Antibodies against human CD38

Abstract

Autoimmunity and (Chronic) To provide isolated monoclonal antibodies that bind to human CD38 and are useful in the treatment of diseases including inflammatory diseases such as type 1 and type 2 diabetics, thyroiditis, Graves' disease, arthritides, neuroinflammation and asthmatics.SOLUTION: There are provided antibodies binding to human CD38 in which Asp at a specific position of an amino acid sequence is substituted with Gly.EFFECT: Antibodies that bind to human CD38 are those that do not bind to the human CD38 mutant to the same extent that they bind to wild-type human CD38, and have a strong stimulatory effect on the cADPR hydrolase activity of CD38, leading to reduced levels of cADPR and inhibiting the capability of CD38 to catalyse the formation of nicotinic acid adenine dinucleotide 2 ' - phosphate (NAADP) via a base exchange reaction.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to antibodies against human CD38 and the use of such antibodies, particularly in therapeutic applications. [Background technology]

[0002] Background of the Invention CD38 is a type II transmembrane glycoprotein normally found on hematopoietic cells and in solid tissues. On hematopoietic cells, the majority of medullary thymocytes express CD38 + and resting and circulating T and B cells are CD38 - and activated cells are CD38 + CD38 is also expressed on approximately 80% of resting NK cells and monocytes, as well as on lymph node germinal center lymphoblasts, plasma B cells, and some intrafollicular cells. CD38 can also be expressed by dendritic cells. Certain progenitor cells, a significant proportion of normal bone marrow cells, express CD38. In addition, 50-80% of umbilical cord blood cells express CD38. + and remains so in human blood for the first 2-3 years of life. In addition to lymphoid progenitor cells, CD38 is also expressed on red blood cells and platelets. In solid tissues, CD38 is expressed by intraepithelial cells and lamina propria lymphocytes in the intestine, Purkinje cells and neurofibrillary tangles in the brain, epithelial cells in the prostate, beta cells in the pancreas, osteoclasts in bone, retinal cells in the eye, and sarcolemmal membranes of smooth and striated muscle.

[0003] CD38 is also expressed in a variety of hematological malignancies, including multiple myeloma, B-cell chronic lymphocytic leukemia, B-cell acute lymphocytic leukemia, Waldenström's macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, prolymphocytic / myelocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, follicular lymphoma, NK-cell leukemia, and plasma cell leukemia. CD38 expression has been described on epithelial / endothelial cells of various origins, including the glandular epithelium of the prostate, pancreatic islet cells, ductal epithelium of glands including the parotid gland, bronchial epithelial cells, cells within the testis and ovaries, and tumor epithelium of colorectal adenocarcinoma. Other diseases in which CD38 expression may be involved include, for example, bronchial epithelial carcinoma of the lung, breast cancer (arising from malignant proliferation of the epithelial lining in the ducts and lobules of the breast), pancreatic tumors arising from B cells (insulinoma), tumors arising from the epithelium in the intestine (e.g., adenocarcinoma and squamous cell carcinoma), carcinoma of the prostate, seminoma of the testes and ovarian cancer. In the CNS, neuroblastoma expresses CD38.

[0004] Other disclosures also suggest a role for CD38 in autoimmune diseases such as Graves' disease and thyroiditis (Antonelli A, et al., Clin. Exp. Immunol. 126, 426-431, 2001) and type 1 and type 2 diabetes (Mallone R and Perin PC, Diabetes Metab Res Rev 2006; 22: 284-294), as well as inflammation of airway smooth muscle cells during asthma (Desphande et al. 2004 am J Respir Cell Mol Biol 31: 36-42).

[0005] CD38 is a multifunctional protein. Functions ascribed to CD38 include both receptor-mediated and (ecto)enzymatic activity in adhesion and signaling events. As an ectoenzyme, CD38 mediates the synthesis of cyclic ADP-ribose (cADPR) and ADPR, as well as NAD as a substrate for the formation of nicotinamide and nicotinic acid adenine dinucleotide phosphate (NAADP). +cADPR is used to release Ca from the endoplasmic reticulum. 2+ The CD38 / cyclic ADP-ribose system has been shown to act as a second messenger for mobilizing intracellular Ca in the lungs. 2+ 1) contributes to airway smooth muscle tone and responsiveness through its effect on agonist-induced increases in IL-1 (Desphande et al. 2005 Am J physiol Lung cell Mol Physiol 288: L773-L788 (Non-Patent Document 4)); 2) regulates neutrophil chemotactic migration toward bacterial chemoattractants, migration of DC precursors from the blood to peripheral sites, and migration of mature DCs from inflammatory sites to lymph nodes (Partida-Sanchez et al. Nat Med 7: 1209-121, 2001 (Non-Patent Document 5); Morita et al. 2008 J Pharmacol Sci. 2008 Mar;106(3):492-504 (Non-Patent Document 6); Partida-Sanchez et al. Immunity 20: 279-291, 2004 (Non-Patent Document 7)), 3) is involved in astrocytic calcium signaling, which is closely related to neuroinflammation and HIV-1-associated dementia (Banerjee S. et. al., J. Neurimmune Pharmacol., 3, 154-164 (2008) (Non-Patent Document 8)), 4) regulates FcγR-mediated phagocytosis in murine macrophages (Song E., et. al., Biochem. and Biophys. Res. Comm., 367, 156-161, (2008) (Non-Patent Document 9)), 5) is related to insulin secretion (Okamoto, Molecular and Cellular Biochemistry, 193, 115-118, 1999 (Non-Patent Document 10)), and 6) plays an essential role in regulating neuropeptide release and maternal and social behavior (Jin D et al. Nature 446: 41-45, 2007 (Non-patent Document 11)). Ca 2+In addition to signaling through CD38, CD38 signaling occurs through crosstalk with antigen-receptor complexes or other types of receptor complexes, such as MHC molecules, on T and B cells, and thus participates in several cellular responses and in the switching and secretion of IgG1.

[0006] Several anti-CD38 antibodies have been described in the literature, for example, Lande R, et al., Cell Immunol. 220(1), 30-8 (2002) (Non-Patent Document 12), Ausiello CM, et al., Tissue Antigens. 56(6), 539-47 (2000) (Non-Patent Document 13), and Cotner T, et al., Int J Immunopharmacol. 3(3), 255-68 (1981) (Non-Patent Document 14). Antibodies that bind to CD38 can have various effects on the function of CD38. For example, the murine anti-CD38 antibody IB4 inhibits Ca2+ expression in Jurkat cells. 2+ It has been shown that IL-6 induces T cell activation as indicated by mobilization (Zubiaur M, et al., J Immunol. 159(1), 193-205 (1997) (Non-Patent Document 15)), induces significant proliferation of peripheral blood mononuclear cells (PBMC), induces the release of significant IL-6 levels, and induces the release of detectable IFN-γ levels (Lande, Zubiaur Morra, Ansiello supra). Hara-Yokoyama et al. Int Immunopharmacol 8, 59-70 (2008) (Non-Patent Document 16) reported that CD38 NAD + An anti-mouse CD38 antibody (CS / 2) inhibits glycohydrolase activity and NAD of the isolated extracellular domain of CD38. + Stimulates glycohydrolase activity but not NAD on cell surface CD38 + Another anti-mouse CD38 antibody (clone 90) has been described that has little effect on glycohydrolase activity. As can be seen from the data presented below, the antibodies of the present invention provide activity on the surface of CD38-positive cells.

[0007] WO2006099875 (Genmab) (Patent Document 1) describes several human anti-CD38 antibodies, including 003 and 005. Antibody 005 inhibits NGD by CD38. + It has been shown to inhibit the production of cGDPR from

[0008] Given the multiple functions of human CD38, there is a need for new therapeutic antibodies that more specifically modulate specific functions of CD38. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2006099875 [Non-patent literature]

[0010] [Non-Patent Document 1] Antonelli A, et al., Clin. Exp. Immunol. 126, 426-431, 2001 [Non-patent document 2] Mallone R and Perin PC, Diabetes Metab Res Rev 2006; 22: 284-294 [Non-patent document 3] Desphande et al. 2004 am J Respir Cell Mol Biol 31: 36-42 [Non-patent document 4] Desphande et al. 2005 Am J physiol Lung cell Mol Physiol 288: L773-L788 [Non-Patent Document 5] Partida-Sanchez et al. Nat Med 7: 1209-121, 2001 [Non-patent document 6] Morita et al. 2008 J Pharmacol Sci. 2008 Mar;106(3):492-504

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Summary of the Invention

[0011] The present invention provides a new class of anti-CD38 antibodies that exert a strong stimulatory effect on the cADPR hydrolase activity of CD38 through interaction with specific amino acids in human CD38, resulting in a decrease in cADPR levels. Furthermore, the anti-CD38 antibodies inhibit the ability of CD38 to catalyze the formation of nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) through a base exchange reaction.

[0012] These antibodies are useful in the treatment of several diseases, including autoimmune and (chronic) inflammatory diseases, such as type 1 and type 2 diabetes, thyroiditis, Graves' disease, arthritis, neuroinflammation and asthma.

[0013] Recent scientific studies have shown that cADPR, synthesized extracellularly by CD38, can be transported into cells via nucleoside transporters and then mobilize Ca(2+) via an FK506-binding protein-dependent process. This process may be involved in fMLP-induced intracellular Ca(2+) signaling and migration in human neutrophils (Morita et al. 2008 J Pharmacol Sci. 2008 Mar;106(3):492-504), migration of DC precursors from the blood to peripheral sites, and migration of mature DCs from inflammatory sites to lymph nodes (Partida-Sanchez et al. Immunity 20: 279-291, 2004). Therefore, without being bound by theory, reduced cADPR levels obtained by treatment with the antibodies of the present invention may reduce neutrophil and dendritic cell migration and have an anti-inflammatory effect. Thus, although the antibodies of the present invention may be useful for numerous purposes, they may be particularly useful in treating inflammation associated with, for example, autoimmune diseases, due to their unique effect on the enzymatic activity of CD38 upon binding to specific sites on CD38. [The present invention 1001] An antibody that binds to human CD38 (SEQ ID NO:52), but does not bind to a mutant of human CD38 in which Asp at position 202 is substituted with Gly to the same extent as it binds to human CD38. [The present invention 1002] EC of antibody binding to a mutant of human CD38 in which Asp at position 202 is replaced by Gly 50 EC of the antibody binding to human CD38 50 1001. An antibody of the invention, wherein the antibody has less than 50%, for example less than 10%, less than 5% or less than 1% of the total antibody content. [The present invention 1003] Any of the antibodies of the present invention, which bind to a human CD38 mutant in which Gln at position 272 is substituted with Arg to the same extent as it binds to human CD38. [The present invention 1004] EC of antibody binding to a mutant of human CD38 in which Gln at position 272 is substituted with Arg 50 EC of the antibody binding to human CD38 50 The antibody of the present invention 1003, wherein the antibody is at least 80%, such as at least 90%, such as at least 95%, such as at least 98% of the total antibody content. [The present invention 1005] Any of the antibodies of the present invention, which bind to a human CD38 mutant in which Ser at position 274 is substituted with Phe to the same extent as it binds to human CD38. [The present invention 1006] EC of antibody binding to human CD38 variants 50 EC of the antibody binding to human CD38 50 an antibody of the invention 1005, wherein the antibody is at least 75%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 98% of the total antibody content. [The present invention 1007] Any of the antibodies of the present invention, which have the following binding properties: (i) it does not bind to a mutant of human CD38 in which Asp at position 202 is substituted with Gly to the same extent as it binds to human CD38; (ii) it binds to a mutant of human CD38 in which Gln at position 272 is substituted with Arg to the same extent as it binds to human CD38; and (iii) it binds to a mutant of human CD38 in which Ser at position 274 is substituted with Phe to the same extent as it binds to human CD38. [The present invention 1008] Any of the antibodies of the invention that bind to human CD38 and have an inhibitory effect on CD38 cyclase activity and a stimulatory effect on CD38 hydrolase activity as measured in the assay of Example 8. [The present invention 1009] The antibody of the present invention, wherein the inhibitory effect on CD38 cyclase activity is at least 50 to 66% of the inhibitory effect on CD38 cyclase activity in the absence of the antibody. [The present invention 1010] An antibody that binds to human CD38, encoded by a human heavy chain nucleic acid comprising in its variable region a nucleotide sequence set forth in SEQ ID NO:1, 6, 11, 16 or 21 and a human light chain nucleic acid comprising in its variable region a nucleotide sequence set forth in SEQ ID NO:26, 31, 36, 41 or 46. [The present invention 1011] The antibody of the present invention 1010, encoded by human heavy chain nucleic acid and human light chain nucleic acid comprising the nucleotide sequences set forth in SEQ ID NOs: 1 and 26, 6 and 31, 11 and 36, 16 and 41, or 21 and 46, respectively, in their variable regions. [The present invention 1012] c) a sequence set forth in SEQ ID NO: 5, 10, 15, 20, or 25; or d) variants of said sequences, e.g., variants having at most one, two, or three amino acid modifications, preferably substitutions, e.g., conservative substitutions An antibody that binds to human CD38, comprising a VH CDR3 comprising: [The present invention 1013] An antibody that binds to human CD38, comprising a VH CDR3 having the sequence set forth in SEQ ID NO:5, 10, 15, 20 or 25 and a VL CDR3 having the sequence set forth in SEQ ID NO:30, 35, 40, 45 or 50. [The present invention 1014] An antibody that binds to human CD38, comprising SEQ ID NO:5 and SEQ ID NO:30, or SEQ ID NO:10 and SEQ ID NO:35, or SEQ ID NO:15 and SEQ ID NO:40, or SEQ ID NO:20 and SEQ ID NO:45, or SEQ ID NO:25 and SEQ ID NO:50 as the VH CDR3 and VL CDR3, respectively. [The present invention 1015] (i) a VH CDR1 having a sequence set forth in any of SEQ ID NOs: 3, 8, 13, 18 and 23; a VH CDR2 having a sequence set forth in any of SEQ ID NOs: 4, 9, 14, 19 and 24; a VH CDR3 having a sequence set forth in any of SEQ ID NOs: 5, 10, 15, 20 and 25; a VL CDR1 having a sequence set forth in any of SEQ ID NOs: 28, 33, 38, 43 and 48; a VL CDR2 having a sequence set forth in any of SEQ ID NOs: 29, 34, 39, 44 and 49; a VL CDR3 having a sequence set forth in any of SEQ ID NOs: 30, 35, 40, 45 and 50; (ii) a VH CDR1 having the sequence set forth in SEQ ID NO: 3, a VH CDR2 having the sequence set forth in SEQ ID NO: 34, a VH CDR3 having the sequence set forth in SEQ ID NO: 5, a VL CDR1 having the sequence set forth in SEQ ID NO: 28, a VL CDR2 having the sequence set forth in SEQ ID NO: 29, and a VL CDR3 having the sequence set forth in SEQ ID NO: 30; (iii) a VH CDR1 having the sequence set forth in SEQ ID NO: 8, a VH CDR2 having the sequence set forth in SEQ ID NO: 9, a VH CDR3 having the sequence set forth in SEQ ID NO: 10, a VL CDR1 having the sequence set forth in SEQ ID NO: 33, a VL CDR2 having the sequence set forth in SEQ ID NO: 34, and a VL CDR3 having the sequence set forth in SEQ ID NO: 35; (iv) a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 14, a VH CDR3 having the sequence set forth in SEQ ID NO: 15, a VL CDR1 having the sequence set forth in SEQ ID NO: 38, a VL CDR2 having the sequence set forth in SEQ ID NO: 39, and a VL CDR3 having the sequence set forth in SEQ ID NO: 40; (v) a VH CDR1 having the sequence set forth in SEQ ID NO: 18, a VH CDR2 having the sequence set forth in SEQ ID NO: 19, a VH CDR3 having the sequence set forth in SEQ ID NO: 20, a VL CDR1 having the sequence set forth in SEQ ID NO: 43, a VL CDR2 having the sequence set forth in SEQ ID NO: 44, and a VL CDR3 having the sequence set forth in SEQ ID NO: 45; (vi) a VH CDR1 having the sequence set forth in SEQ ID NO: 23, a VH CDR2 having the sequence set forth in SEQ ID NO: 24, a VH CDR3 having the sequence set forth in SEQ ID NO: 25, a VL CDR1 having the sequence set forth in SEQ ID NO: 48, a VL CDR2 having the sequence set forth in SEQ ID NO: 49, a VL CDR3 having the sequence set forth in SEQ ID NO: 50, or (vii) A variant of any of the preceding antibodies, preferably having no more than one, two, or three amino acid modifications, and more preferably no more than one, two, or three amino acid substitutions, e.g., conservative amino acid substitutions, in one or more of the sequences. An antibody that binds to CD38, comprising: [The present invention 1016] (i) comprising the sequence of SEQ ID NO: 2, 7, 12, 17 or 22; or (ii) has at least 80% identity, e.g., 90%, or 95%, or 97%, or 98%, or 99%, or 100% identity, to the VH region sequence set forth in SEQ ID NO: 2, 7, 12, 17, or 22. An antibody that binds to CD38, comprising a VH region. [The present invention 1017] (i) comprising the sequence of SEQ ID NO: 27, 32, 37, 42 or 47; or (ii) has at least 80% identity, e.g., 90%, or 95%, or 97%, or 98%, or 99%, or 100% identity, to a VL region sequence selected from the group consisting of SEQ ID NOs: 27, 32, 37, 42, or 47. An antibody that binds to CD38, comprising a VL region. [The present invention 1018] An antibody that binds to CD38, comprising a VH region comprising any of the sequences of SEQ ID NOs: 2, 7, 12, 17, and 22, and a VL region comprising any of the sequences of SEQ ID NOs: 27, 32, 37, 42, and 47. [The present invention 1019] (i) a VH region comprising the sequence set forth in SEQ ID NO:2 and a VL region comprising the sequence set forth in SEQ ID NO:27; (ii) a VH region comprising the sequence set forth in SEQ ID NO:7 and a VL region comprising the sequence set forth in SEQ ID NO:32; (iii) a VH region comprising the sequence set forth in SEQ ID NO: 12 and a VL region comprising the sequence set forth in SEQ ID NO: 37; (iv) a VH region comprising the sequence set forth in SEQ ID NO: 17 and a VL region comprising the sequence set forth in SEQ ID NO: 42; or (v) a VH region comprising the sequence set forth in SEQ ID NO: 22 and a VL region comprising the sequence set forth in SEQ ID NO: 47 An antibody that binds to CD38, comprising: [The present invention 1020] An anti-CD38 antibody according to any one of claims 1010 to 1019 of the present invention, which has the binding property of any one of claims 1001 to 1009 of the present invention. [The present invention 1021] An anti-CD38 antibody that binds to the same epitope on CD38 as any of the anti-CD38 antibodies of the present invention. [The present invention 1022] An antibody having substantially the same specific binding properties with respect to binding to human CD38 as any one of antibodies 1001 to 1019 of the present invention. [The present invention 1023] Preferably, the EC50 of 5 nM or less, such as 1 nM or less, for example 0.2 nM or less, as measured by the method described in Example 6 herein. 50 Any of the anti-CD38 antibodies of the invention, which are capable of inducing antibody-dependent cellular cytotoxicity (ADCC) in, for example, Daudi cells, at a concentration of 1000 uM. [The present invention 1024] Any of the anti-CD38 antibodies of the present inventions 1001 to 1022, which are unable to induce ADCC in Daudi cells according to the method described in Example 6 herein. [The present invention 1025] Any of the anti-CD38 antibodies of the present invention which are unable to induce complement-dependent cytotoxicity (CDC) in CHO-CD38 cells. [The present invention 1026] 10 -8 K below M D , preferably 10 -9 K below M D Any of the anti-CD38 antibodies of the present invention, which bind to human CD38 at the [The present invention 1027] Any of the anti-CD38 antibodies of the present invention, which are human monovalent antibodies. [The present invention 1028] Any of the antibodies of the present invention, characterized in that it is a full-length IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibody, for example an IgG1 antibody, preferably an IgG1,κ antibody or an IgM antibody, preferably an IgM,κ antibody. [The present invention 1029] The anti-CD38 antibody of any one of claims 1001 to 1027, which is an antibody fragment or a single-chain antibody. [The present invention 1030] An anti-CD38 antibody according to any one of 1001 to 1022 and 1024 to 1029 of the present invention, which is an effector function-deficient antibody. [The present invention 1031] The anti-CD38 antibody of the present invention 1030, wherein the effector function-deficient anti-CD38 antibody is a stabilized human IgG4 antibody. [The present invention 1032] 1031. The anti-CD38 antibody of the present invention, wherein the stabilized IgG4 antibody is an antibody in which arginine at position 409 in the heavy chain constant region of human IgG4 is substituted with lysine, threonine, methionine or leucine, preferably lysine. [The present invention 1033] The anti-CD38 antibody of the present invention 1032, which comprises a Lys residue at the position corresponding to position 409, or the CH3 region thereof is substituted with the CH3 region of human IgG1, human IgG2, or human IgG3. [The present invention 1034] The anti-CD38 antibody of any of claims 1032 to 1033, which does not contain a Cys-Pro-Pro-Cys sequence in the hinge region. [This invention 1035] The anti-CD38 antibody of any of claims 1032 to 1033, which comprises a Cys-Pro-Pro-Cys sequence in the hinge region. [The present invention 1036] An anti-CD38 antibody according to any one of claims 1001 to 1035 of the present invention, which is a monovalent antibody. [This invention 1037] The monovalent antibody i) providing a nucleic acid construct encoding the light chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the VL region of SEQ ID NO: 27, 32, 37, 42 or 47 and a nucleotide sequence encoding a constant CL region of an Ig, wherein the nucleotide sequence encoding the VL region of a selected antigen-specific antibody and the nucleotide sequence encoding the CL region of an Ig are operably linked, and in the case of an IgG1 subtype, the nucleotide sequence encoding the CL region has been modified so that the CL region does not contain any amino acids that can form disulfide bonds or covalent bonds with other peptides containing the same amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to an animal or human; ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the VH region of SEQ ID NO: 2, 7, 12, 17 or 22 and a nucleotide sequence encoding the constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified so that a region corresponding to a hinge region and, if required by the Ig subtype, other regions of the CH region, e.g., the CH3 region, do not contain any amino acid residues that are involved in the formation of disulfide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising the same amino acid sequence of the CH region of the human Ig in the presence of polyclonal human IgG or when administered to an animal or human, and wherein the nucleotide sequence encoding the VH region of a selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operably linked; iii) providing a cellular expression system for producing said monovalent antibody; iv) producing the monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii). 1036. An anti-CD38 antibody of the present invention constructed by a method comprising: [The present invention 1038] C H 2 and CH C, which includes three areas H The region corresponds to the hinge region, and if the immunoglobulin is not an IgG4 subtype, the C H Other areas of the region, e.g., C H The three regions were identified as C3 in the presence of polyclonal human IgG. H Disulfide bond with the region or identical C H Any of the anti-CD38 antibodies of the present invention 1036 to 1037, which has been modified so as not to contain any amino acid residues capable of forming other covalent or stable non-covalent inter-heavy chain bonds with the region. [This invention 1039] Monovalent antibodies are of the IgG4 subtype, but C H The anti-CD38 antibody of any one of 1036 to 1038, wherein the three regions have been modified to include one or more of the following amino acid substitutions: Thr (T) at position 366 is replaced by Ala (A); Leu (L) at position 368 is replaced by Ala (A); Leu (L) at position 368 is replaced by Val (V); Phe (F) at position 405 is replaced by Ala (A); Phe (F) at position 405 is replaced by Leu (L); Tyr (Y) at position 407 is replaced by Ala (A); Arg (R) at position 409 is replaced by Ala (A). [The present invention 1040] The anti-CD38 antibody of any one of 1036 to 1039, wherein the heavy chain is modified so that the entire hinge is deleted. [The present invention 1041] 1040. The anti-CD38 antibody of any of claims 1036 to 1040, wherein the sequence of the monovalent antibody has been modified so that it does not contain any acceptor sites for N-linked glycosylation. [The present invention 1042] Any of the antibodies of the invention that inhibit CD38-catalyzed synthesis of cGDPR by at least 25%, such as at least 30%, as measured by the spectrophotometric method described in Example 8 herein after 90 minutes at a concentration of 3 μg / ml. [This invention 1043] Any of the antibodies of the present invention that inhibits CD38-catalyzed synthesis of cADPR by at least 25%, for example at least 30%, as measured by the HPLC method described in Munshi et al., J. Biol. Chem. 275, 21566-21571 (2000) after 90 minutes at a concentration of 3 μg / ml. [This invention 1044] Any of the antibodies of the invention which stimulate the hydrolase activity of CD38 by at least 25%. [This invention 1045] Any of the antibodies of the invention which stimulate the NAD hydrolase activity of CD38 by at least 25%. [The present invention 1046] Any of the antibodies of the invention which stimulate the cADPR hydrolase activity of CD38 by at least 25%. [This invention 1047] Any of the antibodies of the invention that inhibit the ability of CD38 to catalyze the formation of NAADP via a base exchange reaction with an IC50 of less than 0.5 μg / mL, for example less than 0.2 μg / mL, by the method described in Example 8 of the present specification. [This invention 1048] An antibody-drug conjugate comprising any of the antibodies of the present invention, wherein the antibody is conjugated to a cytotoxic agent, a radioisotope, or a drug. [This invention 1049] 1048. The antibody drug conjugate of the present invention, wherein the antibody is conjugated to an auristatin or a functional peptide analog or derivative thereof via a linker. [The present invention 1050] A bispecific antibody comprising any one of the antibodies of the present invention 1001 to 1049 and a second binding specificity for a human effector cell or a cancer antigen. [This invention 1051] A bispecific molecule comprising an antibody of the invention 1050, wherein the second binding specificity is for a human Fc receptor or a T cell receptor, such as CD3. [This invention 1052] An isolated nucleic acid encoding any of the antibodies of the present invention. [This invention 1053] An expression vector comprising a nucleotide sequence encoding one or more of the amino acid sequences of any one of 1010 to 1019 of the present invention. [This invention 1054] An expression vector of the present invention 1053 further comprising a nucleotide sequence encoding the constant region of the light chain, the heavy chain, or both the light and heavy chains of a human antibody. [This invention 1055] A recombinant eukaryotic or prokaryotic host cell that produces any one of the antibodies of the present inventions 1001 to 1047. [The present invention 1056] A pharmaceutical composition comprising any one of the antibodies of the present inventions 1001 to 1047, any one of the immunoconjugates of the present inventions 1048 to 1049, any one of the bispecific antibodies of the present inventions 1050 to 1051, or any one of the expression vectors of the present inventions 1053 to 1054, and a pharmaceutically acceptable carrier. [This invention 1057] An antibody according to any one of claims 1001 to 1047 of the present invention for use as a pharmaceutical. [This invention 1058] Any of the antibodies of the present inventions 1001 to 1047 for use in inhibiting the growth and / or proliferation, migration of tumor cells expressing CD38, or inducing phagocytosis of tumor cells expressing CD38. [This invention 1059] An antibody according to any one of claims 1001 to 1047 of the present invention for use in treating rheumatoid arthritis. [The present invention 1060] 10. The antibody of any of claims 1001 to 1047 for use in the treatment of a disorder selected from chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (adult) (AML), mantle cell lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. [The present invention 1061] An antibody according to any one of claims 1001 to 1047 of the present invention for use in treating multiple myeloma. [This invention 1062] a) culturing a host cell of the present invention; and b) Purifying anti-CD38 antibodies from the culture medium A method for producing the anti-CD38 antibody of any one of claims 1001 to 1047, comprising: [This invention 1063] A diagnostic composition comprising any one of the antibodies 1001 to 1047 of the present invention. [This invention 1064] contacting the sample with an anti-CD38 antibody of any of claims 1001 to 1047 under conditions that allow the formation of a complex between the antibody or bispecific molecule and CD38; Analyzing whether a complex is formed 1. A method for detecting the presence of the CD38 antigen, i.e., cells expressing CD38, in a sample, comprising: [This invention 1065] A kit for detecting the presence of CD38 antigen, i.e., cells expressing CD38, in a sample, comprising an anti-CD38 antibody according to any one of claims 1001 to 1047 and instructions for use of the kit. [The present invention 1066] An anti-idiotype antibody that binds to any one of the anti-CD38 antibodies of the present invention Nos. 1001 to 1047. [This invention 1067] Administration of any of the antibodies of the present inventions 1001 to 1047, any of the immunoconjugates of the present inventions 1048 to 1049, any of the bispecific antibodies of the present inventions 1050 to 1051, any of the expression vectors of the present inventions 1053 to 1054, or the pharmaceutical composition of the present invention 1056, so that the growth and / or proliferation, migration or phagocytosis of cells expressing CD38 is inhibited. 10. A method for inhibiting the growth and / or proliferation, migration of cells expressing CD38, or for inducing phagocytosis of cells expressing CD38, comprising: [The present invention 1068] Administering any one of the antibodies of the present inventions 1001 to 1047, any one of the immunoconjugates of the present inventions 1048 to 1049, any one of the bispecific antibodies of the present inventions 1050 to 1051, any one of the expression vectors of the present inventions 1053 to 1054, or the pharmaceutical composition of the present invention 1056 to a subject in need thereof. 10. A method of treating a disease or disorder involving cells expressing CD38 in a subject, comprising: [The present invention 1069] The method of any of claims 1067 to 1068, wherein the disease or disorder is rheumatoid arthritis. [The present invention 1070] 9. The method of any of claims 1067 to 1068, wherein the disease or disorder is selected from chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (adult) (AML), mantle cell lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. [This invention 1071] The method of any of claims 1067 to 1068, wherein the disease or disorder is multiple myeloma. [This invention 1072] 2. The method of any of claims 1067 to 1071, comprising administration to the subject of one or more additional therapeutic agents. [This invention 1073] The method of claim 1072, wherein the one or more additional therapeutic agents are selected from a chemotherapeutic agent, an anti-inflammatory agent, or an immunosuppressant and / or immunomodulatory agent. [This invention 1074] 1073. The method of claim 1073, wherein the one or more additional therapeutic agents are selected from the group consisting of cisplatin, gefitinib, cetuximab, rituximab, ofatumumab, bevacizumab, erlotinib, bortezomib, thalidomide, pamidronate, zoledronic acid, clodronate, risedronate, ibandronate, etidronate, alendronate, tiludronate, arsenic trioxide, lenalidomide, dexamethasone, prednisolone, filgrastim, pegfilgrastim, sargramostim, suberoylanilide hydroxamic acid, and SCIO-469. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows a cross-blocking study of antibodies of the present invention. More specifically, the figure shows the binding of 005-FITC to CHO-CD38 cells treated with an excess of unlabeled CD38-specific antibodies 025, 026, 028, 049 and 056. [Figure 2-01] Figure 2(A) shows the binding of anti-CD38 antibodies of the invention to wild-type (WT) and mutant (T237A, Q272R, and S274F) CD38. [Figure 2-02] This is a figure showing the continuation of Figure 2-01. [Figure 2-03] Figure 2(B) shows the binding of anti-CD38 antibodies of the invention to wild-type (WT) and mutant (D202G) CD38. [Figure 2-04] This is a figure showing the continuation of Figure 2-03. [Figure 3-01] Binding of the antibodies of the invention to Daudi-luc cells and CHO-CD38 cells is shown. [Figure 3-02] This is a figure showing the continuation of Figure 3-01. [Figure 3-03] This is a figure showing the continuation of Figure 3-02. [Figure 3-04] This is a figure showing the continuation of Figure 3-03. [Figure 4-01] 1 shows ADCC-mediated lysis of Daudi-luc cells caused by an anti-CD38 antibody of the invention and an anti-KLH antibody (HuMab-KLH) as an isotype control. [Figure 4-02] This is a figure showing the continuation of Figure 4-01. [Figure 5] CDC-mediated lysis of CHO-CD38 cells caused by anti-CD38 antibodies of the invention. [Figure 6A]Figure 6 shows the inhibition of cGDPR production by His-tagged CD38 protein and cell-expressed CD38 in the presence of anti-CD38 antibodies of the present invention. Figure 6(A) shows the percent inhibition of cGDPR production (by recombinant human CD38 protein) in the presence of CD38-specific antibodies 025, 026, 028, 049, and 056 (3 μg / mL). [Figure 6B] Figure 6(B) shows the inhibition of cGDPR production by His-tagged CD38 protein and cell-expressed CD38 in the presence of an anti-CD38 antibody of the present invention. Figure 6(B) shows the effect of anti-CD38 antibody on cGDPR production over time. Anti-CD38 antibody was used at a final concentration of 10 μg / ml. [Figure 6C] Figure 6(C) shows the inhibition of cGDPR production by His-tagged CD38 protein and cell-expressed CD38 in the presence of anti-CD38 antibodies of the invention. Figure 6(D) shows the effect of anti-CD38 antibodies on cGDPR production using serial dilutions (0.01-30 μg / mL) of 028 or the isotype control HuMab-KLH. [Figure 6D] Figure 6(D) shows the inhibition of cGDPR production by His-tagged CD38 protein and cell-expressed CD38 in the presence of anti-CD38 antibodies of the present invention. Figure 6(D) shows the percent inhibition of cGDPR production (by cell-expressed CD38 (CHO-CD38 cells)) in the presence of serial dilutions (0.01-30 μg / mL) of 028 or the IgG1 isotype control HuMab-KLH. [Figure 7] The effect of antibody 028 of the present invention on 8NH2-cADPR production is shown. The products of each reaction were analyzed by HPLC. Figure 7(A) shows the elution positions of the product and substrate. Figure 7(B) shows the antibody concentration dependence on 8NH2-cADPR production. HuMab-KLH (○), mAb-028 (●). [Figure 8A]The effect of antibody 028 of the present invention on cADPR hydrolase and NADase activity, more specifically, the effect of mAb-028 on cADPR hydrolase (Panel A, upper panel, B and C) and NADase (Panel A, lower panel) activity, is shown. Figure 8(A) shows the results of incubating CD38 recombinant protein with cADPR or NAD in the presence of 10 μg HuMab-KLH (CD38 + 10 μg HuMab-KLH), 10 μg Ab028 (CD38 + 10 μg Ab028), or in the absence of antibody (CD38 control). The products of each reaction were analyzed by HPLC. [Figure 8B] The effect of antibody 028 of the present invention on cADPR hydrolase and NADase activity, more specifically, the effect of mAb-028 on cADPR hydrolase (Figure A, upper panel, B and C) and NADase (Figure A, lower panel) activity, is shown. Figure 8(B) shows the titer of CD38 antibody at various concentrations on cADPR hydrolase activity analyzed by HPLC. HuMab-KLH (○), mAb-028 (●). [Figure 8C] The effect of antibody 028 of the present invention on cADPR hydrolase and NADase activity, more specifically the effect of mAb-028 on cADPR hydrolase (Figure A, upper panel, B and C) and NADase (Figure A, lower panel) activity, is shown. Figure 8(C) shows the results of incubating CD38 recombinant protein with 32P-cADPR in the presence of mAb-003, mAb-028, daratumumab (005), or HuMab-KLH. The products were analyzed by thin-layer chromatography. HuMab-KLH (○), mAb-028 (●). [Figure 9A] 9 shows the effect of anti-CD38 antibodies of the present invention on the base exchange activity of CD38. Figure 9(A) shows the effect of antibodies on NAADP production at the indicated concentrations. [Figure 9B] Figure 9(B) shows the effect of anti-CD38 antibodies of the present invention on the base exchange activity of CD38. Figure 9(B) shows the effect of mAb-028 titer on the rate of NAADP formation. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention definition The term "human CD38", as used herein, includes any variants, isoforms and species homologs of human CD38 (Swiss-Prot: locus CD38_HUMAN, accession number P28907) that are naturally expressed by cells or expressed on cells transfected with the human CD38 gene.

