD28supxCD3mid trispecific binding proteins, and benchmark anti-CD38xanti-CD3 bispecific antibody using the human PBMCs used in the in vivo study. Human PBMCs from two donor humanized NSG mice were used after 24h. incubation with an effectonPBMC ratio of 10: 1.

[0067] FIG. 23C shows superior in vivo anti-tumor activity of

CD38vHixCD28supxCD3mid and CD38hhyi37oxCD28supxCD3mid trispecific binding proteins, as compared to benchmark anti-CD38xanti-CD3 bispecific antibody, administered at the indicated doses in an NCI-H929-Luc disseminated tumor model in PBMC humanized NSG mice. Binding proteins were administered by weekly intraperitoneal (IP) injection at 3( kg.

[0068] FIG. 24A shows the results of a luciferase reporter assay using GloResponse™ IL2-luc2P Jurkat Cells (Promeg °a) ' after stimulation by J CD38 VHl / CD28 sup xCD3 mi .d . and its single binding site KO and triple KO mutants at 10 nM concentration.

[0069] FIG. 24B shows the optimization of anti-CD3xCD28 CODV-Fab antibody. The optimal configuration of the a-CD3 and a-CD28 in alternative positions of the CODV bispecific Fab was evaluated by cytokine release assays using human PBMCs in vitro. Distal-CD28 x proximal CD3 was identified as optimal positioning based on the secretion of IFN-γ and IL-2 in supernatant after 24 hours.

[0070] FIG. 25 shows that CD38W1/ CD28supxCD3mid induced upregulation of Bcl-2 family member Bcl-xL in primary T cells is CD28 dependent.

[0071] FIG. 26 shows that anti-CD28 in the trispecific Ab provided secondary signaling essential for supporting primary T cell proliferation in vitro.

[0072] FIG. 27 shows the configuration of the trispecific antibody, color-coded by parental antibody (left). Dark shades (purple or green) denote heavy chain peptides; light shades denote light chain peptides. Also shown is a structure model of the

CD38vHi/CD28supxCD3mid trispecific Ab based on crystal structures of anti-CD38 VH1 Fab and CD28sup/CD3mid CODV Fab (right).

[0073] FIGS. 28A & 28B show that multiple myeloma (MM) cells with high (RPMI-8226; FIG. 28A) and low (KMS-11; FIG. 28B) CD38 surface expression were lysed efficiently by human PBMCs (E:T=10: 1) incubated with various concentrations of the trispecific Ab. Contribution to the killing activity by each binding site was demonstrated by binding site KO mutations, as indicated.

[0074] FIGS. 29A-29C show that the anti-CD28sup () mutant of the trispecific Ab exhibited markedly reduced anti -tumor activity against 0038^, 0038^ and CD38low

MM cells in vitro. Shown are assays using RPMI-8226 (FIG. 29A), U266 (FIG. 29B), or KMS-11 (FIG. 29C) cells.

[0075] FIG. 30 shows that reduction of tumor burden in CD38vm/

CD28SUpxCD3mid_FALA trispecific antibody treatment groups was dose-dependent and statistically different in a disseminated human multiple myeloma cell line model using an NSG mouse reconstituted with in vitro amplified human primary T cells.

[0076] FIGS. 31A & 31B show vital microscopy analysis of myeloma cell cytolysis by the CD38 trispecific Ab in vitro in the presence of primary human T cells. Time lapse photography of microscopic images was performed using a negative control (triple KO trispecific; FIG. 31 A) or the CD38/CD28xCD3 trispecific Ab (FIG. 31B) using human PBMCs incubated with RPMI-8226 myeloma cell line labeled with CellTracker™ deep red dye. Images presented were collected after 24 hour incubation. Scale bar: 50μπι.

[0077] FIG. 32 shows alternative mutations in the Fc region of IgG4 prepared for analysis in Fc receptor binding assays. Shown are SEQ ID NOs: 111-116 (top to bottom, respectively).

[0078] FIG. 33 shows the results of SPR assays to measure the affinity of the specified IgG4 Fc variants to the indicated human Fc receptors.

