ANTI-CD38 ANTIBODIES AND METHODS OF USE

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

[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: 183952029900seqlist.TXT, date recorded: Oct. 9, 2018, size: 158 KB).

[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.

[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.

[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 K_D of 1.5 nM 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 K_D of 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, 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 K_D of 3.5 nM 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 K_D of 7.5 nM 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 FcγRI and/or FcγRII, 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 K_D of 1.5 nM 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 K_D of 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, 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 K_D of 3.5 nM 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 K_D of 7.5 nM 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 FcγRI and/or FcγRII, 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-α) 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 MHWVKEAPGQGLEWIGY (SEQ ID NO:91); wherein FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO:96). In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:5, and/or the VL domain comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:7 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:17, and/or the VL domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:19 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:21, and/or the VL domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:22 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:23, and/or the VL domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the antigen binding site comprises: (a) an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and (b) an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the VH domain comprises the sequence, 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). 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 (K_D) of 2.1 nM 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 (K_D) of 1.3 nM 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 IgG1 Fc region. In some embodiments, the human IgG1 Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the human IgG1 Fc region comprises amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to 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:

V_L2-L₁-V_L1-L₂-C_L  [I]

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

V_H1-L₃-V_H2-L₄-C_H1-hinge-C_H2-C_H3  [II]

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

V_H3-C_H1-hinge-C_H2-C_H3  [III]

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

V_L3-C_L  [IV]

wherein:

[0013] V_L1 is a first immunoglobulin light chain variable domain;

[0014] V_L2 is a second immunoglobulin light chain variable domain;

[0015] V_L3 is a third immunoglobulin light chain variable domain;

[0016] V_H1 is a first immunoglobulin heavy chain variable domain;

[0017] V_H2 is a second immunoglobulin heavy chain variable domain;

[0018] V_H3 is a third immunoglobulin heavy chain variable domain;

[0019] C_L is an immunoglobulin light chain constant domain;

[0020] C_H1 is an immunoglobulin C_H1 heavy chain constant domain;

[0021] C_H2 is an immunoglobulin C_H2 heavy chain constant domain;

[0022] C_H3 is an immunoglobulin C_H3 heavy chain constant domain;

[0023] hinge is an immunoglobulin hinge region connecting the C_H1 and C_H2 domains; and

[0024] L₁, L₂, L₃ and L₄ are amino acid linkers;

[0025] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair, and

[0026] wherein: (a) the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);

(b) the V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L2 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 V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

V_L2-L₁-V_L1-L₂-C_L  [I]

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

V_H1-L₃-V_H2-L₄-C_H1-hinge-C_H2-C_H3  [II]

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

V_H3-C_H1-hinge-C_H2-C_H3  [III]

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

V_L3-C_L  [IV]

wherein:

[0027] V_L1 is a first immunoglobulin light chain variable domain;

[0028] V_L2 is a second immunoglobulin light chain variable domain;

[0029] V_L3 is a third immunoglobulin light chain variable domain;

[0030] V_H1 is a first immunoglobulin heavy chain variable domain;

[0031] V_H2 is a second immunoglobulin heavy chain variable domain;

[0032] V_H3 is a third immunoglobulin heavy chain variable domain;

[0033] C_L is an immunoglobulin light chain constant domain;

[0034] C_H1 is an immunoglobulin C_H1 heavy chain constant domain;

[0035] C_H2 is an immunoglobulin C_H2 heavy chain constant domain;

[0036] C_H3 is an immunoglobulin C_H3 heavy chain constant domain;

[0037] hinge is an immunoglobulin hinge region connecting the C_H1 and C_H2 domains; and

[0038] L₁, L₂, L₃ and L₄ are amino acid linkers;

[0039] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair, and

[0040] wherein: (a) the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36);

(b) the V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); or
(c) the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); the V_H1 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 V_LA domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); the V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); the V_H2 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 V_L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36); the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L3 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 V_H3 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 V_L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L3 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 V_H3 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 V_L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:5, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:6; the V_H3 domain comprises the amino acid sequence of SEQ ID NO:17, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:18; the V_H3 domain comprises the amino acid sequence of SEQ ID NO:21, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:18; the V_H3 domain comprises the amino acid sequence of SEQ ID NO:23, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:18; or the V_H3 domain comprises the amino acid sequence of SEQ ID NO:13, and the V_L3 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:

V_L2-L₁-V_L1-L₂-C_L  [I]

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

V_H1-L₃-V_H2-L₄-C_H1-hinge-C_H2-C_H3  [II]

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

V_H3-C_H1-hinge-C_H2-C_H3  [III]

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

V_L3-C_L  [IV]

wherein:

[0041] V_L1 is a first immunoglobulin light chain variable domain;

[0042] V_L2 is a second immunoglobulin light chain variable domain;

[0043] V_L3 is a third immunoglobulin light chain variable domain;

[0044] V_H1 is a first immunoglobulin heavy chain variable domain;

[0045] V_H2 is a second immunoglobulin heavy chain variable domain;

[0046] V_H3 is a third immunoglobulin heavy chain variable domain;

[0047] C_L is an immunoglobulin light chain constant domain;

[0048] C_H1 is an immunoglobulin C_H1 heavy chain constant domain;

[0049] C_H2 is an immunoglobulin C_H2 heavy chain constant domain;

[0050] C_H3 is an immunoglobulin C_H3 heavy chain constant domain;

[0051] hinge is an immunoglobulin hinge region connecting the C_H1 and C_H2 domains; and

[0052] L₁, L₂, L₃ and L₄ are amino acid linkers;

[0053] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair, and

[0054] wherein (a) the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the V_L1 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 V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the V_L2 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 V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the V_L3 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 V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the V_L3 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 V_H3 domain comprises the amino acid sequence of SEQ ID NO:9, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:49, the V_L1 domain comprises the amino acid sequence of SEQ ID NO:50, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:53, and the V_L2 domain comprises the amino acid sequence of SEQ ID NO:54; the V_H2 domain comprises the amino acid sequence of SEQ ID NO:49, the V_L2 domain comprises the amino acid sequence of SEQ ID NO:50, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:53, and the V_L1 domain comprises the amino acid sequence of SEQ ID NO:54; the V_H1 domain comprises the amino acid sequence of SEQ ID NO:51, the V_L1 domain comprises the amino acid sequence of SEQ ID NO:52, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:53, and the V_L2 domain comprises the amino acid sequence of SEQ ID NO:54; the V_H2 domain comprises the amino acid sequence of SEQ ID NO:51, the V_L2 domain comprises the amino acid sequence of SEQ ID NO:52, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:53, and the V_L1 domain comprises the amino acid sequence of SEQ ID NO:54 the V_H1 domain comprises the amino acid sequence of SEQ ID NO:49, the V_L1 domain comprises the amino acid sequence of SEQ ID NO:50, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:84, and the V_L2 domain comprises the amino acid sequence of SEQ ID NO:85; the V_H2 domain comprises the amino acid sequence of SEQ ID NO:49, the V_L2 domain comprises the amino acid sequence of SEQ ID NO:50, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:84, and the V_L1 domain comprises the amino acid sequence of SEQ ID NO:85; the V_H1 domain comprises the amino acid sequence of SEQ ID NO:51, the V_L1 domain comprises the amino acid sequence of SEQ ID NO:52, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:84, and the V_L2 domain comprises the amino acid sequence of SEQ ID NO:85; or the V_H2 domain comprises the amino acid sequence of SEQ ID NO:51, the V_L2 domain comprises the amino acid sequence of SEQ ID NO:52, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:84, and the V_L1 domain comprises the amino acid sequence of SEQ ID NO:85. In some embodiments, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:49, the V_L1 domain comprises the amino acid sequence of SEQ ID NO:50, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:53, the V_L2 domain comprises the amino acid sequence of SEQ ID NO:54, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:13, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:14; or the V_H1 domain comprises the amino acid sequence of SEQ ID NO:49, the V_L1 domain comprises the amino acid sequence of SEQ ID NO:50, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:53, the V_L2 domain comprises the amino acid sequence of SEQ ID NO:54, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:9, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, at least one of L₁, L₂, L₃ or L₄ is independently 0 amino acids in length. In some embodiments, L₁, L₂, L₃ or L₄ are each independently at least one amino acid in length. In some embodiments, (a) L₁, L₂, L₃ and L₄ 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) L₁, L₂, L₃ and L₄ each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L₁ comprises the sequence GQPKAAP (SEQ ID NO: 58), L₂ comprises the sequence TKGPS (SEQ ID NO:57), L₃ comprises the sequence S, and L₄ comprises the sequence RT; L₁ comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₂ comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₃ is 0 amino acids in length, and L₄ is 0 amino acids in length; L₁ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₂ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₃ is 0 amino acids in length, and L₄ is 0 amino acids in length; or L₁ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L₂ is 0 amino acids in length, L₃ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), and L₄ is 0 amino acids in length. In some embodiments, the hinge-C_H2-C_H3 domains of the second and the third polypeptide chains are human IgG4 hinge-C_H2-C_H3 domains, and wherein the hinge-C_H2-C_H3 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-C_H2-C_H3 domains of the second and the third polypeptide chains are human IgG4 hinge-C_H2-C_H3 domains, and wherein the hinge-C_H2-C_H3 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-C_H2-C_H3 domains of the second and the third polypeptide chains are human IgG4 hinge-C_H2-C_H3 domains, and wherein the hinge-C_H2-C_H3 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-C_H2-C_H3 domains of the second and the third polypeptide chains are human IgG1 hinge-C_H2-C_H3 domains, and wherein the hinge-C_H2-C_H3 domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the hinge-C_H2-C_H3 domains of the second and the third polypeptide chains are human IgG1 hinge-C_H2-C_H3 domains, and wherein the hinge-C_H2-C_H3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S. In some embodiments, the hinge-C_H2-C_H3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-C_H2-C_H3domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-C_H2-C_H3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-C_H2-C_H3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 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.

[0055] 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:

V_L2-L₁-V_L1-L₂-C_L  [I]

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

V_H1-L₃-V_H2-L₄-C_H1-hinge-C_H2-C_H3  [II]

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

V_H3-C_H1-hinge-C_H2-C_H3  [III]

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

V_L3-C_L  [IV]

wherein:

[0056] V_L1 is a first immunoglobulin light chain variable domain;

[0057] V_L2 is a second immunoglobulin light chain variable domain;

[0058] V_L3 is a third immunoglobulin light chain variable domain;

[0059] V_H1 is a first immunoglobulin heavy chain variable domain;

[0060] V_H2 is a second immunoglobulin heavy chain variable domain;

[0061] V_H3 is a third immunoglobulin heavy chain variable domain;

[0062] C_L is an immunoglobulin light chain constant domain;

[0063] C_H1 is an immunoglobulin C_H1 heavy chain constant domain;

[0064] C_H2 is an immunoglobulin C_H2 heavy chain constant domain;

[0065] C_H3 is an immunoglobulin C_H3 heavy chain constant domain;

[0066] hinge is an immunoglobulin hinge region connecting the C_H1 and C_H2 domains; and

[0067] L₁, L₂, L₃ and L₄ are amino acid linkers;

[0068] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

[0069] wherein (a) the hinge-C_H2-C_H3 domains of the second and the third polypeptide chains are human IgG1 hinge-C_H2-C_H3 domains, and wherein the hinge-C_H2-C_H3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S; or (b) the hinge-C_H2-C_H3 domains of the second and the third polypeptide chains are human IgG4 hinge-C_H2-C_H3 domains, and wherein the hinge-C_H2-C_H3 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-C_H2-C_H3 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 V_H1 and V_L1, V_H2 and V_L2, and V_H3 and V_L3 forms an antigen binding site that binds a CD38 polypeptide. In some embodiments, one, two, or three pairs of V_H1 and V_L1, V_H2 and V_L2, and V_H3 and V_L3 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, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1. In some embodiments, a first pair of V_H1 and V_L1, V_H2 and V_L2, and V_H3 and V_L3 forms an antigen binding site that binds a human CD3 polypeptide, a second pair of V_H1 and V_L1, V_H2 and V_L2, and V_H3 and V_L3 forms an antigen binding site that binds a human CD28 polypeptide, and a third pair of V_H1 and V_L1, V_H2 and V_L2, and V_H3 and V_L3 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, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0143] The disclosure provides binding protein comprising at least one antigen binding site that binds a CD38 polypeptide.

[0144] 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.

[0145] It is understood that aspects and embodiments of the present disclosure described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments.

[0146] 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.

[0147] 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.

[0148] 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).

[0149] 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 (V_H) and three constant domains (C_H1, C_H2, and C_H3), wherein the V_H domain is at the amino-terminus of the polypeptide and the C_H3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (V_L) and a constant domain (C_L), wherein the V_L domain is at the amino-terminus of the polypeptide and the C_L domain is at the carboxyl-terminus.

[0150] 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, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains 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.

[0151] 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 L1, L2, and L3 or H1, 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., Dübel 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.

[0152] 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(1):55-77; www.imgt.org).

[0153] 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., IgG1, IgG2, IgG3, IgA1, 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.

[0154] A F(ab) fragment typically includes one light chain and the V_H and C_H1 domains of one heavy chain, wherein the V_H-C_H1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, 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 C_H1 domain.

[0155] 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 C_H1 and C_H2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab′)₂ molecule.

[0156] 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

[0157] A “recombinant” molecule is one that has been prepared, expressed, created, or isolated by recombinant means.

[0158] 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).

[0159] 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 V_H1 and V_H2 domains, and therefore the order of V_L1 and V_L2 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 V_H and V_L refer only to the domain's location on a particular protein chain in the final format. For example, V_H1 and V_H2 could be derived from V_L1 and V_L2 domains in parent antibodies and placed into the V_H1 and V_H2 positions in the binding protein. Likewise, V_L1 and V_L2 could be derived from V_H1 and V_H2 domains in parent antibodies and placed in the V_H1 and V_H2 positions in the binding protein. Thus, the V_H and V_L designations refer to the present location and not the original location in a parent antibody. V_H and V_L domains are therefore “swappable.”

[0160] 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.

[0161] “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:1) and the extracellular domain of cynomolgus monkey CD38 (e.g., as represented by SEQ ID NO:30).

[0162] 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.

[0163] The term “monospecific binding protein” refers to a binding protein that specifically binds to one antigen target.

[0164] The term “monovalent binding protein” refers to a binding protein that has one antigen binding site.

[0165] 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.

[0166] The term “bivalent binding protein” refers to a binding protein that has two binding sites.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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 ≤10⁻⁹M, and most preferably when the dissociation constant is ≤10⁻¹⁰ M.

[0172] The dissociation constant (K_D) 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, N.J.). 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 “K_D,” as used herein refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

[0173] 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 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² 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.

[0174] 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:1 (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 (Ag1) 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 Ag1 is cross-reactive to Ag2 when the ratio of affinity of Ag2 to affinity of Ag1 is equal or less than 10 (for instance 5, 2, 1 or 0.5), affinities being measured with the same method for both antigens.

[0175] A binding protein binding to Ag1 is “not significantly cross-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 Ag1 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 Ag1 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 Ag1.

[0176] 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 L₁, which is located on the light chain between the C-terminus of the V_L2 and the N-terminus of the V_L1 domain; and L₂, which is located on the light chain between the C-terminus of the V_L1 and the N-terminus of the C_L domain. The heavy chain linkers are known as L₃, which is located between the C-terminus of the V_H1 and the N-terminus of the V_H2 domain; and L₄, which is located between the C-terminus of the V_H2 and the N-terminus of the C_H1 domain.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids; unnatural amino acids and analogs such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the binding proteins. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.

[0182] Naturally occurring residues may be divided into classes based on common side chain properties:

[0183] (1) hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

[0184] (2) polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

[0185] (3) aliphatic: Ala, Gly, Ile, Leu, Val, Pro;

[0186] (4) aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro;

[0187] (5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0188] (6) acidic: Asp, Glu;

[0189] (7) basic: His, Lys, Arg;

[0190] (8) residues that influence chain orientation: Gly, Pro;

[0191] (9) aromatic: His, Trp, Tyr, Phe; and

[0192] (10) aromatic hydrophobic: Phe, Trp, Tyr.

[0193] 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.

[0194] 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.

[0195] The term “patient” as used herein includes human and animal subjects (e.g., mammals, such as dogs, pigs, horses, cats, cows, etc.).

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] One embodiment of the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

[0201] 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.

[0202] 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).

[0203] In some embodiments, the binding proteins comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding proteins comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding proteins comprise 1, 2, 3, 4, 5, or 6 CDRs from an antibody VH and/or VL domain sequence of mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb2×CD28sup×CD3mid IgG4 FALA, mAb2×CD28sup×CD3mid IgG1LALA P329A, mAb2×CD28sup×CD3mid IgG1 NNSA, mAb6×CD28sup×CD3mid IgG4 FALA, mAb6×CD28sup×CD3mid IgG1LALA P329A, or mAb6×CD28sup×CD3mid IgG1 NNSA, as shown in Table G, H, or I.

[0204] In some embodiments, the binding proteins comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).

[0205] In some embodiments, the binding proteins comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding proteins comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In other embodiments, the binding proteins comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding proteins comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).

[0206] 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; where FR1 comprises the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87), or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); where FR2 comprises the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO:91); where FR3 comprises the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); and where FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO:96). In some embodiments, the VL domain comprises the sequence, from N-terminus to C-terminus, FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4; where FR1 comprises the sequence DIVLTQSPATLSLSPGERATISCRAS (SEQ ID NO:97); where FR2 comprises the sequence MHWYQQKPGQPPRLLIY (SEQ ID NO:99); where FR3 comprises the sequence SRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYC (SEQ ID NO:101); and where FR4 comprises the sequence FGGGTKLEIK (SEQ ID NO:103).

[0207] In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5; and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:17; and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:21; and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23; and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13; and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14.

[0208] In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:17; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:21; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14.

[0209] In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:5; and the VL domain comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:17; and the VL domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:21; and the VL domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:23; and the VL domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:13; and the VL domain comprises the amino acid sequence of SEQ ID NO:14.

[0210] In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:7 and/or an antibody light chain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:19 and/or an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:22 and/or an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and/or an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:15 and/or an antibody light chain comprising the amino acid sequence of SEQ ID NO:16.

[0211] In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:7 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:19 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:22 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:15 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:16.

[0212] In some embodiments, the binding proteins comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43); or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46). In some embodiments, the binding proteins comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:46).

[0213] 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; where FR1 comprises the sequence QVQLVESGGGVVQPGRSLRLSCAAS (SEQ ID NO:89); where FR2 comprises the sequence MHWVRQAPGKGLEWVAV (SEQ ID NO:92); where FR3 comprises the sequence YYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:95); and where FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO:96). In some embodiments, the VL domain comprises the sequence, from N-terminus to C-terminus, FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4; where FR1 comprises the sequence AIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO:98); where FR2 comprises the sequence GWYQQKPGKAPKLLIY (SEQ ID NO:100); where FR3 comprises the sequence SLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYC (SEQ ID NO:102); and where FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO:104).

[0214] In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9; and/or the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:10.

[0215] In some embodiments, the VH domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9; and the VL domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:10. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:9; and the VL domain comprises the amino acid sequence of SEQ ID NO:10.

[0216] In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:11 or an antibody light chain comprising the amino acid sequence of SEQ ID NO:12. In some embodiments, a binding protein of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:11 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:12.

[0217] In some embodiments, a binding protein of the present disclosure comprises 1, 2, 3, 4, 5, or 6 CDR sequences of an antibody sequence shown in Table G. In some embodiments, a binding protein of the present disclosure comprises 1, 2, 3, 4, 5, or 6 CDR sequences, a VH domain sequence, and/or a VL domain sequence of an antibody sequence shown in Table H. In some embodiments, a binding protein of the present disclosure comprises 1, 2, 3, 4, 5, or 6 CDR sequences, a VH domain sequence, and/or a VL domain sequence of an antibody sequence shown in Table I. In some embodiments, a binding protein of the present disclosure comprises 1, 2, 3, or 4 polypeptide sequences shown in Table I.

