RU2812910C2 - Antibodies to cd38 and combinations with antibodies to cd3 and cd28 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is presented: a monospecific binding protein that binds a CD38 polypeptide, a conjugate for targeting a cytotoxic agent or tag to cells expressing CD38 on their surface, a polynucleotide encoding a monospecific binding protein, an expression vector containing the polynucleotide and a host cell. A method of producing a monospecific binding protein is also disclosed.

EFFECT: obtaining new antibodies with high affinity for HLA-G.

18 cl, 40 dwg, 27 tbl, 15 ex

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of priority under U.S. Provisional Patent Application Serial No. 62/570655, filed October 10, 2017, U.S. Provisional Patent Application Serial No. 62/570660, filed Oct. 11, 2017, U.S. Provisional Patent Application No. Registration No. 62/676221, filed May 24, 2018, and European Patent Application No. EP 18187186.4, filed August 3, 2018; which are all incorporated herein by reference in their entirety.

REPRESENTATION OF A LIST OF SEQUENCES IN AN ASCII TEXT FILE

The contents of the following ASCII text file are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (File Name: 183952029941seqlist.TXT, Date Compiled: October 8, 2018, Size: 158 kilobytes).

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present disclosure relates to binding proteins that bind CD38 polypeptides (e.g. human and cynomolgus CD38 polypeptides), including monospecific, bispecific or trispecific binding proteins with at least one antigen binding domain that binds the CD38 polypeptide, as well as polynucleotides, host cells, methods of preparation and methods of use associated therewith.

PREREQUISITES FOR CREATION OF THE INVENTION

Biotherapeutics based on monoclonal antibodies have become an important area in the development of new drugs. Monoclonal antibody technology offers specific targeting and precise delivery of signals and/or payload to a specific cell population and provides long-lasting biological effects through Fc-mediated functions. Efforts made in the field of antibody engineering have resulted in the development of multispecific antibodies that combine the specificities of multiple monoclonal antibodies for a variety of biological applications, expanding the scope of antibody drug development.

CD38 is a promising drug target because it is expressed on the cell surface of many lymphoid tumor cells (see Table 1). Stevenson, G.T. (2006)Mol. Med.12:345-346). DARZALEX® (daratumumab) is an anti-CD38 antibody approved for use in the treatment of multiple myeloma. However, there remains a need for CD38-targeting therapeutics with a different mechanism of action and/or improved properties, including, but not limited to, high affinity binding to CD38, cross-reactivity between human and cynomolgus CD38 polypeptides, binding to lymphoma cells (e.g. large B-cell lymphoma and multiple myeloma cell lines) and the ability to induce apoptosis and/or antibody-dependent cell-mediated cytotoxicity (ADCC) and T cell-mediated antitumor activity.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides binding proteins that bind CD38 polypeptides (e.g. human and cynomolgus CD38 polypeptides), including monospecific, bispecific or trispecific binding proteins with at least one antigen binding site that binds the CD38 polypeptide. Advantageously, such binding proteins are characterized by the ability to recruit T cells in close proximity to cancer cells, followed by activation of T cells and stimulation of activated T cells to kill neighboring cancer cells through the granzyme/perforin mechanism, thus providing another mechanism of action for antitumor activity anti-CD38 antibodies such as DARZALEX® (daratumumab). Moreover, the ability to bind human and cynomolgus CD38 polypeptides allows for easy testing of binding proteins in preclinical toxicology studies, e.g. to evaluate their safety profiles for subsequent clinical use.

In some embodiments, provided herein are monospecific binding proteins that bind to human CD38 polypeptide. In some embodiments, the binding proteins cross-react with human and cynomolgus CD38 polypeptides. In some embodiments, the binding proteins bind to human CD38 isoform A and isoform E polypeptides. In some embodiments, the binding proteins have one or more of the following characteristics (in any combination): bind to the extracellular domain of a human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO: 1) as a purified protein according to SPR; bind to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1) as a purified protein with K_D 1.5 nM or less as measured by SPR assay; bind to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1), expressed on the surface of a cell, as measured by flow cytometry analysis; bind to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1), expressed on the cell surface, with an apparent K value_D 20 nM, 15 nM, 10 nM, 5 nM, 1 nM or less as measured by flow cytometry analysis; bind to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) as a purified protein, as measured by SPR analysis; bind to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) as a purified protein with K_D 3.5 nM or less as measured by SPR assay; bind to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) expressed on the cell surface as measured by flow cytometry analysis; bind to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30), expressed on the cell surface, with an apparent K value_D 7.5 nM or less as measured by flow cytometry analysis; bind to the extracellular domain of human CD38 isoform E polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105) as a purified protein, as measured by ELISA analysis; bind to the extracellular domain of human CD38 isoform E polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105) expressed on the cell surface as measured by flow cytometry analysis; induce apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of a cell expressing CD38 on its cell surface; and have one or more mutations (for example, in the Fc region) resulting in decreased binding to FcγRI and/or FcγRII compared to the same binding protein in the absence of one or more mutations. For exemplary assays, see Examples 1, 3, and 4. In some embodiments, KD is measured at 4°C or 25°C.

In some embodiments, provided herein are trispecific binding proteins that bind to human CD38 polypeptide. In some embodiments, trispecific binding proteins bind (e.g. simultaneously) with CD38 polypeptide (for example, expressed on the cell surface) and one or more other target antigens expressed on the surface of the second cell, thereby recruiting the second cell into close proximity to the cell expressing the CD38 polypeptide. In some embodiments, trispecific binding proteins bind (e.g. simultaneously) with CD38 polypeptide (for example, expressed on the cell surface) and one or two target antigens expressed on the surface of the T cell, thereby recruiting the T cell in close proximity to the cell expressing the CD38 polypeptide. In some embodiments, the trispecific binding proteins activate a 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 CD38 polypeptides. In some embodiments, the trispecific binding proteins bind to human CD38 isoform A and isoform E polypeptides. In some embodiments, the trispecific binding proteins have one or more of the following characteristics (in any combination): bind to the extracellular domain of a human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO: 1) as a purified protein according to SPR; bind to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1) as a purified protein with K_D 1.5 nM or less as measured by SPR assay; bind to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1), expressed on the surface of a cell, as measured by flow cytometry analysis; bind to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1), expressed on the cell surface, with an apparent K value_D 20 nM, 15 nM, 10 nM, 5 nM, 1 nM or less as measured by flow cytometry analysis; bind to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) as a purified protein, as measured by SPR analysis; bind to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) as a purified protein with K_D 3.5 nM or less as measured by SPR assay; bind to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) expressed on the cell surface as measured by flow cytometry analysis; bind to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30), expressed on the cell surface, with an apparent K_D 7.5 nM or less as measured by flow cytometry analysis; bind to the extracellular domain of human CD38 isoform E polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105) as a purified protein, as measured by ELISA analysis; bind to the extracellular domain of human CD38 isoform E polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105) expressed on the cell surface as measured by flow cytometry analysis; induce apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of a cell expressing CD38 on its cell surface; and have one or more mutations (for example, in the Fc region) resulting in decreased binding to FcγRI and/or FcγRII compared to the same binding protein in the absence of one or more mutations resulting in decreased binding to FcγRI and/or FcγRII compared to the same binding protein in absence of one or more mutations; induce T cell proliferation (e.g. CD4+ and/or CD8+ T cells); induce expression by T cells (e.g. CD4+ and/or CD8+ T cells) Bcl-xL; induce apoptosis of CD38+ cells; bind to CD38 expressed on the surface of the cell and one or more target T cell antigens expressed on the surface of the T cell; bind to CD38 expressed on the cell surface, CD28 expressed on the surface of the T cell, and CD3 expressed on the surface of the T cell; stimulate T-cell receptor activation; induce costimulation of T cell receptor signaling (e.g. CD28-mediated); and have one or more mutations (for example, in the Fc region), leading to a decrease in the induction of cytokine release (for example, IFN-γ, IL-2 and/or TNF-α) by PBMC compared to the same binding protein in the absence of one or more mutations; induce the release of cytokines (eg IFN-γ and/or IL-6) by PBMC in the presence of the target CD38+ cell. For exemplary assays, see Examples 1, 3, and 4. In some embodiments, KD is measured at 4°C or 25°C.

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) a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and/or (b) an antibody light chain variable domain (VL) comprising a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the antigen binding site comprises: (a) a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), the sequence CDR- H2 containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and the sequence CDR-H3 containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and/or (b) an antibody light chain variable domain (VL) comprising a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the antigen binding site comprises: (a) a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and/or (b) an antibody light chain variable domain (VL) comprising a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the VH domain comprises, in an N-terminal to C-terminal direction, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; where FR1 contains the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87) or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); where FR2 contains the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO:91); where FR3 contains the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); and where FR4 contains the sequence WGQGTLVTVSS (SEQ ID NO:96). In some embodiments, the VH domain contains the amino acid sequence of SEQ ID NO:5, and/or the VL domain contains 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 contains the amino acid sequence of SEQ ID NO:17, and/or the VL domain contains 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 contains the amino acid sequence of SEQ ID NO:21, and/or the VL domain contains 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 contains the amino acid sequence of SEQ ID NO:23, and/or the VL domain contains 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) a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); and (b) an antibody light chain variable domain (VL) comprising a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and the sequence CDR-L3 containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the VH domain comprises, in an N-terminal to C-terminal direction, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; where FR1 contains the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87) or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); where FR2 contains the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO:91); where FR3 contains the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); and where FR4 contains the sequence WGQGTLVTVSS (SEQ ID NO:96). In some embodiments, the VH domain contains the amino acid sequence of SEQ ID NO:13, and/or the VL domain contains 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.

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) a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence containing the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence containing the amino acid sequence ARMFRGAFDY (SEQ ID NO:43); and (b) an antibody light chain variable domain (VL) comprising a CDR-L1 sequence containing the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:45), and the sequence CDR-L3 containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46). In some embodiments, the VH domain contains the amino acid sequence of SEQ ID NO:9, and/or the VL domain contains 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 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 a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1 with an equilibrium dissociation constant ( _KD ) of 2.1 nM or less. In some embodiments, the antigen binding site binds a human CD38 isoform E polypeptide comprising the amino acid sequence of SEQ ID NO:105. In some embodiments, the antigen binding site binds a cynomolgus CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30. In some embodiments, the antigen binding site binds a cynomolgus 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.

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 human monoclonal antibody. In some embodiments, the binding protein comprises one or more full-length antibody heavy chains containing an Fc region. In some embodiments, the Fc region is a human Fc region containing one or more mutations that reduce or eliminate Fc receptor binding and/or Fc region effector function. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region of human IgG1 contains amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG1 according to the EU index, where the amino acid substitutions are L234A, L235A and P329A. In some embodiments, the Fc region of human IgG1 contains amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1 according to the EU index, where 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 Fc region of human IgG4 contains amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P and R409K. In some embodiments, the human IgG4 Fc region contains amino acid substitutions at positions corresponding to human IgG4 positions 234 and 235 according to the EU index, where the amino acid substitutions are F234A and L235A. In some embodiments, the human IgG4 Fc region contains amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index, where the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, The Fc region of human IgG4 contains amino acid substitutions at positions corresponding to positions 233-237 of human IgG4 according to the EU index, where the sequence EFLGG is replaced by the sequence PVAG. In some embodiments, the binding protein comprises an F(ab)-, F(ab')2-, Fab'-SH-, Fv-, or scFv-fragment of an antibody. 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 a first antigen binding site that binds a CD38 polypeptide and a second antigen binding site. In some embodiments, the binding protein is a trispecific binding protein comprising a first antigen binding site that binds a 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 each of the second and third antigen binding sites binds a T cell surface protein. In some embodiments, the first antigen binding site binds an 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 the antigen binding site binds the human CD28 polypeptide.

In some embodiments, provided herein is a binding protein comprising three antigen binding sites, each of which binds 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 macaque CD38 polypeptide. crabeater. 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 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 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 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 three antigen binding sites, wherein the first polypeptide chain contains the structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I];

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

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

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

where (a) domain V _{of H1} contains a CDR-H1 sequence containing the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the _L1 domain V contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO: 36);

(b) the V _H2 domain contains a CDR-H1 sequence containing the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and the CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the _L2 domain V contains the CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36 ); or

(c) domain V of _H3 contains a CDR-H1 sequence containing the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and the CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the _L3 domain V contains the CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36 ). In some embodiments, the binding protein comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain contains the structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I];

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

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

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

where (a) domain V _{of H1} contains a CDR-H1 sequence containing the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the _L1 domain V contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO: 36);

(b) the V _H2 domain contains a CDR-H1 sequence containing the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and the CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the _L2 domain V contains the CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36 ); or

(c) domain V of _H3 contains a CDR-H1 sequence containing the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32), or IYPGQGGT (SEQ ID NO:38), and the CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the _L3 domain V contains the CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36 ). In some embodiments, the _H1 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the V _L1 domain contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36); domain V of _H1 contains a CDR-H1 sequence containing the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and domain V _L1 contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO: 36); domain V of _H2 contains a CDR-H1 sequence containing the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and domain V _L2 contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36); domain V of _H2 contains a CDR-H1 sequence containing the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the _L2 domain contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40) and a CDR-L3 sequence containing amino acid sequence QQNKEDPWT (SEQ ID NO: 36); domain V of _H3 contains a CDR-H1 sequence containing the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence containing the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33), and domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO: 36); or the V domain of _H3 contains a CDR-H1 sequence containing the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY ( SEQ ID NO:33), and domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and a CDR- sequence L3 containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the V _L3 domain contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36); or the V domain of _H3 contains a CDR-H1 sequence containing the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY ( SEQ ID NO:33), and domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and a CDR- sequence L3 containing the amino acid sequence 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 contains the amino acid sequence of SEQ ID NO:6; the V _H3 domain contains the amino acid sequence of SEQ ID NO:17, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:18; the V _H3 domain contains the amino acid sequence of SEQ ID NO:21, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:18; the V _H3 domain contains the amino acid sequence of SEQ ID NO:23, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:18; or the V _H3 domain contains the amino acid sequence of SEQ ID NO:13, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:14. In some embodiments, the binding protein comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain contains the structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I];

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

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

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

where (a) the _H1 domain contains a CDR-H1 sequence containing the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence containing the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence containing the amino acid sequence ARMFRGAFDY (SEQ ID NO:43), and domain V _L1 contains the sequence CDR-L1 containing the amino acid sequence QGIRND (SEQ ID NO:44), the sequence CDR-L2 containing the amino acid sequence AAS (SEQ ID NO:45), and the sequence CDR-L3 containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46);

(b) the V domain of _H2 contains a CDR-H1 sequence containing the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence containing the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence containing the amino acid sequence ARMFRGAFDY (SEQ ID NO:43), and the V _L2 domain contains a CDR-L1 sequence containing the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:45), and the sequence CDR-L3 containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46); or

(c) domain V of _H3 contains a CDR-H1 sequence containing the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence containing the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence containing the amino acid sequence ARMFRGAFDY (SEQ ID NO:43), and domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:45), and the sequence CDR-L3 containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARMFRGAFDY (SEQ ID NO:43), and the V _L3 domain contains a CDR-L1 sequence containing the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:45), and a CDR-L3 sequence containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46). In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:9, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:10. In some embodiments, the V _H1 domain contains the amino acid sequence of SEQ ID NO:49, the V _L1 domain contains the amino acid sequence of SEQ ID NO:50, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L2 domain contains the amino acid sequence under SEQ ID NO:54; the V _H2 domain contains the amino acid sequence of SEQ ID NO:49, the V _L2 domain contains the amino acid sequence of SEQ ID NO:50, the V _H1 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L1 domain contains the amino acid sequence of SEQ ID NO: :54; the V _H1 domain contains the amino acid sequence of SEQ ID NO:51, the V _L1 domain contains the amino acid sequence of SEQ ID NO:52, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L2 domain contains the amino acid sequence of SEQ ID NO: :54; the V _H2 domain contains the amino acid sequence of SEQ ID NO:51, the V _L2 domain contains the amino acid sequence of SEQ ID NO:52, the V _H1 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L1 domain contains the amino acid sequence of SEQ ID NO: :54, the V _H1 domain contains the amino acid sequence of SEQ ID NO:49, the V _L1 domain contains the amino acid sequence of SEQ ID NO:50, the V _H2 domain contains the amino acid sequence of SEQ ID NO:84, and the V _L2 domain contains the amino acid sequence of SEQ ID NO:85; the V _H2 domain contains the amino acid sequence of SEQ ID NO:49, the V _L2 domain contains the amino acid sequence of SEQ ID NO:50, the V _H1 domain contains the amino acid sequence of SEQ ID NO:84, and the V _L1 domain contains the amino acid sequence of SEQ ID NO: :85; the V _H1 domain contains the amino acid sequence of SEQ ID NO:51, the V _L1 domain contains the amino acid sequence of SEQ ID NO:52, the V _H2 domain contains the amino acid sequence of SEQ ID NO:84, and the V _L2 domain contains the amino acid sequence of SEQ ID NO: :85; or the V _H2 domain contains the amino acid sequence of SEQ ID NO:51, the V _L2 domain contains the amino acid sequence of SEQ ID NO:52, the V _H1 domain contains the amino acid sequence of SEQ ID NO:84, and the V _L1 domain contains the amino acid sequence of SEQ ID NO:85. In some embodiments, the V _H1 domain contains the amino acid sequence of SEQ ID NO:49, the V _L1 domain contains the amino acid sequence of SEQ ID NO:50, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, the V _L2 domain contains the amino acid sequence of SEQ ID NO:54, the V _H3 domain contains the amino acid sequence of SEQ ID NO:13, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:14; or the V _H1 domain contains the amino acid sequence of SEQ ID NO:49, the V _L1 domain contains the amino acid sequence of SEQ ID NO:50, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, the V _L2 domain contains the amino acid sequence of SEQ ID NO :54, the V _H3 domain contains the amino acid sequence of SEQ ID NO:9, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:10. In some embodiments, the length of at least one of L ₁ , L ₂ , L ₃ or L ₄ is independently 0 amino acids. In some embodiments, L ₁ , L ₂ , L ₃ , or L ₄ are each independently at least one amino acid long. In some embodiments, (a) each of L ₁ , L ₂ , L ₃ and L ₄ independently has a length of zero amino acids, or contains a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59); or (b) each of L ₁ , L ₂ , L ₃ and L ₄ independently contains a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58) and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L ₁ contains the sequence GQPKAAP (SEQ ID NO: 58), L ₂ contains the sequence TKGPS (SEQ ID NO: 57), L ₃ contains the sequence S, and L ₄ contains the sequence RT; L ₁ contains the sequence GGGGSGGGGS (SEQ ID NO: 55), L ₂ contains the sequence GGGGSGGGGS (SEQ ID NO: 55), the length of L ₃ is 0 amino acids, and the length of L ₄ is 0 amino acids; L ₁ contains the sequence GGSGSSGSGG (SEQ ID NO: 59), L ₂ contains the sequence GGSGSSGSGG (SEQ ID NO: 59), the length of L ₃ is 0 amino acids, and the length of L ₄ is 0 amino acids; or L ₁ contains the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), the length of L ₂ is 0 amino acids, L ₃ contains the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), and the length of L ₄ is 0 amino acids. In some embodiments, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are hinge-C _H2 -C _H3 domains of human IgG4, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding positions 234 and 235 of human IgG4 according to the EU index, where the amino acid substitutions are F234A and L235A. In some embodiments, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are hinge-C _H2 -C _H3 domains of human IgG4, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding positions 233-236 of human IgG4 according to the EU index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are hinge domains -C _H2 -C _H3 human IgG4, and wherein each of the hinge domains -C _H2 -C _H3 contains amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P and R409K . In some embodiments, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are hinge-C _H2 -C _H3 domains of human IgG1, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG1 according to the EU index, where the amino acid substitutions are L234A, L235A and P329A. In some embodiments, the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are hinge-C _H2 -C _H3 domains of human IgG1, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1 according to the EU index, where the amino acid substitutions are S298N, T299A and Y300S. In some embodiments, the hinge-C _H2 -C _H3 domain of the second polypeptide chain contains amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y349C, T366S, L368A and Y407V; and wherein the hinge-C _H2 -C _H3 domain of the third polypeptide chain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-C _H2 -C _H3 domain of the second polypeptide chain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W; and wherein the hinge-C _H2 -C _H3 domain of the third polypeptide chain contains amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V. In some embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:60, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:62, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:63. In some embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:64, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:65, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:63. In some embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:66, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:67, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:63. In some embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:60, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:68, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:69. In some embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:64, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:70, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:69. In some embodiments, the first polypeptide chain contains the amino acid sequence of SEQ ID NO:61, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:66, the third polypeptide chain contains the amino acid sequence of SEQ ID NO:71, and the fourth polypeptide chain contains the amino acid sequence under SEQ ID NO:69.

In some embodiments, provided herein is a binding protein comprising three antigen binding sites, each of which binds one or more target proteins, wherein the binding protein contains four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain contains a structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I];

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

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

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

wherein (a) the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are the hinge-C _H2 -C _H3 domains of human IgG1, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to 298, 299 and 300 human IgG1 according to the EU index, where the amino acid substitutions are S298N, T299A and Y300S; or (b) the hinge-C _H2 -C _H3 domains of the second and third polypeptide chains are hinge-C _H2 -C _H3 domains of human IgG4, and wherein each of the hinge-C _H2 -C _H3 domains contains amino acid substitutions at positions corresponding to 233-236 human IgG4 according to the EU index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the hinge-C _H2 -C _H3 domains of human IgG4 include amino acid substitutions at positions corresponding to 233-237 Human IgG4 according to the EU index, where the sequence EFLGG is replaced by the sequence 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 the 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 a target antigen 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, chitinases, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3 , DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, 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, the 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, the second pair of V _H1 and V _L1 , V _H2 and V _L2 , as well as V _H3 and V _L3, forms an antigen binding site that binds human CD28 polypeptide, and the 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 the target antigen human, 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, chitinases, CLEC9, CLEC91, CRTH2, CSF-1, CSF- 2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM , ICOSLG, IDO, IFNα, IgE, IGF1R, 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, provided herein is a set of polynucleotides comprising: (a) a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:72, a third polynucleotide comprising the sequence of SEQ ID NO: 74, and a fourth polynucleotide containing the sequence of SEQ ID NO:75; (b) a first polynucleotide containing the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:76, a third polynucleotide containing the sequence of SEQ ID NO:77, and a fourth polynucleotide containing the sequence of SEQ ID NO: 75; (c) a first polynucleotide containing the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:78, a third polynucleotide containing the sequence of SEQ ID NO:79, and a fourth polynucleotide containing the sequence of SEQ ID NO: 75; (d) a first polynucleotide containing the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:72, a third polynucleotide containing the sequence of SEQ ID NO:80, and a fourth polynucleotide containing the sequence of SEQ ID NO: 81; (e) a first polynucleotide containing the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:76, a third polynucleotide containing the sequence of SEQ ID NO:82, and a fourth polynucleotide containing the sequence of SEQ ID NO: 81; or (f) a first polynucleotide containing the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:78, a third polynucleotide containing the sequence of SEQ ID NO:83, and a fourth polynucleotide containing the sequence of SEQ ID NO: :81.

In some embodiments, provided herein is a polynucleotide comprising a binding protein according to any of the above embodiments. In some embodiments, provided herein is a vector containing a polynucleotide that contains a binding protein according to any of the above embodiments.

In some embodiments, provided herein is a host cell containing a set of polynucleotides, a polynucleotide, or a vector according to any of the above embodiments. In some embodiments, provided herein is a method for producing a binding protein, the method comprising culturing a host cell in accordance with any of the above embodiments to thereby produce the binding protein. In some embodiments, the method further comprises retrieving the binding protein from the host cell.

In some embodiments, provided herein is a pharmaceutical composition comprising a binding protein in accordance with any of the above embodiments and a pharmaceutically acceptable carrier.

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 in accordance with any of the above embodiments or a pharmaceutical composition in accordance with any of the above embodiments. implementation. 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 B-cell lymphoma. In some embodiments, the patient is a human. In some embodiments, the patient is selected for treatment because the cancer cells express human isoform E CD38 polypeptide (e.g. set forth under SEQ ID NO:105) on its 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.

In some embodiments, provided herein is at least one binding protein in accordance with any of the above embodiments or a pharmaceutical composition in accordance with any of the above embodiments for use in the prevention and/or treatment of cancer in a patient (eg, in a patient in need, such as a patient with cancer). In some embodiments, provided herein is at least one binding protein in accordance with any of the above embodiments or a pharmaceutical composition in accordance with any of the above embodiments for use in the manufacture of a medicament for the prevention and/or treatment of cancer in the patient (for example, in a patient in need, such as a patient with cancer). In some embodiments, in accordance with 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 B-cell lymphoma. In some embodiments, the patient is a human. In some embodiments, the patient is selected for treatment because the cancer cells express human isoform E CD38 polypeptide (e.g. set forth under SEQ ID NO:105) on its 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.

It should be understood that one, more, or all of the features of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to one skilled in the art. These and other embodiments of the present invention are further described in the following detailed description.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

The file for this patent or this application contains at least one graphic material in color. Copies of this patent or patent application publication with color graphic material(s) will be provided by the patent office upon request and upon payment of the required fee.

In fig. 1A shows the binding of anti-CD38 mAb1 (top) and isatuximab (bottom) to SU-DHL-8 human lymphoma cells or MOLP-8 human multiple myeloma cells as determined by flow cytometry analysis.

In fig. 1B shows the results of flow cytometry binding assays that examined the binding of anti-CD38 mAb1 or isatuximab (no binding observed) to cells expressing cynomolgus CD38 on their surface.

In fig. 2A-2I show assay results characterizing the binding of anti-CD38 antibodies to human and cynomolgus CD38 polypeptides. In fig. 2A shows the binding of a humanized anti-CD38 mAb2 to soluble human CD38 (top, “hCD38::His tag”) or cynomolgus CD38 (top, “cynoCD38::His tag”) by ELISA assay, as well as binding of mAb2 to surface of cells expressing human CD83 (as indicated below) or cynomolgus CD38 (as indicated below), as determined by flow cytometry analysis. In fig. 2B shows the binding of mAb2 to human CD38 (top) or cynomolgus CD38 (bottom) as measured by surface plasmon resonance (SPR). In fig. 2C shows the binding of a humanized anti-CD38 mAb3 to soluble human CD38 (top, “hCD38::His tag”) or cynomolgus CD38 (top, “cynoCD38::His tag”) by ELISA assay, as well as binding of mAb3 to surface of cells expressing human CD83 (as indicated below) or cynomolgus CD38 (as indicated below) as determined by flow cytometry analysis. In fig. 2D shows the binding of mAb3 to human CD38 (top) or cynomolgus CD38 (bottom) as determined by SPR. In fig. 2E shows the binding of a humanized anti-CD38 mAb5 to soluble human CD38 (top, “hCD38::His tag”) or cynomolgus CD38 (top, “cynoCD38::His tag”) by ELISA assay, as well as mAb5 binding to surface of cells expressing human CD83 (as indicated below) or cynomolgus CD38 (as indicated below), as determined by flow cytometry analysis. In fig. 2F shows the binding of mAb5 to human CD38 (top) or cynomolgus CD38 (bottom) as determined by SPR. In fig. 2G shows the binding of human anti-CD38 antibody hhy1370 to soluble human CD38 (top, “hCD38::His tag”) or cynomolgus CD38 (top, “cynoCD38::His tag”) by ELISA, as well as binding of hhy1370 to the surface cells expressing human CD83 (as indicated below) or cynomolgus CD38 (as indicated below), as determined by flow cytometry analysis. In fig. 2H shows the binding of hhy1370 to human CD38 (top) or cynomolgus CD38 (bottom) as determined by SPR. In fig. 2I summarizes the binding data from ELISA, SPR and FACS assays and the percentage identity of each antibody VH ("H") or VL ("L") domain to the human V region sequence from top to bottom, corresponding to mAb2, mAb3, mAb 4, mAb 5 and mAb6 in the last line (1195 HHKK-3 is not described by its VL/VH amino acid sequence in the text below).

In fig. 2J shows the concentration-dependent induction of apoptosis of SU-DHL-8 cells by mAb7 and mAb1 after incubation for 72 hours at 37°C.

In fig. 2K shows the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isatuximab and mAb1 against SU-DHL-8 cells in the presence of NK92 cells.

In fig. 2L shows the concentration-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isatuximab (right) and mAb1 (left) against SU-DHL-8 cells in the presence of NK92 cells after 4 hours at 37°C.

Onfig. 2M-2Q shows the results of apoptosis induction assays using the indicated anti-CD38 antibodies against SU-DHL-8 tumor cells. Apoptosis was quantified by paired measurements of annexin V and propidium iodide uptake using flow cytometry. Onfig. 2M the percentage of cells with apoptosis induced by each antibody is shown. Onfig. 2N - 2Q showed dose-dependent induction of apoptosis in SU-DHL-8 lymphoma cells by mAb2 antibodies to CD38 (fig. 2N), mAb3 (fig. 2O), mAb4 (fig. 2P) and mAb5 (fig. 2Q), as well as the IC50 for each antibody.

Onfig. 3Adepicts a schematic representation of a trispecific binding protein containing four polypeptide chains that form three antigen binding sites that bind three target proteins: CD28, CD3 and CD38. The first pair of polypeptides has double variable domains having a crossover orientation (VH1-VH2 and VL2-VL1), forming two antigen-binding sites that recognize CD3 and CD28, and the second pair of polypeptides has one variable domain (VH3 and VL3), forming one antigen-binding site site that recognizes CD38. In the trispecific binding protein shown infig. 3A, the IgG4 constant region is used with a knob-to-trench mutation, where the knob is on the second pair of polypeptides with one variable domain.

In fig. 3B depicts a schematic representation of an SPR-based assay for examining the ability of each antigen binding domain of the anti-CD38/CD28/CD3 trispecific binding proteins to bind its cognate antigen.

In fig. 3C shows the results of SPR-based assays to study the binding of CD38 to CD38/CD28/CD3 trispecific binding proteins. Binding of human CD38 to trispecific binding proteins was examined alone (top left), after prebinding to CD3 (top center), after prebinding to CD28 (top right), after prebinding to CD3 then CD28 (bottom left), or after prebinding binding to CD28, then CD3 (bottom right).

In fig. Figure 4 shows the sequential binding of human CD3, CD28 and CD38 polypeptides to CD38/CD28/CD3 trispecific binding proteins as determined by SPR analysis.

In fig. 5 shows a summary of the binding affinities of these trispecific binding proteins to their cognate antigens (human CD3, CD28 and CD38) as measured by SPR.

In fig. Figure 6A compares the apparent affinity of the antigen binding domain of isatuximab in human IgG1 format (sheet ² ) or trispecific binding protein format with the antigen binding domains of isatuximab, anti-CD28, and anti-CD3 (format as in Fig. 3A ; sheet ¹ ) to bind human (top) or cynomolgus (bottom) CD38 polypeptides as determined by flow cytometry analysis.

In fig. 6B - 6D compare the apparent affinities of trispecific binding protein CD38 _VH1 xCD28 _sup xCD3 _mid , CD38 _VH1 xCD28 _cvn xCD3 _mid , or monospecific anti-CD38 mAb2 to bind to cells expressing human or cynomolgus CD38 polypeptides as determined by flow cytometry analysis. In fig. 6B shows the binding of CD38 trispecific binding protein _VH1 xCD28 _sup xCD3 _mid to cells expressing human (top) or cynomolgus (bottom) CD38 polypeptides. In fig. 6C shows the binding of trispecific binding protein CD38 _VH1 xCD28 _cvn xCD3 _mid to cells expressing human (top) or cynomolgus (bottom) CD38 polypeptides. In fig. 6D shows the binding of monospecific anti-CD38 mAb2 to cells expressing human (top) or cynomolgus (bottom) CD38 polypeptides.

In fig. 6E shows a comparison of the apparent affinities of the CD38 trispecific binding protein _HHY1370 xCD28 _sup xCD3 _mid or the anti-CD38 monospecific antibody mAb6 for binding to cells expressing human (top) or cynomolgus (bottom) CD38 polypeptides as determined by flow cytometry analysis.

In fig. 6F summarizes the binding affinities of the indicated CD38x-CD28x-CD3 trispecific binding proteins for human CD38 as measured by SPR or flow cytometry (FACS).

In fig. 6G shows the apparent affinity of the trispecific binding protein ΔCD38 _VH1 xCD28 _sup xCD3 _mid , which lacks the antigen binding domain of the anti-CD38 antibody, to bind to cells expressing human (top) or cynomolgus (bottom) CD38 polypeptides, as determined by flow cytometry analysis.

In fig. 7A and 7B show the results of ELISA assays for the binding affinities of various anti-CD38 x CD28 x CD3 IgG4 trispecific binding proteins or control antibodies to human and cynomolgus CD3, CD28 and CD38 polypeptides.

In fig. 8A-8D show the results of antibody-mediated specific killing of CD38 ⁺ cells by PBMC from three different human donors using the indicated CD38 x CD28 x CD3 trispecific binding protein antibodies and control antibodies. Representative results using multiple myeloma cell lines RPMI8266 ( Figure 8A ), NCI-H929 ( Figure 8B ), KMS-26 ( Figure 8C ), and KMS-11 cell lines ( Figure 8D ) are shown, and EC50 values are presented in Table N. EC50 values obtained using NCI-H929, KMS-26 and KMS-11 cells are presented in the OQ tables.

In fig. 8E and 8F show the results of antibody-mediated specific killing of CD38 ⁺ cells by PBMC from two different donors using the indicated CD38 x CD28 x CD3 Fc region variant trispecific binding protein antibodies and control antibodies. Representative results using KMS-11 CD38 ⁺ ( Figure 8D ) and U266 ( Figure 8E ) cell lines are shown, and EC50 values are presented in Tables Q2 and Q3.

Onfig. 9A, 9B and 10activation shown (CD69⁺) human T cells treated with various anti-CD38 x CD28 x CD3 trispecific binding protein antibodies or control antibodies for 24 hours. Onfig. 9Aactivation shown (CD69⁺) CD3⁺Human T cells. Onfig. 9Vactivation shown (CD69⁺) CD3⁺ CD4⁺Human T cells. Onfig. 10activation shown (CD69⁺) CD3⁺ CD8⁺Human T cells.

FIG . 11A - 11B show the results of in vitro evaluations of cytokine release from human PBMCs treated with the indicated CD38 x CD28 x CD3 trispecific binding protein antibodies or control antibodies based on the plate-and-dry method as described in Stebbings, R. et al. (2007) J. Immunol. 179:3325–3331. FIG . 11A shows the results obtained using 5 μg/ml of the indicated antibodies. FIG . 11B shows the results obtained using 25 ng/ml of the indicated antibodies.

Onfig.12A - 12E activity shownin vivoCD38 trispecific binding protein_(VHI) xCD28_(sup) xCD3_(mid) in umbilical cord blood CD34+ cells from a humanized NSG mouse model implanted with RPMI-8226 multiple myeloma cells. Onfig.12Ashows changes in tumor volume in mice injected with trispecific binding protein-antibody to CD38_(VHI) xCD28_(sup) xCD3_(mid) at the indicated concentrations, compared with mice treated with a bispecific antibody to CD3/CD38. Onfig. 12Vshows the mean tumor volume at day 18 in mice treated with CD38 trispecific binding protein antibody_(VHI) xCD28_(sup) xCD3_(mid) at the indicated concentrations, compared with mice treated with a bispecific antibody to CD3/CD38. Onfig. 12Cshows the average final tumor weight in mice treated with CD38 trispecific binding protein-antibody_(VHI) xCD28_(sup) xCD3_(mid) at the indicated concentrations, compared with mice treated with a bispecific antibody to CD3/CD38. Onfig. 12Dshows the average tumor growth curve during an experiment in mice injected with trispecific binding protein-antibody to CD38_(VHI) xCD28_(sup) xCD3_(mid) at the indicated concentrations, compared with mice treated with a bispecific antibody to CD3/CD38. Onfig. 12Eshows the average change in body weight at several time points during the experiment in mice injected with CD38 trispecific binding protein antibody_(VHI) xCD28_(sup) xCD3_(mid) at the indicated concentrations, compared with mice treated with a bispecific antibody to CD3/CD38.

In fig. 13A - 13F show the in vivo activity of trispecific binding protein CD38 _(VHI) x CD28 _(sup) x CD3 _(mid) in PBMC cells of the humanized NSG mouse model implanted with RPMI-8226 multiple myeloma cells. FIG . 13A shows the change in tumor volume in mice treated with CD38 trispecific binding protein _(VHI) x CD28 _(sup) x CD3 _(mid) at the indicated concentrations compared to mice treated with CD3/CD38 bispecific antibody. In fig. 13B shows tumor volume at day 4 in mice treated with CD38 trispecific binding protein _(VHI) x CD28 _(sup) x CD3 _(mid) at the indicated concentrations compared to mice treated with CD3/CD38 bispecific antibody. In fig. 13C shows tumor volume at day 21 in mice treated with CD38 trispecific binding protein _(VHI) x CD28 _(sup) x CD3 _(mid) at the indicated concentrations compared to mice treated with CD3/CD38 bispecific antibody. In fig. 13D shows the average tumor volume at day 21 in mice treated with anti-CD38 _(VHI) x CD28 _(sup) x CD3 _(mid) trispecific binding protein antibody at the indicated concentrations, compared with mice treated with anti-CD3/CD38 bispecific antibody. In fig. 13E shows the average final tumor weight of mice treated with anti-CD38 _(VHI) x CD28 _(sup) x CD3 _(mid) trispecific binding protein antibody at the indicated concentrations compared with mice treated with anti-CD3/CD38 bispecific antibody. In fig. 13F shows the mean tumor volume at several time points during the experiment in mice treated with CD38 trispecific binding protein _(VHI) x CD28 _(sup) x CD3 _(mid) at the indicated concentrations, compared with mice treated with anti-CD38 bispecific antibody. CD3/CD38.