[0079] FIG. 34 shows that a CD38 trispecific binding protein with minimal FcR binding reduced non-specific cytokine release by human PBMCs in vitro. Different FcR inactivating mutations (as indicated) were analyzed for their proinflammatory effects in the human IgG4 isotype. Human PBMCs were incubated in media (Unstimulated) or in the presence of the myeloma cells, RPMI-8226 (Stimulated), and bars indicate supernatant IFN-γ levels measured by ELISA.

[0080] FIG. 35 shows that a CD38 trispecific binding protein with minimal FcR binding lysed human multiple myeloma cells with different CD38 expression levels.

Cytolysis of myeloma cells with IgG4 or the indicated Fc mutations was assessed in vitro using human PBMCs with the indicated tumor targets.

[0081] FIG. 36 shows a comparison of in vitro cytolytic activity of the trispecific anti-CD38/CD28/CD3 Ab and the anti-CD38 antibody daratumumab measured using human PBMC as effector cells against cell lines RPMI-8226, U266, and KMS-11(E:T=10: 1).

[0082] FIGS. 37A-37D show the characterization of in vitro T cell subset expansion in response to CD38/CD3xCD28. 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 indicated time points. The triple mutant trispecific ab was used as negative control. Flow cytometry was used to determine central and effector memory CD4 T cells (FIG. 37 A), T helper cells (FIG. 37B), central and effector memory CD 8 T cells (FIG. 37C), and CMV pp65-specific CD 8 cells (FIG. 37D) as described in Example 12. Analysis of CMV-specific pp65 effector cells was performed by pentamer staining of PBMCs from HLA-A2 CMV+ donors treated with the CD38 trispecific or the triple negative control.

[0083] FIG. 38 shows the contribution of CD28 expression on target cells to susceptibility to cytolysis by CD38/CD3xCD28. CD28 was knocked out in KMS-11 cells using CRISPR/Cas 9 gene targeting and used as cytolytic targets in vitro. Compared to parental KMS-11, CD38 expression was preserved while CD28 was eliminated, as demonstrated by flow cytometry (upper panel, KMS-11 vs. KMS-11 (CD28KO). Cytolysis

of the CD28 KO cells was examined with the WT or CD28 null trispecific (lower panel; trispecific vs. trispecific (CD28KO)).

[0084] FIG. 39 shows the cytolytic activity of the CD38/CD28xCD3 trispecific FALA mutant Ab against indicated CD38+CD28" lines, including acute myelocytic leukemia (AML (KG-1)), a B cell lymphoma (OCI-Lyl9), acute T lymphocytic leukemia (ALL (KOPN8)), and chronic lymphocytic lymphoma (CLL(Z-138)).

[0085] FIG. 40 shows that in vitro activation of human PBMC by a-CD28 superagonist requires bivalency of the antibody.

DETAILED DESCRIPTION

[0086] The disclosure provides binding protein comprising at least one antigen binding site that binds a CD38 polypeptide.

I. General Definitions

[0087] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0088] It is understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting," and "consisting essentially of aspects and embodiments.

[0089] The term "polynucleotide" as used herein refers to single-stranded or double-stranded nucleic acid polymers of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or

deoxyribonucleotides or a modified form of either type of nucleotide. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as

phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,

phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term

"polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

[0090] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0091] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free from polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[0092] Naturally occurring antibodies typically comprise a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain" as used herein refer to any

immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CHI, CH2, and Cm), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a

constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.

[0093] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within full-length light and heavy chains, the variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 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 an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0094] The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol Biol. 196: 901-17; Chothia et al, 1989, Nature 342: 877-83) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2, and L3 or HI, H2, and H3 where the "L" and the "H" designates the light chain and the heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs

overlapping with the Kabat CDRs have been described by Padlan, 1995, FASEB J. 9: 133-39; MacCallum, 1996, J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003, Dev. Comp.

Immunol. 27: 55-77. Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs. Identification of predicted CDRs using the amino acid sequence is well known in the field, such as in Martin, A.C. "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2. Kontermann R., Diibel S., eds. Springer- Verlag, Berlin, p. 33-51 (2010). The amino acid sequence of the heavy and/or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional methods, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. The numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments.

