Combination treatment method of CD38-expressing tumors

Abstract

To provide a novel method for treatment of cancer using a combination therapy comprising an antibody that binds CD38, a corticosteroid and a non-corticosteroid chemotherapeutic agent.SOLUTION: A pharmaceutical composition is formed by combining i) a non-agonistic antibody which binds to CD38, ii) at least one glucocorticoid, and iii) at least one non-corticosteroid chemotherapeutic agent including thalidomide or a thalidomide analog, or a proteasome inhibitor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the treatment of cancer with a combination therapy comprising an antibody that binds to CD38, a corticosteroid, and a non-corticosteroid chemotherapeutic agent. [Background technology]

[0002] background Multiple myeloma is a B-cell malignancy characterized by the latent accumulation in the bone marrow of secretory plasma cells with a low proliferation index and extended life span. The disease ultimately attacks the bone and bone marrow, resulting in multiple tumors and lesions throughout the skeletal system.

[0003] Approximately 1% of all cancers and slightly more than 10% of all hematologic malignancies can be attributed to multiple myeloma (MM). The incidence of MM is increasing in an aging population, with a median age at diagnosis of approximately 61 years.

[0004] Currently available treatments for multiple myeloma include chemotherapy, stem cell transplantation, Thalomid® (thalidomide), Velcade® (bortezomib), Aredia® (pamidronate), and Zometa® (zoledronic acid). Current treatment protocols, which include combinations of chemotherapy agents such as vincristine, BCNU, melphalan, cyclophosphamide, adriamycin, and prednisone or dexamethasone, result in only approximately a 5% complete remission rate, with a median survival of approximately 36 to 48 months from the time of diagnosis. Recent advances using high-dose chemotherapy followed by autologous bone marrow or peripheral blood mononuclear cell transplantation have increased the complete remission rate and duration of remission. However, overall survival has only slightly increased, and no evidence of a cure has been obtained. Ultimately, all MM patients relapse, even under maintenance treatment with interferon-alpha (IFN-α) alone or in combination with steroids.

[0005] If the patient is a candidate or potential candidate for autologous transplantation, induction therapy is often accompanied by non-alkylating chemotherapy, in that alkylating agents interfere with harvesting (stem cell recovery). The preferred regimen is VAD, which allows for later harvesting (Wu KL, Clin Lymphoma Myeloma 2005;6:96). Another treatment modality being considered in the pre-transplant induction setting includes thalidomide in combination with dexamethasone (Cavo M Blood 2005;106:35).

[0006] The effectiveness of available chemotherapy treatment regimens for MM is limited by low cell proliferation rates and the development of multidrug resistance. For more than 90% of MM patients, the disease becomes chemoresistant. As a result, alternative treatment regimens aimed at adoptive immunotherapy targeting surface antigens on plasma cells are being explored.

[0007] CD38 is an example of an antigen expressed on such malignant plasma cells and is expressed in a variety of malignant hematological diseases, including, but not limited to, multiple myeloma, B-cell chronic lymphocytic leukemia, B-cell acute lymphocytic leukemia, Waldenstrom'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 different origins, including the glandular epithelium of the prostate, pancreatic islet cells, ductal epithelium of glands, including the parotid gland, bronchial epithelial cells, cells of the testis and ovary, and tumor epithelium of colorectal adenocarcinoma. Diseases in which CD38 expression may be implicated include, but are not limited to, bronchial carcinoma of the lung, breast cancer (arising from malignant proliferation of the epithelial lining of the breast ducts and lobules), pancreatic tumors (insulinomas) arising from B cells, and tumors arising from the intestinal epithelium (e.g., adenocarcinomas and squamous cell carcinomas). In the CNS, neuroblastomas express CD38. Other such diseases include carcinoma of the prostate, seminoma of the testis, and ovarian cancer.

[0008] Normally, CD38 is expressed by hematopoietic cells and in solid tissues. Regarding 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 lymphoblasts in lymph node germinal centers, plasma B cells, and some intrafollicular cells. CD38 can also be expressed by dendritic cells. A significant proportion of normal bone marrow cells, especially precursor cells, express CD38. In addition to lymphoid precursor cells, CD38 is also expressed on erythrocytes and platelets.

[0009] In solid tissues, CD38 is expressed by intraepithelial cells and lamina propria lymphocytes in the intestine, by Purkinje cells and neurofibrillary tangles in the brain, by epithelial cells in the prostate, by beta cells in the pancreas, by osteoclasts in bone, by retinal cells in the eye, and by the sarcolemma of smooth and striated muscle.

[0010] Functions ascribed to CD38 include both receptor-mediated and (ecto)enzymatic activity in adhesion and signaling events. As an ectoenzyme, CD38 mediates the formation of cyclic ADP-ribose (cADPR) and ADPR, as well as NAD as a substrate for the formation of nicotinamide and nicotinic acid-adenosine dinucleotide phosphate (NAADP). + cADPR and NAADP are used. 2+ It has been shown to act as a second messenger for mobilization of NAD+. By converting NAD+ to cADPR, CD38 regulates extracellular NAD+ concentrations and therefore cell survival by modulating NAD-induced cell death (NCID). 2+ In addition to signaling via CD38, CD38 signaling occurs via crosstalk with antigen-receptor complexes or other types of receptor complexes, e.g., MHC molecules, on T and B cells and is thus involved in several cellular responses as well as IgG1 switching and secretion.

[0011] Anti-CD38 antibodies are described in the literature, for example, in Lande R, et al., Cell Immunol. 220 (1), 30-8 (2002) (Non-patent document 3), Ausiello CM, et al., Tissue Antigens. 56 (6), 539-47(2000) (Non-Patent Document 4), and Cotner T, et al., Int J Immunopharmacol. 3 (3), 255-68 (1981) (Non-Patent Document 5), and in International Publication No. 2005 / 103083 (Morphosys) (Patent Document 1). CD38 has many functions, which may or may not be activated by molecules that bind to CD38. For example, the murine anti-CD38 antibody IB4 has agonistic properties with respect to CD38. IB4 inhibits Ca2+ uptake in Jurkat cells. 2+ Induction of T cell activation as indicated by mobilization (Zubiaur M, et al., J Immunol. 159 (1), 193-205 (1997) (Non-Patent Document 6)), and has been shown to induce significant proliferation of peripheral blood mononuclear cells (PBMCs), induce significant release of IL-6 levels, and induce detectable release of IFN-γ levels (Lande, Zubiaur Morra, Ansiello supra (Non-Patent Document 3)).

[0012] Despite recent advances in the discovery and development of anti-cancer drugs, it is clear that many forms of cancer involving CD38-expressing tumors still have poor prognoses. Thus, there is a need for improved methods for treating such forms of cancer. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] WO 2005 / 103083 (Morphosys) [Non-patent literature]

[0014] [Non-Patent Document 1] Wu KL, Clin Lymphoma Myeloma 2005;6:96 [Non-patent document 2] Cavo M Blood 2005;106:35 [Non-patent document 3] Lande R, et al., Cell Immunol. 220(1), 30-8(2002) [Non-patent document 4] Ausiello CM, et al., Tissue Antigens. 56(6), 539-47(2000) [Non-Patent Document 5] Cotner T, et al., Int J Immunopharmacol. 3(3), 255-68(1981) [Non-patent document 6] Zubiaur M, et al., J Immunol. 159(1), 193-205(1997) Summary of the Invention

[0015] It is an object of the present invention to provide improved methods for the treatment of CD38-expressing tumors that result in increased efficacy and / or prolonged survival.