[0016] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains: a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, with all four chains interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain generally contains a heavy chain variable region (referred to herein as V H or VH) and a heavy chain constant region. The heavy chain constant region generally consists of three domains: C H 1. C H 2 and C H Each light chain generally consists of a light chain variable region (referred to herein as V L The light chain constant region generally consists of one domain, C L It is composed of: V H and V L The regions may be further subdivided into regions of hypervariability (or hypervariable regions that may be hypervariable in the sequence and / or configuration of structurally defined loops), also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V Lis generally composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Generally, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). (Herein, phrases such as "as in Kabat" or "variable domain residue numbering according to Kabat" refer to this numbering system for heavy chain variable domains or light chain variable domains.) Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to shortening of, or insertion into, the FRs or CDRs of the variable domain. For example, a heavy chain variable domain may have the following structure: V H It may contain a single amino acid insertion after CDR2 residue 52 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence with a "standard" Kabat numbered sequence at the region of homology.

[0017] The term "antibody" (Ab), in the context of the present invention, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, that has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of a significant period of time, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7, or more days, or any other relevant, functionally defined period of time (e.g., a period of time sufficient to elicit, promote, enhance, and / or modulate a physiological response related to antibody binding to the antigen and / or a period of time sufficient for the antibody to recruit Fc-mediated effector activity). The variable regions of the heavy and light chains of an immunoglobulin molecule contain the binding domains that interact with the antigen. The constant region of an antibody (Ab) can also mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. Anti-CD38 antibodies can also be bispecific antibodies, diabodies, or similar molecules (see, e.g., PNAS USA 90(14), 6444-8 (1993) for a description of diabodies). Indeed, the bispecific antibodies, diabodies, etc. provided by the present invention can bind to any suitable target in addition to portions of CD38. As noted above, the term antibody herein includes fragments of antibodies that retain the ability to specifically bind to an antigen, unless otherwise specified or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antibody" include (i) Fab' or Fab fragments, V L , V H , C L and C H (ii) a monovalent fragment consisting of one domain or a monovalent antibody as described in WO2007059782 (Genmab); (iii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iv) a monovalent fragment consisting essentially of a VH and C H (iv) an Fd fragment consisting essentially of one arm of an antibody; L and V H (v) an Fv fragment consisting essentially of a V domain; H (vi) dAb fragments (Ward et al., Nature 341, 544-546 (1989)), which consist of domains and are also called domain antibodies (Holt et al., Trends Biotechnol. 2003 Nov;21(11):484-90), (vi) camelids or nanobodies (Revets et al., Expert Opin Biol Ther. 2005 Jan;5(1):111-24), and (vii) isolated complementarity-determining regions (CDRs). Furthermore, the two domains V of the Fv fragment L and V H are encoded by separate genes, but using recombinant methods, V L and V HThe regions can also be joined by synthetic linkers that allow them to be assembled as a single protein chain that pairs to form a monovalent molecule (known as single-chain antibodies or single-chain Fvs (scFvs)—see, e.g., Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. While such fragments are generally included within the meaning of antibodies, they collectively and individually exhibit various biological properties and utilities, and are an inherent feature of the present invention. These and other useful antibody fragments in the context of the present invention are described in further detail herein. The term antibody, unless otherwise specified, is also understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant technology, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen. The generated antibody can have any isotype. As used herein, "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by the heavy chain constant region gene. An "anti-CD38 antibody" is an antibody that binds to the antigen CD38.

[0018] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may also contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0019] In a preferred embodiment, the antibody of the present invention is isolated. As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to CD38 is substantially free of antibodies that specifically bind to antigens other than CD38). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of human CD38 may have cross-reactivity with other related antigens, for example, from other species (e.g., homologs of the CD38 species). Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals. In one embodiment of the present invention, a combination of "isolated" monoclonal antibodies with different specificities is combined in a well-defined composition.

[0020] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies may be produced by hybridomas containing B cells obtained from a transgenic or transchromosomal non-human animal, e.g., a transgenic mouse, whose genome contains human heavy chain and light chain transgenes fused to an immortalized cell.

[0021] As used herein, the term "binding" in reference to the binding of an antibody to a given antigen generally refers to a binding of about 10 as determined, for example, by surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less or about 10 -11 K below M D and the K is at least 10 times lower, e.g., at least 100 times lower, e.g., at least 1,000 times lower, e.g., at least 10,000 times lower, e.g., at least 100,000 times lower, e.g., at least 100,000 times lower, than the affinity of binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D The lower affinity portion of the antibody binds to a given antigen with an affinity corresponding to the K D and therefore the K D is very low (i.e., the antibody is very specific), the amount by which the affinity for the antigen is lower than the affinity for a nonspecific antigen may be at least 10,000 times.

[0022] As used herein, the term "k d ”(seconds -1) refers to the dissociation constant of a particular antibody-antigen interaction. This value is also known as k off Also called value.

[0023] As used herein, the term "k a " (M -1 × seconds -1 ) refers to the dissociation constant of a particular antibody-antigen interaction.

[0024] As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0025] As used herein, the term "K A " (M -1 ) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, and k a k d It is obtained by dividing by

[0026] The antibodies of the present invention exert their effects on enzyme systems as described in the Examples section. The antibodies are described by their stimulatory or inhibitory effects on various parameters. Stimulatory and inhibitory effects can also be measured as disclosed in the Examples section of this specification.

[0027] The antibodies described and claimed herein can also be functional variants of any of the specific antibodies described herein. Such variant antibodies are antibodies that differ from the specific antibodies described herein by, for example, one or more appropriate amino acid residue alterations, i.e., substitutions, deletions, insertions, or terminal sequence additions, in the constant and / or variable domains (or any one or more CDRs thereof) of a variant antibody. V as used in connection with anti-CD38 antibodies L , V HAlternatively, functional variants of the CDR regions enable the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity / avidity and / or specificity / selectivity of the parent antibody, and in some cases, such anti-CD38 antibodies may also associate with greater affinity, selectivity and / or specificity than the parent antibody.

[0028] Such functional variants generally retain significant sequence identity with the parent antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., percent homology = number of identical positions / total number of positions x 100). Comparison of sequences and determination of the percent identity between two sequences can also be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0029] The percent identity between two nucleotide sequences can also be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can also be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com) using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0030] The sequences of CDR variants may differ from those of the CDRs of the parent antibody sequence primarily by conservative substitutions. For example, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more (e.g., about 65-99%) of the substitutions in the variant are conservative amino acid residue substitutions. In the context of the present invention, conservative substitutions may be defined by substitutions within classes of amino acids reflected in one or more of the following three tables:

[0031] Conservatively substituted amino acid residue classes TIFF2026021523000001.tif51132

[0032] Alternative conservative amino acid residue substitution classes TIFF2026021523000002.tif39132

[0033] Physical and functional classification of amino acid residue alternatives TIFF2026021523000003.tif82140

[0034] More conservative substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0035] Additional groups of amino acids can also be incorporated using, for example, the principles described in Creighton (1984) Proteins: Structure and Molecular Properties (2d Ed. 1993), WH Freeman and Company.

[0036] As noted above, generally, amino acid sequence changes desirably do not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acid should not tend to disrupt the secondary structure that characterizes the function of the parent sequence), but may be associated with advantageous properties that alter the functional or pharmacokinetic properties of the antibody, such as increasing half-life, altering immunogenicity, providing a site for covalent or non-covalent attachment to another molecule, reducing susceptibility to proteolysis, reducing susceptibility to oxidation, or altering the glycosylation pattern.

[0037] Examples of antibody functional properties that may be altered or retained in the variant anti-CD38 antibodies of the present invention compared to prior art antibodies include, for example: (1) high-affinity binding to CD38 and / or (2) binding to transfected cells expressing CD38, e.g., CHO or HEK293 cells, and / or (3) Induction of CDC and / or (4) induction of ADCC and / or (5) changes in enzyme activity and / or (6) induction of apoptosis after secondary cross-linking and / or (7) Phagocytosis is.

[0038] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural features and specific charge characteristics. Conformational epitopes are distinguished from nonconformational epitopes in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. An epitope can include amino acid residues directly involved in binding (also called the immunodominant component of the epitope) as well as other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked by the specifically antigen-binding peptide (in other words, the amino acid residues are within the footprint of the specifically antigen-binding peptide).

[0039] As used herein, a human antibody is "derived" from a particular germline sequence if the antibody is obtained from a system, for example, by immunizing a transgenic mouse harboring human immunoglobulin genes or by screening a human immunoglobulin gene library, using human immunoglobulin sequences, and the selected human antibody is at least 90%, such as at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or for example at least 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Generally, outside of the heavy chain CDR3, a human antibody derived from a particular human germline sequence will exhibit no more than 20 amino acid differences, for example no more than 10 amino acid differences, for example no more than 5 amino acid differences, for example no more than 4, 3, 2, or 1 amino acid difference, from the amino acid sequence encoded by the germline immunoglobulin gene.

[0040] As used herein, the term "inhibit proliferation" (e.g., with respect to cells such as tumor cells) is intended to include a substantial reduction in proliferation of the same cells when contacted with an anti-CD38 antibody, e.g., an inhibition of proliferation of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%, compared to the proliferation of the same cells not contacted with the anti-CD38 antibody. Such a reduction in cell proliferation can occur by a variety of mechanisms, e.g., effector cell phagocytosis, ADCC, CDC, and / or apoptosis.

[0041] The term "bispecific antibody" is intended to include any antibody having two different binding specificities. The term "bispecific antibody" also includes diabodies (see, e.g., Holliger, P. et al., PNAS USA 90, 6444-6448 (1993); Poljak, RJ. et al., Structure 2, 1121-1123 (1994)).

[0042] An "antibody lacking effector function" or "effector function-deficient antibody" refers to an antibody that has a significantly reduced or no ability to activate one or more immune effector mechanisms, such as complement activation or Fc receptor binding. Thus, an effector function-deficient antibody has a significantly reduced or no ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0043] In the context of the present invention, the term "monovalent antibody" means that the antibody molecule can only bind to one antigen molecule and is therefore incapable of cross-linking antigens.

[0044] As used herein, the term "effector cell" refers to an immune cell involved in the effector phase of an immune response, rather than the recognition and activation phases. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear leukocytes, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, can induce antibody-dependent cellular cytotoxicity (ADCC). For example, FcR-expressing monocytes and macrophages are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system or binding to cells that present antigens. In some embodiments, effector cells can also phagocytose target antigens or target cells.

[0045] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Various types of vectors are well known in the art. One type of vector is a plasmid.

[0046] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which an expression vector has been introduced. Such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain changes may occur in subsequent generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, e.g., CHO cells, HEK293 cells, NS / 0 cells, and lymphocytic cells.

[0047] The term "transgenic non-human animal" refers to a non-human animal having a genome containing one or more human heavy and / or light chain transgenes or transchromosomes (either integrated or not integrated into the animal's native genomic DNA) and capable of fully expressing human antibodies. For example, a transgenic mouse carrying a human light chain transgene and a human heavy chain transgene or human heavy chain transchromosome can produce human anti-CD38 antibodies when immunized with CD38 antigen and / or CD38-expressing cells. The human heavy chain transgene may be integrated into the chromosomal DNA of the mouse, as in the case of a transgenic mouse, e.g., a HuMAb mouse, e.g., HCo7 or HCol2 mouse, or may be maintained extrachromosomally, as in the case of a transchromosomal KM mouse, as described in WO 02 / 43478. Such transgenic and transchromosomic mice (collectively referred to herein as "transgenic mice") are capable of producing multiple isotypes of human monoclonal antibodies (e.g., IgG, IgA, IgM, IgD, and / or IgE) against a given antigen by undergoing VDJ recombination and isotype switching. Transgenic non-human animals can also be used to produce antibodies against a particular antigen by introducing genes encoding such specific antibodies, e.g., by operatively linking these genes to genes that are expressed in the animal's milk.

[0048] In the context of the present invention, the term "B cell neoplasm" or "mature B cell neoplasm" includes small lymphocytic lymphoma, B cell prolymphocytic lymphoma, B cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse large B cell lymphoma, multiple myeloma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell neoplasms such as plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, MALT lymphoma, nodal marginal zone B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma and AIDS-related non-Hodgkin's lymphoma.

[0049] "Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention with the purpose of palliating, ameliorating, suppressing, or eradicating (cure) the symptoms or disease state.

[0050] An "effective amount" refers to an amount effective to achieve a desired therapeutic result, at dosages and for periods of time necessary. A therapeutically effective amount of an anti-CD38 antibody may vary according to factors such as the individual's disease state, age, sex, and weight, and the ability of the anti-CD38 antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0051] An "anti-idiotypic" (Id) antibody is an antibody that recognizes unique determinants generally associated with the antigen-binding site of an antibody.

[0052] Antibodies of the Invention The present invention relates to an antibody that binds to human CD38 (SEQ ID NO:52), but does not bind to a variant of human CD38 in which Asp at position 202 is substituted with Gly to the same extent as it binds to human CD38. In one embodiment, the EC 50 is the EC of antibody binding to human CD38 50 is less than 50%, for example less than 10%, less than 5% or less than 1% of the total.

[0053] In one embodiment, the antibody binds to a variant of human CD38 in which Gln at position 272 is substituted with Arg to the same extent as it binds to human CD38. In one embodiment, the EC 50 is the EC of antibody binding to human CD38 50 at least 80%, such as at least 90%, such as at least 95%, such as at least 98% of the total.

[0054] In one embodiment, the antibody of any of the above embodiments binds to a variant of human CD38 in which Ser at position 274 is substituted with Phe to the same extent as it binds to human CD38. In one embodiment, the EC 50 is the EC of antibody binding to human CD38 50 at least 75%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 98% of the total.

[0055] In one embodiment, the antibody has the following binding properties: (i) it does not bind to a variant of human CD38 in which Asp at position 202 is substituted with Gly to the same extent as it binds to human CD38, (ii) it binds to a variant of human CD38 in which Gln at position 272 is substituted with Arg to the same extent as it binds to human CD38, and (iii) it binds to a variant of human CD38 in which Ser at position 274 is substituted with Phe to the same extent as it binds to human CD38.

[0056] In one embodiment, the antibody described in any of the above embodiments binds to human CD38 and has an inhibitory effect on CD38 cyclase activity and a stimulatory effect on CD38 hydrolase activity, as measured in the assay of Example 8, that is at least 50-66% of the inhibitory effect on CD38 cyclase activity in the absence of the antibody.

[0057] In one embodiment, the antibody of any of the above embodiments is encoded by a human heavy chain nucleic acid comprising in its variable region a nucleotide sequence set forth in SEQ ID NO: 1, 6, 11, 16 or 21, and a human light chain nucleic acid comprising in its variable region a nucleotide sequence set forth in SEQ ID NO: 26, 31, 36, 41 or 46.

[0058] In one embodiment, the antibody according to any of the above embodiments is encoded by human heavy chain nucleic acid and human light chain nucleic acid comprising the nucleotide sequences set forth in SEQ ID NOs: 1 and 26, 6 and 31, 11 and 36, 16 and 41, or 21 and 46 in their variable regions, respectively.

[0059] In one embodiment, the antibody according to any of the above embodiments comprises: a) a sequence as set forth in SEQ ID NO: 5, 10, 15, 20 or 25, or b) variants of said sequences, e.g., having at most one, two or three amino acid modifications, preferably substitutions, e.g., conservative substitutions and a VH CDR3 comprising:

[0060] In one embodiment, the antibody according to any of the above embodiments comprises a VH CDR3 having the sequence set forth in SEQ ID NO:5, 10, 15, 20 or 25 and a VL CDR3 having the sequence set forth in SEQ ID NO:30, 35, 40, 45 or 50.

[0061] In one embodiment, the antibody according to any of the above embodiments comprises SEQ ID NO:5 and SEQ ID NO:30, or SEQ ID NO:10 and SEQ ID NO:35, or SEQ ID NO:15 and SEQ ID NO:40, or SEQ ID NO:20 and SEQ ID NO:45, or SEQ ID NO:25 and SEQ ID NO:50 as the VH CDR3 and VL CDR3, respectively.

[0062] In one embodiment, the antibody described in any of the above embodiments comprises: (i) a VH CDR1 having a sequence set forth in any of SEQ ID NOs: 3, 8, 13, 18 and 23; a VH CDR2 having a sequence set forth in any of SEQ ID NOs: 4, 9, 14, 19 and 24; a VH CDR3 having a sequence set forth in any of SEQ ID NOs: 5, 10, 15, 20 and 25; a VL CDR1 having a sequence set forth in any of SEQ ID NOs: 28, 33, 38, 43 and 48; a VL CDR2 having a sequence set forth in any of SEQ ID NOs: 29, 34, 39, 44 and 49; a VL CDR3 having a sequence set forth in any of SEQ ID NOs: 30, 35, 40, 45 and 50; (ii) a VH CDR1 having the sequence set forth in SEQ ID NO: 3, a VH CDR2 having the sequence set forth in SEQ ID NO: 4, a VH CDR3 having the sequence set forth in SEQ ID NO: 5, a VL CDR1 having the sequence set forth in SEQ ID NO: 28, a VL CDR2 having the sequence set forth in SEQ ID NO: 29, and a VL CDR3 having the sequence set forth in SEQ ID NO: 30; (iii) a VH CDR1 having the sequence set forth in SEQ ID NO: 8, a VH CDR2 having the sequence set forth in SEQ ID NO: 9, a VH CDR3 having the sequence set forth in SEQ ID NO: 10, a VL CDR1 having the sequence set forth in SEQ ID NO: 33, a VL CDR2 having the sequence set forth in SEQ ID NO: 34, and a VL CDR3 having the sequence set forth in SEQ ID NO: 35; (iv) a VH CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 14, a VH CDR3 having the sequence set forth in SEQ ID NO: 15, a VL CDR1 having the sequence set forth in SEQ ID NO: 38, a VL CDR2 having the sequence set forth in SEQ ID NO: 39, and a VL CDR3 having the sequence set forth in SEQ ID NO: 40; (v) a VH CDR1 having the sequence set forth in SEQ ID NO: 18, a VH CDR2 having the sequence set forth in SEQ ID NO: 19, a VH CDR3 having the sequence set forth in SEQ ID NO: 20, a VL CDR1 having the sequence set forth in SEQ ID NO: 43, a VL CDR2 having the sequence set forth in SEQ ID NO: 44, and a VL CDR3 having the sequence set forth in SEQ ID NO: 45; (vi) a VH CDR1 having the sequence set forth in SEQ ID NO: 23, a VH CDR2 having the sequence set forth in SEQ ID NO: 24, a VH CDR3 having the sequence set forth in SEQ ID NO: 25, a VL CDR1 having the sequence set forth in SEQ ID NO: 48, a VL CDR2 having the sequence set forth in SEQ ID NO: 49, a VL CDR3 having the sequence set forth in SEQ ID NO: 50, or (vii) A variant of any of the preceding antibodies, preferably having at most one, two, or three amino acid modifications, and more preferably amino acid substitutions, e.g., conservative amino acid substitutions, in one or more of said sequences.

[0063] In one embodiment, the antibody according to any of the above embodiments comprises: (i) comprising the sequence of SEQ ID NO: 2, 7, 12, 17 or 22; or (ii) has at least 80%, e.g., 90%, or 95%, or 97%, or 98%, or 99%, or 100% identity to the VH region sequence set forth in SEQ ID NO: 2, 7, 12, 17, or 22 Contains the VH region.

[0064] In one embodiment, the antibody according to any of the above embodiments comprises: (i) comprising the sequence of SEQ ID NO: 27, 32, 37, 42 or 47; or (ii) has at least 80%, e.g., 90%, or 95%, or 97%, or 98%, or 99%, or 100% identity to a VL region sequence selected from the group consisting of SEQ ID NOs: 27, 32, 37, 42, or 47. Contains the VL region.

[0065] In one embodiment, the antibody according to any of the above embodiments comprises a VH region comprising any of the sequences of SEQ ID NOs: 2, 7, 12, 17 and 22 and a VL region comprising any of the sequences of SEQ ID NOs: 27, 32, 37, 42 and 47.

[0066] In one embodiment, the antibody described in any of the above embodiments comprises: (i) a VH region comprising the sequence set forth in SEQ ID NO:2 and a VL region comprising the sequence set forth in SEQ ID NO:27; (ii) a VH region comprising the sequence set forth in SEQ ID NO:7 and a VL region comprising the sequence set forth in SEQ ID NO:32; (iii) a VH region comprising the sequence set forth in SEQ ID NO: 12 and a VL region comprising the sequence set forth in SEQ ID NO: 37; (iv) a VH region comprising the sequence set forth in SEQ ID NO: 17 and a VL region comprising the sequence set forth in SEQ ID NO: 42; or (v) a VH region comprising the sequence set forth in SEQ ID NO:22 and a VL region comprising the sequence set forth in SEQ ID NO:47.

[0067] In one embodiment, the invention relates to an anti-CD38 antibody that binds to the same epitope on CD38 as an anti-CD38 antibody described in any one of the above embodiments.

[0068] In one embodiment, the invention relates to an anti-CD38 antibody having substantially the same specific binding characteristics to human CD38 as an antibody described in any one of the above embodiments.

[0069] In one embodiment, the antibody according to any of the above embodiments preferably has an EC of 5 nM or less, such as 1 nM or less, for example 0.2 nM or less, when measured by the method described in Example 6 herein. 50At these levels, it is possible to induce antibody-dependent cellular cytotoxicity (ADCC) in, for example, Daudi cells.

[0070] In one embodiment, the antibody described in any of the above embodiments is unable to induce ADCC in Daudi cells according to the method described in Example 6 herein.

[0071] In one embodiment, the antibody according to any of the above embodiments is unable to induce complement dependent cytotoxicity (CDC) in CHO-CD38 cells.

[0072] In one embodiment, the antibody according to any of the above embodiments is -8 K below M D , preferably 10 -9 K below M D It binds to human CD38.

[0073] In one embodiment, the antibody according to any of the above embodiments is a human monovalent antibody.

[0074] In one embodiment, the antibody according to any of the above embodiments is a full-length IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibody, such as an IgG1 antibody, preferably an IgG1,κ antibody or an IgM antibody, preferably an IgM,κ antibody.

[0075] In one embodiment, the antibody according to any of the above embodiments is an antibody fragment or a single chain antibody.

[0076] In one embodiment, the antibody according to any of the above embodiments is an effector function-deficient antibody, such as a stabilized human IgG4 antibody.

[0077] In one embodiment, such a stabilized IgG4 antibody is an antibody in which arginine at position 409 in the heavy chain constant region of human IgG4 is substituted with lysine, threonine, methionine, or leucine, preferably lysine. In one embodiment, such an antibody contains a Lys residue at the position corresponding to position 409, or the CH3 region of the antibody is substituted with the CH3 region of human IgG1, human IgG2, or human IgG3. In one embodiment, such an antibody does not contain a Cys-Pro-Pro-Cys sequence in the hinge region. In another embodiment, such an antibody contains a Cys-Pro-Pro-Cys sequence in the hinge region.

[0078] In one embodiment, the antibody according to any of the above embodiments is a monovalent antibody.

[0079] In one embodiment, such a monovalent antibody comprises: i) providing a nucleic acid construct encoding the light chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VL region of SEQ ID NO: 27, 32, 37, 42 or 47 and a nucleotide sequence encoding a constant CL region of an Ig, wherein said nucleotide sequence encoding the VL region of a selected antigen-specific antibody and said nucleotide sequence encoding the CL region of an Ig are operably linked, and in the case of an IgG1 subtype, the nucleotide sequence encoding the CL region has been modified so that the CL region does not contain any amino acids that can form disulfide bonds or covalent bonds with other peptides comprising the same amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to an animal or human; ii) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VH region of SEQ ID NO: 2, 7, 12, 17 or 22 and a nucleotide sequence encoding the constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that a region corresponding to the hinge region and, if required by the Ig subtype, another region of the CH region, e.g., the CH3 region, does not contain any amino acid residues that are involved in the formation of disulfide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising the same amino acid sequence of the CH region of a human Ig in the presence of polyclonal human IgG or when administered to an animal or a human, and wherein said nucleotide sequence encoding the VH region of a selected antigen-specific antibody and said nucleotide sequence encoding the CH region of said Ig are operably linked; iii) providing a cellular expression system for producing said monovalent antibody; iv) producing the monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii). The method includes:

[0080] In one embodiment, C H 2 and C H C, which includes three areas H The regions are those corresponding to the hinge region and, if the immunoglobulin is not an IgG4 subtype, C H Other areas of the region, e.g., C H The three regions were identified as C3 in the presence of polyclonal human IgG. H Disulfide bond with the region or identical C H The heavy chains are modified so as not to contain any amino acid residues that can form other covalent or stable non-covalent inter-heavy chain bonds with the region.

[0081] In one embodiment, such a monovalent antibody is of the IgG4 subtype, but HThe three regions have been modified to include one or more of the following amino acid substitutions: Thr (T) at position 366 is replaced by Ala (A), Leu (L) at position 368 is replaced by Ala (A), Leu (L) at position 368 is replaced by Val (V), Phe (F) at position 405 is replaced by Ala (A), Phe (F) at position 405 is replaced by Leu (L), Tyr (Y) at position 407 is replaced by Ala (A), and Arg (R) at position 409 is replaced by Ala (A).

[0082] In one embodiment, the heavy chain of such a monovalent antibody is modified so that the entire hinge is deleted.

[0083] In one embodiment, the sequence of the monovalent antibody is modified so that it does not contain any acceptor sites for N-linked glycosylation.

[0084] In one embodiment, the antibody according to any of the above embodiments inhibits CD38-catalyzed synthesis of cGDPR by at least 25%, such as at least 30%, after 90 minutes at a concentration of 3 μg / ml, as measured by the spectrophotometric method described in Example 8 herein.

[0085] In one embodiment, the antibody described in any of the above embodiments inhibits CD38-catalyzed synthesis of cADPR by at least 25%, e.g., at least 30%, as measured by the HPLC method described in Munshi et al., J. Biol. Chem. 275, 21566-21571 (2000) after 90 minutes at a concentration of 3 μg / ml.

[0086] In one embodiment, the antibody stimulates the hydrolase activity of CD38 by at least 25%.

[0087] In one embodiment, the antibody stimulates the NAD hydrolase activity of CD38 by at least 25%.

[0088] In one embodiment, the antibody according to any of the above embodiments stimulates the cADPR hydrolase activity of CD38 by at least 25%.

[0089] In one embodiment, the antibody described in any of the above embodiments inhibits the ability of CD38 to catalyze the base exchange reaction to form NAADP with an IC50 of less than 0.5 μg / mL, e.g., less than 0.2 μg / mL, by the method described in Example 8 herein.