Onfig. 14A - 14U shows results from a dose escalation study (0.5, 2.5, 12.5 μg/kg) using CD38 trispecific binding protein antibodies in non-human primates_(VHI) xCD28_(sup) xCD3_(mid), CD38_(VHI) xCD28_(cvn) xCD3_(mid), CD38_(hhy1370) xCD28_(sup) xCD3_(mid) and CD38_(hhy1370) xCD28_(cvn) xCD3_(mid). Onfig. 14A shows T cell activation (CD69⁺) circulating CD3⁺ T cells after administration of different doses of trispecific binding protein antibody to CD38_(VHI) xCD28_(sup) xCD3_(mid). Onfig. 14V shows T cell activation (CD69⁺) circulating CD3⁺ T cells after administration of trispecific binding protein antibody to CD38_(VHI) xCD28_(cvn) xCD3_(mid) in different doses. Onfig. 14C shows T cell activation (CD69⁺) circulating CD3⁺ T cells after administration of trispecific binding protein antibody to CD38_(hhy1370) xCD28_(sup) xCD3_(mid) in different doses. Onfig. 14D shows T cell activation (CD69⁺) circulating CD3⁺ T cells after administration of trispecific binding protein antibody to CD38_(hhy1370) xCD28_(cvn) xCD3_(mid) in different doses. Onfig. 14E shows change in percentage of circulating CD4⁺ T cells after administration of trispecific binding protein antibody to CD38_(VHI) xCD28_(sup) xCD3_(mid) in the indicated doses. Onfig. 14F shows change in percentage of circulating CD8⁺ T cells after administration of trispecific binding protein antibody to CD38_(VHI) xCD28_(sup) xCD3_(mid) in the indicated doses. Onfig. 14G shows change in percentage of circulating CD4⁺ T cells after administration of trispecific binding protein antibody to CD38_(VHI) xCD28_(cvn) xCD3_(mid) in the indicated doses. Onfig. 14H shows change in percentage of circulating CD8⁺ T cells after administration of trispecific binding protein antibody to CD38_(VHI) xCD28_(cvn) xCD3_(mid) in the indicated doses. Onfig. 14I shows change in percentage of circulating CD4⁺ T cells after administration of trispecific binding protein antibody to CD38_(hhy1370) xCD28_(sup) xCD3_(mid) in the indicated doses. Onfig. 14J shows change in percentage of circulating CD8⁺ T cells after administration of trispecific binding protein antibody to CD38_(hhy1370) xCD28_(sup) xCD3_(mid) in the indicated doses. Onfig. 14K shows change in percentage of circulating CD4⁺ T cells after administration of trispecific binding protein antibody to CD38_(hhy1370) xCD28_(cvn) xCD3_(mid) in the indicated doses. Onfig. 14L shows change in percentage of circulating CD8⁺ T cells after administration of trispecific binding protein antibody to CD38_(hhy1370) xCD28_(cvn) xCD3_(mid) in the indicated doses. Onfig. 14M shows changes in total CD4 count⁺ T cells 6, 24, and 48 hours after administration of 12.5 μg/kg of the indicated trispecific binding proteins. Onfig. 14N shows changes in total NK cell counts 6, 24, and 48 hours after administration of 12.5 μg/kg of the indicated trispecific binding proteins. Onfig. 14O shows changes in total CD8 count⁺ T cells 6, 24, and 48 hours after administration of 12.5 μg/kg of the indicated trispecific binding proteins. Onfig. 14P shows changes in total B cell counts 6, 24, and 48 hours after administration of 12.5 μg/kg of the indicated trispecific binding proteins. Onfig. 14Q shows changes in cytokine levels 6 hours after administration of CD38 trispecific binding protein antibody_(VH1) xCD28_(sup) xCD3_(mid) in three increasing doses (0.5, 2.5, 12.5 μg/kg) (results using different experimental animals designated as “117065” and “117066”). Onfig. 14R shows changes in cytokine levels 6 hours after administration of CD38 trispecific binding protein antibody_(VH1) xCD28_(cvn) xCD3_(mid) in three increasing doses (0.5, 2.5, 12.5 μg/kg) (results using different experimental animals designated as “117067” and “117068”). Onfig. 14S shows changes in cytokine levels 6 hours after administration of CD38 trispecific binding protein antibody_(hhy1370) xCD28_(sup) xCD3_(mid) in three increasing doses (0.5, 2.5, 12.5 μg/kg) (results using different experimental animals designated as “117069” and “117070”). Onfig. 14T shows changes in cytokine levels 6 hours after administration of CD38 trispecific binding protein antibody_(hhy1370) xCD28_(cvn) xCD3_(mid) in three increasing doses (0.5, 2.5, 12.5 μg/kg) (results using different experimental animals designated as “117071” and “117072”). Onfig. 14U shows changes in cytokine levels 24 hours after administration of the indicated trispecific binding proteins at three increasing doses (0.5, 2.5, 12.5 μg/kg) (results shown for all experimental animals).

In fig. 14V and 4W show that trispecific binding protein antibodies to CD38 _(VHI) × CD28 _(sup) × CD3 _(mid) and CD38 _(VHI) × CD28 _(cvn) × CD3 _(mid) in vivo induced exhaustion of T cells in the blood in non-human primates at higher doses (6 hours after administration).

In fig. 14X - 14Y showed that trispecific binding proteins-antibodies to CD38 _(HHY1370) × CD28 _(sup) × CD3 _(mid) and CD38 _(HHY1370) × CD28 _(cvn) × CD3 _(mid) in vivo induced exhaustion of T cells in the blood in non-human primates at higher doses (6 hours after administration).

In fig. 14Z - 14AA shows the number of blood T cells in non-human primates over time after administration of trispecific binding protein antibodies to CD38 _(VHI) × CD28 _(sup) × CD3 _(mid) or CD38 _(VHI) × CD28 _{(cvn )} × CD3 _(mid) .

In fig. 14AB - 14AC show the number of blood T cells in non-human primates over time after administration of trispecific binding protein antibodies to CD38 _(HHY1370) × CD28 _(sup) × CD3 _(mid) or CD38 _(HHY1370) × CD28 _{(cvn )} × CD3 _(mid) .

In fig. 14AD - 14AE show the number of trispecific binding protein-bound CD4+ T cells following a 100 μg/kg dose in non-human primates.

In fig. 14AF - 14AG shows the number of CD8+ T cells associated with trispecific binding protein after a dose of 100 μg/kg in non-human primates.

In fig. 15A - 15C show the binding or lack of binding of various Fc variants to the human FcγR I ( FIG. 15A ), FcγR IIa ( FIG. 15B ), and FcγR IIIb/c ( FIG. 15C ) Fc receptors. The variants tested were human IgG1, human IgG4, and human IgG4 with FALA mutations.

In fig. 16 shows binding of human IgG4 with FALA mutations or in the absence of mutations to FcRn.

In fig. 17 summarizes the PK parameters of the indicated trispecific binding proteins (CD38 _VH1 ×CD28 _sup ×CD3 _mid IgG4, CD38 _VH1 ×CD28 _sup ×CD3 _mid IgG4 FALA, CD38 _VH1 ×CD28 _sup ×CD3 _mid IgG1 LALA P329A and CD38 _HHY1370 ×CD28 _sup ×CD3 _mid IgG4 FALA) in NSG mice.

In fig. 18A - 18C show Fc/FcR interaction-mediated (nonspecific) release of IFN-γ ( FIG. 18A ), IL-2 ( FIG. 18B ), or TNF-α ( FIG. 18C ) from human PBMCs incubated with Fc trispecific binding proteins -wild type regions or FALA variants.

In fig. 18D shows in vitro activation of human PBMC by trispecific binding proteins anti-CD38 _VH1 x CD28 _sup x CD3 _mid and CD38 _HHY1370 x CD28 _sup x CD3 _{mid and} their Fc variants IgG1 and IgG4.

In fig. 19A - 19B show that induction of Bcl-xL in CD4+ ( FIG. 19A ) or CD8+ ( FIG. 19B ) T cells by the anti-CD38 trispecific binding protein _VH1 ×CD28 _sup ×CD3 _mid requires the CD3 and CD28 antigen binding domains. Histogram=mean and std. deviation from 3 PBMC donors. *p= < 0.009.

In fig. 19C - 19D show that CD38 _VH1 xCD28xCD3 with the IgG4 FALA Fc variant activates Bcl-xL in CD4+ ( FIG. 19C ) or CD8+ ( FIG. 19D ) T cells to a greater extent than the comparative bispecific antibody. Histogram=mean and std. deviation from 3 PBMC donors. *p= < 0.045

In fig. 19E shows that activation of T cells by anti-CD38x-CD28x-CD3 trispecific binding proteins, as determined by IL-2 expression analysis in the Jurkat T-cell reporter line, is dependent on the antigen-binding domain of the anti-CD3 antibody.

In fig. 19F shows the release of the cytokines TNF, IFNg, IL-2, IL-6 and IL-10 by trispecific anti-CD38 binding proteins _VH1 xCD28 _sup xCD3 _mid compared with mutant anti-CD28, CD38 or CD28, CD38 and CD3 binding proteins , as well as control.

In fig. 19G shows the proliferation of T cells activated by trispecific binding protein antibody to CD38x-CD28x-CD3 with Fc variant IgG4 FALA, a comparative bispecific antibody to CD38x-CD3, or an isotype control (trispecific binding protein with Fc variant IgG4 FALA, characterized by mutant binding domains).

In fig. 20 shows the proliferation of T cells activated by trispecific binding protein antibody to CD38x-CD28x-CD3 with Fc variant IgG4 FALA, trispecific binding protein antibody to CD38x-CD28x-CD3 with Fc variant IgG4 FALA and mutation in binding CD38, CD28 or CD3 domain or isotype control (trispecific binding protein with the Fc variant of IgG4 FALA having three mutant binding domains).

In fig. 21 shows the in vivo antitumor activity of the anti-CD38 trispecific binding protein antibody _VH1 xCD28 _sup xCD3 _mid IgG4 FALA, administered at the indicated doses, in the NCI-H929-Luc disseminated tumor model in NSG mice humanized with human PBMC.

In fig. 22 shows the in vivo antitumor activity of the anti-CD38 trispecific binding protein antibody _HHY1370 xCD28 _sup xCD3 _mid IgG4 FALA, administered at the indicated doses, in the NCI-H929-Luc disseminated tumor model in NSG mice humanized with human PBMC.

In fig. 23A - 23B show high in vitro antitumor activity against NCI-929-Luc cells using the trispecific binding proteins CD38 _VH1 xCD28 _sup xCD3 _mid and CD38 _HHY1370 xCD28 _sup xCD3 _mid , as well as a comparative bispecific antibody to CD38x-CD3 using human PBMC, used in the in vivo study. Human PBMCs from two humanized NSG donor mice were used after 24 hours of incubation at an effector:PBMC ratio of 10:1.

In fig. 23C demonstrates in vivo superior antitumor activity of trispecific binding proteins CD38 _VH1 xCD28 _sup xCD3 _mid and CD38 _hhy1370 xCD28 _sup xCD3 _mid compared with the comparative bispecific antibody to CD38x-CD3 administered at the indicated doses in the NCI-H929-Luc disseminated tumor model NSG mice humanized with human PBMC. Binding proteins were administered once a week intraperitoneally (i/p) by injection at a dose of 30 μg/kg.

In fig. 24A shows the results of a luciferase reporter gene assay using GloResponse™ IL2-luc2P Jurkat cells (Promega) after stimulation with CD38 _VH1 /CD28 _sup xCD3 _mid and its single binding site mutant KO and triple mutant KO at a concentration of 10 nM.

In fig. 24B shows optimization of the anti-CD3xCD28 antibody CODV-Fab. The optimal configuration of α-CD3 and α-CD28 at alternative positions of CODV bispecific Fab was assessed using cytokine release assays using in vitro human PBMCs. Distal CD28 and proximal CD3 were identified as the optimal location based on the secretion of IFN-γ and IL-2 in the supernatant at 24 hours.

In fig. 25 shows that CD38 _VH1 /CD28 _sup xCD3 _mid induces activation of the Bcl-xL family member Bcl-2 in primary T cells in a CD28-dependent manner.

In fig. 26 shows that in a trispecific Ab, anti-CD28 provides secondary signaling necessary to support primary T cell proliferation in vitro.

In fig. Figure 27 shows the trispecific antibody configuration with the parent antibody color coded (left). Dark shades (purple or green) indicate heavy chain peptides; Light shades indicate light chain peptides. Also shown is a structural model of the trispecific Ab CD38 _VH1 /CD28 _sup xCD3 _mid based on the crystal structures of the CD38 VH1 Fab and CD28 _sup /CD3 _mid CODV Fab antibodies (right).

In fig. 28A and 28B show that multiple myeloma (MM) cells with high (RPMI-8226; FIG. 28A ) and low (KMS-11; FIG. 28B ) CD38 surface expression were efficiently killed by human PBMC (E:T=10 :1), incubated with trispecific Ab at different concentrations. The contribution to killing activity of each binding site was demonstrated by binding site KO mutations as indicated.

In fig. 29A-29C show that the CD28 _supKO mutant antibody in the trispecific Ab exhibited markedly reduced in vitro antitumor activity against CD38 _high , CD38 _mid , and CD38 _low MM cells. Assays using RPMI-8226 ( Figure 29A ), U266 ( Figure 29B ) or KMS-11 ( Figure 29C ) cells are shown.

In fig. 30 shows that the reduction in tumor burden in the CD38 _VH1 /CD28 _sup xCD3 _mid _FALA trispecific antibody treatment groups was dose dependent and statistically different in the NSG mouse model of human disseminated multiple myeloma cell line reconstituted in vitro with amplified primary human T cells.

In fig. 31A and 31B show vital microscopic analysis of myeloma cell cytolysis mediated by the CD38 trispecific Ab in vitro in the presence of primary human T cells. Time-lapse microscopy imaging was performed using a negative control (trispecific triple KO; Figure 31A ) or trispecific Ab CD38/CD28xCD3 ( Figure 31B ) using human PBMCs incubated with the myeloma cell line RPMI-8226 labeled with CellTracker™ deep red dye. The images shown were collected after 24 hours of incubation . Scale mark: 50 µm

In fig. 32 shows alternative mutations in the Fc region of IgG4 obtained for analysis in Fc receptor binding assays. Shown are SEQ ID NOs:111-116 (from top to bottom, respectively).

In fig. 33 shows the results of SPR assays for measuring the affinity of Fc variants of IgG4 and the indicated human Fc receptors.

In fig. 34 shows that a trispecific anti-CD38 binding protein with minimal FcR binding reduces nonspecific cytokine release in human PBMCs in vitro . Various FcR inactivating mutations (as indicated) were analyzed for their proinflammatory effects on the human IgG4 isotype. Human PBMCs were incubated in medium (unstimulated) or in the presence of myeloma cells, RPMI-8226 (stimulated), and bars indicate supernatant IFN-γ levels measured by ELISA.

In fig. 35 shows that trispecific CD38 binding protein with minimal FcR binding provides lysis of human multiple myeloma cells with different levels of CD38 expression. Cytolysis of myeloma cells with IgG4 or the indicated Fc mutations was assessed in vitro using human PBMC with the indicated tumor targets.

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

In fig. 37A-37D show the in vitro expansion pattern of a subset of T cells in response to CD38/CD3xCD28. T cell subset expansion was assessed by coating wells with 350 ng/well CD38 trispecific Ab in the absence of exogenous cytokines. T cell populations were measured at the indicated time points. Triple mutant trispecific ab was used as a negative control. Flow cytometry was used to identify central and effector memory CD4 T cells ( Figure 37A ), T helper cells ( Figure 37B ), central and effector memory CD8 T cells ( Figure 37C ), and pp65 CMV-specific CD8 cells (Figure 37C). Fig. 37D ) as described in Example 12. Analysis of CMV-specific pp65 effector cells was performed by pentamer staining of PBMC from HLA-A2 CMV+ donors treated with trispecific CD38 or triple negative control.

In fig. 38 shows the contribution of CD28 expression on target cells to susceptibility to cytolysis via CD38/CD3xCD28. CD28 was knocked down in KMS-11 cells using CRISPR/Cas 9 gene targeting and used as cytolytic targets in vitro . Compared to parental KMS-11, CD38 expression was maintained and CD28 was deleted, as demonstrated by flow cytometry (top panel, KMS-11 and KMS-11 (CD28KO). Cytolysis of CD28 KO cells was examined with WT or trispecific CD28 null ( bottom panel; trispecific and trispecific (CD28KO)).

In fig. 39 shows the cytolytic activity of CD38/CD28xCD3 trispecific FALA mutant Ab against the indicated CD38 ⁺ CD28 ^- lines, including acute myelocytic leukemia (AML (KG-1)), B-cell lymphoma (OCI-Ly19), acute T cell lines -lymphocytic leukemia (ALL (KOPN8)) and chronic lymphocytic lymphoma (CLL(Z-138)).

In fig. 40 shows that activation of human PBMC in vitro by the α-CD28 superagonist requires bivalent antibody.

DETAILED DESCRIPTION

The present invention provides a binding protein comprising at least one antigen binding site that binds a CD38 polypeptide.

I. General definitions

As used in accordance with the present invention, the following terms, unless otherwise indicated, should be understood to have the following meanings. Unless the context otherwise requires, terms in the singular will include the plural forms, and terms in the plural will include the singular form. As used in this detailed description and the accompanying claims, the singular form in the singular includes a reference to the plural form unless the content expressly states otherwise. Thus, for example, reference to a “molecule” does not necessarily include a combination of two or more such molecules and the like.

It is understood that aspects and embodiments of the present invention described herein include aspects and embodiments “comprising of,” “consisting of,” and/or “essentially consisting of.”

As used herein, the term “polynucleotide” refers to single-stranded or double-stranded nucleic acid polymers having a length of at least 10 nucleotides. In certain embodiments, the nucleotides constituting a polynucleotide may be ribonucleotides, or deoxyribonucleotides, or a modified form of any 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 phosphothioate, phosphodithioate, phosphoselenoate, phosphodiselenoate, phosphoanilotioate, phosphoaniladate and phosphoamidate. The term "polynucleotide" specifically refers to single-stranded and double-stranded forms of DNA.

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

An "isolated polypeptide" is one that: (1) does not contain at least some of the other polypeptides with which it is commonly found, (2) does not contain substantially other polypeptides from the same source, e.g. from the same species, (3) is expressed by a cell from another species, (4) has been separated from at least approximately 50 percent of the polynucleotides, lipids, carbohydrates or other substances with which it is naturally associated, (5) is not associated ( by covalent or non-covalent interaction) with parts of the polypeptide to which the "isolated polypeptide" is naturally associated, (6) operably linked (by covalent or non-covalent interaction) with a polypeptide to which it is not naturally associated, or (7) does not occur in nature. Such an isolated polypeptide may be encoded by genomic DNA, cDNA, mRNA or other RNA of synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free of polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

Naturally occurring antibodies are typically a tetramer. Each such tetramer typically consists of two identical pairs of polypeptide chains, with each pair having one full-length "light" chain (typically having a molecular weight of approximately 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of approximately 50-70 kDa) . As used herein, the terms “heavy chain” and “light chain” refer to any immunoglobulin polypeptide containing a variable domain sequence sufficient to confer specificity for a target antigen. The amino terminal portion of each light and heavy chain typically contains a variable domain of approximately 100-110 amino acids or more, which is typically responsible for antigen recognition. The carboxy-terminal part of each chain usually defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, the full-length immunoglobulin heavy chain polypeptide contains a variable domain ( _VH ) and three constant domains ( _CH1 , _CH2 and _CH3 ), where the _VH domain is at the amino terminus of the polypeptide and the C _H3 is at the carboxyl terminus, and the full-length immunoglobulin light chain polypeptide contains a variable domain (V _L ) and a constant domain ( _CL ), where the V _L domain is at the amino terminus of the polypeptide and the C _L domain is at the carboxyl terminus.

Human light chains are usually classified as kappa and lambda light chains, and human heavy chains are usually classified as mu, delta, gamma, alpha, or epsilon, and they define the antibody isotype 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 divided into subclasses, including but not limited to IgA1 and IgA2. In full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, with the heavy chain also containing a "D" region of about 10 or more amino acids. Cm., For example, Fundamental Immunology (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light/heavy chain pair typically form an antigen-binding site. The variable domains of naturally occurring antibodies are typically characterized by the same overall structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of the two strands of each pair are typically aligned using framework regions, which may allow binding to a specific epitope. From the amino terminus to the carboxyl terminus, the variable domains of both the light and heavy chains typically contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains.

The term "set of CDRs" refers to a group of three CDRs that are contained in a single variable region capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabatet al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD. (1987) and (1991)), not only provides a unique residue numbering system applicable to any antibody variable region, but also provides precise residue boundaries defining three CDR. These CDRs may be called CDRs according to Kabat. Chothia and colleagues (Chothia and Lesk, 1987,J. Mol. Biol. 196: 901-17; Chothiaet al., 1989,Nature 342: 877-83) found that certain subfragments within the CDRs according to Kabat adopt almost identical peptide backbone conformations, despite the presence of large differences at the amino acid sequence level. These subfragments were designated L1, L2 and L3 or H1, H2 and H3, where “L” and “H” denote the light chain and heavy chain regions, respectively. These areas may be called CDR according to Chothia, the boundaries of which coincide with the CDR according to Kabat. Other boundaries defining the CDR, which coincide with the CDR according to Kabat, have been described by Padlan, 1995.FASEB J. 9: 133-39; MacCallum, 1996J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003,Dev. Comp. Immunol. 27: 55-77. Still other definitions of CDR boundaries may not strictly correspond to one of the systems given in this document, but will nevertheless coincide with CDR according to Kabat, although they may be shortened or lengthened based on predictive or experimental conclusions about whether that specific residues or groups of residues, or even all CDRs, do not significantly affect antigen binding. The methods used herein may use CDRs defined in accordance with any of these systems, although in certain embodiments CDRs defined in accordance with Kabat or Chothia are used. Identification of predicted CDRs using amino acid sequence is well known in the art, for example, Martin, A.C. "Protein sequence and structure analysis of antibody variable domains" onAntibody 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 can also be examined to identify CDR sequences using other conventional methods, for example, by comparison with known amino acid sequences of other variable regions of the heavy and light chains, identifying areas of hypervariability in the sequence. Numbered sequences can be aligned manually or by using an alignment program such as one from the CLUSTAL software package, as described in Thompson, 1994.Nucleic Acids Res. 22: 4673-80. Molecular models are traditionally used to correctly identify framework and CDR regions and thus correct sequence-based alignments.

In some embodiments, the determination of CDR/FR in an immunoglobulin light or heavy chain should be based on the IMGT determination (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org) .

As used herein, the term "Fc" refers to a molecule, whether in monomeric or multimeric form, containing a sequence that is not part of an antigen-binding fragment, which is obtained by cleavage of an antibody or obtained by other means, and may contain a hinge region. The original immunoglobulin source of the native Fc is preferably human and can be any of the immunoglobulins. Fc molecules are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms through covalent (i.e..disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules varies from 1 to 4 depending on the class (for example, IgG, IgA and IgE) or subclass (e.g. IgG1, IgG2, IgG3, IgA1, IgGA2 and IgG4). One example of an Fc is a disulfide-linked dimer resulting from the cleavage of IgG by papain. The term "native Fc" as used herein is intended to cover monomeric, dimeric and multimeric forms.

The F(ab) fragment typically contains one light chain and the V _H and C _H1 domains of one heavy chain, where the V _H -C _H1 portion of the heavy chain of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, an F(ab) fragment may also comprise one light chain comprising 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.

The F(ab') fragment typically contains one light chain and part of one heavy chain, which contains most of the constant region (between the C _H1 and C _H2 domains), whereby an interchain disulfide bond can be formed between the two heavy chains to form an F molecule (ab') ₂ .

As used herein, the term “binding protein” refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen, e.g. CD38 polypeptide of the present invention.

A "recombinant" molecule is a molecule that has been produced, expressed, created or isolated by recombinant methods.

In one embodiment of the present invention, binding proteins are provided that have biological and immunological specificity for one to three target antigens. In another embodiment, the present invention provides nucleic acid molecules containing nucleotide sequences encoding polypeptide chains that form such binding proteins. In another embodiment, the present invention provides expression vectors containing nucleic acid molecules containing nucleotide sequences encoding polypeptide chains that form such binding proteins. In yet another embodiment, the present invention provides host cells that express such binding proteins (i.e. containing nucleic acid molecules or vectors encoding the polypeptide chains that form such binding proteins).

As used herein, the term “exchangeability” refers to the ability to interchange variable domains within a binding protein format while maintaining folding and highest binding affinity. "Full Exchange Capacity" refers to the ability to change the order of domains as V_H1, and V_H2 and therefore the order of domains V_L1 and V_L2 in a polypeptide chain of formula I or a polypeptide chain of formula II (i.e. reverse order) while maintaining full functionality of the binding protein, as evidenced by retention of binding affinity. In addition, it should be noted that the notation V_H and V_L refer only to the position of the domain in a particular protein chain in the final format. For example, V_H1 and V_H2 can be derived from V domains_L1 and V_L2 original antibodies and introduced into positions corresponding to V_H1 and V_H2 binding protein. Same as V_L1 and V_L2 can be derived from V domains_H1 and V_H2 original antibodies and placed in positions corresponding to V_H1 and V_H2 binding protein. Thus, the notation V_H and V_L refer to the currently existing position and not to the original position in the original antibody. Therefore, domains V_H and V_L are "capable of exchange".

As used herein, the term “antigen” or “target antigen” or “target antigen” refers to a molecule or part of a molecule that can be bound by a binding protein and can further be 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. Given that each target antigen is recognized by a binding protein, the binding protein is able to compete with an intact antibody that recognizes the target antigen.

"CD38" stands for cluster of differentiation polypeptide 38 and is a glycoprotein found on the surface of many immune cells. In some embodiments, the binding protein of the present invention binds the extracellular domain of one or more CD38 polypeptides. Exemplary CD38 polypeptide extracellular domain sequences include, but are not limited to, human CD38 extracellular domain (e.g. presented under SEQ ID NO:1) and the extracellular domain of cynomolgus monkey CD38 (e.g. presented under SEQ ID NO:30).

The term "T cell recruiter" refers to binding proteins directed to the host immune system, more specifically to the cytotoxic activity of T cells, and also directed to a tumor protein target.

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

The term "monovalent binding protein" refers to a binding protein that has a single antigen binding site.

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

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

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

The term "trivalent binding protein" refers to a binding protein that has three binding sites. In specific embodiments, the trivalent binding protein may bind to a single target antigen. In other embodiments, the trivalent binding protein can bind to two target antigens. In other embodiments, the trivalent binding protein can bind to three target antigens.

An "isolated" binding protein is a binding protein that has been identified and separated and/or extracted from a component of its natural environment. Contaminants from its natural environment are substances that would be problematic in diagnostic or therapeutic applications of the binding protein, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the binding protein will be purified: (1) to greater than 95% by weight of the antibody, as determined by the Lowry method, and most preferably to greater than 99% by weight, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spin-beaker sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver dye. Isolated binding proteins include the binding protein in situ in recombinant cells, since at least one component of the natural environment of the binding protein will not be present.

As used herein, the terms “substantially pure” or “substantially purified” mean a compound or structure that is the predominant structure present (i.e. per mole it is more abundant than any other single structure in the composition). In some embodiments, the substantially purified fraction is a composition wherein the structure represents at least about 50% (on a mole basis) of all macromolecular structures present. In other embodiments, the substantially pure composition will contain more than about 80%, 85%, 90%, 95%, or 99% of all macromolecular structures present in the composition. In still other embodiments, the structure is purified to the desired homogeneity (contaminant structures cannot be detected in the composition using traditional detection methods), where the composition consists of essentially a single macromolecular structure.

The term "epitope" means any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include reactive surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structure 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 considered to specifically bind an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, a binding protein is considered to specifically bind an antigen if the equilibrium dissociation constant is ≤ 10 ^-8 M, more preferably if the equilibrium dissociation constant is ≤ 10 ^-9 M, and most preferably if the dissociation constant is ≤ 10 ^-10 M.

The dissociation constant (K _D ) of the binding protein can be determined, for example, using surface plasmon resonance. In general, surface plasmon resonance assays measure binding interactions between a ligand (target antigen on a biosensor array) and an analyte (binding protein in solution) in real time using surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, New -Jersey). Surface plasmon resonance analysis can also be performed by immobilizing an analyte (binding protein on a biosensor array) and presenting a ligand (target antigen). As used herein, the term “K _D ” refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

As used herein, the term “binds to” in reference to a binding protein means the ability of the binding protein or antigen binding fragment thereof to bind an epitope-containing antigen with a Kd of at least about 1×10^-6 M, 1×10^-7 M, 1×10^-8 M, 1×10^-9 M, 1×10^-10 M, 1×10^-eleven M, 1×10^-12 M or greater, and/or binds to the epitope with an affinity that is at least twice that of the nonspecific antigen. In some embodiments, the binding protein of the present invention binds to two or more antigens, e.g. human and cynomolgus macaque CD38 polypeptide.

In some embodiments, the antigen binding domain and/or binding protein of the present invention "cross-reacts" with human and cynomolgus CD38 polypeptides, for example, CD38 extracellular domains such as SEQ ID NO:1 (human CD38 isoform A), SEQ ID NO:105 (human CD38 isoform E) and SEQ ID NO:30 (cynomolgus CD38). Binding protein binding to antigen 1 (Ag1) is “cross-reactive” to antigen 2 (Ag2) if the EC50 values are in a similar range for both antigens. As used herein, binding of a binding protein to Ag1 is cross-reactive to Ag2 if the ratio of affinity for Ag2 to affinity for Ag1 is 10 or less (e.g., 5, 2, 1, or 0.5), with affinities measured using one and the same method in the case of both antigens.

Binding protein binding to Ag1 is not “significantly cross-reactive” to Ag2 if the affinities for the two antigens are very different. Affinity for Ag2 may not be measurable if the binding reaction is too weak. In this application, the binding of a binding protein to Ag1 is not significantly cross-reactive with Ag2 if the binding reaction of the binding protein to Ag2 is less than 5% of the binding reaction of the same binding protein to Ag1 under the same experimental conditions and under the same experimental conditions. antibody concentrations. In practice, the concentration of binding protein used may be the EC50 or the concentration required to achieve the saturation plateau observed with Ag1.

As used herein, the term “linker” refers to one or more amino acid residues inserted between immunoglobulin domains to provide mobility sufficient to allow the light and heavy chain domains to fold into immunoglobulins with two variable regions crossover. The linker is inserted into the transition region between variable domains or between variable and constant domains, respectively, at the sequence level. The transition region between the domains can be identified because the approximate size of the immunoglobulin domains is well known. The exact location of the transition region between domains can be determined by determining the position of peptide stretches that do not form secondary structure elements such as beta sheets or alpha helices, as demonstrated by experimental data or as predicted by modeling or secondary structure prediction techniques . The linkers described herein are designated L ₁ , which is located on the light chain between the C-terminus of the V _L2 domain and the N-terminus of the V _L1 domain; and L ₂ , which is located on the light chain between the C-terminus of the V _L1 domain and the N-terminus of the C _L domain. The heavy chain linkers are referred to as L ₃ , which is located between the C-terminus of the V _H1 domain and the N-terminus of the V _H2 domain; and L ₄ , which is located between the C-terminus of the V _H2 domain and the N-terminus of the C _H1 domain.

As used herein, the term “vector” refers to any molecule (e.g. nucleic acid, plasmid, or virus) used to carry encoded information into a host cell. The term "vector" refers to 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 can be inserted. Another type of vector is a viral vector, whereby additional DNA segments can be inserted into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal vectors for mammals). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the host cell genome after introduction into the host cell, and thereby replicate along with the host genome. In addition, certain vectors are capable of controlling the expression of genes to which they are functionally associated. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). Typically, expression vectors useful in recombinant DNA technologies are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably herein since plasmid is the most widely used form of vector. However, other forms of expression vectors are intended to be included in the present invention, such as viral vectors (eg, retroviruses, adenoviruses and replication-defective adeno-associated viruses) that perform equivalent functions.

As used herein, the phrase “recombinant host cell” (or “host cell”) refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cell is intended to refer not only to the particular cell in question, but also to the progeny of such cell. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not actually 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 binding proteins, including bacterial, yeast, baculovirus, and mammalian cell-based expression systems (as well as phage display-based expression systems). An example of a suitable bacterial expression vector is pUC19. For recombinant expression of a binding protein, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding polypeptide chains of the binding protein, whereby the polypeptide chains are expressed in the host cell and preferably secreted into the medium in which the host cells are cultured, in this case, the binding protein can be extracted from this medium.

As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell, and the cell has been transformed if it has been modified to contain new DNA. For example, a cell is transformed if it is genetically modified from its native state. After transformation, the transforming DNA may undergo recombination with the cell's DNA by physical integration into the cell's chromosome, or may be temporarily retained as an episomal element without replication, or may replicate independently as a plasmid. A cell is said to be stably transformed if the DNA is replicated when the cell divides. As used herein, the term "transfection" refers to the uptake of foreign or exogenous DNA into a cell, and the cell has been "transfected" if the exogenous DNA is introduced into 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.

As used herein, the term "naturally occurring" and applied to an item refers to the fact that the item can be found in nature and has not been manipulated by humans. For example, a polynucleotide or polypeptide present in an organism (including viruses), which can be isolated from a natural source and which has not been intentionally modified by humans, is naturally occurring. Likewise, as used herein, the term “non-naturally occurring” refers to an entity that is not found in nature or that has been structurally modified or synthesized by humans.

As used herein, the twenty standard amino acids and their abbreviations are in accordance with generally accepted practice. Stereoisomers (for example, d-amino acids) twenty standard amino acids; non-naturally occurring amino acids and analogs such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid and other non-standard amino acids may also be suitable components of the polypeptide chains of binding proteins. Examples of non-standard amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N, N,N-trimethyllysine, ε-N-acetyl-lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ- N-methylarginine and other similar amino acids and imino acids (for example, 4-hydroxyproline). In the polypeptide naming system used herein, the left direction represents the direction towards the amino terminus and the right direction represents the direction towards the carboxyl terminus, in accordance with standard practice and convention.

Naturally occurring residues can be divided into classes based on general side chain properties:

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

(2) polar hydrophobic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

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

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

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

(6) acidic: Asp, Glu;

(7) basic: His, Lys, Arg;

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

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

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

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

One skilled in the art will be able to determine suitable variants of the polypeptide chains of binding proteins using generally known techniques. For example, one skilled in the art can identify suitable regions in a polypeptide chain that can be altered without interfering with activity by targeting regions that are not expected to be important in terms of activity. Alternatively, one of ordinary skill in the art may identify residues and portions of molecules that are conserved among similar polypeptides. In addition, even regions that may be biologically or structurally important can be subject to conservative amino acid substitutions without impairing biological activity or adversely affecting the structure of the polypeptide.

As used herein, the term “patient” includes humans and animals (eg mammals such as dogs, pigs, horses, cats, cows, etc.).

As used herein, the terms “treatment” or “treat” include both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who have the disorder, as well as those who are predisposed to the disorder, or those in whom the disorder needs to be prevented. In specific embodiments, the binding proteins can be used to treat people with cancer or people susceptible to cancer, or to reduce the severity of cancer in a human subject. Binding proteins can also be used to prevent cancer in a human patient. In specific embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, B-cell germinal center lymphoma, or B- cellular acute lymphoblastic leukemia.

As used herein, the terms “pharmaceutical composition” or “therapeutic composition” mean a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

As used herein, the term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” refers to one or more substances of the composition suitable for achieving or improving the delivery of a binding protein.

The terms "effective amount" and "therapeutically effective amount" when used in relation to a pharmaceutical composition containing one or more binding proteins mean an amount or dose sufficient to obtain the desired therapeutic result. More specifically, a therapeutically effective amount is an amount of binding protein sufficient to inhibit, for a period of time, one or more clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the particular binding protein to be used and also depends on a number 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 , then factors such as the age, body weight and health status of the patient, as well as dose-response curves and toxicity data obtained from a preclinical animal study, will be included. factors taken into account. Determination of the effective amount or therapeutically effective amount of the specified pharmaceutical composition is within the competence of specialists in the art.

In one embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

II. CD38 binding proteins

Certain aspects of the present invention relate to binding proteins that contain an antigen binding site that binds a CD38 polypeptide (e.g. human and cynomolgus CD38 polypeptides). In some embodiments, the binding proteins are monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or polyspecific and/or multivalent.

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

In some embodiments, the binding protein comprises an antigen binding site comprising a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR- sequence H2 containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and the sequence CDR-H3 containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); or a light chain variable domain (VL) of an antibody comprising a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO :35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding protein comprises an antigen binding site comprising a heavy chain variable domain (VH) of an antibody comprising a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR- sequence H2 containing the amino acid sequence IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and the sequence CDR-H3 containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); or a light chain variable domain (VL) of an antibody comprising a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQG (SEQ ID NO:132), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO :35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the binding proteins comprise 1, 2, 3, 4, 5, or 6 CDRs from the VH and/or VL domain sequence from the antibody mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb2xCD28supxCD3mid IgG4 FALA, mAb2xCD28supxCD3mid IgG1LALA P329A, mAb2xCD28supxCD3mid IgG1 NNSA, mAb6xCD28supxCD3mid IgG4 FALA, mAb6xCD28supxCD3mid IgG1LALA P329A, or mAb6xCD28supxCD3mid IgG1 NNSA, as shown in Table G, H, or I.

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

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

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

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 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 contains 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 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 contains 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 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 contains 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 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 contains 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 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 contains 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.

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 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 contains 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 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 contains 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 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 contains 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 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 contains 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 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 contains 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.

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

In some embodiments, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding protein of the present invention 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, the binding protein of the present invention 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.

In some embodiments, the binding protein of the present invention comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:7 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the binding protein of the present invention comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:19 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the binding protein of the present invention comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:22 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the binding protein of the present invention comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:24 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the binding protein of the present invention 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.

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

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

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 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 contains 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 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 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 contains 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 contains the amino acid sequence of SEQ ID NO:9, and the VL domain contains the amino acid sequence of SEQ ID NO:10.