[0016] Thus, in a first main aspect, the present invention provides a method for inhibiting the growth and / or proliferation of tumor cells expressing CD38 in an individual in need thereof, comprising administering to the individual i) a non-agonistic antibody that binds to CD38; ii) at least one corticosteroid, and iii) at least one non-corticosteroid chemotherapy agent The present invention relates to a method comprising administering

[0017] The three drugs may be administered simultaneously or sequentially in any order. Additionally, they may be administered separately or in one or two pharmaceutical compositions.

[0018] Triple therapy may, in some embodiments, allow for the administration of lower amounts of medications than when used as monotherapy or in dual therapy. Such lower amounts may result in fewer side effects and allow for more effective treatment of patients who cannot be treated with higher doses, such as elderly or hypersensitive patients.

[0019] In one embodiment, the non-agonistic antibody that binds CD38 used in the present invention is antibody -005, -003, or -024 described herein. These antibodies have been previously described in patent application PCT / DK2006 / 000166 (WO 2006099875) (Genmab).

[0020] In some embodiments, the at least one non-corticosteroid chemotherapeutic agent is - alkylating agents, such as melphalan, and / or - glutamic acid derivatives, such as thalidomide or lenalidomide and / or - Proteasome inhibitors, such as bortezomib Includes.

[0021] In a similar aspect, the invention relates to a method of treating cancer involving cells expressing CD38 in an individual, the method comprising features of the above-described method.

[0022] In a further aspect, the present invention provides a method for treating cancer involving tumor cells expressing CD38 in an individual in need thereof, comprising administering to the individual i) a non-agonistic antibody that binds to CD38; ii) optionally at least one corticosteroid, and iii) optionally at least one non-corticosteroid chemotherapy agent followed by autologous peripheral stem cell transplantation or bone marrow transplantation.

[0023] Thus, in this method, anti-CD38 antibodies are used in induction therapy prior to autologous peripheral stem cell transplantation or bone marrow transplantation. Without being bound by any particular theory, it is believed that anti-CD38 antibodies are particularly suitable for such induction therapy because they do not have many undesirable side effects and therefore keep patients in good condition before transplantation.