[0090] In one embodiment, the invention relates to an antibody-drug conjugate comprising an antibody according to any of the above embodiments conjugated to a cytotoxic agent, a radioisotope, or a drug, hi one embodiment, the antibody is conjugated to an auristatin or a functional peptide analog or derivative thereof via a linker.

[0091] In one embodiment, the invention relates to a bispecific antibody comprising an antibody according to any of the above embodiments and a second binding specificity for a human effector cell or a cancer antigen. In one embodiment, the second binding specificity is for a human Fc receptor or a T cell receptor, such as CD3.

[0092] In one embodiment, the invention relates to an isolated nucleic acid encoding an antibody according to any of the above embodiments.

[0093] In one aspect, the present invention relates to an expression vector comprising a nucleotide sequence encoding one or more of the amino acid sequences described in any of the above aspects.

[0094] In one embodiment, the expression vector further comprises a nucleotide sequence encoding the constant region of the light chain, the heavy chain, or both the light and heavy chains of a human antibody.

[0095] In one embodiment, the invention relates to a recombinant eukaryotic or prokaryotic host cell that produces an antibody according to any of the above embodiments.

[0096] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody, immunoconjugate, bispecific antibody, or expression vector according to any of the above aspects and a pharmaceutically acceptable carrier.

[0097] In one embodiment, the invention relates to an antibody according to any of the above embodiments for use as a medicament.

[0098] In one embodiment, the invention relates to an antibody as described in any of the above embodiments for use in inhibiting the growth and / or proliferation, migration of, or inducing phagocytosis of, tumor cells expressing CD38.

[0099] In one embodiment, the invention relates to an antibody according to any of the above embodiments for use in the treatment of rheumatoid arthritis.

[0100] In one embodiment, the invention relates to an antibody as described in any of the above embodiments for use in the treatment of a disorder selected from chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (adult) (AML), mantle cell lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma.

[0101] In one embodiment, the invention relates to an antibody according to any of the above embodiments for use in the treatment of multiple myeloma.

[0102] In one embodiment, the present invention relates to a method of producing an anti-CD38 antibody as described in any of the above embodiments, comprising the steps of: a) culturing a host cell according to any of the above embodiments, and b) purifying the anti-CD38 antibody from the culture medium.

[0103] In one embodiment, the invention relates to a diagnostic composition comprising an antibody according to any of the above embodiments.

[0104] In one embodiment, the present invention relates to a method for detecting the presence of CD38 antigen, i.e., cells expressing CD38, in a sample, comprising the steps of: contacting the sample with an anti-CD38 antibody according to any of the above embodiments under conditions that allow for the formation of a complex between the antibody or bispecific molecule and CD38; and Analyzing whether a complex was formed.

[0105] In one embodiment, the present invention relates to a kit for detecting the presence of the CD38 antigen, i.e., cells expressing CD38, in a sample, the kit comprising an anti-CD38 antibody as described in any of the above embodiments and instructions for use of the kit.

[0106] In one embodiment, the invention relates to an anti-idiotypic antibody that binds to an anti-CD38 antibody described in any of the above embodiments.

[0107] In one embodiment, the invention relates to a method of inhibiting the growth and / or proliferation, migration of cells expressing CD38, or inducing phagocytosis of cells expressing CD38, the method comprising administering an antibody, immunoconjugate, bispecific antibody, expression vector, or pharmaceutical composition as described in any of the above embodiments, such that cell growth and / or proliferation, migration, or phagocytosis is inhibited.

[0108] In one embodiment, the invention relates to a method of treating a disease or disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need thereof an antibody, immunoconjugate, bispecific antibody, expression vector or pharmaceutical composition described in any of the above embodiments.

[0109] In one embodiment, the disease or disorder is rheumatoid arthritis.

[0110] In another embodiment, the disease or disorder is selected from chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (adult) (AML), mantle cell lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma.

[0111] In yet another embodiment, the disease or disorder is multiple myeloma.

[0112] In one embodiment, the method of any of the above embodiments includes administering to the subject one or more additional therapeutic agents, e.g., one or more additional therapeutic agents selected from a chemotherapeutic agent, an anti-inflammatory agent, or an immunosuppressant and / or an immunomodulatory agent. In one embodiment, the one or more additional therapeutic agents are selected from the group consisting of cisplatin, gefitinib, cetuximab, rituximab, ofatumumab, bevacizumab, erlotinib, bortezomib, thalidomide, pamidronate, zoledronic acid, clodronate, risedronate, ibandronate, etidronate, alendronate, tiludronate, arsenic trioxide, lenalidomide, dexamethasone, prednisolone, filgrastim, pegfilgrastim, sargramostim, suberoylanilide hydroxamic acid, and SCIO-469.

[0113] An embodiment of the present invention provides antibodies that bind to human CD38 but do not bind to a mutant of CD38 in which Asp at position 202 is substituted with Gly.

[0114] An embodiment of the present invention is a method for determining the EC of binding of an antibody to a variant of human CD38. 50 EC of peptide binding to human CD38 50 The antibody of the above aspect is provided, wherein the nucleotide sequence is less than 50%, for example less than 10%, less than 5%, or less than 1% of the nucleotide sequence.

[0115] An embodiment of the present invention provides an antibody of any of the above embodiments, which binds to a mutant of human CD38 in which Gln at position 272 is substituted with Arg to the same extent as it binds to human CD38.

[0116] An embodiment of the present invention is a method for determining the EC of binding of an antibody to a variant of human CD38. 50 EC of peptide binding to human CD38 50 In some embodiments, the antibody of any of the above aspects is at least 80%, such as at least 90%, such as at least 95%, such as at least 98% of the total antibody fragment length.

[0117] An embodiment of the present invention provides an antibody according to any of the above embodiments, which binds to a variant of human CD38 in which Ser at position 274 is substituted with Phe to the same extent as it binds to human CD38.

[0118] An embodiment of the present invention is a method for determining the EC of binding of an antibody to a variant of human CD38. 50 EC of peptide binding to human CD38 50 The antibody of the above aspect is provided wherein the antibody has at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% of the total antibody content.

[0119] Embodiments of the present invention provide an antibody of any of the above embodiments, having the following binding properties: (i) it does not bind to a variant of human CD38 in which Asp at position 202 is substituted with Gly to the same extent as it binds to human CD38, (ii) it binds to a variant of human CD38 in which Gln at position 272 is substituted with Arg to the same extent as it binds to human CD38, and (iii) it binds to a variant of human CD38 in which Ser at position 274 is substituted with Phe to the same extent as it binds to human CD38.

[0120] An embodiment of the invention provides an antibody of any of the above embodiments, which binds to human CD38 and has an inhibitory effect on CD38 cyclase activity and a stimulatory effect on CD38 hydrolase activity as measured in the assay of Example 8.

[0121] An embodiment of the present invention provides an antibody of the above embodiment, which has an inhibitory effect of at least 50 to 66% compared to CD38 alone. An embodiment of the present invention provides an antibody that binds to human CD38, encoded by a human heavy chain nucleic acid comprising in its variable region the nucleotide sequence set forth in SEQ ID NO: 1, 6, 11, 16, or 21, and a human light chain comprising in its variable region the nucleotide sequence set forth in SEQ ID NO: 26, 31, 36, 41, or 46, and which contains conservative sequence modifications of the above sequences.

[0122] Embodiments of the present invention provide antibodies of the above embodiments, encoded by human heavy chain nucleic acid and human light chain nucleic acid comprising in their variable regions, respectively, the nucleotide sequences set forth in SEQ ID NOs: 1 and 26, 6 and 31, 11 and 36, 16 and 41, or 21 and 46, including conservative sequence modifications of the above sequences.

[0123] An aspect of the present invention is a) a sequence as set forth in SEQ ID NO: 5, 10, 15, 20 or 25, 30, or b) variants of said sequences, e.g., having at most one, two or three amino acid modifications, preferably substitutions, e.g., conservative substitutions The present invention provides an antibody that binds to human CD38, comprising a VH CDR3 region having the following structure:

[0124] An embodiment of the present invention provides an antibody that binds to human CD38, comprising a VH CDR3 region having the sequence set forth in SEQ ID NO:5, 10, 15, 20, 25 or 30 and a VL CDR3 region having the sequence set forth in SEQ ID NO:30, 35, 40, 45 or 50.

[0125] An embodiment of the present invention provides an antibody that binds to human CD38, comprising a VH CDR3 region having the sequence set forth in SEQ ID NO:5 and a VL CDR3 region comprising SEQ ID NO:30 or SEQ ID NO:10 and SEQ ID NO:35 or SEQ ID NO:15 and SEQ ID NO:40 or SEQ ID NO:20 and SEQ ID NO:45 or SEQ ID NO:25 and SEQ ID NO:45 or SEQ ID NO:30 and SEQ ID NO:50 as the VH CDR3 region and VL CDR3 region, respectively.

[0126] An embodiment of the invention provides an anti-CD38 antibody of any of the above embodiments, comprising a VH CDR1 region having the sequence set forth in any of SEQ ID NOs:3, 8, 13, 18 or 23; a VH CDR2 region having the sequence set forth in any of SEQ ID NOs:4, 9, 14, 19 or 24; a VL CDR3 region having the sequence set forth in any of SEQ ID NOs:30, 35, 40, 45 or 50; and a VH CDR3 region having the sequence set forth in SEQ ID NOs:5, 10, 15, 20 or 25.

[0127] Aspects of the present invention provide the following: a VH CDR1 region having the sequence set forth in any of SEQ ID NOs: 3, 8, 13, 18 or 23; a VH CDR2 region having the sequence set forth in any of SEQ ID NOs: 4, 9, 14, 19 or 24; a VH CDR3 region having the sequence set forth in SEQ ID NOs: 5, 10, 15, 20 or 25; a VL CDR1 region set forth in SEQ ID NOs: 28, 33, 38, 43 or 48; a VL CDR2 region set forth in SEQ ID NOs: 29, 34, 39, 44 or 49; and a VL CDR3 region having the sequence set forth in any of SEQ ID NOs: 30, 35, 40, 45 or 50. an antibody that binds to CD38, comprising Preferably, variants of said antibodies have at most one, two or three amino acid modifications, and more preferably amino acid substitutions, eg, conservative amino acid substitutions, in said sequence.

[0128] An embodiment of the invention provides an antibody that binds to CD38, comprising a VH CDR1 region having the sequence set forth in any of SEQ ID NOs:3, 8, 13, 18 or 23; a VH CDR2 region having the sequence set forth in any of SEQ ID NOs:4, 9, 14, 19 or 24; a VH CDR3 region having the sequence set forth in SEQ ID NOs:5, 10, 15, 20 or 25; a VL CDR1 region set forth in SEQ ID NOs:28, 33, 38, 43 or 48; a VL CDR2 region set forth in SEQ ID NOs:29, 34, 39, 44 or 49; and a VL CDR3 region having the sequence set forth in any of SEQ ID NOs:30, 35, 40, 45 or 50.

[0129] An embodiment of the invention provides an anti-CD38 antibody of any of the above embodiments, comprising a VH having at least 80% identity, e.g., 90%, or 95%, or 97%, or 98%, or 99%, or 100% identity, to a VH region sequence set forth in SEQ ID NO:2, 7, 12, 17, or 22.

[0130] An embodiment of the invention provides an anti-CD38 antibody of any of the above embodiments, comprising a VL having at least 80% identity, e.g., 90%, or 95%, or 97%, or 98%, or 99%, or 100% identity, to a VL region sequence selected from the group consisting of SEQ ID NO: 27, 32, 37, 42, or 47.

[0131] An embodiment of the invention provides an anti-CD38 antibody of any of the above embodiments, comprising a VH region comprising the sequence of SEQ ID NO: 2, 7, 12, 17 or 22 and a VL region comprising the sequence of SEQ ID NO: 27, 32, 37, 42 or 47.

[0132] An embodiment of the present invention provides an antibody that competes with an antibody of any of the above embodiments for binding to CD38.

[0133] An embodiment of the invention provides an anti-CD38 antibody that competes for CD38 binding with an anti-CD38 antibody comprising a VH region comprising any of the sequences of SEQ ID NOs: 2, 7, 12, 17, or 22 and a VL region comprising any of the sequences of SEQ ID NOs: 27, 32, 37, 42, or 47.

[0134] An embodiment of the present invention provides an anti-CD38 antibody of any of the above embodiments that binds to the same epitope on CD38 as an anti-CD38 antibody described in any of the above embodiments.

[0135] An embodiment of the present invention provides an antibody having substantially the same specific binding characteristics with respect to binding to human CD38 as the antibody of any of the above embodiments.

[0136] An embodiment of the invention provides an anti-CD38 antibody of any of the above embodiments, which is capable of inducing complement dependent cytotoxicity (CDC) in CHO-CD38 cells.

[0137] An embodiment of the invention provides an anti-CD38 antibody of any of the above embodiments, which is capable of eliciting antibody-dependent cellular cytotoxicity (ADCC).

[0138] Aspects of the invention preferably have an EC50 of 5 nM or less, such as 1 nM or less, for example 0.2 nM or less, as measured by the method described in Example 6 herein. 50 The anti-CD38 antibody of claim 25 induces ADCC in Daudi cells at a value of

[0139] An embodiment of the present invention provides an anti-CD38 antibody of any of the above embodiments that is unable to induce ADCC.

[0140] An embodiment of the invention provides an anti-CD38 antibody of any of the above embodiments that is unable to induce complement dependent cytotoxicity (CDC).

[0141] The present invention is directed to a method for manufacturing a semiconductor device. -8 K below M D , preferably 10 -9 K below M D The anti-CD38 antibody of any of the above aspects is provided, wherein the anti-CD38 antibody binds to human CD38 at

[0142] An aspect of the present invention is a human germline V selected from the group consisting of IGHV1-69*04 and / or IGHJ3*02 H Heavy chain variable region derived from sequence a human germline V selected from the group consisting of IGKV1D-16*01 and / or IGKJ4*01 K Light chain variable region derived from sequence The anti-CD38 antibody of any of the above aspects is provided, comprising:

[0143] An embodiment of the invention provides the anti-CD38 antibody of any of the above embodiments, which is a human antibody.

[0144] An embodiment of the present invention provides the antibody of any of the above embodiments, characterized in that it is a full-length IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibody, such as an IgG1 antibody, preferably an IgG1,κ antibody or an IgM antibody, preferably an IgM,κ antibody.

[0145] An embodiment of the present invention provides an anti-CD38 antibody of any of the above embodiments that is an antibody fragment or a single chain antibody.

[0146] An embodiment of the present invention provides an anti-CD38 antibody of any of the above embodiments that is conjugated to another moiety, eg, a cytotoxic moiety, a radioisotope, or a drug.

[0147] An embodiment of the present invention provides an anti-CD38 antibody of any of the above embodiments, which is an effector function-deficient antibody.

[0148] An embodiment of the present invention provides the anti-CD38 antibody of the above embodiment, wherein the effector function-deficient anti-CD38 antibody is a stabilized human IgG4 antibody.

[0149] An embodiment of the present invention provides the anti-CD38 antibody of the above embodiment, wherein the stabilized human IgG4 antibody is an antibody in which arginine at position 409 in the heavy chain constant region of human IgG4 has been substituted with lysine, threonine, methionine, or leucine, preferably lysine.

[0150] An embodiment of the present invention provides the anti-CD38 antibody of the above embodiment, wherein the antibody comprises a Lys residue at a position corresponding to position 409, or the CH3 region of the antibody has been substituted with the CH3 region of human IgG1, human IgG2, or human IgG3.

[0151] An embodiment of the present invention provides the anti-CD38 antibody of the above embodiment, which does not contain a Cys-Pro-Pro-Cys sequence in the hinge region.

[0152] An embodiment of the present invention provides the anti-CD38 antibody of the above embodiment, which comprises a Cys-Pro-Pro-Cys sequence in the hinge region.

[0153] An embodiment of the invention provides the anti-CD38 antibody of any of the above embodiments, which is a monovalent antibody.

[0154] An embodiment of the present invention is wherein the monovalent antibody i) providing a nucleic acid construct encoding the light chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding a VL region of a selected antigen-specific antibody and a nucleotide sequence encoding a constant CL region of an Ig, wherein the nucleotide sequence encoding the VL region of the selected antigen-specific antibody and the nucleotide sequence encoding the CL region of an Ig are operably linked, and in the case of an IgG1 subtype, the nucleotide sequence encoding the CL region has been modified so that the CL region does not contain any amino acids that can form disulfide bonds or covalent bonds with other peptides comprising the same amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to an animal or human; ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the VH region of a selected antigen-specific antibody and a nucleotide sequence encoding the constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified so that a region corresponding to the hinge region and, if required by the Ig subtype, other regions of the CH region, such as the CH3 region, do not contain any amino acid residues that are involved in the formation of disulfide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising the same amino acid sequence of the CH region of a human Ig in the presence of polyclonal human IgG or when administered to an animal or a human, and wherein the nucleotide sequence encoding the VH region of the selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operably linked; iii) providing a cellular expression system for producing said monovalent antibody; iv) producing the monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii). The anti-CD38 antibody of the above aspect is provided, wherein the antibody is constructed by a method comprising:

[0155] An embodiment of the present invention is a monovalent antibody comprising: (i) a variable region or an antigen-binding portion of said region of an antibody of any of the above embodiments; and (ii) immunoglobulin C H Area or C H 2 and C H a fragment thereof containing the IgG3 region, a region corresponding to the hinge region, and, if the immunoglobulin is not of the IgG4 subtype, C H Other areas of the region, e.g., C H The three regions were identified as C3 in the presence of polyclonal human IgG. H Disulfide bond with the region or identical C H C H Region or its fragment The anti-CD38 antibody of the above aspect is provided, comprising:

[0156] In an embodiment of the invention, the monovalent antibody is of the IgG4 subtype, but H The anti-CD38 antibody of the above embodiment is provided, wherein three regions have been modified to include one or more of the following amino acid substitutions: Thr (T) at position 366 is substituted with Ala (A), Leu (L) at position 368 is substituted with Ala (A), Leu (L) at position 368 is substituted with Val (V), Phe (F) at position 405 is substituted with Ala (A), Phe (F) at position 405 is substituted with Leu (L), Tyr (Y) at position 407 is substituted with Ala (A), and Arg (R) at position 409 is substituted with Ala (A).

[0157] An embodiment of the invention provides an anti-CD38 antibody of any of the above embodiments, wherein the heavy chain is modified such that the entire hinge is deleted.

[0158] An embodiment of the invention provides an anti-CD38 antibody of any of the above embodiments, wherein the sequence of the monovalent antibody has been modified so that it does not contain any acceptor sites for N-linked glycosylation.

[0159] An embodiment of the invention provides an antibody of any of the above embodiments that inhibits the synthesis of cGDPR by at least 25%, such as at least 30%, after 90 minutes, as measured by the spectrophotometric method described in Example 8 herein.

[0160] An embodiment of the present invention provides an antibody of any of the above embodiments that inhibits the synthesis of cADPR by at least 25%, e.g., at least 30%, as measured by the HPLC method described in Munshi et al., J. Biol. Chem. 275, 21566-21571 (2000) after 90 minutes at a concentration of 3 μg / ml.

[0161] An embodiment of the invention provides the antibody of any of the above embodiments, which stimulates the hydrolase activity of CD38 by at least 25%.

[0162] An embodiment of the invention provides the antibody of any of the above embodiments, which stimulates NAD hydrolase activity by at least 25%.

[0163] An embodiment of the invention provides the antibody of any of the above embodiments, which stimulates cADPR hydrolase activity by at least 25%.

[0164] An aspect of the invention provides an isolated nucleic acid encoding the peptide of any of the above aspects.

[0165] An aspect of the invention provides an expression vector comprising a nucleotide sequence encoding one or more of the amino acid sequences of any of the above aspects.

[0166] An aspect of the present invention provides an expression vector of the above aspect, further comprising a nucleotide sequence encoding the constant region of the light chain, the heavy chain, or both the light and heavy chains of a human antibody.

[0167] An aspect of the invention provides a recombinant eukaryotic or prokaryotic host cell that produces an antibody according to any of the above aspects.

[0168] An aspect of the present invention provides a hybridoma that produces an antibody according to any of the above aspects.

[0169] An aspect of the invention provides a pharmaceutical composition comprising an antibody according to any of the above aspects and a pharmaceutically acceptable carrier.

[0170] An aspect of the invention provides an antibody according to any of the above aspects for use as a pharmaceutical.

[0171] An embodiment of the invention provides an antibody according to any of the above embodiments for use in inhibiting the growth and / or proliferation, migration of, or inducing phagocytosis of, tumor cells expressing CD38.

[0172] An aspect of the invention provides an antibody according to any of the above aspects for use in treating rheumatoid arthritis.

[0173] An aspect of the invention provides an antibody according to any of the above aspects for use in the treatment of multiple myeloma.

[0174] An aspect of the invention provides an antibody according to any of the above aspects for use in the treatment of multiple sclerosis.

[0175] An embodiment of the invention provides an antibody as described in any of the above embodiments for use in treating a B cell neoplasm such as any one of small lymphocytic lymphoma, B cell prolymphocytic lymphoma, B cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse large B cell lymphoma, multiple myeloma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell neoplasms, e.g., plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, MALT lymphoma, nodal marginal zone B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma, or AIDS-related non-Hodgkin's lymphoma.

[0176] An embodiment of the invention provides a method of inhibiting the growth and / or proliferation, migration of, or inducing phagocytosis of, tumor cells expressing CD38, the method comprising administering to an individual in need thereof an antibody of any of the above embodiments.

[0177] An embodiment of the present invention provides a method of producing an anti-CD38 antibody of any of the above embodiments, comprising the steps of: a) culturing the host cell of claim 52 or the hybridoma of the above embodiment; and b) purifying the anti-CD38 antibody from the culture medium.

[0178] An aspect of the invention provides a diagnostic composition comprising an antibody according to any of the above aspects.

[0179] An embodiment of the present invention provides a method for detecting the presence of CD38 antigen, i.e., cells expressing CD38, in a sample, comprising the steps of: contacting the sample with an anti-CD38 antibody of any of the above embodiments under conditions that allow for the formation of a complex between the antibody or bispecific molecule and CD38; and Analyzing whether a complex was formed.

[0180] An embodiment of the present invention provides a kit for detecting the presence of CD38 antigen, i.e., cells expressing CD38, in a sample, comprising: an anti-CD38 antibody of any of the above embodiments, and Instructions for use of the kit.

[0181] An embodiment of the present invention provides an anti-idiotypic antibody that binds to the anti-CD38 antibody of any of the above embodiments.

[0182] An embodiment of the present invention relates to a method for inhibiting the growth and / or proliferation of cells expressing CD38, the method comprising administering a peptide of any of the above embodiments, an immunoconjugate of the above embodiments, a pharmaceutical composition of the above embodiments, or an expression vector recited in any of the above embodiments, such that cell growth and / or proliferation, migration, or phagocytosis is inhibited.

[0183] An embodiment of the present invention provides a method of treating a disease or disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need of treatment a peptide of any of the above embodiments, an immunoconjugate of any of the above embodiments, a pharmaceutical composition of any of the above embodiments, or an expression vector of any one of the above embodiments.

[0184] An embodiment of the present invention provides a method for preventing a disease or disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need of treatment a peptide of any of the above embodiments, an immunoconjugate of any of the above embodiments, a pharmaceutical composition of any of the above embodiments, or an expression vector of any one of the above embodiments.

[0185] An embodiment of the present invention provides the method of the above embodiment, wherein the disease or disorder is rheumatoid arthritis.

[0186] An embodiment of the present invention provides a method of the above embodiment, wherein the disease or disorder is a B cell neoplasm such as any one of small lymphocytic lymphoma, B cell prolymphocytic lymphoma, B cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse large B cell lymphoma, multiple myeloma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell neoplasms, e.g., plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, MALT lymphoma, nodal marginal zone B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma, and AIDS-related non-Hodgkin's lymphoma.

[0187] An embodiment of the present invention provides the method of the above embodiment, wherein the disease or disorder is multiple myeloma.

[0188] An embodiment of the present invention provides the method of the above embodiment, wherein the disease or disorder is an autoimmune disease.

[0189] An embodiment of the present invention provides the method of the above embodiment, wherein the disease or disorder is diabetes.

[0190] An embodiment of the present invention provides the method of the above embodiment, wherein the disease or disorder is multiple sclerosis.

[0191] An embodiment of the present invention provides the method of the above embodiment, wherein the disease or disorder is Graves' disease.

[0192] An embodiment of the present invention provides a method of the above embodiment, wherein the disease or disorder is neuroinflammation.

[0193] An embodiment of the present invention provides a method of the above embodiment, wherein the disease or disorder is inflammation of airway smooth muscle in asthma.

[0194] Aspects of the present invention provide methods of the above aspects, comprising administering to the subject one or more additional therapeutic agents.

[0195] An embodiment of the present invention provides the method of the above embodiment, wherein the one or more additional therapeutic agents are selected from a chemotherapeutic agent, an anti-inflammatory agent, or an immunosuppressant and / or immunomodulatory agent.

[0196] Embodiments of the present invention provide the methods of the above embodiments, wherein the one or more additional therapeutic agents are selected from the group consisting of cisplatin, gefitinib, cetuximab, rituximab, bevacizumab, erlotinib, bortezomib, thalidomide, pamidronate, zoledronic acid, clodronate, risedronate, ibandronate, etidronate, alendronate, tiludronate, arsenic trioxide, lenalidomide, filgrastim, pegfilgrastim, sargramostim, suberoylanilide hydroxamic acid, and SCIO-469.

[0197] The monoclonal antibodies of the present invention may be produced, for example, by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may also be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with the antigen of interest in the form of cells expressing the antigen on their surface or with a nucleic acid encoding the antigen of interest.

[0198] In one embodiment, the antibody of the present invention is a human antibody. Human monoclonal antibodies against CD38 can also be generated using transgenic or transchromosomal mice that have parts of the human immune system rather than the mouse immune system. Such transgenic and transchromosomal mice include those referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice."

[0199] HuMAb mice contain human immunoglobulin gene minilocuses encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Thus, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994); Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995); and Harding, F. and Lonberg, N. Ann. NY Acad. Sci 764 536-546 (1995)). The preparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), and Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996).See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98 / 24884, WO 94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918 and WO 01 / 09187.

[0200] HCo7 mice have a JKD disruption in their endogenous light chain (κ) gene (described in Chen et al., ENBO J. 12, 821-830 (1993)), a CMD disruption in their endogenous heavy chain gene (described in Example 1 of WO01 / 14424), a KCo5 human κ light chain transgene (described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)), and an HCo7 human heavy chain transgene (described in US Pat. No. 5,770,429).

[0201] HCo12 mice have a JKD disruption in their endogenous light chain (κ) gene (described in Chen et al., ENBO J. 12, 821-830 (1993)), a CMD disruption in their endogenous heavy chain gene (described in Example 1 of WO01 / 14424), a KCo5 human κ light chain transgene (described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)), and an HCo12 human heavy chain transgene (described in Example 2 of WO01 / 14424).

[0202] In the KM mouse strain, the endogenous mouse kappa light chain gene is homozygously disrupted as described in Chen et al., ENBO J. 12, 811-820 (1993), and the endogenous mouse heavy chain gene is homozygously disrupted as described in Example 1 of WO01 / 01987. This mouse strain harbors a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also harbors a human heavy chain transchromosome composed of chromosome 14 fragment hCF(SC20), as described in WO02 / 43478.

[0203] Splenocytes from these transgenic mice can also be used to generate hybridomas that secrete human monoclonal antibodies, according to well-known techniques.

[0204] Human monoclonal or polyclonal antibodies of the invention, or antibodies of the invention derived from other species, can also be produced transgenically by generating another non-human mammal or plant carrying transgenes for the immunoglobulin heavy and light chain sequences of interest and producing the antibody in a recoverable form therefrom. For transgenically produced mammals, antibodies can also be produced in and recovered from the milk of goats, cows, or other mammals. See, e.g., US 5,827,690, US 5,756,687, US 5,750,172, and US 5,741,957.

[0205] Furthermore, human antibodies of the present invention or antibodies of the present invention from other species can also be generated and identified by display-type technologies, including but not limited to phage display, retroviral display, ribosome display, and other technologies, using techniques well known in the art, and the resulting molecules can be subjected to further maturation, e.g., affinity maturation, as is well known in the art (e.g., Hoogenboom et al., J. Mol. Biol. 227, 381 (1991) (phage display); Vaughan et al., Nature Biotech 14, 309 (1996) (phage display); Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosome display); Parmley and Smith, Gene 73, 305-318 (1988) (phage display); Scott TIBS 17, 241-245 (1992); Cwirla et al., PNAS USA 87, 6378-6382 (1990); Russell et al., Nucl. Acids Research 21, 1081-1085 (1993); Hoogenboom et al., Immunol. Reviews 130, 43-68 (1992); Chiswell and McCafferty TIBTECH 10, 80-84 (1992); and US Pat. No. 5,733,743. When display techniques are used to produce non-human antibodies, such antibodies can also be humanized.

[0206] Competition for binding to CD38 or a portion of CD38 by two or more anti-CD38 antibodies can be determined by any suitable technique. In the context of the present invention, competition refers to a detectably significant decrease in the ability of a particular molecule to bind to a specific binding partner in the presence of another molecule that binds to that specific binding partner. Generally, competition refers to the ability of a given anti-CD38 antibody to bind to a CD38 molecule using sufficient amounts of two or more competing anti-CD38 antibodies and CD38 molecules, as measured, for example, by ELISA or FACS analysis (described in the Examples section). (a) the form of CD38 (e.g., "processed," "mature," "unprocessed," or "immature" CD38); (b) forms of free CD38 (e.g., CD38 fragments produced by in vivo processing); (c) another peptide associated with CD38, e.g., a heterodimeric peptide composed of CD31 associated with CD38; (d) a complex of CD38 with one or more substrates, e.g., cAMP, NAD+ and / or cADPR; (e) a soluble ligand, e.g., a dimer of CD38 dimerized, associated, and / or processed with CD31; or (f) CD38 region " refers to at least about a 10% reduction, e.g., at least about a 15%, or at least about a 20% reduction, in binding between CD38 and another anti-CD38 antibody, caused by the presence of another anti-CD38 antibody. It may also be true that there is competition between anti-CD38 antibodies for more than one form of CD38 and / or portion of CD38, for example, in situations where antibody binding properties of a particular region of CD38 are retained in the fragment, e.g., in the case of a well-represented linear epitope located in the various tested fragments or a sufficiently large CD38 fragment and conformational epitope represented in CD38.