TABLE G
CDR sequences of anti-CD38 binding proteins.
Ab	CDR_H1	CDR_H2	CDR_H3	CDR_L1	CDR_L2	CDR_L3
mAb1	GYTFTSFN	IYPGNGGT	ARTGGLRRAYFTY	ESVDSYGNGF	LAS	QQNKEDPWT
	(SEQ ID NO: 31)	(SEQ ID NO: 32)	(SEQ ID NO: 33)	(SEQ ID NO: 34)	(SEQ ID NO: 35)	(SEQ ID NO: 36)
mAb2	GYTFTSYA	IYPGQGGT	ARTGGLRRAYFTY	QSVSSYGQGF	GAS	QQNKEDPWT
	(SEQ ID NO: 37)	(SEQ ID NO: 38)	(SEQ ID NO: 33)	(SEQ ID NO: 39)	(SEQ ID NO: 40)	(SEQ ID NO: 36)
mAb3	GYTFTSFN	IYPGNGGT	ARTGGLRRAYFTY	ESVDSYGNGF	LAS	QQNKEDPWT
	(SEQ ID NO: 31)	(SEQ ID NO: 32)	(SEQ ID NO: 33)	(SEQ ID NO: 34)	(SEQ ID NO: 35)	(SEQ ID NO: 36)
mAb4	GYTFTSFN	IYPGNGGT	ARTGGLRRAYFTY	ESVDSYGNGF	LAS	QQNKEDPWT
	(SEQ ID NO: 31)	(SEQ ID NO: 32)	(SEQ ID NO: 33)	(SEQ ID NO: 34)	(SEQ ID NO: 35)	(SEQ ID NO: 36)
mAb5	GYTFTSFN	IYPGNGGT	ARTGGLRRAYFTY	ESVDSYGNGF	LAS	QQNKEDPWT
	(SEQ ID NO: 31)	(SEQ ID NO: 32)	(SEQ ID NO: 33)	(SEQ ID NO: 34)	(SEQ ID NO: 35)	(SEQ ID NO: 36)
mAb6	GFTFSSYG	IWYDGSNK	ARMFRGAFDY	QGIRND	AAS	LQDYIYYPT
	(SEQ ID NO: 41)	(SEQ ID NO: 42)	(SEQ ID NO: 43)	(SEQ ID NO: 44)	(SEQ ID NO: 45)	(SEQ ID NO: 46)

TABLE H
Variable domain sequences of anti-CD38
(mAb1-7) and other binding proteins.
Ab	VH (protein)	VL (protein)
mAb1	QVQLQQSGAELVRSGASVKMSCKA	DIVLTQSPASLAVSLGQRATISCR
	SGYTFTSFNMHWVKETPGQGLEWI	ASESVDSYGNGFMHWYQQKPG
	GYIYPGNGGTNYNQKFKGKATLT	QPPKLLIYLASNLESGVPARFSG
	ADTSSSTAYMQISSLTSEDSAVYFC	SGSRTDFTLTIDPVEADDAATYY
	ARTGGLRRAYFTYWGQGTLVTVS	CQQNKEDPWTFGGGTKLEIK
	S (SEQ ID NO: 5)	(SEQ ID NO: 6
mAb2	QVQLVQSGAEVVKPGASVKVSCKA	DIVLTQSPATLSLSPGERATISCR
	SGYTFTSYAMHWVKEAPGQRLEW	ASQSVSSYGQGFMHWYQQKPG
	IGYIYPGQGGTNYNQKFQGRATLT	QPPRLLIYGASSRAT
	ADTSASTAYMELSSLRSEDTAVYFC	GIPARFSGSGSGTDFTLTISPLEPE
	ARTGGLRRAYFTYWGQGTLVTVS	DFAVYYCQQNKEDPWTFGGGT
	S (SEQ ID NO: 13)	KLEIK (SEQ ID NO: 14)
mAb3	QVQLVQSGAEVVKPGASVKVSCKA	DIVLTQSPATLSLSPGERATISCR
	SGYTFTSFNMHWVKEAPGQRLEWI	ASESVDSYGNGFMHWYQQKPG
	GYIYPGNGGTNYNQKFQGRATLTA	QPPRLLIYLASSRAT
	DTSASTAYMELSSLRSEDTAVYFCA	GIPARFSGSGSGTDFTLTISPLEPE
	RTGGLRRAYFTYWGQGTLVTVSS	DFAVYYCQQNKEDPWTFGGGT
	(SEQ ID NO: 17)	KLEIK (SEQ ID NO: 18)
mAb4	QVQLVQSGAEVVKSGASVKVSCKA	DIVLTQSPATLSLSPGERATISCR
	SGYTFTSFNMHWVKEAPGQGLEWI	ASESVDSYGNGFMHWYQQKPG
	GYIYPGNGGTNYNQKFQGRATLTA	QPPRLLIYLASSRAT
	DTSASTAYMEISSLRSEDTAVYFCA	GIPARFSGSGSGTDFTLTISPLEPE
	RTGGLRRAYFTYWGQGTLVTVSS	DFAVYYCQQNKEDPWTFGGGT
	(SEQ ID NO: 21)	KLEIK (SEQ ID NO: 18)
mAb5	QVQLVQSGAEVVKPGASVKMSCK	DIVLTQSPATLSLSPGERATISCR
	ASGYTFTSFNMHWVKEAPGQRLE	ASESVDSYGNGFMHWYQQKPG
	WIGYIYPGNGGTNYNQKFQGRATL	QPPRLLIYLASSRAT
	TADTSASTAYMEISSLRSEDTAVYF	GIPARFSGSGSGTDFTLTISPLEPE
	CARTGGLRRAYFTYWGQGTLVTV	DFAVYYCQQNKEDPWTFGGGT
	SS (SEQ ID NO: 23)	KLEIK (SEQ ID NO: 18)
mAb6	QVQLVESGGGVVQPGRSLRLSCAA	AIQMTQSPSSLSASVGDRVTITC
	SGFTFSSYGMHWVRQAPGKGLEW	RASQGIRNDLGWYQQKPGKAP
	VAVIWYDGSNKYYADSVKGRFTIS	KLLIYAASSLQSGVPSRFSGSGS
	GDNSKNTLYLQMNSLRAEDTAVYY	GTDFTLTISGLQPEDSATYYCLQ
	CARMFRGAFDYWGQGTLVTVSS	DYIYYPTFGQGTKVEIK (SEQ ID
	(SEQ ID NO: 9)	NO: 10)
mAb7	QVQLVQSGAEVAKPGTSVKLSCKA	DIVMTQSHLSMSTSLGDPVSITC
	SGYTFTDYWMQWVKQRPGQGLEW	KASQDVSTVVAWYQQKPGQSP
	IGTIYPGDGDTGYAQKFQGKATLTA	RRLIYSASYRYIGVPDRFTGSGA
	DKSSKTVYMHLSSLASEDSAVYYC	GTDFTFTISSVQAEDLAVYYCQQ
	ARGDYYGSNSLDYWGQGTSVTVSS	HYSPPYTFGGGTKLEIK (SEQ ID
	(SEQ ID NO: 47)	NO: 48)
Anti-	QVQLVQSGAEVVKPGASVKVSCKA	DIQMTQSPSSLSASVGDRVTITC
CD28sup	SGYTFTSYYIHWVRQAPGQGLEWI	QASQNIYVWLNWYQQKPGKAP
	GSIYPGNVNTNYAQKFQGRATLTV	KLLIYKASNLHTGVPSRFSGSGS
	DTSISTAYMELSRLRSDDTAVYYCT	GTDFTLTISSLQPEDIATYYCQQ
	RSHYGLDWNFDVWGKGTTVTVSS	GQTYPYTFGQGTKLEIK (SEQ
	(SEQ ID NO: 49)	ID NO: 50)
Anti-	QVQLQESGPGLVKPSQTLSLTCTVS	DIVLTQSPASLAVSPGQRATITCR
CD28cvn	GFSLSDYGVHWVRQPPGKGLEWL	ASESVEYYVTSLMQWYQQKPG
	GVIWAGGGTNYNPSLKSRKTISKD	QPPKLLIFAASNVESGVPARFSG
	TSKNQVSLKLSSVTAADTAVYYCA	SGSGTDFTLTINPVEANDVANYY
	RDKGYSYYYSMDYWGQGTTVTVS	CQQSRKVPYTFGQGTKLEIK
	S (SEQ ID NO: 51)	(SEQ ID NO: 52)
Anti-	QVQLVESGGGVVQPGRSLRLSCAA	DIVMTQTPLSLSVTPGQPASISCK
CD3mid	SGFTFTKAWMHWVRQAPGKQLE	SSQSLVHNNANTYLSWYLQKP
	WVAQIKDKSNSYATYYADSVKGR	GQSPQSLIYKVSNRFSGVPDRFS
	FTISRDDSKNTLYLQMNSLRAEDTA	GSGSGTDFTLKISRVEAEDVGVY
	VYYCRGVYYALSPFDYWGQGTLV	YCGQGTQYPFTFGSGTKVEIK
	TVSS (SEQ ID NO: 53)	(SEQ ID NO: 54)
Anti-	QVQLVESGGGVVQPGRSLRLSCAA	DIVMTQTPLSLSVTPGQPASISCK
CD3low	SGFTFTKAWMHWVRQAPGKGLE	SSQSLVHNNGNTYLSWYLQKP
	WVAQIKDKSNSYATYYADSVKGR	GQSPQLLIYKVSNRFSGVPDRFS
	FTISRDNSKNTLYLQMNSLRAEDTA	GSGSGTDFTLKISRVEAEDVGVY
	VYYCRGVYYALSPFDYWGQGTLV	YCGQGTQYPFTFGGGTKVEIK
	TVSS (SEQ ID NO: 84)	(SEQ ID NO: 85)
Note:
CDR sequences are bolded and underlined in amino acid sequences above.