[0024] In yet a further aspect, the present invention provides a method for the treatment of rheumatoid arthritis, which is suitable for separate, sequential, and / or simultaneous administration: i) a non-agonistic antibody that binds to CD38; ii) at least one corticosteroid, and iii) at least one non-corticosteroid chemotherapy agent The present invention relates to a therapeutic combination for inhibiting the growth and / or proliferation of tumor cells that express CD38, comprising: [The present invention 1001] for individuals i) a non-agonistic antibody that binds to CD38; ii) at least one corticosteroid, and iii) at least one non-corticosteroid chemotherapy agent including the administration of A method for inhibiting the growth and / or proliferation of tumor cells expressing CD38 in an individual in need thereof. [The present invention 1002] for individuals i) a non-agonistic antibody that binds to CD38; ii) optionally at least one corticosteroid, and iii) optionally at least one non-corticosteroid chemotherapy agent administration of followed by autologous peripheral stem cell transplantation or bone marrow transplantation, A method for treating cancer involving tumor cells that express CD38 in an individual in need thereof. [The present invention 1003] 1003. The method of any one of claims 1001 to 1002, wherein the at least one non-corticosteroid chemotherapeutic agent comprises a cytotoxic agent and / or an angiogenesis inhibitor. [The present invention 1004] Any of the aforementioned methods of the present invention, wherein at least one non-corticosteroid chemotherapeutic agent comprises an alkylating agent. [The present invention 1005] Any of the aforementioned methods of the present invention, wherein the at least one non-corticosteroid chemotherapeutic agent comprises one or more agents selected from the group consisting of melphalan, mechlorethamine, thioepa, chlorambucil, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin. [The present invention 1006] Any of the methods of the preceding invention, wherein the at least one non-corticosteroid chemotherapeutic agent comprises a glutamic acid derivative, such as thalidomide (Thalomid®) or a thalidomide analogue, e.g., CC-5013 (lenalidomide, Revlimid™) or CC4047 (Actimid™). [The present invention 1007] Any of the aforementioned methods of the present invention, wherein the at least one non-corticosteroid chemotherapeutic agent comprises a proteasome inhibitor, such as bortezomib (Velcade®). [The present invention 1008] Any of the aforementioned methods of the present invention, wherein at least one non-corticosteroid chemotherapeutic agent comprises a vinca alkaloid, such as vincristine. [The present invention 1009] Any of the aforementioned methods of the present invention, wherein at least one non-corticosteroid chemotherapeutic agent comprises an anthracycline, such as doxorubicin. [The present invention 1010] Any of the aforementioned methods of the present invention, wherein the at least one corticosteroid comprises a glucocorticoid. [The present invention 1011] Any of the aforementioned methods of the present invention, wherein the at least one corticosteroid comprises prednisone. [The present invention 1012] Any of the aforementioned methods of the present invention, wherein the at least one corticosteroid comprises prednisone and the at least one non-corticosteroid chemotherapeutic agent comprises melphalan. [The present invention 1013] Any of the aforementioned methods of the present invention, wherein the at least one corticosteroid comprises prednisone and the at least one non-corticosteroid chemotherapeutic agent comprises thalidomide. [The present invention 1014] Any of the aforementioned methods of the present invention, wherein the at least one corticosteroid comprises prednisone and the at least one non-corticosteroid chemotherapeutic agent comprises melphalan and thalidomide. [The present invention 1015] Any of the aforementioned methods of the present invention, wherein the at least one corticosteroid comprises dexamethasone. [The present invention 1016] Any of the aforementioned methods of the present invention, wherein the at least one corticosteroid comprises dexamethasone and the at least one non-corticosteroid chemotherapeutic agent comprises thalidomide and / or lenalidomide. [The present invention 1017] Any of the aforementioned methods of the present invention, wherein the at least one corticosteroid comprises dexamethasone and the at least one non-corticosteroid chemotherapeutic agent comprises vincristine and / or doxorubicin. [The present invention 1018] Any of the aforementioned methods of the present invention comprising the further administration of interferon-alpha. [The present invention 1019] Any of the aforementioned methods of the present invention, wherein the antibody is a monoclonal antibody. [The present invention 1020] Any of the aforementioned methods of the present invention, wherein the antibody is a human monoclonal antibody. [The present invention 1021] Any of the aforementioned methods of the present invention, wherein the antibody is an antagonist of CD38. [The present invention 1022] Any of the methods of the present invention, wherein the antibody does not induce significant IL-6 release by human monocytes or peripheral blood mononuclear cells, as determined by the method described in Example 19 herein. [The present invention 1023] Any of the methods of the present invention, wherein the antibody does not induce detectable release of IFN-γ by human T cells or peripheral blood mononuclear cells, as determined by the method described in Example 20 herein. [The present invention 1024] Any of the methods of the present invention, wherein the antibody is an antibody that is internalized by CD38-expressing cells, for example, an antibody that is internalized by CHO-CD38 cells at 37°C within 5 to 15 minutes by the method described in Example 12 of the present specification. [The present invention 1025] The antibody is an antibody that induces ADCC, e.g., an EC200 of less than 15 ng / ml, e.g., less than 10 ng / ml, on Daudi-luc cells, as determined, e.g., by the method described in Example 5 herein. 50 values ​​and an EC of less than 75 ng / ml in MM cells, e.g., less than 50 ng / ml, 30 ng / ml, or 10 ng / ml 50 Any of the aforementioned methods of the present invention, wherein the antibody has a value. [The present invention 1026] The antibody is an antibody that induces CDC in the presence of complement and has an EC of less than 5 μg / ml, e.g., less than 1 μg / ml, on daudi-luc or CD38-CHO cells, e.g., by the method described in Example 6 herein. 50 Any of the aforementioned methods of the present invention, wherein the antibody has a value. [The present invention 1027] Any of the aforementioned methods of the present invention, wherein the antibody is an antibody that inhibits the synthesis of cGDPR. [The present invention 1028] Any of the aforementioned methods of the present invention, wherein the antibody is an antibody that inhibits the synthesis of cADPR. [The present invention 1029] The antibody has a 10 -8 Less than M, e.g. 10 -8 M~10 -11 Within the range of M, e.g., 7 x 10 -9 M~10 -10 Affinity (K D ) an antibody that binds to human CD38. [The present invention 1030] Any of the methods of the invention, wherein the antibody inhibits synthesis of cGDPR by at least 25%, such as at least 30%, after 90 minutes at a concentration of 3 μg / ml, as determined by the spectrophotometric method described in Example 24 herein. [The present invention 1031] Any of the methods of the present invention, wherein the antibody inhibits the synthesis of cADPR by at least 25%, for example at least 30%, after 90 minutes at a concentration of 3 μg / ml, as determined by the HPLC method described in Munshi et al., J. Biol. Chem. 275, 21566-21571 (2000). [The present invention 1032] The antibody comprises a V having the sequence shown in SEQ ID NO: 10. H Any of the aforementioned methods of the invention, wherein the antibody is an antibody that comprises a CDR3 or an antibody that competes with the antibody for CD38 binding, e.g., by binding to the same epitope as the antibody. [The present invention 1033] The antibody has the sequence shown in SEQ ID NO:5 L CDR3 and V having the sequence shown in SEQ ID NO: 10 H Any of the aforementioned methods of the present invention, wherein the antibody comprises a CDR3. [The present invention 1034] the antibody comprises a human light chain and a human heavy chain variable region, The light chain variable region has the sequence shown in SEQ ID NO:3 LCDR1, V having the sequence shown in SEQ ID NO:4 L CDR2, and V having the sequence shown in SEQ ID NO:5 L CDR3 and a heavy chain variable region having the sequence set forth in SEQ ID NO: 8 H CDR1, V having the sequence shown in SEQ ID NO:9 H CDR2, and V having the sequence shown in SEQ ID NO: 10 H an antibody comprising CDR3, Any of the methods of the present invention. [This invention 1035] The antibody has the amino acid sequence shown in SEQ ID NO:2 L a region, or a V region having at least about 90%, e.g., at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO:2 L The method according to any one of claims 1032 to 1034, wherein the antibody comprises the region. [The present invention 1036] The antibody has the amino acid sequence shown in SEQ ID NO:7 H region, or a V having at least about 90%, e.g., at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO:7. H region, or V having 1 to 5, for example 1 to 3 amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 7. H The method according to any one of claims 1032 to 1035, wherein the antibody comprises the region. [This invention 1037] The antibody comprises a V having the sequence shown in SEQ ID NO:20 H The method of any of claims 1001 to 1031, wherein the antibody comprises a CDR3 or competes with said antibody for CD38 binding, for example by binding to the same epitope as said antibody. [The present invention 1038] The antibody comprises a V having the sequence shown in SEQ ID NO: 15. L CDR3 and V having the sequence shown in SEQ ID NO:20 H The method according to any one of claims 1001 to 1031, wherein the antibody comprises CDR3. [This invention 1039] the antibody comprises a human light chain and a human heavy chain variable region, The light chain variable region has the sequence shown in SEQ ID NO: 13. L CDR1, V having the sequence shown in SEQ ID NO: 14 L CDR2, and V having the