In some embodiments, the binding protein of the present invention comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:11, or an antibody light chain comprising the amino acid sequence of SEQ ID NO:12. In some embodiments, the binding protein of the present invention 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 binding protein of the present invention contains 1, 2, 3, 4, 5, or 6 CDR sequences from the antibody sequence shown in Table G. In some embodiments, the binding protein of the present invention contains 1, 2, 3, 4, 5 or 6 CDR sequences, a VH domain sequence, and/or a VL domain sequence from the antibody sequence shown in Table H. In some embodiments, the binding protein of the present invention contains 1, 2, 3, 4, 5, or 6 CDR sequences, a domain sequence VH and/or VL domain sequence from the antibody sequence shown in Table I. In some embodiments, the binding protein of the present invention contains 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
(SEQ ID NO:31) IYPGNGGT
(SEQ ID NO:32) ARTGGLRRAYFTY
(SEQ ID NO:33) ESVDSYGNGF
(SEQ ID NO:34) LAS
(SEQ ID NO:35) QQNKEDPWT
(SEQ ID NO:36) mAb2 GYTFTSYA
(SEQ ID NO:37) IYPGQGGT
(SEQ ID NO:38) ARTGGLRRAYFTY
(SEQ ID NO:33) QSVSSYGQGF
(SEQ ID NO:39) G.A.S.
(SEQ ID NO:40) QQNKEDPWT
(SEQ ID NO:36) mAb3 GYTFTSFN
(SEQ ID NO:31) IYPGNGGT
(SEQ ID NO:32) ARTGGLRRAYFTY
(SEQ ID NO:33) ESVDSYGNGF
(SEQ ID NO:34) LAS
(SEQ ID NO:35) QQNKEDPWT
(SEQ ID NO:36) mAb4 GYTFTSFN
(SEQ ID NO:31) IYPGNGGT
(SEQ ID NO:32) ARTGGLRRAYFTY
(SEQ ID NO:33) ESVDSYGNGF
(SEQ ID NO:34) LAS
(SEQ ID NO:35) QQNKEDPWT
(SEQ ID NO:36) mAb5 GYTFTSFN
(SEQ ID NO:31) IYPGNGGT
(SEQ ID NO:32) ARTGGLRRAYFTY
(SEQ ID NO:33) ESVDSYGNGF
(SEQ ID NO:34) LAS
(SEQ ID NO:35) QQNKEDPWT
(SEQ ID NO:36) mAb6 GFTFSSYG
(SEQ ID NO:41) IWYDGSNK
(SEQ ID NO:42) ARMFRGAFDY
(SEQ ID NO:43) QGIRND
(SEQ ID NO:44) A.A.S.
(SEQ ID NO:45) LQDYIYYPT (SEQ ID NO:46)

Table H. Sequences of the variable domains of anti-CD38 antibody binding proteins (mAb1-7) and other binding proteins

Ab VH (protein) VL (protein) mAb1 QVQLQQSGAELVRSGASVKMSCKASGYTFTSFNMHWVKETPGQGLEWIGYIYPGNGGTNYNQKFKGKATLTADTSSSTAYMQISSLTSEDSAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:5) DIVLTQSPASLAVSLGQRATISCRASESVDSYGNGFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:6 mAb2 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:13) DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:14) mAb3 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:17) DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18) mAb4 QVQLVQSGAEVVKSGASVKVSCKASGYTFTSFNMHWVKEAPGQGLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:21) DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18) mAb5 QVQLVQSGAEVVKPGASVKMSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:23) DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:18) mAb6 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO:9) AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO:10) mAb7 Qvqlvqsgaevakpgtsvklsckasgytftdywmqwvkqrpgqglewigtiypgdgdtgyaqkfqgkatltadkssktvymhlsslasedsavyycargdyygsnsldywgqgtsvtvss (SEq ID NO:47) DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKLEIK (SEQ ID NO:48) Antibody to CD28 _sup qvqlvqsgaevvkpgasvkvsckasgytftsyyihwvrqapgqglewigsiypgnvntnyaqkfqgratltvdtsistaymelsrlrsddtavyyctrshygldwnfdvwgkgttvtvss (SEQ ID NO:49) diqmtqspsslsasvgdrvtitcqasqniyvwlnwyqqkpgkapklliykasnlhtgvpsrfsgsgsgtdftltisslqpediatyycqqgqtypytfgqgtkleik (SEq ID NO:50) Antibody to CD28 _cvn qvqlqesgpglvkpsqtlsltctvsgfslsdygvhwvrqppgkglewlgviwagggtnynpslksrktiskdtsknqvslklssvtaadtavyycardkgysyyysmdywgqgttvtvss (SEQ ID NO:51) divltqspaslavspgqratitcrasesveyyvtslmqwyqqkpgqppkllifaasnvesgvparfsgsgsgtdftltinpveandvanyycqqsrkvpytfgqgtkleik (SEQ ID NO:52) Antibody to CD3 _mid qvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkqlewvaqikdksnsyatyyadsvkgrftisrddskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvss (SEQ ID NO:53) divmtqtplslsvtpgqpasisckssqslvhnnantylswylqkpgqspqsliykvsnrfsgvpdrfsgsgsgtdftlkisrveaedvgvyycgqgtqypftfgsgtkveik (SEQ ID NO:54) Antibody to CD3 _low qvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkglewvaqikdksnsyatyyadsvkgrftisrdnskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvss (SEQ ID NO:84) divmtqtplslsvtpgqpasisckssqslvhnngntylswylqkpgqspqlliykvsnrfsgvpdrfsgsgsgtdftlkisrveaedvgvyycgqgtqypftfgggtkveik (SEq ID NO:85)

Note: In the amino acid sequences above, the CDR sequences are in bold and underlined.

Table I. Full-length binding protein sequences

mAb2xCD28supxCD3mid IgG4 FALA CD28supxCD3mid IgG4(hollow) FALA heavy chain 1
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) Qvqlvqsgaevvkpgasvkvsckasgytftsyyihwvrqapgqglewigsiypgnvntnyaqkfqgratltvdtsistaymelsrlrsddtavyyctrshygldwnfdvwgkgttvtvsssqvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkqlewvaqikdks nsyatyyadsvkgrftisrddskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvssrtastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygppcppc papeaaggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvctlppsqeemtknqvslscavkgfypsdiavewesngqpennykttppvldsdgsfflvskltv dksrwqegnvfscsvmhealhnhytqkslslslg SEQ ID NO: 60 CD28supxCD3mid light chain 1
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) Divmtqtplslsvtpgqpasisckssqslvhnnantylswylqkpgqspqsliykvsnrfsgvpdrfsgsgsgtdftlkisrveaedvgvyycgqgtqypftfgsgtkveikgqpkaapdiqmtqspsslsasvgdrvtitcqasqniyvwlnwyqqkpgk apklliykasnlhtgvpsrfsgsgsgtdftltisslqpediatyycqqgqtypytfgqgtkleiktkgpsrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslslsstltlskadyekhkvyacevthqglsspvtksfnrgec SEQ ID NO: 61 mAb2 IgG4(overhang) FALA heavy chain 2
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) Qvqlvqsgaevvkpgasvkvsckasgytftsyamhwvkeapgqrlewigyiypgqggtnynqkfqgratltadtsastaymelsslrsedtavyfcartgglrrayftywgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqss glyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygppcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvytlppcq eemtknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqegnvfscsvmhealhnhytqkslslslg SEQ ID NO: 62 mAb2 light chain 2
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) Divltqspatlslspgeratiscrasqsvssygqgfmhwyqqkpgqpprlliygassratgiparfsgsgsgtdftltisplepedfavyycqqnkedpwtfgggtkleikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsst ltlskadyekhkvyacevthqglsspvtksfnrgec SEQ ID NO: 63 mAb2xCD28supxCD3mid IgG1LALA P329A CD28supxCD3mid IgG1(hollow) LALA P329A heavy chain 1
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) Qvqlvqsgaevvkpgasvkvsckasgytftsyyihwvrqapgqglewigsiypgnvntnyaqkfqgratltvdtsistaymelsrlrsddtavyyctrshygldwnfdvwgkgttvtvsssqvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkqlewvaqikdks nsyatyyadsvkgrftisrddskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvssrtastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdktht cppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalaapiektiskakgqprepqvctlppsrdeltknqvslscavkgfypsdiavewesngqpennykttppvldsdgsfflvskltvd ksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 64 CD28supxCD3mid light chain 1
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 61 mAb2 IgG1(overhang) LALA P329A heavy chain 2
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) Qvqlvqsgaevvkpgasvkvsckasgytftsyamhwvkeapgqrlewigyiypgqggtnynqkfqgratltadtsastaymelsslrsedtavyfcartgglrrayftywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssg lyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvvsvltvlhqdwlngkeykckvsnkalaapiektiskakgqprepqvytlppcrdel tknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 65 mAb2 light chain 2
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 63 mAb2xCD28supxCD3mid IgG1 NNSA CD28supxCD3mid IgG1(hollow) NNSA heavy chain 1
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) Qvqlvqsgaevvkpgasvkvsckasgytftsyyihwvrqapgqglewigsiypgnvntnyaqkfqgratltvdtsistaymelsrlrsddtavyyctrshygldwnfdvwgkgttvtvsssqvqlvesgggvvqpgrslrlscaasgftftkawmhwvrqapgkqlewvaqikdks nsyatyyadsvkgrftisrddskntlylqmnslraedtavyycrgvyyalspfdywgqgtlvtvssrtastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdktht cppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynnasrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvctlppsrdeltknqvslscavkgfypsdiavewesngqpennykttppvldsdgsfflvskltv dksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 66 CD28supxCD3mid light chain 1
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 61 mAb2 IgG1(overhang) NNSA heavy chain 2
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) qvqlvqsgaevvkpgasvkvsckasgytftsyamhwvkeapgqrlewigyiypgqggtnynqkfqgratltadtsastaymelsslrsedtavyfcartgglrrayftywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqss glyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynnasrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlpp crdeltknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 67 CD38VH1 light chain 2
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 63 mAb6xCD28supxCD3mid IgG4 FALA CD28supxCD3mid IgG4(hollow) FALA heavy chain 1
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 60 CD28supxCD3mid light chain 1
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 61 mAb6 IgG4(overhang) FALA heavy chain 2
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) Qvqlvesgggvvqpgrslrlscaasgftfssygmhwvrqapgkglewvaviwydgsnkyyadsvkgrftisgdnskntlylqmnslraedtavyycarmfrgafdywgqgtlvtvssastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqss glyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygppcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvytlppcq eemtknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqegnvfscsvmhealhnhytqkslslslg SEQ ID NO: 68 mAb6 light 2
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) aiqmtqspsslsasvgdrvtitcrasqgirndlgwyqqkpgkapklliyaasslqsgvpsrfsgsgsgtdftltisglqpedsatyyclqdyiyyptfgqgtkveikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdsty slsstltlskadyekhkvyacevthqglsspvtksfnrgec SEQ ID NO: 69 mAb6xCD28supxCD3mid IgG1LALA P329A CD28supxCD3mid IgG1(hollow) LALA P329A heavy chain 1
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 64 CD28supxCD3mid light chain 1
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 61 mAb6 IgG1(overhang) LALA P329A heavy chain 2
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) qvqlvesgggvvqpgrslrlscaasgftfssygmhwvrqapgkglewvaviwydgsnkyyadsvkgrftisgdnskntlylqmnslraedtavyycarmfrgafdywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqss glyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalaapiektiskakgqprepqvytlppcr deltknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 70 mAb6 light 2
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 69 mAb6xCD28supxCD3mid IgG1 NNSA CD28supxCD3mid IgG1(hollow) NNSA heavy chain 1
(for example, the second polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 66 CD28supxCD3mid light chain 1
(for example, the first polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 61 mAb6 IgG1(overhang) NNSA heavy chain 2
(for example, the third polypeptide chain of the trispecific binding protein of the present invention) Qvqlvesgggvvqpgrslrlscaasgftfssygmhwvrqapgkglewvaviwydgsnkyyadsvkgrftisgdnskntlylqmnslraedtavyycarmfrgafdywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssg lyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynnasrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppcr deltknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 71 mAb6 light 2
(for example, the fourth polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO: 69 Monovalent antibody mAb1 Heavy chain mAb1 QVQLQQSGAELVRSGASVKMSCKASGYTFTSFNMHWVKETPGQGLEWIGYIYPGNGGTNYNQKFKGKATLTADTSSSTAYMQISSLTSEDSAVYFCARTGGLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 7 mAb1 light chain DIVLTQSPASLAVSLGQRATISCRASESVDSYGNGFMHWYQQKPGQPPKLLIYLASNLES
GVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 8 Monovalent antibody mAb2 Heavy chain mAb2 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 15 mAb2 light chain DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 16 Monovalent antibody mAb3 Heavy chain mAb3 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 19 mAb3 light chain DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 20 Monovalent antibody mAb4 Heavy chain mAb4 QVQLVQSGAEVVKSGASVKVSCKASGYTFTSFNMHWVKEAPGQGLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 22 mAb4 light chain DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 20 Monovalent antibody mAb5 Heavy chain mAb5 QVQLVQSGAEVVKPGASVKMSCKASGYTFTSFNMHWVKEAPGQRLEWIGYIYPGNGGTNYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 24 mAb5 light chain DIVLTQSPATLSLSPGERATISCRASESVDSYGNGFMHWYQQKPGQPPRLLIYLASSRAT
GIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 20 Monovalent antibody mAb6 Heavy chain mAb6 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 11 mAb6 light chain AIQMTQSPSSLSSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 Monovalent antibody mAb7 mAb7 heavy chain QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGTIYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDYYGSNSLDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 107 mAb7 light chain DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKLEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 106

Table J. Full-length polynucleotide sequences of binding proteins

mAb2xCD28supxCD3mid IgG4 FALA CD28supxCD3mid IgG4(hollow) FALA heavy chain 1
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaggtcgtgaaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccagctactacatccactgggtgcgccaggcccctggacagggactggaatggatcggcagcatctaccccggcaacgtgaacaccaactacgcccagaagttccagggcagagccaccctgacc gtggacaccagcatcagcaccgcctacatggaactgagccggctgagaagcgacgacaccgccgtgtactactgcacccggtcccactacggcctggattggaacttcgacgtgtggggcaagggcaccaccgtgacagtgtctagcagccaggtgcagctggtggaatctggcggcggagtggtgcagcctggcagaagcctgagactgagct gtgccgccagcggcttcaccttcaccaaggcctggatgcactggggtgcgccaggcccctggaaagcagctggaatgggtggcccagatcaaggacaagagcaacagctacgccacctactacgccgacagcgtgaagggccggttcaccatcagccgggacgacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggac accgccgtgtactactgtcggggcgtgtactatgccctgagccccttcgattactggggccagggaaccctcgtgaccgtgtctagtcggaccgccagcacaaagggcccatcggtgttccctctggccccttgcagcagaagcaccagcgaatctacagccgccctgggctgcctcgtgaaggactactttcccgagcccgtgacc gtgtcctggaactctggcgctctgacaagcggcgtgcacacctttccagccgtgctccagagcagcggcctgtactctctgagcagcgtcgtgacagtgcccagcagcagcctgggcaccaagacctacacctgtaacgtggaccacaagcccagcaacaccaaggtggacaagcgggtggaatctaagtacggccctcc ctgccctccttgcccagcccctgaagctgccggcggaccctccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccaggaagatcccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagagg aacagttcaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcccagctccatcgagaaaaccatcagcaaggccaagggccagccccgcgagcctcaagtgtgtaccctgccccctagccaggaagagatgaccaagaaccaggtgtccct gagctgtgccgtgaaaggcttctaccccagcgacattgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctggtgtccaagctgaccgtggacaagagccggtggcaggaaggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaacc actacacccagaagtccctgtctctgtccctgggc SEQ ID NO:72 CD28supxCD3mid light chain 1
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) gacatcgtgatgacccagacccccctgagcctgagcgtgacacctggacagcctgccagcatcagctgcaagagcagccagagcctggtgcacaacaacgccaacacctacctgagctggtatctgcagaagcccggccagagcccccagtccctgatctacaaggtgtccaacagattcagcggcgtgcccgacagattctccggcag cggctctggcaccgacttcaccctgaagatcagccggggtggaagccgaggacgtgggcgtgtactattgtggccagggcacccagtaccccttcacctttggcagcggcaccaaggtggaaatcaagggccagcccaaggccgcccccgacatccagatgacccagagccccagcagcctgtctgccagcgtgggcgacagagtgaccat cacctgtcaggccagccagaacatctacgtgtggctgaactggtatcagcagaagcccggcaaggcccccaagctgctgatctacaaggccagcaacctgcacaccggcgtgcccagcagattttctggcagcggctccggcaccgacttcaccctgacaatcagctccctgcagcccgaggacattgccacctactactgccagcaggccagacctacccc tacacctttggccaggcaccaagctggaaatcaagaccaagggccccagccgtacggtggccgctcccagcgtgttcatcttcccacctagcgacgagcagctgaagtccggcacagcctctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaagtgcagtggaaggtggacaacgccctgcagagcggcaacag caggaaagcgtgaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgacactgagcaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagagcttcaaccggggcgagtgt SEQ ID NO:73 mAb2 IgG4(overhang) FALA heavy chain 2
(for example, the sequence encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaagtcgtgaaacctggcgcctccgtgaaggtgtcctgcaaggccagcggctacacctttaccagctacgccatgcactgggtcaaagaggcccctggccagagactggaatggatcggctacatctaccccggccaggcggcaccaactacaaccagaagttccagggcagagccaccctgaccg ccgatacaagcgccagcaccgcctacatggaactgagcagcctgcggagcgaggataccgccgtgtacttctgtgccagaacaggcggcctgaggcgggcctactttacctattggggccagggcaccctcgtgaccgtgtctagcgctagcacaaagggccatcggtgttccctctggccccttgcagcagaagcaccagcgaatctac agccgccctgggctgcctcgtgaaggactactttcccgagcccgtgaccgtgtcctggaactctggcgctctgacaagcggcgtgcacacctttccagccgtgctccagagcagcggcctgtactctctgagcagcgtcgtgacagtgcccagcagcagcctgggcaccaagacctacacctgtaacgtggaccaca agcccagcaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccctccttgcccagcccctgaagctgccggcggaccctccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccaggaagatcccgaggtgcagtt caattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcccagctccatcgagaaaaccatcagcaaggccaagggccagccccgcgagcct caagtgtataccctgcccccttgccaggaagagatgaccaagaaccaggtgtccctgtggtgtctcgtgaaaggcttctaccccagcgacattgccgtggaatggggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtactccaagctgaccgtggacaagagccggtggcagg aaggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtctctgtccctgggc SEQ ID NO:74 mAb2 light chain 2
(for example, the sequence encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) gacatcgtgctgacacagagccctgccaccctgtctctgagccctggcgagagagccaccatcagctgtagagccagccagagcgtgtccagctacggccagggcttcatgcactggtatcagcagaagcccggccagccccccagactgctgatctatggcgccagcagcagagccacaggcatccccgccagattttctggctctggcagcggcaccg acttcaccctgacaatcagccccctggaacccgaggacttcgccgtgtactactgccagcagaacaaagaggacccctggaccttcggcggaggcaccaagctggaaatcaagcgtacggtggccgctcccagcgtgttcatcttcccacctagcgacgagcagctgaagtccggcacagcctctgtcgtgtgcctgctgaacaactt ctacccccgcgaggccaaggtgcagtggaaggtggacaatgccctgcagagcggcaacagccaggaaagcgtgaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgaccctgtccaaggccgattacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagagcttca accggggcgagtgc SEQ ID NO:75 mAb2xCD28supxCD3mid IgG1LALA P329A CD28supxCD3mid IgG1(hollow) LALA P329A heavy chain 1
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaggtcgtgaaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccagctactacatccactgggtgcgccaggcccctggacagggactggaatggatcggcagcatctaccccggcaacgtgaacaccaactacgcccagaagttccagggcagagccaccctgacc gtggacaccagcatcagcaccgcctacatggaactgagccggctgagaagcgacgacaccgccgtgtactactgcacccggtcccactacggcctggattggaacttcgacgtgtggggcaagggcaccaccgtgacagtgtctagcagccaggtgcagctggtggaatctggcggcggagtggtgcagcctggcagaagcctgagactgagct gtgccgccagcggcttcaccttcaccaaggcctggatgcactggggtgcgccaggcccctggaaagcagctggaatgggtggcccagatcaaggacaagagcaacagctacgccacctactacgccgacagcgtgaagggccggttcaccatcagccgggacgacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggac accgccgtgtactactgtcggggcgtgtactatgccctgagccccttcgattactggggccagggaaccctcgtgaccgtgtctagtcggaccgccagcacaaagggccccagcgtgttccctctggcccctagcagcaagagcacatctggcggaacagccgccctgggctgcctcgtgaaggactactttcccgagcccgtgaccg tgtcctggaattctggcgccctgaccagcggcgtgcacacctttccagctgtgctgcagtccagcggcctgtacagcctgagcagcgtcgtgacagtgcccagcagctctctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtggacaagaaggtggaacccaagagctgcgacaagac cccacacctgtcccccttgtcctgcccccgaagccgccggaggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagccaaga gaggaacagtacaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctggccgcccccatcgagaaaaccatcagcaaggccaagggccagccccgcgaaccccaggtgtgcacactgcccccaagcagggacgagctgaccaagaaccaggtgtccctga gctgtgccgtgaaaggcttctacccctccgatatcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctggtgtccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttcagctgctccgtgatgcacgaggccctgcaca accactacacccagaagtccctgagcctgagccccggc SEQ ID NO:76 CD28supxCD3mid light chain 1
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:73 mAb2 IgG1(overhang) LALA P329A heavy chain 2
(for example, the sequence encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaagtcgtgaaacctggcgcctccgtgaaggtgtcctgcaaggccagcggctacacctttaccagctacgccatgcactgggtcaaagaggcccctggccagagactggaatggatcggctacatctaccccggccaggcggcaccaactacaaccagaagttccagggcagagccaccctgaccg ccgatacaagcgccagcaccgcctacatggaactgagcagcctgcggagcgaggataccgccgtgtacttctgtgccagaacaggcggcctgaggcgggcctactttacctattggggccagggcaccctcgtgaccgtgtctagcgctagcacaaagggccccagcgtgttccctctggcccctagcagcaagagcacatctggcggaacag ccgccctgggctgcctcgtgaaggactactttcccgagcccgtgaccgtgtcctggaattctggcgccctgaccagcggcgtgcacacctttccagctgtgctgcagtccagcggcctgtacagcctgagcagcgtcgtgacagtgcccagcagctctctgggcacccagacctacatctgcaacgtgaaccaca agcccagcaacaccaaggtggacaagaaggtggaacccaagagctgcgacaagacccacacctgtcccccttgtcctgcccccgaagccgccggaggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagtga agttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagccaagagaggaacagtacaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctggccgcccccatcgagaaaaccatcagcaaggccaagggccagccccgcgaaccccagg tgtacacactgcccccatgcagggacgagctgaccaagaaccaggtgtccctgtggtgtctggtgaaaggcttctacccctccgatatcgccgtggaatggggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagca gggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggc SEQ ID NO:77 mAb2 light chain 2
(for example, the sequence encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:75 mAb2xCD28supxCD3mid IgG1 NNSA CD28supxCD3mid IgG1(hollow) NNSA heavy chain 1
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaggtcgtgaaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccagctactacatccactgggtgcgccaggcccctggacagggactggaatggatcggcagcatctaccccggcaacgtgaacaccaactacgcccagaagttccagggcagagccaccctgacc gtggacaccagcatcagcaccgcctacatggaactgagccggctgagaagcgacgacaccgccgtgtactactgcacccggtcccactacggcctggattggaacttcgacgtgtggggcaagggcaccaccgtgacagtgtctagcagccaggtgcagctggtggaatctggcggcggagtggtgcagcctggcagaagcctgagactgagct gtgccgccagcggcttcaccttcaccaaggcctggatgcactggggtgcgccaggcccctggaaagcagctggaatgggtggcccagatcaaggacaagagcaacagctacgccacctactacgccgacagcgtgaagggccggttcaccatcagccgggacgacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggac accgccgtgtactactgtcggggcgtgtactatgccctgagccccttcgattactggggccagggaaccctcgtgaccgtgtctagtcggaccgccagcacaaagggccccagcgtgttccctctggcccctagcagcaagagcacatctggcggaacagccgccctgggctgcctcgtgaaggactactttcccgagcccgtgaccg tgtcctggaattctggcgccctgaccagcggcgtgcacacctttccagctgtgctgcagtccagcggcctgtacagcctgagcagcgtcgtgacagtgcccagcagctctctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtggacaagaaggtggaacccaagagctgcgacaagac cccacacctgtcccccttgtcctgcccccgaactgctgggaggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagccaagaga ggaacagtacaacaatgcctcccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcccccatcgagaaaaccatcagcaaggccaagggccagccccgcgaaccccaggtgtgcacactgcccccaagcagggacgagctgaccaagaaccaggtgtcc ctgagctgtgccgtgaaaggcttctacccctccgatatcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctggtgtccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttcagctgctccgtgatgcacgaggccctg cacaaccactacacccagaagtccctgagcctgagccccggc SEQ ID NO:78 CD28supxCD3mid light chain 1
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:73 mAb2 IgG1(overhang) NNSA heavy chain 2
(for example, the sequence encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtgcagtctggcgccgaagtcgtgaaacctggcgcctccgtgaaggtgtcctgcaaggccagcggctacacctttaccagctacgccatgcactgggtcaaagaggcccctggccagagactggaatggatcggctacatctaccccggccaggcggcaccaactacaaccagaagttccagggcagagccaccctgaccg ccgatacaagcgccagcaccgcctacatggaactgagcagcctgcggagcgaggataccgccgtgtacttctgtgccagaacaggcggcctgaggcgggcctactttacctattggggccagggcaccctcgtgaccgtgtctagcgctagcacaaagggccatcggtcttccccctggcaccctcctccaagagcacctctggggggcacagc ggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaa ggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtatgttgacgg cgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacaatgcctcccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcc cccatgccgggatgagctgaccaagaatcaagtcagcctgtggtgcctggtaaaaggcttctatcccagcgacatcgccgtggagtggggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctactcaaaactcaccgtggacaagagcaggtggcagcaggggaacgtct tctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt SEQ ID NO:79 mAb2 light chain 2
(for example, the sequence encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:75 mAb6xCD28supxCD3mid IgG4 FALA CD28supxCD3mid IgG4(hollow) FALA heavy chain 1
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) See above. SEQ ID NO:72 CD28supxCD3mid light chain 1
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:73 mAb6 IgG4(overhang) FALA heavy chain 2
(for example, the sequence encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtggaaagcggcggaggcgtggtgcagcctggcaggtctctgagactgagctgtgccgccagcggcttcaccttcagcagctacggaatgcactgggtgcgccaggcccctggcaaaggactggaatgggtggccgtgatttggtacgacggcagcaacaagtactacgccgacagcgtgaaggg ccggttcaccatcagcggcgacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaatgttcagaggcgccttcgactactggggccagggcacactcgtgaccgtgtctagtgcgtcgaccaagggcccatcggtgttccctctggcccctgcagcagaagcacc agcgaatctacagccgccctgggctgcctcgtgaaggactactttcccgagcccgtgaccgtgtcctggaactctggcgctctgacaagcggcgtgcacacctttccagccgtgctccagagcagcggcctgtactctctgagcagcgtcgtgacagtgcccagcagcagcctgggcaccaagacctacacctgtaac gtggaccacaagcccagcaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccctccttgcccagcccctgaagctgccggcggaccctccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccaggaagatcccgagg tgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcccagctccatcgagaaaaccatcagcaaccaagggccagcccc gcgagcctcaagtgtataccctgcccccttgccaggaagagatgaccaagaaccaggtgtccctgtggtgtctcgtgaaaggcttctaccccagcgacattgccgtggaatggggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtactccaagctgaccgtggacaagag ccggtggcaggaaggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtctctgtccctgggc SEQ ID NO:80 mAb6 light 2
(for example, the sequence encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) gccatccagatgacccagagccccagcagcctgtctgccagcgtgggcgacagagtgaccatcacctgtagagccagccagggcatccggaacgacctgggctggtatcagcagaagcctggcaaggcccccaagctgctgatctacgccgctagctctctgcagtccggcgtgcccagcagattttctggcagcggctccggcaccgact tcaccctgacaatctctggcctgcagcccgaggacagcgccacctactactgtctgcaagactacatctactaccccaccttcggccagggcaccaaggtggaaatcaagcgtacggtggccgctcccagcgtgttcatcttcccacctagcgacgagcagctgaagtccggcacagcctctgtcgtgtgcctgctgaacaacttctacccc cgcgaggccaaagtgcagtggaaggtggacaacgccctgcagagcggcaacagccaggaaagcgtgaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgacactgagcaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagagcttcaaccgggg cgagtgt SEQ ID NO:81 mAb6xCD28supxCD3mid IgG1LALA P329A CD28supxCD3mid IgG1(hollow) LALA P329A heavy chain 1
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:76 CD28supxCD3mid light chain 1
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:73 mAb6 IgG1(overhang) LALA P329A heavy chain 2
(for example, the sequence encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtggaaagcggcggaggcgtggtgcagcctggcaggtctctgagactgagctgtgccgccagcggcttcaccttcagcagctacggaatgcactgggtgcgccaggcccctggcaaaggactggaatgggtggccgtgatttggtacgacggcagcaacaagtactacgccgacagcgtgaaggg ccggttcaccatcagcggcgacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaatgttcagaggcgccttcgactactggggccagggcacactcgtgaccgtgtctagtgcgtcgaccaagggccccagcgtgttccctctggcccctagcaagagcacatct ggcggaacagccgccctgggctgcctcgtgaaggactactttcccgagcccgtgaccgtgtcctggaattctggcgccctgaccagcggcgtgcacacctttccagctgtgctgcagtccagcggcctgtacagcctgagcagcgtcgtgacagtgcccagcagctctctgggcacccagacctacatctgcaac gtgaaccacaagcccagcaacaccaaggtggacaagaaggtggaacccaagagctgcgacaagacccacacctgtcccccttgtcctgcccccgaagccgccggaggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgagg accctgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagccaagagaggaacagtacaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctggccgcccccatcgagaaaaccatcagcaaccaagggccagcccc gcgaaccccaggtgtacacactgcccccatgcagggacgagctgaccaagaaccaggtgtccctgtggtgtctggtgaaaggcttctacccctccgatatcgccgtggaatggggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtactccaagctgacagtggacaagtccc ggtggcagcagggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggc SEQ ID NO:82 mAb6 light 2
(for example, the sequence encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:81 mAb6xCD28supxCD3mid IgG1 NNSA CD28supxCD3mid IgG1(hollow) NNSA heavy chain 1
(for example, the sequence encoding the second polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:78 CD28supxCD3mid light chain 1
(for example, the sequence encoding the first polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:73 mAb6 IgG1(overhang) NNSA heavy chain 2
(for example, the sequence encoding the third polypeptide chain of the trispecific binding protein of the present invention) caggtgcagctggtggaaagcggcggaggcgtggtgcagcctggcaggtctctgagactgagctgtgccgccagcggcttcaccttcagcagctacggaatgcactgggtgcgccaggcccctggcaaaggactggaatgggtggccgtgatttggtacgacggcagcaacaagtactacgccgacagcgtgaaggg ccggttcaccatcagcggcgacaacagcaagaacaccctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagaatgttcagaggcgccttcgactactggggccagggcacactcgtgaccgtgtctagtgcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctg ggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagccca gcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtat gttgacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacaatgcctcccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtg tacaccctgcccccatgccgggatgagctgaccaagaatcaagtcagcctgtggtgcctggtaaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctactcaaaactcaccgtggacaagagcaggtggcagcagg ggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt SEQ ID NO:83 mAb6 light 2
(for example, the sequence encoding the fourth polypeptide chain of the trispecific binding protein of the present invention) see above. SEQ ID NO:81

CD38 polypeptides

In some embodiments, the binding protein of the present invention contains an antigen binding site that binds the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus CD38 polypeptide. Exemplary assays for determining whether an antigen binding site binds an antigen are described herein and are known in the art. In some embodiments, binding is determined using an ELISA assay, for example as described below. In some embodiments, binding is determined using an SPR assay, for example as described below. In some embodiments, binding is determined by flow cytometry analysis using cells expressing CD38 polypeptide on their cell surface, for example as described below. See for example examples 1, 3 and 4.

In some embodiments, the binding protein of the present invention binds a purified polypeptide or fragment thereof containing the amino acid sequence of SEQ ID NO: 1 and/or 30 (such as measured by ELISA or SPR). In some embodiments, the binding protein of the present invention binds to a polypeptide or contains the amino acid sequence of SEQ ID NO:1 and/or 30 when expressed on the surface of a cell (such as measured by flow cytometry).

In some embodiments, the binding protein of the present invention binds to CD38 isoform A polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1). In some embodiments, the binding protein of the present invention binds to an isoform E CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105 and not containing the complete amino acid sequence of SEQ ID NO:1, consisting of the amino acid sequence of SEQ ID NO:105, or consisting essentially of the amino acid sequence of SEQ ID NO:105). In some embodiments, the binding protein of the present invention binds to CD38 isoform A polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1) and a CD38 isoform E polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105, and not containing the complete amino acid sequence of SEQ ID NO:1, consisting of the amino acid sequence of SEQ ID NO:105, or consisting essentially of the amino acid sequence of SEQ ID NO:105). Without being bound by theory, it is believed that binding to CD38 isoform E polypeptide may be preferential, e.g. by targeting the binding protein of the present invention to the cell(s) expressing the CD38 isoform E polypeptide.

Polypeptide sequence of the extracellular domain of human CD38 isoform A

RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQT LEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI (SEQ ID NO:1)

Polypeptide sequence of human CD38 isoform E

RWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRHFWECGSP (SEQ ID NO:105)

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 the cynomolgus CD38 polypeptide comprises the amino acid sequence of SEQ ID NO:30.

CD38 polypeptide sequence of cynomolgus macaque

RWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQSVWDAFKGAFISKYPCNITEEDYQPLVKLGTQTVPCNKTLLWSRIKDLAHQFTQVQRDMFTLEDMLLGYLADDLTWCGEFNTFEINYQSCPDWRKDCSNNPVSVFWKTVSRRFAETACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKV QALEAWVIHGGREDSRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKFLQCVKNPEDSSCLSGI (SEQ ID NO:30)

Polyspecific (for example, bispecific, trispecific or multispecific) binding proteins that bind CD38 polypeptides

In some embodiments, the binding protein of the present invention 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 another target antigen. In some embodiments, the binding protein of the present invention 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.

In some embodiments, the binding protein of the present invention 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 polypeptides (e.g. extracellular domain of human and/or cynomolgus CD38 polypeptide), and one or two antigen-binding sites that bind T cell surface protein. In some embodiments, one of the antigen binding sites binds one or more CD38 polypeptides (e.g. extracellular domain of human and/or cynomolgus CD38 polypeptide), one of the antigen binding sites binds human CD3 polypeptide, and one of the antigen binding sites binds human CD28 polypeptide. Human CD3 and CD28 polypeptides are known in the art. Provided herein are the amino acid sequences of illustrative and non-limiting variable domains of antibodies that bind human CD3 and CD28 polypeptides.

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

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

In some embodiments, the binding proteins specifically bind to one or more CD38 polypeptides and one or more target proteins on a T cell, including the T cell receptor complex. These T cell recruiter binding proteins are capable of transiently recruiting T cells to target cells while simultaneously activating T cell cytolytic activity. Examples of T cell target proteins include, but are not limited to, CD3 and CD28. Additional examples of such target antigens or target proteins are given higher. In some embodiments, trispecific binding proteins can be produced by combining the antigen binding domains of two or more monospecific antibodies (parent antibodies) into a single antibody.

Bispecific binding protein formats

In some embodiments, the binding protein of the present invention 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 target antigens or target proteins (e.g. having the structure described in international publication No. WO2012/135345). In some embodiments, the binding protein is bivalent and/or bispecific. In some embodiments, the binding protein is tetravalent and/or tetraspecific. In some embodiments, the binding protein is tetravalent and/or bispecific. In some embodiments, at least one of the antigen binding sites binds a CD38 polypeptide (e.g. extracellular domain of human and/or cynomolgus monkey CD38 polypeptides).

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

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I],

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

V _H2 -L ₃ -V _H1 -L ₄ -C _H1 -Fc [II],

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ;

Fc includes the hinge region of the immunoglobulin and the constant domains C _H2 , C _H3 of the immunoglobulin heavy chain;

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

and wherein the polypeptides of formula I and the polypeptides of formula II form a light chain-heavy chain crossover 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 target antigen. 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 target antigen.

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

V _L1 -L ₁ -V _L2 -L ₂ -CL-Fc,

and the second polypeptide chain contains

V _H2 -L ₃ -V _HI -L ₄ -C _H1 -Fc,

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

CL is the immunoglobulin light chain constant domain;

C _H1 is the CH1 constant domain of the immunoglobulin heavy chain;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

Fc includes the immunoglobulin hinge region and constant domains C _H2 , C _H3 of the immunoglobulin heavy chain; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

where the first and second polypeptides form a light chain-heavy chain crossover 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 target antigen. 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 target antigen.

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

V _L1 -L ₁ -V _L2 -L ₂ -CL,

the second polypeptide chain contains

V _H2 -L ₃ -V _HI -L ₄ -C _H1 -Fc,

the third polypeptide chain contains the Fc region of the antibody,

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

CL is the immunoglobulin light chain constant domain;

C _H1 is the CH1 constant domain of the immunoglobulin heavy chain;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

Fc includes the hinge region of the immunoglobulin and the constant domains C _H2 , C _H3 of the immunoglobulin heavy chain; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

where the first and second polypeptides form a light chain-heavy chain crossover 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 target antigen. 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 target antigen.