[0095] In some embodiments, CDR/FR definition in an immunoglobulin light or heavy chain is to be determined based on IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(l):55-77; www.imgt.org).

[0096] The term "Fc" as used herein refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins. Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent {i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class {e.g., IgG, IgA, and IgE) or subclass {e.g., IgGl, IgG2, IgG3, IgAl, IgGA2, and IgG4). One example of a Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term "native Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.

[0097] A F(ab) fragment typically includes one light chain and the VH and CHI domains of one heavy chain, wherein the VH-CHI heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab) fragment can 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 CHI domain.

[0098] A F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the CHI and Cm domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.

[0099] The term "binding protein" as used herein refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen, e.g., a CD38 polypeptide of the present disclosure

[0100] A "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means.

[0101] One embodiment of the disclosure provides binding proteins having biological and immunological specificity to between one and three target antigens. Another

embodiment of the disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Another

embodiment of the disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Yet another embodiment of the disclosure provides host cells that express such binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).

[0102] The term "swapability" as used herein refers to the interchangeability of variable domains within the binding protein format and with retention of folding and ultimate binding affinity. "Full swapability" refers to the ability to swap the order of both VHI and VH2 domains, and therefore the order of VLI and VL2 domains, in the polypeptide chain of formula I or the polypeptide chain of formula II (i.e., to reverse the order) while maintaining full functionality of the binding protein as evidenced by the retention of binding affinity.

Furthermore, it should be noted that the designations VH and VL refer only to the domain's location on a particular protein chain in the final format. For example, VHI and VH2 could be derived from VLI and VL2 domains in parent antibodies and placed into the VHI and VH2 positions in the binding protein. Likewise, VLI and VL2 could be derived from VHI and VH2 domains in parent antibodies and placed in the Vm and VH2 positions in the binding protein. Thus, the VH and VL designations refer to the present location and not the original location in a parent antibody. VH and VL domains are therefore "swappable. "

[0103] The term "antigen" or "target antigen" or "antigen target" as used herein refers to a molecule or a portion of a molecule that is capable of being bound by a binding protein, and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. With respect to each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen.

[0104] "CD38" is cluster of differentiation 38 polypeptide and is a glycoprotein found on the surface of many immune cells. In some embodiments, a binding protein of the present disclosure binds the extracellular domain of one or more CD38 polypeptide. Exemplary CD38 extracellular domain polypeptide sequences include, but are not limited to, the extracellular domain of human CD38 (e.g., as represented by SEQ ID NO: l) and the extracellular domain of cynomolgus monkey CD38 (e.g., as represented by SEQ ID NO: 30).

[0105] The term "T-cell engager" refers to binding proteins directed to a host's immune system, more specifically the T cells' cytotoxic activity as well as directed to a tumor target protein.

[0106] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

[0107] The term "monovalent binding protein" refers to a binding protein that has one antigen binding site.

[0108] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets. In some embodiments, a bispecific binding protein binds to two different antigens. In some embodiments, a bispecific binding protein binds to two different epitopes on the same antigen.

[0109] The term "bivalent binding protein" refers to a binding protein that has two binding sites.

[0110] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets. In some embodiments, a trispecific binding protein binds to three different antigens. In some embodiments, a trispecific binding protein binds to one, two, or three different epitopes on the same antigen.

[0111] The term "trivalent binding protein" refers to a binding protein that has three binding sites. In particular 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.

[0112] An "isolated" binding protein is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein will be purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated binding proteins include the binding protein in situ within recombinant cells since at least one component of the binding protein's natural environment will not be present.

[0113] The terms "substantially pure" or "substantially purified" as used herein refer to a compound or species that is the predominant species present {i.e., on a molar basis it is more abundant than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition wherein the species comprises at least about 50%) (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise more than about 80%>, 85%>, 90%, 95%, or 99% of all macromolar species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0114] The term "epitope" includes any determinant, preferably a polypeptide

determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody or binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, a binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is < 10"8 M, more

preferably when the equilibrium dissociation constant is < 10"9 M, and most preferably when the dissociation constant is < 10"10 M.

[0115] The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between ligand (a target antigen on a biosensor matrix) and analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). The term "KD," as used herein refers to the

dissociation constant of the interaction between a particular binding protein and a target antigen.