[0207] Assessment of competition generally involves assessing relative inhibitory binding using a first amount of a first molecule, a second amount of a second molecule, and a third amount of a third molecule (or a standard determined by binding experiments to which new binding data for the first and second molecules can be reasonably compared as a surrogate for actual coincidence data), where the first, second, and third amounts are all sufficient to perform a comparison that provides information about the selectivity and / or specificity of the molecule in question relative to other molecules present. The first, second, and third amounts may vary with the nature of the anti-CD38 antibody and its potential target of interest. For example, in an ELISA assay similar to that described in the Examples section, approximately 5-50 μg (e.g., about 10-50 μg, about 20-50 μg, about 5-20 μg, about 10-20 μg, etc.) of anti-CD38 antibody and / or CD38 target is required to assess whether competition exists. Conditions should also be favorable for binding. Typically, physiological or near-physiological conditions (e.g., temperatures of about 20-40°C, pH of about 7-8, etc.) are suitable for anti-CD38 antibody:CD38 binding. Competition is often indicated by a relative inhibition significantly greater than about 5% as measured by ELISA and / or FACS analysis. It may be desirable to establish a higher relative inhibition threshold as a criterion / determinant of an appropriate level of competition in a particular situation (e.g., when using competition assays to select or screen new antibodies designed to have the desired function of blocking binding of another peptide or molecule that binds to CD38 (e.g., CD31, also known as the CD31 antigen, EndoCAM, GPIIA', PECAM-1, platelet / endothelial cell adhesion molecule, or natural anti-CD38 antibodies)). Thus, for example, competitive criteria can be established such that at least about 10% relative inhibition, at least about 15% relative inhibition, or at least about 20% relative inhibition is detected before an antibody is considered sufficiently competitive.When epitopes belonging to competing antibodies are closely located in the antigen, competition may also be significantly indicated by a relative inhibition of CD38 binding of greater than about 40% (e.g., at least about 45% inhibition, such as at least about 50% inhibition, for example at least about 55% inhibition, for example at least about 60% inhibition, such as at least about 65% inhibition, for example at least about 70% inhibition, such as at least about 75% inhibition, for example at least about 80% inhibition, such as at least about 85% inhibition, for example at least about 90% inhibition, for example at least about 95% inhibition or a higher level of relative inhibition).

[0208] Competition can also be considered the reciprocal of cross-reactivity between a molecule and two potential binding partners. In certain embodiments, the anti-CD38 antibodies of the present invention specifically bind to one or more residues or regions in CD38 but do not cross-react with other peptides, peptide regions, or molecules. For example, the present invention provides anti-CD38 antibodies that do not cross-react with proteins homologous to CD38, such as BST-1 (bone marrow stromal cell antigen 1) and Mo5, also known as CD157, or that do not cross-react with CD38 in the context of normal tissue, e.g., tissue not associated with multiple myeloma. Typically, lack of cross-reactivity refers to a relative competitive inhibition of less than about 5% between the molecules, as assessed by ELISA and / or FACS analysis using sufficient amounts of the molecules under appropriate assay conditions.

[0209] In one embodiment, the present invention provides a method for binding to CD38 or a portion thereof, comprising administering to the patient a therapeutically effective amount of ... L Sequence and V of SEQ ID NO:2 H Anti-CD38 antibodies are provided that compete with antibodies having the sequence, for example, antibody 028.

[0210] In one embodiment, the present invention provides a method for binding to CD38 or a portion thereof, comprising administering to the patient a therapeutically effective amount of ... L Sequence and V of SEQ ID NO:7 H Anti-CD38 antibodies are provided that compete with antibodies having the sequence, for example, antibody 025.

[0211] In one embodiment, the present invention provides a method for binding to CD38 or a portion thereof, comprising administering to the patient a therapeutically effective amount of ... L Sequence and V of SEQ ID NO: 12 H Anti-CD38 antibodies are provided that compete with antibodies having the sequence, for example, antibody 026.

[0212] In one embodiment, the present invention provides a method for binding to CD38 or a portion thereof, comprising administering to the patient a therapeutically effective amount of V of SEQ ID NO:42. L Sequence and V of SEQ ID NO: 17 H Anti-CD38 antibodies that compete with antibodies having the sequence, for example, antibody 049, are provided.

[0213] In one embodiment, the present invention provides a method for binding to CD38 or a portion thereof, comprising administering to the patient a therapeutically effective amount of V of SEQ ID NO:47. L Sequence and V of SEQ ID NO:22 H Anti-CD38 antibodies that compete with an antibody having the sequence, for example, antibody 056, are provided.

[0214] As detailed elsewhere herein, unless otherwise stated or clearly contradicted by context, binding of an anti-CD38 antibody to CD38 is understood to refer to binding in an appropriate context, e.g., a conformational context or a linear epitope context in which the structure of CD38 resides. Of course, binding in a limited subset of such contexts may be an important feature for any anti-CD38 antibody provided by the invention.

[0215] Further methods for determining anti-CD38 antibody specificity by competitive inhibition can be found, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley InterScience NY, (1992, 1993), and Muller, Meth. Enzymol. 92, 589-601 (1983).

[0216] Human CD38 contains a number of different epitopes, which may include: (1) peptide antigenic determinants contained within single peptide chains within human CD38; (2) conformational antigenic determinants consisting of one or more non-adjacent amino acids on a particular chain and / or amino acids present on spatially adjacent but separate peptide chains (typically, the amino acid sequence of each chain is located disjointly along the human CD38 polypeptide sequence); (3) post-translational antigenic determinants consisting entirely or partially of molecular structures covalently attached to human CD38, e.g., carbohydrate groups; or (4) combinations of (1)-(3).

[0217] Epitopes in the context of the present invention include any peptide or peptide-derived determinant capable of specific binding to an immunoglobulin. Epitopes can also comprise any suitable number of amino acids in any suitable position (relative to the linear sequence of CD38), orientation (relative to folded CD38 or a fragment thereof), and amino acid composition (and, therefore, at least partially, charge). Thus, for example, an epitope may consist of about 3-10 amino acids, typically 3-8 amino acids, located at one or more contiguous or noncontiguous positions relative to the primary sequence of CD38 (e.g., an epitope may consist essentially of two, three, four, five, six, seven, or eight amino acid residues distributed at one, two, three, four, or five noncontiguous positions in CD38). Alternatively, for example, an epitope can be considered to be defined (alone or in combination with portions of an adjacent CD38 domain) by a region of about 5-40 contiguous amino acid residues in CD38 (e.g., about 7-30 amino acid residues, about 5-20 amino acid residues, or about 3-15 amino acid residues). In some epitopes, one or only a few amino acid residues may be critical to CDR or CDR recognition (and thereby critical to anti-CD38 antibody:CD38 antigen affinity and avidity). As such, an epitope can be characterized based on one or more of such critical residues, with the recognition that other residues may make lesser contributions to the epitope. In the case of an epitope defined by a region of amino acids, one or more amino acids in the region may make a small or negligible contribution to antibody binding, and the residues may be amenable to substitution with a suitable different residue without resulting in "loss" of the epitope for at least some anti-CD38 antibodies specific thereto.

[0218] In some embodiments, the present invention provides an anti-CD38 antibody, e.g., V of SEQ ID NO:27. L Sequence and V of SEQ ID NO:2 HAn antibody having the sequence (e.g., antibody 028) or V of SEQ ID NO:32 L Sequence and V of SEQ ID NO:7 H An antibody having the sequence (e.g., antibody 025) or V of SEQ ID NO:37 L Sequence and V of SEQ ID NO: 12 H An antibody having the sequence (e.g., antibody 026) or V of SEQ ID NO:42 L Sequence and V of SEQ ID NO: 17 H An antibody having the sequence (e.g., antibody 049) or V of SEQ ID NO:47 L Sequence and V of SEQ ID NO:22 H Anti-CD38 antibodies are provided that specifically bind to the CD38 epitope that is specifically bound by an antibody having the sequence (e.g., antibody 056).

[0219] SEQ ID NO:27V L Sequence and V of SEQ ID NO:2 H CDR of an antibody having the sequence V of SEQ ID NO:32 L Sequence and V of SEQ ID NO:7 H CDR of an antibody having the sequence or V of SEQ ID NO:37 L Sequence and V of SEQ ID NO: 12 H CDR of an antibody having the sequence or V of SEQ ID NO:42 L Sequence and V of SEQ ID NO: 17 H CDR of an antibody having the sequence or V of SEQ ID NO:47 L Sequence and V of SEQ ID NO:22 H Further, anti-CD38 antibodies having one or more CDRs different from the CDRs of an antibody having the sequence V of SEQ ID NO:27, respectively. L Sequence and V of SEQ ID NO:2 H CDR of an antibody having the sequence V of SEQ ID NO:32 L Sequence and V of SEQ ID NO:7 H CDR of an antibody having the sequence or V of SEQ ID NO:37 LSequence and V of SEQ ID NO: 12 H CDR of an antibody having the sequence or V of SEQ ID NO:42 L Sequence and V of SEQ ID NO: 17 H CDR of an antibody having the sequence or V of SEQ ID NO:47 L Sequence and V of SEQ ID NO:22 H It is possible to say that the CDRs of an antibody having the sequence V of SEQ ID NO:27 are specific for the same epitope as an antibody having the sequence V of SEQ ID NO:27. L Sequence and V of SEQ ID NO:2 H CDR of an antibody having the sequence V of SEQ ID NO:32 L Sequence and V of SEQ ID NO:7 H CDR of an antibody having the sequence or V of SEQ ID NO:37 L Sequence and V of SEQ ID NO: 12 H CDR of an antibody having the sequence or V of SEQ ID NO:42 L Sequence and V of SEQ ID NO: 17 H CDR of an antibody having the sequence or V of SEQ ID NO:47 L Sequence and V of SEQ ID NO:22 H It may also recognize or be more specific / selective for a particular structure or region of the epitope than an antibody having the CDR of an antibody having the sequence.

[0220] SEQ ID NO:27V L Sequence and V of SEQ ID NO:2 H An antibody having the sequence (e.g., antibody 028) or V of SEQ ID NO:32 L Sequence and V of SEQ ID NO:7 H An antibody having the sequence (e.g., antibody 025) or V of SEQ ID NO:37 L Sequence and V of SEQ ID NO: 12 H An antibody having the sequence (e.g., antibody 026) or V of SEQ ID NO:42 LSequence and V of SEQ ID NO: 17 H An antibody having the sequence (e.g., antibody 049) or V of SEQ ID NO:47 L Sequence and V of SEQ ID NO:22 H The CD38 epitope bound by an antibody having a sequence (e.g., antibody 056) can be identified by standard mapping and characterization techniques, and its further refined molecular structure can be discerned by any suitable technique, of which numerous examples are available to those skilled in the art.

[0221] These techniques can also be used to identify and / or characterize epitopes for anti-CD38 antibodies in general. As an example of such a mapping / characterization method, epitopes for anti-CD38 antibodies can be determined by epitope "footprinting," which uses chemical modification of exposed amines / carboxyls in the CD38 protein. One specific example of such a footprinting technique is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry), in which hydrogen / deuterium exchange, binding, and back-exchange of receptor and ligand protein amide protons occur, where the backbone amide groups involved in protein binding are protected from back-exchange and therefore remain deuterated. In this regard, relevant regions can also be identified by pepsin proteolysis, high-speed microbore high-performance liquid chromatography separation, and / or electrospray ionization mass spectrometry. See, e.g., Ehring H, Analytical Biochemistry, 267(2) 252-259 (1999) and / or Engen, JR and Smith, DL (2001) Anal. Chem. 73, 256A-265A. Another example of a suitable epitope identification technique is nuclear magnetic resonance (NMR) epitope mapping, which typically compares the positions of signals in two-dimensional NMR spectra of free antigen and antigen complexed with an antigen-binding peptide, e.g., an antibody. Antigens are typically selectively mapped so that only signals corresponding to the antigen and no signals from the antigen-binding peptide are seen in the NMR spectrum.15 The antigen-binding peptide is isotopically labeled with N. Typically, the antigen signal arising from the amino acid involved in the interaction with the antigen-binding peptide shifts its position in the spectrum of the complex compared to the spectrum of the free antigen, and the amino acid involved in binding can be identified in this manner. See, for example, Ernst Schering Res Found Workshop. (44), 149-67 (2004); Huang et al., Journal of Molecular Biology 281(1), 61-67 (1998); and Saito and Patterson, Methods. 9(3), 516-24 (1996).

[0222] Epitope mapping / characterization can also be performed using mass spectrometry, see, e.g., Downward, J Mass Spectrom. 35(4), 493-503 (2000) and Kiselar and Downard, Anal Chem. 71(9), 1792-801 (1999).

[0223] Protease digestion techniques can also be useful in connection with epitope mapping and identification. For example, protease digestion using trypsin at a ratio of approximately 1:50 to CD38 at 37°C and pH 7-8 overnight (O / N) can be followed by mass spectrometry (MS) analysis for peptide identification to determine antigenic determinant-associated regions / sequences. Subsequent comparison of samples subjected to trypsin digestion with samples incubated with CD38BP and then subjected to, for example, trypsin digestion (thereby revealing a footprint for the binder) can also identify peptides protected from trypsin cleavage by CD38BP. Additionally or alternatively, other enzymes, such as chymotrypsin, pepsin, etc., can be used in similar epitope characterization methods.

[0224] In these measurements, V of SEQ ID NO:27 L Sequence and V of SEQ ID NO:2H An antibody having the sequence (e.g., antibody 028) or V of SEQ ID NO:32 L Sequence and V of SEQ ID NO:7 H An antibody having the sequence (e.g., antibody 025) or V of SEQ ID NO:37 L Sequence and V of SEQ ID NO: 12 H An antibody having the sequence (e.g., antibody 026) or V of SEQ ID NO:42 L Sequence and V of SEQ ID NO: 17 H An antibody having the sequence (e.g., antibody 049) or V of SEQ ID NO:47 L Sequence and V of SEQ ID NO:22 H Anti-CD38 antibodies that perform significantly the same results as antibodies having sequences V and V of SEQ ID NO:27, respectively (e.g., antibody 056). L Sequence and V of SEQ ID NO:2 H An antibody having the sequence (e.g., antibody 028) or V of SEQ ID NO:32 L Sequence and V of SEQ ID NO:7 H An antibody having the sequence (e.g., antibody 025) or V of SEQ ID NO:37 L Sequence and V of SEQ ID NO: 12 H An antibody having the sequence (e.g., antibody 026) or V of SEQ ID NO:42 L Sequence and V of SEQ ID NO: 17 H An antibody having the sequence (e.g., antibody 049) or V of SEQ ID NO:47 L Sequence and V of SEQ ID NO:22 H It is considered to be an antibody that binds to the same epitope as an antibody having the sequence (e.g., antibody 056). For a detailed description of similar technology, see, for example, Manca, Ann Ist Super Sanita. 27(1), 15-9 (1991).

[0225] Epitope mapping by competitive binding to CD38 with two antibodies, one of which is biotinylated, is another method for identifying related antigenic determinant regions. Antibody binding to linear and loop peptides of CD38 by PEPSCAN-based enzyme-linked immunoassay is another method for identifying related antigenic determinant regions. See, e.g., Slootstra-JW et al., Mol-Divers. 1, 87-96 (1996).

[0226] Site-directed mutagenesis is another method for identifying relevant antigenic determinant regions. See, for example, Polyak and Deans, Blood 99, 3956-3962 (2002). Epitopes can also be identified using various phage display techniques. See, for example, Wang and Yu, Curr Drug Targets. 5(1), 1-15 (2004); Burton, Immunotechnology. 1(2), 87-94 (1995 Aug); Cortese et al., Immunotechnology. 1(2), 87-94 (1995) and Irving et al., Curr Opin Chem Biol. 5(3), 314-24 (2001). Consensus epitopes can also be identified by improved phage display related techniques (see http: / / www.cs.montana.edu / ~mumey / papers / jcb03.pdf for a detailed description).

[0227] Other potentially useful methods for epitope mapping include crystallography, X-ray diffraction (e.g., the X-ray diffraction / sequence study technique developed by Poljak et al. in the 1970s and 1980s), and the application of multi-pin peptide synthesis. Sequence analysis and computer-based methods such as three-dimensional structural analysis and docking can also be used to identify antigenic determinants. For example, epitopes can also be determined by molecular modeling using the structure of CD38 along with the docking of the structures of the Fab fragments of individual monoclonal antibodies. These and other mapping methods are detailed in Epitope Mapping: A Practical Approach (Westwood and Hay, Eds.), 2001, Oxford University Press.

[0228] In one embodiment, the present invention provides a method for the preparation of a nucleic acid sequence comprising the V L Sequence and V of SEQ ID NO:2 H An antibody having the sequence (e.g., antibody 028) or V of SEQ ID NO:32 L Sequence and V of SEQ ID NO:7 H An antibody having the sequence (e.g., antibody 025) or V of SEQ ID NO:37 L Sequence and V of SEQ ID NO: 12 H An antibody having the sequence (e.g., antibody 026) or V of SEQ ID NO:42 L Sequence and V of SEQ ID NO: 17 H An antibody having the sequence (e.g., antibody 049) or V of SEQ ID NO:47 L Sequence and V of SEQ ID NO:22 H Anti-CD38 antibodies are provided that have substantially the same specific CD38 binding characteristics of one or more mAbs selected from the antibody having the sequence (e.g., antibody 056).

[0229] Mapping experiments have shown that several monoclonal antibodies raised against human CD38 bind to epitopes in the C-terminal region (220-296) of CD38 (Hoshino et al. and Ferrero et al.). Within this region, three amino acid differences were found between the human and cynomolgus monkey CD38 sequences: T237, Q272, and S274 in humans correspond to A238, R273, and F275 in cynomolgus monkeys. A limited number of amino acid differences exist between the human and monkey CD38 sequences, for example, in the carboxy-terminal end of the protein. For example, the following three amino acid differences exist between the human and cynomolgus CD38 sequences: T237, Q272, and S274 of human CD38 correspond to A238, R273, and F275 of cynomolgus CD38 (compare SEQ ID NO:21 and SEQ ID NO:22).

[0230] The antibodies of the present invention do not bind to a human CD38 mutant in which the aspartic acid at position 202 is substituted with glycine to the same extent as they bind to human CD38. The present invention provides antibodies that bind to human CD38 and to mutant human CD38 in which the serine residue at position 274 is substituted with a phenylalanine residue. The antibodies of the present invention also bind to a human CD38 mutant in which the glutamine at position 272 is substituted with arginine. The antibodies of the present invention also bind to a human CD38 mutant in which the threonine at position 237 is substituted with alanine.

[0231] The term "does not bind to the same extent" should be interpreted to mean that the binding of the antibody to mutant human CD38 is significantly lower than that of the antibody to wild-type human CD38. The term "binds to the same extent" should be interpreted to mean that the binding of the antibody to mutant human CD38 is substantially the same as that of the antibody to wild-type human CD38. Binding of peptides to CD38 molecules (wild-type and mutant) can be determined in several ways, and it is within the general knowledge of one skilled in the art to determine whether binding to a mutant is "significantly lower" than binding to the wild-type. Many different techniques for determining binding of one peptide to another are available to those skilled in the art, such as ELISA, radioimmunoassay, BIAcore, or flow cytometry.

[0232] One way to determine binding is to measure the EC of antibody binding to the mutant and wild-type proteins. 50 Another method for determining binding is by examining the magnitude of binding at saturating concentrations (e.g., plateau of binding signal), or by comparing the kinetic constant K on and K. off is determined by, for example, BIAcore.

[0233] In one embodiment, binding of the antibody in question to the CD38 protein (mutant or wild-type) is by use of an ELISA as described in Example 4.

[0234] In a further embodiment, the antibody of the invention an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 10, 15, 20 and 25; or Any variant of said sequence, preferably having only conservative amino acid substitutions The human heavy chain variable region (VH) CDR3 sequence comprises:

[0235] In one embodiment, the variant consists essentially of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, for example at least about 95% amino acid sequence identity to any one of SEQ ID NOs:5, 10, 15, 20 and 25.

[0236] In further embodiments, the variant has at most one, two or three amino acid modifications, eg, amino acid substitutions, preferably conservative substitutions, compared to the sequence.

[0237] In a preferred embodiment, the antibody comprises a human heavy chain variable region CDR3 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 10, 15, 20 and 25.

[0238] In still further embodiments, the antibody of the invention comprises: a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 3, 4 and 5; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 8, 9 and 10; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 13, 14 and 15; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 18, 19 and 20; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 23, 24 and 25; or A variant of any of the foregoing VH regions, preferably having only conservative amino acid substitutions.

[0239] In one embodiment, the variant comprises a VH CDR1 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 3, 8, 13, 18 or 23.

[0240] In one embodiment, the variant comprises a VH CDR2 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs:4, 9, 14, 19 or 24.

[0241] In one embodiment, the variant comprises a VH CDR3 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs:5, 10, 15, 20 or 25.

[0242] In another embodiment, the antibody comprises: a) a VL CDR3 region having the sequence set forth in SEQ ID NO:30 and a VH CDR3 region having a sequence selected from the group consisting of SEQ ID NO:5; b) a VL CDR3 region having the sequence set forth in SEQ ID NO:35 and a VH CDR3 region having the sequence set forth in SEQ ID NO:10; c) a VL CDR3 region having the sequence set forth in SEQ ID NO:40 and a VH CDR3 region having the sequence set forth in SEQ ID NO:15; d) a VL CDR3 region having the sequence set forth in SEQ ID NO:45 and a VH CDR3 region having the sequence set forth in SEQ ID NO:20; e) a VL CDR3 region having the sequence set forth in SEQ ID NO:50 and a VH CDR3 region having the sequence set forth in SEQ ID NO:25; f) any of the preceding variants, preferably having only conservative substitutions in said sequence Includes:

[0243] In one embodiment, the variant comprises a VH CDR3 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to the sequence of any one of SEQ ID NOs: 5, 10, 15, 20 or 25. In one embodiment, the variant comprises a VL CDR3 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to the sequence of any one of SEQ ID NOs: 30, 35, 40, 45 or 50.

[0244] In a further embodiment, the antibody of the invention comprises: a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 3, 4 and 5 and a VL region comprising the CDR3 sequence of SEQ ID NO: 30; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 8, 9 and 10 and a VL region comprising the CDR3 sequence of SEQ ID NO: 35; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 13, 14 and 15 and a VL region comprising the CDR3 sequence of SEQ ID NO: 40; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 18, 19 and 20 and a VL region comprising the CDR3 sequence of SEQ ID NO: 45; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 23, 24 and 25 and a VL region comprising the CDR3 sequence of SEQ ID NO: 50; or A variant of any of the preceding antibodies, which preferably only has conservative amino acid substitutions in said sequence.

[0245] In one embodiment, the variant comprises a VH CDR1 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 3, 8, 13, 18 or 23.

[0246] In one embodiment, the variant comprises a VH CDR2 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs:4, 9, 14, 19 or 24.

[0247] In one embodiment, the variant comprises a VH CDR3 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs:5, 10, 15, 20 or 25.

[0248] In one embodiment, the variant comprises a VL CDR1 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 28, 33, 38, 43 or 48.

[0249] In one embodiment, the variant comprises a VL CDR2 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 29, 34, 39, 44 or 49.

[0250] In one embodiment, the variant comprises a VL CDR3 consisting essentially of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 30, 35, 40, 45 or 50.

[0251] In a further embodiment, the antibody of the invention comprises: a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 3, 4 and 5 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 28, 29 and 30; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 8, 9 and 10 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 33, 34 and 35; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 13, 14 and 15 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 38, 39 and 40; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 18, 19 and 20 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 43, 44 and 45; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 23, 24 and 25 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 48, 49 and 50; or A variant of any of the preceding antibodies, which preferably only has conservative amino acid modifications in said sequence.

[0252] In one embodiment, the variant comprises a VH CDR1 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 3, 8, 13, 18 or 23.

[0253] In one embodiment, the variant comprises a VH CDR2 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs:4, 9, 14, 19 or 24.

[0254] In one embodiment, the variant comprises a VH CDR3 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs:5, 10, 15, 20 or 25.

[0255] In one embodiment, the variant comprises a VL CDR1 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 28, 33, 38, 43 or 48.

[0256] In one embodiment, the variant comprises a VL CDR2 essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 29, 34, 39, 44 or 49.

[0257] In one embodiment, the variant comprises a VL CDR3 consisting essentially of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 30, 35, 40, 45 or 50.

[0258] In still further embodiments, the antibody of the invention comprises: a VH region comprising the sequence of SEQ ID NO:2 and a VL region comprising the sequence of SEQ ID NO:27; or a VH region comprising the sequence of SEQ ID NO:7 and a VL region comprising the sequence of SEQ ID NO:32; or a VH region comprising the sequence of SEQ ID NO: 12 and a VL region comprising the sequence of SEQ ID NO: 37; or a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 42; or a VH region comprising the sequence of SEQ ID NO:22 and a VL region comprising the sequence of SEQ ID NO:47; or Any of the foregoing variants preferably having only conservative modifications.

[0259] In one embodiment, the variant comprises a VH region consisting essentially of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 2, 7, 12, 17 or 22.

[0260] In one embodiment, the variant comprises a VL region essentially consisting of a sequence having at least about 50%, such as at least 60%, for example at least about 70%, such as at least about 75%, for example at least about 80%, such as at least about 85%, for example at least about 90%, such as at least about 95% amino acid sequence identity to any one of SEQ ID NOs: 27, 32, 37, 42 or 47.

[0261] In a further embodiment, an antibody of the invention comprises a VH having at least 80% identity, such as 90% or 95% or 97% or 98% or 99% identity, to a VH region sequence selected from the group consisting of SEQ ID NO:2, 7, 12, 17 or 22.

[0262] In a further embodiment, an antibody of the invention comprises a VL having at least 80% identity, such as 90% or 95% or 97% or 98% or 99% identity, to a VL region sequence selected from the group consisting of SEQ ID NO: 27, 32, 37, 42 or 47.

[0263] In still further embodiments, the antibody of the invention comprises a VH region selected from the group consisting of SEQ ID NO:2, 7, 12, 17 or 22.

[0264] In still further embodiments, the antibody of the invention comprises a VL region selected from the group consisting of SEQ ID NOs: 27, 32, 37, 42 or 47.

[0265] In still further embodiments, the antibody of the invention comprises: a VH region comprising the sequence of SEQ ID NO:2 and a VL region comprising the sequence of SEQ ID NO:27; or a VH region comprising the sequence of SEQ ID NO:7 and a VL region comprising the sequence of SEQ ID NO:32; or a VH region comprising the sequence of SEQ ID NO: 12 and a VL region comprising the sequence of SEQ ID NO: 37; or a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 42; or A VH region comprising the sequence of SEQ ID NO:22 and a VL region comprising the sequence of SEQ ID NO:47.

[0266] The present invention also provides, in one aspect, a VH region comprising the sequence of SEQ ID NO:2 and a VL region comprising the sequence of SEQ ID NO:27; or a VH region comprising the sequence of SEQ ID NO:7 and a VL region comprising the sequence of SEQ ID NO:32; or a VH region comprising the sequence of SEQ ID NO: 12 and a VL region comprising the sequence of SEQ ID NO: 37; or a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 42; or A VH region comprising the sequence of SEQ ID NO: 22 and a VL region comprising the sequence of SEQ ID NO: 47 The present invention provides anti-CD38 antibodies characterized for their ability to compete with antibodies having the following structure:

[0267] The present invention also provides a VH region comprising the sequence of SEQ ID NO:2 and a VL region comprising the sequence of SEQ ID NO:27; or a VH region comprising the sequence of SEQ ID NO:7 and a VL region comprising the sequence of SEQ ID NO:32; or a VH region comprising the sequence of SEQ ID NO: 12 and a VL region comprising the sequence of SEQ ID NO: 37; or a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 42; or A VH region comprising the sequence of SEQ ID NO: 22 and a VL region comprising the sequence of SEQ ID NO: 47 The present invention relates to an anti-CD38 antibody that binds to the same epitope as an antibody having the formula:

[0268] The antibodies of the present invention may be of any isotype. The choice of isotype is typically dictated by the desired effector function, such as ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the kappa or lambda human light chain constant region may be used. If necessary, the class of the anti-CD38 antibodies of the present invention may be switched by known methods. For example, an originally IgM antibody of the present invention may be class switched to an IgG antibody of the present invention. Furthermore, class switching technology can be used to convert one IgG subclass to another, e.g., IgG1 to IgG2. Thus, for various therapeutic applications, the effector function of the antibodies of the present invention can be altered by isotype switching, for example, to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. In one embodiment, the antibody of the present invention is an IgG1 antibody, e.g., IgG1κ.

[0269] In one embodiment, the antibody of the invention is a full-length antibody. In another embodiment, the antibody of the invention is an antibody fragment or a single chain antibody.

[0270] Antibody fragments can be obtained, for example, by fragmentation using conventional techniques, and the fragments can be screened for utility in the same manner as described herein for intact antibodies. For example, F(ab')2 fragments can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Fab fragments can also be obtained by treating an IgG antibody with papain, or by pepsin digestion of an IgG antibody. F(ab')2 fragments can also be produced by linking the Fab' described below via a thioether bond or a disulfide bond. Fab' fragments are antibody fragments obtained by cleaving the disulfide bond in the hinge region of F(ab')2. Fab' fragments can also be obtained by treating an F(ab')2 fragment with a reducing agent such as dithiothreitol. Antibody fragments can also be produced by expressing nucleic acids encoding such fragments in recombinant cells (see, e.g., Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a chimeric gene encoding a portion of the F(ab')2 fragment can be produced by expressing the C of the H chain. H The DNA sequence encoding the antibody fragment fragment 1 domain and hinge region may also include a translation stop codon following the sequence, resulting in such a truncated antibody fragment molecule.

[0271] In one embodiment, the anti-CD38 antibody is a monovalent antibody, preferably a monovalent antibody as described in WO2007059782 (Genmab), which is incorporated herein by reference. Thus, in one embodiment, the antibody is a monovalent antibody, and the anti-CD38 antibody is i) providing a nucleic acid construct encoding the light chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding a VL region of a selected antigen-specific anti-CD38 antibody and a nucleotide sequence encoding a constant CL region of an Ig, wherein the nucleotide sequence encoding the VL region of the selected antigen-specific antibody and the nucleotide sequence encoding the CL region of an Ig are operably linked, and in the case of an IgG1 subtype, the nucleotide sequence encoding the CL region has been modified so that the CL region does not contain any amino acids that can form disulfide bonds or covalent bonds with other peptides comprising the same amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to an animal or human; ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the VH region of a selected antigen-specific antibody and a nucleotide sequence encoding the constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that a region corresponding to the hinge region and, if required by the Ig subtype, another region of the CH region, e.g., the CH3 region, does not contain any amino acid residues that are involved in the formation of disulfide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising the same amino acid sequence of the CH region of a human Ig in the presence of polyclonal human IgG or when administered to an animal or a human, and the nucleotide sequence encoding the VH region of the selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operably linked; iii) providing a cellular expression system for producing said monovalent antibody; iv) producing the monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii). The method includes:

[0272] Similarly, in one embodiment, the anti-CD38 antibody is (i) a variable region or an antigen-binding portion of an antibody of the invention as described herein; and (ii) immunoglobulin C H Area or C H 2 and C H a fragment thereof containing the IgG3 region, a region corresponding to the hinge region, and, if the immunoglobulin is not of the IgG4 subtype, C H Other areas of the region, e.g., C H The three regions were identified as C3 in the presence of polyclonal human IgG. H Disulfide bond with the region or identical C H C H Region or its fragment is a monovalent antibody comprising:

[0273] In a further embodiment, the heavy chain of the monovalent anti-CD38 antibody is modified to delete the entire hinge.

[0274] In a further embodiment, the monovalent antibody is of the IgG4 subtype but is not C H The three regions have been modified to include one or more of the following amino acid substitutions: Thr (T) at position 366 is replaced by Ala (A), Leu (L) at position 368 is replaced by Ala (A), Leu (L) at position 368 is replaced by Val (V), Phe (F) at position 405 is replaced by Ala (A), Phe (F) at position 405 is replaced by Leu (L), Tyr (Y) at position 407 is replaced by Ala (A), and Arg (R) at position 409 is replaced by Ala (A).