In some embodiments, the binding protein comprises a first polypeptide chain containing the structure represented by the formula:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I],

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

V _H2 -L ₃ -V _H1 -L ₄ -C _H1 [II],

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a light chain-heavy chain crossover 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 target antigen. 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 target antigen.

In any embodiment of the bispecific binding proteins described above, the non-CD38 target antigen may be any of the following typical target antigens: 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, 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, IFNα, 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 receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as integrin b4), ITK , KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II molecule, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as 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 coreceptor for IL7Ra) , TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more of the above target antigens are human target antigens.

In any of the bispecific binding proteins describedhigher, any linker or combination of linkers described herein may be used. For example, in some embodiments, the length of at least one of L₁, L₂, L₃ or L₄ independently constitutes 0 amino acids. In some embodiments, the length of each of L₁, L₂, L₃ or L₄ independently constitutes at least one amino acid. In some embodiments, each of L₁, L₂, L₃and L₄ independently has a length of zero amino acids, or contains a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, each of L₁, L₂, L₃and L₄ independently contains a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L₁ contains the sequence GQPKAAP (SEQ ID NO: 58), L₂ contains the sequence TKGPS (SEQ ID NO: 57), L₃ contains a sequence of S, and L₄ contains the RT sequence. In some embodiments, L₁ contains the sequence GGGGSGGGGS (SEQ ID NO: 55), L₂ contains the sequence GGGGSGGGGS (SEQ ID NO: 55), length L₃ is 0 amino acids, and length L₄ is 0 amino acids. In some embodiments, L₁ contains the sequence GGSGSSGSGG (SEQ ID NO: 59), L₂ contains the sequence GGSGSSGSGG (SEQ ID NO: 59), length L₃ is 0 amino acids, and length L₄ is 0 amino acids. In some embodiments, L₁ contains the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), length L₂ is 0 amino acids, L₃ contains the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), and length L₄ is 0 amino acids.

Trispecific binding proteins that bind to CD38 polypeptides

In some embodiments, the binding protein of the present invention 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 target antigens or target proteins. In some embodiments, at least one of the antigen binding sites binds a CD38 polypeptide (e.g. extracellular domain of human and/or cynomolgus monkey CD38 polypeptides). In some embodiments, the first polypeptide chain comprises a structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I];

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

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

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

In some embodiments, the binding protein of the present invention 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 target antigens or target proteins. In some embodiments, at least one of the antigen binding sites binds a CD38 polypeptide (e.g. extracellular domain of human and/or cynomolgus monkey CD38 polypeptides). In some embodiments, the first polypeptide chain comprises a structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I];

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

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

and wherein the polypeptide of formula I and the polypeptide of formula II form a light chain-heavy chain crossover pair. In some embodiments, the second and third polypeptide chains further comprise a _CH1 -linked Fc region, wherein the Fc regions comprise an immunoglobulin hinge region and immunoglobulin heavy chain constant domains _CH2 and _CH3 .

In some embodiments, the first polypeptide chain and the second polypeptide chain have a crossover orientation such that they form two different antigen binding sites. In some embodiments, VH1 and VL1 form a binding pair and form a first antigen binding site. In some embodiments, VH2 and VL2 form a binding pair and form a second antigen binding site. In some embodiments, the first antigen binding site binds a CD3 polypeptide (e.g. human CD3), and a second antigen binding site binds the 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 the CD28 polypeptide (e.g. human CD28). In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen binding site. In some embodiments, VH3 and VL3 form a binding pair and form a third antigen binding site. In some embodiments, the third antigen binding site binds a CD38 polypeptide (e.g. CD38 of humans and not necessarily cynomolgus macaques). Exemplary binding protein formats with crossover orientations contemplated for use herein are also described in US Patent Application Serial No. 15/487243 and International Application Serial No. PCT/US2017/027488.

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

In some embodiments, the _H1 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARMFRGAFDY (SEQ ID NO:43), and/or domain V _L1 contains the sequence CDR-L1 containing the amino acid sequence QGIRND (SEQ ID NO:44), the sequence CDR-L2 containing the amino acid sequence AAS (SEQ ID NO:45) , and a CDR-L3 sequence containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46). In some embodiments, the _H2 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARMFRGAFDY (SEQ ID NO:43), and/or domain V _L2 contains the sequence CDR-L1 containing the amino acid sequence QGIRND (SEQ ID NO:44), the sequence CDR-L2 containing the amino acid sequence AAS (SEQ ID NO:45) and a CDR-L3 sequence containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARMFRGAFDY (SEQ ID NO:43), and/or domain V _L3 contains the sequence CDR-L1 containing the amino acid sequence QGIRND (SEQ ID NO:44), the sequence CDR-L2 containing the amino acid sequence AAS (SEQ ID NO:45) , and a CDR-L3 sequence containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46).

In some embodiments, the _H1 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARMFRGAFDY (SEQ ID NO:43), and the V _L1 domain contains a CDR-L1 sequence containing the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:45), and a CDR-L3 sequence containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46). In some embodiments, the _H2 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARMFRGAFDY (SEQ ID NO:43), and the V _L2 domain contains a CDR-L1 sequence containing the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:45), and a CDR-L3 sequence containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARMFRGAFDY (SEQ ID NO:43), and the V _L3 domain contains a CDR-L1 sequence containing the amino acid sequence QGIRND (SEQ ID NO:44), a CDR-L2 sequence containing the amino acid sequence AAS (SEQ ID NO:45), and a CDR-L3 sequence containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46).

In some embodiments, the _H1 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and/or domain V _L1 contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) , and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H1 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and/or domain V _L1 contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40) , and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H2 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and/or domain V _L2 contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) , and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H2 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and/or domain V _L2 contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40) , and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and/or domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) , and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and/or domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40) , and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36).

In some embodiments, the _H1 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the V _L1 domain contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H1 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the V _L1 domain contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H2 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the _L2 domain V contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H2 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the V _L2 domain contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and the V _L3 domain contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence sequence ARTGGLRRAYFTY (SEQ ID NO:33), and domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36).

In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and/or domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence containing the amino acid sequence LAS (SEQ ID NO:35) , and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARTGGLRRAYFTY (SEQ ID NO:33), and/or domain V _L3 contains a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40) , and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36).

In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:5, or the V _L3 domain contains the amino acid sequence of SEQ ID NO:6. In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:17, or the V _L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:21, or the V _L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:23, or the V _L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:13, or the V _L3 domain contains the amino acid sequence of SEQ ID NO:14.

In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:5, and/or the V _L3 domain contains the amino acid sequence of SEQ ID NO:6. In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:17, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:21, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:23, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:13, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:14.

In some embodiments, the _H3 domain V comprises a CDR-H1 sequence comprising the amino acid sequence GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence the sequence ARMFRGAFDY (SEQ ID NO:43), and/or domain V _L3 contains the sequence CDR-L1 containing the amino acid sequence QGIRND (SEQ ID NO:44), the sequence CDR-L2 containing the amino acid sequence AAS (SEQ ID NO:45) , and a CDR-L3 sequence containing the amino acid sequence LQDYIYYPT (SEQ ID NO:46).

In some embodiments, the V _H3 domain contains the amino acid sequence of SEQ ID NO:9, and/or the V _L3 domain contains the amino acid sequence of SEQ ID NO:10.

In some embodiments of any of the trispecific binding proteins of the present invention, one antigen binding domain binds a CD3 polypeptide (e.g. human CD3), and one antigen binding domain binds the CD28 polypeptide (e.g. human CD28). In some embodiments, domain V_H1 contains three CDRs from SEQ ID NO:49 or 51, as shown in Table H, and domain V_L1 contains three CDRs from SEQ ID NO:50 or 52, as shown in Table H. In some embodiments, domain V_H2 contains three CDRs from SEQ ID NO:49 or 51, as shown in Table H, and domain V_L2 contains three CDRs from SEQ ID NO:50 or 52, as shown in Table H. In some embodiments, domain V_H1 contains three CDRs from SEQ ID NO:53 or 84, as shown in Table H, and domain V_L1 contains three CDRs from SEQ ID NO:54 or 85, as shown in Table H. In some embodiments, domain V_H2 contains three CDRs from SEQ ID NO:53 or 84, as shown in Table H, and domain V_L2 contains three CDRs from SEQ ID NO:54 or 85, as shown in Table H.

In some embodiments, the V _H1 domain contains the amino acid sequence of SEQ ID NO:49, the V _L1 domain contains the amino acid sequence of SEQ ID NO:50, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L2 domain contains the amino acid sequence under SEQ ID NO:54. In some embodiments, the V _H2 domain contains the amino acid sequence of SEQ ID NO:49, the V _L2 domain contains the amino acid sequence of SEQ ID NO:50, the V _H1 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L1 domain contains the amino acid sequence under SEQ ID NO:54. In some embodiments, the V _H1 domain contains the amino acid sequence of SEQ ID NO:51, the V _L1 domain contains the amino acid sequence of SEQ ID NO:52, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L2 domain contains the amino acid sequence under SEQ ID NO:54. In some embodiments, the V _H2 domain contains the amino acid sequence of SEQ ID NO:51, the V _L2 domain contains the amino acid sequence of SEQ ID NO:52, the V _H1 domain contains the amino acid sequence of SEQ ID NO:53, and the V _L1 domain contains the amino acid sequence under SEQ ID NO:54.

In some embodiments, the V _H1 domain contains the amino acid sequence of SEQ ID NO:49, the V _L1 domain contains the amino acid sequence of SEQ ID NO:50, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, the V _L2 domain contains the amino acid sequence of SEQ ID NO:54, the V _H3 domain contains the amino acid sequence of SEQ ID NO:13, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:14. In some embodiments, the V _H1 domain contains the amino acid sequence of SEQ ID NO:49, the V _L1 domain contains the amino acid sequence of SEQ ID NO:50, the V _H2 domain contains the amino acid sequence of SEQ ID NO:53, the V _L2 domain contains the amino acid sequence of SEQ ID NO:54, the V _H3 domain contains the amino acid sequence of SEQ ID NO:9, and the V _L3 domain contains the amino acid sequence of SEQ ID NO:10.

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

In any of the trispecific binding proteins describedhigher, the non-CD38 target antigen may be any of the following typical target antigens: 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 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, 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 known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, 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 receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as integrin b4), ITK, KIR , LAG3, LAMP1, leptin, LPFS2, MHC class II molecules, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as receptor for IL33), STEAP2, Syk kinases, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a coreceptor for IL7Ra), TSLPR , TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more of the above target antigens are human target antigens.

In some embodiments, the binding protein of the present invention is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody.

The binding proteins of the present invention can be prepared using domains or sequences derived from or derived from any human or non-human antibody, including, for example, human, mouse, or humanized antibodies.

Linkers

In some embodiments, the length of the linkers L ₁ , L ₂ , L ₃ and L ₄ ranges from zero amino acids (length=0) to about 100 amino acids or less, 50, 40, 30, 20 or 15 amino acids or less. The length of linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid. All of the L ₁ , L ₂ , L ₃ and L ₄ in one binding protein may have the same amino acid sequence, or they may all have different amino acid sequences.

Examples of suitable linkers include a single glycine (Gly) residue; diglycine peptide (Gly-Gly); tripeptide (Gly-Gly-Gly); peptide of four glycine residues; peptide of five glycine residues; peptide of six glycine residues; a peptide of seven glycine residues and a peptide of eight glycine residues. Other combinations of amino acid residues can be used, such as peptide GGGGSGGGGS (SEQ ID NO: 55), peptide GGGGSGGGGSGGGGS (SEQ ID NO: 56), peptide TKGPS (SEQ ID NO: 57), peptide GQPKAAP (SEQ ID NO: 58) , as well as the peptide GGSGSSGSGG (SEQ ID NO:59). The above examples are not intended to limit the scope of the present invention in any way, and it has been shown that linkers containing 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 are suitable for binding proteins. For further description of linker sequences, see, e.g. in WO2012135345 and international application No. PCT/US2017/027488.

The composition and sequence of amino acid residues in the linker may vary depending on the type of secondary structure element that needs to be achieved by the linker. For example, glycine, serine and alanine are the most suitable linkers for maximum flexibility. Some combinations of glycine, proline, threonine and serine are useful if a stiffer, longer linker is needed. If desired, any amino acid residue can be considered as a linker in combination with other amino acid residues to construct longer peptide linkers depending on the desired properties.

In some embodiments, the length of at least one of L₁, L₂, L₃ or L₄ independently constitutes 0 amino acids. In some embodiments, the length of each of L₁, L₂, L₃ or L₄ independently constitutes at least one amino acid. In some embodiments, the length L₁ at least twice the length L₃. In some embodiments, the length L₂ at least twice the length L₄. In some embodiments, the length L₁ at least twice the length L₃, and length L₂ at least twice the length L₄. In some embodiments, the length L₁ is 3-12 amino acid residues, length L₂ is 3-14 amino acid residues, length L₃ is 1-8 amino acid residues, and length L₄ consists of 1-3 amino acid residues. In some embodiments, the length L₁ is 5-10 amino acid residues, length L₂ is 5-8 amino acid residues, length L₃ is 1-5 amino acid residues, and length L₄ consists of 1-2 amino acid residues. In some embodiments, the length L₁ is 7 amino acid residues, length L₂ is 5 amino acid residues, length L₃ is 1 amino acid residue, and length L₄ consists of 2 amino acid residues. In some embodiments, the length L₁ is 10 amino acid residues, length L₂ is 10 amino acid residues, length L₃ is 0 amino acid residues, and length L₄ consists of 0 amino acid residues. In some embodiments, the length of each of L₁, L₂, L₃ and L₄ independently ranges 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), where at least two linkers are 1-15 amino acids in length (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). In some embodiments, the length of each of L₁, L₂, L₃ and L₄ is 0 amino acids.

In some embodiments, L₁, L₂, L₃ and/or L₄ contain a sequence derived from a naturally occurring sequence at the junction between the variable domain of an antibody and the constant domain of an antibody (e.g. described in WO2012/135345). For example, in some embodiments, the linker comprises a sequence located in the transition region between the endogenous V domains_H and C_H1 or between endogenous V domains_L and C_L (For example, kappa or lambda chains). In some embodiments, the linker comprises a sequence located in the transition region between the endogenous V domains_H and C_H1 human or between endogenous V domains_L and C_L person (for example, human kappa or lambda chains).

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

Fc regions and constant domains

In some embodiments, the binding protein of the present invention comprises a full-length antibody heavy chain or a polypeptide chain containing an Fc region. In some embodiments, the Fc region is a human Fc region, for example, Fc region of human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region includes a hinge region, C_H1, C_H2, C_H3 and optionally C domains_H4. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region contains one or more mutations described below.

In some embodiments, the binding protein of the present invention contains one or two Fc variants. The term "Fc variant" as used herein refers to a molecule or sequence that is modified from the native Fc but still contains a binding site for the FcRn recycling receptor (neonatal Fc receptor). Exemplary Fc variants and their interaction with the salvage receptor are known in the art. Thus, the term "Fc variant" may include a molecule or sequence resulting from the humanization of a native Fc of non-human origin. In addition, the native Fc contains 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 present invention. Thus, the term "Fc variant" refers to a molecule or sequence that lacks one or more native Fc regions or residues or in which one or more Fc regions or residues have been modified to affect or involve: (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) heterogeneity of N-termini when expressed in the selected host cell, (4) glycosylation, (5) interaction with the complement system, (6) binding to an Fc receptor other than the receptor recycling, or (7) antibody-dependent cellular cytotoxicity (ADCC).

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

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

In some embodiments, the Fc region is a human IgG4 Fc region containing 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 containing 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 human IgG4 positions 228 and/or 409 according to the EU index. In some embodiments, the amino acid substitutions are S228P and/or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to human IgG4 positions 234 and/or 235 according to the EU index. In some embodiments, the amino acid substitutions are F234A and/or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to human IgG4 EU index positions 228, 234, 235 and/or 409. 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 human IgG4 positions 233-236 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at positions corresponding to positions 228, 233-236, and/or or 409 human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and deletion at position 236; and/or R409K.

In some embodiments, the binding protein of the present invention contains one or more mutations to improve purification, for example, by modulating the affinity for the purification reagent. For example, it is known that heterodimeric binding proteins can be selectively purified from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains mutation(s) that weaken or eliminate binding to protein A, since the heterodimeric form will have intermediate affinity for the purification agent based on Protein A over any homodimeric form and can be selectively eluted from Protein A, e.g. by using a different pH value (see e.g.Smith, E. J. et al. (2015) Sci. Rep.5:17943). In some embodiments, the mutation involves substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where 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, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain, and wherein the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; and wherein only one of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein of the present invention contains knob and valley mutations and one or more mutations to improve clearance. 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.

To increase the yield of certain binding proteins (e.g. bispecific or trispecific binding proteins) domains C_H3 can be modified using ridge-in-groove technology, which is described in detail with several examples, for example in International Publication No. WO 96/027011, Ridgwayet al., 1996,Protein Eng. 9: 617-21; and Merchantet al., 1998,Nat. Biotechnol. 16: 677-81. In particular, the interacting surfaces of two domains C_H3 modified to increase heterodimerization of both heavy chains containing these two C domains_H3. Each of the two C domains_H3 (of two heavy chains) may represent a "bump" while the other represents a "valley". The introduction of a disulfide bridge further stabilizes heterodimers (Merchantet al., 1998; Atwellet al., 1997,J. Mol. Biol. 270: 26-35) and increases yield. In certain embodiments, the overhang is on a second pair of single variable domain polypeptides. In other embodiments, the protrusion is on the first pair of polypeptides characterized by cross orientation. In yet other embodiments, domains C_H3 do not contain ridge-in-groove.

In some embodiments, the binding protein of the present invention (for example, trispecific binding protein) contains a knob mutation on the second polypeptide chain and a trench mutation on the third polypeptide chain. In some embodiments, the binding protein of the present invention comprises a knob mutation on a third polypeptide chain and a trench mutation on a second polypeptide chain. In some embodiments, the overhang mutation involves substitution(s) at positions corresponding to positions 354 and/or 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the overhang mutation involves substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W. In some embodiments, the hole mutation involves substitution(s) at positions corresponding to positions 407 and optionally 349, 366 and/or 368 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S and/or L368A. In some embodiments, the hole mutation involves substitutions at positions corresponding to human IgG1 or IgG4 EU index positions 349, 366, 368 and 407. In some embodiments, 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, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises an amino acid substitution(s). substitutions) at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4 in accordance with the EU index, where 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, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366 and/or 368 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S and/or L368A.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises an amino acid substitution(s). substitutions) at positions corresponding to positions 407 and optionally 349, 366 and/or 368 of human IgG1 or IgG4 according to the EU index, where 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, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, 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 the EU index, where the amino acid substitutions are T366W or T366Y and optionally S354C.

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

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises an amino acid substitution(s). substitutions) at positions corresponding to positions 366, 368 and/or 407 of human IgG1 or IgG4 according to the EU index, where 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, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises an amino acid substitution at a position corresponding to position 366 Human IgG1 or IgG4 according to the EU index, where the amino acid substitution is T366W.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349 , 366, 368 and 407 human IgG1 or IgG4 according to the EU index, where 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, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, where 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, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 human IgG1 or IgG4 according to the EU index, where 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.

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 immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 228, 354, 366 and 409 of human IgG4 in accordance with the EU index, where 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 the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to positions 228, 349, 366, 368, 407 and 409 of human IgG4 according to the EU index, where 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 immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407 and 409 of human IgG4 in accordance with the EU index, where 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 the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to positions 228, 354, 366 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, S354C, T366W 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 immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 234, 235, 354 and 366 of human IgG4 in accordance with the EU index, where 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 the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to positions 234, 235, 349, 366, 368 and 407 of human IgG4 according to the EU index, where 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 immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368 and 407 of human IgG4 in accordance with the EU index, where 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 the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to positions 234, 235, 354 and 366 of human IgG4 according to the EU index, where the amino acid substitutions are F234A, L235A, S354C and T366W.

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 immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366 and 409 of human IgG4 in accordance with the EU index, where 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 the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to EU index human IgG4 positions 228, 234, 235, 349, 366, 368, 407 and 409, where 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 immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region contains amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407 and 409 of the human IgG4 according to the EU index, where 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 the CH2 and CH3 constant domains of an immunoglobulin heavy chain, wherein the second Fc region comprises amino acids substitutions at positions corresponding to EU index human IgG4 positions 228, 234, 235, 354, 366 and 409, where the amino acid substitutions are S228P, F234A, L235A, S354C, T366W and R409K.

In some embodiments, the binding protein of the present invention contains 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 involves substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first CH1-linked Fc region, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region contains an immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first and/or second Fc regions contain amino acid substitutions at positions corresponding to positions 428 and 434 of IgG1 or human IgG4 according to the EU index, where the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein of the present invention contains knob and valley 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.

In some embodiments, the binding protein of the present invention contains one or more mutations to improve stability, for example, hinge region and/or contact surface of the IgG4 dimer (see, for example, Spiess, C.et al.(2013)J Biol. Chem.288:26583-26593). In some embodiments, the mutation involves substitutions at positions corresponding to human IgG4 EU index positions 228 and 409, where 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, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain, and wherein the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein each of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein of the present invention contains knob and trench mutations and one or more mutations to improve stability. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

In some embodiments, the binding protein of the present invention contains one or more mutations to improve purification, for example, by modulating the affinity for the purification reagent. For example, it is known that heterodimeric binding proteins can be selectively purified from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains mutation(s) that weaken or eliminate binding to protein A, since the heterodimeric form will have intermediate affinity for the purification agent based on Protein A over any homodimeric form and can be selectively eluted from Protein A, e.g. by using a different pH value (see e.g.Smith, E. J. et al. (2015) Sci. Rep.5:17943). In some embodiments, the mutation involves substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where 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, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain, and wherein the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; and wherein only one of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein of the present invention contains knob and valley mutations and one or more mutations to improve clearance. 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.

In some embodiments, the binding protein of the present invention contains 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 involves substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first CH1-linked Fc region, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region contains an immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first and/or second Fc regions contain amino acid substitutions at positions corresponding to positions 428 and 434 of IgG1 or human IgG4 according to the EU index, where the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein of the present invention contains knob and valley 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.

In some embodiments, the binding protein of the present invention contains one or more mutations to reduce effector function, for example, antibody-dependent cellular phagocytosis mediated by Fc receptors (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, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; where the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein each of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, where the amino acid substitutions are L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are Fc regions of human IgG1, and wherein each of the Fc regions contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitutions the replacements are L234A and L235A. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to C_H1, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; where the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein each of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG1 according to the EU index, where the amino acid substitutions are L234A, L235A and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and wherein each of the Fc regions comprises amino acid substitutions at positions corresponding to human IgG1 positions 234, 235, and 329 according to the EU index, wherein the amino acid substitutions are L234A, L235A and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG4 Fc regions, and each of the Fc regions comprises amino acid substitutions at positions corresponding to human IgG4 positions 234 and 235 according to the EU index, wherein the amino acid substitutions represent 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, while the first Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain, and wherein the third polypeptide chain additionally contains a second Fc region linked to C_H1, while the second Fc region contains the immunoglobulin hinge region and C constant domains_H2 and C_H3 immunoglobulin heavy chain; and wherein each of the first and second Fc regions contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, where the amino acid substitutions are F234A and L235A.

In some embodiments, the binding protein of the present invention contains knob and trench 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, for example, Shields, R.L.et al.(2001)J Biol. Chem.276:6591-6604 and WO 1999051642.

In some embodiments, the types of mutations described above can be combined in any order or combination. For example, the binding protein of the present invention may contain two or more knob and valley mutations, one or more mutations to improve serum half-life, one or more mutations to improve IgG4 stability, one or more mutations to improve purifications and/or one or more mutations to reduce effector function described higher.

In some embodiments, the binding protein of the present invention comprises an antibody fragment, including, without limitation, F(ab)-, F(ab')2-, Fab'-SH-, Fv- or scFv-antibody fragments.

Analyzes

The present invention relates to antigen binding proteins that bind human and/or cynomolgus CD38 polypeptides and induce T cell proliferation (e.g. CD4+ and/or CD8+ T cells) and/or induce apoptosis of CD38+ cells. Presented herein are exemplary assays for measuring such parameters and identifying said binding proteins. For example, in some embodiments, the binding affinity between the binding protein or antigen binding fragment thereof and the purified CD38 polypeptide is measured using SPR (e.g. as describedbelow), and the binding affinity between the binding protein or antigen-binding fragment thereof and the CD38 polypeptide expressed on the cell surface is measured by flow cytometry (e.g. as describedbelow).

In some embodiments, the antigen binding site of the binding protein of the present invention binds a 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 flow-through cytometry using cells that express human CD38 polypeptide containing the amino acid sequence of SEQ ID NO:1 on their cell surface, for example as describedbelow. In some embodiments, the antigen binding site binds a cynomolgus 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 flow-through cytometry using cells that express the cynomolgus CD38 polypeptide containing the amino acid sequence of SEQ ID NO:30 on their cell surface, for example, like describedbelow. In some embodiments, the antigen binding site binds a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1 with an equilibrium dissociation constant (K_D), being 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, or 0.83 nM or less, as measured by SPR using a human CD38 polypeptide containing the amino acid sequence of SEQ ID NO:1, for example, like describedbelow. In some embodiments, the antigen binding site binds a cynomolgus CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30 with an equilibrium dissociation constant (K_D), being 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, which measured by SPR assay using the cynomolgus CD38 polypeptide containing the amino acid sequence of SEQ ID NO:30, for example as describedbelow. As illustrated herein, in some embodiments, a binding protein of the present invention may have one or more of the exemplary binding properties described herein. In some embodiments, KD is measured at 4°C or 25°C.

In some embodiments, the monospecific binding protein of the present invention is characterized by one or more of the following: binds to the extracellular domain of a human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO: 1) as a purified protein according to SPR or ELISA; binds to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1) as a purified protein with K_D 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 measured by SPR assay or ELISA; binds to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1) expressed on the cell surface as measured by flow cytometry analysis; binds to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1), expressed on the cell surface, with an apparent K value_D 20 nM or less, 15 nM or less, 12 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less, as measured by flow cytometry analysis; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) as a purified protein, as measured by SPR analysis or ELISA; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) as a purified protein with K_D 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 measured by SPR assay or ELISA; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) expressed on the cell surface as measured by flow cytometry analysis; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30), expressed on the cell surface, with an apparent K value_D 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 measured by flow cytometry analysis; binds to the extracellular domain of human CD38 isoform E polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105) as a purified protein, as measured by SPR analysis or ELISA; binds to the extracellular domain of human CD38 isoform E polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105) expressed on the cell surface as measured by flow cytometry analysis; induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of a cell expressing CD38 on its cell surface; and has one or more mutations (for example, in the Fc region) resulting in decreased binding to FcγRI and/or FcγRII compared to the same binding protein in the absence of one or more mutations. In some embodiments, the binding protein of the present invention binds a CD38 polypeptide (e.g. human or cynomolgus monkey) expressed on the cell surface 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 measured by flow cytometry analysis. In some embodiments, the binding protein of the present invention 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, which measured by ELISA assay. In some embodiments, KD is measured at 4°C or 25°C.

In some embodiments, the trispecific binding protein of the present invention is characterized by one or more of the following: induces T cell proliferation (e.g. CD4+ and/or CD8+ T cells); induces expression by T cells (e.g. CD4+ and/or CD8+ T cells) Bcl-xL; induces apoptosis of CD38+ cells (e.g. measured by annexin V staining and/or propidium iodide uptake assay); binds to CD38 expressed on the cell surface and one or more target T cell antigens expressed on the surface of the T cell; binds to CD38 expressed on the cell surface, CD28 expressed on the surface of T cells, and CD3 expressed on the surface of T cells; stimulates T cell receptor activation (e.g. measured by CD69 expression); induces costimulation of T-receptor signaling (e.g. CD28-mediated); has one or more mutations (for example, in the Fc region), leading to a decrease in the induction of cytokine release (for example, IFN-γ, IL-2 and/or TNF-α) PBMC compared to the same binding protein in the absence of one or more mutations; induces the release of cytokines (eg IFN-γ and/or IL-6) PBMC in the presence of a CD38+ target cell (e.g. according to immunoassay); binds to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO: 1) as a purified protein, as measured by SPR analysis or ELISA; binds to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1) as a purified protein with K_D 1.5 nM or less, as measured by SPR assay or ELISA; binds to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO: 1) expressed on the surface of a cell, as measured by flow cytometry analysis; binds to the extracellular domain of human CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:1), expressed on the cell surface, with an apparent K_D 12 nM or less by flow cytometry; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) as a purified protein, as measured by SPR analysis or ELISA; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) as a purified protein with K_D 3.5 nM or less as measured by SPR assay or ELISA; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30) expressed on the cell surface as measured by flow cytometry analysis; binds to the extracellular domain of the cynomolgus monkey CD38 polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:30), expressed on the cell surface, with an apparent K_D 7.5 nM or less as measured by flow cytometry analysis; binds to the extracellular domain of human CD38 isoform E polypeptide (e.g. containing the amino acid sequence of SEQ ID NO:105) as a purified protein, as measured by SPR analysis or ELISA; binds to the extracellular domain of human CD38 isoform E polypeptide (e.g. comprising the amino acid sequence of SEQ ID NO:105) expressed on the cell surface as measured by flow cytometry analysis; induces apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of a cell expressing CD38 on its cell surface; and has one or more mutations (for example, in the Fc region) resulting in decreased binding to FcγRI and/or FcγRII compared to the same binding protein in the absence of one or more mutations. In some embodiments, the binding protein of the present invention binds a CD38 polypeptide (e.g. human or cynomolgus monkey) expressed on the cell surface 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 measured by flow cytometry analysis. In some embodiments, the binding protein of the present invention 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, which measured by ELISA assay.

Nucleic acids

Standard recombinant DNA technologies are used to construct polynucleotides that encode polypeptides that form binding proteins, insert these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. Cm., eg Sambrooket al., 2001, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification procedures can be carried out in accordance with manufacturers' specifications that are routinely performed in the art or as described herein. Unless specific definitions are provided, the terminology used in the context of analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry, as well as the laboratory procedures and techniques described herein, are well known and widely used in the art. Likewise, conventional techniques can be used for chemical synthesis options, chemical analysis options, pharmaceutical preparation, formulation, delivery and patient treatment.

Other aspects of the present invention relate to isolated nucleic acid molecules containing a nucleotide sequence encoding any of the binding proteins described herein. In some embodiments, the isolated nucleic acid molecules contain a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 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 sequences under SEQ ID NO:60-83 and/or shown in table J.

Certain aspects of the present invention relate to sets of polynucleotides. In some embodiments, the one or more polynucleotides are a vector (for example, expression vector). The kits may find use, among other things, in the production of one or more of the binding proteins described herein, for example, trispecific binding protein of the present invention. In some embodiments, the kit contains one, two, three, or four polynucleotides shown in Table J (e.g. mAb2xCD28supxCD3mid IgG4 FALA, mAb2xCD28supxCD3mid IgG1LALA P329A, mAb2xCD28supxCD3mid IgG1 NNSA, mAb6xCD28supxCD3mid IgG4 FALA, mAb6xCD28supxCD3mid IgG1LALA P329A or mAb6xCD28supxCD3mid IgG1 NN SA). In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:72, a third polynucleotide containing the sequence of SEQ ID NO:74, and a fourth polynucleotide containing the sequence of SEQ ID NO:75. In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:76, a third polynucleotide containing the sequence of SEQ ID NO:77, and a fourth polynucleotide containing the sequence of SEQ ID NO:75. In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:78, a third polynucleotide containing the sequence of SEQ ID NO:79, and a fourth polynucleotide containing the sequence of SEQ ID NO:75. In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:72, a third polynucleotide containing the sequence of SEQ ID NO:80, and a fourth polynucleotide containing the sequence of SEQ ID NO:81. In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:76, a third polynucleotide containing the sequence of SEQ ID NO:82, and a fourth polynucleotide containing the sequence of SEQ ID NO:81. In some embodiments, the set of polynucleotides comprises a first polynucleotide comprising the sequence of SEQ ID NO:73, a second polynucleotide containing the sequence of SEQ ID NO:78, a third polynucleotide containing the sequence of SEQ ID NO:83, and a fourth polynucleotide containing the sequence of SEQ ID NO:81.

In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to drive transcription of the nucleic acid sequence encoding the binding protein. A promoter may refer to nucleic acid control sequences that direct transcription of the nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence, wherein the first nucleic acid sequence is operably linked to a second nucleic acid sequence. For example, a promoter is operably linked to a binding protein coding sequence if the promoter affects transcription or expression of the coding sequence. Examples of promoters may include, but are not limited to, promoters derived from the genomes of viruses (such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.), heterologous eukaryotic promoters (such as actin gene promoter, immunoglobulin gene promoter, heat shock protein gene promoters, etc.), CAG promoter (Niwa et al., Gene 108(2 ):193-9, 1991), phosphoglycerate kinase (PGK) gene promoter, tetracycline inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005), lac system, trp system, tac system, trc- system, the main operator and promoter regions of lambda phage, the promoter of the 3-phosphoglycerate kinase gene, the promoters of the yeast acid phosphatase gene and the promoter of the yeast mating factor alpha gene. The polynucleotides encoding the proteins of the present invention may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein, or other suitable promoter that is readily recognized by one of ordinary skill in the art.

In some embodiments, the isolated nucleic acid is inserted into a vector. In some embodiments, the vector is an expression vector. Expression vectors may contain one or more regulatory sequences operably 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, etc.) and enhancer sequences from eukaryotic cell virus (such as the SV40 late origin enhancer (100-270 bp), early promoter/enhancer of cytomegalovirus, polyoma enhancer at the late origin of replication, enhancers of adenoviruses, etc.). Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g. vectors based on adenovirus, vaccinia virus, Sindbis virus, measles virus, herpes virus, lentivirus, retrovirus, adeno-associated virus, 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 DNA 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.

Other aspects of the present invention relate to a vector system comprising one or more vectors encoding the first, second, third and fourth polypeptide chains of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding a first polypeptide chain of the binding protein, a second vector encoding a second polypeptide chain of the binding protein, a third vector encoding a third polypeptide chain of the binding protein, and a fourth vector encoding a 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.

Isolated host cells

Other aspects of the present invention relate to an isolated host cell containing one or more isolated polynucleotides, sets of polynucleotides, vectors and/or vector systems described herein. In some embodiments, the host cell is a bacterial cell (e.g. cellE. coli). In some embodiments, the host cell is a yeast cell (e.g. cellS. cerevisiae). In some embodiments, the host cell is an insect cell. Examples of insect host cells may include, for example, cellsDrosophila (For example, S2 cells), cellsTrichoplusia ni (For example, High Five™ cages) and cagesSpodoptera frugiperda (For example, 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), Expi293™ cells, CHO cells, neonatal 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, human hepatoma cell line (e.g. Hep G2) and myeloma cells (eg NS0 and Sp2/0 cells).

Other aspects of the present invention relate to a method for producing any of the binding proteins described herein. In some embodiments, the method comprises: a) culturing a host cell (e.g. any of the host cells described herein) containing 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 in which the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods for culturing host cells under conditions that allow protein expression are well known to one skilled in the art. Methods for isolating proteins from cultured host cells are well known to one of ordinary skill in the art, including, for example, affinity chromatography (e.g. two-step affinity chromatography, including protein A affinity chromatography followed by size exclusion chromatography).

In some embodiments, the binding protein of the present invention is purified using Protein A affinity chromatography, kappa light chain affinity chromatography (e.g. using KappaSelect resin in accordance with the manufacturer's instructions; GE Healthcare) and optionally lambda light chain affinity chromatography (e.g. using LambdaFabSelect resin in accordance with the manufacturer's instructions; GE Healthcare). In some embodiments, the binding protein of the present invention is purified using Protein A affinity chromatography, lambda light chain affinity chromatography (e.g. using LambdaFabSelect resin in accordance with the manufacturer's instructions; GE Healthcare) and optionally kappa light chain affinity chromatography (e.g. using KappaSelect resin in accordance with the manufacturer's instructions; GE Healthcare). In some embodiments, the binding protein contains two Fc regions, each of which contains a C domain_H3, and only one of the domains C_H3 contains amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein of the present invention is purified sequentially by protein A affinity chromatography followed by kappa light chain affinity chromatography (e.g. using KappaSelect resin in accordance with the manufacturer's instructions; GE Healthcare) and then optionally lambda light chain affinity chromatography (e.g. using LambdaFabSelect resin in accordance with the manufacturer's instructions; GE Healthcare). In some embodiments, the binding protein of the present invention is purified by protein A affinity chromatography followed by lambda light chain affinity chromatography (e.g. using LambdaFabSelect resin in accordance with the manufacturer's instructions; GE Healthcare) and then optionally kappa light chain affinity chromatography (e.g. using KappaSelect resin in accordance with the manufacturer's instructions; GE Healthcare). For example, in some embodiments, the binding protein is contacted with Protein A, eluted from Protein A under conditions suitable for separating the binding protein from binding proteins containing 0 or 2 C domains_H3, containing the amino acid substitutions H435R and Y436F, are brought into contact with a kappa light chain affinity environment (e.g. used with KappaSelect resin; GE Healthcare) and elute from kappa light chain affinity medium under conditions suitable for separating the binding protein from binding proteins containing only C domains_L lambda circuits (for example, according to manufacturer's instructions). Conditions suitable for elution from Protein A are known in the art, including, but not limited to, pH step gradient elution of 4.5-2.8. In some embodiments, Protein A or a variant of Protein A useful for protein purification is used. In some embodiments, Protein A is attached to a support or resin, for example, 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. used with LambdaFabSelect resin; GE Healthcare) and elute from lambda light chain affinity medium under conditions suitable for separating the binding protein from binding proteins containing only C domains_L kappa chains (for example, according to manufacturer's instructions). In some embodiments, the binding protein of the present invention is detected using HIC chromatography. In some embodiments, the binding protein comprises a first polypeptide chain that contains a C domain_L lambda circuits; domain C_H3 a second polypeptide chain that contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W; domain C_H3 a third polypeptide chain that contains amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435 and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R and Y436F; and a fourth polypeptide chain, which contains domain C_L kappa chains. In some embodiments, the binding protein is produced by the host cell. In some embodiments, the binding protein is purified from cell culture medium or host cell extract. In some embodiments, the binding proteins are secreted by the host cell or produced by and extracted from the host cell (e.g. before being brought into contact with protein A). In some embodiments, the binding protein is present in a cell culture medium or host cell extract when contacted with protein A. In some embodiments, the binding protein is purified from other binding proteins, polypeptides, and/or other cellular components.