[0116] The term "binds to" as used herein in reference to a binding protein refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with an 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 more, and/or to bind to an epitope with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen. In some

embodiments, a binding protein of the present disclosure binds to two or more antigens, e.g., a human and a cynomologus monkey CD38 polypeptide.

[0117] In some embodiments, an antigen binding domain and/or binding protein of the present disclosure "cross reacts" with human and cynomolgus monkey CD38 polypeptides, e.g., CD38 extracellular domains, such as SEQ ID NO: l (human CD38 isoform A), SEQ ID NO: 105 (human CD38 isoform E) and SEQ ID NO:30 (cynomolgus monkey CD38). A binding protein binding to antigen 1 (Agl) is "cross-reactive" to antigen 2 (Ag2) when the EC50S are in a similar range for both antigens. In the present application, a binding protein binding to Agl is cross-reactive to Ag2 when the ratio of affinity of Ag2 to affinity of Agl is equal or less than 10 (for instance 5, 2, 1 or 0.5), affinities being measured with the same method for both antigens.

[0118] A binding protein binding to Agl is "notsignificantlycross-reactive" to Ag2 when the affinities are very different for the two antigens. Affinity for Ag2 may not be measurable if the binding response is too low. In the present application, a binding protein binding to Agl is not significantly cross-reactive to Ag2, when the binding response of the binding protein to Ag2 is less than 5% of the binding response of the same binding protein to Agl in the same experimental setting and at the same antibody

concentration. In practice, the binding protein concentration used can be the EC50 or the concentration required to reach the saturation plateau obtained with Agl .

[0119] The term "linker" as used herein refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into cross over dual variable region immunoglobulins. A linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level. The transition between domains can be identified because the approximate size of the immunoglobulin domains are well understood. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction. The linkers described herein are referred to as Li, which is located on the light chain between the C-terminus of the VL2 and the N-terminus of the VLI domain; and L2, which is located on the light chain between the C-terminus of the VLi and the N-terminus of the CL domain. The heavy chain linkers are known as L3, which is located between the C-terminus of the Vm and the N-terminus of the VH2 domain; and L4, which is located between the C-terminus of the Vm and the N-terminus of the CHI domain.

[0120] The term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted. Another type of vector is a viral vector, wherein additional DNA segments may be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably herein, as a plasmid is the most commonly used form of vector. However, the 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 serve equivalent functions.

[0121] The phrase "recombinant host cell" (or "host cell") as used herein refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express the binding proteins, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To express a binding protein recombinantly, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the binding protein can be recovered.

[0122] The term "transformation" as used herein refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transformation, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been stably transformed when the DNA is replicated with the division of the cell. The term "transfection" as used herein 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 inside the cell membrane. A number of transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells.

[0123] The term "naturally occurring" as used herein and applied to an object refers to the fact that the object can be found in nature and has not been manipulated by man. For example, a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally-occurring. Similarly, "non-naturally occurring" as used herein refers to an object that is not found in nature or that has been structurally modified or synthesized by man.

[0124] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids; unnatural amino acids and analogs such as α-, α-di substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the binding proteins. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-Ν-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.

[0125] Naturally occurring residues may be divided into classes based on common side chain properties:

(1) hydrophobic: Met, Ala, Val, Leu, He, Phe, Trp, Tyr, Pro;

(2) polar hydrophilic: Arg, Asn, Asp, Gin, Glu, His, Lys, Ser, Thr ;

(3) aliphatic: Ala, Gly, He, Leu, Val, Pro;

(4) aliphatic hydrophobic: Ala, He, Leu, Val, Pro;

(5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(6) acidic: Asp, Glu;

(7) basic: His, Lys, Arg;

(8) residues that influence chain orientation: Gly, Pro;

(9) aromatic: His, Trp, Tyr, Phe; and

(10) aromatic hydrophobic: Phe, Trp, Tyr.

[0126] Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.