sequence shown in SEQ ID NO: 15 L CDR3 and a heavy chain variable region having the sequence set forth in SEQ ID NO: 18 H CDR1, V having the sequence shown in SEQ ID NO: 19 H CDR2, and V having the sequence shown in SEQ ID NO:20 H an antibody comprising CDR3, Any of the methods of 1001 to 1031 of the present invention. [The present invention 1040] The antibody has the amino acid sequence shown in SEQ ID NO: 12 L a region, or a V region having at least about 90%, for example at least about 95%, amino acid sequence identity with a sequence according to SEQ ID NO: 12 L The method according to any one of claims 1037 to 1039, wherein the antibody comprises the region. [The present invention 1041] The antibody has the amino acid sequence shown in SEQ ID NO: 17. H a region, or a V region having at least about 90%, e.g., at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO: 17 H region, or V having 1 to 5, for example 1 to 3 amino acid substitutions, deletions, or additions compared to the sequence set forth in SEQ ID NO: 17. H The method according to any one of claims 1037 to 1040, wherein the antibody comprises the region. [The present invention 1042] The antibody comprises a V having the sequence shown in SEQ ID NO: 30 H The method of any of claims 1001 to 1031, wherein the antibody comprises a CDR3 or competes with said antibody for CD38 binding, for example by binding to the same epitope as said antibody. [This invention 1043] The antibody comprises a V LCDR3 and V having the sequence shown in SEQ ID NO: 30 H The method according to any one of claims 1001 to 1031, wherein the antibody comprises CDR3. [This invention 1044] the antibody comprises a human light chain and a human heavy chain variable region, The light chain variable region has the sequence shown in SEQ ID NO: 23. L CDR1, V having the sequence shown in SEQ ID NO:24 L CDR2, and V having the sequence shown in SEQ ID NO: 25 L a heavy chain variable region comprising a V CDR3 and having the sequence set forth in SEQ ID NO: 28; H CDR1, V having the sequence shown in SEQ ID NO:29 H CDR2, and V having the sequence shown in SEQ ID NO: 30 H an antibody comprising CDR3, Any of the methods of 1001 to 1031 of the present invention. [This invention 1045] The antibody has the amino acid sequence shown in SEQ ID NO: 22 L region, or a V having at least about 90%, for example at least about 95%, amino acid sequence identity with a sequence according to SEQ ID NO: 22. L The method according to any one of claims 1042 to 1044, wherein the antibody comprises the region. [The present invention 1046] The antibody has the amino acid sequence set forth in SEQ ID NO:27 H region, or a V having at least about 90%, e.g., at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO:27. H region, or V having 1 to 5, for example 1 to 3 amino acid substitutions, deletions, or additions compared to the sequence set forth in SEQ ID NO: 27. H The method according to any one of claims 1042 to 1045, wherein the antibody comprises the region. [This invention 1047] Any of the aforementioned methods of the present invention, wherein the antibody 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. [This invention 1048] The antibody, (i) Human Hv1263 / 3M28(V H I) a heavy chain variable region amino acid sequence derived from a germline sequence and a light chain variable region amino acid sequence derived from a human L15 (VκI) germline sequence; or (ii) Human V H 3-DP-47 / V3-23(V H III) Heavy chain variable region amino acid sequence derived from germline sequence and light chain variable region amino acid sequence derived from human L6 (VκI) germline sequence is a human monoclonal antibody comprising Any of the methods of the present invention. [This invention 1049] Any of the aforementioned methods of the invention, wherein the antibody is an antibody fragment or a single chain antibody. [The present invention 1050] Any of the aforementioned methods of the invention, wherein the antibody is conjugated to a cytotoxic agent, radioisotope, or drug. [This invention 1051] The antibody, an antibody as defined in any of the preceding aspects of the invention, and Binding specificity to human effector cells is a bispecific or multispecific molecule comprising Any of the methods of the present invention. [This invention 1052] The antibody, an antibody as defined in any of the preceding aspects of the invention, and Binding specificity for CD3, CD4, CD138, IL-15R, membrane-bound or receptor-bound TNF-α, human Fc receptor, or membrane-bound or receptor-bound IL-15 is a bispecific or multispecific molecule comprising Any of the methods of the present invention. [This invention 1053] Any of the aforementioned methods of the present invention, wherein the tumor cells are multiple myeloma cells or chronic lymphocytic leukemia cells. [This invention 1054] Any of the aforementioned methods of the present invention, wherein the tumor cells are relapsed or refractory tumor cells. [This invention 1055] Any of the aforementioned methods of the present invention, wherein the individual is 65 years of age or older. [This invention 1056] The method of any one of claims 1001 to 1054, wherein the individual is under 65 years of age. [This invention 1057] Any of the aforementioned methods of the present invention, wherein the individual has not received prior anti-cancer treatment for the same cancer. [This invention 1058] The method of any of claims 1001 to 1056, wherein the individual has not responded to prior anti-cancer treatment for the same cancer. [This invention 1059] The method of any of claims 1001 to 1056 or 1058, wherein the individual has previously undergone an autologous peripheral stem cell transplant or bone marrow transplant. [The present invention 1060] The method of any of claims 1001 or 1003-1059, wherein the individual is enrolled to undergo a subsequent autologous peripheral stem cell transplant or bone marrow transplant. [This invention 1061] Any of the aforementioned methods of the invention, wherein the antibody, at least one corticosteroid, and at least one non-corticosteroid chemotherapeutic agent are administered simultaneously. [This invention 1062] The method of any of claims 1001 to 1060, wherein the antibody, at least one corticosteroid, and at least one non-corticosteroid chemotherapeutic agent are administered sequentially. [This invention 1063] Any of the aforementioned methods of the invention, wherein the antibody, at least one corticosteroid, and at least one non-corticosteroid chemotherapeutic agent are all administered separately. [This invention 1064] The method of any of claims 1001 to 1060, wherein the antibody, at least one corticosteroid, and at least one non-corticosteroid chemotherapeutic agent are co-administered as one or two pharmaceutical compositions. [This invention 1065] The method of claim 1062, wherein the antibody is administered at least 1 day, such as at least 2 days, such as at least 1 week, before the administration of the at least one corticosteroid and the at least one non-corticosteroid chemotherapeutic agent. [The present invention 1066] Any of the aforementioned methods of the present invention, wherein the antibody is administered at a dose of 1 mg / kg or more, for example, at a dose of 1 to 20 mg / kg, for example, at a dose of 5 to 20 mg / kg, for example, at a dose of 8 mg / kg. [This invention 1067] Any of the methods of the present invention, wherein the antibody is administered once a week for 2 to 12 weeks, for example, 3 to 10 weeks, for example, 4 to 8 weeks. [The present invention 1068] A method of treating cancer involving cells expressing CD38 in an individual, comprising any one or more features of the invention. [This invention 1069] The method of claim 1068, wherein the cancer is multiple myeloma or chronic lymphocytic leukemia. [The present invention 1070] 10. Use of an antibody that binds CD38 in the manufacture of a medicament for the treatment of cancer, which is or is to be administered in combination therapy with at least one corticosteroid and at least one non-corticosteroid chemotherapeutic agent. [This invention 1071] Use of invention 1070, including any one or more features of inventions 1001-1067. [This invention 1072] Suitable for separate, sequential and / or simultaneous administration; i) a non-agonistic antibody that binds to CD38; ii) at least one corticosteroid, and iii) at least one non-corticosteroid chemotherapy agent 10. A therapeutic combination for inhibiting the growth and / or proliferation of tumor cells that express CD38, comprising: [This invention 1073] The therapeutic combination of claim 1072, wherein the composition comprises one or more features of any one of claims 1001 to 1067. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1A shows the binding of -003, -005, and the isotype control antibody HuMab-KLH to CD38-transfected CHO (CHO-CD38) cells as measured by flow cytometry. The experimental set-up is described in Example 4. Figure 1B shows the binding of -024 and HuMab-KLH to CD38-transfected CHO (CHO-CD38) cells as measured by flow cytometry. The experimental set-up is described in Example 4. [Figure 2] Figure 2A shows the binding of -003, -005, and HuMab-KLH to Daudi cells as measured by flow cytometry. The experimental setup is described in Example 4. Figure 2B shows the binding of -024 and HuMab-KLH to Daudi cells as measured by flow cytometry. The experimental setup is described in Example 4. [Figure 3] Binding of -003, -005, -024, and HuMab-KLH to multiple myeloma cells is shown. The experimental setup is described in Example 4. [Figure 4] Figure 4A shows the ability of -003 and -005 to induce lysis of Daudi cells by ADCC compared to rituximab and HuMab-KLH. The experimental setup is described in Example 5. Figure 4B shows the ability of -024 to induce lysis of Daudi cells by ADCC compared to HuMab-KLH. The experimental setup is described in Example 5. [Figure 5]Figure 5A shows the ability of -003, -005, and -024 to induce lysis of fresh multiple myeloma tumor cells by ADCC compared to HuMab-KLH. The experimental setup is described in Example 5. Figure 5B shows the ability of -003, -005, and -024 to induce lysis of fresh plasma cell leukemia tumor cells by ADCC compared to HuMab-KLH. The experimental setup is described in Example 5. [Figure 6] Figure 1 shows the ability of -003 and -005 to induce lysis of JK6L (multiple myeloma cell line) by ADCC compared to HuMab-KLH. The experimental setup is described in Example 