III. Trispecific binding proteins

In some embodiments, the binding protein of the present invention 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 target antigens or target proteins. In some embodiments, the first polypeptide chain comprises a structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I];

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

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge-C _H2 -C _H3 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 -hinge-C _H2 -C _H3 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ;

C _H2 is the immunoglobulin heavy chain C _H2 constant domain;

C _H3 is the immunoglobulin heavy chain C _H3 constant domain;

the hinge is the hinge region of an immunoglobulin connecting the C _H1 and C _H2 domains; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a light chain-heavy chain crossover pair. In some embodiments, the first polypeptide chain and the second polypeptide chain have a crossover orientation such that they form two different antigen binding sites. As described above, the second and third polypeptide chains include an Fc region (e.g. containing hinge-C domains_H2-C_H3). In some embodiments, one or both Fc regions are human IgG4 Fc regions containing amino acid substitutions at positions corresponding to human IgG4 positions 233-236 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the Fc regions are human IgG4 Fc regions containing amino acid mutations at positions corresponding to positions 228, 233-236, and/or or 409 human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and deletion at position 236; and/or R409K. In some embodiments, one or both Fc regions are human IgG1 Fc regions containing one or more amino acid substitutions at positions corresponding to human IgG1 positions 234, 235 and/or 329 according to the 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 human IgG1 positions 298, 299 and/or 300 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.

In some embodiments, the binding protein of the present invention 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 target antigens or target proteins. In some embodiments, at least one of the antigen binding sites binds a CD38 polypeptide (eg, the extracellular domain of human and/or cynomolgus CD38 polypeptides). In some embodiments, the first polypeptide chain comprises a structure represented by the formula:

V _L2 -L ₁ -V _L1 -L ₂ -C _L [I];

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

V _H1 -L ₃ -V _H2 -L ₄ -C _H1 [II];

and the third polypeptide chain contains the structure represented by the formula:

V _H3 -C _H1 [III];

and the fourth polypeptide chain contains the structure represented by the formula:

V _L3 -C _L [IV];

Where

V _L1 is the first variable domain of the immunoglobulin light chain;

V _L2 is the second variable domain of the immunoglobulin light chain;

V _L3 is the third variable domain of the immunoglobulin light chain;

V _H1 is the first variable domain of the immunoglobulin heavy chain;

V _H2 is the second variable domain of the immunoglobulin heavy chain;

V _H3 is the third variable domain of the immunoglobulin heavy chain;

C _L is an immunoglobulin light chain constant domain;

C _H1 is the constant domain of immunoglobulin heavy chain C _H1 ; And

L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers;

and wherein the polypeptide of formula I and the polypeptide of formula II form a light chain-heavy chain crossover pair. In some embodiments, the second and third polypeptide chains further comprise a _CH1 -linked Fc region, wherein the Fc regions comprise an immunoglobulin hinge region and immunoglobulin heavy chain constant domains _CH2 and _CH3 . In some embodiments, one or both Fc regions are human IgG4 Fc regions containing amino acid substitutions at positions corresponding to human IgG4 positions 233-236 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the Fc regions are human IgG4 Fc regions containing amino acid mutations at positions corresponding to positions 228, 233-236, and/or or 409 human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and deletion at position 236; and/or R409K. In some embodiments, one or both Fc regions are human IgG1 Fc regions containing one or more amino acid substitutions at positions corresponding to human IgG1 positions 234, 235 and/or 329 according to the 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 human IgG1 positions 298, 299 and/or 300 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.

In some embodiments, VH1 and VL1 form a binding pair and form a first antigen binding site. In some embodiments, VH2 and VL2 form a binding pair and form a second antigen binding site. In some embodiments, VH3 and VL3 form a binding pair and form a third antigen binding site. Binding proteins can also be used to activate cells, target tumors, neutralize forms of cytokine activity, neutralize viral infection, combine multiple signal transduction events to treat cancer, arthritis and/or inflammatory disorders. For example, in some embodiments, the binding protein specifically binds one, two, or three target antigens 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 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, 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, IFNα, 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 receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as integrin b4), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II molecules, 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 coreceptor for IL7Ra ), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more of the above target antigens are human target antigens.

In some embodiments, one of the three antigen binding sites binds a CD3 polypeptide (e.g. human CD3 polypeptide), one of the three antigen-binding sites binds the CD28 polypeptide (e.g. human CD28 polypeptide) and one of the three antigen-binding sites binds the third polypeptide. In some embodiments, an antigen binding site that specifically binds a target antigen other than CD3 or CD28 binds a target antigen 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-1d), CCL17 (also known as 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, 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, IFNα, 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 receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as integrin b4) , ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II molecules, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as 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 co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more of the above target antigens are human target antigens.

Any of the trispecific binding proteins described above may use any linker or combination of linkers described herein. For example, in some embodiments, the length of at least one of L ₁ , L ₂ , L ₃ or L ₄ is independently 0 amino acids. In some embodiments, L ₁ , L ₂ , L ₃ , or L ₄ are each independently at least one amino acid long. In some embodiments, L ₁ , L ₂ , L ₃ , and L ₄ are each independently zero amino acids in length or contain a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO:59). In some embodiments, L ₁ , L ₂ , L ₃ , and L ₄ each independently 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, L ₁ contains the sequence GQPKAAP (SEQ ID NO: 58), L ₂ contains the sequence TKGPS (SEQ ID NO: 57), L ₃ contains the sequence S, and L ₄ contains the sequence RT. In some embodiments, L ₁ contains the sequence GGGGSGGGGS (SEQ ID NO: 55), L ₂ contains the sequence GGGGSGGGGS (SEQ ID NO: 55), L ₃ is 0 amino acids long, and L ₄ is 0 amino acids long. In some embodiments, L ₁ contains the sequence GGSGSSGSGG (SEQ ID NO: 59), L ₂ contains the sequence GGSGSSGSGG (SEQ ID NO: 59), L ₃ is 0 amino acids long, and L ₄ is 0 amino acids long. In some embodiments, L ₁ contains the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), L ₂ is 0 amino acids long, L ₃ contains the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56), and L ₄ is 0 amino acids long.

IV. Application options for binding proteins

Binding proteins can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays to detect and quantify one or more target antigens. Binding proteins will bind one or more target antigens with an affinity that is appropriate for the assay used.

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

Binding proteins are also useful for in vivo imaging. A binding protein labeled with a detectable fragment can be administered to an animal, preferably into the bloodstream, and the presence and position of the labeled antibody in the host analyzed. The binding protein can be labeled with any moiety that can be detected in the animal by nuclear magnetic resonance, x-ray, or other detection methods known in the art.

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

The present invention also provides a kit containing a binding protein and other reagents useful for detecting levels of target antigens in biological samples. Such reagents may include detection tag, serum blocking, positive and negative control samples, and detection reagents. In some embodiments, the kit contains a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein. In some embodiments, the kit includes a container and a label or package insert on or with the container. Suitable containers include, for example, bottles, vials, syringes, IV bags, etc. Containers can be made from a number of materials, such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective for treating, preventing and/or diagnosing a condition and may have a sterile opening (for example, the container may be a bag of intravenous solution or a vial with a stopper that can be pierced with a hypodermic needle). In some embodiments, the label or package insert states that the composition is used to prevent, diagnose, and/or treat a preferred condition. Alternatively or additionally, the article or kit may further comprise a second (or third) container containing 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 substances required from a commercial and consumer point of view, including other buffers, diluents, fillers, needles and syringes.

In some embodiments, the binding protein of the present invention is administered to a patient in need thereof to treat or prevent cancer. In some embodiments, the present invention provides a method for preventing and/or treating a proliferative disease or disorder (eg, cancer). In some embodiments, the method comprises administering to a patient a therapeutically effective amount of at least one of the binding proteins or related pharmaceutical compositions described herein. In some embodiments, the present invention relates to the use of at least one of the binding proteins or related pharmaceutical compositions described herein for the prevention and/or treatment of a proliferative disease or disorder (eg, cancer) in a patient in need. In some embodiments, the present invention provides at least one of the binding proteins or related pharmaceutical compositions described herein for use in the manufacture of a medicament for the prevention and/or treatment of a proliferative disease or disorder (eg, cancer) in a patient in need. 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, such as, for example, described in section IIhigher. 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.

In some embodiments, cancer cells express human isoform A CD38 polypeptide on their cell surface (e.g. containing the amino acid sequence of SEQ ID NO:1). In some embodiments, cancer cells express human isoform E CD38 polypeptide on their cell surface (e.g. containing the amino acid sequence of SEQ ID NO:105). In some embodiments, a patient is selected for treatment based on the cancer cells expressing human isoform E CD38 polypeptide on their cell surface (e.g. containing 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.

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, B-cell germinal center lymphoma, 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 B-cell lymphoma.

In certain embodiments, the cancer is multiple myeloma. Antibodies to CD38 have been tested for use in the treatment of multiple myeloma, such as daratumumab and isatuximab. However, although multiple myeloma is considered curable, relapse is inevitable in almost all patients, leading to the development of treatment-resistant disease. In some embodiments, the cancer is relapsed or refractory multiple myeloma. In some embodiments, the patient has been treated with a prior course of treatment for multiple myeloma. In some embodiments, the binding protein of the present invention is administered to a patient as a treatment for multiple myeloma 1^th, 2^th or 3^thlines. Without being bound by theory, it is believed that the anti-CD38x-CD28x-CD3 antibody binding protein of the present invention may be useful in the treatment of multiple myeloma, for example, through recruitment of T cells to tumor cells by anti-CD38 (or anti-CD38/CD28), activation of recruited T cells by anti-CD3/CD28, and/or killing of tumor cells via perforin/granzyme-based mechanisms. CD28 has been reported to be a novel cancer marker in multiple myeloma. Cm. Nair, J.R.et al.(2011)J. Immunol.187:1243–1253.

In some embodiments, the at least one binding protein is (or needs to be) administered in combination with one or more anticancer therapeutic agents (e.g. any anti-cancer therapeutic agent known in the art, such as a chemotherapeutic agent or therapy). In some embodiments, the at least one binding protein is administered (or needs to be administered) before one or more anticancer therapeutic agents. In some embodiments, the at least one binding protein is administered (or needs to be administered) concomitantly with one or more anticancer therapeutic agents. In some embodiments, the at least one binding protein is administered (or needs to be administered) after one or more antiretroviral therapeutic agents.

V. Therapeutic compositions based on binding proteins and their administration

Therapeutic or pharmaceutical compositions containing binding proteins are within the scope of the present invention. Such therapeutic or pharmaceutical compositions may contain a therapeutically effective amount of a binding protein or binding protein-drug conjugate in admixture with a pharmaceutically or physiologically acceptable formulation agent selected in accordance with the route of administration.

Suitable formulation agents are preferably non-toxic to patients receiving them at the doses and concentrations used.

The pharmaceutical composition may contain formulation agents intended to modify, maintain or maintain, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. Suitable formulation agents include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite or sodium 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 ethylenediaminetetraacetic 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), colors, flavors and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides , salt-forming counterions (such as sodium ion), 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; sorbitol esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancers (such as sucrose and sorbitol), tonicity enhancers (such as alkali metal halides - preferably sodium or potassium chloride - or mannitol, sorbitol), delivery media, diluents, excipients and/ or pharmaceutical adjuvants (see for example, Remington's Pharmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990) and its subsequent editions, which are incorporated herein by reference for all purposes).

The optimal pharmaceutical composition will be determined by one skilled in the art depending, for example, on the intended route of administration, delivery format, and dosage required. Such compositions may be influenced by the physical state, stability, in vivo release rate, and in vivo release rate of the binding protein.

The basic medium or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or vehicle for injection may be water, physiological saline, or artificial cerebrospinal fluid, possibly with the addition of other substances conventional for compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary media. Other exemplary pharmaceutical compositions contain a Tris buffer with a pH of approximately 7.0-8.5, or an acetate buffer with a pH of approximately 4.0-5.5, which may further include sorbitol or a suitable substitute. In one embodiment of the present invention, binding protein compositions can be prepared for storage in the form of a lyophilized mass or an aqueous solution by mixing the selected composition at the required degree of purity with optional formulation aids. In addition, the binding protein can be formulated in the form of a lyophilisate using suitable excipients such as sucrose.

The pharmaceutical compositions of the present invention may be intended for parenteral or subcutaneous delivery. Alternatively, the compositions may be intended for inhalation or for delivery through the digestive tract, for example, orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the person skilled in the art.

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

If parenteral administration is contemplated, the therapeutic compositions to be used may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, with which the binding protein is formulated as a sterile isotonic solution with suitable preservatives. Another formulation may be formulated based on the desired molecule with a vehicle, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), granules or liposomes, which provides a controlled or sustained release of the product, which can then be delivered via depot injection. Hyaluronic acid can also be used and may have the effect of providing a long stay in the bloodstream. Other suitable means for administering the desired molecule include implantable drug delivery devices.

In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, the binding protein may 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, the solutions can be sprayed.

It is also contemplated that certain formulations may be administered orally. In one embodiment of the present invention, the binding proteins that are administered in this manner can be formulated with or without such carriers as are typically used in the formulation of solid dosage forms such as tablets and capsules. For example, the capsule may be designed to release the active portion of the formulation upon entry into the digestive tract, where bioavailability is maximized and disintegration prior to entry into the systemic circulation is minimized. Additional substances may be included to facilitate absorption of the binding protein. Diluents, flavoring agents, low-melting waxes, vegetable oils, lubricants, suspending agents, disintegrating agents and binders may also be used.

Another pharmaceutical composition may include an effective amount of binding proteins in admixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water or other suitable medium, 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 binders such as starch, gelatin or gum acacia; or lubricants such as magnesium stearate, stearic acid or talc.

Additional pharmaceutical compositions of the present invention will be apparent to those skilled in the art, including formulations comprising binding proteins in a sustained or controlled delivery form. Techniques for formulating a number of other delayed or controlled delivery agents, such as liposomal carriers, biodegradable microparticles or porous granules and depot injection forms, are also known to those skilled in the art. Additional examples of sustained release formulations include semi-permeable polymer matrices in the form of shaped articles, e.g. films or microcapsules. Sustained release matrices may 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 may also include liposomes, which can be prepared by any of several methods known in the art.

Pharmaceutical compositions to be used for in vivo administration generally must be sterile. This can be achieved by filtration through sterile filtration membranes. If the composition is lyophilized, then sterilization using this method can be carried out either before or after lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in solution form. In addition, compositions for parenteral administration are typically placed in a container having a sterile entry port, such as an intravenous solution bag or vial with a stopper that can be pierced with a hypodermic injection needle.

Once the pharmaceutical composition is 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 ready-to-use form or in a form (e.g. lyophilized), requiring reconstitution before administration.

The present invention also covers kits for preparing a single dose unit. Each of the kits may contain both a first container with a dried protein and a second container with an aqueous composition. Also included within the scope of the present invention are kits containing single and multi-chamber prefilled syringes (e.g. syringes with liquid and syringes with lyophilisate).

The effective amount of the binding protein pharmaceutical composition to be used therapeutically will depend, for example, on the therapeutic case and purpose. One skilled in the art will appreciate that suitable dosage levels for treatment will therefore vary depending in part on the molecule being delivered, the indication for which the binding protein will be used, the route of administration and metric characteristics (body weight, body surface area or organ size) and condition (age and general health) of the patient. Accordingly, the doctor can select the dose and modify the route of administration to obtain the optimal therapeutic effect.

The frequency of dosing will depend on the pharmacokinetic parameters of the binding protein in the formulation to be used. Typically, the physician will administer the composition until the dosage reaches a level at which the desired effect is achieved. Therefore, the composition can 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 a period of time, or as a continuous infusion via an implantable device or catheter. Further determination of the appropriate dosage is performed in a standard manner by those of ordinary skill in the art and is within the range of tasks that they typically perform. Suitable dosages can be determined using appropriate dose-response data.

The route of administration of the pharmaceutical composition corresponds to known methods, for example, orally; by injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; through sustained release systems or through implantable devices. If necessary, the compositions can be administered as a bolus injection or continuously by infusion or via an implantable device.

The composition can also be administered topically by implantation of a membrane, sponge, or other suitable material into which the desired molecule has been absorbed or encapsulated. If an implantable device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be through diffusion, bolus administration with time-controlled release, or continuous administration.

EXAMPLES

The following examples illustrate specific embodiments of the present invention and various applications thereof. They are provided for explanatory purposes only and should not be construed as limiting the scope of the present invention in any way.

The following terminology may be used interchangeably in the examples and graphics provided herein to refer to specific antigen binding domains or antibodies to CD38:

antibody CD38_C2-CD38-1: mAb1

antibody CD38_C2-CD38-1_VH1-VL1 or CD38 _VH1 : mAb2

antibody CD38_C2-CD38-1_VH3-VL3: mAb3

antibody CD38_C2-CD38-1_VH5-VL3: mAb4

antibody CD38_C2-CD38-1_VH6-VL3: mAb5

antibody CD38_1370 or CD38 _HHY1370 : mAb6

CD38_SB19 antibody or isatuximab: mAb7.

Example 1. Production and characterization of monoclonal antibodies to CD38

The following examples describe the production and characterization of anti-CD38 monoclonal antibodies. Advantageously, the antibodies presented herein cross-react with human and monkey CD38 proteins to produce molecules that can be used for both safety studies and clinical trials. Such antibodies are also capable of killing CD38+ cells through apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC).

This example describes an efficient sequence for generating CD38-specific and cross-reactive monoclonal antibodies from individual murine B cells.

Materials and methods

Preparation of monoclonal antibodies

Anti-human CD38 antibodies were generated using the extracellular domain of human CD38 R45-I300 (SEQ ID NO:1). Cm. 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 an immune response to either normal BalbC mice or Trianni™ transgenic mice (Trianni, San Francisco, CA) containing DNA encoding the variable regions of the human immunoglobulin kappa heavy and light chains.

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

Immunization of mice and selection of individual B cells

Antibodies to CD38 were also isolated directly from antigen-positive B cells without fusion with myeloma cells. More anti-CD38 antibodies, such as mAb1, have been produced using this method (see SEQ ID NO: 5 and 6 for the VH and VL sequences, respectively, and SEQ ID NO: 7 and 8 for the heavy chain and light chain sequences, respectively). Briefly, each 6- to 8-week-old female BALB/c mouse (S082342; Charles River Labs, Bar Harbor, ME) was given three cycles of immunization over 41 days using the classical method described by A. Wennerberget al.(1993 Am. J. Pathol. 143:1050-1054). The antigen was administered intraperitoneally from the ventral side of mice. Three days after the last injection, mice were sacrificed and spleens were collected aseptically and washed with fresh RPMI medium. Lymphocytes were isolated from the spleen and the single cell suspension was washed twice with RPMI before sorting using a four-color sorting strategy including a panel of fluorescent antibodies and human and monkey CD38 dual proteins and then separated using flow cytometric cell sorting to isolate cross-reactive IgG-CD38 -specific human/monkey B cells. Individual cells were directly sorted into PCR tubes to amplify the cognate pair of VH and VL genes using RT-PCR (T. Tiller, C. Busse and H. Wardemann 2009 J. Immunol. Methods 350:183-193). The resulting DNA was subjected to sequencing.

The resulting DNA was cloned into a mammalian expression vector encoding human IgG1 or human Ck domains, respectively, for transient expression in HEK293 cells using the FreeStyle™ MAX 293 expression system according to the manufacturer's instructions. Batches were purified using Protein A affinity chromatography (MabSelect, GE Healthcare). The eluate was dialyzed against PBS before sterile filtration and stored at 4°C.

Obtaining antibodies by immunizing transgenic mice with human immunoglobulin and selection using hybridoma technology

Immunization, fusion and screening were performed using human CD38 extracellular domain P3X63-Ag8.653 myeloma cells as described in Kilpatricket al.1997 Hybridoma 16: 381389. Using the RIMMS method described by Kilpatricket al.,Each of the 6-8 week old transgenic Trianni Mice™ females containing DNA encoding the variable regions of the heavy and light kappa chains of human immunoglobulin was given four cycles of immunization over 14 days at intervals of 3-4 days. CD38 protein emulsified in RIBI adjuvant (Sigma #T2684) was injected subcutaneously at six sites proximal to the draining lymph nodes along the back of mice and at six adjacent sites along the abdomen. Four days after the last injection, mice were sacrificed. Bilateral popliteal, superficial inguinal, axillary and bronchial lymph nodes were aseptically removed and washed with fresh RPMI medium. Lymphocytes were recovered from lymph nodes and the single-cell suspension was washed twice with RPMI medium before fusion with P3X63-AG8.653 myeloma cells using polyethylene glycol. After confluence, the cell mixture was incubated in an incubator at 37°C for 16-24 hours. The resulting cell preparation was transferred to a selective semisolid medium and aseptically seeded into Petri dishes with a diameter of 100 mm and incubated at 37°C. Ten days after the start of selection, the plates were examined for hybridoma growth, and visible colonies were collected and plated into 96-well plates containing 200 μl of growth medium. 96-well plates were stored in an incubator at 37°C for 2-4 days. Using this technique and the immunogen described above, several chimeric CD38 antibodies have been produced, for example, mAb 6 (see SEQ ID NOs: 9 and 10 for the VH and VL sequences, respectively, and SEQ ID NOs: 11 and 12 for the heavy chain and light chain sequences, respectively. The VH and VL sequences were obtained by RT-PCR, and mAb 6 was obtained by transient expression as described above.

Binding affinity for soluble extracellular domains of CD38

The binding properties of anti-human CD38 antibodies were assessed using BIAcore 2000 (BIAcore Inc., Uppsala, NJ). Briefly, the CM5 BIAcore biosensor chip was fixed in this instrument and activated with 250 μL of 1:1 NHS/EDC at room temperature. Mouse anti-human IgG1 Fc (GE Healthcare #BR-1008-39) (13.5 μg/ml in 0.05 M acetate buffer, pH 5) was immobilized on activated chips in flow cells 1. Immobilization was performed at a flow rate of 5 μl /min. The chip was then blocked by injecting 55 μl ethanolamine-HCl, pH 8.5, followed by washing five times with 50 mM NaOH, 1 M NaCl. To measure the binding of anti-CD38 antibodies to human CD38 or cynomolgus CD38, antibodies were used at a concentration of 2 μg/ml in BIAcore running buffer (HBS-EP). Antigens (human CD38-his-tag (ID2) or cynomolgus CD38-his-tag (ID3)) were administered at 3 to 1000 nM. After completion of the injection phase, dissociation was monitored in BIAcore running buffer at the same flow rate for 360 s. The surface was regenerated between injections using 30 μl of 50 mM NaOH-1 M NaCl. Individual sensorgrams were analyzed using BIAsimulation software.

Binding affinity for B cell progenitors expressing human CD38

The binding of anti-CD38 antibodies to CD38 expressed on the surface of recombinant mouse preB::300.19 cells was determined by flow cytometry. The recombinant cell line was described in J. Deckket et al. 2014 Clin. Cancer Res 20:4574–4583. Mouse preB::300.19 CD38-expressing cells were transferred at 40,000 cells/well into a 96-well High Bind plate (MSD L15XB-3) and 100 μl/well of anti-CD38 antibody was added for 45 minutes at 4°C and washed three times. using PBS 1% BSA. 100 μl/well of goat anti-human IgG conjugated to Alexa488 (Jackson ImmunoResearch; #109-545-098) was added over 45 min at 4°C and washed three times with PBS 1% BSA. Antibody binding was assessed after centrifugation and resuspension of cells by adding 200 μl/well of 1% BSA PBS and reading using a Guava® easyCyte™ 8HT flow cytometry system. Apparent KD and EC50 values were estimated using BIOST@T-BINDING and BIOST@T-SPEED programs, respectively.

results

Binding of the newly isolated mAb1 to human SU-DHL-8 or MOLP-8 cells was compared with that of isatuximab using flow cytometry ( Figure 1A ). Both antibodies showed high affinity binding to both cell lines. However, only mAb1 was able to bind to cells expressing cynomolgus CD38 on its surface ( Figure 1B ).

Binding to the soluble extracellular domains of human and cynomolgus CD38 polypeptides was examined with newly isolated antibodies mAb1 and mAb6 using surface plasmon resonance (SPR). The SPR results are shown in Table A.

Table A. Antibody binding affinities to the soluble extracellular domain of hCD38 and cCD38 as determined by SPR analysis

hCD38-his (SEQ ID NO:2) cCD38-his (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

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

Table B. Binding affinity of antibodies to hCD38 or cCD38 expressed by mouse preB::300.10 cells determined by flow cytometry

Apparent KD FACS values (M) hCD38-expressing cells cCD38-expressing cells mAb1 2.80E-10 2.20E-10 mAb6 2.07E-09 1.14E-09

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

Example 2: Construction in silico variants of humanized antibodies to CD38

This example describes the humanization of antibodies obtained in example 1.

The mAb1 variable domain sequences were analyzed in silico. The ImMunoGeneTics International Immunoglobulin Information System (IMGT) was used to identify complementarity determining regions (CDRs).

First, a parallel search was performed to determine the closest combination of mouse germline and human germline protein sequence for each variable chain. This was performed using Basic Local Alignment Search Tool (BLAST) (Nucleic Acids Res. 25:3389–3402) by comparison with mouse and human germline protein sequence databases. For each antibody chain, the closest protein sequences of mouse and human V and J were identified. The fitness of a given high-identity protein sequence was measured by percent V-region identity. The mAb1 light and heavy chain search results are presented in Tables C and D, respectively.

Table C. mAb1 variable light chains of the closest human and mouse germline protein sequences (V and J).

View Allele V Region V identity (%) Allele J [Homo sapiens] IGKV4-1*01 (imgt) 66.34% IGKJ1*01 (imgt) Best functional germlines Functional Functional [Mus musculus] IGKV3-10*01 (imgt) 96.97% IGKJ1*01 (imgt) Best functional germlines Functional Functional

Table D. mAb1 variable heavy chains of the closest human and mouse germline protein sequences (V and J).

View Allele V Region V identity (%) Allele J [Homo sapiens] IGHV1-3*01 (imgt) 69.39% IGHJ4*01 (imgt) Best functional germlines Functional Functional [Mus musculus] IGHV1-12*01 (imgt) 87.76% IGHJ3*01 (imgt) Best functional germ lines Functional Functional

The mAb1 variable domain sequences were then screened for the presence of sequence instability sites susceptible to, for example, deamidation, acid cleavage, oxidation, or formation of isoaspartate sites. The structure of mAb1 was analyzed using 3D homology modeling to determine how amino acid residues interact within or between molecules. This step led to the identification of a group of amino acid residues that are structurally important for mAb1 functionality such as CDR conformation and antigen binding. These amino acid residues were selected to be present in the variable regions of the humanized mAb1 sequences.

CDRs from the parental mouse mAb1 were grafted onto the corresponding scaffolds. Based on the above analyzeshuman IGKV3-20*02 V combined withIGKJ1*01 Andhuman IGHV1-3*01 in combination withIGHJ4*01 chosen in as the basis for humanizing the mAb1 light chain variable region and heavy chain variable region, respectively. The light chain of mAb1 shows 64.52% identity in the V region with the selected human germline IGKV3-20*02. The heavy chain of mAb1 shows 69.39% identity in the V region with the selected human germline IGHV1-3*01. During the humanization process, parental murine mAb1 CDRs were transplanted between selected germline frameworks to recombine standard antibody variable region sequences. Attention was drawn to a previously identified group of amino acid residues in mAb1 that are structurally important for its functionality, as noted above. When necessary, these amino acid residues were replaced in the newly generated sequences with their exact corresponding mAb1 residue. This corresponds to a reverse mutation step to incorporate the corresponding amino acid residues of the parent sequence. Some mutations in the CDRs have been introduced both to humanize and to prevent sites of sequence instability in the parental CDRs. The newly generated light and heavy chain variable region sequences were used to generate three-dimensional variable region homology models of the humanized mAb1. 3D models were built using the Model Antibody Framework from the BIOVIA Discovery Studio kit.

Based on the CDR grafting approach, two light chain variable region variants (VL1 and VL3) and four heavy chain variable region variants (VH1, VH3, VH5 and VH6) were generated. The specific combination of amino acid residues that vary between the light and heavy chain variable regions of mAb1 and their humanized variants are presented in Tables E and F, respectively.

Table E. Sequence Differences Between mAb1 Light Chain Variable Regions and Humanized Variants

Ig regions (IMGT) VL of parent mAb1 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 heavy chain variable regions and humanized variants

Ig regions (IMGT) VH of parent mAb1 Humanizi
roved VH1 Humanizi
rated VH3 Humanizi
rated VH5 Humanizi
rated 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

The humanized VL1 variant SEQ ID NO: 14 exhibits a total of 22 mutations (18 in FR and 4 in CDR ) compared to the parental VL in the mAb1 sequence SEQ ID NO: 6. This variant is derived from IGKV3-20 germline frameworks *02 human combined with IGKJ1*01 with 8 reverse mutations made due to the risk of negative effects on mAb structure, CDR conformation and therefore binding to its target. Four positions in the parental CDRs were mutated to increase the level of humanization or to avoid the occurrence of regions of instability in the sequence.

The humanized variant VL3 SEQ ID NO: 18 shows a total of 18 mutations (18 in FR ) compared to the parental VL in the mAb1 sequence SEQ ID NO: 6. This variant is derived from human IGKV3-20*02 germline frameworks in combined with IGKJ1*01 with 8 reverse mutations performed due to the risk of negative effects on mAb structure, CDR conformation and therefore binding to its target.

The humanized VH1 variant SEQ ID NO: 13 exhibits a total of 17 mutations (14 in FR and 3 in CDR ) compared to the parental VH in the mAb1 sequence SEQ ID NO: 5. This variant is derived from IGHV1-3 germline frameworks *01 human combined with IGHJ4*01 with 11 reverse mutations performed due to the risk of negative effects on the mAb structure, CDR conformation and therefore binding to its target. Three positions in the parental CDRs were mutated to increase the level of humanization or to avoid the occurrence of regions of instability in the sequence.

The humanized variant VH3 SEQ ID NO: 17 exhibits a total of 14 mutations (14 in FR ) compared to the parental VH mAb1 sequence SEQ ID NO: 5. This variant is derived from human IGHV1-3*01 germline frameworks in combination with IGHJ4*01 with 11 back mutations performed due to the risk of negative effects on mAb structure, CDR conformation, and therefore binding to its target.

The humanized variant VH5 SEQ ID NO: 21 exhibits a total of 11 mutations (11 in FR ) compared to the parental VH in the mAb1 sequence SEQ ID NO: 5. This variant is derived from human IGHV1-3*01 germline frameworks in combined with IGHJ4*01 with 14 reverse mutations performed due to the risk of negative effects on mAb structure, CDR conformation and therefore binding to its target.

The humanized variant VH6 SEQ ID NO: 23 exhibits a total of 12 mutations (12 in FR ) compared to the parental VH in the mAb1 sequence SEQ ID NO: 5. This variant is derived from human IGHV1-3*01 germline frameworks in combined with IGHJ4*01 with 13 reverse mutations performed due to the risk of negative effects on mAb structure, CDR conformation and therefore binding to its target.

The resulting humanized light and heavy chain variable region sequences were purified to determine sequence similarity to the Immune Epitope Database (IEDB) database ((PLos Biol (2005) 3 (3) e91) www.iedb.org) to ensure that that none of the sequences contained any known B- or T-cell epitope set forth in this database.

The complete amino acid sequences of the mAb1 variable region and the humanized light and heavy chain variable domains are shown in Table G. These humanized light and heavy chain variable domains were combined to create several humanized variants of the parent mAb1 variable domains. The variable domains of mAb2 correspond to the association of humanized VH1 in combination with humanized VL1. The variable domains of mAb3 correspond to the association of humanized VH3 in combination with humanized VL3. The variable domains of mAb4 correspond to the association of humanized VH5 in combination with humanized VL3. The variable domains of mAb5 correspond to the association of humanized VH6 in combination with humanized VL3. The trispecific binding proteins shown in Table G are described in more detail in Example 4.

The corresponding DNA coding sequences of the humanized variants VH and VL described above were cloned into a mammalian expression vector encoding human IgG1 or human Ck domains, respectively, for transient expression and purification as described in Example 1. The amino acid sequences of the full-length humanized variants of anti-CD38 antibodies, derived from mAb1 are given as mAb2 (heavy chain: HC1, SEQ ID NO:15; light chain: LC1, SEQ ID NO:16), mAb3 (heavy chain: HC3, SEQ ID NO:19; light chain: LC3, SEQ ID NO:20), mAb4 (heavy chain: HC5, SEQ ID NO:22; light chain: LC3, SEQ ID NO:20) and mAb5 (heavy chain: HC6, SEQ ID NO:24; light chain: LC3, SEQ ID NO:20).

Example 3: Cross-reactivity and induction of apoptosis by anti-CD38 antibodies

The humanized anti-CD38 antibody variants produced in Example 2 were then characterized for binding to human and cynomolgus CD38 polypeptides and inducing apoptosis.

Materials and methods

Apoptosis Induction Assay

Cells were incubated at ^2x105 cells/ml in complete medium (RPMI-1640, 10% FBS, 2 mM L-glutamine) with 1.5 μg/ml (10 nM) of the indicated antibodies for 20 hours at 37°C with 5% CO2. Cells were stained with Annexin V-FITC according to the manufacturer's instructions (Life Technologies). Samples were analyzed by flow cytometry on a BD FACSAria™ flow cytometer using BD FACSDiva software to control data acquisition and analysis (both BD Biosciences).

results

The binding properties of selected anti-human CD38 antibodies prepared as described above were examined ( FIGS. 2A-2H ). Antibody binding to soluble human CD38 and cynomolgus CD38 was examined using ELISA and SPR assays. Data from the ELISA assay were used to determine the EC50 binding of human and cynomolgus CD38 antibodies for the humanized anti-CD38 mAb2 ( Figure 2A ), mAb3 ( Figure 2C ), mAb4, mAb5 ( Figure 2E ), and human anti-CD38 mAb6 (Figure 2A). Fig. 2I ).

The binding of humanized anti-CD38 antibody variants or human anti-CD38 mAb to CD38 was also assessed using the SPR assay described above. SPR assay data were used to determine the _KD and _koff of human and cynomolgus CD38 antibody binding for the humanized anti-CD38 mAb2 ( Figure 2B ), mAb3 ( Figure 2D ), mAb4, mAb5 ( Figure 2F ) and human antibodies anti-CD38 mAb6 ( Figure 2H ). The binding data summarized in Table K shows that all CD38 mAbs bind to CD38 with similar binding characteristics.

Table K. Binding affinity of CD38 mAb to the soluble extracellular domain of human CD38 and cynomolgus CD38 determined by surface plasmon resonance

hCD38-his (SEQ ID NO:2) cCD38-his (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 mAb2 3.90E-04 3.32E-10 7.84E-04 3.44E-09 mAb3 2.83E-04 4.83E-10 1.29E-04 7.10E-10 mAb4 5.29E-04 8.22E-10 2.01E-04 1.14E-09 mAb5 3.33E-04 3.12E-10 1.25E-04 5.63E-10 mAb6 2.03E-04 1.44E-09 1.90E-04 1.38E-09

The ability of the humanized anti-CD38 antibody variants to bind to CD38-expressing cells was assessed using the FACS-based binding assay described above. Data from FACS analysis were used to determine human and cynomolgus CD38 antibody binding EC50 for humanized anti-CD38 mAb2 ( Figure 2A ), mAb3 ( Figure 2C ), mAb4, mAb5 ( Figure 2E ), and human anti-CD38 mAb6 (Figure 2A). Fig. 2G ). The binding assay data shown in Table L shows that all humanized anti-CD38 antibody variants exhibited similar binding affinities to cell surface CD38.

Table L. Binding affinity of CD38 mAb to CD38 expressed in murine preB::300.19 cells

FACS Apparent KD Values (M) hCD38-expressing cells cCD38-expressing cells mAb1 2.80E-10 2.20E-10 mAb2 3.30E-10 7.50E-10 mAb3 7.80E-10 1.31E-09 mAb4 5.50E-10 1.15E-09 mAb5 6.80E-10 1.07E-09 mAb6 2.07E-09 1.14E-09

Binding assay data from the three assays are summarized in FIG. 2I along with the sequence identity of the VH and VL domains with the human V regions.

The ability of the parent antibody mAb1 and mAb7 to induce apoptosis was then examined. Both antibodies provided enhanced annexin V staining and propidium iodide (PI) uptake. 40% of cells became double positive for Annexin V and PI staining after mAb7 treatment, whereas 60% of cells treated with mAb1 were double positive. Both antibodies exhibited similar concentration-dependent apoptotic effects in SU-DHL-8 cells ( Figure 2J ). Similarly, both antibodies stimulated ADCC activity against SU-DHL-8 cells in the presence of NK92 cells ( Figure 2K ), providing up to 60% cytotoxicity and an IC50 of 4–6 μM after 4 hours at 37°C ( Figure 2L ).

CD38 isoform E was identified in silico and confirmed at the transcript level from NK cells, PBMC and BMMC from multiple myeloma patients and cancer cell lines (MOLP-8, CU1702 and CU2332). CD38 isoform E was confirmed by nucleic acid sequence database screening using BLASTN 2.2.26 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.) in the Human RefSeq transcript database release 20131216. The BLASTN program was used without masking low complexity regions in sequence and having at least: 98.5% identity with the nucleotide sequence of human CD38 isoform A over a region of at least 100 nucleic acid residues. The sequences isolated from this screen were re-aligned to the CD38 gene locus to confirm that they were transcriptional forms of the human CD38 gene (same intron-exon genomic structure). The nucleic sequence of CD38 isoform E was one of the sequences confirmed to be a transcriptional form of the human CD38 gene.