TABLE I
Full-length sequences of binding proteins.
mAb2xCD28supxCD3mid IgG4 FALA
CD28supxCD3mid	QVQLVQSGAEVVKPGASVKVSCKASGYTF	SEQ ID
IgG4(hole) FALA Heavy	TSYYIHWVRQAPGQGLEWIGSIYPGNVNTN	NO: 60
Chain 1	YAQKFQGRATLTVDTSISTAYMELSRLRSD
(e.g., a second polypeptide	DTAVYYCTRSHYGLDWNFDVWGKGTTVT
chain of a trispecific binding	VSSSQVQLVESGGGVVQPGRSLRLSCAASG
protein of the present	FTFTKAWMHWVRQAPGKQLEWVAQIKDK
disclosure)	SNSYATYYADSVKGRFTISRDDSKNTLYLQ
	MNSLRAEDTAVYYCRGVYYALSPFDYWG
	QGTLVTVSSRTASTKGPSVFPLAPCSRSTSE
	STAALGCLVKDYFPEPVTVSWNSGALTSGV
	HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY
	TCNVDHKPSNTKVDKRVESKYGPPCPPCPA
	PEAAGGPSVFLFPPKPKDTLMISRTPEVTCV
	VVDVSQEDPEVQFNWYVDGVEVHNAKTK
	PREEQFNSTYRVVSVLTVLHQDWLNGKEY
	KCKVSNKGLPSSIEKTISKAKGQPREPQVCT
	LPPSQEEMTKNQVSLSCAVKGFYPSDIAVE
	WESNGQPENNYKTTPPVLDSDGSFFLVSKL
	TVDKSRWQEGNVFSCSVMHEALHNHYTQ
	KSLSLSLG
CD28supxCD3mid Light	DIVMTQTPLSLSVTPGQPASISCKSSQSLVH	SEQ ID
Chain 1	NNANTYLSWYLQKPGQSPQSLIYKVSNRFS	NO: 61
(e.g., a first polypeptide	GVPDRFSGSGSGTDFTLKISRVEAEDVGVY
chain of a trispecific binding	YCGQGTQYPFTFGSGTKVEIKGQPKAAPDI
protein of the present	QMTQSPSSLSASVGDRVTITCQASQNIYVW
disclosure)	LNWYQQKPGKAPKLLIYKASNLHTGVPSRF
	SGSGSGTDFTLTISSLQPEDIATYYCQQGQT
	YPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPP
	SDEQLKSGTASVVCLLNNFYPREAKVQWK
	VDNALQSGNSQESVTEQDSKDSTYSLSSTL
	TLSKADYEKHKVYACEVTHQGLSSPVTKSF
	NRGEC
mAb2 IgG4(knob) FALA	QVQLVQSGAEVVKPGASVKVSCKASGYTF	SEQ ID
Heavy Chain 2	TSYAMHWVKEAPGQRLEWIGYIYPGQGGT	NO: 62
(e.g., a third polypeptide	NYNQKFQGRATLTADTSASTAYMELSSLRS
chain of a trispecific binding	EDTAVYFCARTGGLRRAYFTYWGQGTLVT
protein of the present	VSSASTKGPSVFPLAPCSRSTSESTAALGCL
disclosure)	VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
	SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP
	SNTKVDKRVESKYGPPCPPCPAPEAAGGPS
	VFLFPPKPKDTLMISRTPEVTCVVVDVSQED
	PEVQFNWYVDGVEVHNAKTKPREEQFNST
	YRVVSVLTVLHQDWLNGKEYKCKVSNKG
	LPSSIEKTISKAKGQPREPQVYTLPPCQEEM
	TKNQVSLWCLVKGFYPSDIAVEWESNGQP
	ENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
	QEGNVFSCSVMHEALHNHYTQKSLSLSLG
mAb2 Light Chain 2	DIVLTQSPATLSLSPGERATISCRASQSVSSY	SEQ ID
(e.g., a fourth polypeptide	GQGFMHWYQQKPGQPPRLLIYGASSRATGI	NO: 63
chain of a trispecific binding	PARFSGSGSGTDFTLTISPLEPEDFAVYYCQ
protein of the present	QNKEDPWTFGGGTKLEIKRTVAAPSVFIFPP
disclosure)	SDEQLKSGTASVVCLLNNFYPREAKVQWK
	VDNALQSGNSQESVTEQDSKDSTYSLSSTL
	TLSKADYEKHKVYACEVTHQGLSSPVTKSF
	NRGEC
mAb2xCD28supxCD3mid IgG1LALA P329A
CD28supxCD3mid	QVQLVQSGAEVVKPGASVKVSCKASGYTF	SEQ ID
IgG1(hole) LALA P329A	TSYYIHWVRQAPGQGLEWIGSIYPGNVNTN	NO: 64
Heavy Chain 1	YAQKFQGRATLTVDTSISTAYMELSRLRSD
(e.g., a second polypeptide	DTAVYYCTRSHYGLDWNFDVWGKGTTVT
chain of a trispecific binding	VSSSQVQLVESGGGVVQPGRSLRLSCAASG
protein of the present	FTFTKAWMHWVRQAPGKQLEWVAQIKDK
disclosure)	SNSYATYYADSVKGRFTISRDDSKNTLYLQ
	MNSLRAEDTAVYYCRGVYYALSPFDYWG
	QGTLVTVSSRTASTKGPSVFPLAPSSKSTSG
	GTAALGCLVKDYFPEPVTVSWNSGALTSG
	VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
	YICNVNHKPSNTKVDKKVEPKSCDKTHTCP
	PCPAPEAAGGPSVFLFPPKPKDTLMISRTPE
	VTCVVVDVSHEDPEVKFNWYVDGVEVHN
	AKTKPREEQYNSTYRVVSVLTVLHQDWLN
	GKEYKCKVSNKALAAPIEKTISKAKGQPRE
	PQVCTLPPSRDELTKNQVSLSCAVKGFYPS
	DIAVEWESNGQPENNYKTTPPVLDSDGSFF
	LVSKLTVDKSRWQQGNVFSCSVMHEALHN
	HYTQKSLSLSPG
CD28supxCD3mid Light	See above.	SEQ ID
Chain 1		NO: 61
(e.g., a first polypeptide
chain of a trispecific binding
protein of the present
disclosure)
mAb2 IgG1(knob) LALA	QVQLVQSGAEVVKPGASVKVSCKASGYTF	SEQ ID
P329A Heavy Chain 2	TSYAMHWVKEAPGQRLEWIGYIYPGQGGT	NO: 65
(e.g., a third polypeptide	NYNQKFQGRATLTADTSASTAYMELSSLRS
chain of a trispecific binding	EDTAVYFCARTGGLRRAYFTYWGQGTLVT
protein of the present	VSSASTKGPSVFPLAPSSKSTSGGTAALGCL
disclosure)	VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
	SSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
	SNTKVDKKVEPKSCDKTHTCPPCPAPEAAG
	GPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
	HEDPEVKFNWYVDGVEVHNAKTKPREEQY
	NSTYRVVSVLTVLHQDWLNGKEYKCKVSN
	KALAAPIEKTISKAKGQPREPQVYTLPPCRD
	ELTKNQVSLWCLVKGFYPSDIAVEWESNG
	QPENNYKTTPPVLDSDGSFFLYSKLTVDKS
	RWQQGNVFSCSVMHEALHNHYTQKSLSLS
	PG
mAb2 Light Chain 2	See above.	SEQ ID
(e.g., a fourth polypeptide		NO: 63
chain of a trispecific binding
protein of the present
disclosure)
mAb2xCD28supxCD3mid IgG1 NNSA
CD28supxCD3mid	QVQLVQSGAEVVKPGASVKVSCKASGYTF	SEQ ID
IgG1(hole) NNSA Heavy	TSYYIHWVRQAPGQGLEWIGSIYPGNVNTN	NO: 66
Chain 1	YAQKFQGRATLTVDTSISTAYMELSRLRSD
(e.g., a second polypeptide	DTAVYYCTRSHYGLDWNFDVWGKGTTVT
chain of a trispecific binding	VSSSQVQLVESGGGVVQPGRSLRLSCAASG
protein of the present	FTFTKAWMHWVRQAPGKQLEWVAQIKDK
disclosure)	SNSYATYYADSVKGRFTISRDDSKNTLYLQ
	MNSLRAEDTAVYYCRGVYYALSPFDYWG
	QGTLVTVSSRTASTKGPSVFPLAPSSKSTSG
	GTAALGCLVKDYFPEPVTVSWNSGALTSG
	VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
	YICNVNHKPSNTKVDKKVEPKSCDKTHTCP
	PCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
	TCVVVDVSHEDPEVKFNWYVDGVEVHNA
	KTKPREEQYNNASRVVSVLTVLHQDWLNG
	KEYKCKVSNKALPAPIEKTISKAKGQPREPQ
	VCTLPPSRDELTKNQVSLSCAVKGFYPSDIA
	VEWESNGQPENNYKTTPPVLDSDGSFFLVS
	KLTVDKSRWQQGNVFSCSVMHEALHNHY
	TQKSLSLSPG
CD28supxCD3mid Light	See above.	SEQ ID
Chain 1		NO: 61
(e.g., a first polypeptide
chain of a trispecific binding
protein of the present
disclosure)
mAb2 IgG1(knob) NNSA	QVQLVQSGAEVVKPGASVKVSCKASGYTF	SEQ ID
Heavy Chain 2	TSYAMHWVKEAPGQRLEWIGYIYPGQGGT	NO: 67
(e.g., a third polypeptide	NYNQKFQGRATLTADTSASTAYMELSSLRS
chain of a trispecific binding	EDTAVYFCARTGGLRRAYFTYWGQGTLVT
protein of the present	VSSASTKGPSVFPLAPSSKSTSGGTAALGCL
disclosure)	VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
	SSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
	SNTKVDKKVEPKSCDKTHTCPPCPAPELLG
	GPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
	HEDPEVKFNWYVDGVEVHNAKTKPREEQY
	NNASRVVSVLTVLHQDWLNGKEYKCKVS
	NKALPAPIEKTISKAKGQPREPQVYTLPPCR
	DELTKNQVSLWCLVKGFYPSDIAVEWESN
	GQPENNYKTTPPVLDSDGSFFLYSKLTVDK
	SRWQQGNVFSCSVMHEALHNHYTQKSLSL
	SPG
CD38VH1 Light Chain 2	See above.	SEQ ID
(e.g., a fourth polypeptide		NO: 63
chain of a trispecific binding
protein of the present
disclosure)
mAb6xCD28supxCD3mid IgG4 FALA
CD28supxCD3mid	See above.	SEQ ID
IgG4(hole) FALA Heavy		NO: 60
Chain 1
(e.g., a second polypeptide
chain of a trispecific binding
protein of the present
disclosure)
CD28supxCD3mid Light	See above.	SEQ ID
Chain 1		NO: 61
(e.g., a first polypeptide
chain of a trispecific binding
protein of the present
disclosure)
mAb6 IgG4(knob) LALA	QVQLVESGGGVVQPGRSLRLSCAASGFTFS	SEQ ID
Heavy Chain 2	SYGMHWVRQAPGKGLEWVAVIWYDGSNK	NO: 68
(e.g., a third polypeptide	YYADSVKGRFTISGDNSKNTLYLQMNSLRA
chain of a trispecific binding	DETAVYYCARMFRGAFDYVVGQGTLVTVSS
protein of the present	ASTKGPSVFPLAPCSRSTSESTAALGCLVKD
disclosure)	YFPEPVTVSWNSGALTSGVHTFPAVLQSSG
	LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT
	KVDKRVESKYGPPCPPCPAPEAAGGPSVFL
	FPPKPKDTLMISRTPEVTCVVVDVSQEDPEV
	QFNWYVDGVEVHNAKTKPREEQFNSTYRV
	VSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
	EKTISKAKGQPREPQVYTLPPCQEEMTKNQ
	VSLWCLVKGFYPSDIAVEWESNGQPENNY
	KTTPPVLDSDGSFFLYSKLTVDKSRWQEGN
	VFSCSVMHEALHNHYTQKSLSLSLG
mAb6 Light2	AIQMTQSPSSLSASVGDRVTITCRASQGIRN	SEQ ID
(e.g., a fourth polypeptide	DLGWYQQKPGKAPKLLIYAASSLQSGVPSR	NO: 69
chain of a trispecific binding	FSGSGSGTDFTLTISGLQPEDSATYYCLQDY
protein of the present	IYYPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
disclosure)	LKSGTASVVCLLNNFYPREAKVQWKVDNA
	LQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
	DYEKHKVYACEVTHQGLSSPVTKSFNRGEC
mAb6xCD28supxCD3mid IgG1LALA P329A
CD28supxCD3mid	See above.	SEQ ID
IgG1(hole) LALA P329A		NO: 64
Heavy Chain 1
(e.g., a second polypeptide
chain of a trispecific binding
protein of the present
disclosure)
CD28supxCD3mid Light	See above.	SEQ ID
Chain 1		NO: 61
(e.g., a first polypeptide
chain of a trispecific binding
protein of the present
disclosure)
mAb6 IgG1(knob) LALA	QVQLVESGGGVVQPGRSLRLSCAASGFTFS	SEQ ID
P329A Heavy Chain 2	SYGMHWVRQAPGKGLEWVAVIWYDGSNK	NO: 70
(e.g., a third polypeptide	YYADSVKGRFTISGDNSKNTLYLQMNSLRA
chain of a trispecific binding	EDTAVYYCARMFRGAFDYVVGQGTLVTVSS
protein of the present	ASTKGPSVFPLAPSSKSTSGGTAALGCLVK
disclosure)	DYFPEPVTVSWNSGALTSGVHTFPAVLQSS
	GLYSLSSVVTVPSSSLGTQTYICNVNHKPSN
	TKVDKKVEPKSCDKTHTCPPCPAPEAAGGP
	SVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
	DPEVKFNWYVDGVEVHNAKTKPREEQYNS
	TYRVVSVLTVLHQDWLNGKEYKCKVSNK
	ALAAPIEKTISKAKGQPREPQVYTLPPCRDE
	LTKNQVSLWCLVKGFYPSDIAVEWESNGQ
	PENNYKTTPPVLDSDGSFFLYSKLTVDKSR
	WQQGNVFSCSVMHEALHNHYTQKSLSLSPG
mAb6 Light2	See above.	SEQ ID
(e.g., a fourth polypeptide		NO: 69
chain of a trispecific binding
protein of the present
disclosure)
mAb6xCD28supxCD3mid IgG1 NNSA
CD28supxCD3mid	See above.	SEQ ID
IgG1(hole) NNSA Heavy		NO: 66
Chain 1
(e.g., a second polypeptide
chain of a trispecific binding
protein of the present
disclosure)
CD28supxCD3mid Light	See above.	SEQ ID
Chain 1		NO: 61
(e.g., a first polypeptide
chain of a trispecific binding
protein of the present
disclosure)
mAb6 IgG1(knob) NNSA	QVQLVESGGGVVQPGRSLRLSCAASGFTFS	SEQ ID
Heavy Chain 2	SYGMHWVRQAPGKGLEWVAVIWYDGSNK	NO: 71
(e.g., a third polypeptide	YYADSVKGRFTISGDNSKNTLYLQMNSLRA
chain of a trispecific binding	EDTAVYYCARMFRGAFDYVVGQGTLVTVSS
protein of the present	ASTKGPSVFPLAPSSKSTSGGTAALGCLVK
disclosure)	DYFPEPVTVSWNSGALTSGVHTFPAVLQSS
	GLYSLSSVVTVPSSSLGTQTYICNVNHKPSN
	TKVDKKVEPKSCDKTHTCPPCPAPELLGGP
	SVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
	DPEVKFNWYVDGVEVHNAKTKPREEQYN
	NASRVVSVLTVLHQDWLNGKEYKCKVSN
	KALPAPIEKTISKAKGQPREPQVYTLPPCRD
	ELTKNQVSLWCLVKGFYPSDIAVEWESNG
	QPENNYKTTPPVLDSDGSFFLYSKLTVDKS
	RWQQGNVFSCSVMHEALHNHYTQKSLSLS
	PG
mAb6 Light2	See above.	SEQ ID
(e.g., a fourth polypeptide		NO: 69
chain of a trispecific binding
protein of the present
disclosure)
mAb1 monovalent antibody
mAb1 heavy chain	QVQLQQSGAELVRSGASVKMSCKASGYTF	SEQ ID
	TSFNMHWVKETPGQGLEWIGYIYPGNGGT	NO: 7
	NYNQKFKGKATLTADTSSSTAYMQISSLTS
	EDSAVYFCARTGGLRRAYFTYWGQGTLVT
	VSSASTKGPSVFPLAPSSKSTSGGTAALGCL
	VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
	SSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
	SNTKVDKKVEPKSCDKTHTCPPCPAPELLA
	GPDVFLFPPKPKDTLMISRTPEVTCVVVDVS
	HEDPEVKFNWYVDGVEVHNAKTKPREEQY
	NSTYRVVSVLTVLHQDWLNGKEYKCKVSN
	KALPLPEEKTISKAKGQPREPQVYTLPPSRD
	ELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
	PENNYKTTPPVLDSDGSFFLYSKLTVDKSR
	WQQGNVFSCSVMHEALHNHYTQKSLSLSPG
mAb1 light chain	DIVLTQSPASLAVSLGQRATISCRASESVDS	SEQ ID
	YGNGFMHWYQQKPGQPPKLLIYLASNLES	NO: 8
	GVPARFSGSGSRTDFTLTIDPVEADDAATY
	YCQQNKEDPWTFGGGTKLEIKRTVAAPSVF
	IFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
	WKVDNALQSGNSQESVTEQDSKDSTYSLSS
	TLTLSKADYEKHKVYACEVTHQGLSSPVTK
	SFNRGEC
mAb2 monovalent antibody
mAb2 heavy chain	QVQLVQSGAEVVKPGASVKVSCKASGYTF	SEQ ID
	TSYAMHWVKEAPGQRLEWIGYIYPGQGGT	NO: 15
	NYNQKFQGRATLTADTSASTAYMELSSLRS
	EDTAVYFCARTGGLRRAYFTYWGQGTLVT
	VSSASTKGPSVFPLAPSSKSTSGGTAALGCL
	VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
	SSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
	SNTKVDKKVEPKSCDKTHTCPPCPAPELLA
	GPDVFLFPPKPKDTLMISRTPEVTCVVVDVS
	HEDPEVKFNWYVDGVEVHNAKTKPREEQY
	NSTYRVVSVLTVLHQDWLNGKEYKCKVSN
	KALPLPEEKTISKAKGQPREPQVYTLPPSRD
	ELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
	PENNYKTTPPVLDSDGSFFLYSKLTVDKSR
	WQQGNVFSCSVMHEALHNHYTQKSLSLSPG
mAb2 light chain	DIVLTQSPATLSLSPGERATISCRASQSVSSY	SEQ ID
	GQGFMHWYQQKPGQPPRLLIYGASSRAT	NO: 16
	GIPARFSGSGSGTDFTLTISPLEPEDFAVYYC
	QQNKEDPWTFGGGTKLEIKRTVAAPSVF
	IFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
	WKVDNALQSGNSQESVTEQDSKDSTYSLSS
	TLTLSKADYEKHKVYACEVTHQGLSSPVTK
	SFNRGEC
mAb3 monovalent antibody
mAb3 heavy chain	QVQLVQSGAEVVKPGASVKVSCKASGYTF	SEQ ID
	TSFNMHWVKEAPGQRLEWIGYIYPGNGGT	NO: 19
	NYNQKFQGRATLTADTSASTAYMELSSLRS
	EDTAVYFCARTGGLRRAYFTYWGQGTLVT
	VSSASTKGPSVFPLAPSSKSTSGGTAALGCL
	VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
	SSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
	SNTKVDKKVEPKSCDKTHTCPPCPAPELLA
	GPDVFLFPPKPKDTLMISRTPEVTCVVVDVS
	HEDPEVKFNWYVDGVEVHNAKTKPREEQY
	NSTYRVVSVLTVLHQDWLNGKEYKCKVSN
	KALPLPEEKTISKAKGQPREPQVYTLPPSRD
	ELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
	PENNYKTTPPVLDSDGSFFLYSKLTVDKSR
	WQQGNVFSCSVMHEALHNHYTQKSLSLSPG
mAb3 light chain	DIVLTQSPATLSLSPGERATISCRASESVDSY	SEQ ID
	GNGFMHWYQQKPGQPPRLLIYLASSRAT	NO: 20
	GIPARFSGSGSGTDFTLTISPLEPEDFAVYYC
	QQNKEDPWTFGGGTKLEIKRTVAAPSVF
	IFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
	WKVDNALQSGNSQESVTEQDSKDSTYSLSS
	TLTLSKADYEKHKVYACEVTHQGLSSPVTK
	SFNRGEC
mAb4 monovalent antibody
mAb4 heavy chain	QVQLVQSGAEVVKSGASVKVSCKASGYTF	SEQ ID
	TSFNMHWVKEAPGQGLEWIGYIYPGNGGT	NO: 22
	NYNQKFQGRATLTADTSASTAYMEISSLRS
	EDTAVYFCARTGGLRRAYFTYWGQGTLVT
	VSSASTKGPSVFPLAPSSKSTSGGTAALGCL
	VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
	SSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
	SNTKVDKKVEPKSCDKTHTCPPCPAPELLA
	GPDVFLFPPKPKDTLMISRTPEVTCVVVDVS
	HEDPEVKFNWYVDGVEVHNAKTKPREEQY
	NSTYRVVSVLTVLHQDWLNGKEYKCKVSN
	KALPLPEEKTISKAKGQPREPQVYTLPPSRD
	ELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
	PENNYKTTPPVLDSDGSFFLYSKLTVDKSR
	WQQGNVFSCSVMHEALHNHYTQKSLSLSPG
mAb4 light chain	DIVLTQSPATLSLSPGERATISCRASESVDSY	SEQ ID
	GNGFMHWYQQKPGQPPRLLIYLASSRAT	NO: 20
	GIPARFSGSGSGTDFTLTISPLEPEDFAVYYC
	QQNKEDPWTFGGGTKLEIKRTVAAPSVF
	IFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
	WKVDNALQSGNSQESVTEQDSKDSTYSLSS
	TLTLSKADYEKHKVYACEVTHQGLSSPVTK
	SFNRGEC
mAb5 monovalent antibody
mAb5 heavy chain	QVQLVQSGAEVVKPGASVKMSCKASGYTF	SEQ ID
	TSFNMHWVKEAPGQRLEWIGYIYPGNGGT	NO: 24
	NYNQKFQGRATLTADTSASTAYMEISSLRS
	EDTAVYFCARTGGLRRAYFTYWGQGTLVT
	VSSASTKGPSVFPLAPSSKSTSGGTAALGCL
	VKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
	SSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
	SNTKVDKKVEPKSCDKTHTCPPCPAPELLA
	GPDVFLFPPKPKDTLMISRTPEVTCVVVDVS
	HEDPEVKFNWYVDGVEVHNAKTKPREEQY
	NSTYRVVSVLTVLHQDWLNGKEYKCKVSN
	KALPLPEEKTISKAKGQPREPQVYTLPPSRD
	ELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
	PENNYKTTPPVLDSDGSFFLYSKLTVDKSR
	WQQGNVFSCSVMHEALHNHYTQKSLSLSPG
mAb5 light chain	DIVLTQSPATLSLSPGERATISCRASESVDSY	SEQ ID
	GNGFMHWYQQKPGQPPRLLIYLASSRAT	NO: 20
	GIPARFSGSGSGTDFTLTISPLEPEDFAVYYC
	QQNKEDPWTFGGGTKLEIKRTVAAPSVF
	IFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
	WKVDNALQSGNSQESVTEQDSKDSTYSLSS
	TLTLSKADYEKHKVYACEVTHQGLSSPVTK
	SFNRGEC
mAb6 monovalent antibody
mAb6 heavy chain	QVQLVESGGGVVQPGRSLRLSCAASGFTFS	SEQ ID
	SYGMHWVRQAPGKGLEWVAVIWYDGSNK	NO: 11
	YYADSVKGRFTISGDNSKNTLYLQMNSLRA
	EDTAVYYCARMFRGAFDYWGQGTLVTVSS
	ASTKGPSVFPLAPSSKSTSGGTAALGCLVK
	DYFPEPVTVSWNSGALTSGVHTFPAVLQSS
	GLYSLSSVVTVPSSSLGTQTYICNVNHKPSN
	TKVDKKVEPKSCDKTHTCPPCPAPELLAGP
	DVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
	DPEVKFNWYVDGVEVHNAKTKPREEQYNS
	TYRVVSVLTVLHQDWLNGKEYKCKVSNK
	ALPLPEEKTISKAKGQPREPQVYTLPPSRDE
	LTKNQVSLTCLVKGFYPSDIAVEWESNGQP
	ENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
	QQGNVFSCSVMHEALHNHYTQKSLSLSPG
mAb6 light chain	AIQMTQSPSSLSASVGDRVTITCRASQGIRN	SEQ ID
	DLGWYQQKPGKAPKLLIYAASSLQSGVPS	NO: 12
	RFSGSGSGTDFTLTISGLQPEDSATYYCLQD
	YIYYPTFGQGTKVEIKRTVAAPSVFIFPP
	SDEQLKSGTASVVCLLNNFYPREAKVQWK
	VDNALQSGNSQESVTEQDSKDSTYSLSSTL
	TLSKADYEKHKVYACEVTHQGLSSPVTKSF
	NRGEC
mAb7 monovalent antibody
mAb7 heavy chain	QVQLVQSGAEVAKPGTSVKLSCKASGYTFT	SEQ ID
	DYWMQWVKQRPGQGLEWIGTIYPGDGDT	NO: 107
	GYAQKFQGKATLTADKSSKTVYMHLSSLA
	SEDSAVYYCARGDYYGSNSLDYWGQGTSV
	TVSSASTKGPSVFPLAPSSKSTSGGTAALGC
	LVKDYFPEPVTVSWNSGALTSGVHTFPAVL
	QSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
	PSNTKVDKKVEPKSCDKTHTCPPCPAPELL
	GGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
	SHEDPEVKFNWYVDGVEVHNAKTKPREEQ
	YNSTYRVVSVLTVLHQDWLNGKEYKCKVS
	NKALPAPIEKTISKAKGQPREPQVYTLPPSR
	DELTKNQVSLTCLVKGFYPSDIAVEWESNG
	QPENNYKTTPPVLDSDGSFFLYSKLTVDKS
	RWQQGNVFSCSVMHEALHNHYTQKSLSLS
	PGK
mAb7 light chain	DIVMTQSHLSMSTSLGDPVSITCKASQDVST	SEQ ID
	VVAWYQQKPGQSPRRLIYSASYRYIGVPDR	NO: 106
	FTGSGAGTDFTFTISSVQAEDLAVYYCQQH
	YSPPYTFGGGTKLEIKRTVAAPSVFIFPP
	SDEQLKSGTASVVCLLNNFYPREAKVQWK
	VDNALQSGNSQESVTEQDSKDSTYSLSSTL
	TLSKADYEKHKVYACEVTHQGLSSPVTKSF
	NRGEC