5. [Figure 7] Figure 1 shows the ability of -003 and -005 to induce lysis of AMO-1 (a multiple myeloma cell line) by ADCC compared to HuMab-KLH. The experimental setup is described in Example 5. [Figure 8] Figure 1 shows CDC-mediated lysis of Daudi-luc cells induced by -003 and -005 compared to HuMab-KLH. The experimental setup is described in Example 6. [Figure 9] Figure 9A shows CDC-mediated lysis of CHO-CD38 cells induced by -003 and -005 compared to HuMab-KLH. The experimental setup is described in Example 6. Figure 9B shows CDC-mediated lysis of CHO-CD38 cells induced by -024 compared to HuMab-KLH. The experimental setup is described in Example 6. [Figure 10A] Figure 1 shows CDC-mediated lysis of 3% refractory tumor cells in the presence of -003, -005, and HuMab-KLH. The experimental setup is described in Example 6. [Figure 10B] Figure 1 shows CDC-mediated lysis of 9% refractory tumor cells in the presence of -003, -005, and HuMab-KLH. The experimental setup is described in Example 6. [Figure 10C] Figure 1 shows CDC-mediated lysis of 30-40% tumor cells in the presence of -003, -005, and HuMab-KLH. The experimental setup is described in Example 6. [Figure 10D] Figure 1 shows CDC-mediated 70% tumor cell lysis in the presence of -003, -005, and HuMab-KLH. The experimental setup is described in Example 6. [Figure 10E] Figure 1 shows CDC-mediated lysis of multiple myeloma cells in the presence of -024 and HuMab-KLH. The experimental setup is described in Example 6. [Figure 11] This shows that -003 and -005 do not cross-block binding to CD38. The experimental setup is described in Example 7. [Figure 12] Figure 12A shows immunohistological staining of macrophages, lymphocytes, and plasma B cells with -003. The experimental setup is described in Example 10. Figure 12B shows immunohistological staining of bronchial epithelium with -003. The experimental setup is described in Example 10. Figure 12C shows immunohistological staining of muscle cells with -003. The experimental setup is described in Example 10. Figure 12D shows immunohistological staining of cynomolgus monkey lymphoid tissue with -003. The experimental setup is described in Example 10. [Figure 13] Figure 13A shows immunohistological staining of macrophages, lymphocytes, and plasma B cells with -005. The experimental setup is described in Example 10. Figure 13B shows immunohistological staining of bronchial epithelium with -005. The experimental setup is described in Example 10. Figure 13C shows immunohistological staining of muscle cells with -005. The experimental setup is described in Example 10. Figure 13D shows immunohistological staining of cynomolgus monkey lymphoid tissue with -005. The experimental setup is described in Example 10. [Figure 14]Figure 14A shows immunohistological staining of liver endothelium with CD31. The experimental setup is described in Example 10. Figure 14B shows immunohistological staining of liver endothelium with vWF. The experimental setup is described in Example 10. Figure 14C shows immunohistological staining of liver endothelium with anti-KLH. The experimental setup is described in Example 10. Figure 14D shows immunohistological staining of liver endothelium with -003. The experimental setup is described in Example 10. Figure 14E shows immunohistological staining of liver endothelium with -005. The experimental setup is described in Example 10. [Figure 15A] Figure 1 shows the cross-reactivity of -003 and -005 compared to HuMab-KLH on cynomolgus monkey lymphocytes as measured by flow cytometry. The experimental set-up is described in Example 11. [Figure 15B] Figure 1 shows the cross-reactivity of -003 and -005 compared to HuMab-KLH on cynomolgus monkey monocytes as measured by flow cytometry. The experimental set-up is described in Example 11. [Figure 15C] 1 shows the cross-reactivity of -003 and -005 compared to HuMab-KLH on rhesus monkey PBMCs as measured by flow cytometry. The experimental set-up is described in Example 11. [Figure 16] Figure 16A shows the internalization of -003 as measured by EtBr-quenching. The experimental set-up is described in Example 12. Figure 16B shows the internalization of -005 as measured by EtBr-quenching. The experimental set-up is described in Example 12. [Figure 17A] Figure 1 shows the inhibition of tumor cell growth caused by -003 and -005 compared to anti-CD20 monoclonal antibody (rituximab) and HuMab-KLH in a prophylactic setting, as measured by in vivo SCID luciferase imaging. The experimental setup is described in Example 13. [Figure 17B]Figure 1 shows the inhibition of tumor cell growth caused by -003 and -005 compared to an anti-CD20 monoclonal antibody (rituximab) and HuMab-KLH in therapeutic setting I, as measured by in vivo SCID luciferase imaging. The experimental setup is described in Example 13. [Figure 17C] Figure 1 shows the inhibition of tumor cell growth caused by -003 and -005 compared to anti-CD20 monoclonal antibody (rituximab) and HuMab-KLH in therapeutic setting II, as measured by in vivo SCID luciferase imaging. The experimental setup is described in Example 13. [Figure 17D] Figure 1 shows the inhibition of tumor cell growth by -003 and -024 compared to HuMab-KLH in therapeutic setting III as measured by in vivo SCID luciferase imaging. The experimental setting is described in Example 13. [Figure 18] Figure 1 shows the induction of apoptosis by -003 and -005 compared to anti-CD20 monoclonal antibody (rituximab) and HuMab-KLH without or with cross-linking. The experimental setup is described in Example 14. [Figure 19] Figure 1 shows the histological score for CD38-positive cells in transplanted RA-SCID mouse xenografts on day 14 after treatment with anti-KLH (HuMab-KLH) or -005. Methods are described in Example 15. [Figure 20] 1 shows the histological score for CD138-positive cells in transplanted RA-SCID mouse xenografts on day 14 after treatment with anti-KLH or -005. Methods are described in Example 15. [Figure 21] CD38 staining of B cells in xenografts before transplantation (A) or after treatment with anti-KLH (B) or -005 (C) is shown. Methods are described in Example 15. [Figure 22]CD138 staining of B cells in xenografts before transplantation (A) or after treatment with anti-KLH (B) or -005 (C) is shown. Methods are described in Example 15. [Figure 23A] Figure 2 shows the binding of -003 and -005 to wild-type and mutant human CD38 as measured by ELISA. 23A: Binding of -003 and -005 to T237A mutant human CD38. 23B: Binding of -003 and -005 to Q272R mutant human CD38. 23C: Binding of -003 and -005 to S274F mutant human CD38. Methods are described in Example 17. [Figure 23B] Figure 2 shows the binding of -003 and -005 to wild-type and mutant human CD38 as measured by ELISA. 23A: Binding of -003 and -005 to T237A mutant human CD38. 23B: Binding of -003 and -005 to Q272R mutant human CD38. 23C: Binding of -003 and -005 to S274F mutant human CD38. Methods are described in Example 17. [Figure 23C] Figure 2 shows the binding of -003 and -005 to wild-type and mutant human CD38 as measured by ELISA. 23A: Binding of -003 and -005 to T237A mutant human CD38. 23B: Binding of -003 and -005 to Q272R mutant human CD38. 23C: Binding of -003 and -005 to S274F mutant human CD38. Methods are described in Example 17. [Figure 24] The effects of -003 and -005 compared to HuMab-KLH on human PBMC proliferation (A), IL-6 production (B), and IFN-γ production (C) are shown. Methods are described in Examples 18, 19, and 20, respectively. [Figure 25A] Enzymatic production of cGDP-ribose in the presence of various concentrations of -003 (B), -005 (C), -024 (D), or anti-KLH (A) is shown. Methods are described in Example 23. [Figure 25B]Enzymatic production of cGDP-ribose in the presence of various concentrations of -003 (B), -005 (C), -024 (D), or anti-KLH (A) is shown. Methods are described in Example 23. [Figure 25C] Enzymatic production of cGDP-ribose in the presence of various concentrations of -003 (B), -005 (C), -024 (D), or anti-KLH (A) is shown. Methods are described in Example 23. [Figure 25D] Enzymatic production of cGDP-ribose in the presence of various concentrations of -003 (B), -005 (C), -024 (D), or anti-KLH (A) is shown. Methods are described in Example 23. [Figure 26A] 26A shows a comparison between -003 and -005 and Morphosys antibody TH-3079 in CDC on CHO-CD38 cells (26A), CDC on Daudi cells (26B), and ADCC on Daudi cells (26C). Methods are described in Example 24. [Figure 26B] 26A shows a comparison between -003 and -005 and Morphosys antibody TH-3079 in CDC on CHO-CD38 cells (26A), CDC on Daudi cells (26B), and ADCC on Daudi cells (26C). Methods are described in Example 24. [Figure 26C] 26A shows a comparison between -003 and -005 and Morphosys antibody TH-3079 in CDC on CHO-CD38 cells (26A), CDC on Daudi cells (26B), and ADCC on Daudi cells (26C). Methods are described in Example 24. [Figure 27] Figure 1 shows the binding of -005 and the isotype control antibody HuMab-KLH to EBV-transformed chimpanzee B cells as measured by flow cytometry. The experimental set-up is described in Example 26. [Figure 28] We demonstrate the ability of -005, alone and in combination with other compounds (dexamethasone (Dex) and bortezomib (Bor)), to induce cell death in the multiple myeloma cell line UM6 in vitro. [Figure 29-1] 1 shows a sequence listing of the sequences of the present invention. [Figure 29-2] FIG. 29-2 is a continuation of FIG. 29-1. [Figure 29-3] FIG. 29-3 is a continuation of FIG. 29-2. [Figure 29-4] FIG. 29-4 is a continuation of FIG. 29-3. [Figure 29-5] FIG. 29-5 is a continuation of FIG. 29-4. [Figure 29-6] FIG. 29-6 is a continuation of FIG. 29-5. [Figure 29-7] FIG. 29-7 is a continuation of FIG. 29-6. [Figure 29-8] FIG. 29-8 is a continuation of FIG. 29-7. [Figure 29-9] Figure 29-9 is a continuation of Figure 29-8. [Figure 29-10] Figure 29-10 is a continuation of Figure 29-9. [Figure 29-11] Figure 29-11 is a continuation of Figure 29-10. [Figure 29-12] FIG. 29-12 is a continuation of FIG. 29-11. [Figure 29-13] FIG. 29-13 is a continuation of FIG. 29-12. [Figure 29-14] FIG. 29-14 is a continuation of FIG. 29-13. [Figure 29-15] Figure 29-15 is a continuation of Figure 29-14. [Figure 29-16] Figure 29-16 is a continuation of Figure 29-15. [Figure 29-17] Figure 29-17 is a continuation of Figure 29-16. [Figure 29-18] Figure 29-18 is a continuation of Figure 29-17. [Figure 29-19] Figure 29-19 is a continuation of Figure 29-18. [Figure 29-20] Figure 29-20 is a continuation of Figure 29-19.