The ability of anti-CD38 antibodies to bind to both isoforms A and E of human CD38 was also examined. To assess binding to CD38 isoform A and isoform E, an enzyme-linked immunosorbent assay (ELISA) was performed using isoform A and isoform E proteins (prepared as described in Example 1) as capture antigen. 96-well plates were coated by adding each antibody isoform at a concentration of 0.5 μg/well in PBS and 100 μl/well. The plate was incubated at 37°C for 1 hour and washed five times with PBS containing 0.05% Tween-20 (PBS-T). Then, 100 μl of a 1:25,000 dilution of horseradish peroxidase-conjugated anti-human IgG antibody (Jackson Ref: 109-035-098) was added to each well. After incubation at 37°C for 1 h in the dark, the plates were washed with PBS-T five times. Antibody binding was visually monitored by adding TMB-H2O2 buffer and read at 450 nm. EC50 values were estimated using BIOST@T-SPEED software.

The binding affinities of various antibodies to CD38 isoform A (SEQ ID NO:1) and isoform E (SEQ ID NO:105) were determined as shown in Table L2. Table M provides a comparison of binding properties for various anti-CD38 antibodies.

Table L2. Binding affinity of anti-CD38 antibodies to CD38 isoforms A and E based on EC50 according to ELISA analysis

Antibody CD38 isoform A, EC50 (nM) CD38 isoform E, EC50 (nM) mAb1 0.11 (CV 9%) 0.08 (CV 7%) mAb2 0.14 (CV 13%) 0.10 (CV 12%) mAb6 0.47 (CV 3.7%) 0.32 (CV 5%) mAb7 0.10 (CV 7.1%) No binding

Table M. Binding characteristics of various CD38 antibodies.

Antibody to CD38 H11 (Santa Cruz) Daratumumab mAb7 mAb1 mAb6 Binding to human CD38 isoform A + + + + + Binding to human CD38 isoform E + - - + + Binding to CD38 in cynomolgus monkey + - - + +

In conclusion, mAb7 and mAb1 induced similar apoptosis in human MOLP-8 multiple myeloma cells, a similar concentration-dependent apoptotic effect in SU-DHL-8 cells, and a similar concentration-dependent ADCC activity in SU-DHL-8 cells. However, only mAb1 binds to human and cynomolgus CD38 with subnanomolar affinity and binds CD38 isoforms A and E.

The ability of humanized anti-CD38 antibody variants to induce apoptosis was also assessed using fluorescence-activated cell sorting (FACS) as described above. Several anti-CD38 antibodies with different functional properties have been identified previously, while some may mediate in vitro killing of CD38+ cell lines through direct effects on cell proliferation or apoptosis. The results of the apoptosis induction assays are shown in FIG. 2M . All antibodies obtained in examples 1 and 2 were able to induce apoptosis, except for mAb6. Antibodies to CD38, mAb2, mAb3, mAb4 and mAb5, led to a dose-dependent induction of apoptosis in SU-DHL-8 lymphoma cells; The IC50 for each antibody is shown in FIG. 2N - 2Q .

Example 4: Preparation of Trispecific CD38 Binding Proteins

The binding properties of the antigen binding domains of the selected anti-CD38 antibodies described in Examples 1-3 were then analyzed in the trispecific format shown in FIG. 3A.

Using SPR, antigen-binding domains to CD38 were examined in a trispecific format (anti-CD38x-CD28x-CD3) for the ability to bind CD38 when other antigen-binding domains are bound to their cognate ligands. The sequential ligand binding assay is shown in FIG. 3B . To sequentially bind the three antigens to each trispecific Ab, a saturating concentration (>10 kDa) of each antigen was administered for 8 min, followed by dissociation for 5 min. Surface regeneration was performed by injecting 10 mM glycine-HCl pH 2.5 for 60 s at 30 μL/min. Data were fitted to a 1:1 kinetic binding model and analyzed using Biacore S200 Evaluation Software v 1.0. The equilibrium dissociation constant (K _D ) was calculated using the association rate constant (k _on ) and the dissociation rate constant (k _off ).

As shown in FIG. 3C , this SPR-based assay showed that the trispecific binding proteins were able to bind CD38 regardless of whether the CD3 and/or CD28 antigen binding domains were also bound to their cognate antigen. The results of an exemplary sequential binding assay are shown in FIG. 4 . Kinetic parameters measured by SPR are presented in Table M2.

Table M2. Binding of CD38x-CD28x-CD3 trispecific binding protein antibodies to 1, 2 or 3 cognate antigens.

State of the binding protein before CD38 binding k _a (M ^-1 s ^-1 ) k _d (s ^-1 ) _KD (M) No pre-binding 9.02E+05 1.42E-03 1.57E-09 Pre-binding to CD3 8.35E+05 1.24E-03 1.48E-09 Prebinding to CD28 7.39E+05 1.32E-03 1.79E-09 Pre-binding CD3 then CD28 8.18E+05 1.23E-03 1.50E-09 Pre-binding CD28 then CD3 8.37E+05 1.23E-03 1.47E-09

These results demonstrate that all three targets can simultaneously bind to trispecific binding proteins. Prebinding trispecific binding proteins to CD28, CD3, or both (in any order) did not alter binding kinetics or binding affinity to CD38.

Each antigen binding domain of the trispecific binding protein CD38 _SB19 xCD28 _sup xCD3 _mid was then assessed by SPR for the ability to bind the cognate antigen with and without the other two antigen binding domains at saturation. Tables M3 and M4 show the results of these analyses.

Table M3. Target binding in the absence of other targets

Target k _a (M ^-1 s ^-1 ) k _d (s ^-1 ) _KD (M) CD38 8.04E+05 1.41E-03 1.75E-09 CD28 1.16E+05 3.14E-04 2.71E-09 CD3 2.90E+04 6.73E-04 2.32E-08

Table M4. Binding of a target in the presence of other targets at saturation

Target k _a (M ^-1 s ^-1 ) k _d (s ^-1 ) _KD (M) CD38 5.93E+05 1.44E-03 2.42E-09 CD28 1.05E+05 3.96E-04 3.77E-09 CD3 1.27E+05 2.36E-03 1.86E-08

As shown in Tables M3 and M4, the presence of two targets enriched by antigen prebinding did not affect the kinetics or binding affinity of the third target for CD38 or CD28. In the case of CD3 binding, prebound CD38 and/or CD28 resulted in faster kinetics (approximately 4-fold effect on k _on and k _off values).

The antigen-binding domains of anti-CD38 antibodies were examined in a trispecific format with two antigen-binding domains of anti-CD28 antibody (superagonist, “sup” and traditional agonist, “cvn”) and two antigen-binding domains of anti-CD3 antibody (“mid” and “low”). The variable domain sequences for these antigen binding domains are as follows: CD28 _sup antibody: SEQ ID NO:49 (VH) and SEQ ID NO:50 (VL); CD28 _cvn antibody: SEQ ID NO:51 (VH) and SEQ ID NO:52 (VL); CD3 _mid antibody: SEQ ID NO:53 (VH) and SEQ ID NO:54 (VL); CD3 _low antibody: SEQ ID NO:84 (VH) and SEQ ID NO:85 (VL). The results of SPR assays assessing the binding of trispecific binding proteins are shown in FIG. 5 . The three binding domains of the CD38 antibody had approximately the same binding affinities in the trispecific binding protein format as in the monospecific format. Both CD3-binding domains had approximately the same binding affinities in mono-, bi- and tri-specific formats. CD28 binding domains should have slightly reduced (but still nanomolar) binding affinity in the bi- or trispecific format compared to the monospecific format. When the other two antigen-binding domains were saturated, the CD38 _SB19 antibody and the CD28 _sup -binding domains had similar binding affinities compared to when the other two antigen-binding domains were not bound to antigen. However, the CD3 _mid -binding domain showed faster kinetics when the other two antigen-binding domains were in a saturated state. These results demonstrate that the CD38, CD28, and CD3 binding domains are compatible for use in a trispecific binding protein format.

The anti-CD38 antigen-binding domains generated herein were also compared to the existing anti-CD38 mAb7 antigen-binding domain (see SEQ ID NO:47 for VH and SEQ ID NO:48 for VL sequences, respectively). Binding of the trispecific molecules to CD38 expressed on the surface of recombinant mouse preB::300.19 cells was determined by flow cytometry, and the corresponding monovalent anti-CD38 antibodies were analyzed in parallel. The recombinant cell line was described in J. Deckket et al., 2014 Clin. Cancer Res 20:4574–4583. Mouse preB::300.19 CD38-expressing cells were transferred at 40,000 cells/well into a 96-well High Bind plate (MSD L15XB-3), and 100 μl/well of trispecific molecules were added for 45 minutes at 4°C and washed three times with PBS 1% BSA. 100 μl/well of goat anti-human IgG conjugated to Alexa488 (Jackson ImmunoResearch; #109-545-098) was added over 45 min at 4°C and washed three times with PBS 1% BSA. Antibody binding was assessed after centrifugation and resuspension of cells by adding 200 μl/well of 1% BSA PBS and reading using a Guava® easyCyte™ 8HT flow cytometry system. Apparent KD and EC50 values were estimated using BIOST@T-BINDING and BIOST@T-SPEED programs, respectively.

Flow cytometry was used as described above to assess the binding of mAb7 or the trispecific binding protein to the antigen binding domain of the anti-CD38 antibody mAb7 to murine pre-B cells expressing human or cynomolgus CD38 polypeptide on their cell surface. In fig. 6A shows that the trispecific binding protein CD38xCD28 _sup xCD3 _mid with the antigen binding domain of the anti-CD38 antibody mAb7 binds to cells expressing human CD38 (top left) with an apparent affinity that is 8-fold less than that of the monospecific antibody mAb7 (top right). Neither the monospecific antibody mAb7 (bottom right) nor the trispecific binding protein with the antigen-binding domain of the anti-CD38 antibody mAb7 (bottom left) binds to cynomolgus monkey CD38-expressing cells.

The binding domain of the humanized anti-CD38 mAb2 was also tested in trispecific formats for binding to cells expressing human or cynomolgus CD38 polypeptides. In fig. 6B - 6D show that, in contrast to mAb7, the trispecific binding proteins CD38xCD28 _sup xCD3 _mid and CD38xCD28 _cvn xCD3 _mid with the antigen binding domain of the anti-CD38 mAb2 antibody, as well as the monospecific antibody mAb2, were able to bind human and cynomolgus CD38 polypeptides. The trispecific binding protein CD38xCD28 _cvn xCD3 _mid antigen binding domain anti-CD38 mAb2 binds to cells expressing human CD38 with an apparent affinity that is 9-fold less than that of the parent mAb2 antibody (comparing top of Fig. 6C and top of Fig. 6D ) . The CD38xCD28 _cvn xCD3 _mid trispecific binding protein with the anti-CD38 mAb2 antigen-binding domain binds to cells expressing cynomolgus CD38 with an apparent affinity that is 7.5-fold less than that of the parent mAb2 antibody (comparing the bottom panel of Fig. 6C and the bottom panel Fig. 6D ). The CD38xCD28 _sup xCD3 _mid trispecific binding protein with the anti-CD38 mAb2 antigen-binding domain binds to cells expressing human CD38 with an apparent affinity that is 2.5 times less than that of the CD38xCD28 _cvn xCD3 _mid trispecific binding protein with the anti-CD38 mAb2 antigen-binding domain (upper comparison). part of Fig. 6C and the top of Fig. 6B ).

The binding domain of the humanized anti-CD38 mAb6 was also compared with the monospecific mAb6 for binding to cells expressing human or cynomolgus CD38 polypeptides. Although the monospecific antibody mAb6 binds to cells expressing human (top right) or cynomolgus (bottom right) CD38 polypeptides in the nM range ( Figure 6E ), the trispecific binding protein CD38xCD28 _sup xCD3 _mid with the anti-CD38 antigen binding domain of mAb6 binds to cells expressing human (top left) or cynomolgus (bottom left) CD38 polypeptides in the absence of saturation.

In conclusion, the binding affinity of the trispecific binding protein CD38 _SB19 xCD28 _sup xCD3 _mid to human CD38 was found to be in the same range regardless of whether binding to recombinant human CD38 by SPR or to human CD38 expressed on cell surface using flow cytometry ( Figure 6F ). Similarly, the affinity of the trispecific binding proteins CD38 _VH1 xCD28 _sup xCD3 _mid/low (mAb2 CD38 binding domain) and CD38 _VH1 xCD28 _cvn xCD3 _mid/low (mAb2 CD38 binding domain) for binding to human CD38 was also in the same range range in both analyses. For CD38 _HHY1370 xCD28 _sup xCD3 _mid (mAb6 anti-CD38 binding domain), the _KD for human CD38 binding determined by SPR was 1 nM, whereas the exact EC50 could not be estimated by flow cytometry. A summary description of the apparent KD values (obtained by FACS analysis) for trispecific binding proteins with different anti-CD38 binding domains is presented in Table M5.

Table M5. Summary description of apparent KD values obtained by flow cytometry analysis

Apparent KD FACS values (M) hCD38-expressing cells cCD38-expressing cells Trispecific with mAb2 to CD38 4.4 nM 7.5 nM Trispecific with mAb6 to CD38 No saturation No saturation Trispecific with mAb7 to CD38 4 nM No binding hCD38-expressing cells cCD38-expressing cells mAb2 0.5 nM 1 nM mAb6 11.2 nM 6.6 nM mAb7 0.5 nM No binding

As expected, the trispecific binding protein ΔCD38xCD28 _sup xCD3 _mid , which lacks the anti-CD38 antibody binding domain, did not bind to cells expressing human or cynomolgus CD38 polypeptides ( Fig. 6G ). This indicates that the binding observed in this assay was specific to the antigen binding domains of the anti-CD38 antibody.

Example 5 Characterization in vitro And in vivo trispecific binding proteins containing domains from mAb2 to CD38 and mAb6 to CD38

The following example describes experiments performed to characterize the stability, binding properties, and activity of novel T cell recruiters that contain variable domains derived from the antibodies mAb2 and mAb6. Additional anti-CD38 x CD28 x CD3 antibodies were generated containing variant arms of the anti-CD38, CD3 and CD28 trispecific binding proteins. New antibodies to CD38/CD3/CD28 differ in the following: 1) CD38 binding domain (mAb2 or mAb6); 2) CD3 binding domain (CD3 _high or CD3 _low ; see SEQ ID NO: 84 and 85 for VH and VL sequences to CD3 _low , respectively); 3) CD28 binding domain (CD28 _sup or CD28 _cvn ). Within the range of possible combinations, a collection of anti-CD38 x CD28 x CD3 antibodies have been designed, produced and subsequently tested for various functions.

Materials and methods

Preparation and purification of trispecific binding proteins

Trispecific binding proteins were produced by transiently transfecting Expi293 cells with 4 expression plasmids using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, 25% (w/w) of each plasmid was diluted in Opti-MEM, mixed with prediluted ExpiFectamine reagent for 20-30 minutes at room temperature (RT) and added to Expi293 cells (2.5 x 10 ⁶ cells /ml). Transfection optimization to determine the best ratio of plasmids has often been used to obtain trispecific binding protein in good yield and purity.

Four to five days after transfection, the supernatant of the transfected cell culture was collected and filtered through a 0.45-μm filter block (Nalgene). Trispecific binding protein in the supernatant was purified using a 3-step procedure. In the first step, protein A affinity purification was used and bound Ab was eluted using “IgG elution buffer” (Thermo Fisher Scientific). In the second step, the product was dialyzed against PBS (pH 7.4) overnight with 2 changes of PBS buffer. Any precipitate was cleared by filtration through a 0.45 μm filter block (Nalgene) before the next step. In the third step, size exclusion chromatography (SEC) purification (Hiload 16/600 Superdex 200 pg or Hiload 26/600 Superdex 200 pg, GE Healthcare) was used to remove aggregates and other structures in the sample. Before pooling, fractions were analyzed by SDS-PAGE under reducing and non-reducing conditions to identify fractions containing monomeric trispecific binding protein. The purified antibody can be aliquoted and stored at -80°C long-term.

ELISA tests

The binding properties of the purified antibodies were analyzed using ELISA or SPR methods. For ELISA, the corresponding antigens for each binding site in the trispecific binding protein were used to cover a 96-well Immuno Plate (Nunc 439454, Thermo Fisher Scientific) overnight at 4°C using 2 μg/ml of each antigen in PBS (pH 7.4 ). The coated plate was blocked with 5% skim milk+2% BSA in PBS for one hour at RT, followed by three washes in PBS+0.25% Tween 20 (Aqua Max 400, Molecular Devices). Antibodies (trispecific and control Abs) were prepared in serial dilutions and added to ELISA plates (100 μl/well in duplicate), incubated at room temperature for an hour, followed by washing 5 times with PBS + 0.25% Tween 20.

After washing, HRP-conjugated anti-human Fab secondary antibody (1:5000, cat. no. 109-035-097, Jackson ImmunoResearch Inc) was added to each well and incubated at room temperature for 30 minutes. After washing 5 times with PBS+0.25% Tween 20, 100 μl of TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD, USA) was added to each well. The reaction was stopped by adding 50 μl of 1 MH ₂ SO ₄ and OD ₄₅₀ was measured using SpectraMax M5 (Molecular Devices) and analyzed using SoftMax Pro6.3 software (Molecular Devices). The final data were transferred to GraphPad Prism software (GraphPad Software, California, USA) and plotted as shown. EC50 was calculated using the same software.

To determine the binding of trispecific antibodies to CD38xCD28xCD3 or isotype control antibodies (human IgG4) to human CD3 (Cambridge Biologics LLC, cat. no. 03-01-0051), CD28 (Cambridge Biologics LLC, cat. no. 03-01-0303) and CD38 (Cambridge Biologics LLC, cat. no. 03-01-0369) ELISA assay was used. Bound antibodies were detected using horseradish peroxidase (HRP)-conjugated anti-Fab secondary antibody (Jackson ImmunoResearch Inc., #109-035-097).

In vitro cell killing assay

Purified human PBMCs were used for in vitro killing assays of different cancer cells by different trispecific binding proteins. Briefly, the killing assay was performed in a 96-well plate with a V-shaped well bottom. 40 ml of PBMC from each donor were seeded onto each plate at 2 x 10 ⁶ cells/ml and 30 ml of PKH26-labeled target cells (Sigma, no. MINI26) were prepared at 2.5 x 10 ⁵ cells/ml (4 µl of dye for staining up to 1×10 ⁷ cells). 20 µl/well of test proteins at various concentrations or PMA were first added to each well, followed by the addition of 80 µl/well of labeled target cells to each well (2 x 10^4 cells/well). Then 100 μl of PBMC was added to each well to achieve a well E:T=10:1 (2×10 ⁵ cells/well) and incubated for 24 hours in an incubator at 37°C and 5% CO ₂ . Cells were pelleted by centrifugation and the supernatant was collected to measure cytokine release or discarded. Cells were stained with Vivid LIVE/DEAD™ Fixed Violet Dead Cell Staining Buffer (Life Technology, #L34955) (staining buffer was prepared by adding 60 μl of Vivid reagent to 60 ml of PBS). Cells were resuspended in 100 μl of staining buffer by incubating for 15 min at room temperature. In the dark. After washing the cells in 1x PBS, cells were resuspended in 200 μl PBS with 0.5% paraformaldehyde, and data on PKH26+ Vivid+ cancer cells were obtained using a Fortessa flow cytometer (Beckton Dickinson, San Jose, CA) followed by analysis using software FlowJo. The percentage killing was calculated as “specific killing-spontaneous killing/total cell count” and plotted as shown.

Cytokine Release Assay

To measure inflammatory cytokine concentrations in in vitro activation assays, in vitro killing assays, in vivo activation assays in NSG mice humanized with CD34+ cord blood cells, and toxicity studies, cell culture supernatants were collected and serum samples were diluted according to protocol manufacturer using the highly sensitive 13-plex Milliplex human T cell kit (EMD Millipore). They were then analyzed using the EMD Millipore MAGPIX® system and MILLIPLEX® Analyst 5.1 software.

Mouse models and in vivo efficacy studies

NSG mice transplanted with human CD34+ hematopoietic stem cells (hu-CD34) were used as a mouse modelin vivo. These mice develop human mixed immune cells and provide a validated platform for immuno-oncology efficacy studies (see e.g. Shultz, L.D.et al.(2014)Cold Spring Harb. Protoc.2014:694-708). Hu-CD34⁺ NSG mice are generated by injection of CD34⁺ hematopoietic stem cells, which demonstrates the effective transplantation of mixed human immune cell populations, including T cells, B cells and some other populations (McDermott, S.P.et al.(2010)Blood116:193-200). Mixed type hematopoiesis occurs over a period of 12 weeks. The transplantation is stable for more than one year without graft-versus-host disease.

For efficacy studies using NSG hu-CD34 mice, mice were purchased from The Jackson Laboratory (Maine, USA), and human cell populations were validated prior to use. Typically, 5×10 ⁶ tumor cells mixed with Matrigel (BD Biosciences) (50% v/v) were used for tumor inoculation for each mouse. After the tumor reached a size in the range of 100-150 mm, ³ mice were selected and randomized into each group for the study. Antibodies were administered intravenously in the indicated doses 3 times a week. Body weight was recorded 1-3 times a week. Tumor size was measured by measuring the tumor with a caliper 1–3 times/week. All mice were sacrificed when the tumor size reached 1500 mm ³ or 24 hours after the last dose. Terminal blood samples (0.3 ml) were collected in serum separation tubes, mixed by gentle inversion five times, and placed in a tube rack. Terminal tumors were also collected and weighed before placement in fixative for immunohistochemical analysis.

NSG mice humanized with human PBMC (hu-PBMC) were used as another in vivo mouse model. These mice are generated by injection of purified human PBMC from healthy donors, which have the shortest engraftment times using mature peripheral blood mononuclear cells and allow for short-term studies requiring enhanced function of effector and memory T cells and NK cells, and they are suitable for short-term efficacy studies (3-4 weeks) due to graft-versus-host disease.

For the efficacy study using NSG mice, hu-PBMCs from 8- to 10-week-old NSG mice (Cat. No.: 005557, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) were purchased from The Jackson Laboratory (Maine, USA). Each mouse was inoculated subcutaneously with 5 x 10 ⁶ tumor cells mixed with Matrigel (BD Biosciences) (50% v/v). Once the tumor reached a size in the range of 50-100 mm ^{3 ,} each mouse was reconstituted with 10×10 ⁶ human PBMC from healthy donors intraperitoneally (i/p). Recovery using human cells was confirmed the next day. After the tumor reached a size in the range of 100-150 mm, ³ mice were selected and randomized into each group for the study. Antibodies were administered intravenously in the indicated doses 3 times a week. Body weight was recorded 1-3 times a week. Tumor size was measured by measuring the tumor with a caliper 1–3 times/week. All mice were sacrificed when the tumor size reached 1500 mm ³ or 24 hours after the last dose. Terminal blood samples (0.3 ml) were collected in serum separation tubes, mixed by gentle inversion five times, and placed in a tube rack. Terminal tumors were also collected and weighed before placement in fixative for immunohistochemical analysis.

The NSG hu-PMBC mouse model with disseminated tumor was treated with 1-5 x 10 ⁶ cells intravenously on day 0. On day 3, fluorescence imaging at baseline was used as the basis for randomization. On day 4, each mouse was reconstituted with 10×10 ⁶ human PBMC from healthy donors intraperitoneally (i/p). Mice were treated once weekly on days 5, 12, 19 at the indicated doses. To monitor tumor volume in each animal, weekly fluorescence body imaging was used on days 10, 17, 24. At the end of the study, blood, spleen, bone, and bone marrow were collected for histopathological examination.

results

The ability of the antibodies to bind to each of the three target antigens was examined using an ELISA assay. CD38 _VH1 xCD28 _sup xCD3 _mid IgG4, CD38 _VH1 xCD28 _sup xCD3 _low IgG4, CD38 _VH1 xCD28 _cvn xCD3 _mid IgG4, CD38 _VH1 xCD28 _cvn xCD3 _low IgG4 showed similar binding affinities to human and monkey CD3 and CD38, but varying binding affinities to CD28 ( humans and monkeys have an identical extracellular domain) with CD28 _sup showing better affinity ( Figure 7A ). CD38 _VH1 xCD28 _sup xCD3 _mid and CD38 _HHY1370 xCD28 _sup xCD3 _mid IgG4 FALA variant and IgG1 LALA variants P329A and NNAS all showed similar binding to human CD3, CD28 and CD38 ( Figure 7B ).

CD38xCD3xCD28 trispecific binding protein variants were then examined for their ability to induce T cell activation and antibody-mediated tumor cell killing ( Figures 8A-8D ). In fig. 8A shows the activity of CD38 _VH1 xCD28 _sup xCD3 _mid IgG4, CD38 _hhy1370 xCD28 _sup xCD3 _mid IgG4, CD38 _VH1 xCD28 _cvn xCD3 _mid IgG4, CD38 _hhy1370 xCD28 _cvn xCD3 _mid IgG4 towards in vitro killing of the human multiple myeloma cell line RPMI-8226 using PBMC. from various donors at E:T=10. The EC50 of killing activity was calculated and summarized in Table N. Both CD38xCD3xCD28 trispecific binding protein antibodies containing CD28 _sup showed better killing activity. The in vitro killing activity of these molecules was also examined using the cell lines NCI-H929 ( Fig. 8B ), KMS-26 ( Fig. 8C ), and KMS-11 ( Fig. 8D ).

Trispecific binding protein antibodies to CD38x-CD3x-CD28 with IgG1 variants of LALA P329A and NNAS or IgG4 variant of FALA also exhibit killing activity in vitro against multiple myeloma cells KMS-11 ( Fig. 8E ) and U266 ( Fig. 8F ). The EC50 for each antibody against each cell line was calculated and the values were summarized in Table Q2 (KMS-11) and Q3 (U266).

In fig. 9A, 9B and 10 show the in vitro T cell activation profile for CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 and CD38 _VH1 xCD28 _sup xCD3 _low IgG4. Both antibodies showed similar activation activities against CD4 and CD8 T cells. The EC50 for each antibody against each cell line was calculated and the values were summarized in Table N (RPMI-8226), Table O (NCI-H929), Table P (KMS-26) and Table Q (KMS-11).

Table N. Antibody-Mediated Specific Killing of CD38+ RPMI8266 Cells by PBMC from Different Donors

Antibody EC50 (ng/ml) Donor #65 Donor #67 Donor #68 CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 5.30 1.95 3.3 CD38 _hhy1370 xCD28 _sup xCD3 _mid IgG4 2.24 3.42 3.4 CD38 _VH1 xCD28 _cvn xCD3 _mid IgG4 8.31 29.52 46.8 CD38 _hhy1370 xCD28 _cvn xCD3 _mid IgG4 4.62 8.98 16.5

Table O. Antibody-Mediated Specific Killing of CD38+ Cells NCI-H929 via PBMC from different donors

EC50 (ng/ml) Donor #65 Donor #67 Donor #68 CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 0.51 0.48 0.9 CD38 _hhy1370 xCD28 _sup xCD3 _mid IgG4 0.94 1.84 2.5 CD38 _VH1 xCD28 _cvn xCD3 _mid IgG4 3.70 9.47 10.4 CD38 _hhy1370 xCD28 _cvn xCD3 _mid IgG4 2.31 6.39 7.2

Table P. Antibody-Mediated Specific Killing of CD38+ KMS-26 Cells via PBMC from different donors

EC50 (ng/ml) Donor #65 Donor #67 Donor #68 CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 1.03 0.91 1.3 CD38 _hhy1370 xCD28 _sup xCD3 _mid IgG4 3.95 5.06 5.3 CD38 _VH1 xCD28 _cvn xCD3 _mid IgG4 10.73 28.17 22.4 CD38 _hhy1370 xCD28 _cvn xCD3 _mid IgG4 15.84 32.10 25.7

Table Q. Antibody-Mediated Specific Killing of CD38+ KMS-11 Cells via PBMC from different donors

EC50 (ng/ml) Donor #65 Donor #67 Donor #68 CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 3.05 3.40 14.7 CD38 _hhy1370 xCD28 _sup xCD3 _mid IgG4 4.17 7.74 25.1 CD38 _VH1 xCD28 _cvn xCD3 _mid IgG4 18.14 98.83 566.8 CD38 _hhy1370 xCD28 _cvn xCD3 _mid IgG4 16.30 27.63 139.2

Table Q2. Antibody-mediated specific killing of CD38+ KMS-11 cells via PBMC from different donors (IgG1/4 Fc variants)

EC50 (pM)
(KMS11) CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 FALA CD38 _VH1 xCD28 _sup xCD3 _mid
IgG1 LALA P329A CD38 _VH1 xCD28 _sup xCD3 _mid
IgG1 NNAS CD38 _hhy1370 xCD28 _sup xCD3 _mi
IgG4 FALA CD38 _hhy1370 xCD28 _sup xCD3 _mi
IgG1 LALA P329A CD38 _hhy1370 xCD28 _sup xCD3 _mi
IgG1 NNAS KP50901 3.879 4.375 4,411 7,731 9,311 17.71 KP50904 4,379 6,739 8,644 19.01 18.88 24.39

Table Q3. Antibody-mediated specific killing of CD38+ U266 cells via PBMC from different donors (IgG1/4 Fc variants)

EC50 (pM)
(U266) CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 FALA CD38 _VH1 xCD28 _sup xCD3 _mid
IgG1 LALA P329A CD38 _VH1 xCD28 _sup xCD3 _mid
IgG1 NNAS CD38 _hhy1370 xCD28 _sup xCD3 _mi
IgG4 FALA CD38 _hhy1370 xCD28 _sup xCD3 _mi
IgG1 LALA P329A CD38 _hhy1370 xCD28 _sup xCD3 _mi
IgG1 NNAS KP50901 1.879 1.031 1,150 2,691 1,597 2,816 KP50904 0.8657 3,570 2,018 2.112 0.8375 3.527

Cytokine production induced by trispecific binding protein variants was also examined ( FIGS. 11A and 11B ) using the method described in Stebbings, R. et al. (2007) ( J. Immunol. 179:3325-3331), by coating the plate with the indicated antibodies followed by incubation with human PBMC for 24 hours using 2 concentrations of the test antibodies (5 μg/ml and 25 ng/ml). The 24-hour culture supernatant was collected and used to measure the concentrations of IL2, IL6, IL10, IL12, and TNF-α, IFN-γ in the supernatants as described above. All of CD38 _VH1 xCD28 _sup xCD3 _mid IgG4, CD38 _hhy1370 xCD28 _sup xCD3 _mid IgG4, CD38 _VH1 xCD28 _cvn xCD3 _mid IgG4, CD38 _hhy1370 xCD28 _cvn xCD3 _mid IgG4 stimulated the production of significant amounts of IL2, TNF-α and IFN-γ at 5 µg/ ml, but did not induce measurable levels of either cytokine at 25 ng/ml, a dose shown to be effective in vivo in 2 humanized NSG mouse models.

Antitumor activity was tested in in vivo mouse models for trispecific binding protein variants ( FIGS. 12A-13F ) as described above. In fig. 12A-12E show the results of an in vivo efficacy study using an NSG (hu-CD34) mouse model implanted with human CD34+ stem cells with the MM cell line RPMI-8226. To treat mice with tumors, CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 and comparative marker control bispecific antibodies CD38xCD3 were used at the indicated doses 3QW (6 doses in total). Body weight and tumor growth of each mouse were measured and plotted ( Fig. 12A-12E ). The average tumor growth curve ( Fig. 12D ), tumor volume at day 18 ( Fig. 12B ), and final D19 tumor weight ( Fig. 12C ) for each group were also plotted. All groups treated with CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 not only demonstrated statistical efficacy compared to the PBS control, but also showed statistically better efficacy at 1 μg/kg compared to the CD38xCD3 bispecific antibody control.

In fig. 13A-13F show the results of an in vivo efficacy study using a PBMC humanized NSG mouse model implanted with human RPMI-8226 MM cells. To treat tumor-bearing mice, CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 and comparative control bispecific antibodies CD38xCD3 were used at the indicated doses 3QW (8 doses in total). Tumor growth in each mouse was measured and plotted ( Fig. 13A ). Average tumor growth curve ( Fig. 13F ), tumor volume on day 4 (day of start of treatment; Fig. 13B ), tumor volume on day 21 (day of last treatment; Fig. 13C ), mean tumor volume on day 21 ( Fig. 13D ) and final tumor weight at day 22 ( Fig. 13E ) for each group were also plotted. All groups treated with CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 not only demonstrated statistical efficacy compared to the PBS control, but also showed statistically superior multiple dose efficacy compared to the CD38xCD3 bispecific antibody control, indicating superior in vivo antitumor activity of the trispecific antibody to CD38/CD3/CD28.

The comparative control bispecific antibody CD38xCD3 contained the following sequences:

SEQ ID NO 108 : comparative heavy chain 1 (binds CD38),

evqlvesggglvqpggslrlscaasgftfsyswmnwvrqapgkglewvseinpqsstinyatsvkgrftisrdnskntlylqmnslraedtavyycarygnwfpywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglysls svvtvpssslgtqtyicnvnhkpsdtkvdkkvepkscdkthtcppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvkhedpevkfnwyvdgvevhnaktkpreeeynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemt knqvsltcdvsgfypsdiavewesdgqpennykttppvldsdgsfflyskltvdksrweqgdvfscsvmhealhnhytqkslslspgk

SEQ ID NO 109 : comparative heavy chain 2 (binds CD3),

evqlvesggglvqpggslrlscaasgftfstyamnwvrqapgkglewvgrirskynnyatyyadsvkgrftisrddskntlylqmnslraedtavyycvrhgnfgdsyvswfaywgqgtlvtvssgkpgsgkpgsgkpgsgkpgsqavvtqepsltvspggtvtltcgsstgavtt snyanwvqqkpgksprgliggtnkrapgvparfsgsllggkaaltisgaqpedeadyycalwysnhwvfgggtkltvlepkssdkthtcppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvkhedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeyk ckvsnkalpapiektiskakgqprepqvytlppsreqmtknqvkltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk

SEQ ID NO 110 : comparative heavy chain (binds CD38).

divmtqspsslsasvgdrvtitcrasqnvdtwvawyqqkpgqspkaliysasyrysgvpdrftgsgsgtdftltisslqpedfatyfcqqydsypltfgggtkleikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdsty slsstltlskadyekhkvyacevthqglsspvtksfnrgec

Example 6: Dose escalation study of trispecific binding proteins containing domains from mAb2 and mAb6 to CD38

Materials and methods

All NHP studies were performed at Covance (Princeton, NJ, USA) according to the IACUC Covance protocol. All studies used male cynomolgus macaques that were drug and protein naïve or protein naïve. According to the study design, monkeys were selected and assigned to d groups corresponding to each trispecific binding protein. The antibody was administered by intravenous infusion into the saphenous vein over 1 hour. Increasing doses were administered on consecutive days for low doses (<10 μg/kg) but 1–2 days apart for higher doses (>10 μg/kg) for observation purposes. Blood samples were collected at 0 hours (day 1 only), 0.5 hours (during infusion), 1 and 6 hours after the start of infusion from all animals after each dose as indicated. Additional unscheduled blood samples were collected at the discretion of the study director, pathologist, and/or veterinary clinician. All animals were returned to the colony on day 60. PBMC and serum were obtained from blood samples using standard methods and stored for further analysis.

Blood from treated non-human primates was stained with fluorescently conjugated antibodies to T cell markers and human IgG Fcγ fragment and analyzed on a flow cytometer.

results

Dose escalation toxicity studies were performed in non-human primates using mAb2-containing CD38 _VH1 xCD28 _sup xCD3 _mid IgG4, mAb6-containing CD38 _hhy1370 xCD28 _sup xCD3 _mid IgG4, mAb2-containing CD38 _VH1 xCD28 _cvn xCD3 _mid IgG4, and mAb6- containing CD38 _hhy1370 xCD28 _cvn xCD3 _mid IgG4 trispecific binding proteins. All three binding domains in the 4 binding proteins cross-react with cynomolgus monkey CD38/CD3/CD28 polypeptides. The study was designed to evaluate the potential toxicity profile of the molecule. Blood samples were collected for serum and PBMC extraction procedures. Following each dosing, circulating T cell populations were examined ( Figures 14E-14H ) along with activation of T cell subsets (CD69+) ( Figures 14A-14D ). The percentage of CD4 and CD8 T cells in the circulation decreased with increasing dose. Significant activation of CD4 and CD8 T cells was noticeable starting at doses of 2.5 μg/kg, indicating high activating capacity for mAb2- and mAb6-containing molecules. Significant depletion of circulating T cells was observed when total proteins were used at 12.5 μg/kg ( Figures 14I-14L ), again correlating with efficacy. The depletion of circulating T cells was quite transient, with a return to pre-treatment levels within 24-48 hours ( Figures 14M-14P ). Serum levels of several cytokines were also measured. Significant release of IL-6 and IL-10 was observed at 12.5 μg/kg using all molecules, which was quite transient, returning to pre-treatment levels after 24 hours ( Figure 14U ).

Both CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 ( Figure 14V ) and CD38 _VH1 xCD28 _cvn xCD3 _mid IgG4 ( Figure 14W ) induced T cell depletion in the blood at higher doses. Similarly, CD38 _hhy1370 xCD28 _sup xCD3 _mid IgG4 ( Figure 14X ) and CD38 _hhy1370 xCD28 _cvn xCD3 _mid IgG4 ( Figure 14Y ) also induced T cell depletion in the blood at higher doses. However, T cells began to reappear in the blood 24 hours after treatment with any of the four trispecific binding proteins ( Figures 14Z-14AC ). In fig. 14AD and 14AE show the number of CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 bound to CD4+ T cells following a 100 μg/kg dose. In fig. 14AF and 14AG show the amount of CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 associated with CD8+ T cells after a dose of 100 μg/kg. These data clearly demonstrate that trispecific Ab CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 can bind to T cells in vivo for extended periods of time (48-72 hours).