TABLE J
Full-length polynucleotide sequences of binding proteins.
mAb2xCD28supxCD3mid IgG4 FALA
CD28supxCD3mid	CAGGTGCAGCTGGTGCAGTCTGGCGCCGA	SEQ ID
IgG4(hole) FALA Heavy	GGTCGTGAAACCTGGCGCCTCTGTGAAGG	NO: 72
Chain 1	TGTCCTGCAAGGCCAGCGGCTACACCTTT
(e.g., encoding a second	ACCAGCTACTACATCCACTGGGTGCGCCA
polypeptide chain of a	GGCCCCTGGACAGGGACTGGAATGGATC
trispecific binding protein of	GGCAGCATCTACCCCGGCAACGTGAACA
the present disclosure)	CCAACTACGCCCAGAAGTTCCAGGGCAG
	AGCCACCCTGACCGTGGACACCAGCATCA
	GCACCGCCTACATGGAACTGAGCCGGCTG
	AGAAGCGACGACACCGCCGTGTACTACT
	GCACCCGGTCCCACTACGGCCTGGATTGG
	AACTTCGACGTGTGGGGCAAGGGCACCA
	CCGTGACAGTGTCTAGCAGCCAGGTGCAG
	CTGGTGGAATCTGGCGGCGGAGTGGTGC
	AGCCTGGCAGAAGCCTGAGACTGAGCTG
	TGCCGCCAGCGGCTTCACCTTCACCAAGG
	CCTGGATGCACTGGGTGCGCCAGGCCCCT
	GGAAAGCAGCTGGAATGGGTGGCCCAGA
	TCAAGGACAAGAGCAACAGCTACGCCAC
	CTACTACGCCGACAGCGTGAAGGGCCGG
	TTCACCATCAGCCGGGACGACAGCAAGA
	ACACCCTGTACCTGCAGATGAACAGCCTG
	CGGGCCGAGGACACCGCCGTGTACTACTG
	TCGGGGCGTGTACTATGCCCTGAGCCCCT
	TCGATTACTGGGGCCAGGGAACCCTCGTG
	ACCGTGTCTAGTCGGACCGCCAGCACAAA
	GGGCCCATCGGTGTTCCCTCTGGCCCCTT
	GCAGCAGAAGCACCAGCGAATCTACAGC
	CGCCCTGGGCTGCCTCGTGAAGGACTACT
	TTCCCGAGCCCGTGACCGTGTCCTGGAAC
	TCTGGCGCTCTGACAAGCGGCGTGCACAC
	CTTTCCAGCCGTGCTCCAGAGCAGCGGCC
	TGTACTCTCTGAGCAGCGTCGTGACAGTG
	CCCAGCAGCAGCCTGGGCACCAAGACCT
	ACACCTGTAACGTGGACCACAAGCCCAG
	CAACACCAAGGTGGACAAGCGGGTGGAA
	TCTAAGTACGGCCCTCCCTGCCCTCCTTG
	CCCAGCCCCTGAAGCTGCCGGCGGACCCT
	CCGTGTTCCTGTTCCCCCCAAAGCCCAAG
	GACACCCTGATGATCAGCCGGACCCCCGA
	AGTGACCTGCGTGGTGGTGGATGTGTCCC
	AGGAAGATCCCGAGGTGCAGTTCAATTG
	GTACGTGGACGGCGTGGAAGTGCACAAC
	GCCAAGACCAAGCCCAGAGAGGAACAGT
	TCAACAGCACCTACCGGGTGGTGTCCGTG
	CTGACCGTGCTGCACCAGGACTGGCTGAA
	CGGCAAAGAGTACAAGTGCAAGGTGTCC
	AACAAGGGCCTGCCCAGCTCCATCGAGA
	AAACCATCAGCAAGGCCAAGGGCCAGCC
	CCGCGAGCCTCAAGTGTGTACCCTGCCCC
	CTAGCCAGGAAGAGATGACCAAGAACCA
	GGTGTCCCTGAGCTGTGCCGTGAAAGGCT
	TCTACCCCAGCGACATTGCCGTGGAATGG
	GAGAGCAACGGCCAGCCCGAGAACAACT
	ACAAGACCACCCCCCCTGTGCTGGACAGC
	GACGGCTCATTCTTCCTGGTGTCCAAGCT
	GACCGTGGACAAGAGCCGGTGGCAGGAA
	GGCAACGTGTTCAGCTGCTCCGTGATGCA
	CGAGGCCCTGCACAACCACTACACCCAG
	AAGTCCCTGTCTCTGTCCCTGGGC
CD28supxCD3mid Light	GACATCGTGATGACCCAGACCCCCCTGAG	SEQ ID
Chain 1	CCTGAGCGTGACACCTGGACAGCCTGCCA	NO: 73
(e.g., encoding a first	GCATCAGCTGCAAGAGCAGCCAGAGCCT
polypeptide chain of a	GGTGCACAACAACGCCAACACCTACCTG
trispecific binding protein of	AGCTGGTATCTGCAGAAGCCCGGCCAGA
the present disclosure)	GCCCCCAGTCCCTGATCTACAAGGTGTCC
	AACAGATTCAGCGGCGTGCCCGACAGATT
	CTCCGGCAGCGGCTCTGGCACCGACTTCA
	CCCTGAAGATCAGCCGGGTGGAAGCCGA
	GGACGTGGGCGTGTACTATTGTGGCCAGG
	GCACCCAGTACCCCTTCACCTTTGGCAGC
	GGCACCAAGGTGGAAATCAAGGGCCAGC
	CCAAGGCCGCCCCCGACATCCAGATGACC
	CAGAGCCCCAGCAGCCTGTCTGCCAGCGT
	GGGCGACAGAGTGACCATCACCTGTCAG
	GCCAGCCAGAACATCTACGTGTGGCTGAA
	CTGGTATCAGCAGAAGCCCGGCAAGGCC
	CCCAAGCTGCTGATCTACAAGGCCAGCAA
	CCTGCACACCGGCGTGCCCAGCAGATTTT
	CTGGCAGCGGCTCCGGCACCGACTTCACC
	CTGACAATCAGCTCCCTGCAGCCCGAGGA
	CATTGCCACCTACTACTGCCAGCAGGGCC
	AGACCTACCCCTACACCTTTGGCCAGGGC
	ACCAAGCTGGAAATCAAGACCAAGGGCC
	CCAGCCGTACGGTGGCCGCTCCCAGCGTG
	TTCATCTTCCCACCTAGCGACGAGCAGCT
	GAAGTCCGGCACAGCCTCTGTCGTGTGCC
	TGCTGAACAACTTCTACCCCCGCGAGGCC
	AAAGTGCAGTGGAAGGTGGACAACGCCC
	TGCAGAGCGGCAACAGCCAGGAAAGCGT
	GACCGAGCAGGACAGCAAGGACTCCACC
	TACAGCCTGAGCAGCACCCTGACACTGAG
	CAAGGCCGACTACGAGAAGCACAAGGTG
	TACGCCTGCGAAGTGACCCACCAGGGCCT
	GTCTAGCCCCGTGACCAAGAGCTTCAACC
	GGGGCGAGTGT
mAb2 IgG4(knob) FALA	CAGGTGCAGCTGGTGCAGTCTGGCGCCGA	SEQ ID
Heavy Chain 2	AGTCGTGAAACCTGGCGCCTCCGTGAAGG	NO: 74
(e.g., encoding a third	TGTCCTGCAAGGCCAGCGGCTACACCTTT
polypeptide chain of a	ACCAGCTACGCCATGCACTGGGTCAAAG
trispecific binding protein of	AGGCCCCTGGCCAGAGACTGGAATGGAT
the present disclosure)	CGGCTACATCTACCCCGGCCAGGGCGGCA
	CCAACTACAACCAGAAGTTCCAGGGCAG
	AGCCACCCTGACCGCCGATACAAGCGCC
	AGCACCGCCTACATGGAACTGAGCAGCCT
	GCGGAGCGAGGATACCGCCGTGTACTTCT
	GTGCCAGAACAGGCGGCCTGAGGCGGGC
	CTACTTTACCTATTGGGGCCAGGGCACCC
	TCGTGACCGTGTCTAGCGCTAGCACAAAG
	GGCCCATCGGTGTTCCCTCTGGCCCCTTG
	CAGCAGAAGCACCAGCGAATCTACAGCC
	GCCCTGGGCTGCCTCGTGAAGGACTACTT
	TCCCGAGCCCGTGACCGTGTCCTGGAACT
	CTGGCGCTCTGACAAGCGGCGTGCACACC
	TTTCCAGCCGTGCTCCAGAGCAGCGGCCT
	GTACTCTCTGAGCAGCGTCGTGACAGTGC
	CCAGCAGCAGCCTGGGCACCAAGACCTA
	CACCTGTAACGTGGACCACAAGCCCAGC
	AACACCAAGGTGGACAAGCGGGTGGAAT
	CTAAGTACGGCCCTCCCTGCCCTCCTTGC
	CCAGCCCCTGAAGCTGCCGGCGGACCCTC
	CGTGTTCCTGTTCCCCCCAAAGCCCAAGG
	ACACCCTGATGATCAGCCGGACCCCCGAA
	GTGACCTGCGTGGTGGTGGATGTGTCCCA
	GGAAGATCCCGAGGTGCAGTTCAATTGGT
	ACGTGGACGGCGTGGAAGTGCACAACGC
	CAAGACCAAGCCCAGAGAGGAACAGTTC
	AACAGCACCTACCGGGTGGTGTCCGTGCT
	GACCGTGCTGCACCAGGACTGGCTGAAC
	GGCAAAGAGTACAAGTGCAAGGTGTCCA
	ACAAGGGCCTGCCCAGCTCCATCGAGAA
	AACCATCAGCAAGGCCAAGGGCCAGCCC
	CGCGAGCCTCAAGTGTATACCCTGCCCCC
	TTGCCAGGAAGAGATGACCAAGAACCAG
	GTGTCCCTGTGGTGTCTCGTGAAAGGCTT
	CTACCCCAGCGACATTGCCGTGGAATGGG
	AGAGCAACGGCCAGCCCGAGAACAACTA
	CAAGACCACCCCCCCTGTGCTGGACAGCG
	ACGGCTCATTCTTCCTGTACTCCAAGCTG
	ACCGTGGACAAGAGCCGGTGGCAGGAAG
	GCAACGTGTTCAGCTGCTCCGTGATGCAC
	GAGGCCCTGCACAACCACTACACCCAGA
	AGTCCCTGTCTCTGTCCCTGGGC
mAb2 Light Chain 2	GACATCGTGCTGACACAGAGCCCTGCCAC	SEQ ID
(e.g., encoding a fourth	CCTGTCTCTGAGCCCTGGCGAGAGAGCCA	NO: 75
polypeptide chain of a	CCATCAGCTGTAGAGCCAGCCAGAGCGT
trispecific binding protein of	GTCCAGCTACGGCCAGGGCTTCATGCACT
the present disclosure)	GGTATCAGCAGAAGCCCGGCCAGCCCCC
	CAGACTGCTGATCTATGGCGCCAGCAGCA
	GAGCCACAGGCATCCCCGCCAGATTTTCT
	GGCTCTGGCAGCGGCACCGACTTCACCCT
	GACAATCAGCCCCCTGGAACCCGAGGAC
	TTCGCCGTGTACTACTGCCAGCAGAACAA
	AGAGGACCCCTGGACCTTCGGCGGAGGC
	ACCAAGCTGGAAATCAAGCGTACGGTGG
	CCGCTCCCAGCGTGTTCATCTTCCCACCT
	AGCGACGAGCAGCTGAAGTCCGGCACAG
	CCTCTGTCGTGTGCCTGCTGAACAACTTC
	TACCCCCGCGAGGCCAAGGTGCAGTGGA
	AGGTGGACAATGCCCTGCAGAGCGGCAA
	CAGCCAGGAAAGCGTGACCGAGCAGGAC
	AGCAAGGACTCCACCTACAGCCTGAGCA
	GCACCCTGACCCTGTCCAAGGCCGATTAC
	GAGAAGCACAAGGTGTACGCCTGCGAAG
	TGACCCACCAGGGCCTGTCTAGCCCCGTG
	ACCAAGAGCTTCAACCGGGGCGAGTGC
mAb2xCD28supxCD3mid IgG1LALA P329A
CD28supxCD3mid	CAGGTGCAGCTGGTGCAGTCTGGCGCCGA	SEQ ID
IgG1(hole) LALA P329A	GGTCGTGAAACCTGGCGCCTCTGTGAAGG	NO: 76
Heavy Chain 1	TGTCCTGCAAGGCCAGCGGCTACACCTTT
(e. g., encoding a second	ACCAGCTACTACATCCACTGGGTGCGCCA
polypeptide chain of a	GGCCCCTGGACAGGGACTGGAATGGATC
trispecific binding protein of	GGCAGCATCTACCCCGGCAACGTGAACA
the present disclosure)	CCAACTACGCCCAGAAGTTCCAGGGCAG
	AGCCACCCTGACCGTGGACACCAGCATCA
	GCACCGCCTACATGGAACTGAGCCGGCTG
	AGAAGCGACGACACCGCCGTGTACTACT
	GCACCCGGTCCCACTACGGCCTGGATTGG
	AACTTCGACGTGTGGGGCAAGGGCACCA
	CCGTGACAGTGTCTAGCAGCCAGGTGCAG
	CTGGTGGAATCTGGCGGCGGAGTGGTGC
	AGCCTGGCAGAAGCCTGAGACTGAGCTG
	TGCCGCCAGCGGCTTCACCTTCACCAAGG
	CCTGGATGCACTGGGTGCGCCAGGCCCCT
	GGAAAGCAGCTGGAATGGGTGGCCCAGA
	TCAAGGACAAGAGCAACAGCTACGCCAC
	CTACTACGCCGACAGCGTGAAGGGCCGG
	TTCACCATCAGCCGGGACGACAGCAAGA
	ACACCCTGTACCTGCAGATGAACAGCCTG
	CGGGCCGAGGACACCGCCGTGTACTACTG
	TCGGGGCGTGTACTATGCCCTGAGCCCCT
	TCGATTACTGGGGCCAGGGAACCCTCGTG
	ACCGTGTCTAGTCGGACCGCCAGCACAAA
	GGGCCCCAGCGTGTTCCCTCTGGCCCCTA
	GCAGCAAGAGCACATCTGGCGGAACAGC
	CGCCCTGGGCTGCCTCGTGAAGGACTACT
	TTCCCGAGCCCGTGACCGTGTCCTGGAAT
	TCTGGCGCCCTGACCAGCGGCGTGCACAC
	CTTTCCAGCTGTGCTGCAGTCCAGCGGCC
	TGTACAGCCTGAGCAGCGTCGTGACAGTG
	CCCAGCAGCTCTCTGGGCACCCAGACCTA
	CATCTGCAACGTGAACCACAAGCCCAGC
	AACACCAAGGTGGACAAGAAGGTGGAAC
	CCAAGAGCTGCGACAAGACCCACACCTG
	TCCCCCTTGTCCTGCCCCCGAAGCCGCCG
	GAGGCCCTTCCGTGTTCCTGTTCCCCCCA
	AAGCCCAAGGACACCCTGATGATCAGCC
	GGACCCCCGAAGTGACCTGCGTGGTGGTG
	GATGTGTCCCACGAGGACCCTGAAGTGA
	AGTTCAATTGGTACGTGGACGGCGTGGAA
	GTGCACAACGCCAAGACCAAGCCAAGAG
	AGGAACAGTACAACAGCACCTACCGGGT
	GGTGTCCGTGCTGACCGTGCTGCACCAGG
	ACTGGCTGAACGGCAAAGAGTACAAGTG
	CAAGGTGTCCAACAAGGCCCTGGCCGCCC
	CCATCGAGAAAACCATCAGCAAGGCCAA
	GGGCCAGCCCCGCGAACCCCAGGTGTGC
	ACACTGCCCCCAAGCAGGGACGAGCTGA
	CCAAGAACCAGGTGTCCCTGAGCTGTGCC
	GTGAAAGGCTTCTACCCCTCCGATATCGC
	CGTGGAATGGGAGAGCAACGGCCAGCCC
	GAGAACAACTACAAGACCACCCCCCCTGT
	GCTGGACAGCGACGGCTCATTCTTCCTGG
	TGTCCAAGCTGACAGTGGACAAGTCCCGG
	TGGCAGCAGGGCAACGTGTTCAGCTGCTC
	CGTGATGCACGAGGCCCTGCACAACCACT
	ACACCCAGAAGTCCCTGAGCCTGAGCCCC
	GGC
CD28supxCD3mid Light	See above.	SEQ ID
Chain 1		NO: 73
(e.g., encoding a first
polypeptide chain of a
trispecific binding protein of
the present disclosure)
mAb2 IgG1(knob) LALA	CAGGTGCAGCTGGTGCAGTCTGGCGCCGA	SEQ ID
P329A Heavy Chain 2	AGTCGTGAAACCTGGCGCCTCCGTGAAGG	NO: 77
(e.g., encoding a third	TGTCCTGCAAGGCCAGCGGCTACACCTTT
polypeptide chain of a	ACCAGCTACGCCATGCACTGGGTCAAAG
trispecific binding protein of	AGGCCCCTGGCCAGAGACTGGAATGGAT
the present disclosure)	CGGCTACATCTACCCCGGCCAGGGCGGCA
	CCAACTACAACCAGAAGTTCCAGGGCAG
	AGCCACCCTGACCGCCGATACAAGCGCC
	AGCACCGCCTACATGGAACTGAGCAGCCT
	GCGGAGCGAGGATACCGCCGTGTACTTCT
	GTGCCAGAACAGGCGGCCTGAGGCGGGC
	CTACTTTACCTATTGGGGCCAGGGCACCC
	TCGTGACCGTGTCTAGCGCTAGCACAAAG
	GGCCCCAGCGTGTTCCCTCTGGCCCCTAG
	CAGCAAGAGCACATCTGGCGGAACAGCC
	GCCCTGGGCTGCCTCGTGAAGGACTACTT
	TCCCGAGCCCGTGACCGTGTCCTGGAATT
	CTGGCGCCCTGACCAGCGGCGTGCACACC
	TTTCCAGCTGTGCTGCAGTCCAGCGGCCT
	GTACAGCCTGAGCAGCGTCGTGACAGTGC
	CCAGCAGCTCTCTGGGCACCCAGACCTAC
	ATCTGCAACGTGAACCACAAGCCCAGCA
	ACACCAAGGTGGACAAGAAGGTGGAACC
	CAAGAGCTGCGACAAGACCCACACCTGT
	CCCCCTTGTCCTGCCCCCGAAGCCGCCGG
	AGGCCCTTCCGTGTTCCTGTTCCCCCCAA
	AGCCCAAGGACACCCTGATGATCAGCCG
	GACCCCCGAAGTGACCTGCGTGGTGGTGG
	ATGTGTCCCACGAGGACCCTGAAGTGAA
	GTTCAATTGGTACGTGGACGGCGTGGAAG
	TGCACAACGCCAAGACCAAGCCAAGAGA
	GGAACAGTACAACAGCACCTACCGGGTG
	GTGTCCGTGCTGACCGTGCTGCACCAGGA
	CTGGCTGAACGGCAAAGAGTACAAGTGC
	AAGGTGTCCAACAAGGCCCTGGCCGCCCC
	CATCGAGAAAACCATCAGCAAGGCCAAG
	GGCCAGCCCCGCGAACCCCAGGTGTACA
	CACTGCCCCCATGCAGGGACGAGCTGACC
	AAGAACCAGGTGTCCCTGTGGTGTCTGGT
	GAAAGGCTTCTACCCCTCCGATATCGCCG
	TGGAATGGGAGAGCAACGGCCAGCCCGA
	GAACAACTACAAGACCACCCCCCCTGTGC
	TGGACAGCGACGGCTCATTCTTCCTGTAC
	TCCAAGCTGACAGTGGACAAGTCCCGGTG
	GCAGCAGGGCAACGTGTTCAGCTGCTCCG
	TGATGCACGAGGCCCTGCACAACCACTAC
	ACCCAGAAGTCCCTGAGCCTGAGCCCCGGC
mAb2 Light Chain 2	See above.	SEQ ID
(e.g., encoding a fourth		NO: 75
polypeptide chain of a
trispecific binding protein of
the present disclosure)
mAb2xCD28supxCD3mid IgG1 NNSA
CD28supxCD3mid	CAGGTGCAGCTGGTGCAGTCTGGCGCCGA	SEQ ID
IgG1(hole) NNSA Heavy	GGTCGTGAAACCTGGCGCCTCTGTGAAGG	NO: 78
Chain 1	TGTCCTGCAAGGCCAGCGGCTACACCTTT
(e.g., encoding a second	ACCAGCTACTACATCCACTGGGTGCGCCA
polypeptide chain of a	GGCCCCTGGACAGGGACTGGAATGGATC
trispecific binding protein of	GGCAGCATCTACCCCGGCAACGTGAACA
the present disclosure)	CCAACTACGCCCAGAAGTTCCAGGGCAG
	AGCCACCCTGACCGTGGACACCAGCATCA
	GCACCGCCTACATGGAACTGAGCCGGCTG
	AGAAGCGACGACACCGCCGTGTACTACT
	GCACCCGGTCCCACTACGGCCTGGATTGG
	AACTTCGACGTGTGGGGCAAGGGCACCA
	CCGTGACAGTGTCTAGCAGCCAGGTGCAG
	CTGGTGGAATCTGGCGGCGGAGTGGTGC
	AGCCTGGCAGAAGCCTGAGACTGAGCTG
	TGCCGCCAGCGGCTTCACCTTCACCAAGG
	CCTGGATGCACTGGGTGCGCCAGGCCCCT
	GGAAAGCAGCTGGAATGGGTGGCCCAGA
	TCAAGGACAAGAGCAACAGCTACGCCAC
	CTACTACGCCGACAGCGTGAAGGGCCGG
	TTCACCATCAGCCGGGACGACAGCAAGA
	ACACCCTGTACCTGCAGATGAACAGCCTG
	CGGGCCGAGGACACCGCCGTGTACTACTG
	TCGGGGCGTGTACTATGCCCTGAGCCCCT
	TCGATTACTGGGGCCAGGGAACCCTCGTG
	ACCGTGTCTAGTCGGACCGCCAGCACAAA
	GGGCCCCAGCGTGTTCCCTCTGGCCCCTA
	GCAGCAAGAGCACATCTGGCGGAACAGC
	CGCCCTGGGCTGCCTCGTGAAGGACTACT
	TTCCCGAGCCCGTGACCGTGTCCTGGAAT
	TCTGGCGCCCTGACCAGCGGCGTGCACAC
	CTTTCCAGCTGTGCTGCAGTCCAGCGGCC
	TGTACAGCCTGAGCAGCGTCGTGACAGTG
	CCCAGCAGCTCTCTGGGCACCCAGACCTA
	CATCTGCAACGTGAACCACAAGCCCAGC
	AACACCAAGGTGGACAAGAAGGTGGAAC
	CCAAGAGCTGCGACAAGACCCACACCTG
	TCCCCCTTGTCCTGCCCCCGAACTGCTGG
	GAGGCCCTTCCGTGTTCCTGTTCCCCCCA
	AAGCCCAAGGACACCCTGATGATCAGCC
	GGACCCCCGAAGTGACCTGCGTGGTGGTG
	GATGTGTCCCACGAGGACCCTGAAGTGA
	AGTTCAATTGGTACGTGGACGGCGTGGAA
	GTGCACAACGCCAAGACCAAGCCAAGAG
	AGGAACAGTACAACAATGCCTCCCGGGT
	GGTGTCCGTGCTGACCGTGCTGCACCAGG
	ACTGGCTGAACGGCAAAGAGTACAAGTG
	CAAGGTGTCCAACAAGGCCCTGCCTGCCC
	CCATCGAGAAAACCATCAGCAAGGCCAA
	GGGCCAGCCCCGCGAACCCCAGGTGTGC
	ACACTGCCCCCAAGCAGGGACGAGCTGA
	CCAAGAACCAGGTGTCCCTGAGCTGTGCC
	GTGAAAGGCTTCTACCCCTCCGATATCGC
	CGTGGAATGGGAGAGCAACGGCCAGCCC
	GAGAACAACTACAAGACCACCCCCCCTGT
	GCTGGACAGCGACGGCTCATTCTTCCTGG
	TGTCCAAGCTGACAGTGGACAAGTCCCGG
	TGGCAGCAGGGCAACGTGTTCAGCTGCTC
	CGTGATGCACGAGGCCCTGCACAACCACT
	ACACCCAGAAGTCCCTGAGCCTGAGCCCC
	GGC
CD28supxCD3mid Light	See above.	SEQ ID
Chain 1		NO: 73
(e.g., encoding a first
polypeptide chain of a
trispecific binding protein of
the present disclosure)
mAb2 IgG1(knob) NNSA	CAGGTGCAGCTGGTGCAGTCTGGCGCCGA	SEQ ID
Heavy Chain 2	AGTCGTGAAACCTGGCGCCTCCGTGAAGG	NO: 79
(e.g., encoding a third	TGTCCTGCAAGGCCAGCGGCTACACCTTT
polypeptide chain of a	ACCAGCTACGCCATGCACTGGGTCAAAG
trispecific binding protein of	AGGCCCCTGGCCAGAGACTGGAATGGAT
the present disclosure)	CGGCTACATCTACCCCGGCCAGGGCGGCA
	CCAACTACAACCAGAAGTTCCAGGGCAG
	AGCCACCCTGACCGCCGATACAAGCGCC
	AGCACCGCCTACATGGAACTGAGCAGCCT
	GCGGAGCGAGGATACCGCCGTGTACTTCT
	GTGCCAGAACAGGCGGCCTGAGGCGGGC
	CTACTTTACCTATTGGGGCCAGGGCACCC
	TCGTGACCGTGTCTAGCGCTAGCACAAAG
	GGCCCATCGGTCTTCCCCCTGGCACCCTC
	CTCCAAGAGCACCTCTGGGGGCACAGCG
	GCCCTGGGCTGCCTGGTCAAGGACTACTT
	CCCCGAACCGGTGACGGTGTCGTGGAACT
	CAGGCGCCCTGACCAGCGGCGTGCACAC
	CTTCCCGGCTGTCCTACAGTCCTCAGGAC
	TCTACTCCCTCAGCAGCGTGGTGACCGTG
	CCCTCCAGCAGCTTGGGCACCCAGACCTA
	CATCTGCAACGTGAATCACAAGCCCAGCA
	ACACCAAGGTGGACAAGAAAGTTGAGCC
	CAAATCTTGTGACAAAACTCACACATGCC
	CACCGTGCCCAGCACCTGAACTCCTGGGG
	GGACCGTCAGTCTTCCTCTTCCCCCCAAA
	ACCCAAGGACACCCTCATGATCTCCCGGA
	CCCCTGAGGTCACATGCGTGGTGGTGGAC
	GTGAGCCACGAAGACCCTGAGGTCAAGT
	TCAACTGGTATGTTGACGGCGTGGAGGTG
	CATAATGCCAAGACAAAGCCGCGGGAGG
	AGCAGTACAACAATGCCTCCCGTGTGGTC
	AGCGTCCTCACCGTCCTGCACCAGGACTG
	GCTGAATGGCAAGGAGTACAAGTGCAAG
	GTCTCCAACAAAGCCCTCCCAGCCCCCAT
	CGAGAAAACCATCTCCAAAGCCAAAGGG
	CAGCCCCGAGAACCACAGGTGTACACCCT
	GCCCCCATGCCGGGATGAGCTGACCAAG
	AATCAAGTCAGCCTGTGGTGCCTGGTAAA
	AGGCTTCTATCCCAGCGACATCGCCGTGG
	AGTGGGAGAGCAATGGGCAGCCGGAGAA
	CAACTACAAGACCACGCCTCCCGTGCTGG
	ACTCCGACGGCTCCTTCTTCCTCTACTCAA
	AACTCACCGTGGACAAGAGCAGGTGGCA
	GCAGGGGAACGTCTTCTCATGCTCCGTGA
	TGCATGAGGCTCTGCACAACCACTACACG
	CAGAAGAGCCTCTCCCTGTCTCCGGGT
mAb2 Light Chain 2	See above.	SEQ ID
(e.g., encoding a fourth		NO: 75
polypeptide chain of a
trispecific binding protein of
the present disclosure)
mAb6xCD28supxCD3mid IgG4 FALA
CD28supxCD3mid	See above.	SEQ ID
IgG4(hole) FALA Heavy		NO: 72
Chain 1
(e.g., encoding a second
polypeptide chain of a
trispecific binding protein of
the present disclosure)
CD28supxCD3mid Light	See above.	SEQ ID
Chain 1		NO: 73
(e.g., encoding a first
polypeptide chain of a
trispecific binding protein of
the present disclosure)
mAb6 IgG4(knob) FALA	CAGGTGCAGCTGGTGGAAAGCGGCGGAG	SEQ ID
Heavy Chain 2	GCGTGGTGCAGCCTGGCAGGTCTCTGAGA	NO: 80
(e.g., encoding a third	CTGAGCTGTGCCGCCAGCGGCTTCACCTT
polypeptide chain of a	CAGCAGCTACGGAATGCACTGGGTGCGC
trispecific binding protein of	CAGGCCCCTGGCAAAGGACTGGAATGGG
the present disclosure)	TGGCCGTGATTTGGTACGACGGCAGCAAC
	AAGTACTACGCCGACAGCGTGAAGGGCC
	GGTTCACCATCAGCGGCGACAACAGCAA
	GAACACCCTGTACCTGCAGATGAACAGCC
	TGCGGGCCGAGGACACCGCCGTGTACTAC
	TGCGCCAGAATGTTCAGAGGCGCCTTCGA
	CTACTGGGGCCAGGGCACACTCGTGACCG
	TGTCTAGTGCGTCGACCAAGGGCCCATCG
	GTGTTCCCTCTGGCCCCTTGCAGCAGAAG
	CACCAGCGAATCTACAGCCGCCCTGGGCT
	GCCTCGTGAAGGACTACTTTCCCGAGCCC
	GTGACCGTGTCCTGGAACTCTGGCGCTCT
	GACAAGCGGCGTGCACACCTTTCCAGCCG
	TGCTCCAGAGCAGCGGCCTGTACTCTCTG
	AGCAGCGTCGTGACAGTGCCCAGCAGCA
	GCCTGGGCACCAAGACCTACACCTGTAAC
	GTGGACCACAAGCCCAGCAACACCAAGG
	TGGACAAGCGGGTGGAATCTAAGTACGG
	CCCTCCCTGCCCTCCTTGCCCAGCCCCTG
	AAGCTGCCGGCGGACCCTCCGTGTTCCTG
	TTCCCCCCAAAGCCCAAGGACACCCTGAT
	GATCAGCCGGACCCCCGAAGTGACCTGC
	GTGGTGGTGGATGTGTCCCAGGAAGATCC
	CGAGGTGCAGTTCAATTGGTACGTGGACG
	GCGTGGAAGTGCACAACGCCAAGACCAA
	GCCCAGAGAGGAACAGTTCAACAGCACC
	TACCGGGTGGTGTCCGTGCTGACCGTGCT
	GCACCAGGACTGGCTGAACGGCAAAGAG
	TACAAGTGCAAGGTGTCCAACAAGGGCC
	TGCCCAGCTCCATCGAGAAAACCATCAGC
	AAGGCCAAGGGCCAGCCCCGCGAGCCTC
	AAGTGTATACCCTGCCCCCTTGCCAGGAA
	GAGATGACCAAGAACCAGGTGTCCCTGT
	GGTGTCTCGTGAAAGGCTTCTACCCCAGC
	GACATTGCCGTGGAATGGGAGAGCAACG
	GCCAGCCCGAGAACAACTACAAGACCAC
	CCCCCCTGTGCTGGACAGCGACGGCTCAT
	TCTTCCTGTACTCCAAGCTGACCGTGGAC
	AAGAGCCGGTGGCAGGAAGGCAACGTGT
	TCAGCTGCTCCGTGATGCACGAGGCCCTG
	CACAACCACTACACCCAGAAGTCCCTGTC
	TCTGTCCCTGGGC
mAb6 Light2	GCCATCCAGATGACCCAGAGCCCCAGCA	SEQ ID
(e.g., encoding a fourth	GCCTGTCTGCCAGCGTGGGCGACAGAGTG	NO: 81
polypeptide chain of a	ACCATCACCTGTAGAGCCAGCCAGGGCAT
trispecific binding protein of	CCGGAACGACCTGGGCTGGTATCAGCAG
the present disclosure)	AAGCCTGGCAAGGCCCCCAAGCTGCTGAT
	CTACGCCGCTAGCTCTCTGCAGTCCGGCG
	TGCCCAGCAGATTTTCTGGCAGCGGCTCC
	GGCACCGACTTCACCCTGACAATCTCTGG
	CCTGCAGCCCGAGGACAGCGCCACCTACT
	ACTGTCTGCAAGACTACATCTACTACCCC
	ACCTTCGGCCAGGGCACCAAGGTGGAAA
	TCAAGCGTACGGTGGCCGCTCCCAGCGTG
	TTCATCTTCCCACCTAGCGACGAGCAGCT
	GAAGTCCGGCACAGCCTCTGTCGTGTGCC
	TGCTGAACAACTTCTACCCCCGCGAGGCC
	AAAGTGCAGTGGAAGGTGGACAACGCCC
	TGCAGAGCGGCAACAGCCAGGAAAGCGT
	GACCGAGCAGGACAGCAAGGACTCCACC
	TACAGCCTGAGCAGCACCCTGACACTGAG
	CAAGGCCGACTACGAGAAGCACAAGGTG
	TACGCCTGCGAAGTGACCCACCAGGGCCT
	GTCTAGCCCCGTGACCAAGAGCTTCAACC
	GGGGCGAGTGT
mAb6xCD28supxCD3mid IgG1LALA P329A
CD28supxCD3mid	See above.	SEQ ID
IgG1(hole) LALA P329A		NO: 76
Heavy Chain 1
(e.g., encoding a second
polypeptide chain of a
trispecific binding protein of
the present disclosure)
CD28supxCD3mid Light	See above.	SEQ ID
Chain 1		NO: 73
(e.g., encoding a first
polypeptide chain of a
trispecific binding protein of
the present disclosure)
mAb6 IgG1(knob) LALA	CAGGTGCAGCTGGTGGAAAGCGGCGGAG	SEQ ID
P329A Heavy Chain 2	GCGTGGTGCAGCCTGGCAGGTCTCTGAGA	NO: 82
(e.g., encoding a third	CTGAGCTGTGCCGCCAGCGGCTTCACCTT
polypeptide chain of a	CAGCAGCTACGGAATGCACTGGGTGCGC
trispecific binding protein of	CAGGCCCCTGGCAAAGGACTGGAATGGG
the present disclosure)	TGGCCGTGATTTGGTACGACGGCAGCAAC
	AAGTACTACGCCGACAGCGTGAAGGGCC
	GGTTCACCATCAGCGGCGACAACAGCAA
	GAACACCCTGTACCTGCAGATGAACAGCC
	TGCGGGCCGAGGACACCGCCGTGTACTAC
	TGCGCCAGAATGTTCAGAGGCGCCTTCGA
	CTACTGGGGCCAGGGCACACTCGTGACCG
	TGTCTAGTGCGTCGACCAAGGGCCCCAGC
	GTGTTCCCTCTGGCCCCTAGCAGCAAGAG
	CACATCTGGCGGAACAGCCGCCCTGGGCT
	GCCTCGTGAAGGACTACTTTCCCGAGCCC
	GTGACCGTGTCCTGGAATTCTGGCGCCCT
	GACCAGCGGCGTGCACACCTTTCCAGCTG
	TGCTGCAGTCCAGCGGCCTGTACAGCCTG
	AGCAGCGTCGTGACAGTGCCCAGCAGCTC
	TCTGGGCACCCAGACCTACATCTGCAACG
	TGAACCACAAGCCCAGCAACACCAAGGT
	GGACAAGAAGGTGGAACCCAAGAGCTGC
	GACAAGACCCACACCTGTCCCCCTTGTCC
	TGCCCCCGAAGCCGCCGGAGGCCCTTCCG
	TGTTCCTGTTCCCCCCAAAGCCCAAGGAC
	ACCCTGATGATCAGCCGGACCCCCGAAGT
	GACCTGCGTGGTGGTGGATGTGTCCCACG
	AGGACCCTGAAGTGAAGTTCAATTGGTAC
	GTGGACGGCGTGGAAGTGCACAACGCCA
	AGACCAAGCCAAGAGAGGAACAGTACAA
	CAGCACCTACCGGGTGGTGTCCGTGCTGA
	CCGTGCTGCACCAGGACTGGCTGAACGGC
	AAAGAGTACAAGTGCAAGGTGTCCAACA
	AGGCCCTGGCCGCCCCCATCGAGAAAAC
	CATCAGCAAGGCCAAGGGCCAGCCCCGC
	GAACCCCAGGTGTACACACTGCCCCCATG
	CAGGGACGAGCTGACCAAGAACCAGGTG
	TCCCTGTGGTGTCTGGTGAAAGGCTTCTA
	CCCCTCCGATATCGCCGTGGAATGGGAGA
	GCAACGGCCAGCCCGAGAACAACTACAA
	GACCACCCCCCCTGTGCTGGACAGCGACG
	GCTCATTCTTCCTGTACTCCAAGCTGACA
	GTGGACAAGTCCCGGTGGCAGCAGGGCA
	ACGTGTTCAGCTGCTCCGTGATGCACGAG
	GCCCTGCACAACCACTACACCCAGAAGTC
	CCTGAGCCTGAGCCCCGGC
mAb6 Light2	See above.	SEQ ID
(e.g., encoding a fourth		NO: 81
polypeptide chain of a
trispecific binding protein of
the present disclosure)
mAb6xCD28supxCD3mid IgG1 NNSA
CD28supxCD3mid	See above.	SEQ ID
IgG1(hole) NNSA Heavy		NO: 78
Chain 1
(e.g., encoding a second
polypeptide chain of a
trispecific binding protein of
the present disclosure)
CD28supxCD3mid Light	See above.	SEQ ID
Chain 1		NO: 73
(e.g., encoding a first
polypeptide chain of a
trispecific binding protein of
the present disclosure)
mAb6 IgG1(knob) NNSA	CAGGTGCAGCTGGTGGAAAGCGGCGGAG	SEQ ID
Heavy Chain 2	GCGTGGTGCAGCCTGGCAGGTCTCTGAGA	NO: 83
(e.g., encoding a third	CTGAGCTGTGCCGCCAGCGGCTTCACCTT
polypeptide chain of a	CAGCAGCTACGGAATGCACTGGGTGCGC
trispecific binding protein of	CAGGCCCCTGGCAAAGGACTGGAATGGG
the present disclosure)	TGGCCGTGATTTGGTACGACGGCAGCAAC
	AAGTACTACGCCGACAGCGTGAAGGGCC
	GGTTCACCATCAGCGGCGACAACAGCAA
	GAACACCCTGTACCTGCAGATGAACAGCC
	TGCGGGCCGAGGACACCGCCGTGTACTAC
	TGCGCCAGAATGTTCAGAGGCGCCTTCGA
	CTACTGGGGCCAGGGCACACTCGTGACCG
	TGTCTAGTGCGTCGACCAAGGGCCCATCG
	GTCTTCCCCCTGGCACCCTCCTCCAAGAG
	CACCTCTGGGGGCACAGCGGCCCTGGGCT
	GCCTGGTCAAGGACTACTTCCCCGAACCG
	GTGACGGTGTCGTGGAACTCAGGCGCCCT
	GACCAGCGGCGTGCACACCTTCCCGGCTG
	TCCTACAGTCCTCAGGACTCTACTCCCTC
	AGCAGCGTGGTGACCGTGCCCTCCAGCAG
	CTTGGGCACCCAGACCTACATCTGCAACG
	TGAATCACAAGCCCAGCAACACCAAGGT
	GGACAAGAAAGTTGAGCCCAAATCTTGT
	GACAAAACTCACACATGCCCACCGTGCCC
	AGCACCTGAACTCCTGGGGGGACCGTCA
	GTCTTCCTCTTCCCCCCAAAACCCAAGGA
	CACCCTCATGATCTCCCGGACCCCTGAGG
	TCACATGCGTGGTGGTGGACGTGAGCCAC
	GAAGACCCTGAGGTCAAGTTCAACTGGTA
	TGTTGACGGCGTGGAGGTGCATAATGCCA
	AGACAAAGCCGCGGGAGGAGCAGTACAA
	CAATGCCTCCCGTGTGGTCAGCGTCCTCA
	CCGTCCTGCACCAGGACTGGCTGAATGGC
	AAGGAGTACAAGTGCAAGGTCTCCAACA
	AAGCCCTCCCAGCCCCCATCGAGAAAACC
	ATCTCCAAAGCCAAAGGGCAGCCCCGAG
	AACCACAGGTGTACACCCTGCCCCCATGC
	CGGGATGAGCTGACCAAGAATCAAGTCA
	GCCTGTGGTGCCTGGTAAAAGGCTTCTAT
	CCCAGCGACATCGCCGTGGAGTGGGAGA
	GCAATGGGCAGCCGGAGAACAACTACAA
	GACCACGCCTCCCGTGCTGGACTCCGACG
	GCTCCTTCTTCCTCTACTCAAAACTCACCG
	TGGACAAGAGCAGGTGGCAGCAGGGGAA
	CGTCTTCTCATGCTCCGTGATGCATGAGG
	CTCTGCACAACCACTACACGCAGAAGAG
	CCTCTCCCTGTCTCCGGGT
mAb6 Light2	See above.	SEQ ID
(e.g., encoding a fourth		NO: 81
polypeptide chain of a
trispecific binding protein of
the present disclosure)