[0026] Sequence Listing Free Text SEQ ID NO: 1 V of antibody-003 L Nucleotide sequence of the region. SEQ ID NO: 2 V of antibody-003 L Amino acid sequence of the region. SEQ ID NO: 3 V of antibody-003 comprising aa 24 to 34 of SEQ ID NO: 2 L Amino acid sequence of CDR1. SEQ ID NO: 4 V of antibody-003 comprising aa 50 to 56 of SEQ ID NO: 2 L Amino acid sequence of CDR2. SEQ ID NO: 5 V of antibody-003 comprising aa 89 to 97 of SEQ ID NO: 2 L Amino acid sequence of CDR3. SEQ ID NO: 6 V of antibody-003 H Nucleotide sequence of the region. SEQ ID NO: 7 V of antibody-003 H Amino acid sequence of the region. SEQ ID NO: 8 V of antibody-003 comprising aa 31 to 35 of SEQ ID NO: 7 H Amino acid sequence of CDR1. SEQ ID NO: 9 V of antibody-003 comprising aa 50 to 66 of SEQ ID NO: 7 H Amino acid sequence of CDR2. SEQ ID NO: 10 V of antibody-003 comprising aa 99 to 109 of SEQ ID NO: 7 H Amino acid sequence of CDR3. SEQ ID NO: 11 V of antibody-005 L Nucleotide sequence of the region. SEQ ID NO: 12 V of antibody-005 L Amino acid sequence of the region. SEQ ID NO: 13 V of antibody-005 comprising aa 24 to 34 of SEQ ID NO: 12 L Amino acid sequence of CDR1. SEQ ID NO: 14 V of antibody-005 comprising aa 50 to 56 of SEQ ID NO: 12 L Amino acid sequence of CDR2. SEQ ID NO: 15 V of antibody-005 comprising aa 89 to 97 of SEQ ID NO: 12 L Amino acid sequence of CDR3. SEQ ID NO: 16 V of antibody-005H Nucleotide sequence of the region. SEQ ID NO: 17 V of antibody-005 H Amino acid sequence of the region. SEQ ID NO: 18 V of antibody-005 comprising aa 31 to 35 of SEQ ID NO: 17 H Amino acid sequence of CDR1. SEQ ID NO: 19 V of antibody-005 comprising aa 50 to 66 of SEQ ID NO: 17 H Amino acid sequence of CDR2. SEQ ID NO: 20 V of antibody-005 comprising aa 99 to 111 of SEQ ID NO: 17 H Amino acid sequence of CDR3. SEQ ID NO: 21 V of antibody-024 L Nucleotide sequence of the region. SEQ ID NO: 22 V of antibody-024 L Amino acid sequence of the region. SEQ ID NO: 23 V of antibody-024 comprising aa 24 to 34 of SEQ ID NO: 22 L Amino acid sequence of CDR1. SEQ ID NO: 24 V of antibody-024 comprising aa 50 to 56 of SEQ ID NO: 22 L Amino acid sequence of CDR2. SEQ ID NO: 25 V of antibody-024 comprising aa 89 to 97 of SEQ ID NO: 22 L Amino acid sequence of CDR3. SEQ ID NO: 26 V of antibody-024 H Nucleotide sequence of the region. SEQ ID NO: 27 V of antibody-024 H Amino acid sequence of the region. SEQ ID NO: 28 V of antibody-024 comprising aa 31 to 35 of SEQ ID NO: 27 H Amino acid sequence of CDR1. SEQ ID NO: 29 V of antibody-024 comprising aa 50 to 66 of SEQ ID NO: 27 H Amino acid sequence of CDR2. SEQ ID NO: 30 V of antibody-024 comprising aa 99 to 111 of SEQ ID NO: 27 H Amino acid sequence of CDR3. SEQ ID NO: 31 Sequence of human CD38. SEQ ID NO: 32 Sequence of mutant human CD38, in which the threonine residue at position 237 is replaced with an alanine residue. SEQ ID NO: 33 Sequence of mutant human CD38, in which the glutamine residue at position 272 is replaced with an arginine residue. SEQ ID NO: 34 Sequence of mutant human CD38, in which the serine residue at position 274 is replaced with a phenylalanine residue. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description of the Invention definition As used herein, a "non-agonistic antibody that binds to CD38" or an "anti-CD38 antibody" refers to an antibody that does not induce significant proliferation of peripheral blood mononuclear cells upon binding to CD38 compared to the proliferation induced by an isotype control antibody or medium alone (e.g., as assayed as described in Example 18 herein below). In certain embodiments, the anti-CD38 antibodies used in the present invention are not only non-agonistic but also antagonistic of CD38.