[0275] In another further embodiment, the sequence of the monovalent antibody is modified so that it does not contain any acceptor sites for N-linked glycosylation.

[0276] The anti-CD38 antibodies of the present invention also include single-chain antibodies. A single-chain antibody is a peptide in which the Fv regions of the heavy and light chains are connected. In one embodiment, the present invention provides single-chain Fvs (scFvs) in which the heavy and light chains in the Fv of an anti-CD38 antibody of the present invention are linked into a single peptide chain by a flexible peptide linker (generally about 10, 12, 15, or more amino acid residues). Methods for producing such antibodies are described, for example, in U.S. Pat. No. 4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et al., Science 242, 423-426 (1988), Huston et al., PNAS USA 85, 5879-5883 (1988), and McCafferty et al., Nature 348, 552-554 (1990). Single-chain antibodies are composed of a single V H and V L It may be monovalent if only one V is used, and two V H and V L may be bivalent if used, and may be bivalent if used with three or more V H and V L may be multivalent if used.

[0277] In one embodiment, the anti-CD38 antibody of the present invention is an effector function-deficient antibody. Such antibodies are particularly useful when used to stimulate and dampen the immune system by blocking the inhibitory effects of CD38. For such applications, it may be advantageous for the antibody to lack effector functions such as ADCC, as this may result in undesirable cytotoxicity.

[0278] In one embodiment, the effector function-deficient anti-CD38 antibody is a stabilized IgG4 antibody. An example of a suitable stabilized IgG4 antibody is an antibody in which arginine at position 409 in the heavy chain constant region of human IgG4, as shown in the EU index of Kabat et al., is substituted with lysine, threonine, methionine, or leucine, preferably lysine (described in WO2006033386 (Kirin)). Preferably, the antibody contains a Lys or Ala residue at the position corresponding to position 409, or the CH3 region of the antibody is substituted with the CH3 region of human IgG1, human IgG2, or human IgG3.

[0279] In a further embodiment, the stabilized IgG4 anti-CD38 antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of Lys, Ala, Thr, Met, and Leu at the position corresponding to position 409 and / or a residue selected from the group consisting of Ala, Val, Gly, Ile, and Leu at the position corresponding to position 405, and the antibody optionally comprises one or more further substitutions, deletions, and / or insertions, but does not comprise a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, the antibody comprises a Lys or Ala residue at the position corresponding to position 409, or the CH3 region of the antibody has been replaced by the CH3 region of human IgG1, human IgG2, or human IgG3.

[0280] In yet a further embodiment, the stabilized IgG4 anti-CD38 antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of Lys, Ala, Thr, Met, and Leu at the position corresponding to position 409 and / or a residue selected from the group consisting of Ala, Val, Gly, Ile, and Leu at the position corresponding to position 405, and wherein the antibody optionally comprises one or more further substitutions, deletions, and / or insertions, and comprises a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, the antibody comprises a Lys or Ala residue at the position corresponding to position 409, or the CH3 region of the antibody has been replaced by the CH3 region of human IgG1, human IgG2, or human IgG3.

[0281] In a further embodiment, the effector function-deficient anti-CD38 antibody is a non-IgG4 type, e.g., IgG1, IgG2, or IgG3, antibody that has been mutated to reduce or eliminate its ability to mediate effector functions such as ADCC. Examples of such mutations are described in Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.;75(24):12161-12168 (2001).

[0282] Conjugates In a further embodiment, the antibody of the invention is conjugated to another moiety, eg, a cytotoxic moiety, a radioisotope, or a drug.

[0283] Such antibodies can also be produced by chemically conjugating other moieties to the N- or C-terminus of an anti-CD38 antibody or fragment thereof (e.g., anti-CD38 antibody H chain, L chain, or anti-CD38-specific / selective fragment thereof) (see, e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated antibody derivatives can also be produced by conjugation at internal residues or sugars, as appropriate.

[0284] The anti-CD38 antibodies described herein may also be modified by including any suitable number of modified amino acids. Suitability in this regard is typically determined by the ability to at least substantially retain the CD38 selectivity and / or specificity associated with the underivatized parent anti-CD38 antibody. The inclusion of one or more modified amino acids may be advantageous, for example, to increase polypeptide serum half-life, reduce polypeptide antigenicity, or enhance polypeptide storage stability. Amino acids may be modified during recombinant production, e.g., co- or post-translationally (e.g., N-linked glycosylation at NXS / T motifs during expression in mammalian cells), or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, prenylated (e.g., farnesylated, geranylgeranylated) amino acids, acetylated amino acids, acylated amino acids, PEGylated amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, and the like. Relevant references are readily available to guide those skilled in the art of amino acid modifications. Exemplary protocols can be found in Walker (1998) Protein Protocols On Cd-Rom, Humana Press, Towata, NJ.

[0285] Anti-CD38 antibodies may also be chemically modified by covalent conjugation to polymers, e.g., to increase their circulating half-life. Exemplary polymers and methods for attaching them to peptides are shown, for example, in US 4,766,106, US 4,179,337, US 4,495,285, and US 4,609,546.

[0286] In one embodiment, the present invention provides an anti-CD38 antibody conjugated to a second molecule selected from a radionuclide, an enzyme, an enzyme substrate, a cofactor, a fluorescent marker, a chemiluminescent marker, a peptide tag, or a magnetic particle. In one embodiment, the anti-CD38 antibody may be conjugated to one or more antibody fragments, nucleic acids (oligonucleotides), nucleases, hormones, immunomodulators, chelators, boron compounds, photoactive agents, dyes, and the like. These and other suitable agents may be directly or indirectly conjugated to the anti-CD38 antibody of the present invention. One example of indirect conjugation of a second agent is via a spacer moiety.

[0287] In one embodiment, an anti-CD38 antibody is provided that contains one or more radiolabeled amino acids. The radiolabeled anti-CD38 antibody may be used for diagnostic or therapeutic purposes (conjugation to a radiolabeled molecule is another possible feature). Non-limiting examples of labels for polypeptides include, but are not limited to, 3H, 14C, 15N, 35S, 90Y, 99Tc, 125I, 131I, and 186Re. Methods for preparing radiolabeled amino acid and related peptide derivatives are known in the art (see, e.g., Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)) and US 4,681,581, US 4,735,210, US 5,101,827, US 5,102,990 (US RE35,500), US 5,648,471 and US 5,697,902).

[0288] In one embodiment, the anti-CD38 antibody of the present invention is conjugated to a radioisotope or a radioisotope-containing chelate. For example, the anti-CD38 antibody may be conjugated to a chelator linker, such as DOTA, DTPA, or tiuxetan, that allows the anti-CD38 antibody to be complexed with a radioisotope. Additionally or alternatively, the anti-CD38 antibody may contain or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Radiolabeled anti-CD38 antibodies may be used for diagnostic or therapeutic purposes. Non-limiting examples of radioisotopes include H, C, N, S, Y, Tc, I, In, I, Re, Bs, Ac, and Th.

[0289] In one embodiment, the anti-CD38 antibody of the present invention is conjugated to an auristatin or an auristatin peptide analog or derivative (US Pat. No. 5,635,483, US Pat. No. 5,780,588). Auristatins have been shown to interfere with microtubule movement, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), have anticancer properties (US Pat. No. 5,663,149), and have antifungal properties (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42:2961-2965). The auristatin drug moiety can also be attached to the antibody via a linker through the N-terminus or C-terminus of the peptide drug moiety.

[0290] Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF disclosed in Senter et al., Proceedings of the American Association for Cancer Research. Volume 45, abstract number 623, presented March 28, 2004, and described in US 2005 / 0238648.

[0291] An exemplary auristatin embodiment is MMAE (monomethylauristatin E), where the wavy line indicates the covalent attachment to the linker (L) of the antibody drug conjugate. TIFF2026021523000004.tif27142

[0292] Another exemplary auristatin embodiment is MMAF (monomethylauristatin F), where the wavy line indicates the covalent attachment to the linker (L) of the antibody drug conjugate (US2005 / 0238649). TIFF2026021523000005.tif26134

[0293] The anti-CD38 antibody drug conjugate of the present invention comprises a linker unit between the cytostatic drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody into the intracellular environment. In yet other embodiments, the linker unit is non-cleavable, and the drug is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker can be a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, for example, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids or at least three amino acids in length. Cleavage agents can include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs within target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).In certain embodiments, the peptidyl linker that can be cleaved by intracellular proteases is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (see, for example, US Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a Val-Cit linker).One advantage of using intracellular proteolytic release of therapeutic agents is that the drug is generally attenuated when conjugated, and the serum stability of the conjugate is generally high.

[0294] In yet another embodiment, the linker unit is non-cleavable, and the drug is released by antibody degradation (see US2005 / 0238649). Generally, such linkers are substantially insensitive to the extracellular environment. As used herein with respect to a linker, "substantially insensitive to the extracellular environment" means that when the antibody-drug conjugate compound is present in an extracellular environment (e.g., plasma), no more than 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers in a sample of the antibody-drug conjugate compound are cleaved. Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate compound with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma.

[0295] Further exemplary embodiments comprising MMAE or MMAF and various linker components have the following structures, where Ab refers to antibody and p, representing drug loading (or average number of cytostatic drugs per molecule), is 1 to about 8:

[0296] Examples of cleavable linkers combined with auristatins include vcMMAF and vcMMAE (vc is an abbreviation for Val-Cit (valine-citrulline) based linker). TIFF2026021523000006.tif58136

[0297] Other examples include auristatins coupled with non-cleavable linkers, such as mcMMAF (mc is an abbreviation for maleimidocaproyl). TIFF2026021523000007.tif34134

[0298] Cytostatic drug loading is represented by p, the average number of cytostatic drug moieties per antibody molecule (also called the drug / antibody ratio, DAR). Cytostatic drug loading can range from 1 to 20 drug moieties per antibody and can occur on amino acids with useful functional groups, such as amino or sulfhydryl groups, as in lysine or cysteine.

[0299] Depending on the conjugation method, p may be limited by the number of binding sites on the antibody, for example, when the linkage is a cysteine ​​thiol, as in the present invention. Generally, because most cysteine ​​thiol residues in antibodies exist as disulfide bridges, antibodies do not contain many free reactive cysteine ​​thiol groups that can also be conjugated to drug moieties. Therefore, in certain embodiments, antibodies can be reduced with reducing agents such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partially or fully reducing conditions to generate reactive cysteine ​​thiol groups. In certain embodiments, after (partial) reduction of the antibody, up to eight free cysteine ​​thiol groups become available (there are eight cysteines involved in interchain disulfide bonds), and thus the drug loading of the ADCs of the present invention ranges from 1 to about 8.

[0300] In one embodiment, the drug linker moiety is vcMMAE. vcMMAE drug linker moieties and conjugation methods are disclosed in WO2004010957, US7659241, US7829531, US7851437 and US11 / 833,028 (Seattle Genetics, Inc.), which are incorporated herein by reference, and the vcMMAE drug linker moiety is attached to the anti-CD38 antibody at a cysteine ​​using methods similar to those disclosed herein.

[0301] In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF drug linker moiety and conjugation methods are disclosed in US7498298, US11 / 833,954 and WO2005081711 (Seattle Genetics, Inc.), which are incorporated herein by reference, and the mcMMAF drug linker moiety is attached to the anti-CD38 antibody at a cysteine ​​using methods similar to those disclosed herein.

[0302] Once the anti-CD38 antibody drug conjugates are purified, they can be formulated into pharmaceutical compositions using well-known pharmaceutical carriers or excipients.

[0303] In one embodiment, the anti-CD38 antibody is conjugated to a functional nucleic acid molecule. Functional nucleic acids include antisense molecules, interfering nucleic acid molecules (e.g., siRNA molecules), aptamers, ribozymes, triplex-forming molecules, and external guide sequences. An external guide sequence (EGS) is a molecule that binds to a target nucleic acid molecule to form a complex that is recognized by RNase P, which cleaves the target molecule. Functional nucleic acid molecules may act as agonists, inhibitors, regulators, and stimulators of specific activities possessed by the target molecule, or may possess new activities independent of other molecules. Representative examples of methods and technologies supporting the design and use of antisense molecules can also be found in the following non-limiting list of U.S. patents: US5,135,917, US5,294,533, US5,627,158, US5,641,754, US5,691,317, US5,780,607, US5 ,786,138, US5,849,903, US5,856,103, US5,919,772, US5,955,590, US5,990,088, US5,994 ,320, US5,998,602, US6,005,095, US6,007,995, US6,013,522, US6,017,898, US6,018,042, US6,025,198, US6,033,910, US6,040,296, US6,046,004, US6,046,319 and US6,057,437.

[0304] Methods known in the art for conjugating a conjugated molecule, such as an anti-CD38 antibody, to the above-mentioned molecule may be used, including those described by Hunter et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain et al., J. Immunol. Meth. 40, 219 (1981), and Nygren, J. Histchem. and Cytochem. 30, 407 (1982). The binding / conjugation may be carried out by any suitable method. For example, the covalent bond may take the form of a disulfide bond (the anti-CD38 antibody may be engineered to contain an extra cysteine ​​codon, if necessary and appropriate). A toxin molecule derivatized with a sulfhydryl group that reacts with the cysteine ​​of the modified anti-CD38 antibody may also form an immunoconjugate with the anti-CD38 antibody. Alternatively, sulfhydryl groups can be introduced directly into anti-CD38 antibodies using solid-phase polypeptide techniques. For example, introduction of sulfhydryl groups into peptides is described in Hiskey, Peptides 3, 137 (1981). Introduction of sulfhydryl groups into proteins is described in Maasen et al., Eur. J. Biochem. 134, 32 (1983).

[0305] Many types of cytotoxic compounds can also be attached to proteins by using reactive groups on the cytotoxic compound or by using cross-linking agents. A common reactive group that forms stable covalent bonds with amines in vivo is isothiocyanate (Means et al., Chemical Modifications of Proteins (Holden-Day, San Francisco 1971) pp. 105-110). This group reacts preferentially with the ε-amine group of lysine. Maleimide is a commonly used reactive group to form stable covalent bonds in vivo with sulfhydryl groups on cysteine ​​(Ji., Methods Enzymol 91, 580-609 (1983)). Monoclonal antibodies generally cannot form covalent bonds with radioactive metal ions, but they can be indirectly attached to the antibody through the use of chelating agents covalently bound to the antibody. Chelating agents can also be attached through the amines (Meares et al., Anal. Biochem. 142, 68-78 (1984)) and sulfhydral groups (Koyama, Chem. Abstr. 120, 217262t (1994)) of amino acid residues, as well as carbohydrate groups (Rodwell et al., PNAS USA 83, 2632-2636 (1986); Quadri et al., Nucl. Med. Biol. 20, 559-570 (1993)). Additionally or alternatively, therapeutic or diagnostic agents can be attached to the hinge region of a reduced antibody component via disulfide bond formation.

[0306] In one embodiment, the present invention provides an anti-CD38 antibody, e.g., a human anti-CD38 antibody, conjugated to a therapeutic moiety, e.g., a cytotoxin, a chemotherapeutic agent, an immunosuppressant, or a radioisotope. Such conjugates are referred to herein as "immunoconjugates." Immunoconjugates containing one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. For a description of these classes of drugs and their mechanisms of action, which are well known in the art, see Goodman et al., Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 8th Ed., Macmillan Publishing Co., 1990. Further techniques for preparing antibody immunotoxins are provided, for example, in Vietta, *Immunol. Today* 14, 252 (1993) and US Pat. No. 5,194,594.

[0307] Suitable therapeutic agents for forming immunoconjugates of the invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, and the like. fluticasone, propranolol, and puromycin, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decaffeinated, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromide, anticoagulants (e.g., thiamin ... toxins (e.g., diphtheria A chain and its active fragments and hybrid molecules), ricin toxin (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas aeruginosa exotoxin, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, xeronine, mitogenin, restrictocin, phenomycin, enomycin toxin, calicheamicin, and duocarmycin. Therapeutic agents that can be administered in combination with the anti-CD38 antibodies of the invention, as described elsewhere herein, may also be candidates for therapeutic moieties useful for conjugation to the anti-CD38 antibodies of the invention.

[0308] As mentioned above, the drug moiety need not be construed as being limited to classical chemical therapeutic agents. For example, the drug moiety can also be a protein or polypeptide possessing a desired biological activity. In one embodiment, the anti-CD38 antibody of the present invention is linked to a chelator linker, such as tiuxetan, that allows the antibody to be conjugated to a radioisotope.

[0309] bispecific antibody In a further aspect, the present invention relates to bispecific molecules comprising a first antigen-binding site from an anti-CD38 antibody of the invention as described herein above and a second antigen-binding site with a different binding specificity, e.g., binding specificity for a non-overlapping epitope of human effector cells, a human Fc receptor, a T cell receptor, a B cell receptor, or CD38, i.e., bispecific antibodies in which the first and second antigen-binding sites do not cross-block each other for binding to CD38, e.g., when tested as described in Example 3.

[0310] Exemplary bispecific antibody molecules of the invention include: (i) two antibodies conjugated together, one with specificity for CD38 and the other with specificity for a second target; (ii) a single antibody with one chain or arm specific for CD38 and a second chain or arm specific for a second molecule; (iii) a single-chain antibody with specificity for CD38 and also specificity for a second molecule, e.g., via two scFvs linked in tandem by an extra peptide linker; and (iv) a dual variable domain antibody (DVD-Ig), in which the light and heavy chains each contain two variable domains in tandem via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)), (v) chemically linked bispecific (Fab') fragments, (vi) TandAbs, which are the fusion of two single-chain diabodies to generate tetravalent bispecific antibodies with two binding sites for each target antigen, (vii) Flexibodies, which are the combination of scFvs and diabodies to generate multivalent molecules, (viii) so-called "dock-and-lock" molecules based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fabs, can generate trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments, (ix) so-called Scorpion molecules, which comprise, for example, two scFvs fused to opposite ends of a human Fc region, and (x) diabodies. In one embodiment, the bispecific antibodies of the present invention are diabodies, crossbodies, or bispecific antibodies obtained via controlled Fab arm exchange as described herein.

[0311] Examples of platforms useful for preparing bispecific antibodies include, but are not limited to, BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab arm exchange; Genmab, present application).

[0312] Examples of different classes of bispecific antibodies include: asymmetric IgG-like molecules, in which one side of the molecule contains the Fab region or part of the Fab region of at least one antibody and the other side of the molecule contains the Fab region or part of the Fab region of at least one other antibody (in this class there is asymmetry in the Fc region, which can be used for specific binding of the two parts of the molecule); a symmetric IgG-like molecule, each of the two sides of which contains the Fab region or part of the Fab region of at least two different antibodies; IgG fusion molecules in which a full-length IgG antibody is fused to an extra Fab region or part of a Fab region, Fc fusion molecules in which a single chain Fv molecule or a stabilized diabody is fused to an Fcγ region or part thereof; Fab fusion molecules in which different Fab fragments are fused together, ScFv and diabody-based molecules in which different single-chain Fv molecules or different diabodies are fused to each other or to another protein or carrier molecule Including, but not limited to:

[0313] Examples of asymmetric IgG-like molecules include, but are not limited to, Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche) and electrostatically matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (EMD Serono), Biclonic (Merus) and DuoBody (Genmab A / S).

[0314] Examples of symmetric IgG-like molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), and CovX-body (CovX / Pfizer).

[0315] Examples of IgG fusion molecules include, but are not limited to, Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (ImClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche).

[0316] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine - China).

[0317] Examples of Class V bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / Amgen), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0318] Examples of ScFv and diabody-based molecules include, but are not limited to, Bispecific T Cell Engager (BiTE) (Micromet9), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack), and COMBODY (Epigen Biotech).

[0319] In a further aspect, the present invention relates to a bispecific molecule comprising an anti-CD38 antibody of the present invention as described hereinabove and a second binding specificity, e.g., a binding specificity for a human cytokine. In one embodiment, the cytokine is an anti-inflammatory cytokine, e.g., IL-1ra, IL-4, IL-6, IL-10, IL-11, IL-13, IL-16, IFN-α, and TGF-β. In another embodiment, the cytokine is a pro-inflammatory cytokine, e.g., IL-1α, IL-1β, and IL-6. In some embodiments, the binding specificity is for a human effector cell, a human Fc receptor, or a T cell receptor. In one embodiment, the T cell receptor is CD3. In another embodiment, the human Fc receptor is human FcγRI (CD64), human FcγRII (CD32), FcγRIII (CD16), or human Fcα receptor (CD89). Bispecific molecules of the invention can further comprise a third binding specificity, in addition to an anti-CD38 binding specificity and a binding specificity for a human effector cell, a human Fc receptor, or a T cell receptor.

[0320] Exemplary bispecific antibody molecules of the invention include (i) two antibodies conjugated together, one with specificity for CD38 and the other with specificity for a second target, (ii) a single antibody with one chain specific for CD38 and a second chain specific for a second molecule, and (iii) a single chain antibody with specificity for CD38 and a second molecule. In one embodiment, the second molecule is a cancer antigen / tumor-associated antigen, such as CD20, carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), RAGE (renal antigen), alpha-fetoprotein, CAMEL (a CTL-recognized antigen in melanoma), CT antigens (e.g., MAGE-B5, -B6, -C2, -C3 and D, Mage-12, CT10, NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin CA125, etc.), ganglioside antigens, tyrosinase, gp75, C-myc, Mart1, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, and Ep-CAM. In one embodiment, the second molecule is a cancer-associated integrin, such as alpha5beta3 integrin. In one embodiment, the second molecule is an angiogenic factor or other cancer-associated growth factor, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), angiogenin and its receptor, particularly a receptor associated with cancer progression (e.g., one of the HER1 to HER4 receptors). Other cancer progression-associated proteins detailed herein may also be suitable second molecules.

[0321] In an embodiment of the invention, the antibody is a single antibody (non-competing antibody) having one chain specific for a CD38 epitope described herein that contains an aspartic acid at position 202 and a second chain specific for a CD38-specific epitope that does not contain an aspartic acid at position 202. Such an antibody is described, for example, as antibody 003 in WO2006099875.

[0322] In one embodiment, the bispecific antibody of the invention is a diabody.

[0323] Generation of bispecific antibodies by 2-MEA-induced Fab arm exchange An in vitro method for producing bispecific antibodies is described in WO2008119353 (Genmab) and reported by van der Neut-Kolfschoten et al. (Science. 2007 Sep 14;317(5844):1554-7). In this method, bispecific antibodies are formed by "Fab arm" or "half molecule" exchange (exchange of heavy chains and associated light chains) between two monospecific IgG4 or IgG4-like antibodies by incubation under mildly reducing conditions. This Fab arm exchange reaction is the result of a disulfide bond isomerization reaction in which the inter-heavy chain disulfide bond in the hinge region of a monospecific antibody is reduced, and the resulting free cysteine ​​forms a new inter-heavy chain disulfide bond with a cysteine ​​residue of another antibody molecule with a different specificity. The resulting product is a bispecific antibody with two Fab arms of different sequences.

[0324] In a novel invention, knowledge of this natural IgG4 Fab arm exchange is applied to create a method for producing stable IgG1-based bispecific antibodies. The bispecific antibody products generated by this method, described below, no longer involve IgG4 Fab arm exchange. The basis of this method is the use of complementary CH3 domains that promote heterodimer formation under specific assay conditions. To enable the production of bispecific antibodies by this method, IgG1 molecules were generated with specific mutations in the CH3 domain in one of the parent IgG1 antibodies: T350I, K370T, and F405L mutations in the other parent IgG1 antibody, and K409R mutation.

[0325] To generate bispecific antibodies, these two parent antibodies were incubated with 25 mM 2-mercaptoethylamine HCl (2-MEA) at a final concentration of 0.5 mg / mL (equimolar) in a total volume of 100 μL of TE for 90 minutes at 37°C. The reducing agent 2-MEA was removed using a spin column (Microcon centrifugal filter, 30k, Millipore) according to the manufacturer's protocol, and the reduction reaction was stopped. This method can also be used to generate the following bispecific antibodies: A bispecific antibody wherein the anti-CD38 antibody is 025, 026, 028, 049, or 056, and the second binding moiety is an anti-CD3 antibody. A bispecific antibody wherein the anti-CD38 antibody is 025, 026, 028, 049, or 056, and the second binding moiety is an anti-CD20 antibody, e.g., ofatumumab. A bispecific antibody wherein the anti-CD38 antibody is 025, 026, 028, 049, or 056, and the second binding moiety is an anti-CD16 antibody. A bispecific antibody wherein the anti-CD38 antibody is 025, 026, 028, 049, or 056, and the second binding moiety is an anti-CD32 antibody. A bispecific antibody wherein the anti-CD38 antibody is 025, 026, 028, 049, or 056, and the second binding moiety is an anti-CD64 antibody.

[0326] Nucleic acids, vectors, host cells, and methods for producing antibodies of the invention In a further aspect, the present invention relates to nucleic acids encoding (parts of) the antibodies of the present invention and expression vectors comprising such nucleic acids.

[0327] In one embodiment, an expression vector of the present invention comprises a nucleotide sequence encoding one or more of the amino acid sequences selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:5.

[0328] In a further embodiment, the expression vector further comprises a nucleotide sequence encoding the constant region of the light chain, the heavy chain, or both the light and heavy chains of an antibody, eg, a human antibody.

[0329] Such expression vectors can also be used for the recombinant production of the antibodies of the invention.

[0330] Expression vectors in the context of the present invention can be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising an appropriate set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the anti-CD38 antibody is contained in a naked DNA or RNA vector, including, for example, a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compact nucleic acid vector (e.g., as described in US 6,077,835 and / or WO 00 / 70087), a plasmid vector such as pBR322, pUC19 / 18, or pUC118 / 119, a "midge" minimal size nucleic acid vector (e.g., as described in Schakowski et al., Mol Thr 3, 793-800 (2001)), or a precipitated nucleic acid vector construct, e.g., a CaPO4 precipitated construct (e.g., as described in WO 00 / 46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725-730 (2001)). (1978) and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, e.g., US 5,589,466 and US 5,973,972).

[0331] In one embodiment, the vector is suitable for expressing an anti-CD38 antibody in bacterial cells. In another embodiment, the expression vector can be a vector suitable for expression in a yeast system. Most commonly, the vector is a vector suitable for expressing an antibody of the invention in mammalian cells, such as CHO, HEK, or PER.C6® cells (human cell lines developed by DSM and Crucell NV, the Netherlands). Another suitable vector system is the glutamine synthetase (GS) vector system developed by Lonza Biologics (see, e.g., EP 216846, US 5981216, WO 8704462, EP 323997, US 5591639, US 5658759, EP 338841, US 5879936, and US 5891693).

[0332] In the expression vectors of the invention, the nucleic acid encoding the anti-CD38 antibody may contain or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include a strong expression promoter (e.g., the human CMV IE promoter / enhancer and the RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), an efficient poly(A) termination sequence, an origin of replication of the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also contain an inducible promoter, as opposed to a constitutive promoter such as CMV IE.

[0333] In one embodiment, an expression vector encoding an anti-CD38 antibody may be placed in and / or delivered to a host cell or animal via a viral vector.

[0334] In yet a further aspect, the present invention relates to recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the antibodies of the present invention described herein. Exemplary host cells include yeast, bacteria, and mammalian cells, such as CHO, HEK, or PER.C6® cells. For example, in one embodiment, the present invention provides a cell that contains a nucleic acid stably integrated into the cellular genome, comprising a sequence coding for expression of an anti-CD38 antibody of the present invention. In another embodiment, the present invention provides a cell that contains a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising a sequence coding for expression of an anti-CD38 antibody of the present invention.

[0335] In a further aspect, the present invention relates to hybridomas that produce the antibodies of the present invention described herein. In yet a further aspect, the present invention relates to transgenic non-human animals that contain nucleic acids encoding a human heavy chain and a human light chain, and the animals or plants that produce the antibodies of the present invention. The generation of such hybridomas and transgenic animals is described above.

[0336] In a further aspect, the present invention relates to a method of producing an anti-CD38 antibody of the present invention, comprising the steps of: a) culturing the hybridoma or host cell of the invention as described herein above, and b) purifying the antibody of the invention from the culture medium.

[0337] Pharmaceutical Compositions In yet a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: an anti-CD38 antibody described herein, and Pharmaceutically acceptable carrier The present invention relates to a pharmaceutical composition comprising:

[0338] Pharmaceutical compositions can be formulated with pharmaceutically acceptable carriers or diluents and other known adjuvants and excipients according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Pharmaceutical compositions of the present invention can also include diluents, fillers, salts, buffers, surfactants (e.g., non-ionic surfactants such as Tween-20 or Tween-80), stabilizers (e.g., sugars or non-protein amino acids), preservatives, tissue fixatives, solubilizing agents and / or other materials suitable for inclusion in pharmaceutical compositions.

[0339] Pharmaceutically acceptable carriers or diluents and other known adjuvants and excipients should be suitable for the selected compound of the present invention and the selected administration form. The suitability of carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative effect on the desired biological properties of the selected compound of the present invention or pharmaceutical composition (e.g., substantially less effect on antigen binding (relative inhibition rate of 10% or less, relative inhibition rate of 5% or less, etc.)).

[0340] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to provide an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and administration form without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention or its amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0341] The pharmaceutical composition can be administered by any suitable route and form. Suitable routes for administering the compounds of the present invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art.

[0342] In one embodiment, the pharmaceutical compositions of the present invention are administered parenterally.

[0343] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include epithelial, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intrathecal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion.

[0344] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0345] In one embodiment, the compounds of the invention are administered in crystalline form by subcutaneous injection. See Yang et al., PNAS USA, 100(12), 6934-6939 (2003).

[0346] Pharmaceutically acceptable carriers include all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption delaying agents, and the like that are physiologically compatible with the compounds of the present invention.

[0347] Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils, carboxymethylcellulose, colloidal solutions, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical arts.

[0348] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium bisulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0349] The pharmaceutical compositions of the present invention may also contain isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride.

[0350] The pharmaceutical compositions of the present invention can also contain one or more adjuvants suitable for the selected route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, antiseptics or buffers, which may increase the shelf life or expiration date of the pharmaceutical compositions.The compounds of the present invention can also be prepared with carriers that protect the compound from rapid release, such as sustained-release formulations, including implants, transdermal patches and microencapsulated delivery systems.Methods for preparing such formulations are generally known to those skilled in the art.See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0351] The pharmaceutical compositions of the present invention may contain one antibody of the present invention, or may contain a combination of two or more antibodies of the present invention.

[0352] therapeutic use In another aspect, the present invention relates to an antibody of the present invention as described herein for use as a medicament.

[0353] The anti-CD38 antibodies of the present invention have numerous therapeutic uses, including the treatment of disorders involving cells expressing CD38. For example, the antibodies may be administered to cells in culture, e.g., in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat or prevent various disorders. As used herein, the term "subject" is intended to include both human and non-human animals that respond to the antibodies. A subject may also include a human patient with a disorder that may be corrected or ameliorated by modulating CD38 function, such as enzyme activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation and / or induction of lysis and / or elimination / reduction of the number of CD38-expressing cells.