[0218] CD38 Polypeptides

[0219] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site that binds an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide. Exemplary assays for determining whether an antigen binding site binds an antigen are described herein and known in the art. In some embodiments, binding is determined by ELISA assay, e.g., as described infra. In some embodiments, binding is determined by SPR assay, e.g., as described infra. In some embodiments, binding is determined by flow cytometry assay using cells expressing a CD38 polypeptide on their cell surface, e.g., as described infra. See, e.g., Examples 1, 3, and 4.

[0220] In some embodiments, a binding protein of the present disclosure binds a purified polypeptide or fragment thereof comprising the amino acid sequence of SEQ ID NO:1 and/or 30 (e.g., as measured by ELISA or SPR). In some embodiments, a binding protein of the present disclosure binds a polypeptide or comprising the amino acid sequence of SEQ ID NO:1 and/or 30 when expressed on the surface of a cell (e.g., as measured by flow cytometry).

[0221] In some embodiments, a binding protein of the present disclosure binds to a CD38 isoform A polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1). In some embodiments, a binding protein of the present disclosure binds to a CD38 isoform E polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:105 and not comprising the full amino acid sequence of SEQ ID NO:1, consisting of the amino acid sequence of SEQ ID NO:105, or consisting essentially of the amino acid sequence of SEQ ID NO:105). In some embodiments, a binding protein of the present disclosure binds to a CD38 isoform A polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) and a CD38 isoform E polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:105 and not comprising the full amino acid sequence of SEQ ID NO:1, consisting of the amino acid sequence of SEQ ID NO:105, or consisting essentially of the amino acid sequence of SEQ ID NO:105). Without wishing to be bound to theory, it is thought that binding to a CD38 isoform E polypeptide can be advantageous, e.g., in targeting a binding protein of the present disclosure to cell(s) expressing a CD38 isoform E polypeptide.

Human CD38 isoform A extracellular domain

polypeptide sequence

(SEQ ID NO: 1)

RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAF

ISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDM

FTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKT

VSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEA

WVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVK

NPEDSSCTSEI

Human CD38 isoform E polypeptide sequence

(SEQ ID NO: 105)

RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAF

ISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDM

FTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKT

VSRRHFWECGSP

[0222] In some embodiments, the extracellular domain of a human CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the extracellular domain of a cynomolgus monkey CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:30.

Cynomolgus monkey CD38 polypeptide sequence

(SEQ ID NO: 30)

RWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQSVWDAFKGAF

ISKYPCNITEEDYQPLVKLGTQTVPCNKTLLWSRIKDLAHQFTQVQRDM

FTLEDMLLGYLADDLTWCGEFNTFEINYQSCPDWRKDCSNNPVSVFWKT

VSRRFAETACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQALEA

WVIHGGREDSRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKFLQCVK

NPEDSSCLSGI

[0223] Multispecific (e.g., Bispecific, Trispecific, or Multispecific) Binding Proteins that Bind CD38 Polypeptides

[0224] In some embodiments, a binding protein of the present disclosure is a bispecific binding protein comprising an antigen binding site that binds one or more CD38 polypeptides and a second antigen binding site that binds a different target antigen. In some embodiments, a binding protein of the present disclosure is a bispecific binding protein comprising an antigen binding site that binds one or more CD38 polypeptides and a second antigen binding site that binds one or more CD38 polypeptides.

[0225] In some embodiments, a binding protein of the present disclosure is a trispecific binding protein comprising an antigen binding site that binds one or more CD38 polypeptides, a second antigen binding site, and a third antigen binding site. For example, in some embodiments, one of the antigen binding sites binds one or more CD38 polypeptide(s) (e.g., the extracellular domain of a human and/or cynomolgus monkey CD38 polypeptide), and one or two of the antigen binding sites binds a T-cell surface protein. In some embodiments, one of the antigen binding sites binds one or more CD38 polypeptide(s) (e.g., the extracellular domain of a human and/or cynomolgus monkey CD38 polypeptide), one of the antigen binding sites binds a human CD3 polypeptide, and one of the antigen binding sites binds a human CD28 polypeptide. Human CD3 and CD28 polypeptides are known in the art. The amino acid sequences of exemplary and non-limiting antibody variable domains that bind human CD3 and CD28 polypeptides are provided herein.

[0226] In some embodiments, provided herein are binding proteins comprising three antigen binding sites that each bind one or more target proteins. In some embodiments, at least one of the three antigen binding sites binds an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide. In some embodiments, the human CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:1, and/or the cynomolgus monkey CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:30. In some embodiments, the binding protein comprises an antigen binding site that binds 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 (e.g., a human CD28 polypeptide and/or a human CD3 polypeptide).

[0227] In some embodiments, a binding protein of the present disclosure binds one or more tumor target proteins (e.g., one or more CD38 polypeptide(s)) and one or more T cell target proteins. In some embodiments, the binding protein is capable of binding one tumor target protein (e.g., one or more CD38 polypeptide(s)) and two different epitopes on a single T cell target protein. In some embodiments, the binding protein is capable of binding one tumor target protein (e.g., one or more CD38 polypeptide(s)) and two different T cell target proteins (e.g., CD28 and CD3). In some embodiments, the binding protein is capable of binding one T cell target protein and two different epitopes on a single tumor target protein (e.g., one or more CD38 polypeptide(s)). In some embodiments, the binding protein is capable of binding one T cell target protein and two different tumor target proteins (e.g., one or more CD38 polypeptide(s) and another tumor target protein). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen binding sites that target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that binds one or more CD38 polypeptide(s). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen binding sites that target two tumor target proteins (e.g., one or more CD38 polypeptide(s) and another tumor target protein), and the third and fourth polypeptide chains of the binding protein form an antigen binding site that binds a T cell target protein. In some embodiments, the one or more T cell target proteins are one or more of CD3 and CD28.

[0228] In some embodiments, the binding proteins specifically bind to one or more CD38 polypeptide(s) and one or more target protein on a T-cell including a T cell receptor complex. These T-cell engager binding proteins are capable of recruiting T cells transiently to target cells and, at the same time, activating the cytolytic activity of the T cells. Examples of target proteins on T cells include but are not limited to CD3 and CD28, among others. Further examples of such antigen targets or target proteins are provided supra. In some embodiments, the trispecific binding proteins may be generated by combining the antigen binding domains of two or more monospecific antibodies (parent antibodies) into one antibody.

[0229] Bispecific Binding Protein Formats

[0230] In some embodiments, the binding protein of the disclosure is a bispecific and/or bivalent binding protein comprising four polypeptide chains that form four antigen binding sites that bind one or more (e.g., two) different antigen targets or target proteins (e.g., having a structure described in International Publication No. WO2012/135345). In some embodiments, the binding protein is bivalent and/or bispecific. In some embodiments, the binding protein is tetravalent and/or tetraspecific. In some embodiments, the binding protein is tetravalent and/or bispecific. In some embodiments, at least one of the antigen binding sites binds a CD38 polypeptide (e.g., the extracellular domain of human and/or cynomolgus monkey CD38 polypeptides).

[0231] In some embodiments, the binding protein comprises two polypeptide chains having a structure represented by the formula:

V_L1-L₁-V_L2-L₂-C_L  [I]

and two polypeptide chains have a structure represented by the formula:

V_H2-L₃-V_H1-L₄-C_H1-Fc  [II]

wherein:

• [0232] V_L1 is a first immunoglobulin light chain variable domain;
• [0233] V_L2 is a second immunoglobulin light chain variable domain;
• [0234] V_H1 is a first immunoglobulin heavy chain variable domain;
• [0235] V_H2 is a second immunoglobulin heavy chain variable domain;
• [0236] C_L is an immunoglobulin light chain constant domain;
• [0237] C_H1 is the immunoglobulin C_H1 heavy chain constant domain;
• [0238] Fc comprises an immunoglobulin hinge region and C_H2, C_H3 immunoglobulin heavy chain constant domains;
• [0239] L₁, L₂, L₃, and L₄ are amino acid linkers;
• [0240] and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair. In some embodiments, V_H1 and V_L1 form an antigen binding domain that binds a CD38 polypeptide, and V_H2 and V_L2 form an antigen binding domain that binds another antigen target. In some embodiments, V_H2 and V_L2 form an antigen binding domain that binds a CD38 polypeptide, and V_H1 and V_L1 form an antigen binding domain that binds another antigen target.