[0127] A skilled artisan will be able to determine suitable variants of the polypeptide chains of the binding proteins using well-known techniques. For example, one skilled in the art may identify suitable areas of a polypeptide chain that may be changed without destroying activity by targeting regions not believed to be important for activity. Alternatively, one skilled in the art can identify residues and portions of the molecules that are conserved among similar polypeptides. In addition, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0128] The term "patient" as used herein includes human and animal subjects (e.g., mammals, such as dogs, pigs, horses, cats, cows, etc.).

[0129] The terms "treatment" or "treat" as used herein refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those having a disorder as well as those prone to have the disorder or those in which the disorder is to be prevented. In particular embodiments, binding proteins can be used to treat humans with cancer, or humans susceptible to cancer, or ameliorate cancer in a human subject. The binding proteins can also be used to prevent cancer in a human patient. In particular 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 lympohoma or B-cell acute

lymphoblastic leukemia.

[0130] The terms "pharmaceutical composition" or "therapeutic composition" as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0131] The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a binding protein.

[0132] The terms "effective amount" and "therapeutically effective amount" when used in reference to a pharmaceutical composition comprising one or more binding proteins refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of a binding protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific binding protein that is being used, and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo, factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the ability of those skilled in the art.

[0133] One embodiment of the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

II. Anti-CD38 Binding Proteins

[0134] Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds a CD38 polypeptide {e.g., human and cynomolgus monkey CD38 polypeptides). In some embodiments, the binding proteins are monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent.

[0135] A variety of features of exemplary monospecific, bispecific, or trispecific binding proteins are described herein. For example, in some embodiments, a binding protein or antigen-binding fragment thereof cross-reacts with human CD38 {e.g., a human CD38 isoform A and/or isoform E polypeptide) and cynomolgus monkey CD38. In some embodiments, a binding protein induces apoptosis of a CD38+ cell. In some embodiments, a binding protein recruits a T cell to a CD38+ cell and optionally activates the T cell {e.g., though TCR stimulation and/or costimulation).

CLAIMS

What is claimed is:

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

(a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and

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

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 comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and

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

3. The binding protein of claim 1 or claim 2, wherein the VH domain comprises the sequence, from N-terminus to C-terminus, FR1— CDR-H1— FR2— CDR-H2— FR3— CDR-H3— FR4; wherein FR1 comprises the sequence 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 comprising the amino acid sequence of SEQ ID NO:7 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:8.

6. 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 comprising the amino acid sequence of SEQ ID NO: 19 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20.

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

9. The binding protein of claim 8, wherein the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:22 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20.

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

11. The binding protein of claim 10, wherein the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20.

12. 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 comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and

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

13. The binding protein of claim 1 or claim 12, wherein the VH domain comprises the sequence, from N-terminus to C-terminus, FR 1— CDR-H 1— FR2— CDR-H2— FR3— CDR-H3— FR4; wherein FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87), or

QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); wherein FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHW VKE APGQ GLEWIGY (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).

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

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

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

(a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence

comprising the amino acid sequence of IWYDGS K (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43); and

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

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

18. The binding protein of claim 16 or claim 17, wherein the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 12.

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

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

21. The binding protein of claim 20, wherein the antigen binding site binds the human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO: l with an equilibrium dissociation constant (KD) of 2.1nM or less.

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

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

24. The binding protein of claim 23, wherein the antigen binding site binds the cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30 with an equilibrium dissociation constant (KD) of 1.3nM or less.

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

26. The binding protein of any one of claims 16-24, wherein the binding protein is a human antibody.

27. The binding protein of any one of claims 1-24, wherein the binding protein is a monoclonal antibody.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

48. The binding protein of any one of claims 43-47, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

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

VHi-L3-VH2-L4-CHi-hinge-CH2-CH3 [Π]

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

Vm-Cm-hinge-Cm-Cro [HI]

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

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHi heavy chain constant domain;

CH2 is an immunoglobulin Cm heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and

Li, L2, L3 and L4 are amino acid linkers;

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

wherein:

(a) the VHI domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or lYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VLi domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQ KEDPWT (SEQ ID NO:36);

(b) the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or lYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or

(c) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or lYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence

of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).

49. The binding protein of claim 48, wherein

(a) the VHI domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VLi domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);

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

(c) the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL2 domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);

(d) the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL2 domain comprises a CDR-Ll sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); (e) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or

(h) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-Ll sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).