Example 7 Optimization of pharmacokinetics/pharmacodynamics using Fc variants

Materials and methods

C-terminal 6-His tagged recombinant human Fcγ RI (R&D Systems, #1257-FC-050), C-terminal 10-His tagged recombinant human Fcγ RIIA (R&D Systems, #1330-CD-050/CF) and C- terminally HPC4-tagged recombinant human FcγRIII (V158 or F158) was captured onto a Biacore chip. Antibodies at concentrations of 200, 100 and 50 nM were injected for 2 minutes followed by dissociation for 2 minutes in HBS-P+ buffer, 2 mM CaCl2 pH 7.4 at a flow rate of 30 μl/min using Biacore 200. The binding curve is shown for 200 nM.

An ELISA assay using C-terminally HPC4-labeled recombinant human neonatal Fc receptor (FcRn) was used to measure the binding properties of trispecific binding proteins with different Fc modifications. Briefly, recombinant human neonatal Fc receptor (FcRn) was used to coat an ELISA plate (Nunc 80040LE)(2 μg/ml in PBS) overnight. Serially diluted trispecific binding proteins with different Fc modifications were added to each well and incubated at room temperature for one hour, followed by washing and incubation with HRP-labeled anti-human IgG secondary antibody.

results

The trispecific binding protein variants described above were then analyzed for binding to different Fc receptors to optimize pharmacokinetics (PK)/pharmacodynamics (PD).

Human Fc variants were characterized for binding to FcγR I ( FIG. 15A ), FcγR IIa ( FIG. 15B ), and FcγR IIIb/c ( FIG. 15C ) as described above. The variants tested were human IgG1, human IgG4, and human IgG4 with FALA mutations (F234A and L235A according to the EU index) with a control trispecific binding protein.

As shown in FIG. 15A-15C , wild-type IgG1 and IgG4 were able to bind FcγR I and FcγR IIa, but not FcγR IIIb c, as previously reported. FALA mutations in IgG4 abolished the binding of FcγR I and FcγR IIa. Without being bound by theory, it is believed that eliminating the binding of FcγR I and FcγR IIa may improve PK/PD by removing unwanted clustering through Fc/FcγR interactions.

The IgG4 FALA variant was then examined for FcRn binding. This variant, as well as wild-type IgG4, bind to FcRn ( Fig. 16 ). These results demonstrate that IgG4 FALA mutations do not affect the interactions between IgG4 Fc and FcRn, implying minimal impact on the half-life of the binding protein.

The PK parameters of CD38 _VH1 xCD28 _sup xCD3 _mid IgG4, CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 FALA, CD38 _VH1 xCD28 _sup xCD3 _mid IgG1 LALA P329A, and CD38 _HHY1370 xCD28 _sup xCD3 _mid IgG4 FALA determined in NSG mice are summarized in FIG. 17 . Fc modifications of IgG4 showed significantly improved half-life and AUC in NSG mice by eliminating binding to NSG-specific FcγRI, which is abundantly present on macrophages.

Example 8: Activation in vitro and antitumor efficacy in vivo trispecific binding proteins to CD38

Materials and methods

Release of cytokines from human PBMCs

Human PBMCs were incubated with 100 μM binding proteins for 24 h. Supernatants were collected and cytokine levels were measured by Luminex using Drop Array plates (in triplicate). For experiments using targeted tumor cells, human PMBCs were incubated with 100 μM antibodies and with or without RPMI-8226 targeting cells at an E:T ratio of 10:1 for 24 hours. Supernatants were collected after incubation of PMBCs with trispecific proteins, analyzed for cytokines using the MILLIPLEX® MAP kit, and analyzed in the MAGPIX® system.

Bcl-xL assay

Negatively sorted T cells from PBMCs (magnetic) were incubated with 100 nM plate-bound Ab for 1 day. T cells were then washed and stained with fluorescently conjugated antibodies specific for T cell markers and Bcl-xL and analyzed on a flow cytometer.

T cell proliferation

Negatively sorted T cells from PBMCs (magnetic bead cell sorting) were incubated with Ab-coupled plates (100 nM) for 1–6 days. Total cell numbers were counted by flow cytometry using counting beads on specific days after the start of incubation. Fold changes were calculated based on the number of cells on day 0 (500 cells/μl). Results are presented from 3 PBMC donors.

IL-2 expression

Jurkat IL2-luc2P GloResponse™ cells were incubated with trispecific proteins for 6 hours, then the Bio-Glo™ Luciferase Assay System was used to detect luciferase reporter gene expression.

PBMC activation in vitro

Activation (CD69 ⁺ ) of human PBMC cells treated with trispecific antibodies to CD38xCD28xCD3 or control IgG4 binding protein was determined.

In vivo efficacy in a disseminated tumor model

The NSG hu-PMBC mouse model with disseminated tumor was treated with 1-5 x 10 ⁶ cells intravenously on day 0. On day 3, fluorescence imaging at baseline was used as the basis for randomization. On day 4, each mouse was reconstituted with 10×10 ⁶ human PBMC from healthy donors intraperitoneally (i/p). Mice were treated once weekly on days 5, 12, 19 at the indicated doses. To monitor tumor volume in each animal, weekly fluorescence body imaging was used on days 10, 17, 24. At the end of the study, blood, spleen, bone, and bone marrow were collected for histopathological examination.

results

mAb2-containing anti-CD38x-CD28x-CD3 trispecific binding proteins with the FALA IgG4 Fc region variant caused markedly reduced levels of nonspecific IFN-γ release in human PBMCs compared to similar wild-type Fc region binding proteins ( Figure 18A ). . Similar results were observed for the release of IL-2 ( Fig. 18B ) and TNF-α ( Fig. 18C ). Importantly, cytokine release was significantly lower for these Fc variant trispecific binding proteins compared to the comparative anti-CD38x-CD3 bispecific antibody. These results suggest an improved safety profile of trispecific binding proteins with Fc variants due to reduced nonspecific cytokine release.

Fc variants IgG1 and IgG4 of both mAb2-containing CD38 _VH1 xCD28 _sup xCD3 _mid and mAb6-containing CD38 _HH71370 xCD28 _sup xCD3 _mid also resulted in in vitro activation of human PBMC ( Fig. 18D ).

Immune co-stimulation by CD28 ligation is essential for T cell survival and proliferation; TCR signaling alone does not lead to T cell proliferation (Sharmee and Allison (2015) Science 348:56-61). The trispecific binding protein CD38 _VH1 xCD28 _sup xCD3 _mid resulted in the induction of Bcl-xL in both CD4+ and CD8+ T cells, whereas deletion of the CD28 or CD3 antigen-binding domains abolished this effect ( Figures 19A and 19B ). These results demonstrate, as expected, that the binding arms of both CD3 and CD28 are required to induce Bcl-xL in T cells via trispecific anti-CD38 binding proteins, thereby mediating the delivery of a pro-survival signal to T cells. The anti-CD28 antibody arm of the trispecific binding protein is critical for the activation of pro-survival Bcl-xL in T cells. Compared to the comparative CD38x-CD3 bispecific antibody, the CD38 _VH1 xCD28xCD3 trispecific binding protein resulted in greater Bcl-xL activation in CD4+ and CD8+ T cells ( Figures 19C and 19D ).

To determine the primary driver of T cell activation through trispecific binding proteins CD38 _VH1 xCD28 _sup xCD3 _mid , IL-2 expression in the Jurkat T cell reporter line containing the human IL-2 promoter driving the luciferase reporter gene was used to measure activation. A knockout mutation of the anti-CD3 antibody binding domain resulted in abolition of T cell activation, demonstrating that T cell activation is a specific effect of CD3 ligation ( Figure 19E ). These results indicate that the anti-CD3 domain is a major factor in T cell activation by trispecific binding proteins and that the anti-CD28 domain also makes a significant contribution to the T cell activation signal. Primary cytokine releasing factor was also examined using human PBMC ( Figure 19F ). When studying the release of TNF, IFNg, IL-2, IL-6 and IL-10, CD3 was found to be the main determinant of cytokine release. It was found that CD28 makes the least contribution to this process. Trispecific CD38 _VH1 domain binding proteins were also found to induce less cytokine release than the comparator.

T cell proliferation was also assessed. Activation of T cells using the anti-CD38x-CD28x-CD3 trispecific binding protein antibody with the IgG4 FALA Fc variant resulted in greater proliferation than either the comparator or isotype control ( Figure 19G ). This activation was reduced when the antigen-binding domains of the anti-CD28 antibody or the anti-CD3 antibody were mutated ( Fig. 20 ).

A humanized NSG mouse model was then used to experiment with solid tumor growth in vivo . Mice 6-8 weeks of age were implanted with human myeloma cells RPMI8226. At week 9, human PBMCs were administered by IP injection, hPBMCs were reconstituted, and antitumor therapy was administered at weeks 10–13. 56 PBMC humanized female NSG mice were implanted with 5 million RPMI8226 cells in 50% Matrigel, and mice were recruited and randomized for the study on day 5 or 6 postimplantation. This model serves as an in vivo tumor model in mice with mature human T cells.

Trispecific binding proteins were also analyzed in the NSG mouse model of disseminated tumor growth using NCI-H929-Luc human myeloma cells and bioluminescence tumor growth assay. Trispecific binding protein CD38 _VH1 xCD28 _sup xCD3 _mid with Fc variant IgG4 FALA showed statistically significant dose-dependent antitumor efficacy in this disseminated tumor model at 30 μg/kg ( Fig. 21 ). The CD38 trispecific binding protein _HHY1370 xCD28 _sup xCD3 _mid with the IgG4 FALA Fc variant also showed statistically significant dose-dependent antitumor efficacy in this disseminated tumor model at 30 μg/kg and 10 μg/kg ( Fig. 22 ).

Trispecific binding proteins, mAb2-containing CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 FALA and mAb6-containing CD38 _hhy1370 xCD28 _sup xCD3 _mid IgG4 FALA, demonstrated high in vitro antitumor activity against NCI-H929-Luc myeloma cells using human PBMC from two humanized NSG mouse donors ( FIGS. 23A and 23B ). The trispecific binding protein CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 FALA demonstrated superior antitumor activity in vivo compared to a comparative bispecific antibody in a disseminated tumor model ( Figure 23C ). P-values for comparison with vehicle control (PBS) were determined as follows: CD38 _VH1 xCD28 _sup xCD3 _mid IgG4 FALA: p=0.0023; CD38 _HHY1370 xCD28 _sup xCD3 _mid IgG4 FALA: p=0.0088; comparative bispecific construct CD38xCD3 Fab-scFv-Fc: p=0.6045.

Taken together, these results demonstrate an improved Fc region variant CD38 antibody candidate that exhibits increased half-life, reduced release of nonspecific cytokines, and high antitumor efficacy in vivo .

Example 9: Further Characterization of Anti-CD38 Trispecific Binding Proteins

To stimulate T cells, two signals are required for optimal effector function and sustained proliferation. Activation mediated by the T receptor (TCR)-CD3 complex induces transcriptional activation leading to cytokine secretion. Recruitment of a second membrane surface protein, CD28, stimulates an alternative signal transduction pathway and suppresses programmed cell death (Esensten JH, Helou YA, et al. Immunity 44 :973-88(2016); Hui E, Cheung J, et al. Science 355 : 1428-1433(2017)). In the absence of a second signal, stimulation of T cells with CD3 alone usually results in activation-induced cell death (Chai JG, Lechler RI. Int Immunol. 9 :935-44 (1997)). It has been hypothesized that T cell proliferation may be enhanced by combining specificity for these two different targets.

Onfig. 24A shows the results of a luciferase reporter gene assay using GloResponse™ IL2-luc2P Jurkat cells (Promega) after stimulation with CD38_VH1/CD28_supxCD3_mid and its single-binding-site mutant KO and triple-mutant KO at 10 nM concentration. These data illustrate the contribution of CD28 to T cell stimulation (e.g. NFAT signaling).

Using the crossover double variable region (CODV) bispecific Ab format (Steinmetz A, et al. MAbs 8 :867–878 (2016)), combinations of α-CD3ε (signal 1) and α-CD28 (signal 2) Fv were assessed to determine whether the dual interaction of these cell surface molecules can stimulate and maintain T cell activation ( Figure 24B ). Medium affinity CD3ε Abs (K _D ~ 20 nM) were used to avoid high affinity T cell receptor stimulation, which causes high levels of cytokine release and increases the risk of cytokine release syndrome. This CD3 agonist was tested in both positions of the double arm in combination with a CD28 superagonist Ab, which has previously been shown to mediate T cell proliferation (Waibler Z, Sender LY, et al. PLoS One 3:e1708 (2008)). These monovalent bispecific antibodies were incubated with human PBMC in vitro , and T cell stimulation was measured by interferon-γ and IL-2 secretion. Although both orientations were active, optimal release of these cytokines was observed with CD3 in the proximal and CD28 in the distal position ( Fig. 24B ). This double arm was then selected for pairing with anti-CD38 antibody in trispecific Ab format (Xu L, Pegu A, et al. Science 358 :85-90 (2017)).

In fig. 25 shows that activation of the Bcl-2 family member Bcl-xL in primary T cells induced by CD38 _VH1 /CD28 _sup xCD3 _mid is CD28-dependent. These data illustrate the contribution of CD28 to T cell survival.

In fig. 26 shows that in a trispecific Ab, anti-CD28 provides secondary signaling necessary to support primary T cell proliferation in vitro . These data illustrate the contribution of CD28 to T cell proliferation.

Taken together, these data indicate that the anti-CD28 _sup domain in the CD38 _VH1 /CD28 _sup xCD3 _mid trispecific antibody provides significant functionality in T cell activation, survival, and proliferation in vitro.

In fig. 27 shows the configuration of the trispecific antibody indicating the parent antibody. Also shown is a structural model of the trispecific Ab CD38 _VH1 /CD28 _sup xCD3 _mid based on the crystal structures of CD38 VH1 Fab and CD28 _sup /CD3 _mid CODV Fab (right).

In fig. 28A and 28B show that multiple myeloma (MM) cells with high (RPMI-8226; FIG. 28A ) and low (KMS-11; FIG. 28B ) CD38 surface expression were efficiently killed by human PBMC (E:T=10 :1), incubated with trispecific Ab at different concentrations. The contribution to killing activity of each binding site was demonstrated through KO mutations of the binding site. These data demonstrate that the CD38 trispecific antibody mediated the lysis of human MM cells by recognizing both CD38 and CD28. CD28, expressed on multiple myeloma cells, provides an additional target for the trispecific antibody CD38 _VH1 /CD28 _sup xCD3 _{mid_FALA} , significantly enhancing its killing activity.

In fig. 29A-29C show that the anti-CD28 _supKO mutant antibody in the trispecific Ab exhibited markedly reduced antitumor activity in vitro against CD38 _high , CD38 _mid , and CD38 _low MM cells. Assays using RPMI-8226 ( Figure 29A ), U266 ( Figure 29B ) or KMS-11 ( Figure 29C ) cells are shown. Expression of CD28 on MM cells increased their sensitivity to CD38 trispecific antibody-mediated cytolysis.

In fig. 30 shows the results of an in vivo efficacy study in a human disseminated multiple myeloma cell line model using NSG mice reconstituted in vitro with expanded primary human T cells. The reduction in tumor burden in the CD38 _VH1 /CD28 _sup xCD3 _mid _FALA trispecific antibody treatment groups was dose-dependent and statistically different. Thus, a trispecific antibody to CD38 provides protection against the growth of disseminated human tumor MM cells in vivo.

The microscopy study also demonstrated trispecific antibody-mediated killing of cancer cells by primary human T cells in vitro. In fig. 31A and 31B are microscopy images demonstrating lysis of RPMI-8226 (human MM cells labeled with CellTracker Deep Red) by human PBMC mediated by the CD38 _VH1 /CD28 _sup xCD3 _{mid_FALA} trispecific antibody. In fig. 31A shows control, while FIG. 31B shows lysis mediated by the CD38 _VH1 /CD28 _sup xCD3 _{mid_} FALA trispecific antibody. An E:T ratio of 10:1 was used for 24-h incubation. Exposure to the control triple-null Ab mutant induced minimal changes in tumor cell viability over 24 hours ( Figure 31A ). In contrast, incubation with active trispecific Ab to CD38 induced significant clustering of T cells around tumor cells, resulting in almost complete lysis ( Fig. 31B ). These data demonstrate that the CD38/CD3xCD28 trispecific antibody mediates the cytolysis of myeloma cells through T cells.

Example 10. Modification of Fc receptor binding sites in the IgG4 Fc region of anti-CD38 trispecific binding proteins reduces nonspecific inflammation

The Fc region of antibodies binds to cellular receptors on monocytes and NK cells, which induces nonspecific inflammation, which may predispose treated subjects to cytokine release syndrome. The role of Fc receptors in promoting cytokine release was assessed by examining mutants that abolished the binding of FcγI, FcγIIa, b, and FcγIII. For this purpose, the IgG4 isotype was used, which does not fix complement.

Surface plasmon resonance (SPR) binding assays using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ) were used to measure the affinity of the indicated IgG4 Fc variants to the indicated human Fc receptors immobilized on the chip. IgG4 Fc variants were used at 150 nM. Binding to human FcRn was performed using ELISA by coating the plate with human FcRn antigen.

A mutation (“FALA”) in the Fc region, which was previously described (Alegre ML, Peterson LJ, et al.Transplantation 57:1537-43(1994); cm. also examples 7-9), eliminated binding to all Fc receptors (fig. 32 and 33). When tested for nonspecific cytokine release, this mutation eliminated IFN-γ release in unstimulated PBMCs while providing maximal stimulation in the presence of tumor targets that expressed CD38 (fig. 34, left and right, FALA). This Fc mutant retained its cytolytic activity against myeloma tumor targets, comparable to the control wild-type Fc in cells expressing different levels of CD38 (fig. 35). Similarly, another mutant Fc region of IgG4 (“PVA”, cm.fig. 32), which ensured the elimination of binding to Fc receptors (fig. 33), also abolished IFN-γ release in unstimulated PBMCs while simultaneously providing maximal stimulation in the presence of tumor targets that expressed CD38 (fig. 34, left and right, PVA), with retention of cytolytic activity against myeloma tumor targets comparable to control wild-type Fc in cells expressing varying levels of CD38 (fig. 35).

Example 11: Contribution of Anti-CD38 Antibody and Anti-CD28 Antibody Domains to the Cytolytic Activity of Trispecific Binding Proteins

For each binding site or combination of binding sites, trispecific mutant Abs were generated and tested for cytolytic activity against multiple myeloma cell lines with high (RPMI-8226) or low (KMS-11) surface expression of CD38 and CD28.

As shown above in FIG. 28A and 28B , a CD3 specific binding mutation eliminated cytolysis, whereas null mutations in the anti-CD38 and anti-CD28 antibody domains showed markedly reduced killing activity while retaining some residual function, indicating that both the anti-CD38 and anti-CD38 antibody domains to CD28 contributed to the destruction of tumor cells.

Compared with daratumumab (an anti-α-CD38 monoclonal antibody approved for the treatment of multiple myeloma; see McKeage K.Drugs. 76:275-81(2016)) trispecific Ab demonstrated high killing activityin vitro, which is 3 to 4 logs higher for a variety of CD38-expressing cell lines, including CD38 multiple myeloma cells_high and CD38_low (fig. 36).

Example 12: CD38/CD3xCD28 Ab Stimulates CD4 and CD8 Central Memory Cells, Th1 and Antigen-Specific Responses

To determine whether trispecific Ab CD38/CD3xCD28 could enhance cellular immune function, the phenotype of in vitro expanded T cells was assessed.

Materials and methods

Peripheral blood mononuclear cells were isolated from healthy donor blood collected by Research Blood Components, LLC (Boston, MA). PBMCs were added to antibody-coated plates (350 ng/well) (5 x 10 ⁵ cells/ml) as described above and incubated at 37°C for 3 and 7 days. Cells were collected at specific time points and analyzed by flow cytometry for T cell subsets: naïve (CCR7+ CD45RO-), Tcm (CCR7+ CD45RO+), Tem (CCR7- CD45RO+), Treg (CD4+ Foxp3+ CD25hi). Cells were also treated with monensin (GolgiStop) (BD Biosciences, California) for at least 6 hours before staining for flow cytometry to determine the expression of intracellular cytokines: Th1 (CD4+ IFN-γ+), Th2 (CD4+ IL-4+) and Th17 (CD4+ IL-17+). CMV pp65-specific CD8+ T cells were detected using a fluorescent-conjugated pentamer, HLA-restricted to donor PBMCs (A*02:01/NLVPMVATV) (ProImmune, Oxford, UK). PBMCs were obtained from HemaCare (Van Nuys, CA) from donors with known CMV-positive populations and HLA types. PMBC from donors negative for the restrictive HLA type were used as negative controls. Staining was performed according to the manufacturer's protocol.

results

Human PBMCs were incubated for 7 days with trispecific Ab or triple mutant negative control in the absence of cytokines. Analysis of CD4 subsets revealed the greatest proliferation in the pool of central memory cells, with a smaller increase in the number of effector memory cells ( Figure 37A ). Analysis of CD4 subsets also revealed the greatest proliferation of Th1 cells (>6-fold) compared to Th2 or Th17 cells ( Figure 37B ). In the CD8 subpopulation, a 150-fold increase in the CD8 subpopulation of central memory cells was observed by day 7, with a smaller increase in the number of effector memory cells ( Fig. 37C ). Importantly, pre-existing antigen-specific CD8 responses to CMV targeting the pp65 epitope in HLA-A2 seropositive donors using tetramer staining (Gratama JW, van Esser JW, et al. Blood 98 :1358-1364(2001)) increased >44-fold in the presence of CD38 trispecific protein antibody compared to the negative control ( Figure 37D ).

Taken together, these data indicate that CD38 trispecific Ab stimulates Th1 function and protective memory CD8 T cell responses that may enhance antitumor immunity in vivo .

Example 13: CD28 on Multiple Myeloma Cells Enhances T Cell Recognition and Cytolysis

Materials and methods

Flow cytometry

Levels of CD28 and CD38 on the indicated tumor cell lines were determined by flow cytometry using a QIFI kit (Dako, Denmark, cat. no. K007811) as described above (S4). Briefly, mouse anti-human CD38 (clone AT13/5, mouse IgG1, Santa Cruz Biotech, cat. no. 59028) and anti-human CD28 (clone CD28.2, mouse IgG1, k, BioLegend, cat. no. 3029123) were used in as a primary antibody (at a concentration of 10 μg/ml) to detect surface CD38 and CD28 on cell lines using the manufacturer's protocol. Surface concentrations were calculated using calibration standards from the QIFI kit and the composition provided in the kit.

Generation of CD28 KO human multiple myeloma cell lines using CRISPR-mediated knockout

For the KMS-11 cell line, CD28 knockout was performed using a human CD28 CRISPR knockout kit (Origene). The kit contains a donor vector with a functional GFP-puromycin cassette and two separate Cas-9 vectors, each with a different pre-designed guide RNA (gRNA 1: TACCTGTTACTTGAATTGAA (SEQ ID NO:117) and gRNA 2: ATTTTTTGAGGTCTTCCAAT (SEQ ID NO:118), which target exon 1 and intron 1 of CD28, respectively). Cells were seeded in a 6-well plate at a density of 3.10 ⁵ cells/well in RPMI 1640 GlutaMAX medium supplemented with 10% FBS and 24 hours later transfected simultaneously with all three vectors using Lipofectamine™ 3000 transfection reagent (ThermoFisher Scientific) according to with the manufacturer's instructions. Puromycin was added 48 hours after transfection at a final concentration of 0.5 μg/ml. Transfection efficiency was 30% using 6.25 μl/well Lipofectamine™ 3000, 1.25 μg of each Cas-9/gRNA vector, and 2.5 μg of GFP-puromycin donor vector. CD28 knockout in the RPMI 8226 cell line was performed using the CD28 sgRNA CRISPR All-in-One Lentivirus kit (Applied Biological Materials Inc.). It consists of a set of 3 all-in-one lentiviral vectors expressing a Cas9 gene, a puromycin resistance gene and another sgRNA (sgRNA 1: ATTGTCGTACGTACAAGCA (SEQ ID NO:119) targeting exon 2 and CD28 sgRNA 2: CAAAAGGGCTTAGAAGGTCC (SEQ ID NO:120), and sgRNA 3: CTATAGCTTGCTAGTAACAG (SEQ ID NO:121), targeting exon 3 of CD28). Cells were infected using a centrifugal inoculation protocol with 2.10 ⁵ cells mixed with lentiviral particles (10 IU/cell), 8 μg/ml polybrene, and 1:100 ViralPlus transduction enhancer (Applied Biological Materials Inc.). The mixture was pre-incubated for 20 minutes at room temperature, then centrifuged for 30 minutes at 32°C and 800×g and seeded into a 12-well plate. Puromycin was added 48 hours after infection at a final concentration of 0.25 μg/ml. Both cell lines were seeded at least 5 times (or until cell viability was stable) before confirming CD28 knockdown at the protein level by flow cytometry (anti-CD28 antibody PE Clone 28.2, BioLegend). CD28-negative cells were then sorted on a Sony SH800S cell sorter and cultured in media supplemented with 0.25 μg/ml or 0.5 μg/ml puromycin, 100 U/ml penicillin, and 100 μg/ml streptomycin (ThermoFisher Scientific). Cells were then cloned by limiting dilution and CD28 knockout confirmed by flow cytometry, PCR and Sanger sequencing using the forward and reverse primers below.

Table R. List of forward and reverse primers

Cell line Amplicon Primers Amplicon size Comments KMS-11 CD28_GFP_left F: CACAACCTGTCCCCATCCTATGAA (SEQ ID NO:122)
R: cAAGCTGCCATCCAGATCGTTATCG (SEQ ID NO:123) 1475 p.o. Confirms insertion of the GFP-puromycin cassette (left side) CD28_PURO_right F: CCAATTAAGGGCCAGCTCATTCC (SEQ ID NO:124)
R: ACCTGTACATCCTTGGGCAAATCC (SEQ ID NO:125) 1233 p.o. Confirms insertion of the GFP-puromycin cassette (right side) CD28_exon_1 F: GTCAGGATGCCTTGTGGTTTGAGT (SEQ ID NO:126)
R: CAGAGCTTCCAGAGCCAATCTAC (SEQ ID NO:127) Variable; WT=720 Region surrounding the CD28 exon1-intron1 junction corresponding to the site of GFP-puromycin cassette insertion and/or insertion/deletion after CRISPR cleavage RPMI 8226 Exon_2 F: CCATGTACTGCCTTCTGGGTGAAA
(SEQ ID NO:128)
R: CCACTGACCACAACCCAGTTTT
(SEQ ID NO:129) Variable; WT=682 bp Region surrounding CD28 exon 2 corresponding to CRISPR cleavage sites Exon_3 F: AGGCCATTGGAAGTCACCGTTT
(SEQ ID NO:130)
R: GCCAACATTGTCCATTGGCTTCAG
(SEQ ID NO:131) Variable; WT=816 bp Region surrounding CD28 exon 3 corresponding to CRISPR cleavage sites

results

CD28 was incorporated into a trispecific Ab to improve T cell proliferation and survival; however, when assessing cell surface markers on multiple myeloma cells, the majority of cell lines (>95%) were found to express the CD28 glycoprotein (Table S).

Table S. Cell Line Surface Expression of CD28 and CD38

human multiple myeloma.

Cell line CD28 CD38 JJN3 1,040 50 KMS-11 (p17) 35.345 2.845 U266 170.525 13,825 L-363 9.145 17,500 MM.1S 56,560 44,535 MM.1R 66,220 49,160 KMS-12BM 0 62,845 RPMI 8226 83,915 136.255 NCI-H929 22,065 153,620 LP-1 7,420 372.835 U266-CD38++ 126.465 633.805 JJN3-CD38 0 714.805 MOLP-8 245 885,030 NCI-H929-CD38++ 22,935 981.310 RPMI 8226-CD38++ 86,320 1,419,120

It has been reported that CD28 expression, although absent on normal plasma cells (Almeida J, et al. British J. of Haematol. 107 :121-131(1999)), can be detected on primary myeloma plasma cells in approximately one third of patients with recently diagnosed (Mateo G, et al. Clin. Cancer Res. 11 :3661-3667(2005)). In addition, it increases in frequency as myeloma progresses and is correlated with poor prognosis and aggressive features of myeloma (Robillard N, Jego G, et al. Clin Cancer Res. 4 :1521-6(1998); Nair JR, Carlson LM, et al J Immunol 187 :1243-53(2011)).

To determine whether expression of CD28 on target cells could improve recognition and lysis by T cells, the activity of wild-type and trispecific non-mutant Ab to CD28 was compared in three independent myeloma lines with varying degrees of CD38 and CD28 expression. As noted above in Example 9 and shown in FIG. 29A - 29C , cytolysis was observed in all three lines, regardless of the level of CD28 expression by trispecific Ab to CD38; however, across all cell lines, the cytolytic activity of the CD28 trispecific non-mutant Ab was reduced 30- to 100-fold, with the most significant reduction in the KMS-11 cell line, which exhibited the least CD38 expression (compare WT and ΔCD28, Fig. 29C and Fig. 29A or 29B ).

The contribution of CD28 to tumor cell recognition and lysis was further confirmed using CRISPR-mediated knockout of CD28 on a myeloma cell target. CD28 expression was not detected on CD28KO KMS-11 cells compared to the parental line ( Fig. 38 , top panel, right and left). The sensitivity of CD28KO KMS-11 cells to cytolysis by T cells was concomitantly reduced 10- to 100-fold ( Fig. 38 , bottom panel, left). Consistent with this effect, there was no difference in cytolysis with trispecific CD38 versus trispecific non-mutant CD28 on target CD38 KO cells ( Figure 38 , bottom panel, right).

Example 14 Cytolysis by anti-CD38 trispecific Ab against CD38 ⁺ hematologic cancer cell lines

The previous examples demonstrate CD38/CD3/CD28 trispecific antibodies that promote cytolysis of multiple myeloma cells. The ability of these antibodies to stimulate cytolysis of other cancer cell lines was studied.

As shown in FIG. 39 , a trispecific Ab to CD38/CD28xCD3, FALA mutant, was characterized by cytolytic activity against the indicated CD38 ⁺ CD28 ^- lines, including acute myelocytic leukemia cell lines (AML (KG-1)), B-cell lymphoma (OCI-Ly19 ), acute T-lymphocytic leukemia (ALL (KOPN8)) and chronic lymphocytic lymphoma (CLL(Z-138)). This demonstrates that CD38/CD3/CD28 trispecific antibodies have activity against other CD38+ hematologic cancer cell lines, including those that are CD28−.

Example 15. Activation of human PBMCs in vitro via α-CD28 superagonist requires antibody bivalence

In vitro activation of human PBMCs with TGN1412 (TGN), one TGN1412 binding arm (TGNslg), and one anti-CD28 _sup binding arm (CD28 _sup slg) was performed as described (Findlay L, Eastwood D, et al . J Immunol Methods 352:1-12 (2010)). 10 ⁵ human PBMCs were seeded onto polypropylene 96-well plates coated with the indicated antibodies and dried (1 μg/well) for 24 hours as previously described. Cytokines such as IFN-γ, TNF-α and IL-2 in the supernatant were measured using Luminex as described in Example 8.

Inclusion of a single CD28 superagonist specificity also reduced the nonspecific cytokine release observed with the CD28 superagonist mAb ( Figure 40 ), previously associated with its adverse effects as a native IgG in humans (Suntharalingam G, Perry MR, et al. N Engl J Med 355 :1018-1028(2006)). In contrast, co-stimulation with trispecific Ab increased the activity and survival of T cells that lyse and protect against tumors. More interestingly, CD38 trispecific Abs preferentially expand Th1 and Tcm cell populations in vitro , exhibiting an important feature that distinguishes them from α-CD3 agonist or α-CD28 superagonist monoclonal Abs used to expand Tregs to induce tolerance. (Penaranda C1, Tang Q, Bluestone JA. J Immunol. 187 :2015-22.(2011); Tabares P, Berr S, et al. Eur J Immunol. 44:1225-36(2014)).

Taken together, the data presented in Examples 1-15 demonstrate CD38/CD3/CD28 trispecific antibodies that stimulate potent antitumor immunity by recruiting two T cell signaling receptors and enhancing target cell recognition. Binding of CD3 and CD28 triggers T cell receptor signaling that activates Bcl-xl and inhibits T cell apoptosis, enhancing T cell effector function and survival. The third arm of the trispecific Ab interacts with CD38, which is expressed at high levels on multiple myeloma cells. Surprisingly, CD28 is also expressed on most myeloma cells; therefore, the trispecific Ab provided improved targeting while improving T cell activation and cytolysis. The optimized trispecific antibody was >1000-fold more effective in killing myeloma cells than the CD38 therapeutic mAb daratumumab and demonstrated potent antitumor activity in vivo in a humanized mouse model.

Although the present invention includes various embodiments, it should be understood that variations and modifications will be apparent to those skilled in the art. Thus, the appended claims are intended to cover all such equivalent variations that fall within the scope of the present invention. In addition, the section titles used herein are provided for purposes of organization only and should not be construed as limiting in any way the subject matter of the present invention.

Each embodiment described herein may be combined with any other embodiment or embodiments unless expressly stated otherwise. In particular, any feature or embodiment as preferred or advantageous may be combined with another feature or features or embodiment or embodiments stated as preferred or advantageous unless expressly stated to the contrary.

All literature cited in this application is expressly incorporated by reference into this document.