[0241] In some embodiments, the binding protein comprises two polypeptide chains that form two antigen binding sites, wherein a first polypeptide chain comprises

V_L1-L₁-V_L2-L₂-CL-Fc

and a second polypeptide chain comprises

V_H2-L₃-V_H1-L₄-C_H1-Fc

wherein:

[0242] V_L1 is a first immunoglobulin light chain variable domain;

[0243] V_L2 is a second immunoglobulin light chain variable domain;

[0244] V_H1 is a first immunoglobulin heavy chain variable domain;

[0245] V_H2 is a second immunoglobulin heavy chain variable domain;

[0246] CL is an immunoglobulin light chain constant domain;

[0247] C_H1 is the immunoglobulin CH1 heavy chain constant domain;

[0248] C_H2 is an immunoglobulin C_H2 heavy chain constant domain;

[0249] C_H3 is an immunoglobulin C_H3 heavy chain constant domain;

[0250] Fc comprises an immunoglobulin hinge region and C_H2, C_H3 immunoglobulin heavy chain constant domains; and

[0251] L₁, L₂, L₃, and L₄ are amino acid linkers;

• • [0252] wherein the first and second polypeptides form a cross-over light chain-heavy chain pair. In some embodiments, V_H1 and V_L1 form an antigen binding domain that binds a CD38 polypeptide, and V_H2 and V_L2 form an antigen binding domain that binds another antigen target. In some embodiments, V_H2 and V_L2 form an antigen binding domain that binds a CD38 polypeptide, and V_H1 and V_L1 form an antigen binding domain that binds another antigen target.

[0253] In some embodiments, the binding protein comprises three polypeptide chains that form two antigen binding sites, wherein a first polypeptide chain comprises

V_L1-L₁-V_L2-L₂-CL

a second polypeptide chain comprises

V_H2-L₃-V_H1-L₄-C_H1-Fc

a third polypeptide chain comprises an antibody Fc region
wherein:

[0254] V_L1 is a first immunoglobulin light chain variable domain;

[0255] V_L2 is a second immunoglobulin light chain variable domain;

[0256] V_H1 is a first immunoglobulin heavy chain variable domain;

[0257] V_H2 is a second immunoglobulin heavy chain variable domain;

[0258] CL is an immunoglobulin light chain constant domain;

[0259] C_H1 is the immunoglobulin CH1 heavy chain constant domain;

[0260] C_H2 is an immunoglobulin C_H2 heavy chain constant domain;

[0261] C_H3 is an immunoglobulin C_H3 heavy chain constant domain;

[0262] Fc comprises an immunoglobulin hinge region and C_H2, C_H3 immunoglobulin heavy chain constant domains; and

[0263] L₁, L₂, L₃, and L₄ are amino acid linkers;

[0264] wherein the first and second polypeptides form a cross-over light chain-heavy chain pair. In some embodiments, V_H1 and V_L1 form an antigen binding domain that binds a CD38 polypeptide, and V_H2 and V_L2 form an antigen binding domain that binds another antigen target. In some embodiments, V_H2 and V_L2 form an antigen binding domain that binds a CD38 polypeptide, and V_H1 and V_L1 form an antigen binding domain that binds another antigen target.

[0265] In some embodiments, the binding protein comprises a first polypeptide chain comprising a structure represented by the formula:

V_L1-L₁-V_L2-L₂-C_L  [I]

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

V_H2-L₃-V_H1-1₄-C_H1  [II]

wherein:

[0266] V_L1 is a first immunoglobulin light chain variable domain;

[0267] V_L2 is a second immunoglobulin light chain variable domain;

[0268] V_H1 is a first immunoglobulin heavy chain variable domain;

[0269] V_H2 is a second immunoglobulin heavy chain variable domain;

[0270] C_L is an immunoglobulin light chain constant domain;

[0271] C_H1 is an immunoglobulin C_H1 heavy chain constant domain; and

[0272] L₁, L₂, L₃ and L₄ are amino acid linkers;

• • [0273] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair. In some embodiments, V_H1 and V_L1 form an antigen binding domain that binds a CD38 polypeptide, and V_H2 and V_L2 form an antigen binding domain that binds another antigen target. In some embodiments, V_H2 and V_L2 form an antigen binding domain that binds a CD38 polypeptide, and V_H1 and V_L1 form an antigen binding domain that binds another antigen target.

[0274] In any of the bispecific binding proteins described supra, the target antigen other than CD38 can be any of the following exemplary antigen targets: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, a receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPRS/CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

[0275] In any of the bispecific binding proteins described supra, any linker or combination of linkers described herein may be used. For example, in some embodiments, at least one of L₁, L₂, L₃ or L₄ is independently 0 amino acids in length. In some embodiments, L₁, L₂, L₃ or L₄ are each independently at least one amino acid in length. In some embodiments, L₁, 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). In some embodiments, L₁, L₂, L₃ and L₄ each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L₁ comprises the sequence GQPKAAP (SEQ ID NO: 58), L₂ comprises the sequence TKGPS (SEQ ID NO:57), L₃ comprises the sequence S, and L₄ comprises the sequence RT. In some embodiments, L₁ comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₂ comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₃ is 0 amino acids in length, and L₄ is 0 amino acids in length. In some embodiments, L₁ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₂ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₃ is 0 amino acids in length, and L₄ is 0 amino acids in length. In some embodiments, L₁ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L₂ is 0 amino acids in length, L₃ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), and L₄ is 0 amino acids in length.

[0276] Trispecific Binding Proteins that Bind to CD38 Polypeptides

[0277] In some embodiments, the binding protein of the disclosure is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one of the antigen binding sites binds a CD38 polypeptide (e.g., the extracellular domain of human and/or cynomolgus monkey CD38 polypeptides). In some embodiments, a first polypeptide chain comprises a structure represented by the formula:

V_L2-L₁-V_L1-L₂-C_L  [I]

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

V_H1-L₃-V_H2-L₄-C_H1-hinge-C_H2-C_H3  [II]

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

V_H3-C_H1-hinge-C_H2-C_H3  [III]

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

V_L3-C_L  [IV]

wherein:

[0278] V_L1 is a first immunoglobulin light chain variable domain;

[0279] V_L2 is a second immunoglobulin light chain variable domain;

[0280] V_L3 is a third immunoglobulin light chain variable domain;

[0281] V_H1 is a first immunoglobulin heavy chain variable domain;

[0282] V_H2 is a second immunoglobulin heavy chain variable domain;

[0283] V_H3 is a third immunoglobulin heavy chain variable domain;

[0284] C_L is an immunoglobulin light chain constant domain;

[0285] C_H1 is an immunoglobulin C_H1 heavy chain constant domain;

[0286] C_H2 is an immunoglobulin C_H2 heavy chain constant domain;

[0287] C_H3 is an immunoglobulin C_H3 heavy chain constant domain;

[0288] hinge is an immunoglobulin hinge region connecting the C_H1 and C_H2 domains; and

[0289] L₁, L₂, L₃ and L₄ are amino acid linkers;

[0290] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair.

[0291] In some embodiments, the binding protein of the disclosure is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one of the antigen binding sites binds a CD38 polypeptide (e.g., the extracellular domain of human and/or cynomolgus monkey CD38 polypeptides). In some embodiments, a first polypeptide chain comprises a structure represented by the formula:

V_L2-L₁-V_L1-L₂-C_L  [I]

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

V_H1-L₃-V_H2-L₄-C_H1  [II]

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

V_H3-C_H1  [III]

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

V_L3-C_L  [IV]

wherein:

[0292] V_L1 is a first immunoglobulin light chain variable domain;

[0293] V_L2 is a second immunoglobulin light chain variable domain;

[0294] V_L3 is a third immunoglobulin light chain variable domain;

[0295] V_H1 is a first immunoglobulin heavy chain variable domain;

[0296] V_H2 is a second immunoglobulin heavy chain variable domain;

[0297] V_H3 is a third immunoglobulin heavy chain variable domain;

[0298] C_L is an immunoglobulin light chain constant domain;

[0299] C_H1 is an immunoglobulin C_H1 heavy chain constant domain; and

[0300] L₁, L₂, L₃ and L₄ are amino acid linkers;

• [0301] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair. In some embodiments, the second and the third polypeptide chain further comprise an Fc region linked to C_H1, the Fc regions comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains.

[0302] In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross-over orientation that forms two distinct antigen binding sites. In some embodiments, the VH1 and VL1 form a binding pair and form the first antigen binding site. In some embodiments, the VH2 and VL2 form a binding pair and form the second antigen binding site. In some embodiments, the first antigen binding site binds a CD3 polypeptide (e.g., human CD3), and the second antigen binding site binds a CD28 polypeptide (e.g., human CD28). In some embodiments, the second antigen binding site binds a CD3 polypeptide (e.g., human CD3), and the first antigen binding site binds a CD28 polypeptide (e.g., human CD28). In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen binding site. In some embodiments, the VH3 and VL3 form a binding pair and form the third antigen binding site. In some embodiments, the third antigen binding site binds a CD38 polypeptide (e.g., human and optionally cynomolgus monkey CD38). Exemplary binding protein formats with cross-over orientations contemplated for use herein are also described in U.S. patent application Ser. No. 15/487,243 and International Application No. PCT/US2017/027488.

[0303] In some embodiments in any of the bispecific, trispecific, or multispecific binding proteins described herein, an antigen binding site binds a CD38 polypeptide (e.g., human and optionally cynomolgus monkey CD38). In some embodiments, other (e.g., not binding to CD38) antigen binding site(s) of any of the bispecific, trispecific, or multispecific binding proteins described herein bind to CD28 or CD3. In some embodiments, the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36).

[0304] In some embodiments, the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and/or the V_L1 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 V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and/or the V_L2 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 V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and/or the V_L3 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).

[0305] In some embodiments, the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the V_L1 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 V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the V_L2 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 V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and the V_L3 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).

[0306] In some embodiments, the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or the V_L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or the V_L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or the V_L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or the V_L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or the V_L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or the V_L3 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).

[0307] In some embodiments, the V_H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H1 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 V_L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H2 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 V_L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and the V_L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H3 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 V_L3 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).

[0308] In some embodiments, the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or the V_L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO:36). In some embodiments, the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO:33), and/or the V_L3 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).

[0309] In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:5, or the V_L3 domain comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:17, or the V_L3 domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:21, or the V_L3 domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:23, or the V_L3 domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:13, or the V_L3 domain comprises the amino acid sequence of SEQ ID NO:14.

[0310] In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:5, and/or the V_L3 domain comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:17, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:21, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:23, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:13, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:14.

[0311] In some embodiments, the V_H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:43), and/or the V_L3 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).

[0312] In some embodiments, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:9, and/or the V_L3 domain comprises the amino acid sequence of SEQ ID NO:10.

[0313] In some embodiments of any of the trispecific binding proteins of the present disclosure, one antigen binding domain binds to a CD3 polypeptide (e.g., human CD3) and one antigen binding domain binds to a CD28 polypeptide (e.g., human CD28). In some embodiments, the V_H1 domain comprises three CDRs from SEQ ID NOs:49 or 51 as shown in Table H, and the V_L1 domain comprises three CDRs from SEQ ID NOs:50 or 52 as shown in Table H. In some embodiments, the V_H2 domain comprises three CDRs from SEQ ID NOs:49 or 51 as shown in Table H, and the V_L2 domain comprises three CDRs from SEQ ID NOs:50 or 52 as shown in Table H. In some embodiments, the V_H1 domain comprises three CDRs from SEQ ID NOs:53 or 84 as shown in Table H, and the V_L1 domain comprises three CDRs from SEQ ID NOs:54 or 85 as shown in Table H. In some embodiments, the V_H2 domain comprises three CDRs from SEQ ID NOs:53 or 84 as shown in Table H, and the V_L2 domain comprises three CDRs from SEQ ID NOs:54 or 85 as shown in Table H.

[0314] In some embodiments, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:49, the V_L1 domain comprises the amino acid sequence of SEQ ID NO:50, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:53, and the V_L2 domain comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:49, the V_L2 domain comprises the amino acid sequence of SEQ ID NO:50, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:53, and the V_L1 domain comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:51, the V_L1 domain comprises the amino acid sequence of SEQ ID NO:52, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:53, and the V_L2 domain comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:51, the V_L2 domain comprises the amino acid sequence of SEQ ID NO:52, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:53, and the V_L1 domain comprises the amino acid sequence of SEQ ID NO:54.

[0315] In some embodiments, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:49, the V_L1 domain comprises the amino acid sequence of SEQ ID NO:50, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:53, the V_L2 domain comprises the amino acid sequence of SEQ ID NO:54, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:13, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the V_H1 domain comprises the amino acid sequence of SEQ ID NO:49, the V_L1 domain comprises the amino acid sequence of SEQ ID NO:50, the V_H2 domain comprises the amino acid sequence of SEQ ID NO:53, the V_L2 domain comprises the amino acid sequence of SEQ ID NO:54, the V_H3 domain comprises the amino acid sequence of SEQ ID NO:9, and the V_L3 domain comprises the amino acid sequence of SEQ ID NO:10.

[0316] In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:60, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:62, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:64, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:65, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:66, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:60, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:68, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:64, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:70, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69. In certain embodiments, the first polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:66, the third polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain comprises a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69.

[0317] In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:62, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:65, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:63. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:60, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:64, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:70, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69. In certain embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:69.

[0318] In any of the trispecific binding proteins described supra, the target antigen other than CD38 can be any of the following exemplary antigen targets: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, a receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPRS/CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

[0319] In some embodiments, a binding protein of the present disclosure is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody.

[0320] The binding proteins of the disclosure may be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.

[0321] Linkers

[0322] In some embodiments, the linkers L₁, L₂, L₃ and L₄ range from no amino acids (length=0) to about 100 amino acids long, or less than 100, 50, 40, 30, 20, or 15 amino acids or less. The linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids long. L₁, L₂, L₃ and L₄ in one binding protein may all have the same amino acid sequence or may all have different amino acid sequences.

[0323] Examples of suitable linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues; a peptide with five glycine residues; a peptide with six glycine residues; a peptide with seven glycine residues; and a peptide with eight glycine residues. Other combinations of amino acid residues may be used such as the peptide GGGGSGGGGS (SEQ ID NO: 55), the peptide GGGGSGGGGSGGGGS (SEQ ID NO: 56), the peptide TKGPS (SEQ ID NO: 57), the peptide GQPKAAP (SEQ ID NO:58), and the peptide GGSGSSGSGG (SEQ ID NO:59). The examples listed above are not intended to limit the scope of the disclosure in any way, and linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable in the binding proteins. For additional descriptions of linker sequences, see, e.g., WO2012135345 and International Application No. PCT/US2017/027488.

[0324] The identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element necessary to achieve in the linker. For example, glycine, serine, and alanine are best for linkers having maximum flexibility. Some combination of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is necessary. Any amino acid residue may be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as necessary depending on the desired properties.

[0325] In some embodiments, at least one of L₁, L₂, L₃ or L₄ is independently 0 amino acids in length. In some embodiments, L₁, L₂, L₃ or L₄ are each independently at least one amino acid in length. In some embodiments, the length of L₁ is at least twice the length of L₃. In some embodiments, the length of L₂ is at least twice the length of L₄. In some embodiments, the length of L₁ is at least twice the length of L₃, and the length of L₂ is at least twice the length of L₄. In some embodiments, L₁ is 3 to 12 amino acid residues in length, L₂ is 3 to 14 amino acid residues in length, L₃ is 1 to 8 amino acid residues in length, and L₄ is 1 to 3 amino acid residues in length. In some embodiments, L₁ is 5 to 10 amino acid residues in length, L₂ is 5 to 8 amino acid residues in length, L₃ is 1 to 5 amino acid residues in length, and L₄ is 1 to 2 amino acid residues in length. In some embodiments, L₁ is 7 amino acid residues in length, L₂ is 5 amino acid residues in length, L₃ is 1 amino acid residue in length, and L₄ is 2 amino acid residues in length. In some embodiments, L₁ is 10 amino acid residues in length, L₂ is 10 amino acid residues in length, L₃ is 0 amino acid residue in length, and L₄ is 0 amino acid residues in length. In some embodiments, L₁, L₂, L₃, and L₄ each have an independently selected length from 0 to 15 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids), wherein at least two of the linkers have a length of 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). In some embodiments, L₁, L₂, L₃, and L₄ are each 0 amino acids in length.

[0326] In some embodiments, L₁, L₂, L₃, and/or L₄ comprise a sequence derived from a naturally occurring sequence at the junction between an antibody variable domain and an antibody constant domain (e.g., as described in WO2012/135345). For example, in some embodiments, the linker comprises a sequence found at the transition between an endogenous V_H and C_H1 domain, or between an endogenous V_L and C_L domain (e.g., kappa or lambda). In some embodiments, the linker comprises a sequence found at the transition between an endogenous human V_H and C_H1 domain, or between an endogenous human V_L and C_L domain (e.g., human kappa or lambda).

[0327] In some embodiments, L₁, 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). In some embodiments, L₁, L₂, L₃ and L₄ each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59).

[0328] In some embodiments, L₁ comprises the sequence GQPKAAP (SEQ ID NO: 58), L₂ comprises the sequence TKGPS (SEQ ID NO:57), L₃ comprises the sequence S, and L₄ comprises the sequence RT. In some embodiments, L₁ comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₂ comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₃ is 0 amino acids in length, and L₄ is 0 amino acids in length. In some embodiments, L₁ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₂ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₃ is 0 amino acids in length, and L₄ is 0 amino acids in length. In some embodiments, L₁ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L₂ is 0 amino acids in length, L₃ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), and L₄ is 0 amino acids in length.

[0329] Fc Regions and Constant Domains

[0330] In some embodiments, a binding protein of the present disclosure comprises a full-length antibody heavy chain or a polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region includes an antibody hinge, C_H1, C_H2, C_H3, and optionally C_H4 domains. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region includes one or more of the mutations described infra.

[0331] In some embodiments, a binding protein of the present disclosure includes one or two Fc variants. The term “Fc variant” as used herein refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art. Thus, the term “Fc variant” can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0332] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and/or effector function of the Fc region (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC)).

[0333] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.

[0334] In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and/or FcγII binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and/or FcγII binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228 and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S228P and/or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 234 and/or 235 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are F234A and/or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A, and/or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and/or R409K.

[0335] In some embodiments, a binding protein of the present disclosure comprises one or more mutations to improve purification, e.g., by modulating the affinity for a purification reagent. For example, it is known that heterodimeric binding proteins can be selectively purified away from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains mutation(s) that reduce or eliminate binding to Protein A, because the heterodimeric form will have an intermediate affinity for Protein A-based purification than either homodimeric form and can be selectively eluted from Protein A, e.g., by use of a different pH (See e.g., Smith, E. J. et al. (2015) Sci. Rep. 5:17943). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C_H1, the first Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C_H1, the second Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains; and wherein only one of the first and the second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve purification. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0336] To improve the yields of some binding proteins (e.g., bispecific or trispecific binding proteins), the C_H3 domains can be altered by the “knob-into-holes” technology which is described in detail with several examples in, for example, International Publication No. WO 96/027011, Ridgway et al., 1996, Protein Eng. 9: 617-21; and Merchant et al., 1998, Nat. Biotechnol. 16: 677-81. Specifically, the interaction surfaces of the two C_H3 domains are altered to increase the heterodimerisation of both heavy chains containing these two C_H3 domains. Each of the two C_H3 domains (of the two heavy chains) can be the “knob,” while the other is the “hole.” The introduction of a disulfide bridge further stabilizes the heterodimers (Merchant et al., 1998; Atwell et al., 1997, J. Mol. Biol. 270: 26-35) and increases the yield. In particular embodiments, the knob is on the second pair of polypeptides with a single variable domain. In other embodiments, the knob is on the first pair of polypeptides having the cross-over orientation. In yet other embodiments, the C_H3 domains do not include a knob in hole.

[0337] In some embodiments, a binding protein of the present disclosure (e.g., a trispecific binding protein) comprises a “knob” mutation on the second polypeptide chain and a “hole” mutation on the third polypeptide chain. In some embodiments, a binding protein of the present disclosure comprises a “knob” mutation on the third polypeptide chain and a “hole” mutation on the second polypeptide chain. In some embodiments, the “knob” mutation comprises substitution(s) at positions corresponding to positions 354 and/or 366 of human IgG1 or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the “knob” mutation comprises substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S354C and T366W. In some embodiments, the “hole” mutation comprises substitution(s) at positions corresponding to positions 407 and, optionally, 349, 366, and/or 368 and of human IgG1 or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A. In some embodiments, the “hole” mutation comprises substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

[0338] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366, and/or 368 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A.

[0339] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366, and/or 368 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C.

[0340] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution at position corresponding to position 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V.

[0341] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution at position corresponding to position 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitution is T366W.

[0342] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0343] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K.

[0344] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W.

[0345] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K.