[0028] The terms "CD38" and "CD38 antigen" are used interchangeably herein and include any variant, isoform, and species homolog of human CD38 that is naturally expressed by cells or expressed on cells transfected with the CD38 gene. Art-recognized synonyms for CD38 include ADP-ribosyl cyclase 1, cADPr hydrolase 1, Cd38-rs1, cyclic ADP-ribose hydrolase 1, I-19, and NIM-R5 antigen.

[0029] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains: one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four of which are interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, e.g., Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)). Briefly, each heavy chain typically comprises a heavy chain variable region (herein referred to as V H The heavy chain constant region typically consists of three domains: C H 1. C H 2, and C H Each light chain is typically composed of a light chain variable region (referred to herein as V L The light chain constant region typically consists of one domain, C L It consists of: V H and V L The regions can be further subdivided into regions of hypervariability (or hypervariable regions where the sequence and / or shape of structurally defined loops can be hypervariable), also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).

[0030] Each V H and V L is typically composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Chothia and Lesk J. Mol. Biol. 196, pp. 901-917 (1987). Typically, the amino acid residues in this region are numbered according to 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). (Phrases such as "like Kabat" or "variable domain residue numbering according to Kabat" refer to this numbering system for heavy chain variable domains or light chain variable domains herein.) Using this numbering system, the actual linear amino acid sequence of the peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or CDRs of the variable domain. For example, a heavy chain variable domain may have the V H It may also include a single amino acid insertion after residue 52 in CDR2 (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 the "standard" Kabat numbering sequence at the regions of homology.

[0031] 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 for a substantial period of time, such as 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 days or more, etc., or any other relevant, functionally defined period of time (such as a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen).

[0032] The variable regions of the heavy and light chains of an immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of an antibody (Ab) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (CIq) of the classical complement system.

[0033] The anti-CD38 antibody may be a bispecific antibody, diabody, or similar molecule (for a description of diabodies, see, e.g., PNAS USA 90 (14), 6444-8 (1993). Indeed, the bispecific antibodies, diabodies, etc. provided by the present invention may bind to any suitable target in addition to a portion of CD38.

[0034] As indicated above, the term antibody as used 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 within the term "antibody" include: (i) V L , V H , C L , and C H (ii) a Fab fragment, which is a monovalent fragment consisting of one domain; (iii) a F(ab)2 and F(ab')2 fragment, which are bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; H and C H (iv) a single-arm V of an antibody; L and V H (v) an Fv fragment consisting essentially of a V domain; H A dAb fragment consisting essentially of a domain (Ward et al., Nature 341 (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs, optionally connected by a synthetic linker. Furthermore, the two domains of the Fv fragment, V Land V H are encoded by separate genes, but V L and V H The domains are paired to form monovalent molecules (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 They can be joined using recombinant methods by synthetic linkers that allow them to be produced as a single protein chain forming a single chain (see, e.g., J. Am. Chem. Soc., 5879-5883 (1988)). Unless otherwise stated or clearly indicated by context, such single-chain antibodies are encompassed within the term antibody. Other forms of single-chain antibodies, such as diabodies, are included within the term antibody. Although such fragments are typically included within the meaning of an antibody, they, collectively and each independently, are unique features of the present invention and exhibit different biological properties and utilities. These and other useful antibody fragments in the context of the present invention are further discussed herein.

[0035] It should also be understood that the term antibody generally includes antibody-like polypeptides, such as polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, and humanized antibodies, anti-idiotypic (anti-Id) antibodies to antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to antigens, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Antibodies as generated can possess any isotype.

[0036] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of a group of chemically active surface molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. Epitopes may include amino acid residues directly involved in binding (also called immunodominant components 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, amino acid residues within the footprint of the specifically antigen-binding peptide).

[0037] The term "bispecific molecule" is intended to include any agent, such as a protein, peptide, or protein or peptide complex, that has two different binding specificities. For example, the molecule may bind to or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "multispecific molecule" is intended to include any agent, such as a protein, peptide, or protein or peptide complex, that has more than two different binding specificities. For example, the molecule may bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Thus, the present invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules directed against CD38 and against other cell surface antigens or targets, such as Fc receptors on effector cells.

[0038] The term "bispecific antibody" is intended to include any anti-CD38 antibody that is a bispecific molecule. The term "bispecific antibody" also includes diabodies. Diabodies are antibodies that bind to a V H and V LThe domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, so the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites; they are bivalent, bispecific antibodies (see, e.g., Holliger, P. et al., PNAS USA 90 , 6444-6448(1993), Poljak, RJ et al., Structure 2 , 1121-1123 (1994).

[0039] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (such as B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells can induce antibody-dependent cellular cytotoxicity (ADCC), such as neutrophils, which can induce ADCC. For example, monocytes and macrophages, which express FcRs, are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system, or in binding to cells that present antigens. In some embodiments, effector cells may phagocytose target antigens, target cells, or microorganisms. The expression of specific FcRs on effector cells can be regulated by humoral factors such as cytokines. For example, FcγRI expression has been shown to be upregulated by interferon-γ (IFN-γ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. Effector cells can phagocytose or lyse target antigens or target cells.

[0040] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" 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.

[0041] As used herein, a human antibody is "derived from" a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, e.g., by immunizing transgenic mice harboring human immunoglobulin genes or by screening a human immunoglobulin gene library, and the selected human antibody is at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% identical in amino acid sequence to the amino acid sequence encoded by the germline VH or VL variable region gene segment. Typically, a human antibody derived from a particular human germline VH or VL variable region gene segment sequence will exhibit no more than 10 amino acid residue differences, e.g., no more than 5 amino acid residue differences, e.g., no more than 4, 3, 2, or 1 amino acid difference, from the amino acid sequence encoded by the germline immunoglobulin gene.

[0042] A "chimeric" antibody is an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies from another species. A monovalent chimeric antibody is a dimer (HL) formed by a chimeric H chain associated through a disulfide bridge with a chimeric L chain. A divalent chimeric antibody is a tetramer (H2L2) formed by two HL dimers associated through at least one disulfide bridge. Polyvalent chimeric antibodies can also be produced, for example, by utilizing CH regions (e.g., from an IgM H chain or μ chain) that oligomerize. Typically, a chimeric antibody refers to an antibody in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as to fragments of such antibodies, so long as they exhibit the desired biological activity (see, for example, U.S. Pat. No. 4,816,567 and Morrison et al., PNAS USA 81 , 6851-6855 (1984). Chimeric antibodies are produced by recombinant processes well known in the art (see, e.g., Cabilly et al., PNAS USA 81 , 3273-3277(1984), Morrison et al., PNAS USA 81 , 6851-6855(1984), Boulianne et al., Nature 312 , 643-646(1984), European Patent No. 125023, Neuberger et al., Nature 314 , 268-270(1985), European Patent No. 171496, European Patent No. 173494, International Publication No. 86 / 01533, European Patent No. 184187, Sahagan et al., J. Immunol. 137 , 1066-1074(1986), International Publication No. 87 / 02671, Liu et al., PNAS USA 84, 3439-3443(1987), Sun et al., PNAS USA 84 , 214-218(1987), Better et al., Science 240 , 1041-1043 (1988), and Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

[0043] "Humanized" antibodies are antibodies derived from non-human species in which certain amino acids in the framework and constant domains of the heavy and light chains have been mutated to avoid or abrogate an immune response in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies containing minimal sequence derived from non-human immunoglobulins. Typically, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable region are replaced with residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired antigen-binding characteristics, such as specificity and affinity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody. These modifications are made to further optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 , 522-525(1986), Riechmann et al., Nature332 , 323-329(1988), and Presta, Curr. Op. Struct. Biol. 2 , 593-596 (1992).

[0044] As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to a preparation of antibody molecules of 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 in hybridomas comprising B cells obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes, fused with an immortalized cell. Monoclonal antibody may also be abbreviated as mAb.