50. The binding protein of claim 49, wherein

(a) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-Ll sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or

(b) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the VL3 domain comprises a CDR-Ll sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).

5 1. The binding protein of claim 50, wherein

(a) the VH3 domain comprises the amino acid sequence of SEQ ID NO: 5, and the VL3 domain comprises the amino acid sequence of SEQ ID NO:6;

(b) the VH3 domain comprises the amino acid sequence of SEQ ID NO: 17, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 18;

(c) the VH3 domain comprises the amino acid sequence of SEQ ID NO:21 , and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 18;

(d) the VH3 domain comprises the amino acid sequence of SEQ ID NO:23, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 18; or

(e) the VH3 domain comprises the amino acid sequence of SEQ ID NO: 13, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 14.

52. The binding protein of any one of claims 43-47, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

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

VHi-L3-VH2-L4-CHi-hinge-CH2-CH3 [Π]

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

VH3-CHi-hinge-CH2-CH3 [HI] and a fourth polypeptide chain comprises a structure represented by the formula:

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHI heavy chain constant domain;

CH2 is an immunoglobulin Cm heavy chain constant domain;

CH3 is an immunoglobulin Cm heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHi and CH2 domains; and

Li, L2, L3 and L4 are amino acid linkers;

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

wherein:

(a) the VHI domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the VLi domain comprises a CDR-Ll sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46);

(b) the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the VL2 domain comprises a CDR-Ll sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46); or

(c) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the VL3 domain comprises a CDR-Ll sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46).

53. The binding protein of claim 52, wherein the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2

sequence comprising the amino acid sequence of IWYDGS K (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIR D (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46).

54. The binding protein of claim 53, wherein the VH3 domain comprises the amino acid sequence of SEQ ID NO: 9, and the VL3 domain comprises the amino acid sequence of SEQ ID NCv lO.

55. The binding protein of any one of claims 48-54, wherein

(a) the VHI domain comprises the amino acid sequence of SEQ ID NO:49, the VLi domain comprises the amino acid sequence of SEQ ID NO:50, the VH2 domain comprises the amino acid sequence of SEQ ID NO:53, and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 54;

(b) the VH2 domain comprises the amino acid sequence of SEQ ID NO:49, the VL2 domain comprises the amino acid sequence of SEQ ID NO:50, the VHI domain comprises the amino acid sequence of SEQ ID NO:53, and the VLi domain comprises the amino acid sequence of SEQ ID NO: 54;

(c) the VHI domain comprises the amino acid sequence of SEQ ID NO:51, the VLi domain comprises the amino acid sequence of SEQ ID NO:52, the VH2 domain comprises the amino acid sequence of SEQ ID NO:53, and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 54;

(d) the VH2 domain comprises the amino acid sequence of SEQ ID NO:51, the VL2 domain comprises the amino acid sequence of SEQ ID NO:52, the VHI domain comprises the amino acid sequence of SEQ ID NO:53, and the VLi domain comprises the amino acid sequence of SEQ ID NO: 54

(e) the VHI domain comprises the amino acid sequence of SEQ ID NO:49, the VLI domain comprises the amino acid sequence of SEQ ID NO:50, the VH2 domain comprises the amino acid sequence of SEQ ID NO:84, and the VL2 domain comprises the amino acid sequence of SEQ ID NO:85;

(f) the VH2 domain comprises the amino acid sequence of SEQ ID NO:49, the VL2 domain comprises the amino acid sequence of SEQ ID NO:50, the VHi domain comprises the amino acid sequence of SEQ ID NO:84, and the VLi domain comprises the amino acid sequence of SEQ ID NO:85;

(g) the VHI domain comprises the amino acid sequence of SEQ ID NO:51, the VLI domain comprises the amino acid sequence of SEQ ID NO:52, the VH2 domain comprises the amino acid sequence of SEQ ID NO:84, and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 85; or

(h) the VH2 domain comprises the amino acid sequence of SEQ ID NO:51, the VL2 domain comprises the amino acid sequence of SEQ ID NO:52, the VHI domain comprises the amino acid sequence of SEQ ID NO:84, and the VLI domain comprises the amino acid sequence of SEQ ID NO:85.