[0346] In some embodiments, a binding protein of the present disclosure comprises one or more mutations to improve serum half-life (See e.g., Hinton, P. R. et al. (2006) J. Immunol. 176(1):346-56). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first and/or second Fc regions comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve serum half-life. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0347] In some embodiments, a binding protein of the present disclosure comprises one or more mutations to improve stability, e.g., of the hinge region and/or dimer interface of IgG4 (See e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutation comprises 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 binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C_H1, the first Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C_H1, the second Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and the second Fc regions 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, a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve stability. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0348] In some embodiments, a binding protein of the present disclosure comprises one or more mutations to improve purification, e.g., by modulating the affinity for a purification reagent. For example, it is known that heterodimeric binding proteins can be selectively purified away from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains mutation(s) that reduce or eliminate binding to Protein A, because the heterodimeric form will have an intermediate affinity for Protein A-based purification than either homodimeric form and can be selectively eluted from Protein A, e.g., by use of a different pH (See e.g., Smith, E. J. et al. (2015) Sci. Rep. 5:17943). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C_H1, the first Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C_H1, the second Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains; and wherein only one of the first and the second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve purification. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0349] In some embodiments, a binding protein of the present disclosure comprises one or more mutations to improve serum half-life (See e.g., Hinton, P. R. et al. (2006) J. Immunol. 176(1):346-56). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first and/or second Fc regions comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve serum half-life. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[0350] In some embodiments, a binding protein of the present disclosure comprises one or more mutations to reduce effector function, e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain further comprises a first Fc region linked to C_H1, the first Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to C_H1, the second Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to C_H1, the first Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to C_H1, the second Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgG4 Fc regions, and the Fc regions 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 binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C_H1, the first Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C_H1, the second Fc region comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains; and wherein the first and the second Fc regions 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.

[0351] In some embodiments, a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to reduce effector function. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions. For further description of Fc mutations at position 329, see, e.g., Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604 and WO1999051642.

[0352] In some embodiments, the types of mutations described supra can be combined in any order or combination. For example, a binding protein of the present disclosure can comprise two or more of the “knob” and “hole” mutations, the one or more mutations to improve serum half-life, the one or more mutations to improve IgG4 stability, the one or more mutations to improve purification, and/or the one or more mutations to reduce effector function described supra.

[0353] In some embodiments, a binding protein of the present disclosure comprises an antibody fragment, including but not limited to antibody F(ab), F(ab′)2, Fab′-SH, Fv, or scFv fragments.

[0354] Assays

[0355] The present disclosure provides antigen binding proteins that bind human and/or cynomolgus CD38 polypeptides, induce T cell (e.g., CD4+ and/or CD8+ T cell) proliferation, and/or induce apoptosis of CD38+ cells. Exemplary assays for measuring these parameters and identifying such binding proteins are provided herein. For example, in some embodiments, binding affinity between a binding protein or antigen-binding fragment thereof and a purified CD38 polypeptide is measured by SPR (e.g., as described infra), and binding affinity between a binding protein or antigen-binding fragment thereof and a CD38 polypeptide expressed on the surface of a cell is measured by flow cytometry (e.g., as described infra).

[0356] In some embodiments, the antigen binding site of a binding protein of the present disclosure binds the human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1 with an equilibrium dissociation constant (K_D) of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, or 0.8 nM or less, as measured by a flow cytometry assay using cells that express the human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1 on their cell surface, e.g., as described infra. 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 (K_D) of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, or 0.75 nM or less, as measured by a flow cytometry assay using cells that express the cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30 on their cell surface, e.g., as described infra. 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 (K_D) of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, or 0.83 nM or less, as measured by an SPR assay using the human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1, e.g., as described infra. 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 (K_D) of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 3.5 nM or less, 1.5 nM or less, or 1.0 nM or less, as measured by an SPR assay using the cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30, e.g., as described infra. As demonstrated herein, in some embodiments, a binding protein of the present disclosure may possess one or more of the exemplary binding properties described herein. In some embodiments, the KD is measured at 4° C. or 25° C.

[0357] In some embodiments, a monospecific binding protein of the present disclosure possesses one or more of the following features: binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purified protein, as assayed by SPR or ELISA; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purified protein with a K_D of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1.5 nM or less, as assayed by SPR or ELISA; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) expressed on the surface of a cell, as 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 K_D of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less, as 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 or ELISA; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30) as a purified protein with a K_D of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, as assayed by SPR or ELISA; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30) expressed on the surface of a cell, as 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 K_D of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 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 SPR or ELISA; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:105) expressed on the surface of a cell, as 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 FcγRI and/or FcγRII, as compared to the same binding protein without the one or more mutations. In some embodiments, a binding protein of the present disclosure binds a CD38 polypeptide (e.g., human or cynomolgus monkey) expressed on the surface of a cell with an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less, as assayed by flow cytometry. In some embodiments, a binding protein of the present disclosure binds a CD38 polypeptide (e.g., human or cynomolgus monkey) as a purified protein with an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less, as assayed by ELISA. In some embodiments, the KD is measured at 4° C. or 25° C.

[0358] In some embodiments, a trispecific binding protein of the present disclosure possesses one or more of the following features: 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 (e.g., as measured by Annexin V staining and/or propidium iodide uptake); 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 (e.g., as measured by CD69 expression); induces costimulation of T cell receptor signaling (e.g., as mediated by CD28); 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-α) 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 (e.g., as measured by immunoassay); 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 or ELISA; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) as a purified protein with a K_D of 1.5 nM or less, as assayed by SPR or ELISA; binds to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:1) expressed on the surface of a cell, as 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 K_D of 12 nM 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 or ELISA; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30) as a purified protein with a K_D of 3.5 nM or less, as assayed by SPR or ELISA; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:30) expressed on the surface of a cell, as 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 K_D of 7.5 nM 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 SPR or ELISA; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO:105) expressed on the surface of a cell, as 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 FcγRI and/or FcγRII, as compared to the same binding protein without the one or more mutations. In some embodiments, a binding protein of the present disclosure binds a CD38 polypeptide (e.g., human or cynomolgus monkey) expressed on the surface of a cell with an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less, as assayed by flow cytometry. In some embodiments, a binding protein of the present disclosure binds a CD38 polypeptide (e.g., human or cynomolgus monkey) as a purified protein with an EC50 of 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less, as assayed by ELISA.

[0359] Nucleic Acids

[0360] Standard recombinant DNA methodologies are used to construct the polynucleotides that encode the polypeptides which form the binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See e.g., Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Similarly, conventional techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0361] Other aspects of the present disclosure relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein. In some embodiments, the isolated nucleic acid molecules comprise a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs:60-83 and/or shown in Table J.

[0362] Certain aspects of the present disclosure relate to kits of polynucleotides. In some embodiments, one or more of the polynucleotides is a vector (e.g., an expression vector). The kits may find use, inter alia, in producing one or more of the binding proteins described herein, e.g., a trispecific binding protein of the present disclosure. In some embodiments, the kit comprises one, two, three, or four polynucleotides shown in Table J (e.g., of mAb2×CD28sup×CD3mid IgG4 FALA, mAb2×CD28sup×CD3mid IgG1LALA P329A, mAb2×CD28sup×CD3mid IgG1 NNSA, mAb6×CD28sup×CD3mid IgG4 FALA, mAb6×CD28sup×CD3mid IgG1LALA P329A, or mAb6×CD28sup×CD3mid IgG1 NNSA). In some embodiments, a kit of polynucleotides comprises: a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:72, a third polynucleotide comprising the sequence of SEQ ID NO:74, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75. In some embodiments, a kit of polynucleotides comprises: a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:76, a third polynucleotide comprising the sequence of SEQ ID NO:77, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75. In some embodiments, a kit of polynucleotides comprises: a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:78, a third polynucleotide comprising the sequence of SEQ ID NO:79, and a fourth polynucleotide comprising the sequence of SEQ ID NO:75. In some embodiments, a kit of polynucleotides comprises: a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:72, a third polynucleotide comprising the sequence of SEQ ID NO:80, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81. In some embodiments, a kit of polynucleotides comprises: a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:76, a third polynucleotide comprising the sequence of SEQ ID NO:82, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81. In some embodiments, a kit of polynucleotides comprises: a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide comprising the sequence of SEQ ID NO:78, a third polynucleotide comprising the sequence of SEQ ID NO:83, and a fourth polynucleotide comprising the sequence of SEQ ID NO:81.

[0363] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct transcription of the binding protein-coding nucleic acid sequence. A promoter may refer to nucleic acid control sequences which direct transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence of a binding protein if the promoter affects the transcription or expression of the coding sequence. Examples of promoters may include, but are not limited to, promoters obtained from the genomes of viruses (such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), and the like), from heterologous eukaryotic promoters (such as the actin promoter, an immunoglobulin promoter, from heat-shock promoters, and the like), the CAG-promoter (Niwa et al., Gene 108(2):193-9, 1991), the phosphoglycerate kinase (PGK)-promoter, a tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005), the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, and the promoter of the yeast alpha-mating factors. Polynucleotides encoding binding proteins of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or other suitable promoter that will be readily recognized by one skilled in the art.

[0364] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. Expression vectors may include one or more regulatory sequences operatively linked to the polynucleotide to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, α-fetoprotein, insulin and the like), and enhancer sequences from a eukaryotic cell virus (such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and the like). Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral, vaccinia viral, Sindbis-viral, measles, herpes viral, lentiviral, retroviral, adeno-associated viral vectors, etc.). Expression vectors can be used to transfect host cells, such as, for example, bacterial cells, yeast cells, insect cells, and mammalian cells. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art, and can be used to transfect any cell of interest.

[0365] Other aspects of the present disclosure relate to a vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein, and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein, and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein, and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. The one or more vectors of the vector system may be any of the vectors described herein. In some embodiments, the one or more vectors are expression vectors.

[0366] Isolated Host Cells

[0367] Other aspects of the present disclosure relate to an isolated host cell comprising one or more isolated polynucleotides, polynucleotide kits, vectors, and/or vector systems described herein. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g., an S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells may include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells may include, for example, human embryonic kidney cells (e.g., 293 or 293 cells subcloned for growth in suspension culture), Expi293TM cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, a human hepatoma line (e.g., Hep G2), and myeloma cells (e.g., NSO and Sp2/0 cells).

[0368] Other aspects of the present disclosure relate to a method of producing any of the binding proteins described herein. In some embodiments, the method includes a) culturing a host cell (e.g., any of the host cells described herein) comprising an isolated nucleic acid, vector, and/or vector system (e.g., any of the isolated nucleic acids, vectors, and/or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods of culturing host cells under conditions to express a protein are well known to one of ordinary skill in the art. Methods of isolating proteins from cultured host cells are well known to one of ordinary skill in the art, including, for example, by affinity chromatography (e.g., two step affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography).

[0369] In some embodiments, a binding protein of the present disclosure is purified by protein A affinity chromatography, kappa light chain affinity chromatography (e.g., using a KappaSelect resin according to manufacturer's instructions; GE Healthcare), and optionally lambda light chain affinity chromatography (e.g., using a LambdaFabSelect resin according to manufacturer's instructions; GE Healthcare). In some embodiments, a binding protein of the present disclosure is purified by Protein A affinity chromatography, lambda light chain affinity chromatography (e.g., using a LambdaFabSelect resin according to manufacturer's instructions; GE Healthcare), and optionally kappa light chain affinity chromatography (e.g., using a KappaSelect resin according to manufacturer's instructions; GE Healthcare). In some embodiments, the binding protein comprises two Fc regions, each comprising a C_H3 domain, and only one of the C_H3 domains comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, a binding protein of the present disclosure is purified by protein A affinity chromatography, then kappa light chain affinity chromatography (e.g., using a KappaSelect resin according to manufacturer's instructions; GE Healthcare), then optionally lambda light chain affinity chromatography (e.g., using a LambdaFabSelect resin according to manufacturer's instructions; GE Healthcare) in sequence. In some embodiments, a binding protein of the present disclosure is purified by Protein A affinity chromatography, then lambda light chain affinity chromatography (e.g., using a LambdaFabSelect resin according to manufacturer's instructions; GE Healthcare), then optionally kappa light chain affinity chromatography (e.g., using a KappaSelect resin according to manufacturer's instructions; GE Healthcare) in sequence. For example, in some embodiments, the binding protein is contacted with Protein A, eluted from Protein A under conditions suitable for isolating the binding protein away from binding proteins comprising either 0 or 2 C_H3 domains comprising the amino acid substitutions are H435R and Y436F, contacted with a kappa light chain affinity medium (e.g., as used in the KappaSelect resin; GE Healthcare), and eluted from the kappa light chain affinity medium under conditions suitable for isolating the binding protein away from binding proteins comprising only lambda C_L domains (e.g., according to manufacturer's instructions). Conditions suitable for the Protein A elution are known in the art, including without limitation a stepwise elution gradient from pH4.5-2.8. In some embodiments, Protein A or a Protein A variant useful for protein purification is employed. In some embodiments, the Protein A is attached to a substrate or resin, e.g., as part of a chromatography medium. In some embodiments, after elution from the kappa light chain affinity medium, the binding protein is contacted with a lambda light chain affinity medium (e.g., as used in the LambdaFabSelect resin; GE Healthcare), and eluted from the lambda light chain affinity medium under conditions suitable for isolating the binding protein away from binding proteins comprising only kappa C_L domains (e.g., according to manufacturer's instructions). In some embodiments, a binding protein of the present disclosure is detected using HIC chromatography. In some embodiments, the binding protein comprises: a first polypeptide chain that comprises a lambda C_L domain; a C_H3 domain of a second polypeptide chain that comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; a C_H3 domain of a third polypeptide chain that comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F; and a fourth polypeptide chain that comprises a kappa C_L domain. In some embodiments, the binding protein is produced by a host cell. In some embodiments, the binding protein is purified from a cell culture medium or host cell extract. In some embodiments, the binding proteins are secreted by a host cell or produced and extracted from a host cell (e.g., before being contacted with Protein A). In some embodiments, the binding protein is in a cell culture medium or host cell extract when contacted with Protein A. In some embodiments, the binding protein is purified away from other binding proteins, polypeptides, and/or other cellular components.

[0370] In some embodiments, the binding protein of the disclosure is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, a first polypeptide chain comprises a structure represented by the formula:

V_L2-L₁-V_L1-L₂-C_L  [I]

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

V_H1-L₃-V_H2-L₄-C_H1-hinge-C_H2-C_H3  [II]

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

V_H3-C_H1-hinge-C_H2-C_H3  [III]

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

V_L3-C_L  [IV]

wherein:

[0371] V_L1 is a first immunoglobulin light chain variable domain;

[0372] V_L2 is a second immunoglobulin light chain variable domain;

[0373] V_L3 is a third immunoglobulin light chain variable domain;

[0374] V_H1 is a first immunoglobulin heavy chain variable domain;

[0375] V_H2 is a second immunoglobulin heavy chain variable domain;

[0376] V_H3 is a third immunoglobulin heavy chain variable domain;

[0377] C_L is an immunoglobulin light chain constant domain;

[0378] C_H1 is an immunoglobulin C_H1 heavy chain constant domain;

[0379] C_H2 is an immunoglobulin C_H2 heavy chain constant domain;

[0380] C_H3 is an immunoglobulin C_H3 heavy chain constant domain;

[0381] hinge is an immunoglobulin hinge region connecting the C_H1 and C_H2 domains; and

[0382] L₁, L₂, L₃ and L₄ are amino acid linkers;

[0383] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair. In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross-over orientation that forms two distinct antigen binding sites. As described above, the second and third polypeptide chains include an Fc region (e.g., comprising the hinge-C_H2-C_H3 domains). In some embodiments, one or both Fc regions are human IgG4 Fc regions comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc regions are human IgG4 Fc regions comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and/or R409K. In some embodiments, one or both Fc regions are human IgG1 Fc regions comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc regions are human IgG1 Fc regions comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.

[0384] In some embodiments, the binding protein of the disclosure is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one of the antigen binding sites binds a CD38 polypeptide (e.g., the extracellular domain of human and/or cynomolgus monkey CD38 polypeptides). In some embodiments, a first polypeptide chain comprises a structure represented by the formula:

V_L2-L₁-V_L1-L₂-C_L  [I]

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

V_H1-L₃-V_H2-1₄-C_H1  [II]

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

V_H3-C_H1  [III]

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

V_L3-C_L  [IV]

wherein:

[0385] V_L1 is a first immunoglobulin light chain variable domain;

[0386] V_L2 is a second immunoglobulin light chain variable domain;

[0387] V_L3 is a third immunoglobulin light chain variable domain;

[0388] V_H1 is a first immunoglobulin heavy chain variable domain;

[0389] V_H2 is a second immunoglobulin heavy chain variable domain;

[0390] V_H3 is a third immunoglobulin heavy chain variable domain;

[0391] C_L is an immunoglobulin light chain constant domain;

[0392] C_H1 is an immunoglobulin C_H1 heavy chain constant domain; and

[0393] L₁, L₂, L₃ and L₄ are amino acid linkers;

and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair. In some embodiments, the second and the third polypeptide chain further comprise an Fc region linked to C_H1, the Fc regions comprising an immunoglobulin hinge region and C_H2 and C_H3 immunoglobulin heavy chain constant domains. In some embodiments, one or both Fc regions are human IgG4 Fc regions comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc regions are human IgG4 Fc regions comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and/or R409K. In some embodiments, one or both Fc regions are human IgG1 Fc regions comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc regions are human IgG1 Fc regions comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.

[0394] In some embodiments, the V_H1 and VL1 form a binding pair and form a first antigen binding site. In some embodiments, the V_H2 and VL2 form a binding pair and form a second antigen binding site. In some embodiments, the V_H3 and VL3 form a binding pair and form a third antigen binding site. The binding proteins can also be used for cell activation, tumor targeting, neutralization of cytokine activities, neutralization of viral infection, combination of multiple signaling events, to treat cancer, arthritis, and/or inflammatory disorders. For example, in some embodiments, a binding protein specifically binds one, two, or three antigen targets selected from A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-id), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, a receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPRS/CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

[0395] In some embodiments, one of the three antigen binding sites binds a CD3 polypeptide (e.g., a human CD3 polypeptide), one of the three antigen binding sites binds a CD28 polypeptide (e.g., a human CD28 polypeptide), and one of the three antigen binding sites binds a third polypeptide. In some embodiments, the antigen binding site that specifically binds an antigen target other than CD3 or CD28 binds an antigen target selected from A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-id), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD19, CD20, CD23 (also known as FCER2, a receptor for IgE), CD24, CD27, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPRS/CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

[0396] In any of the trispecific binding proteins described supra, any linker or combination of linkers described herein may be used. For example, in some embodiments, at least one of L₁, L₂, L₃ or L₄ is independently 0 amino acids in length. In some embodiments, L₁, L₂, L₃ or L₄ are each independently at least one amino acid in length. In some embodiments, L₁, 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). In some embodiments, L₁, L₂, L₃ and L₄ each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58), and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L₁ comprises the sequence GQPKAAP (SEQ ID NO: 58), L₂ comprises the sequence TKGPS (SEQ ID NO:57), L₃ comprises the sequence S, and L₄ comprises the sequence RT. In some embodiments, L₁ comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₂ comprises the sequence GGGGSGGGGS (SEQ ID NO:55), L₃ is 0 amino acids in length, and L₄ is 0 amino acids in length. In some embodiments, L₁ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₂ comprises the sequence GGSGSSGSGG (SEQ ID NO:59), L₃ is 0 amino acids in length, and L₄ is 0 amino acids in length. In some embodiments, L₁ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), L₂ is 0 amino acids in length, L₃ comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:56), and L₄ is 0 amino acids in length.

[0397] The binding proteins can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for the detection and quantitation of one or more target antigens. The binding proteins will bind the one or more target antigens with an affinity that is appropriate for the assay method being employed.

[0398] For diagnostic applications, in certain embodiments, binding proteins can be labeled with a detectable moiety. The detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety can be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²⁵I, ⁹⁹Tc, ¹¹¹In, or ⁶⁷Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0399] The binding proteins are also useful for in vivo imaging. A binding protein labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host assayed. The binding protein can be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

[0400] For clinical or research applications, in certain embodiments, binding proteins can be conjugated to a cytotoxic agent. A variety of antibodies coupled to cytotoxic agents (i.e., antibody-drug conjugates) have been used to target cytotoxic payloads to specific tumor cells. Cytotoxic agents and linkers that conjugate the agents to an antibody are known in the art; see, e.g., Parslow, A. C. et al. (2016) Biomedicines 4:14 and Kalim, M. et al. (2017) Drug Des. Devel. Ther. 11:2265-2276.

[0401] The disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting target antigen levels in biological samples. Such reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein. In some embodiments, the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing a condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the label or package insert indicates that the composition is used for preventing, diagnosing, and/or treating the condition of choice. Alternatively, or additionally, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0402] In some embodiments, a binding protein of the present disclosure is administered to a patient in need thereof for the treatment or prevention of cancer. In some embodiments, the present disclosure relates to a method of preventing and/or treating a proliferative disease or disorder (e.g., cancer). In some embodiments, the method comprises administering to a patient a therapeutically effective amount of at least one of the binding proteins, or pharmaceutical compositions related thereto, described herein. In some embodiments, the present disclosure relates to uses of at least one of the binding proteins, or pharmaceutical compositions related thereto, described herein for preventing and/or treating a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the present disclosure relates to at least one of the binding proteins, or pharmaceutical compositions related thereto, described herein for use in the manufacture of a medicament for preventing and/or treating a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the patient is a human. In some embodiments, the binding protein comprises one antigen binding site that binds a T-cell surface protein and another antigen binding site that binds the extracellular domain of a human CD38 polypeptide, e.g., as described in section II supra. In some embodiments, the binding protein comprises an antigen binding site that binds the extracellular domain of a human CD38 polypeptide, an antigen binding site that binds a human CD28 polypeptide, and an antigen binding site that binds a human CD3 polypeptide.