[0354] For example, anti-CD38 antibodies can also be used to induce one or more of the following biological activities in vivo or in vitro: modulation of CD38 function (e.g., enzymatic activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation, and / or induction of lysis), killing of CD38-expressing cells, mediating phagocytosis or ADCC of CD38-expressing cells in the presence of human effector cells, and killing of CD38-expressing cells by mediating CDC of CD38-expressing cells or apoptosis in the presence of complement.

[0355] The present invention provides methods for treating or preventing a disorder involving cells expressing CD38 in a subject, the methods comprising administering to a subject in need thereof a therapeutically effective amount of an anti-CD38 antibody of the present invention. Such methods comprise administering to the subject an amount of an anti-CD38 antibody of the present invention effective to treat or prevent the disorder.

[0356] In one embodiment of the invention, the disorder involving cells expressing CD38 can also be a tumorigenic disorder such as cancer, i.e., a disorder characterized by the presence of tumor cells expressing CD38, e.g., B-cell lymphoma, plasma cell malignancies, T / NK-cell lymphoma, and myeloid malignancies.

[0357] Examples of such tumorigenic diseases are B-cell lymphomas / leukemias, including precursor B-cell lymphoblastic leukemia / lymphoma and B-cell non-Hodgkin's lymphoma, acute promyelocytic leukemia, acute lymphoblastic leukemia, and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), and leukemia-associated lymphomas (LEMs). ), follicular lymphoma (FL) including low-, intermediate-, and high-grade forms, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT, nodal, and splenic types), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasma cell myeloma, plasma cell leukemia, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, plasma cell leukemia, and anaplastic large cell lymphoma (ALCL).

[0358] In one embodiment, the disorder involving cells expressing CD38 is multiple myeloma.

[0359] In one embodiment, the disorder involving cells expressing CD38 is selected from chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (adult) (AML), mantle cell lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma.

[0360] In one embodiment, the disorder involving cells expressing CD38 is non-small cell lung cancer (NSCLC).

[0361] Examples of B-cell non-Hodgkin's lymphomas are lymphomatoid granulomatosis, primary effusion lymphoma, intravascular large B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain disease (including gamma, mu, and alpha disease), lymphomas induced by immunosuppressant treatment, such as cyclosporine-induced lymphoma and methotrexate-induced lymphoma.

[0362] In one embodiment of the invention, the disorder involving cells expressing CD38 is Hodgkin's lymphoma.

[0363] Other examples of disorders involving cells expressing CD38 include malignancies derived from T and NK cells, such as mature T cell and NK cell neoplasms including T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia / lymphoma, extranodal NK / T cell lymphoma, nasal type, enteropathy-associated T cell lymphoma, hepatosplenic T cell lymphoma, subcutaneous panniculitis-like T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders (primary cutaneous anaplastic large cell lymphoma C-ALCL, lymphomatoid papulosis, borderline lesions), angioimmunoblastic T lymphoma, peripheral T cell lymphoma, unspecified, and anaplastic large cell lymphoma.

[0364] Examples of malignant diseases derived from myeloid cells include acute myeloid leukemia, including acute promyelocytic leukemia, and chronic myeloproliferative disorders, including chronic myelogenous leukemia.

[0365] In another embodiment of the present invention, the disorder involving CD38-expressing cells is an immune disorder involving CD38-expressing B cells, macrophages, plasma cells, monocytes and T cells, such as inflammatory and / or autoimmune diseases. Examples of immune disorders involving CD38-expressing B cells, plasma cells, monocytes and T cells include autoimmune disorders such as psoriasis, psoriatic arthritis, dermatitis, systemic sclerosis and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjogren's syndrome, juvenile-onset diabetes, Reiter's disease, Behcet's disease, These include immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune thrombocytopenias such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omenn's syndrome, chronic renal failure, acute infectious mononucleosis, multiple sclerosis, HIV, and herpesvirus-related diseases. Further examples include severe acute respiratory distress syndrome and chorioretinitis. Additionally, other diseases and disorders caused by or mediated by infection of B cells by viruses such as Epstein-Barr virus (EBV) are included.

[0366] In one embodiment, the disorder involving cells expressing CD38 is rheumatoid arthritis.

[0367] Further examples of inflammatory, immune and / or autoimmune disorders in which autoantibodies and / or excessive B and T lymphocyte activity are prominent that may also be treated in accordance with the present invention include vasculitides and other vascular disorders, such as microscopic polyvasculitis, Churg-Strauss syndrome and other ANCA-associated vasculitides, polyarteritis nodosa, essential cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, Kawasaki disease, Takayasu's arteritis, giant cell arteritis, Henoch-Schönlein purpura, primary or isolated cerebral vasculitis, erythema nodosum, thromboarteritis obliterans, thrombotic thrombocytopenic purpura (including hemolytic uremic syndrome), and secondary vasculitides, such as cutaneous leukocytoclastic vasculitis (e.g., secondary to hepatitis B, hepatitis C, Waldenstrom's macroglobulinemia, B-cell neoplasia, rheumatoid arthritis, Sjogren's syndrome, or systemic lupus erythematosus). Further examples include erythema nodosum, allergic vasculitis, panniculitis, Weber-Christian disease, hyperglobulinemic purpura and Buerger's disease, skin disorders such as contact dermatitis, linear IgA dermatosis, vitiligo, pyoderma gangrenosum, epidermolysis bullosa acquisita, pemphigus vulgaris (including cicatricial pemphigoid and bullous pemphigoid), alopecia areata (including alopecia universalis and alopecia totalis), dermatitis herpetiformis, erythema multiforme and chronic autoimmune urticaria (angioneurotic edema and seizures). Examples of such disorders include: immune cytopenias, such as autoimmune neutropenia and pure red cell aplasia, connective tissue disorders, such as CNS lupus, discoid lupus erythematosus, CREST syndrome, mixed connective tissue disease, polymyositis / dermatomyositis, inclusion body myositis, secondary amyloidosis, cryoglobulinemia types I and II, fibromyalgia, phospholipid syndrome, secondary hemophilia, relapsing polychondritis, sarcoidosis, stiff man syndrome, and rheumatic fever. Further examples are eosinophilic fasciitis, arthritis, such as ankylosing spondylitis, juvenile chronic arthritis, adult Still's disease, and SAPHO syndrome. Further examples are sacroiliitis, reactive arthritis, Still's disease and gout, blood disorders such as aplastic anemia, primary hemolytic anemia (including cold agglutinin syndrome), hemolytic anemia secondary to CLL or systemic lupus erythematosus, POEMS syndrome, pernicious anemia and Waldenstrom's hyperglobulinemic purpura.Further examples are agranulocytosis, autoimmune neutropenia, Franklin's disease, Seligman's disease, gamma heavy chain disease, paraneoplastic syndromes secondary to thymoma and lymphoma, paraneoplastic syndromes secondary to thymoma and lymphoma, and factor VIII inhibitor formation, endocrine disorders such as polyendocrine disorders and Addison's disease. Further examples are autoimmune hypoglycemia, autoimmune hypothyroidism, autoimmune insulin syndrome, De Quervain's thyroiditis and insulin receptor antibody-mediated insulin resistance, hepatic and gastrointestinal disorders such as celiac disease, Whipple's disease, primary biliary cirrhosis, chronic active hepatitis, and primary sclerosing cholangitis. Further examples are autoimmune gastritis, renal disorders such as rapidly progressive glomerulonephritis, poststreptococcal nephritis, Goodpasture's syndrome, membranous glomerulonephritis, and cryoglobulinemia nephritis. Further examples include minimal change disease, neurological disorders such as autoimmune neuropathy, mononeuritis multiplex, Lambert-Eaton myasthenic syndrome, Sydenham chorea, tabes dorsalis, and Guillain-Barré syndrome. Further examples include myelopathy / tropical spastic paraparesis, myasthenia gravis, acute inflammatory demyelinating polyneuropathy and chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, cardiac and pulmonary disorders such as COPD, fibrosing alveolitis, bronchiolitis obliterans, allergic aspergillosis, cystic fibrosis, Löffler's syndrome, myocarditis, and pericarditis. Further examples include hypersensitivity pneumonitis and paraneoplastic syndromes secondary to lung cancer, and allergic disorders such as bronchial asthma and hyper-IgE syndrome. Further examples are amaurosis fugax, ophthalmologic disorders such as idiopathic chorioretinitis, infectious diseases such as parvovirus B infection (including hands-and-socks syndrome), gynecological disorders such as recurrent miscarriage, recurrent fetal loss, and intrauterine growth retardation. Further examples are paraneoplastic syndromes secondary to gynecological neoplasms, male genital disorders such as paraneoplastic syndromes secondary to testicular neoplasms, and transplant-derived disorders such as allograft and xenograft rejection and graft-versus-host disease.

[0368] Dosage regimens in the above-described methods of treatment and use are adjusted to provide the optimum desired response (e.g., therapeutic response), e.g., a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation.

[0369] The effective dosage and administration regimen of the anti-CD38 antibody depends on the disease or condition being treated and can be determined by one of ordinary skill in the art. An exemplary, non-limiting range for a therapeutically effective amount of a compound of the invention is about 0.005-100 mg / kg, e.g., 0.05-100 mg / kg or 1-100 mg / kg, e.g., about 0.1-50 mg / kg, e.g., about 0.1-20 mg / kg, e.g., about 0.1-10 mg / kg, e.g., about 0.1, 0.3, about 0.5, about 1, 2, 3, 4, 8, 16, or 24 mg / kg.

[0370] Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and can be administered, for example, in close proximity to the target site. If desired, the effective daily amount of the pharmaceutical composition can be administered as two, three, four, five, six, or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms.

[0371] In one embodiment, the anti-CD38 antibody is administered at a dose of 10 to 500 mg / m 2 , e.g., 200-400 mg / m 2 Such administration may be repeated, for example, 1 to 8 times, for example, 3 to 5 times. Administration may also be by continuous infusion over a period of 2 to 24 hours, for example, 2 to 12 hours.

[0372] In one embodiment, the anti-CD38 antibody may be administered by slow continuous infusion over an extended period of time, for example, greater than 24 hours, to reduce toxic side effects.

[0373] In one embodiment, the anti-CD38 antibody may be administered in a weekly dose of 250 mg to 2000 mg, e.g., 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg, or 2000 mg, up to 8 times, e.g., 4 to 6 times. Administration may also be by continuous infusion over a period of 2 to 24 hours, e.g., 2 to 12 hours. Such a regimen may be repeated, e.g., one or more times as needed, e.g., after 6 or 12 months.

[0374] In one embodiment, the anti-CD38 antibody can also be administered as a maintenance therapy, eg, once a week for a period of six months or more.

[0375] In another embodiment, the anti-CD38 antibodies can be administered in a regimen comprising a single infusion of an anti-CD38 antibody of the invention, followed by an infusion of an anti-CD38 antibody of the invention conjugated to a radioisotope, which regimen can be repeated, for example, 7-9 days later.

[0376] As a non-limiting example, treatments of the present invention may be administered at a daily dose of about 0.1 to 100 mg / kg, e.g., 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg of a compound of the present invention per day, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg of a compound of the present invention per day, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, It may also be provided using single or divided doses every 24, 12, 8, 6, 4 or 2 hours, or any combination thereof, for at least one day on day 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or for at least one week on week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof.

[0377] An "effective amount" of a tumor treatment can also be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer can also be evaluated in animal model systems predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by testing the compound's ability to inhibit cell proliferation or induce apoptosis using in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound may reduce tumor size or otherwise ameliorate symptoms in a patient.

[0378] A "therapeutically effective amount" for rheumatoid arthritis is a dose that produces at least an ACR 20 Preliminary remission definition, e.g., at least ACR 50 Preliminary remission definition, e.g., at least ACR 70 This may also provide a preliminary definition of remission.

[0379] ACR 20 The preliminary remission definition is defined as follows: ≥ 20% remission in tender joint counts (TJC) and swollen joint counts (SJC) and ≥ 20% remission in three of the following five assessments: patient pain assessment (VAS), patient global assessment (VAS), physician global assessment (VAS), patient self-rated disability (HAQ), and acute phase reactants (CRP or ESR). ACR 50 and ACR 70 are defined as ≧50% remission and ≧70% remission, respectively. For further details, see Felson et al., American College of Rheumatology Preliminary Definition of Improvement in Rheumatoid Arthritis; Arthritis Rheumatism 38, 727-735 (1995).

[0380] Alternatively, a therapeutically effective amount for rheumatoid arthritis may be measured by the DAS (Disease Activity Score), including DAS28 and / or DAS56, as defined by EULAR.

[0381] Anti-CD38 antibodies can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events during cancer progression, and / or reduce the risk of recurrence if the cancer is in remission. This can be particularly useful in patients in whom other biological factors make it difficult to detect tumors known to be present.

[0382] Combination therapy The anti-CD38 antibodies of the invention can also be administered in combination therapy, i.e., in conjunction with other therapeutic agents relevant to the disease or condition being treated. Such administration can be simultaneous, separate, or sequential. When administered simultaneously, the agents can be administered as a single composition or as separate compositions, as appropriate.

[0383] Accordingly, the present invention provides methods for treating disorders involving cells expressing CD38, as described above, which methods comprise administering an anti-CD38 antibody of the present invention in combination with one or more additional therapeutic agents as described below.

[0384] In an embodiment of the present invention, the antibody of the present invention is administered in combination with another anti-CD38 antibody. Such antibodies are described in the present invention and in the prior art. Specifically, the antibody is described in WO2006099875. More specifically, the combination of the present anti-CD38 antibody with a non-cross-blocking anti-CD38 antibody, such as antibody 003 described in WO2006099875, is an embodiment of the present invention.

[0385] In one embodiment, the combination therapy may also include administration of a composition of the invention with at least one cytotoxic agent, at least one chemotherapeutic agent, at least one anti-angiogenic agent, at least one anti-inflammatory agent, and / or at least one immunosuppressant and / or immunomodulatory agent.

[0386] In one embodiment, the invention provides a method of treating a disorder involving cells expressing CD38, e.g., cancer, in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the invention and at least one chemotherapeutic agent.

[0387] In one embodiment, the present invention provides a method of treating multiple myeloma, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the present invention and at least one chemotherapeutic agent.

[0388] In one embodiment, such chemotherapeutic agents may be selected from antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine, and the like.

[0389] In one embodiment, such chemotherapeutic agents may be selected from alkylating agents such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin and similar agents.

[0390] In one embodiment, such chemotherapeutic agents may be selected from antibiotics such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC), and the like.

[0391] In one embodiment, such chemotherapeutic agents may be selected from antimitotic agents, such as taxanes, eg, docetaxel and paclitaxel, and vinca alkaloids, eg, vindesine, vincristine, vinblastine, and vinorelbine.

[0392] In one embodiment, such chemotherapeutic agents may be selected from topoisomerase inhibitors, such as topotecan.

[0393] In one embodiment, such chemotherapeutic agents can be selected from growth factor inhibitors, such as inhibitors of ErbB1 (EGFR) (e.g., gefitinib (Iressa®), cetuximab (Erbitux®), erlotinib (Tarceva®), HuMax-EGFr (zalutumumab, 2F8 disclosed in WO 2002 / 100348) and similar agents), inhibitors of ErbB2 (Her2 / neu) (e.g., trastuzumab (Herceptin®) and similar agents), and similar agents. In one embodiment, such growth factor inhibitors can be farnesyltransferase inhibitors, such as SCH-66336 and R115777. In one embodiment, such growth factor inhibitors can be vascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab (Avastin®).

[0394] In one embodiment, such chemotherapeutic agents can also be tyrosine kinase inhibitors, such as imatinib (Gleevec, Gleevec STI571), lapatinib, PTK787 / ZK222584 and the like.

[0395] In one embodiment, such chemotherapeutic agents can also be histone deacetylase inhibitors. Examples of such histone deacetylase inhibitors include hydroxamic acid-based hybrid polar compounds, such as SAHA (suberoylanilide hydroxamic acid).

[0396] In one embodiment, such a chemotherapeutic agent may be a P38a MAP kinase inhibitor, such as SCIO-469.

[0397] In one embodiment, the present invention provides a method of treating a disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the present invention and at least one inhibitor of angiogenesis, neovascularization and / or other angiogenesis.

[0398] In one embodiment, the present invention provides a method of treating multiple myeloma, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the present invention and at least one inhibitor of angiogenesis, neovascularization and / or other angiogenesis.

[0399] Examples of such angiogenesis inhibitors include urokinase inhibitors, matrix metalloproteinase inhibitors (e.g., marimastat, neovastat, BAY 12-9566, AG 3340, BMS-275291, and similar agents), inhibitors of endothelial cell migration and proliferation (e.g., TNP-470, squalamine, 2-methoxyestradiol, combretastatin, endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison, NJ), R115777 (Janssen Pharmaceutica Inc, Titusville, NJ) and similar agents), antagonists of angiogenic growth factors (e.g., ZD6474, SU6668, antibodies against angiogenic factors and / or their receptors (e.g., VEGF, bFGF, and angiopoietin-1), thalidomide (Thalomid®), thalidomide analogs (e.g., CC-5013 (lenalidomide, Revlimid™) and CC4047 (Actimid™), Sugen 5416, SU5402, anti-angiogenic ribozymes (e.g., angiozyme), interferon alpha (e.g., interferon alpha 2a), suramin and the like), VEGF-R kinase inhibitors and other anti-angiogenic tyrosine kinase inhibitors (e.g., SU011248), inhibitors of endothelial-specific integrin / survival signaling (e.g., vitaxin and the like), copper antagonists / chelators (e.g., tetrathiomolybdate, captopril and the like), carboxyamidotriazole (CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E, and nucleotide molecules that inhibit angiogenesis (e.g., antisense VEGF-cDNA, cDNA encoding angiostatin, cDNA encoding p53, and cDNA encoding defective VEGF receptor-2) and the like.

[0400] Other examples of such inhibitors of angiogenesis, neovascularization and / or other angiogenesis are anti-angiogenic heparin derivatives and related molecules (e.g., heparinase III), temozolomide, NK4, macrophage migration inhibitory factor (MIF), cyclooxygenase-2 inhibitors, inhibitors of hypoxia inducible factor 1, anti-angiogenic soy isoflavones, oltipraz, fumagillin and its analogs, somatostatin analogs, pentosan polysulfate, tecogalan sodium, dalteparin, tumstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin, antibodies against other related targets (e.g., anti-α-v / β-3 integrin and anti-quininostatin mAbs) and similar agents.

[0401] In one embodiment, the invention provides use of an anti-CD38 antibody of the invention for the preparation of a pharmaceutical composition to be administered in combination with thalidomide (Thalomid®), a thalidomide analog (e.g., CC-5013 (lenalidomide, Revlimid™) and / or CC4047 (Actimid™). In a further embodiment, the invention provides use of an anti-CD38 antibody of the invention for the preparation of a pharmaceutical composition to be administered in combination with thalidomide.

[0402] In one embodiment, the invention provides use of an anti-CD38 antibody of the invention for the preparation of a pharmaceutical composition to be administered in combination with an anti-CD20 antibody, such as rituximab (Rituxan®, MabThera®), a human monoclonal anti-CD20 antibody disclosed in WO2004 / 035607, such as 11B8, 2F2 (ofatumumab, Alzera®), or 7D8.

[0403] In one embodiment, a therapeutic agent for combination with an anti-CD38 antibody of the invention to treat disorders such as those described above can also be a proteosome inhibitor, such as bortezomib (Velcade®).

[0404] In one embodiment, a therapeutic agent for use in combination with an anti-CD38 antibody of the present invention to treat disorders such as those described above can be a corticosteroid, such as prednisone, prednisolone, dexamethasone, and the like.

[0405] In one embodiment, an anti-CD38 antibody of the invention is used in combination with lenalidomide and dexamethasone to treat a disorder as described above, eg, multiple myeloma, eg, relapsed multiple myeloma.

[0406] In one embodiment, an anti-CD38 antibody of the invention is used in combination with bortezomib and dexamethasone to treat a disorder such as those described above, eg, multiple myeloma, eg, relapsed multiple myeloma.

[0407] In one embodiment, an anti-CD38 antibody of the invention is used in combination with bortezomib and prednisolone to treat a disorder such as those described above, eg, multiple myeloma, eg, relapsed multiple myeloma.

[0408] In one embodiment, a therapeutic agent used in combination with an anti-CD38 antibody of the invention to treat such disorders can also be an anti-cancer immunogen, such as a cancer antigen / tumor-associated antigen (e.g., epithelial cell adhesion molecule (EpCAM / TACSTD1), mucin 1 (MUC1), oncofetal antigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, a cancer-associated viral vaccine (e.g., human papillomavirus vaccine), tumor-derived heat shock protein, and the like. Additionally or alternatively, many other suitable cancer antigens / tumor-associated antigens described elsewhere herein and the like molecules known in the art can also be used in such embodiments. Anti-cancer immunogenic peptides also include anti-idiotype "vaccines," such as BEC2 anti-idiotype antibody, mitumomab, CeaVac and related anti-idiotype antibodies, anti-idiotype antibodies against the MG7 antibody, and other anti-cancer anti-idiotype antibodies (e.g., Birebent et al. al., Vaccine. 21(15), 1601-12 (2003), Li et al., Chin Med J. (Engl). 114(9), 962-6 (2001), Schmitt et al., Hydridoma. 13(5), 389-96 (1994), Maloney et al., Hybridoma. 4(3), 191-209 (1985), Raychardhuri et al., J Immunol. 137(5), 1743-9 (1986), Pohl et al., Int J Cancer. 50(6), 958-67 (1992), Bohlen et al., Cytokines Mol Ther. 2(4), 231-8 (1996) and Maruyama, J Immunol Method. 264(1-2), 121-33 (2002).Such anti-idiotype antibodies may optionally be conjugated to a carrier, which may be a synthetic (usually inert) molecular carrier, a protein (e.g., keyhole limpet hemocyanin (KLH)) (see, e.g., Ochi et al., Eur J Immunol. 17(11), 1645-8 (1987)), or a cell (e.g., an erythrocyte; see, e.g., Wi et al., J Immunol Methods. 122(2), 227-34 (1989)).

[0409] In one embodiment, therapeutic agents for use in combination with the anti-CD38 antibodies of the invention to treat disorders such as those described above can be bisphosphonates. Examples of potentially suitable bisphosphonates are pamidronate (Alegica®), zoledronic acid (Zometa®), clodronate (Bonefos®), risedronate (Actonel®), ibandronate (Boniva®), etidronate (Didronel®), alendronate (Fosamax®), tiludronate (Skelid®), incadronate (Yamanouchi Pharmaceutical), and minodronate (YM529, Yamanouchi).

[0410] In one embodiment, a therapeutic agent for use in combination with an anti-CD38 antibody of the invention to treat such disorders can also be a colony-stimulating factor. Examples of suitable colony-stimulating factors are granulocyte colony-stimulating factors (G-CSFs), such as filgrastim (Neupogen®) and pegfilgrastim (Neulasta®), and granulocyte-macrophage colony-stimulating factors (GM-CSFs), such as sargramostim (Leukine®).

[0411] In one embodiment, a therapeutic agent for use in combination with an anti-CD38 antibody of the invention to treat a disorder such as those described above can also be an erythropoietic agent. Examples of suitable erythropoietic agents are erythropoietin (EPO), such as epoetin alfa (e.g., Procrit®, Epogen®, and Eprex®) and epoetin beta (e.g., Neorecormon®), and erythropoiesis-stimulating protein (e.g., Aranesp®).

[0412] In one embodiment, therapeutic agents for use in combination with the anti-CD38 antibodies of the present invention to treat disorders such as those described above can be anti-cancer cytokines, chemokines, or combinations thereof. Examples of suitable cytokines and growth factors include IFNγ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα (e.g., IFNα2b), IFNβ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα. Suitable chemokines can also include Glu-Leu-Arg (ELR)-negative chemokines, such as IP-10, MCP-3, MIG, and SDF-1α of the human CXC and CC chemokine families. Suitable cytokines include cytokine derivatives, cytokine mutants, cytokine fragments, and cytokine fusion proteins. Alternatively or additionally, these and other methods or uses involving naturally occurring peptide-encoding nucleic acids herein may be carried out by "gene activation" and homologous recombination gene upregulation techniques such as those described in US 5,968,502, US 6,063,630 and US 6,187,305 and EP 0505500.

[0413] In one embodiment, a therapeutic agent for use in combination with an anti-CD38 antibody of the invention to treat such disorders can also be an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fcα or Fcγ receptors. Examples of agents suitable for this use include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte colony-stimulating factors (G-CSFs) such as filgrastim (Neupogen®) and pegfilgrastim (Neulasta®), and granulocyte-macrophage colony-stimulating factors (GM-CSFs) such as sargramostim (Leukine®), interferon-γ (IFN-γ), and tumor necrosis factors (TNFs).

[0414] In one embodiment, a therapeutic agent for use in combination with an anti-CD38 antibody of the present invention to treat disorders such as those described above can also be a cell cycle control / apoptosis regulator (or "regulator"). Cell cycle control / apoptosis regulators can also include (i) cell cycle control / apoptosis regulators, such as molecules that target and modulate cdc-25 (e.g., NSC 663284), (ii) cyclin-dependent kinase regulators that overstimulate the cell cycle (e.g., flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (e.g., BIBR1532, SOT-095, GRN163, and compositions described in, for example, US Pat. No. 6,440,735 and US Pat. No. 6,713,055). Non-limiting examples of molecules that interfere with the apoptotic pathway include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), agents that induce NF-κB blockade resulting in inhibition of IL-6 production, antibodies that activate the TRAIL receptor, IFN, antisense Bcl-2, and As2O3 (arsenic trioxide, Trisenox®).

[0415] In one embodiment, therapeutic agents for use in combination with the anti-CD38 antibodies of the invention to treat disorders such as those described above can also be hormone modulating agents, such as agents useful in anti-androgen and anti-estrogen therapy. Examples of such hormone-modulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / estinyl, antiandrogens (e.g., flutamide / eulexin), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone / provera, megestrol acepate / megace), corticosteroids (e.g., hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs thereof and other LHRH antagonists, e.g., buserelin and goserelin), aromatase inhibitors (e.g., anastrazole / arimidex, aminoglutethimide / citraden, exemestane), hormone inhibitors (e.g., octreotide / sandostatin) and similar agents.

[0416] In one embodiment, a therapeutic agent for use in combination with an anti-CD38 antibody of the present invention to treat such disorders can also be an anti-allergic agent (e.g., a small molecule compound, protein, glycoprotein, or antibody that reduces tolerance to tumor and cancer antigens). An example of such a compound is a molecule that blocks the activity of CTLA-4, such as MDX-010 (Phan et al., PNAS USA 100, 8372 (2003)).

[0417] In one embodiment, a therapeutic agent for use in combination with an anti-CD38 antibody of the invention to treat such disorders can also be a nucleic acid or vector comprising a tumor suppressor gene, such as a replication-deficient adenovirus encoding human recombinant wild-type p53 / SCH58500, an antisense nucleic acid targeted to an oncogene, a mutated gene, or a deregulated gene, or an siRNA targeted to a mutated gene or a deregulated gene. Examples of tumor suppressor targets include, for example, BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1, and DCC.

[0418] In one embodiment, a therapeutic agent for use in combination with an anti-CD38 antibody of the invention to treat disorders such as those described above can also be an anti-cancer nucleic acid, such as Genasense (augmelocene) / G3139), LY900003 (ISIS 3521), ISIS 2503, OGX-011 (ISIS 112989), LE-AON / LEraf-AON (liposome-encapsulated c-raf antisense oligonucleotide / ISIS-5132), MG98, and other antisense nucleic acids targeting PKCα, clusterin, IGFBP, protein kinase A, cyclin D1, or Bcl-2h.

[0419] In one embodiment, a therapeutic agent for use in combination with an anti-CD38 antibody of the present invention to treat such disorders can also be an anti-cancer inhibitory RNA molecule (see, e.g., Lin et al., Curr Cancer Drug Targets. 1(3), 241-7 (2001); Erratum in: Curr Cancer Drug Targets. 3(3), 237 (2003); Lima et al., Cancer Gene Ther. 11(5), 309-16 (2004); Grzmil et al., Int J Oncol. 4(1), 97-105 (2004); Collis et al., Int J Radiat Oncol Biol Phys. 57(2 Suppl), S144 (2003); Yang et al., Oncogene. 22(36), 5694-701 (2003); and Zhang et al., Biochem Biophys Res Commun. 303(4), 1169-78 (2003).

[0420] The compositions and combined administration methods of the present invention also include the administration of nucleic acid vaccines, such as naked DNA vaccines, encoding such cancer antigens / tumor-associated antigens (see, e.g., US 5,589,466, US 5,593,972, US 5,703,057, US 5,879,687, US 6,235,523 and US 6,387,888). In one embodiment, the combined administration method and / or combination composition comprises an autologous vaccine composition. In one embodiment, the combination composition and / or combination administration method comprises a whole cell vaccine or cytokine-expressing cells (e.g., recombinant IL-2-expressing fibroblasts, recombinant cytokine-expressing dendritic cells, etc.) (see, e.g., Kowalczyk et al., Acta Biochim Pol. 50(3), 613-24 (2003); Reilly et al., Methods Mol Med. 69, 233-57 (2002); and Tirapu et al., Curr Gene Ther. 2(1), 79-89 (2002)). Another example of such an autologous cell approach that may be useful in the combination methods of the present invention is MyVax® Personalized Immunotherapy (formerly known as GTOP-99) (Genitope Corporation—Redwood City, CA, USA).

[0421] In one embodiment, the present invention provides combination compositions and methods of administration in which an anti-CD38 antibody is combined or co-administered with an oncolytic virus.

[0422] The combination compositions and combined administration methods of the present invention can also include "whole cell" and "adoptive" immunotherapy. For example, such methods involve targeting immune system cells (e.g., tumor-infiltrating lymphocytes (TILs), e.g., CD4 + and / or CD8 +These methods and compositions may also include the infusion or reinfusion of T cells (e.g., T cells expanded by tumor-specific antigens and / or genetic enhancement), antibody-expressing B cells or other antibody-producing / presenting cells, dendritic cells (e.g., anti-cytokine-expressing recombinant dendritic cells, dendritic cells cultured with DC expansion agents such as GM-CSF and / or Flt3-L and / or dendritic cells loaded with tumor-associated antigens), anti-tumor NK cells, so-called hybrid cells, or combinations thereof. Cell lysates may also be useful in such methods and compositions. Cellular "vaccines" in clinical trials that may be useful in such aspects include Canvaxin™, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and Melasin® cell lysates. Antigens released from cancer cells and mixtures thereof (see, e.g., Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001), optionally in admixture with adjuvants such as alum, can also be components in such methods and combination compositions.

[0423] In one embodiment, the anti-CD38 antibody of the present invention can also be delivered to patients in combination with the application of internal vaccination. Internal vaccination refers to the induced death of tumor or cancer cells in patients, such as drug-induced or radiation-induced tumor cell death, which generally results in the development of an immune response against (i) tumor cells as a whole or (ii) parts of tumor cells, including (a) secreted proteins, glycoproteins, or other products, (b) membrane-associated proteins or glycoproteins, or other components associated with or inserted into the membrane, and / or (c) intracellular proteins or other intracellular components. The immune response induced by internal vaccination can be humoral (i.e., antibody- or complement-mediated) or cell-mediated (e.g., the generation and / or increase of endogenous cytotoxic T lymphocytes that recognize internally killed tumor cells or parts thereof). In addition to radiation therapy, non-limiting examples of drugs that may be used to induce tumor cell death and internal vaccination include conventional chemotherapeutic agents, cell cycle inhibitors, antiangiogenic agents, monoclonal antibodies, apoptosis inducers, and signal transduction inhibitors.