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LIST OF SEQUENCES

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Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp

65 70 75 80

Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 7

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of HC mAb1

<400> 7

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1. 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 8

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of LC mAb1

<400> 8

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1. 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp

65 70 75 80

Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 9

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of VH mAb6

<400> 9

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 10

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of VL mAb6

<400> 10

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1. 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Ile Tyr Tyr Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 11

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of HC mAb6

<400> 11

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Asp Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 12

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of LC mAb6

<400> 12

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1. 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Ile Tyr Tyr Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 13

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of VH1 mAb2

<400> 13

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Gln Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL1 amino sequence of the mAb2

<400> 14

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1. 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Gly Gln Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 15

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HC1 amino sequence of the mAb2

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Gln Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 16

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LC1 amino sequence of the mAb2

<400> 16

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1. 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Gly Gln Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 17

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH3 amino sequence of the mAb3

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of VL3 mAb3, and mAb4, and mAb5

<400> 18

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1. 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 19

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HC3 amino sequence of the mAb3

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 20

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of LC3 mAb3, and mAb4, and mAb5

<400> 20

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1. 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 21

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH5 amino sequence of the mAb4

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Ser Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Ile Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HC5 amino sequence of the mAb4

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Ser Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Ile Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 23

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH6 amino sequence of the mAb5

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Ile Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 24

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HC6 amino sequence of the mAb5

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe

20 25 30

Asn Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Ile Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly

225 230 235 240

Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 25

<400> 25

000

<210> 26

<400> 26

000

<210> 27

<400> 27

000

<210> 28

<400> 28

000

<210> 29

<400> 29

000

<210> 30

<211> 256

<212> PROTEIN

<213> Macaca fascicularis

<220>

<223> Extracellular domain of CD38

<400> 30

Arg Trp Arg Gln Gln Trp Ser Gly Ser Gly Thr Thr Ser Arg Phe Pro

1.5 10 15

Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Val His Pro Glu

20 25 30

Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala

35 40 45

Phe Ile Ser Lys Tyr Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro

50 55 60

Leu Val Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Thr Leu Leu

65 70 75 80

Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg

85 90 95

Asp Met Phe Thr Leu Glu Asp Met Leu Leu Gly Tyr Leu Ala Asp Asp

100 105 110

Leu Thr Trp Cys Gly Glu Phe Asn Thr Phe Glu Ile Asn Tyr Gln Ser

115 120 125

Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe

130 135 140

Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Thr Ala Cys Gly Val Val

145 150 155 160

His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser

165 170 175

Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln

180 185 190

Ala Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp

195 200 205

Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys

210 215 220

Arg Asn Ile Arg Phe Phe Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe

225 230 235 240

Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Leu Ser Gly Ile

245 250 255

<210> 31

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR-H1 mAb1/mAb3/mAb4/mAb5

<400> 31

Gly Tyr Thr Phe Thr Ser Phe Asn

15

<210> 32

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR-H2 mAb1/mAb3/mAb4/mAb5

<400> 32

Ile Tyr Pro Gly Asn Gly Gly Thr

15

<210> 33

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR-H3 mAb1/mAb2/mAb3/mAb4/mAb5

<400> 33

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr

1.5 10

<210> 34

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR-L1 mAb1/mAb3/mAb4/mAb5

<400> 34

Glu Ser Val Asp Ser Tyr Gly Asn Gly Phe

1.5 10

<210> 35

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR-L2 mAb1/mAb3/mAb4/mAb5

<400> 35

Leu Ala Ser

1

<210> 36

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR-L3 mAb1/mAb2/mAb3/mAb4/mAb5

<400> 36

Gln Gln Asn Lys Glu Asp Pro Trp Thr

15

<210> 37

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 CDR-H1

<400> 37

Gly Tyr Thr Phe Thr Ser Tyr Ala

15

<210> 38

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 CDR-H2

<400> 38

Ile Tyr Pro Gly Gln Gly Gly Thr

15

<210> 39

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 CDR-L1

<400> 39

Gln Ser Val Ser Ser Tyr Gly Gln Gly Phe

1.5 10

<210> 40

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 CDR-L2

<400> 40

Gly Ala Ser

1

<210> 41

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb6 CDR-H1

<400> 41

Gly Phe Thr Phe Ser Ser Tyr Gly

15

<210> 42

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb6 CDR-H2

<400> 42

Ile Trp Tyr Asp Gly Ser Asn Lys

15

<210> 43

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb6 CDR-H3

<400> 43

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr

1.5 10

<210> 44

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb6 CDR-L1

<400> 44

Gln Gly Ile Arg Asn Asp

15

<210> 45

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb6 CDR-L2

<400> 45

Ala Ala Ser

1

<210> 46

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb6 CDR-L3

<400> 46

Leu Gln Asp Tyr Ile Tyr Tyr Pro Thr

15

<210> 47

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH domain mAb7

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Thr

1. 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr

65 70 75 80

Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 48

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL domain mAb7

<400> 48

Asp Ile Val Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly

1. 5 10 15

Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 49

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH domain of CD28sup antibody

<400> 49

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 50

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL domain of CD28sup antibody

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1. 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Tyr Val Trp

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 51

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH domain of CD28cvn antibody

<400> 51

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1. 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Lys Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 52

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL domain of CD28cvn antibody

<400> 52

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1. 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asn Asp Val Ala Asn Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 53

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH domain of CD3mid antibody

<400> 53

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Lys Ala

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gln Leu Glu Trp Val

35 40 45

Ala Gln Ile Lys Asp Lys Ser Asn Ser Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Val Tyr Tyr Ala Leu Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 54

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL domain of CD3mid antibody

<400> 54

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1. 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val His Asn

20 25 30

Asn Ala Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Ser Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly

85 90 95

Thr Gln Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 55

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 55

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1.5 10

<210> 56

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 56

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1. 5 10 15

<210> 57

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 57

Thr Lys Gly Pro Ser

15

<210> 58

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 58

Gly Gln Pro Lys Ala Ala Pro

15

<210> 59

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 59

Gly Gly Ser Gly Ser Ser Gly Ser Gly Gly

1.5 10

<210> 60

<211> 570

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence CD28supxCD3mid IgG4(hollow) FALA heavy chain 1

<400> 60

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ser Gln Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Thr Lys Ala Trp Met His Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gln Leu Glu Trp Val Ala Gln Ile Lys Asp Lys Ser

165 170 175

Asn Ser Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Arg Gly Val Tyr Tyr

210 215 220

Ala Leu Ser Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Arg Thr Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

340 345 350

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

355 360 365

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

370 375 380

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

385 390 395 400

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

405 410 415

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

420 425 430

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

435 440 445

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

450 455 460

Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu

465 470 475 480

Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe

485 490 495

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

500 505 510

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

515 520 525

Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

530 535 540

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

545 550 555 560

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

565 570

<210> 61

<211> 338

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence CD28supxCD3mid light chain 1

<400> 61

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1. 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val His Asn

20 25 30

Asn Ala Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Ser Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly

85 90 95

Thr Gln Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gln Pro Lys Ala Ala Pro Asp Ile Gln Met Thr Gln Ser Pro Ser

115 120 125

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala

130 135 140

Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln Gln Lys Pro Gly

145 150 155 160

Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr Gly

165 170 175

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

180 185 190

Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln

195 200 205

Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

210 215 220

Ile Lys Thr Lys Gly Pro Ser Arg Thr Val Ala Ala Pro Ser Val Phe

225 230 235 240

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

245 250 255

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

260 265 270

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

275 280 285

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

290 295 300

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

305 310 315 320

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

325 330 335

Glu Cys

<210> 62

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence CD38VH1 IgG4(overhang) FALA heavy chain 2

<400> 62

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Gln Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

<210> 63

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD38VH1 Light2 polypeptide sequence

<400> 63

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1. 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Gly Gln Gly Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Lys

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 64

<211> 573

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence CD28supxCD3mid IgG1(hollow) LALA P329A heavy chain 1

<400> 64

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ser Gln Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Thr Lys Ala Trp Met His Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gln Leu Glu Trp Val Ala Gln Ile Lys Asp Lys Ser

165 170 175

Asn Ser Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Arg Gly Val Tyr Tyr

210 215 220

Ala Leu Ser Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Arg Thr Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

340 345 350

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

355 360 365

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

370 375 380

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

385 390 395 400

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

405 410 415

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

420 425 430

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

435 440 445

Lys Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser

450 455 460

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

465 470 475 480

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

485 490 495

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

500 505 510

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

515 520 525

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

530 535 540

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

545 550 555 560

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

565 570

<210> 65

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence CD38VH1 IgG1(overhang) LALA P329A heavy chain 2

<400> 65

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Gln Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 66

<211> 573

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence CD28supxCD3mid IgG1(hollow) NNSA heavy chain 1

<400> 66

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ser Gln Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Thr Lys Ala Trp Met His Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gln Leu Glu Trp Val Ala Gln Ile Lys Asp Lys Ser

165 170 175

Asn Ser Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Arg Gly Val Tyr Tyr

210 215 220

Ala Leu Ser Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Arg Thr Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

340 345 350

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

355 360 365

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

370 375 380

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

385 390 395 400

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

405 410 415

Lys Pro Arg Glu Glu Gln Tyr Asn Asn Ala Ser Arg Val Val Ser Val

420 425 430

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

435 440 445

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

450 455 460

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

465 470 475 480

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

485 490 495

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

500 505 510

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

515 520 525

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

530 535 540

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

545 550 555 560

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

565 570

<210> 67

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence CD38VH1 IgG1(overhang) NNSA heavy chain 2

<400> 67

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Gly Gln Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Gly Leu Arg Arg Ala Tyr Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Asn Ala Ser Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 68

<211> 443

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence CD38hhy1370 IgG4(overhang) FALA P329A heavy chain 2

<400> 68

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 69

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD38hhy1370 Light2 polypeptide sequence

<400> 69

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1. 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Ile Tyr Tyr Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 70

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence CD38hhy1370 IgG1(overhang) LALA P329A heavy chain 2

<400> 70

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 71

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Polypeptide sequence CD38hhy1370 IgG1(overhang) NNSA heavy chain 2

<400> 71

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Asn Ala Ser Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 72

<211> 1710

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide sequence CD28supxCD3mid IgG4(hollow) FALA heavy chain 1

<400> 72

caggtgcagc tggtgcagtc tggcgccgag gtcgtgaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctactaca tccactgggt gcgccaggcc 120

cctggacagg gactggaatg gatcggcagc atctaccccg gcaacgtgaa caccaactac 180

gcccagaagt tccagggcag agccaccctg accgtggaca ccagcatcag caccgcctac 240

atggaactga gccggctgag aagcgacgac accgccgtgt actactgcac ccggtcccac 300

tacggcctgg attggaactt cgacgtgtgg ggcaagggca ccaccgtgac agtgtctagc 360

agccaggtgc agctggtgga atctggcggc ggagtggtgc agcctggcag aagcctgaga 420

ctgagctgtg ccgccagcgg cttcaccttc accaaggcct ggatgcactg ggtgcgccag 480

gcccctggaa agcagctgga atgggtggcc cagatcaagg acaagagcaa cagctacgcc 540

acctactacg ccgacagcgt gaagggccgg ttcaccatca gccgggacga cagcaagaac 600

accctgtacc tgcagatgaa cagcctgcgg gccgaggaca ccgccgtgta ctactgtcgg 660

ggcgtgtact atgccctgag ccccttcgat tactggggcc agggaaccct cgtgaccgtg 720

tctagtcgga ccgccagcac aaagggccca tcggtgttcc ctctggcccc ttgcagcaga 780

agcaccagcg aatctacagc cgccctgggc tgcctcgtga aggactactt tcccgagccc 840

gtgaccgtgt cctggaactc tggcgctctg acaagcggcg tgcacacctt tccagccgtg 900

ctccagagca gcggcctgta ctctctgagc agcgtcgtga cagtgcccag cagcagcctg 960

ggcaccaaga cctacacctg taacgtggac cacaagccca gcaacaccaa ggtggacaag 1020

cgggtggaat ctaagtacgg ccctccctgc cctccttgcc cagcccctga agctgccggc 1080

ggaccctccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 1140

cccgaagtga cctgcgtggt ggtggatgtg tcccaggaag atcccgaggt gcagttcaat 1200

tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcccagaga ggaacagttc 1260

aacagcacct accgggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 1320

aaagagtaca agtgcaaggt gtccaacaag ggcctgccca gctccatcga gaaaaccatc 1380

agcaaggcca agggccagcc ccgcgagcct caagtgtgta ccctgccccc tagccaggaa 1440

gagatgacca agaaccaggt gtccctgagc tgtgccgtga aaggcttcta ccccagcgac 1500

attgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1560

gtgctggaca gcgacggctc attcttcctg gtgtccaagc tgaccgtgga caagagccgg 1620

tggcaggaag gcaacgtgtt cagctgctcc gtgatgcacg aggccctgca caaccactac 1680

acccagaagt ccctgtctct gtccctgggc 1710

<210> 73

<211> 1014

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide sequence CD28supxCD3mid light chain 1

<400> 73

gacatcgtga tgacccagac ccccctgagc ctgagcgtga cacctggaca gcctgccagc 60

atcagctgca agagcagcca gagcctggtg cacaacaacg ccaacaccta cctgagctgg 120

tatctgcaga agcccggcca gagcccccag tccctgatct acaaggtgtc caacagattc 180

agcggcgtgc ccgacagatt ctccggcagc ggctctggca ccgacttcac cctgaagatc 240

agccgggtgg aagccgagga cgtgggcgtg tactattgtg gccagggcac ccagtacccc 300

ttcacctttg gcagcggcac caaggtggaa atcaagggcc agcccaaggc cgcccccgac 360

atccagatga cccagagccc cagcagcctg tctgccagcg tgggcgacag agtgaccatc 420

acctgtcagg ccagccagaa catctacgtg tggctgaact ggtatcagca gaagcccggc 480

aaggccccca agctgctgat ctacaaggcc agcaacctgc acaccggcgt gcccagcaga 540

ttttctggca gcggctccgg caccgacttc accctgacaa tcagctccct gcagcccgag 600

gacattgcca cctactactg ccagcagggc cagacctacc cctacacctt tggccagggc 660

accaagctgg aaatcaagac caagggcccc agccgtacgg tggccgctcc cagcgtgttc 720

atcttcccac ctagcgacga gcagctgaag tccggcacag cctctgtcgt gtgcctgctg 780

aacaacttct acccccgcga ggccaaagtg cagtggaagg tggacaacgc cctgcagagc 840

ggcaacagcc aggaaagcgt gaccgagcag gacagcaagg actccaccta cagcctgagc 900

agcaccctga cactgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 960

acccaccagg gcctgtctag ccccgtgacc aagagcttca accggggcga gtgt 1014

<210> 74

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide sequence CD38VH1 IgG4 (overhang) FALA heavy chain 2

<400> 74

caggtgcagc tggtgcagtc tggcgccgaa gtcgtgaaac ctggcgcctc cgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctacgcca tgcactgggt caaagaggcc 120

cctggccaga gactggaatg gatcggctac atctaccccg gccagggcgg caccaactac 180

aaccagaagt tccagggcag agccaccctg accgccgata caagcgccag caccgcctac 240

atggaactga gcagcctgcg gagcgaggat accgccgtgt acttctgtgc cagaacaggc 300

ggcctgaggc gggcctactt tacctattgg ggccagggca ccctcgtgac cgtgtctagc 360

gctagcacaa agggcccatc ggtgttccct ctggcccctt gcagcagaag caccagcgaa 420

tctacagccg ccctgggctg cctcgtgaag gactactttc ccgagcccgt gaccgtgtcc 480

tggaactctg gcgctctgac aagcggcgtg cacacctttc cagccgtgct ccagagcagc 540

ggcctgtact ctctgagcag cgtcgtgaca gtgcccagca gcagcctggg caccaagacc 600

tacacctgta acgtggacca caagcccagc aacaccaagg tggacaagcg ggtggaatct 660

aagtacggcc ctccctgccc tccttgccca gcccctgaag ctgccggcgg accctccgtg 720

ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 780

tgcgtggtgg tggatgtgtc cccaggaagat cccgaggtgc agttcaattg gtacgtggac 840

ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagttcaa cagcacctac 900

cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 960

tgcaaggtgt ccaacaaggg cctgcccagc tccatcgaga aaaccatcag caaggccaag 1020

ggccagcccc gcgagcctca agtgtatacc ctgccccctt gccaggaaga gatgaccaag 1080

aaccaggtgt ccctgtggtg tctcgtgaaa ggcttctacc ccagcgacat tgccgtggaa 1140

tggggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 1200

gacggctcat tcttcctgta ctccaagctg accgtggaca agagccggtg gcaggaaggc 1260

aacgtgttca gctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1320

ctgtctctgt ccctggggc 1338

<210> 75

<211> 654

<212> DNA

<213> Artificial sequence

<220>

<223> CD38VH1 Light2 polynucleotide sequence

<400> 75

gacatcgtgc tgacacagag ccctgccacc ctgtctctga gccctggcga gagagccacc 60

atcagctgta gagccagcca gagcgtgtcc agctacggcc agggcttcat gcactggtat 120

cagcagaagc ccggccagcc ccccagactg ctgatctatg gcgccagcag cagagccaca 180

ggcatccccg ccagattttc tggctctggc agcggcaccg acttcaccct gacaatcagc 240

cccctggaac ccgaggactt cgccgtgtac tactgccagc agaacaaaga ggacccctgg 300

accttcggcg gaggcaccaa gctggaaatc aagcgtacgg tggccgctcc cagcgtgttc 360

atcttcccac ctagcgacga gcagctgaag tccggcacag cctctgtcgt gtgcctgctg 420

aacaacttct acccccgcga ggccaaggtg cagtggaagg tggacaatgc cctgcagagc 480

ggcaacagcc aggaaagcgt gaccgagcag gacagcaagg actccaccta cagcctgagc 540

agcaccctga ccctgtccaa ggccgattac gagaagcaca aggtgtacgc ctgcgaagtg 600

acccaccagg gcctgtctag ccccgtgacc aagagcttca accggggcga gtgc 654

<210> 76

<211> 1719

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide sequence CD28supxCD3mid IgG1(hollow) LALA P329A heavy chain 1

<400> 76

caggtgcagc tggtgcagtc tggcgccgag gtcgtgaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctactaca tccactgggt gcgccaggcc 120

cctggacagg gactggaatg gatcggcagc atctaccccg gcaacgtgaa caccaactac 180

gcccagaagt tccagggcag agccaccctg accgtggaca ccagcatcag caccgcctac 240

atggaactga gccggctgag aagcgacgac accgccgtgt actactgcac ccggtcccac 300

tacggcctgg attggaactt cgacgtgtgg ggcaagggca ccaccgtgac agtgtctagc 360

agccaggtgc agctggtgga atctggcggc ggagtggtgc agcctggcag aagcctgaga 420

ctgagctgtg ccgccagcgg cttcaccttc accaaggcct ggatgcactg ggtgcgccag 480

gcccctggaa agcagctgga atgggtggcc cagatcaagg acaagagcaa cagctacgcc 540

acctactacg ccgacagcgt gaagggccgg ttcaccatca gccgggacga cagcaagaac 600

accctgtacc tgcagatgaa cagcctgcgg gccgaggaca ccgccgtgta ctactgtcgg 660

ggcgtgtact atgccctgag ccccttcgat tactggggcc agggaaccct cgtgaccgtg 720

tctagtcgga ccgccagcac aaagggcccc agcgtgttcc ctctggcccc tagcagcaag 780

agcacatctg gcggaacagc cgccctgggc tgcctcgtga aggactactt tcccgagccc 840

gtgaccgtgt cctggaattc tggcgccctg accagcggcg tgcacacctt tccagctgtg 900

ctgcagtcca gcggcctgta cagcctgagc agcgtcgtga cagtgcccag cagctctctg 960

ggcacccaga cctacatctg caacgtgaac cacaagccca gcaacaccaa ggtggacaag 1020

aaggtggaac ccaagagctg cgacaagacc cacacctgtc ccccttgtcc tgcccccgaa 1080

gccgccggag gcccttccgt gttcctgttc cccccaaagc ccaaggacac cctgatgatc 1140

agccggaccc ccgaagtgac ctgcgtggtg gtggatgtgt cccacgagga ccctgaagtg 1200

aagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gccaagagag 1260

gaacagtaca acagcaccta ccgggtggtg tccgtgctga ccgtgctgca ccaggactgg 1320

ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg ccctggccgc ccccatcgag 1380

aaaaccatca gcaaggccaa gggccagccc cgcgaacccc aggtgtgcac actgccccca 1440

agcagggacg agctgaccaa gaaccaggtg tccctgagct gtgccgtgaa aggcttctac 1500

ccctccgata tcgccgtgga atgggagagc aacggccagc ccgagaacaa ctacaagacc 1560

accccccctg tgctggacag cgacggctca ttcttcctgg tgtccaagct gacagtggac 1620

aagtcccggt ggcagcaggg caacgtgttc agctgctccg tgatgcacga ggccctgcac 1680

aaccactaca cccagaagtc cctgagcctg agccccggc 1719

<210> 77

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide sequence CD38VH1 IgG1(overhang) LALA P329A heavy chain 2

<400> 77

caggtgcagc tggtgcagtc tggcgccgaa gtcgtgaaac ctggcgcctc cgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctacgcca tgcactgggt caaagaggcc 120

cctggccaga gactggaatg gatcggctac atctaccccg gccagggcgg caccaactac 180

aaccagaagt tccagggcag agccaccctg accgccgata caagcgccag caccgcctac 240

atggaactga gcagcctgcg gagcgaggat accgccgtgt acttctgtgc cagaacaggc 300

ggcctgaggc gggcctactt tacctattgg ggccagggca ccctcgtgac cgtgtctagc 360

gctagcacaa agggccccag cgtgttccct ctggccccta gcagcaagag cacatctggc 420

ggaacagccg ccctgggctg cctcgtgaag gactactttc ccgagcccgt gaccgtgtcc 480

tggaattctg gcgccctgac cagcggcgtg cacacctttc cagctgtgct gcagtccagc 540

ggcctgtaca gcctgagcag cgtcgtgaca gtgcccagca gctctctggg cacccagacc 600

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggaaccc 660

aagagctgcg acaagaccca cacctgtccc ccttgtcctg cccccgaagc cgccggaggc 720

ccttccgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatcag ccggaccccc 780

gaagtgacct gcgtggtggt ggatgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 840

tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc caagagagga acagtacaac 900

agcacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 960

gagtacaagt gcaaggtgtc caacaaggcc ctggccgccc ccatcgagaa aaccatcagc 1020

aaggccaagg gccagccccg cgaaccccag gtgtacacac tgcccccatg cagggacgag 1080

ctgaccaaga accaggtgtc cctgtggtgt ctggtgaaag gcttctaccc ctccgatatc 1140

gccgtggaat gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg 1200

ctggacagcg acggctcatt cttcctgtac tccaagctga cagtggacaa gtcccggtgg 1260

cagcagggca acgtgttcag ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1320

cagaagtccc tgagcctgag ccccggc 1347

<210> 78

<211> 1719

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide sequence CD28supxCD3mid IgG1(hollow) NNSA heavy chain 1

<400> 78

caggtgcagc tggtgcagtc tggcgccgag gtcgtgaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctactaca tccactgggt gcgccaggcc 120

cctggacagg gactggaatg gatcggcagc atctaccccg gcaacgtgaa caccaactac 180

gcccagaagt tccagggcag agccaccctg accgtggaca ccagcatcag caccgcctac 240

atggaactga gccggctgag aagcgacgac accgccgtgt actactgcac ccggtcccac 300

tacggcctgg attggaactt cgacgtgtgg ggcaagggca ccaccgtgac agtgtctagc 360

agccaggtgc agctggtgga atctggcggc ggagtggtgc agcctggcag aagcctgaga 420

ctgagctgtg ccgccagcgg cttcaccttc accaaggcct ggatgcactg ggtgcgccag 480

gcccctggaa agcagctgga atgggtggcc cagatcaagg acaagagcaa cagctacgcc 540

acctactacg ccgacagcgt gaagggccgg ttcaccatca gccgggacga cagcaagaac 600

accctgtacc tgcagatgaa cagcctgcgg gccgaggaca ccgccgtgta ctactgtcgg 660

ggcgtgtact atgccctgag ccccttcgat tactggggcc agggaaccct cgtgaccgtg 720

tctagtcgga ccgccagcac aaagggcccc agcgtgttcc ctctggcccc tagcagcaag 780

agcacatctg gcggaacagc cgccctgggc tgcctcgtga aggactactt tcccgagccc 840

gtgaccgtgt cctggaattc tggcgccctg accagcggcg tgcacacctt tccagctgtg 900

ctgcagtcca gcggcctgta cagcctgagc agcgtcgtga cagtgcccag cagctctctg 960

ggcacccaga cctacatctg caacgtgaac cacaagccca gcaacaccaa ggtggacaag 1020

aaggtggaac ccaagagctg cgacaagacc cacacctgtc ccccttgtcc tgcccccgaa 1080

ctgctgggag gcccttccgt gttcctgttc cccccaaagc ccaaggacac cctgatgatc 1140

agccggaccc ccgaagtgac ctgcgtggtg gtggatgtgt cccacgagga ccctgaagtg 1200

aagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gccaagagag 1260

gaacagtaca acaatgcctc ccgggtggtg tccgtgctga ccgtgctgca ccaggactgg 1320

ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg ccctgcctgc ccccatcgag 1380

aaaaccatca gcaaggccaa gggccagccc cgcgaacccc aggtgtgcac actgccccca 1440

agcagggacg agctgaccaa gaaccaggtg tccctgagct gtgccgtgaa aggcttctac 1500

ccctccgata tcgccgtgga atgggagagc aacggccagc ccgagaacaa ctacaagacc 1560

accccccctg tgctggacag cgacggctca ttcttcctgg tgtccaagct gacagtggac 1620

aagtcccggt ggcagcaggg caacgtgttc agctgctccg tgatgcacga ggccctgcac 1680

aaccactaca cccagaagtc cctgagcctg agccccggc 1719

<210> 79

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide sequence CD38VH1 IgG1(overhang) NNSA heavy chain 2

<400> 79

caggtgcagc tggtgcagtc tggcgccgaa gtcgtgaaac ctggcgcctc cgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctacgcca tgcactgggt caaagaggcc 120

cctggccaga gactggaatg gatcggctac atctaccccg gccagggcgg caccaactac 180

aaccagaagt tccagggcag agccaccctg accgccgata caagcgccag caccgcctac 240

atggaactga gcagcctgcg gagcgaggat accgccgtgt acttctgtgc cagaacaggc 300

ggcctgaggc gggcctactt tacctattgg ggccagggca ccctcgtgac cgtgtctagc 360

gctagcacaa agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tatgttgacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

aatgcctccc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1080

ctgaccaaga atcaagtcag cctgtggtgc ctggtaaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac tcaaaactca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccggggt 1347

<210> 80

<211> 1329

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide sequence CD38hhy1370 IgG4(overhang) FALA P329A heavy chain 2

<400> 80

caggtgcagc tggtggaaag cggcggaggc gtggtgcagc ctggcaggtc tctgagactg 60

agctgtgccg ccagcggctt caccttcagc agctacggaa tgcactgggt gcgccaggcc 120

cctggcaaag gactggaatg ggtggccgtg atttggtacg acggcagcaa caagtactac 180

gccgacagcg tgaagggccg gttcaccatc agcggcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc cagaatgttc 300

agaggcgcct tcgactactg gggccagggc acactcgtga ccgtgtctag tgcgtcgacc 360

aagggcccat cggtgttccc tctggcccct tgcagcagaa gcaccagcga atctacagcc 420

gccctgggct gcctcgtgaa ggactacttt cccgagcccg tgaccgtgtc ctggaactct 480

ggcgctctga caagcggcgt gcacaccttt ccagccgtgc tccagagcag cggcctgtac 540

tctctgagca gcgtcgtgac agtgcccagc agcagcctgg gcaccaagac ctacacctgt 600

aacgtggacc acaagcccag caacaccaag gtggacaagc gggtggaatc taagtacggc 660

cctccctgcc ctccttgccc agcccctgaa gctgccggcg gaccctccgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 780

gtggatgtgt cccaggaaga tcccgaggtg cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgcccag ctccatcgag aaaaccatca gcaaggccaa gggccagccc 1020

cgcgagcctc aagtgtatac cctgccccct tgccaggaag agatgaccaa gaaccaggtg 1080

tccctgtggt gtctcgtgaa aggcttctac cccagcgaca ttgccgtgga atggggagagc 1140

aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggctca 1200

ttcttcctgt actccaagct gaccgtggac aagagccggt ggcaggaagg caacgtgttc 1260

agctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtctctg 1320

tccctgggc 1329

<210> 81

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> CD38hhy1370 Light2 polynucleotide sequence

<400> 81

gccatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60

atcacctgta gagccagcca gggcatccgg aacgacctgg gctggtatca gcagaagcct 120

ggcaaggccc ccaagctgct gatctacgcc gctagctctc tgcagtccgg cgtgcccagc 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatctctgg cctgcagccc 240

gaggacagcg ccacctacta ctgtctgcaa gactacatct actaccccac cttcggccag 300

ggcaccaagg tggaaatcaa gcgtacggtg gccgctccca gcgtgttcat cttcccacct 360

agcgacgagc agctgaagtc cggcacagcc tctgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaagtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gaaagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgaca 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gt 642

<210> 82

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide sequence CD38hhy1370 IgG1(overhang) LALA P329A heavy chain 2

<400> 82

caggtgcagc tggtggaaag cggcggaggc gtggtgcagc ctggcaggtc tctgagactg 60

agctgtgccg ccagcggctt caccttcagc agctacggaa tgcactgggt gcgccaggcc 120

cctggcaaag gactggaatg ggtggccgtg atttggtacg acggcagcaa caagtactac 180

gccgacagcg tgaagggccg gttcaccatc agcggcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc cagaatgttc 300

agaggcgcct tcgactactg gggccagggc acactcgtga ccgtgtctag tgcgtcgacc 360

aagggcccca gcgtgttccc tctggcccct agcagcaaga gcacatctgg cggaacagcc 420

gccctgggct gcctcgtgaa ggactacttt cccgagcccg tgaccgtgtc ctggaattct 480

ggcgccctga ccagcggcgt gcacaccttt ccagctgtgc tgcagtccag cggcctgtac 540

agcctgagca gcgtcgtgac agtgcccagc agctctctgg gcacccagac ctacatctgc 600

aacgtgaacc acaagcccag caacaccaag gtggacaaga aggtggaacc caagagctgc 660

gacaagaccc acacctgtcc cccttgtcct gcccccgaag ccgccggagg cccttccgtg 720

ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 780

tgcgtggtgg tggatgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 840

ggcgtggaag tgcacaacgc caagaccaag ccaagagagg aacagtacaa cagcacctac 900

cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 960

tgcaaggtgt ccaacaaggc cctggccgcc cccatcgaga aaaccatcag caaggccaag 1020

ggccagcccc gcgaacccca ggtgtacaca ctgcccccat gcagggacga gctgaccaag 1080

aaccaggtgt ccctgtggtg tctggtgaaa ggcttctacc cctccgatat cgccgtggaa 1140

tggggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 1200

gacggctcat tcttcctgta ctccaagctg acagtggaca agtcccggtg gcagcagggc 1260

aacgtgttca gctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1320

ctgagcctga gccccggc 1338

<210> 83

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> Polynucleotide sequence CD38hhy1370 IgG1(overhang) NNSA heavy chain 2

<400> 83

caggtgcagc tggtggaaag cggcggaggc gtggtgcagc ctggcaggtc tctgagactg 60

agctgtgccg ccagcggctt caccttcagc agctacggaa tgcactgggt gcgccaggcc 120

cctggcaaag gactggaatg ggtggccgtg atttggtacg acggcagcaa caagtactac 180

gccgacagcg tgaagggccg gttcaccatc agcggcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc cagaatgttc 300

agaggcgcct tcgactactg gggccagggc acactcgtga ccgtgtctag tgcgtcgacc 360

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtatgttgac 840

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa caatgcctcc 900

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat gccgggatga gctgaccaag 1080

aatcaagtca gcctgtggtg cctggtaaaa ggcttctatc ccagcgacat cgccgtggag 1140

tggggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200

gacggctcct tcttcctcta ctcaaaactc accgtggaca agagcaggtg gcagcagggg 1260

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320

ctctccctgt ctccggggt 1338

<210> 84

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH domain of CD3low antibody

<400> 84

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Lys Ala

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Lys Asp Lys Ser Asn Ser Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Val Tyr Tyr Ala Leu Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 85

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL domain of CD3low antibody

<400> 85

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1. 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val His Asn

20 25 30

Asn Gly Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly

85 90 95

Thr Gln Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 86

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW1 VH mAb2/mAb3

<400> 86

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 87

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW1 VH mAb4

<400> 87

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Ser Gly Ala

1. 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 88

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW1 VH mAb5

<400> 88

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1. 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser

20 25

<210> 89

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW1 VH mAb6

<400> 89

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 90

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW2 VH mAb2/mAb3/mAb5

<400> 90

Met His Trp Val Lys Glu Ala Pro Gly Gln Arg Leu Glu Trp Ile Gly

1. 5 10 15

Tyr

<210> 91

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW2 VH mAb4

<400> 91

Met His Trp Val Lys Glu Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly

1. 5 10 15

Tyr

<210> 92

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW2 VH mAb6

<400> 92

Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

1. 5 10 15

Val

<210> 93

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW3 VH mAb2/mAb3

<400> 93

Asn Tyr Asn Gln Lys Phe Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr

1. 5 10 15

Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Phe Cys

35

<210> 94

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW3 VH mAb4/mAb5

<400> 94

Asn Tyr Asn Gln Lys Phe Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr

1. 5 10 15

Ser Ala Ser Thr Ala Tyr Met Glu Ile Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Val Tyr Phe Cys

35

<210> 95

<211> 38

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW3 VH mAb6

<400> 95

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn

1. 5 10 15

Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 96

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW4 VH mAb2/mAb3/mAb4/mAb5/mAb6

<400> 96

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1.5 10

<210> 97

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW1 VL mAb2/mAb3/mAb4/mAb5

<400> 97

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1.5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser

20 25

<210> 98

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW1 VL mAb6

<400> 98

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1. 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

20 25

<210> 99

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW2 VL mAb2/mAb3/mAb4/mAb5

<400> 99

Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile

1. 5 10 15

Tyr

<210> 100

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW2 VL mAb6

<400> 100

Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

1.5 10 15

<210> 101

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW3 VL mAb2/mAb3/mAb4/mAb5

<400> 101

Ser Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

1. 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Pro Leu Glu Pro Glu Asp Phe Ala

20 25 30

Val Tyr Tyr Cys

35

<210> 102

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW3 VL mAb6

<400> 102

Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1. 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro Glu Asp Ser Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 103

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW4 VL mAb2/mAb3/mAb4/mAb5

<400> 103

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1.5 10

<210> 104

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FW4 VL mAb6

<400> 104

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1.5 10

<210> 105

<211> 159

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Sequence of CD38 isoform E

<400> 105

Arg Trp Arg Gln Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro

1. 5 10 15

Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu

20 25 30

Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala

35 40 45

Phe Ile Ser Lys His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro

50 55 60

Leu Met Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu

65 70 75 80

Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg

85 90 95

Asp Met Phe Thr Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp

100 105 110

Leu Thr Trp Cys Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser

115 120 125

Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe

130 135 140

Trp Lys Thr Val Ser Arg Arg His Phe Trp Glu Cys Gly Ser Pro

145 150 155

<210> 106

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb7 light chain

<400> 106

Asp Ile Val Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly

1. 5 10 15

Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 107

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain mAb7

<400> 107

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Thr

1. 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr

65 70 75 80

Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 108

<211> 445

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Comparative heavy chain 1 (binds CD38)

<400> 108

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Glu Ile Asn Pro Gln Ser Ser Thr Ile Asn Tyr Ala Thr Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Asn Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asp Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Lys His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Glu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Asp Val Ser

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asp Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Glu

405 410 415

Gln Gly Asp Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 109

<211> 485

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Comparative heavy chain 2 (binds CD3)

<400> 109

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1. 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asp Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Lys Pro

115 120 125

Gly Ser Gly Lys Pro Gly Ser Gly Lys Pro Gly Ser Gly Lys Pro Gly

130 135 140

Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly

145 150 155 160

Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr

165 170 175

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ser Pro Arg

180 185 190

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg

195 200 205

Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Ser Gly

210 215 220

Ala Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser

225 230 235 240

Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Glu Pro

245 250 255

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro

260 265 270

Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

275 280 285

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

290 295 300

Lys His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

305 310 315 320

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

325 330 335

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

340 345 350

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

355 360 365

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

370 375 380

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Gln Met Thr Lys Asn Gln

385 390 395 400

Val Lys Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

405 410 415

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

420 425 430

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

435 440 445

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

450 455 460

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

465 470 475 480

Leu Ser Pro Gly Lys

485

<210> 110

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Comparative light chain (binds CD38)

<400> 110

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1. 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Val Asp Thr Trp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Asp Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 111

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 111

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

1.5 10

<210> 112

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 112

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

1.5 10

<210> 113

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 113

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

1.5 10

<210> 114

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 114

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

1.5 10

<210> 115

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 115

Cys Pro Pro Cys Pro Ala Pro Val Ala Gly Pro Ser

1.5 10

<210> 116

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 116

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

1.5 10

<210> 117

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 117

tacctgttac ttgaattgaa 20

<210> 118

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 118

attttttgag gtcttccaat 20

<210> 119

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 119

attgtcgtac gctacaagca 20

<210> 120

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 120

caaaagggct tagaaggtcc 20

<210> 121

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 121

ctatagcttg ctagtaacag 20

<210> 122

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 122

cacaacctgt ccccatccta tgaa 24

<210> 123

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 123

aagctgccat cgatcgtt atcg 24

<210> 124

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 124

ccaaattaag ggccagctca ttcc 24

<210> 125

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 125

acctgtacat ccttgggcaa atcc 24

<210> 126

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 126

gtcaggatgc cttgtggttt gagt 24

<210> 127

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 127

cagagcttcc agagccaatc tac 23

<210> 128

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 128

ccatgtactg ccttctgggt gaaa 24

<210> 129

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 129

ccactgacca caacccagtt tt 22

<210> 130

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 130

aggccattgg aagtcaccgt tt 22

<210> 131

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 131

gccaacattg tccatggct tcag 24

<210> 132

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 132

Gln Ser Val Ser Ser Tyr Gly Gln Gly

15

<---

Claims (27)

1. A monospecific binding protein that binds a CD38 polypeptide, wherein said monospecific binding protein comprises: (a) an antibody heavy chain that contains a heavy chain variable domain (VH) containing a CDR-H1 sequence containing the amino acid sequence GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence containing the amino acid sequence IYPGQGGT (SEQ ID NO:38 ), and a CDR-H3 sequence containing the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO:33); And (b) an antibody light chain that contains a light chain variable domain (VL) containing a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence containing the amino acid sequence GAS (SEQ ID NO:40 ), and a CDR-L3 sequence containing the amino acid sequence QQNKEDPWT (SEQ ID NO:36). 2. Monospecific binding protein according to claim 1, where the VH domain contains, in the direction from the N-terminus to the C-terminus, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; where FR1 contains the sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87) or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO:88); where FR2 contains the sequence MHWVKEAPGQRLEWIGY (SEQ ID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO:91); where FR3 contains the sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO:94); and where FR4 contains the sequence WGQGTLVTVSS (SEQ ID NO:96). 3. Monospecific binding protein according to claim 1, where: the VH domain contains the amino acid sequence of SEQ ID NO:13, and the VL domain contains the amino acid sequence of SEQ ID NO:14. 4. Monospecific binding protein according to claim 3, where: the antibody heavy chain contains the amino acid sequence of SEQ ID NO:15, and the antibody light chain contains the amino acid sequence of SEQ ID NO:16. 5. Monospecific binding protein according to any one of paragraphs. 1-4, wherein said binding protein cross-reacts with an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus CD38 polypeptide; where optionally said binding protein binds a human CD38 polypeptide containing the amino acid sequence of SEQ ID NO:1; and where optionally said binding protein binds a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:1 with an equilibrium dissociation constant (KD) of 2.1 nM or less. 6. The monospecific binding protein of claim 5, wherein said binding protein binds a human CD38 isoform E polypeptide containing the amino acid sequence of SEQ ID NO:105. 7. The monospecific binding protein according to claim 5 or 6, wherein said binding protein binds a cynomolgus monkey CD38 polypeptide containing the amino acid sequence of SEQ ID NO:30; wherein optionally said binding protein binds a cynomolgus CD38 polypeptide comprising the amino acid sequence of SEQ ID NO:30 with an equilibrium dissociation constant (KD) of 1.3 nM or less. 8. Monospecific binding protein according to any one of paragraphs. 1-7, wherein said monospecific binding protein is a chimeric or humanized antibody. 9. Monospecific binding protein according to any one of paragraphs. 1-7, wherein said monospecific binding protein is a monoclonal antibody. 10. Monospecific binding protein according to any one of paragraphs. 1-7, wherein said monospecific binding protein comprises one or more full-length antibody heavy chains containing an Fc region; where optionally the Fc region is a human Fc region containing one or more mutations that weaken or eliminate Fc receptor binding and/or Fc region effector function. 11. The monospecific binding protein according to claim 10, wherein the Fc region is a human IgG1 Fc region; where optional: The Fc region of human IgG1 contains amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG1 according to the EU index, where the amino acid substitutions are L234A, L235A and P329A; or The Fc region of human IgG1 contains amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1 according to the EU index, where the amino acid substitutions are S298N, T299A and Y300S. 12. The monospecific binding protein according to claim 10, wherein the Fc region is a human IgG4 Fc region; where optional: The Fc region of human IgG4 contains amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P and R409K, The Fc region of human IgG4 contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, where the amino acid substitutions are F234A and L235A, or The Fc region of human IgG4 contains amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index, where the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. 13. Monospecific binding protein according to any one of paragraphs. 1-7, where the specified monospecific binding protein contains an F(ab)-, F(ab')2-, Fab'-SH-, Fv- or scFv-fragment of the antibody. 14. A conjugate for targeting a cytotoxic agent or label to cells expressing CD38 on their surface, containing a binding protein according to any one of claims. 1-13, conjugated to a cytotoxic agent or label. 15. A polynucleotide encoding a monospecific binding protein according to any one of claims. 1-13. 16. An expression vector containing the polynucleotide according to claim 15. 17. Host cell for producing a monospecific binding protein according to any one of paragraphs. 1-13, containing the polynucleotide according to claim 15 or the expression vector according to claim 16. 18. A method for producing a monospecific binding protein according to any one of claims. 1-13, comprising culturing a host cell according to claim 17, thereby obtaining a monospecific binding protein, optionally wherein the method further comprises recovering the monospecific binding protein from the host cell.

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