[0403] In some embodiments, cells of the cancer express a human CD38 isoform A polypeptide on their cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:1). In some embodiments, cells of the cancer express a human CD38 isoform E polypeptide on their cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:105). In some embodiments, the patient is selected for treatment on the basis that the cells of the cancer express a human CD38 isoform E polypeptide on their cell surface (e.g., comprising the amino acid sequence of SEQ ID NO:105). In some embodiments, the cancer cells express CD38 and CD28. In some embodiments, the cancer cells express CD38 and do not express CD28.

[0404] In some embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, germinal center B-cell lympohoma or B-cell acute lymphoblastic leukemia. In certain embodiments, the cancer is multiple myeloma. In certain embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or a B cell lymphoma.

[0405] In certain embodiments, the cancer is multiple myeloma. Anti-CD38 antibodies have been tested for the treatment of multiple myeloma, such as daratumumab and isatuximab. However, while multiple myeloma is considered treatable, relapse is inevitable in almost all patients, leading to the development of treatment-refractory disease. In some embodiments, the cancer is relapsed or refractory multiple myeloma. In some embodiments, the patient has been treated with a prior multiple myeloma treatment. In some embodiments, a binding protein of the present disclosure is administered to the patient as a 1^st, 2^nd or 3^rd line treatment for multiple myeloma. Without wishing to be bound to theory, it is thought that an anti-CD38×anti-CD28×anti-CD3 binding protein of the present disclosure may be useful in treating multiple myeloma, e.g., by recruiting T cells to tumor cells via anti-CD38 (or anti-CD2⁸/anti-CD38), activation of engaged T cells via anti-CD3/anti-CD28, and/or killing of tumor cells through perforin/granzyme-based mechanisms. CD28 has been reported as a novel cancer marker for multiple myeloma. See Nair, J. R. et al. (2011) J. Immunol. 187:1243-1253.

[0406] In some embodiments, the at least one binding protein is administered (or is to be administered) in combination with one or more anti-cancer therapies (e.g., any anti-cancer therapy known in the art, such as a chemotherapeutic agent or therapy). In some embodiments, the at least one binding protein is administered (or is to be administered) before the one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered (or is to be administered) concurrently with the one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered (or is to be administered) after the one or more anti-retroviral therapies.

[0407] Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the disclosure. Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of a binding protein, or binding protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.

[0408] Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.

[0409] The pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides—preferably sodium or potassium chloride—or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A. R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions of the same, incorporated herein by reference for any purpose).

[0410] The optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.

[0411] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the disclosure, binding protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the binding protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0412] The pharmaceutical compositions of the disclosure can be selected for parenteral delivery or subcutaneous. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

[0413] The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

[0414] When parenteral administration is contemplated, the therapeutic compositions for use can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding protein is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

[0415] In one embodiment, a pharmaceutical composition can be formulated for inhalation. For example, a binding protein can be formulated as a dry powder for inhalation. Binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions can be nebulized.

[0416] It is also contemplated that certain formulations can be administered orally. In one embodiment of the disclosure, binding proteins that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

[0417] Another pharmaceutical composition can involve an effective quantity of binding proteins in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[0418] Additional pharmaceutical compositions of the disclosure will be evident to those skilled in the art, including formulations involving binding proteins in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyric acid. Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.

[0419] Pharmaceutical compositions to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0420] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

[0421] The disclosure also encompasses kits for producing a single-dose administration unit. The kits can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this disclosure are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0422] The effective amount of a binding protein pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the binding protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0423] Dosing frequency will depend upon the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.

[0424] The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.

[0425] The composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

[0426] The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only, and should not be construed as limiting the scope of the invention in any way.

[0427] The following terminology may be used interchangeably in the Examples and Drawings herein to refer to specific anti-CD38 antigen binding domains or antibodies:

[0428] antiCD38_C2-CD38-1: mAb1

[0429] antiCD38_C2-CD38-1_VH1-VL1 or CD38_VH1: mAb2

[0430] antiCD38_C2-CD38-1_VH3-VL3: mAb3

[0431] antiCD38_C2-CD38-1_VH5-VL3: mAb4

[0432] antiCD38_C2-CD38-1_VH6-VL3: mAb5

[0433] antiCD38_1370 or CD38_HHY1370: mAb6

[0434] antiCD38_SB19 or isatuximab: mAb7.

[0435] The Examples that follow describe the generation and characterization of monoclonal anti-CD38 antibodies. Advantageously, antibodies provided herein cross-react with human and monkey CD38 proteins, thereby providing molecules that can be used for both safety and clinical studies. These antibodies are also capable of killing CD38+ cells by apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0436] This Example describes an efficient workflow for generating CD38-specific and cross-reactive monoclonal antibodies from single murine B cells.

[0437] Materials and Methods

[0438] Antibodies to human CD38 were generated using the human CD38 extracellular domain R45-I300 (SEQ ID NO:1). See Q. Liu, I. Krilksunov, R. Graeff, C. Munshi, H. C. Lee, and Q. Hao 2005 Structure 13::1331-1339. The immunogen was administered directly, with an adjuvant to stimulate the immune response, to either normal BalbC mice or transgenic Trianni Mice™ (Trianni, San Francisco, Calif.) comprising DNA encoding human Immunoglobulin heavy and kappa light chain variable regions.

[0439] Various recombinant CD38 proteins derived from isoform A with different tag and point mutations were used (SEQ ID NOs:2, 3, 4, and 28), and a tagged version of CD38 isoform E (SEQ ID NO: 105) encompassing CD38 extracellular domain from R45-P203. The proteins were produced by transient expression in mammalian cells. Coding DNA sequences were cloned into mammalian expression plasmids under CMV enhancer/promoter and SV40 polyA signals. HEK293 cells (Invitrogen; #K9000-10) were transiently transfected with the expression plasmids using FreeStyle™ MAX 293 Expression System according to the manufacturer's instructions.

[0440] Anti-CD38 antibodies were also isolated directly from antigen-positive B cells without fusion to myeloma cells. Using this method, several anti-CD38 antibodies were obtained, such as mAb1 (see SEQ ID Nos: 5 and 6 for VH and VL sequences, respectively, and SEQ ID Nos: 7 and 8 for heavy chain and light chain sequences, respectively). Briefly, 6-8 weeks old female BALB/c mice (S082342; Charles River Labs, Bar Harbor, Me.) each received three rounds of immunization over a course of 41 days using the classical method as described by A. Wennerberg et al. (1993 Am. J. Pathol. 143:1050-1054). Antigen was administered intraperitoneally to ventral site of mice. Three days after the last injection, mice were sacrificed and spleens were isolated aseptically and washed with fresh RPMI medium. Lymphocytes were released from the spleens and single-cell suspension was washed twice with RPMI medium before being sorted using a four colour sorting strategy including a panel of fluorescent antibodies and dual human and monkey CD38 proteins and then were separated using flow cytometric cell sorting to isolate human/monkey cross reactive IgG-CD38 specific B cells. Single cells were directly sorted into PCR tubes to amplify cognate pair of VH and VL genes by RT-PCR (T. Tiller, C. Busse and H. Wardemann 2009 J. Immunol. Methods 350:183-193). Resulting DNA was sequenced.

[0441] Resulting DNA was cloned into a mammalian expression vector encoding respectively the human IgG1 or human Ck domains for transient expression in HEK293 cells using FreeStyle™ MAX 293 Expression System according to the manufacturer's instructions. Batches were purified by protein A affinity chromatography (MabSelect, GE Heathcare). The eluate was dialyzed against PBS before sterile filtration and storage at 4° C.

[0442] Immunizations, fusion and screening were performed using P3×63-Ag8.653 myeloma cells with the extracellular domain of human CD38 as described in Kilpatrick et al. 1997 Hybridoma 16: 381389. Using the RIMMS method described by Kilpatrick et al., 6-8 week old female transgenic Trianni Mice™ comprising DNA encoding human Immunoglobulin heavy and kappa light chain variable regions each received four rounds of immunization over a course of 14 days at intervals of 3-4 days. CD38 protein emulsified in RIBI's adjuvant (Sigma #T2684) was administered subcutaneously to six sites proximal to draining lymph nodes, along the back of the mice and to six juxtaposed sites along abdomen. Four days after the last injection, mice were sacrificed. Bilateral popliteal, superficial inguinal, axillary and branchial lymph nodes were isolated aseptically and washed with fresh RPMI medium. Lymphocytes were released from the lymph nodes and single-cell suspension was washed twice with RPMI medium before being fused with P3×63-AG8.653 myeloma cells using polyethylene glycol. After fusion, the cell mixture was incubated in an incubator at 37° C. for 16-24 hours. The resulting cell preparation was transferred into selective semi-solid medium and aseptically plated out into 100 mm Petri plates and incubated at 37° C. Ten days after initiation of selection, the plates were examined for hybridoma growth, and visible colonies were picked-up and placed into 96-well plates containing 200 μL of growth medium. The 96-well plates were kept in an incubator at 37° C. for 2 to 4 days. Using this technique, and the immunogen described above, several anti-CD38 chimeric antibodies were obtained such as mAb 6 (see SEQ ID Nos: 9 and 10 for VH and VL sequences, respectively, and SEQ ID Nos: 11 and 12 for heavy chain and light chain sequences, respectively. The VH and VL sequences were retrieved by RT-PCR and mAb 6 was produced by transient expression as described above.

[0443] The binding properties of the anti-huCD38 mabs were evaluated using a BIAcore 2000 (BIAcore Inc., Uppsala, N.J.). Briefly, a CMS BIAcore biosensor chip was docked into the instrument and activated with 2504 of 1:1 NHS/EDC at room temperature. A mouse anti-human Fc IgG1 (GE Healthcare #BR-1008-39) (13.5 μg/mL in 0.05M acetate buffer, pH5) were immobilized on the activated chips in flow cells 1. The immobilization was carried out at a flow rate of 54/min. The chip was then blocked by injection of 554 of ethanolamine-HCl, pH8.5, followed by five washes with 50 mM NaOH, 1M NaCl. To measure the binding of anti-CD38 mabs to the human CD38 protein or cyno CD38 protein, antibodies were used at 2 μg/mL in BIAcore running buffer (HBS-EP). Antigens (human CD38-histag (ID2) or cyno CD38-histag (ID3)) were injected from 3 to 1000 nM. Following completion of the injection phase, dissociation was monitored in a BIAcore running buffer at the same flow rate for 360 sec. The surface was regenerated between injections using 30 μL of 50 mM NaOH-1 M NaCl. Individual sensorgrams were analyzed using BIAsimulation software.

[0444] The binding of anti-CD38 antibodies to CD38 expressed on the surface of recombinant murine preB::300.19 cells was determined by flow cytometry. The recombinant cell line was described by J. Deckket et al. 2014 Clin. Cancer Res 20:4574-4583. Murine preB::300.19 CD38-expressing cells were coated at 40,000 cells/well on 96-well High Bind plate (MSD L15XB-3) and 100 μL/well of anti-CD38 antibodies were added for 45 min at 4° C. and washed three times with PBS 1% BSA. 100 μL/well of goat anti-human IgG conjugated with Alexa488 (Jackson ImmunoResearch; #109-545-098) was added for 45 min at 4° C. and washed three times with PBS 1% BSA. Antibody binding was evaluated after centrifugation and resuspension of cells by adding 200 μL/well PBS 1% BSA and read using Guava® easyCyte™ 8HT Flow Cytometry System. Apparent KD and EC50 values were estimated using BIOST@T-BINDING and BIOST@T-SPEED software, respectively.

[0445] Results

[0446] Binding of newly isolated mAb1 to human SU-DHL-8 or MOLP-8 cells was compared to that of isatuximab using flow cytometry (FIG. 1A). Both antibodies exhibited high-affinity binding to both cell lines. However, only mAb1 was able to bind to cells expressing cynomolgus monkey CD38 on their surface (FIG. 1B).

[0447] Binding to soluble extracellular domains of human and cynomolgus monkey CD38 polypeptides was examined for newly isolated antibodies mAb1 and mAb6 using surface plasmon resonance (SPR). The SPR results are summarized in Table A.

TABLE A
Binding affinity of antibodies to the soluble extracellular domain
of hCD38 and cCD38 as determined by SPR assay.
	hCD38-his		cCD38-his
	(SEQ ID NO: 2)		(SEQ ID NO: 4)
	Kd (s−1)	KD (M)	Kd (s−1)	KD (M)
	mAb1	2.66E−04	3.36E−10	9.85E−05	3.90E−10
	mAb6	2.03E−04	1.44E−09	1.90E−04	1.38E−09

[0448] Flow cytometry was also used to examine binding of mAb1 and mAb6 anti-CD38 antibodies to murine pre-B cells expressing human or cynomolgus monkey CD38 polypeptide on their cell surface. The results are shown in Table B. Both antibodies tested exhibited high affinity binding to preB::300.19 huCD38- or cCD38-expressing cells.

TABLE B
Binding affinity of antibodies to hCD38 or cCD38 expressed
by murine preB::300.10 cells as determined by flow cytometry.
	Apparent KD FACS (M)
	hCD38-expressing cells	cCD38-expressing cells
	mAb1	2.80E−10	2.20E−10
	mAb6	2.07E−09	1.14E−09

[0449] These results demonstrate the generation of antibodies that bind with high affinity to both human and cynomolgus CD38 polypeptides. These antibodies, unlike other anti-CD38 antibodies, cross-react with human and cynomologus CD38 polypeptides in soluble extracellular form or expressed on the surface of mammalian cells.

[0450] This Example describes the humanization of antibodies generated in Example 1.

[0451] Sequences of mAb1 variable domains were analysed in-silico. The International ImMunoGeneTics information system for immunoglobulins (IMGT) definition was used to identify Complementarity Determining Regions (CDRs).

[0452] First, in parallel, searches were undertaken to identify the closest Mouse germline and Human germline protein sequence combination for each variable chain. This was performed using a Basic Local Alignment Search Tool (BLAST) (Nucleic Acids Res. 25:3389-3402) search against Mouse and Human germline protein sequence databases. For each antibody chain, closest V and J mouse and human protein sequences were found. The qualification of this high protein sequence identity was measured by the V region identity percentage. Results of the searches for mAb1 light and heavy chain are presented in Tables C and D, respectively.

TABLE C
mAb1 variable light chain closest human and mouse
(V&J) germline protein sequences.
		V Region
Species	V Allele	Identity (%)	J Allele
[Homo sapiens]	IGKV4-1*01 (imgt)	66.34%	IGKJ1*01 (imgt)
Best Functional	Functional		Functional
Germlines
[Mus musculus]	IGKV3-10*01	96.97%	IGKJ1*01 (imgt)
Best Functional	(imgt) Functional		Functional
Germlines

TABLE D
mAb1 variable heavy chain closest human and mouse
(V&J) germline protein sequences.
		V Region
Species	V Allele	Identity (%)	J Allele
[Homo sapiens]	IGHV1-3*01 (imgt)	69.39%	IGHJ4*01 (imgt)
Best Functional	Functional		Functional
Germlines
[Mus musculus]	IGHV1-12*01	87.76%	IGHJ3*01 (imgt)
Best Functional	(imgt) Functional		Functional
Germlines

[0453] Next, mAb1 variable domain sequences were screened for sequence liabilities, such as deamidation, acidic cleavage, oxidation, or iso-aspartate formation sites. mAb1 structure has been analysed by 3D modelling by homology in order to define how amino acid residues interact in intra or intermolecular ways. This step led to the identification of a group of amino acid residues that are structurally important for mAb1 functionalities as CDRs conformation and antigen binding. These amino acid residues were selected to be present in the humanized mAb1 variable sequences.

[0454] CDRs from the parental murine mAb1 were grafted onto the relevant frameworks. Based on the above analyses, human IGKV3-20*02 coupled with IGKJ1*01 and human IGHV1-3*01 coupled with IGHJ4*01 were selected to be foundation for mAb1 variable light chain and variable heavy chain humanization, respectively. The mAb1 light chain displayed 64.52% identity over the V region with the selected human IGKV3-20*02 germline. The mAb1 heavy chain displayed 69.39% identity over the V region with the selected human IGHV1-3*01 germline. During humanization process, the parental murine mAb1 CDRs were transplanted between selected germline frameworks in order to recompose standard antibody variable sequences. Attention was provided to the previously identified group of mAb1 amino acid residues that are structurally important for its functionalities, as noted above. If needed those amino acid residues were replaced in the newly created sequences by their exact corresponding mAb1 residue. This corresponds to a back-mutation step to incorporate adequate parental sequence amino acid residues. Some CDR mutations were incorporated for both humanizing and avoiding sequence liabilities in parental CDRs. The newly created light and heavy variable sequences were used to generate 3D homology models of the humanized mAb1 variable region. The 3D models were built using Model Antibody Framework from BIOVIA Discovery Studio suite.

[0455] Based on CDR grafting approach, two variants for the variable light chain (VL1 and VL3) and four variants for the variable heavy chain (V_H1, V_H3, V_H5 and V_H6) were generated. The particular combination of amino acid residues which vary between mAb1 variable light and heavy sequences and their humanized versions are set forth in Tables E and Table F, respectively.

TABLE E
Sequence differences between mAb1 variable light chain
and humanized variants.
Ig Regions
(IMGT)	parental mAb1 VL	humanized VL1	humanized VL3
FR1	S10	T	T
	A12	S	S
	V13	L	L
	L15	P	P
	Q17	E	E
CDR1	E27	Q	E
	D30	S	D
	N34	Q	N
FR2	K49	R	R
CDR2	L54	G	L
FR3	N57	S	S
	L58	R	R
	E59	A	A
	S60	T	T
	V62	I	I
	R72	G	G
	D80	S	S
	V82	L	L
	A84	P	P
	D85	E	E
	A87	F	F
	T89	V	V

TABLE F
Sequence differences between mAb1 variable heavy chain
and humanized variants.
Ig Regions	parental	humanized	humanized	humanized	humanized
(IMGT)	mAb1 VH	VH1	VH3	VH5	VH6
FR1	Q5	V	V	V	V
	L11	V	V	V	V
	R13	K	K	K	K
	S14	P	P	S	P
	M20	V	V	V	M
CDR1	F32	Y	F	F	F
	N33	A	N	N	N
FR2	T40	A	A	A	A
	G44	R	R	G	R
CDR2	N55	Q	N	N	N
FR3	K65	Q	Q	Q	Q
	K67	R	R	R	R
	S76	A	A	A	A
	Q82	E	E	E	E
	I83	L	L	I	I
	T87	R	R	R	R
	S91	T	T	T	T

[0456] The humanized VL1 variant with SEQ ID NO: 14 displays a total of 22 mutations (18 in FRs and 4 in CDRs) compared to the parental VL of mAb1 sequence with SEQ ID NO: 6. This variant derived from frameworks of human germlines IGKV3-20*02 coupled with IGKJ1*01 with 8 back-mutations done due to the risk of negative impact on mAb structure, CDRs conformation and therefore, on binding to its target. Four positions of the parental CDRs were mutated in order to increase humanization rate or avoid sequence liabilities.

[0457] The humanized VL3 variant with SEQ ID NO: 18 displays a total of 18 mutations (18 in FRs) compared to the parental VL of mAb1 sequence with SEQ ID NO: 6. This variant derived from frameworks of human germlines IGKV3-20*02 coupled with IGKJ1*01 with 8 back-mutations done due to the risk of negative impact on mAb structure, CDRs conformation and therefore, on binding to its target.

[0458] The humanized V_H1 variant with SEQ ID NO: 13 displays a total of 17 mutations (14 in FRs and 3 in CDRs) compared to the parental VH of mAb1 sequence with SEQ ID NO: 5. This variant derived from frameworks of human germlines IGHV1-3*01 coupled with IGHJ4*01 with 11 back-mutations done due to the risk of negative impact on mAb structure, CDRs conformation and therefore, on binding to its target. Three positions of the parental CDRs were mutated in order to increase humanization rate or avoid sequence liabilities.

[0459] The humanized V_H3 variant with SEQ ID NO: 17 displays a total of 14 mutations (14 in FRs) compared to the parental VH mAb1 sequence with SEQ ID NO: 5. This variant derived from frameworks of human germlines IGHV1-3*01 coupled with IGHJ4*01 with 11 back-mutations done due to the risk of negative impact on mAb structure, CDRs conformation and therefore, on binding to its target.

[0460] The humanized V_H5 variant with SEQ ID NO: 21 displays a total of 11 mutations (11 in FRs) compared to the parental VH of mAb1 sequence with SEQ ID NO: 5. This variant derived from frameworks of human germlines IGHV1-3*01 coupled with IGHJ4*01 with 14 back-mutations done due to the risk of negative impact on mAb structure, CDRs conformation and therefore, on binding to its target.

[0461] The humanized V_H6 variant with SEQ ID NO: 23 displays a total of 12 mutations (12 in FRs) compared to the parental VH of mAb1 sequence with SEQ ID NO: 5. This variant derived from frameworks of human germlines IGHV1-3*01 coupled with IGHJ4*01 with 13 back-mutations done due to the risk of negative impact on mAb structure, CDRs conformation and therefore, on binding to its target.

[0462] The resulting light and heavy humanized variable sequences were blasted for sequence similarity against the Immune Epitope Data Base (IEDB) database ((PLos Biol (2005) 3(3)e91) www.iedb.org) to ensure that none of the sequences contained any known B- or T-cell epitope listed therein.