[0424] Examples of other anti-cancer agents that may be suitable as therapeutic agents for use in combination with the anti-CD38 antibodies of the invention to treat disorders such as those described above include differentiation inducers, retinoic acid and retinoic acid analogs (e.g., all-trans retinoic acid, 13-cis retinoic acid, and the like), vitamin D analogs (e.g., seocalcitol and the like), inhibitors of ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa, PDGFRβ, Flk2, Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC, c-met, Ron, Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7, and the like.

[0425] Examples of other anti-cancer agents that may be suitable as therapeutic agents for combination with the anti-CD38 antibodies of the invention to treat disorders such as those described above are modulators of cathepsin B, cathepsin D dehydrogenase activity, glutathione-S-transferases (e.g., glutasylcysteine ​​synthase and lactate dehydrogenase), estramustine, epirubicin, HSP90 inhibitors, e.g., 17-allylaminogeldanamycin, antibodies against tumor antigens such as PSA, CA125, KSA, etc., inhibitors of integrins, e.g., integrin β1, VCAM, and the like.

[0426] Examples of other anti-cancer agents that may be suitable as therapeutic agents for combination with the anti-CD38 antibodies of the invention to treat disorders such as those described above are calcineurin inhibitors (e.g., valspodar, PSC 833 and other MDR-1 or p-glycoprotein inhibitors), TOR inhibitors (e.g., sirolimus, everolimus and rapamycin) and inhibitors of the "lymphocyte homing" mechanism (e.g., FTY720) and agents that affect cell signaling, such as adhesion molecule inhibitors (e.g., anti-LFA).

[0427] In one embodiment, the invention provides a method of treating a disorder involving cells expressing CD38, e.g., cancer, in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the invention and radiation therapy.

[0428] In one embodiment, the present invention provides a method of treating multiple myeloma, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the present invention and radiation therapy.

[0429] Radiation therapy may involve the administration of radiation or radiopharmaceuticals to a patient. The source of radiation may be external or internal to the patient receiving treatment (radiation treatment may be in the form of, for example, external beam radiation therapy (EBRT), brachytherapy (BT), or skeletal-targeted radiation therapy). Radioactive elements that may be used to perform such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and indium-111.

[0430] In one embodiment, the invention provides a method of treating a disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the invention in combination with autologous peripheral stem cell or bone marrow transplantation.

[0431] In one embodiment, the invention provides a method of treating multiple myeloma, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the invention in combination with autologous peripheral stem cell or bone marrow transplantation.

[0432] In one embodiment, the invention provides a method of treating a disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the invention in combination with orthopedic surgical intervention.

[0433] In one embodiment, the invention provides a method of treating a disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the invention and at least one anti-inflammatory agent.

[0434] In one embodiment, such anti-inflammatory agents may be selected from steroids and NSAIDs (non-steroidal anti-inflammatory drugs).

[0435] In one embodiment, such anti-inflammatory agents include aspirin and other salicylates, Cox-2 inhibitors (e.g., rofecoxib and celecoxib), NSAIDs (e.g., ibuprofen, fenoprofen, naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac, oxaprozin, and indomethacin), anti-IL6R antibodies, anti-IL8 antibodies (e.g., antibodies described in WO2004058797, e.g., 10F8), anti-IL15 antibodies (e.g., antibodies described in WO03017935 and WO2004076620), and the like. ), anti-IL15R antibodies, anti-CD4 antibodies (e.g., zanolimumab), anti-CD11a antibodies (e.g., efalizumab), anti-α4 / β1 integrin (VLA4) antibodies (e.g., natalizumab), CTL4-Ig for the treatment of inflammatory diseases, prednisolone, prednisone, disease-modifying antirheumatic drugs (DMARDs) such as methotrexate, hydroxychloroquine, sulfasalazine, pyrimidine synthesis inhibitors (e.g., leflunomide), IL-1 receptor blockers (e.g., anakinra), TNF-α blockers (e.g., etanercept, infliximab, and adalimumab), and the like.

[0436] In one embodiment, the invention provides a method of treating a disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the invention and at least one immunosuppressant and / or immunomodulatory agent.

[0437] In one embodiment, such immunosuppressive and / or immunomodulatory agents may be selected from cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids such as prednisone, methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualin, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, antithymocyte globulin, thymopentin, thymosin-α, and the like.

[0438] In one embodiment, such immunosuppressive and / or immunomodulatory agents may also be selected from immunosuppressive antibodies, e.g., antibodies that bind to p75 of the IL-2 receptor, or antibodies that bind to, e.g., MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFNγ, TNF-α, IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11a, or CD58, or antibodies that bind to their ligands.

[0439] In one embodiment, such immunosuppressive and / or immunomodulatory agents may be selected from soluble IL-15R, IL-10, B7 molecules (B7-1, B7-2, variants thereof and fragments thereof), ICOS and OX40, inhibitors of negative T cell regulators (e.g., antibodies against CTLA4) and the like.

[0440] In one embodiment, the anti-CD38 antibodies of the invention may also be administered in combination with two or more immunosuppressive and / or immunomodulatory agents, for example, in combination with prednisone and cyclosporine, in combination with prednisone, cyclosporine and azathioprine, or in combination with prednisone, cyclosporine and mycophenolate mofetil.

[0441] In one embodiment, the invention provides a method of treating a disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody and an anti-C3b(i) antibody of the invention.

[0442] In one embodiment, the present invention provides a method of treating a disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need thereof therapeutically effective amounts of an anti-CD38 antibody and an anti-C32b antibody of the present invention. In one embodiment of the present invention, the anti-C32b antibody is selected from HuMab-016, -020, -022, -024, 026, 028, -034, -038, or -053, all of which are disclosed in WO2009 / 083009.

[0443] In one embodiment, therapeutic agents for use in combination with the anti-CD38 antibodies of the present invention to treat disorders such as those described above may also be selected from histone deacetylase inhibitors (e.g., phenylbutyrate) and / or DNA repair agents (e.g., DNA repair enzymes and related compositions, e.g., dimelysin).

[0444] Methods of the invention for treating disorders such as those described above, comprising administration of a therapeutically effective amount of an anti-CD38 antibody of the invention, can also include photodynamic anti-cancer therapy (e.g., laser anti-cancer therapy, which may optionally be performed using photosensitizers; see, e.g., Zhang et al., J Control Release. 93(2), 141-50 (2003)), acoustic and shock wave anti-cancer therapy (see, e.g., Kambe et al., Hum Cell. 10(1), 87-94 (1997)), and / or nutritional supplemental anti-cancer therapy (see, e.g., Roudebush et al., Vet Clin North Am Small Anim Pract. 34(1), 249-69, viii (2004) and Rafi, Nutrition. 20(1), 78-82 (2004)). Similarly, the anti-CD38 antibodies of the present invention can also be used for the preparation of pharmaceutical compositions for treating such disorders, administered in conjunction with photodynamic anti-cancer therapy (e.g., laser anti-cancer therapy, which may optionally be performed using photosensitizers), acoustic and shock wave anti-cancer therapy, and / or nutritional supplemental anti-cancer therapy.

[0445] In a further embodiment, the anti-CD38 antibodies of the invention are administered in conjunction with complement.

[0446] As noted above, the pharmaceutical compositions of the present invention may be administered in combination therapy, i.e., in separate pharmaceutical compositions, in combination with one or more agents appropriate for the disease or condition being treated, or as a compound of the present invention formulated with one or more additional therapeutic agents as described above. Such combination therapy may require lower dosages of the compound of the present invention and / or the co-administered agent, thus avoiding possible toxicities or complications associated with the various monotherapies.

[0447] In one embodiment, the invention provides a method of treating a disorder involving cells expressing CD38 in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD38 antibody of the invention and at least one immunosuppressant and / or immunomodulatory agent.

[0448] Diagnostic Use The anti-CD38 antibodies of the present invention can also be used for diagnostic purposes. Thus, in a further aspect, the present invention relates to diagnostic compositions comprising the anti-CD38 antibodies described herein.

[0449] In one embodiment, the anti-CD38 antibodies of the present invention can also be used in vivo or in vitro to diagnose diseases in which activated cells expressing CD38 play an active role in the pathogenesis by detecting levels of CD38 or levels of cells containing CD38 on their membrane surface. This can be accomplished, for example, by contacting the sample to be tested, optionally along with a control sample, with an anti-CD38 antibody under conditions that allow the formation of a complex between the antibody and CD38. Complex formation is then detected (e.g., using ELISA). When a control sample is used along with the test sample, complexes are detected in both samples, and a statistically significant difference in complex formation between the samples indicates the presence of CD38 in the test sample.

[0450] Therefore, in a further aspect, the present invention relates to a method for detecting the presence of CD38 antigen, i.e. cells expressing CD38, in a sample, comprising the steps of: contacting the sample with an anti-CD38 antibody of the invention or a bispecific molecule of the invention under conditions that allow the formation of a complex between the antibody and CD38; and Analyzing whether a complex was formed.

[0451] In one embodiment, the method is performed in vitro.

[0452] More specifically, the present invention provides methods for the identification and diagnosis of invasive cells and tissues and other cells targeted by the anti-CD38 antibodies of the present invention, as well as methods for monitoring the progress of therapeutic treatment, post-treatment status, risk of carcinogenesis, cancer progression, and the like.

[0453] In one example of such a diagnostic assay, the invention provides a method for diagnosing the level of invasive cells in a tissue, the method comprising forming an immune complex between an anti-CD38 antibody and a potential CD38-containing tissue, and detecting the formation of the immune complex, which correlates with the presence of invasive cells in the tissue. Contacting can be performed in vivo using a labeled isolated antibody and standard imaging techniques, or in vitro on a tissue sample.

[0454] Examples of conventional immunoassays provided by the present invention include, but are not limited to, ELISA, RIA, FACS assay, plasmon resonance assay, chromatographic assay, immunohistochemical staining, Western blot, and / or immunoprecipitation using anti-CD38 antibodies. Suitable labels for anti-CD38 antibodies and / or secondary antibodies used in such techniques include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials.

[0455] Anti-CD38 antibodies are particularly useful in in vivo imaging of tumors. In vivo imaging of tumors associated with CD38 can be performed by any suitable technique. For example, 99Anti-CD38 antibodies in tumors or secondary labeled (e.g., FITC-labeled) anti-CD38 antibody:CD38 complexes from tumors can also be labeled with Tc or another gamma-emitting isotope and imaged with a gamma scintillation camera (e.g., an Elscint Apex 409 ECT instrument), typically using a low-energy, high-resolution collimator or a low-energy, general-purpose collimator. The stained tissue can then be evaluated for radioactivity counts as an indicator of the amount of CD38-associated peptide in the tumor. Images obtained using such techniques can also be used to assess the biodistribution of CD38 in patients, mammals, or tissues, for example, with respect to using CD38 or CD38 fragments as biomarkers for the presence of invasive cancer cells. Modifications to this technique can include the use of magnetic resonance imaging (MRI), which provides improved imaging over gamma camera techniques. Similar immunoscintigraphy methods and principles are described, for example, in Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging And Therapy (Plenum Press 1988), Chase, "Medical Applications of Radioisotopes" in Remington's Pharmaceutical Sciences, 18th Edition, Gennaro et al., (ed.), pp. 624-652 (Mack Publishing Co., 1990), and Brown, "Clinical Use of Monoclonal Antibodies" in Biotechnology And Pharmacy 227-49, Pezzuto et al., (ed.) (Chapman & Hall 1993).

[0456] In a further aspect, the present invention relates to a kit for detecting the presence of the CD38 antigen, i.e., cells expressing CD38, in a sample, comprising: an anti-CD38 antibody of the invention or a bispecific molecule of the invention, and Instructions for use of the kit.

[0457] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container containing an anti-CD38 antibody and one or more reagents for detecting binding of the anti-CD38 antibody to a CD38 peptide. The reagents can include, for example, fluorescent tags, enzyme tags, or other detectable tags. The reagents can also include secondary or tertiary antibodies or reagents for enzymatic reactions, which produce products that can be visualized. In one embodiment, the present invention provides a diagnostic kit comprising one or more anti-CD38 antibodies of the present invention in labeled or unlabeled form in a suitable container, reagents for incubation for an indirect assay, and, depending on the nature of the label, a substrate or derivatizing agent for detection in such an assay. Control reagents may also be included.

[0458] In a further aspect, the present invention relates to an anti-idiotypic antibody that binds to an anti-CD38 antibody of the present invention described herein.

[0459] Anti-idiotypic (Id) antibodies are antibodies that recognize unique determinants generally associated with the antigen-binding site of an antibody. Id antibodies can also be prepared by immunizing an animal of the same species and genetic type as the source of the anti-CD38 mAb with the mAb from which the anti-Id is prepared. The immunized animal is generally able to recognize the idiotypic determinants of the immunizing antibody and respond to it by producing antibodies against those idiotypic determinants (anti-Id antibodies). Such antibodies are described, for example, in U.S. Pat. No. 4,699,880.

[0460] Anti-Id antibodies can also be used as "immunogens" to elicit immune responses in yet another animal, producing so-called anti-anti-Id antibodies. Anti-anti-Id antibodies can also be epitopically identical to the original mAb that elicited the anti-Id. Thus, by using antibodies against the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity. Anti-Id antibodies can also be mutated (thereby producing anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein for the anti-CD38 antibodies of the invention.

[0461] The present invention is further illustrated by the following examples which should not be construed as further limiting. [Example]

[0462] Example 1 Antibody generation HCo12 mice were immunized with 20 μg of purified HA-CD38 and NIH-3T3-CD38 transfected cells alternately every 2 weeks. The first immunization consisted of 5 × 10 cells in 100 μl PBS mixed with 100 μl CFA. 6 Immunizations were performed intraperitoneally with 100 μl of HA-CD38 cells, and subsequent immunizations were performed subcutaneously with HA-CD38 in the presence of 100 μl PBS mixed with 100 μl IFA. Subsequent immunizations with transfected cells were performed in the presence of 200 μl PBS. After titer development, mice were boosted intravenously with 20 μg of HA-CD38 in PBA.

[0463] Spleens were harvested from these mice, splenocytes were isolated, and fused to a mouse myeloma cell line by PEG using standard protocols, and the resulting hybridomas were screened for human antibody production by ELISA, for CD38 specificity by FACS analysis using human CD38-transfected NS / 0 cells, and for recombinant HA-CD38 protein binding by ELISA.

[0464] Sequence analysis of anti-CD38 HuMab variable domains and cloning into expression vectors 5 × 10 cDNAs were amplified using the SMART RACE cDNA Amplification Kit (Clontech) according to the manufacturer's instructions. 6 Total RNA of anti-CD38 HuMab was prepared from 100 ng of hybridoma cells, and 5'-RACE complementary DNA (cDNA) was prepared from 100 ng of total RNA.

[0465] The VH and VL coding regions were amplified by PCR and cloned into the pCR-Blunt II-TOPO vector (Invitrogen) using a Zero Blunt PCR cloning kit (Invitrogen). For each HuMab, 16 VL clones and 8 VH clones were sequenced.

[0466] The VL and VH coding regions were cloned into pκ and pG1f vectors.

[0467] The CDR regions are shown according to IMGT. (http: / / imgt.cines.fr / IMGT_vquest / vquest?livret=0&Option=humanIg)

[0468] The following IgG1,κ human monoclonal antibodies were identified: TIFF2026021523000008.tif39161

[0469] Example 2 Electrospray ionization quadrupole time-of-flight mass spectrometry of anti-CD38 antibodies Complete molecular weight data for anti-CD38 antibodies 025, 057 (same amino acid sequence as antibody 026), 028, 049, and 056 were obtained using nanospray electrospray MS on a Q-TOF mass spectrometer. Aliquots of each antibody sample were desalted offline using a C4 Microtap cartridge and eluted in propanol / trifluoroacetic acid solvent. The instrument was calibrated using glu-fibrinopeptide fragment ions in MS / MS mode. The resulting multiply charged data were deconvoluted using MassLynx 4.0 software.

[0470] After reduction with dithiothreitol and analysis as described above, information regarding the molecular weights of the light and heavy chain components of these antibodies was obtained.

[0471] Table 1: Mass of CD38 antibody (in daltons) TIFF2026021523000009.tif35128

[0472] Example 3 Cross-block experiments using FACS CHO-CD38 cells were incubated with an excess amount of unlabeled CD38-specific antibody (4°C, 15 min), and then treated with FITC-labeled 005 antibody (concentration EC 90 The cells were incubated with 005-FITC labeled antibody (005 is disclosed in WO2006099875) at 4°C for 45 minutes. After washing the cells twice with PBS-BSA, fluorescence was measured by flow cytometry. 005-FITC labeled antibody binding was blocked by excess unlabeled antibodies 025, 026, 028, 049, and 056, indicating that these antibodies have overlapping epitopes. 005-FITC binding was not blocked by excess unlabeled 003 (disclosed in WO2006099875), providing evidence for binding to a different epitope.

[0473] Cross-block experiments using ELISA Soluble human CD38 was coated onto the surface of an ELISA plate. The coated CD38 was incubated with an excess amount of unlabeled CD38-specific antibody for approximately 15 minutes, and then biotinylated CD38-specific antibody was added (at a concentration of EC 90 (Approx. 1 h at room temperature). After washing three times with PBS / Tween, horseradish peroxidase (HRP)-conjugated streptavidin was added, and the mixture was incubated for 1 h at room temperature. The complex was detected by the addition of ABTS solution, and HRP-mediated substrate conversion was measured at OD 405 nm using an ELISA reader.

[0474] Cross-block experiments using sandwich ELISA Anti-CD38 antibody was coated onto the surface of an ELISA plate. The plate-bound antibody was incubated with biotinylated soluble CD38 in the presence of excess anti-CD38 antibody in the fluid phase. After washing with PBS / Tween, bound biotinylated CD38 was detected with HRP-conjugated streptavidin for 1 hour at room temperature. The complex was detected by adding ABTS solution (after washing with PBS / Tween), and HRP-mediated substrate conversion was measured at OD 405 nm using an ELISA reader.

[0475] Example 4 Epitope mapping Construction of HA-CD38 and His-CD38 expression vectors The coding sequence for the extracellular domain of human CD38 (identical to amino acids 45-300 from GenBank entry AAA68482) was amplified from the plasmid pCIpuroCD38 (obtained from Prof. M. Glennie, Tenovus Research Laboratory, Southampton General Hospital, Southampton, UK) using PCR to introduce restriction sites, an ideal Kozak sequence (GCCGCCACC), and sequences encoding a signal peptide and an N-terminal HA tag (ypydvpdya). This construct was cloned into the mammalian expression vector pEE13.4 (Lonza Biologies). This construct was designated pEE13.4HACD38.

[0476] A similar construct was synthesized and fully codon-optimized (GeneArt, Regensburg, Germany) by replacing the HA tag coding region with a His tag (HHHHHH). This construct was cloned into pEE13.4 and designated pEE13.4HisCD38.

[0477] Site-directed mutagenesis Several mutations were introduced into the putative antibody binding site on the CD38 molecule.

[0478] DNA substitutions leading to T237A, Q272R, or S274F amino acid substitutions were generated in the pEE13.4HACD38 vector using the QuickChange II XL site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands). Similarly, the D202G coding substitution was introduced into the pEE13.4HisCD38 vector.

[0479] Transient expression in HEK-293F cells Freestyle™ 293-F (a HEK-293 subclone (HEK-293F) adapted to suspension culture and chemically defined Freestyle medium) cells were obtained from Invitrogen and transfected with pEE13.4HACD38, pEE13.4HisCD38, or the four mutant constructs using 293fectin (Invitrogen) according to the manufacturer's protocol. Culture supernatants from transfected cells were used in ELISA for anti-CD38 binding experiments.

[0480] Anti-CD38 antibody binding Mutations T237A, Q272R, and S274F: ELISA plates (Greiner, #655092) were coated with 1 μg anti-HA antibody (Sigma, #H-9658) overnight at 4°C and then blocked with 2% chicken serum. Culture supernatants from transfected HEK293F cells were diluted and applied to the ELISA plates, followed by incubation at room temperature for 1 hour. After washing, serial dilutions of anti-CD38 antibody were added and incubated at room temperature for 1 hour. Bound antibody was detected with HRP-conjugated goat anti-human IgG antibody. The assay was developed with ABTS (Roche, #1112597), and the absorbance was measured at 405 nm using a spectrophotometer.

[0481] Mutation D202G: ELISA plates (Greiner, #655092) were coated with 1 μg penta-His (Qiagen #34660) overnight at 4°C and then blocked with 2% PBS / BSA. Culture supernatants from transfected HEK293F cells were diluted and applied to the ELISA plates, followed by incubation at room temperature for 2 hours. After washing, serial dilutions of anti-CD38 antibody were added and incubated at room temperature for 1 hour. Bound antibody was detected with HRP-conjugated goat anti-human IgG antibody. The assay was developed with ABTS (Roche, #1112597), and absorbance was measured at 405 nm using a spectrophotometer.

[0482] This experiment revealed that the binding of 025, 026, 028, and 049 was insensitive to the mutations T237A, Q272R, S274F, and A199T, but was severely affected by D202G (025, 028, 049) (Figure 2).

[0483] Example 5 Binding of anti-CD38 antibodies to CD38-transfected CHO (CHO-CD38) cells and Daudi-luc cells After harvesting and counting, Daudi-luc cells, CD38-transfected CHO cells, and control CHO cells were resuspended in PBS (1 × 10 6 Cells were transferred to a 96-well V-bottom plate (100 μL / well) and washed twice in PBS-BSA (PBS supplemented with 0.1% BSA and 0.02% Na azide). 50 μL of antibody in PBS-BSA was added as a three-fold dilution ranging from 0.3 to 30 μg / mL (4°C, 30 min). After three washes in PBS-BSA, 50 μL of rabbit anti-human IgG-FITC in PBS-BSA (1:400 dilution) was added (4°C, 30 min in the dark). Cells were washed three times, and specific binding of CD38 antibody to CHO-CD38 and Daudi-luc cells was detected by flow cytometry.

[0484] FIG. 3 shows that 025, 026, 028, 049 and 056 bind to CHO-CD38 cells and Daudi-luc cells.

[0485] Example 6 Antibody-dependent cytotoxicity (ADCC) The ability of anti-CD38 antibodies to mediate ADCC of Daudi-luc cells was measured as follows: Peripheral blood mononuclear cells from healthy volunteers (UMC Utrecht, The Netherlands) were used as effector cells.

[0486] Daudi-luc cells were cultured in RPMI ++The cells were collected (5 × 10 6 pcs), plus 100μCi 51 Cr (chromium 51, Amersham Biosciences, Europe GmbH, Roosendaal, The Netherlands) was added and the mixture was incubated for 1 hour in a water bath at 37°C. After washing the cells (twice in PBS, 1500 rpm, 5 minutes), the cells were resuspended in RPMI ++ The cells were resuspended in 1×10 PBS and counted by trypan blue exclusion. 5 The concentration was adjusted to 1 / mL.

[0487] Preparation of effector cells Fresh peripheral blood mononuclear cells (healthy volunteers, UMC Utrecht, Utrecht, The Netherlands) were isolated from 40 ml of heparinized blood by Ficoll (BioWhittaker, Lymphocyte Separation Medium, cat. 17-829E) according to the manufacturer's instructions. Cells were stored in RPMI ++ After resuspension in PBS, cells were counted by trypan blue exclusion and counted at 1 x 10 7 The concentration was adjusted to 1 / ml.

[0488] ADCC setup 51 Pipette 50 μl of Cr-labeled target cells into a 96-well plate, add 50 μl of antibody, and incubate in RPMI ++ The cells were incubated (15 min at room temperature) and 50 μl effector cells were added to give an effector:target ratio of 100:1 (for determination of maximum lysis, 100 μl 5% Triton-X100 was added instead of effector cells, and for determination of spontaneous lysis, 50 μl target cells and 100 μl RPMI were added). ++The cells were centrifuged (500 rpm, 5 min) and incubated (37°C, 5% CO2, 4 h). After centrifuging (1500 rpm, 5 min), 100 μL of the supernatant was collected in a micronic tube and counted in a gamma counter. The specific lysis rate was calculated as follows: (cpm sample - cpm target cells only) / (cpm maximal lysis - cpm target cells only) where cpm is counts per minute.

[0489] 025, 026, 028, 049 and 056 induced ADCC-mediated lysis in Daudi cells (Fig. 4).

[0490] Example 7 Complement-dependent cytotoxicity (CDC) After harvesting and counting Daudi-luc cells, cell viability should be ≥ 90%. After washing (PBS), cells were cultured at 2.0 × 10 in RPMI-B (RPMI supplemented with 1% BSA). 6 The cells were then resuspended at 1 x 10 cells / ml. 5 The wells were plated with 50 μL of antibody per well (50 μL per well). Then, 50 μL of antibody was added to each well (final concentration range: 0–100 μg / ml, 3-fold dilutions in RPMI-B). After incubation (room temperature, 15 min), 11 μL of pooled human serum (from 18 healthy donors) was added to each well (37°C, 45 min). The wells were resuspended once, and 120 μL was transferred to a FACS tube (Greiner). Then, 10 μL of propidium iodide (PI, Sigma-Aldrich Chemie BV) (10 μg / ml solution) was added to the suspension. Lysis was detected by flow cytometry (FACScalibur™, Becton Dickinson, San Diego, CA, USA) by counting the percentage of dead cells (corresponding to PI-positive cells).

[0491] Figure 5 shows CDC-mediated CHO-CD38 cell lysis caused by anti-CD38 antibodies 025, 026, 028, 049 and 056. These anti-CD38 antibodies were unable to induce CDC in Daudi-luc cells.

[0492] Example 8 Enzyme activity The effect of anti-CD38 antibodies on the enzymatic activity of CD38 was measured. CD38 is known to catalyze various enzymatic reactions, including the cyclase reaction that converts NAD to cyclic ADP-ribose (cADPR), the hydrolase reaction that converts NAD or cADPR to ADP-ribose, and the base exchange reaction that generates nicotinic acid adenine dinucleotide 2'-phosphate (NAADP).

[0493] cyclase activity NGD assay The ability of anti-CD38 antibodies to interfere with the cyclase activity of CD38 was measured using NGD as a substrate in an assay essentially as described by Graeff et al., J. Biol. Chem. 269, 30260-30267 (1994).

[0494] Briefly, the substrate NGD + (80 μM) was incubated with CD38 (His-tagged extracellular domain of human CD38 at 0.6 μg / ml in 20 mM Tris-HCl buffer at pH 7.0; see Example 3 of WO2006099875 for purification of His-CD38). cGDPR production can be monitored spectrophotometrically at an emission wavelength of 410 nm (excitation at 300 nm). In this example, an excitation filter of 340±60 nm and an emission filter of 430±8 nm were used.

[0495] To test the effects of 025, 026, 028, 049, and 056 on the enzymatic activity of CD38, the substrate NGD +Recombinant His-CD38 protein was preincubated with 3 μg / ml of antibody for 15 min at room temperature before addition of 1 μg / ml of antibody. After 90 min, cyclic GDP-ribose (cGDPR) production was recorded.

[0496] Figure 6A shows that antibodies 025, 026, 028, 049, and 056 exerted a significant inhibitory effect on cGDPR production. After 90 minutes, 3 μg / ml of antibody (025, 026, 028, 049, or 056) caused a 53-66% decrease in cGDPR production. A time course experiment demonstrated a decreased rate of cGDPR production in samples treated with the CD38-specific antibody mAb 028 compared to cGDPR production in the presence of HuMab-KLH or untreated CD38 controls (Figure 6B). Figure 6C shows a dose-response curve (0.01-30 μg / ml) for antibody 028. In this experiment, a maximum 41% decrease in cGDPR production was observed.

[0497] To test the effect of O28 on the enzymatic activity of cell-expressed CD38, CHO-CDC38 cells were preincubated with serial dilutions of O28 (0.01–30 μg / ml) at room temperature for 30 min, followed by incubation with the substrate NGD. + After 90 minutes, the production of cyclic GDP-ribose (cGDPR) was recorded. As shown in Figure 6D, antibody 028 inhibited the production of cGDPR in a concentration-dependent manner.

[0498] Reverse cyclase reaction The effect of mAb 028 on CD38-mediated cADPR production from NAD was measured by the reverse cyclase reaction. This assay is based on the reversibility of the reaction catalyzed by CD38. In the presence of high concentrations of nicotinamide and cADPR, ADP-ribosyl cyclase can generate NAD. The antibody was diluted to 10 μg / ml in 20 mM Tris-HCl, 0.01% (v / v) BSA, pH 7.2 (Tris / BSA). Human recombinant CD38 was diluted to 2 μg / ml in Tris / BSA. The antibody was preincubated with CD38 for 10 minutes by mixing an equal volume (50 μL) of diluted antibody with the diluted CD38. Preincubation was performed at room temperature. The reaction was initiated by transferring 25 μL of the CD38 / antibody mixture to 25 μL of a solution containing 1 mM cADPR and 10 mM nicotinamide. The reaction was allowed to proceed for 1–20 min at room temperature, and at the appropriate time point, the reaction was stopped by filtering the entire sample through a Millipore MultiScreen-IP Filter 96-well plate to remove protein. The NAD produced was measured by the method of Graeff and Lee (1). A control containing nicotinamide without cADPR was run to estimate the amount of NAD contaminating the reagent. In these experiments, contaminating NAD was undetectable.

[0499] Table 2 shows that 1 μg / ml mAb-028 reduced cADPR production from NAD by 67%. mAb-KLH had no effect on cADPR production from NAD.

[0500] Table 2: Effect of antibody 028 on cADPR production from NAD TIFF2026021523000010.tif27149

[0501] 8-amino-NAD (8NH2-NAD) assay Because cADPR production accounts for only approximately 1% of the products generated from NAD by CD38 (ADPR accounts for the remainder), ribosyl cyclase activity was similarly assessed using 8-amino-NAD (8NH2-NAD) as a substrate. Unlike NAD, a significantly larger amount (approximately 8%) of the 8NH2-NAD substrate is cyclized to 8-amino-cADPR (8NH2-cADPR), detectable by HPLC analysis. Briefly, antibodies were diluted to 10 μg / mL in 20 mM Tris-HCl, 0.01% (v / v) BSA, pH 7.2 (Tris / BSA). Human recombinant CD38 was diluted to 2 μg / mL in Tris / BSA. The antibodies were preincubated with CD38 for 10 minutes by mixing an equal volume (50 μL) of diluted antibody with the diluted CD38. Preincubation was performed at room temperature. The reaction was initiated by transferring 25 μL of the CD38 / antibody mixture to 75 μL of 0.5 mM 8NH2-NAD. The reaction proceeded for 10 minutes at room temperature, and at the appropriate time point, the reaction was stopped by filtering the entire sample through a Millipore MultiScreen-IP Filter 96-well plate to remove protein. The reaction products (8NH2-cADPR and 8NH2-ADPR) were analyzed by reverse-phase HPLC as follows: The column was a 0.46 × 15 cm LC18-T reverse-phase column from Supelco. Solvent A consisted of 20 mM KH2P04, 5 mM tetrabutylammonium phosphate pH 6, and solvent B consisted of 50% A and 50% methanol. The flow rate was 1 mL / min, and the initial solvent composition was 15% B. Separation of substrate and product was achieved using the following gradient: Substrate and product were eluted using a gradient of 0–3.5 min (15% B), 3.5–5.5 min (15–32.5% B), 5.5–9 min (32.5–40% B), 9–11.5 min (40–50% B), and 16–18 min (50–15% B). Samples were prepared by adding 400 μL of solvent A to 100 μL of filtered sample. The entire sample was injected. Flow rate and buffer composition were controlled by a Beckman 125 HPLC pump and System Gold software, and peak detection was performed with a Beckman 166 UV detector.The areas of the 8NH2-NAD, 8NH2-cADPR, and 8NH2-ADPR peaks were used to calculate the amount of 8NH2-cADPR produced in the assay. The HPLC system was based on that described by Schweitzer et al. (2).