56. The binding protein of claim 55, wherein

(a) the VHI domain comprises the amino acid sequence of SEQ ID NO:49, the VLI domain comprises the amino acid sequence of SEQ ID NO:50, the VH2 domain comprises the amino acid sequence of SEQ ID NO:53, the VL2 domain comprises the amino acid sequence of SEQ ID NO:54, the VH3 domain comprises the amino acid sequence of SEQ ID NO: 13, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 14; or

(b) the VHI domain comprises the amino acid sequence of SEQ ID NO:49, the VLI domain comprises the amino acid sequence of SEQ ID NO:50, the VH2 domain comprises the amino acid sequence of SEQ ID NO:53, the VL2 domain comprises the amino acid sequence of SEQ ID NO:54, the VH3 domain comprises the amino acid sequence of SEQ ID NO:9, and the VL3 domain comprises the amino acid sequence of SEQ ID NO: 10.

57. The binding protein of any one of claims 48-56, wherein at least one of Li, L2, L3 or L4 is independently 0 amino acids in length.

58. The binding protein of any one of claims 48-56, wherein Li, L2, L3 or L4 are each independently at least one amino acid in length.

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

GGSGSSGSGG (SEQ ID NO:59).

60. The binding protein of any one of claims 48-56, wherein

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

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

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

(d) Li comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID

NO:56), and L4 is 0 amino acids in length.

61. The binding protein of any one of claims 48-60, wherein the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A.

62. The binding protein of any one of claims 48-60, wherein the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions

corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.

63. The binding protein of any one of claims 48-62, wherein the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.

64. The binding protein of any one of claims 48-60, wherein the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgGl hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A.

65. The binding protein of any one of claims 48-60, wherein the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgGl hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S.

66. The binding protein of any one of claims 48-65, wherein the hinge-Cm-Cm domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-Cm-Cmdomain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W.

67. The binding protein of any one of claims 48-65, wherein the hinge-Cm-Cm domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-Cm-Cm domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

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

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

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

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

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

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

74. A binding protein comprising three antigen binding sites that each bind one or more target proteins, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-LI-VLI-L2-Cl [I]

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

VHi-L3-VH2-L4-CHi-hinge-CH2-CH3 [Π]

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

Vm-Cm-hinge-Cm-Cro [HI]

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

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHI heavy chain constant domain;

CH2 is an immunoglobulin Cm heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHi and CH2 domains; and

Li, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair; and

wherein:

(a) the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgGl hinge-Cm-Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S; or

(b) the hinge-Cm-Cm domains of the second and the third polypeptide chains are human IgG4 Cm domains, and wherein the hinge-Cm-Cm domains each comprise amino

acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.

75. The binding protein of claim 74, wherein at least one pair of VHi and VLi, VH2 and VL2, and VH3 and VL3 forms an antigen binding site that binds a CD38 polypeptide.

76. The binding protein of claim 74, wherein one, two, or three pairs of Vm and VLI, VH2 and VL2, and VH3 and VL3 form an antigen binding site that binds an antigen target selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD 122, CD 137, CD137L, CD 152, CD 154, CD 160, 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, FCER1 A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, ILIA, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhILlO, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, KG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TFM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, T Fa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.

77. The binding protein of claim 74, wherein a first pair of VHi and VLi, VH2 and VL2, and VH3 and VL3 forms an antigen binding site that binds a human CD3 polypeptide, a second pair of VHI and VLI, VH2 and VL2, and VH3 and VL3 forms an antigen binding site that binds a human CD28 polypeptide, and a third pair of VHI and VLI, VH2 and VL2, and VH3 and VL3 forms an antigen binding site that binds a human antigen target selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD 19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70,

CD80, CD86, CD 122, CD 137, CD137L, CD 152, CD 154, CD 160, 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, HMGB 1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, ILIA, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhILlO, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, SI 52, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, ΤΊΜ3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.

78. 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.

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

80. A vector comprising the polynucleotide of claim 79.

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

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

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

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

85. 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 claims 1-73 or the pharmaceutical composition of claim 84.

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

87. The method of claim 86, wherein 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.

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

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

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

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

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