[0502] Figure 7A shows that mAb-028 inhibits 8NH2-cADPR by 78%. mAb-028 inhibits 8NH2-cADPR production in a concentration-dependent manner (Figure 7B).

[0503] Thus, mAb-028 inhibits the ADP-ribosyl cyclase reaction of CD38 as assayed by three different methods.

[0504] Hydrolase activity Hydrolase activity analysis by HPLC Hydrolase activity was measured by measuring the amount of ADPR produced from cADPR or NAD by HPLC. Antibodies were diluted or titrated to 10 μg / mL in 20 mM Tris-HCl, 0.01% (v / v) BSA, pH 7.2 (Tris / BSA). Human recombinant CD38 was diluted to 2 μg / mL in Tris / BSA. Antibodies were preincubated with CD38 for 10 minutes by mixing an equal volume (50 μL) of diluted antibody with diluted CD38. Preincubation was performed at room temperature. For the HPLC-based method, the cADPR hydrolase reaction was initiated by transferring 40 μL of the CD38 / antibody mixture to 10 μL of 4.3 mM cADPR, and the NADase reaction was initiated by transferring 40 μL of the CD38 / antibody mixture to 10 μL of 1 mM NAD. The reactions were allowed to proceed at room temperature and stopped at the appropriate time by adding 25 μL of 1 M HCl. Protein was removed by filtering the entire sample through a Millipore MultiScreen-IP Filter 96-well plate. Each filtrate was neutralized by adding 15 μL of 2 M Tris base and kept on ice until analysis by HPLC. Analysis of hydrolase activity was based on the HPLC assay developed by Lee and Aarhus (3). Samples were analyzed on a 0.5 x 5 cm column of AG MP-1 (trifluoroacetic acid form) eluted with a concave upward gradient of 0 to 150 mM trifluoroacetic acid (TFA) at 3 mL / min for 10 min. Flow rate and buffer composition were controlled by a Beckman 125 HPLC pump and System Gold software, and peak detection was performed with a Beckman 166 UV detector. The areas of NAD, cADPR, and ADPR were used to calculate the amount of ADPR produced in the assay.

[0505] At a concentration of 10 μg / mL, mAb-028 stimulated cADPR hydrolase activity by 62% and NAD hydrolase activity by 37% compared to the CD38 control, whereas mAb-KLH did not (Figure 8A). Figure 8B shows that mAb-028 stimulated cADPR hydrolysis in a dose-dependent manner. At a concentration of 30 μg / mL, mAb-028 stimulated hydrolase activity by 78%.

[0506] Hydrolase activity assay by thin-layer chromatography (TLC) 32 Produced from P-cADPR 32 Hydrolase activity was measured by measuring the amount of p-ADPR by thin-layer chromatography (4). 20 μL of the CD38 / antibody mixture (described above) was diluted with 0.5 mM cADPR and approximately 0.1 μCi of 32 By adding it to 5 μL of the mixture containing P-cADPR, 32 P-based cADPR hydrolase reactions were initiated. The reactions were allowed to proceed at room temperature, and at the appropriate time points, 5 μL of the reaction was stopped by adding 5 μL of 150 mM TFA. The reactions were analyzed by PEI-cellulose thin-layer chromatography (TLC). 1 μL of each stopped reaction sample was placed at the beginning of a PEI-cellulose TLC plate (10 × 20 cm). The plate was developed with 0.2 M NaCl in 30% (v / v) ethanol. The plate was dried and exposed to a phosphorimage screen. The screen was analyzed on a Packard Cyclone Phosphorimager to determine the amount of ATP produced. 32 The amount of P-ADPR was measured.

[0507] Figure 8C shows that mAb-028 32 These results were similar to the cADPR hydrolase activity measured by HPLC (see Figure 8B).

[0508] Base exchange activity The effect of CD38 antibodies on nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) synthesis via the base exchange activity of CD38 was evaluated. Antibodies (mAb-KLH and 028) were diluted to 40 μg / mL in 20 mM Hepes, pH 7.3, 0.01% (v / v) BSA (Hepes / BSA). Human recombinant CD38 was diluted to 2 μg / mL in Hepes / BSA. Antibodies were preincubated with CD38 for 10 minutes by mixing an equal volume (90 μL) of diluted antibody with diluted CD38. Preincubation was performed at room temperature. The base exchange reaction was initiated by transferring 50 μL of the CD38 / antibody mixture to 50 μL of a reaction mixture containing 200 mM sodium acetate, pH 4.0, 25 mM nicotinic acid, and 2 mM nicotinamide adenine dinucleotide 2'-phosphate (NADP). The reaction proceeded for 30 min at room temperature and was terminated by filtering the entire sample through a Millipore MultiScreen-IP Filter 96-well plate to remove protein. The reaction products in the filtrate were measured by anion-exchange HPLC on a 0.5 x 5 cm column of AG MP-1 (trifluoroacetic acid form) eluting with a 0-150 mM trifluoroacetic acid (TFA) concave ascending gradient at 1 mL / min for 30 min (5). Just prior to injection, the filtrate (50 μL) was neutralized by adding 5 μL of 2 M Tris base. Flow rate and buffer composition were controlled by a Beckman 125 HPLC pump and System Gold software, and peak detection was performed with a Beckman 166 UV detector. The areas of the NADP, NAADP (base exchange product), and adenosine diphosphoribose 2'-phosphate (ADPR-P, hydrolysis product) peaks were used to calculate the rates of NAADP synthesis and NADP hydrolysis.

[0509] Figure 9 shows that mAb-028 inhibits the ability of CD38 to catalyze the formation of NAADP. Inhibition of NAADP production by mAb-028 is concentration-dependent (Figure 9B), with an IC50 of 0.14 μg / mL.

[0510] List of References TIFF2026021523000011.tif54156

[0511] TIFF2026021523000012.tif113156TIFF2026021523000013.tif207156TIFF2026021523000014.tif201155 TIFF2026021523000015.tif186156TIFF2026021523000016.tif216155TIFF2026021523000017.tif129155

[0512] The CDR regions are shown according to IMGT.

[0513] The sequence of human CD38 is set forth in SEQ ID NO: 52. A human CD38 mutation at position 274 resulting in an S to F mutation is set forth in WO2006099875 as SEQ ID NO: 34, and a human CD38 mutation at position 272 resulting in a Q to R mutation is set forth in WO2006099875 as SEQ ID NO: 33. A human CD38 mutation at position 202 resulting in a D to G mutation is set forth above as SEQ ID NO: 51.

[0514] Sequence information SEQUENCE LISTING <110> Genmab A / S <120> ANTIBODIES AGAINST HUMAN CD38 <150> US 60 / 353,082 <151> 2010-06-09 <150> DK PA 2010 00498 <151> 2010-06-09 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 363 <212> DNA <213> homo sapiens <400> 1 caggtccaac tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttttggagg caccttcagc agctacgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcatccgtt tccttggtat agcaaactac 180 gcacagaagt tccagggcag agtcacgctt atcgcggaca aatccacgaa cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgttt attactgtgc gggggaacct 300 ggggagcggg accccgatgc tgttgatatc tggggccaag ggacaatggt caccgtctct 360 tca 363 <210> 2 <211> 121 <212> PRT <213> homo sapiens <400> 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Phe Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Arg Phe Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Ile Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Glu Pro Gly Glu Arg Asp Pro Asp Ala Val Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 3 <211> 9 <212> PRT <213> homo sapiens <400> 3 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 4 <211> 8 <212> PRT <213> homo sapiens <400> 4 Ile Ile Arg Phe Leu Gly Ile Ala 1 5 <210> 5 <211> 14 <212> PRT <213> homo sapiens <400> 5 Ala Gly Glu Pro Gly Glu Arg Asp Pro Asp Ala Val Asp Ile 1 5 10 <210> 6 <211> 364 <212> DNA <213> Homo sapiens <400> 6 caggtccaac tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttttggagg caccttcagc agctatgcta tcagctgggt acgacaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcatccgtt tccttggtaa agcaaatcac 180 gcacagaagt tccagggcag agtcacgctt accgcggaca aatccacgaa cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgttt attactgtgc gggggaacct 300 ggggatcggg accccgatgc tgttgatatc tggggccaag ggacaatggt caccgtctct 360 tcag 364 <210> 7 <211> 121 <212> PRT <213> Homo sapiens <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Phe Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Arg Phe Leu Gly Lys Ala Asn His Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Glu Pro Gly Asp Arg Asp Pro Asp Ala Val Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 8 <211> 8 <212> PRT <213> homo sapiens <400> 8 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 9 <211> 8 <212> PRT <213> homo sapiens <400> 9 Ile Ile Arg Phe Leu Gly Lys Ala 1 5 <210> 10 <211> 14 <212> PRT <213> Homo sapiens <400> 10 Ala Gly Glu Pro Gly Asp Arg Asp Pro Asp Ala Val Asp Ile 1 5 10 <210> 11 <211> 364 <212> DNA <213> Homo sapiens <400> 11 caggtccaac tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttttggagg caccttcagc agttatgcta ttagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcatccgtt tccttggtaa aacaaatcac 180 gcacagaagt tccagggcag agtcacactt accgcggaca aatccacgaa cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgttt attactgtgc gggggaacct 300 ggggatcggg accccgatgc tgttgatatc tggggccaag ggacaatggt caccgtctct 360 tcag 364 <210> 12 <211> 121 <212> PRT <213> Homo sapiens <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Phe Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Arg Phe Leu Gly Lys Thr Asn His Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Glu Pro Gly Asp Arg Asp Pro Asp Ala Val Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 13 <211> 8 <212> PRT <213> homo sapiens <400> 13 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 14 <211> 8 <212> PRT <213> homo sapiens <400> 14 Ile Ile Arg Phe Leu Gly Lys Thr 1 5 <210> 15 <211> 14 <212> PRT <213> homo sapiens <400> 15 Ala Gly Glu Pro Gly Asp Arg Asp Pro Asp Ala Val Asp Ile 1 5 10 <210> 16 <211> 364 <212> DNA <213> homo sapiens <400> 16 caggtccagc tggtgcagtc tggggctgag gtgatgaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttccggagg caccttccgc agctatgcta tcagttgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcatcgttt tccttggtaa aacaaactac 180 gcacagaagt tccagggcag agtcacgctt accgcggaca aatccacgac cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtac gggggaacct 300 ggggctcggg accccgacgc ttttgatatc tggggccaag ggacaatggt caccgtctct 360 tcag 364 <210> 17 <211> 121 <212> PRT <213> homo sapiens <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Val Phe Leu Gly Lys Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Gly Glu Pro Gly Ala Arg Asp Pro Asp Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 18 <211> 8 <212> PRT <213> homo sapiens <400> 18 Gly Gly Thr Phe Arg Ser Tyr Ala 1 5 <210> 19 <211> 8 <212> PRT <213> homo sapiens <400> 19 I...

Claims

1. An antibody that binds to human CD38 (SEQ ID NO: 52), but does not bind to a mutant of human CD38 in which Asp at position 202 is substituted with Gly to the same extent as it binds to human CD38.

2. EC of antibody binding to a mutant of human CD38 in which Asp at position 202 is replaced by Gly 50 EC of the antibody binding to human CD38 50 2. The antibody of claim 1, wherein the antibody has a nucleotide sequence that is less than 50%, such as less than 10%, less than 5% or less than 1% of the nucleotide sequence.

3. The antibody of any one of the preceding claims, which binds to a mutant of human CD38 in which Gln at position 272 is substituted with Arg to the same extent as it binds to human CD38.

4. EC of antibody binding to a mutant of human CD38 in which Gln at position 272 is substituted with Arg 50 EC of the antibody binding to human CD38 50 4. The antibody of claim 3, wherein the antibody is at least 80%, such as at least 90%, such as at least 95%, such as at least 98% of the total antibody content.

5. The antibody of any one of the preceding claims, which binds to a mutant of human CD38 in which Ser at position 274 is substituted with Phe to the same extent as it binds to human CD38.

6. EC of antibody binding to human CD38 variants 50 EC of the antibody binding to human CD38 50 6. The antibody of claim 5, wherein the antibody is at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% of the total antibody content.

7. The antibody of any one of the preceding claims, having the following binding properties: (i) it does not bind to a variant of human CD38 in which Asp at position 202 is substituted with Gly to the same extent as it binds to human CD38; (ii) it binds to a variant of human CD38 in which Gln at position 272 is substituted with Arg to the same extent as it binds to human CD38; and (iii) it binds to a variant of human CD38 in which Ser at position 274 is substituted with Phe to the same extent as it binds to human CD38.

8. 10. The antibody of any one of the preceding claims, which binds to human CD38 and has an inhibitory effect on CD38 cyclase activity and a stimulatory effect on CD38 hydrolase activity as measured in the assay of Example 8.

9. The antibody of claim 8, wherein the inhibitory effect is at least 50 to 66% compared to the inhibitory effect on CD38 cyclase activity in the absence of the antibody.

10. An antibody that binds to human CD38, encoded by a human heavy chain nucleic acid comprising in its variable region a nucleotide sequence set forth in SEQ ID NO: 1, 6, 11, 16 or 21 and a human light chain nucleic acid comprising in its variable region a nucleotide sequence set forth in SEQ ID NO: 26, 31, 36, 41 or 46.

11. 11. The antibody of claim 10, wherein the antibody is encoded by a human heavy chain nucleic acid and a human light chain nucleic acid comprising the nucleotide sequences set forth in SEQ ID NOs: 1 and 26, 6 and 31, 11 and 36, 16 and 41, or 21 and 46, respectively, in their variable regions.

12. c) a sequence set forth in SEQ ID NO: 5, 10, 15, 20, or 25; or d) variants of said sequences, e.g., variants having at most one, two, or three amino acid modifications, preferably substitutions, e.g., conservative substitutions An antibody that binds to human CD38, comprising a VH CDR3 comprising:

13. An antibody that binds to human CD38, comprising a VH CDR3 having the sequence set forth in SEQ ID NO: 5, 10, 15, 20 or 25 and a VL CDR3 having the sequence set forth in SEQ ID NO: 30, 35, 40, 45 or 50.

14. An antibody that binds to human CD38, comprising SEQ ID NO: 5 and SEQ ID NO: 30, or SEQ ID NO: 10 and SEQ ID NO: 35, or SEQ ID NO: 15 and SEQ ID NO: 40, or SEQ ID NO: 20 and SEQ ID NO: 45, or SEQ ID NO: 25 and SEQ ID NO: 50 as the VH CDR3 and VL CDR3, respectively.

15. (i) a VH CDR1 having a sequence set forth in any of SEQ ID NOs: 3, 8, 13, 18 and 23; a VH CDR2 having a sequence set forth in any of SEQ ID NOs: 4, 9, 14, 19 and 24; a VH CDR3 having a sequence set forth in any of SEQ ID NOs: 5, 10, 15, 20 and 25; a VL CDR1 having a sequence set forth in any of SEQ ID NOs: 28, 33, 38, 43 and 48; a VL CDR2 having a sequence set forth in any of SEQ ID NOs: 29, 34, 39, 44 and 49; a VL CDR3 having a sequence set forth in any of SEQ ID NOs: 30, 35, 40, 45 and 50; (ii) a VH CDR1 having the sequence set forth in SEQ ID NO: 3, a VH CDR2 having the sequence set forth in SEQ ID NO: 34, a VH CDR3 having the sequence set forth in SEQ ID NO: 5, a VL CDR1 having the sequence set forth in SEQ ID NO: 28, a VL CDR2 having the sequence set forth in SEQ ID NO: 29, and a VL CDR3 having the sequence set forth in SEQ ID NO: 30; (iii) VH CDR1 having the sequence set forth in SEQ ID NO: 8, VH CDR2 having the sequence set forth in SEQ ID NO: 9, VH CDR3 having the sequence set forth in SEQ ID NO: 10, VL CDR1 having the sequence set forth in SEQ ID NO: 33, VL CDR2 having the sequence set forth in SEQ ID NO: 34, VL CDR3 having the sequence set forth in SEQ ID NO: 35; (iv) VH CDR1 having the sequence set forth in SEQ ID NO: 13, VH CDR2 having the sequence set forth in SEQ ID NO: 14, VH CDR3 having the sequence set forth in SEQ ID NO: 15, VL CDR1 having the sequence set forth in SEQ ID NO: 38, VL CDR2 having the sequence set forth in SEQ ID NO: 39, VL CDR3 having the sequence set forth in SEQ ID NO: 40; (v) VH CDR1 having the sequence set forth in SEQ ID NO: 18, VH CDR2 having the sequence set forth in SEQ ID NO: 19, VH CDR3 having the sequence set forth in SEQ ID NO: 20, VL CDR1 having the sequence set forth in SEQ ID NO: 43, VL CDR2 having the sequence set forth in SEQ ID NO: 44, VL CDR3 having the sequence set forth in SEQ ID NO: 45; (vi) a VH CDR1 having the sequence set forth in SEQ ID NO: 23, a VH CDR2 having the sequence set forth in SEQ ID NO: 24, a VH CDR3 having the sequence set forth in SEQ ID NO: 25, a VL CDR1 having the sequence set forth in SEQ ID NO: 48, a VL CDR2 having the sequence set forth in SEQ ID NO: 49, a VL CDR3 having the sequence set forth in SEQ ID NO: 50, or (vii) A variant of any of the preceding antibodies, preferably having no more than one, two, or three amino acid modifications, and more preferably amino acid substitutions, e.g., conservative amino acid substitutions, in one or more of the sequences. An antibody that binds to CD38, comprising:

16. (i) comprising the sequence of SEQ ID NO: 2, 7, 12, 17 or 22; or (ii) has at least 80% identity, e.g., 90%, or 95%, or 97%, or 98%, or 99%, or 100% identity, to the VH region sequence set forth in SEQ ID NO: 2, 7, 12, 17, or 22. An antibody that binds to CD38, comprising a VH region.

17. (i) comprising the sequence of SEQ ID NO: 27, 32, 37, 42 or 47; or (ii) has at least 80% identity, e.g., 90%, or 95%, or 97%, or 98%, or 99%, or 100% identity, to a VL region sequence selected from the group consisting of SEQ ID NOs: 27, 32, 37, 42, or 47. An antibody that binds to CD38, comprising a VL region.

18. An antibody that binds to CD38, comprising a VH region comprising any of the sequences of SEQ ID NOs: 2, 7, 12, 17 and 22, and a VL region comprising any of the sequences of SEQ ID NOs: 27, 32, 37, 42 and 47.

19. (i) a VH region comprising the sequence set forth in SEQ ID NO: 2 and a VL region comprising the sequence set forth in SEQ ID NO: 27; (ii) a VH region comprising the sequence set forth in SEQ ID NO: 7 and a VL region comprising the sequence set forth in SEQ ID NO: 32; (iii) a VH region comprising the sequence set forth in SEQ ID NO: 12 and a VL region comprising the sequence set forth in SEQ ID NO: 37; (iv) a VH region comprising the sequence set forth in SEQ ID NO: 17 and a VL region comprising the sequence set forth in SEQ ID NO: 42; or (v) a VH region comprising the sequence set forth in SEQ ID NO: 22 and a VL region comprising the sequence set forth in SEQ ID NO: 47 An antibody that binds to CD38, comprising:

20. 20. An anti-CD38 antibody according to any one of claims 10 to 19, having a binding characteristic according to any one of claims 1 to 9.

21. An anti-CD38 antibody that binds to the same epitope on CD38 as the anti-CD38 antibody of any one of the preceding claims.

22. An antibody having substantially the same specific binding characteristics with respect to binding to human CD38 as the antibody of any one of claims 1 to 19.

23. Preferably, the EC50 of 5 nM or less, such as 1 nM or less, for example 0.2 nM or less, as measured by the method described in Example 6 herein. 50 10. The anti-CD38 antibody of claim 1, wherein the antibody is capable of inducing antibody-dependent cellular cytotoxicity (ADCC) in Daudi cells at a concentration of 0.1% or more.

24. 23. The anti-CD38 antibody of any one of claims 1 to 22, which is unable to induce ADCC in Daudi cells according to the method described in Example 6 herein.

25. 10. The anti-CD38 antibody of any one of the preceding claims, which is unable to induce complement-dependent cytotoxicity (CDC) in CHO-CD38 cells.

26. 10 -8 K below M D , preferably 10 -9 K below M D 6. The anti-CD38 antibody of claim 1, wherein the antibody binds to human CD38 at a specific agonist level.

27. 10. The anti-CD38 antibody of any one of the preceding claims, which is a human monovalent antibody.

28. 10. The antibody according to any one of the preceding claims, characterized in that it is a full-length IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibody, such as an IgG1 antibody, preferably an IgG1,κ antibody or an IgM antibody, preferably an IgM,κ antibody.

29. 28. The anti-CD38 antibody of any one of claims 1 to 27, which is an antibody fragment or a single chain antibody.

30. 30. The anti-CD38 antibody of any one of claims 1 to 22 or 24 to 29, which is an effector function-deficient antibody.

31. 31. The anti-CD38 antibody of claim 30, wherein the effector function-deficient anti-CD38 antibody is a stabilized human IgG4 antibody.

32. The anti-CD38 antibody of claim 31, wherein the stabilized IgG4 antibody is an antibody in which arginine at position 409 in the heavy chain constant region of human IgG4 has been substituted with lysine, threonine, methionine, or leucine, preferably lysine.

33. 33. The anti-CD38 antibody of claim 32, wherein said antibody comprises a Lys residue at the position corresponding to position 409, or wherein said CH3 region is substituted with the CH3 region of human IgG1, human IgG2, or human IgG3.

34. The anti-CD38 antibody of any one of claims 32 to 33, which does not contain a Cys-Pro-Pro-Cys sequence in the hinge region.

35. 34. The anti-CD38 antibody of any one of claims 32 to 33, comprising a Cys-Pro-Pro-Cys sequence in the hinge region.

36. 36. The anti-CD38 antibody of any one of claims 1 to 35, which is a monovalent antibody.

37. The monovalent antibody i) providing a nucleic acid construct encoding the light chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the VL region of SEQ ID NO: 27, 32, 37, 42 or 47 and a nucleotide sequence encoding a constant CL region of an Ig, wherein the nucleotide sequence encoding the VL region of a selected antigen-specific antibody and the nucleotide sequence encoding the CL region of an Ig are operably linked, and in the case of an IgG1 subtype, the nucleotide sequence encoding the CL region has been modified so that the CL region does not contain any amino acids that can form disulfide bonds or covalent bonds with other peptides containing the same amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to an animal or human; ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the VH region of SEQ ID NO: 2, 7, 12, 17 or 22 and a nucleotide sequence encoding the constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified so that a region corresponding to a hinge region and, if required by the Ig subtype, other regions of the CH region, e.g., the CH3 region, do not contain any amino acid residues that are involved in the formation of disulfide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising the same amino acid sequence of the CH region of the human Ig in the presence of polyclonal human IgG or when administered to an animal or human, and wherein the nucleotide sequence encoding the VH region of a selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operably linked; iii) providing a cellular expression system for producing said monovalent antibody; iv) producing the monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).

37. The anti-CD38 antibody of claim 36, constructed by a method comprising:

38. C H 2 and C H C, which includes three areas H The region corresponds to the hinge region, and if the immunoglobulin is not an IgG4 subtype, the C H Other areas of the region, e.g., C H The three regions were identified as identical C H Disulfide bond with the region or identical C H 38. The anti-CD38 antibody of any one of claims 36-37, wherein said antibody is modified so as to not contain any amino acid residues capable of forming other covalent or stable non-covalent inter-heavy chain bonds with said region.

39. Monovalent antibodies are of the IgG4 subtype, but C H The anti-CD38 antibody of any one of claims 36-38, wherein said three regions are modified to include one or more of the following amino acid substitutions: Thr (T) at position 366 is replaced by Ala (A); Leu (L) at position 368 is replaced by Ala (A); Leu (L) at position 368 is replaced by Val (V); Phe (F) at position 405 is replaced by Ala (A); Phe (F) at position 405 is replaced by Leu (L); Tyr (Y) at position 407 is replaced by Ala (A); Arg (R) at position 409 is replaced by Ala (A).

40. 40. The anti-CD38 antibody of any one of claims 36 to 39, wherein said heavy chain is modified to delete the entire hinge.

41. The anti-CD38 antibody of any one of claims 36 to 40, wherein the sequence of said monovalent antibody is modified so that it does not contain any acceptor sites for N-linked glycosylation.

42. 10. The antibody of any one of the preceding claims, which inhibits CD38-catalyzed synthesis of cGDPR by at least 25%, such as at least 30%, as measured by the spectrophotometric method described in Example 8 herein after 90 minutes at a concentration of 3 μg / ml.

43. An antibody described in any one of the preceding claims, which inhibits CD38-catalyzed synthesis of cADPR by at least 25%, for example at least 30%, as measured by the HPLC method described in Munshi et al., J. Biol. Chem. 275, 21566-21571 (2000) after 90 minutes at a concentration of 3 μg / ml.

44. 10. The antibody of any one of the preceding claims, which stimulates the hydrolase activity of CD38 by at least 25%.

45. 10. The antibody of any one of the preceding claims, which stimulates the NAD hydrolase activity of CD38 by at least 25%.

46. The antibody of any one of the preceding claims, which stimulates the cADPR hydrolase activity of CD38 by at least 25%.

47. An antibody described in any one of the preceding claims, which inhibits the ability of CD38 to catalyze the formation of NAADP through a base exchange reaction with an IC50 of less than 0.5 μg / mL, for example less than 0.2 μg / mL, by the method described in Example 8 of the present specification.

48. An antibody drug conjugate comprising the antibody of any one of the preceding claims, wherein the antibody is conjugated to a cytotoxic agent, a radioisotope, or a drug.

49. 49. The antibody drug conjugate of claim 48, wherein the antibody is conjugated to the auristatin or functional peptide analog or derivative thereof via a linker.

50. 50. A bispecific antibody comprising the antibody of any one of claims 1 to 49 and a second binding specificity for a human effector cell or a cancer antigen.

51. 51. A bispecific molecule comprising the antibody of claim 50, wherein the second binding specificity is for a human Fc receptor or a T cell receptor, such as CD3.

52. An isolated nucleic acid encoding the antibody of any one of the preceding claims.

53. 20. An expression vector comprising a nucleotide sequence encoding one or more of the amino acid sequences according to any one of claims 10 to 19.

54. 54. The expression vector of claim 53, further comprising a nucleotide sequence encoding the constant region of a human antibody light chain, a heavy chain, or both the light and heavy chains.

55. A recombinant eukaryotic or prokaryotic host cell producing the antibody of any one of claims 1 to 47.

56. 55. A pharmaceutical composition comprising an antibody of any one of claims 1 to 47, an immunoconjugate of any one of claims 48 to 49, a bispecific antibody of any one of claims 50 to 51, or an expression vector of any one of claims 53 to 54, and a pharmaceutically acceptable carrier.

57. 48. The antibody of any one of claims 1 to 47 for use as a medicament.

58. 48. The antibody of any one of claims 1 to 47 for use in inhibiting the growth and / or proliferation, migration of tumor cells expressing CD38, or inducing phagocytosis of tumor cells expressing CD38.

59. 48. The antibody of any one of claims 1 to 47 for use in the treatment of rheumatoid arthritis.

60. 48. The antibody of any one of claims 1 to 47 for use in the treatment of a disorder selected from chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (adult) (AML), mantle cell lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma.

61. 48. The antibody of any one of claims 1 to 47 for use in the treatment of multiple myeloma.

62. a) culturing the host cell of claim 55; and b) Purifying anti-CD38 antibodies from the culture medium 48. A method for producing the anti-CD38 antibody of any one of claims 1 to 47, comprising:

63. A diagnostic composition comprising an antibody according to any one of claims 1 to 47.

64. contacting the sample with the anti-CD38 antibody of any one of claims 1 to 47 under conditions that allow the formation of a complex between said antibody or bispecific molecule and CD38; and Analyzing whether a complex is formed 1. A method for detecting the presence of the CD38 antigen, i.e., cells expressing CD38, in a sample, comprising:

65. 48. A kit for detecting the presence of the CD38 antigen, i.e., cells expressing CD38, in a sample, comprising an anti-CD38 antibody according to any one of claims 1 to 47 and instructions for use of the kit.

66. An anti-idiotypic antibody that binds to the anti-CD38 antibody of any one of claims 1 to 47.

67. Administration of an antibody of any one of claims 1 to 47, an immunoconjugate of any one of claims 48 to 49, a bispecific antibody of any one of claims 50 to 51, an expression vector of any one of claims 53 to 54, or a pharmaceutical composition of claim 56, such that growth and / or proliferation, migration or phagocytosis of cells expressing CD38 is inhibited.

10. A method for inhibiting the growth and / or proliferation, migration of cells expressing CD38, or for inducing phagocytosis of cells expressing CD38, comprising:

68. Administering to a subject in need thereof the antibody of any one of claims 1 to 47, the immunoconjugate of any one of claims 48 to 49, the bispecific antibody of any one of claims 50 to 51, the expression vector of any one of claims 53 to 54, or the pharmaceutical composition of claim 56.

10. A method of treating a disease or disorder involving cells expressing CD38 in a subject, comprising:

69. 69. The method of any one of claims 67-68, wherein the disease or disorder is rheumatoid arthritis.

70. 69. The method of any one of claims 67-68, wherein the disease or disorder is selected from chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (adult) (AML), mantle cell lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma.

71. 69. The method of any one of claims 67-68, wherein the disease or disorder is multiple myeloma.

72. 72. The method of any one of claims 67-71, comprising administration of one or more additional therapeutic agents to the subject.

73. 73. The method of claim 72, wherein the one or more additional therapeutic agents are selected from a chemotherapeutic agent, an anti-inflammatory agent, or an immunosuppressant and / or immunomodulatory agent.

74. 74. The method of claim 73, wherein the one or more additional therapeutic agents are selected from the group consisting of cisplatin, gefitinib, cetuximab, rituximab, ofatumumab, bevacizumab, erlotinib, bortezomib, thalidomide, pamidronate, zoledronic acid, clodronate, risedronate, ibandronate, etidronate, alendronate, tiludronate, arsenic trioxide, lenalidomide, dexamethasone, prednisolone, filgrastim, pegfilgrastim, sargramostim, suberoylanilide hydroxamic acid, and SCIO-469.

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