[0045] As used herein, "specific binding" refers to an antibody that binds to a predetermined antigen. Typically, the antibody binds to a specific antigen with a specific binding activity of about 10 as determined by surface plasmon resonance (SPR) technology on a BIAcore3000 instrument using recombinant CD38 as the ligand and the antibody as the analyte. -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or even less K D The antibody binds with an affinity corresponding to a K that is at least 10 times lower, e.g., at least 100 times lower, e.g., at least 1000 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 its affinity for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). DAmounts with lower affinities may bind to a given antigen with an affinity corresponding to the K D is dependent on the K D is very low (i.e., the antibody is highly specific), the amount by which the affinity for the antigen is lower than the affinity for a nonspecific antigen is at least 10,000-fold.

[0046] The term specificity, as used herein, refers to the ability of a CD38-binding peptide, such as an anti-CD38 antibody, to recognize an epitope within CD38 while having little or no detectable reactivity with other portions of CD38 (including other epitopes bound by other anti-CD38 antibodies). Specificity can be relatively determined by a competitive assay as described herein. More specifically, specificity can be determined by any of the epitope identification / characterization techniques described herein or their equivalents known in the art.

[0047] Antibodies specific for a particular antigenic determinant may nevertheless cross-react with other biological molecules that may be present in some biological context with CD38. More typically, anti-CD38 antibodies may cross-react with CD38 homologs from other species. In one or both contexts, such cross-reactive antibodies are typically selective for human CD38 with respect to relevant structural and / or environmental factors.

[0048] The term "selectivity" as used herein refers to the preferential binding of an anti-CD38 antibody to a particular region, target, or peptide; typically, a region or epitope in CD38, as opposed to one or more other biological molecules, structures, cells, tissues, etc. In certain embodiments, the anti-CD38 antibodies used in the present invention are selective for a portion of CD38 in the context of a colon cancer cell (i.e., the anti-CD38 antibody will preferentially bind to a portion of CD38 over other components of a colon cancer cell).

[0049] As used herein, "kd " (sec -1 The term k ) is intended to refer to the dissociation equilibrium rate constant of a particular antibody-antigen interaction. off Also called the value.

[0050] As used herein, "k a " (M -1 ×sec -1 The term ) is intended to refer to the association equilibrium rate constant of a particular antibody-antigen interaction.

[0051] As used herein, "K D The term "" (M) is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0052] As used herein, "K A " (M -1 ) is intended to refer to the association equilibrium constant of a particular antibody-antigen interaction, k a k d It is obtained by dividing by

[0053] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by heavy chain constant region genes.

[0054] "Target cells" refers to any unwanted cells in an individual. In some embodiments, target cells are cells that express or overexpress CD38. Cells that express CD38 typically include hematopoietic cells, such as medullary thymocytes, activated T and B cells, 80% of resting NK cells and monocytes, lymph node germinal center lymphoblasts, plasma B cells and some intrafollicular cells, dendritic cells, normal bone marrow cells, certain precursor cells, 50-80% of umbilical cord blood cells, erythrocytes, and platelets. CD38 may also be expressed by nonhematopoietic cells, such as intraepithelial cells and lamina propria lymphocytes in the intestine, by 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 the sarcolemma of smooth and striated muscle. With respect to malignant cells, CD38 is expressed in a variety of hematological malignancies, including, but not limited to, multiple myeloma, primary or secondary plasma cell leukemia, B-cell chronic lymphocytic leukemia, B-cell acute lymphocytic leukemia, Waldenstrom's macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, prolymphocytic / myelocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, follicular lymphoma, and NK-cell leukemia.

[0055] As used herein, the term "individual" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0056] "Treatment" means administration of an effective amount of a therapeutically active compound of the present invention with the purpose of alleviating, ameliorating, or eradicating (curing) the symptoms or disease state.

[0057] Aspects and Embodiments of the Invention In a first main aspect, the present invention provides a method for inhibiting the growth and / or proliferation of tumor cells expressing CD38 in an individual in need thereof, comprising administering to the individual i) a non-agonistic antibody that binds, i.e. specifically binds, to CD38; ii) at least one corticosteroid, and iii) at least one non-corticosteroid chemotherapy agent The present invention relates to a method comprising administering

[0058] In a further main aspect, the present invention provides a method for treating cancer involving tumor cells expressing CD38 in an individual in need thereof, comprising administering to the individual i) a non-agonistic antibody that binds, i.e. specifically binds, to CD38; ii) optionally at least one corticosteroid, and iii) optionally at least one non-corticosteroid chemotherapeutic agent, such as a non-alkylating non-corticosteroid chemotherapeutic agent; followed by autologous peripheral stem cell transplantation or bone marrow transplantation.

[0059] In certain embodiments of the above methods of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises a cytotoxic agent and / or an angiogenesis inhibitor. In further embodiments, the at least one non-corticosteroid chemotherapeutic agent comprises an alkylating agent.

[0060] In still further embodiments, the at least one non-corticosteroid chemotherapeutic agent comprises one or more agents selected from the group consisting of melphalan, mechlorethamine, thioepa, chlorambucil, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin.

[0061] In further embodiments, the at least one non-corticosteroid chemotherapeutic agent comprises a glutamic acid derivative, such as thalidomide (Thalomid®) or a thalidomide analogue, e.g., CC-5013 (lenalidomide, Revlimid™) or CC4047 (Actimid™).

[0062] In still further embodiments, the at least one non-corticosteroid chemotherapeutic agent comprises a proteasome inhibitor, such as bortezomib (Velcade®), or a vinca alkaloid, such as vincristine, or an anthracycline, such as doxorubicin.

[0063] In certain embodiments of the methods of the present invention, the at least one corticosteroid comprises a glucocorticoid. In further embodiments, the at least one corticosteroid comprises prednisone or dexamethasone.

[0064] In a further embodiment of the invention, the at least one corticosteroid comprises prednisone and the at least one non-corticosteroid chemotherapeutic agent comprises melphalan.

[0065] In still further embodiments of the invention, the at least one corticosteroid comprises prednisone and the at least one non-corticosteroid chemotherapeutic agent comprises thalidomide.

[0066] In still further embodiments of the invention, the at least one corticosteroid comprises prednisone and the at least one non-corticosteroid chemotherapeutic agent comprises melphalan and thalidomide.

[0067] In still further embodiments of the invention, the at least one corticosteroid comprises dexamethasone and the at least one non-corticosteroid chemotherapeutic agent comprises thalidomide and / or lenalidomide.

[0068] In still further embodiments of the invention, the at least one corticosteroid comprises dexamethasone and the at least one non-corticosteroid chemotherapeutic agent comprises vincristine and / or doxorubicin.

[0069] In certain embodiments of the methods of the present invention, the non-agonistic antibody that binds to CD38 is a monoclonal antibody, such as a human monoclonal antibody.

[0070] In a further embodiment of the method of the invention, the antibody is an antagonist of CD38.

[0071] In a further embodiment of the invention, the antibody - an antibody that does not induce significant IL-6 release by human monocytes or peripheral blood mononuclear cells, as determined by the method described in Example 19 herein. and / or - an antibody that does not induce detectable release of IFN-γ by human T cells or peripheral blood mononuclear cells, as determined by the method described in Example 20 herein. and / or - an antibody that is internalized by CD38-expressing cells, such as an antibody that is internalized by CHO-CD38 cells within 5 to 15 minutes at 37°C by the method described in Example 12 herein; and / or an EC of less than 15 ng / ml, such as less than 10 ng / ml, in Daudi-luc cells as determined by the method described in Example 5 herein; 50 values ​​and an EC of less than 75 ng / ml in MM cells, e.g., less than 50 ng / ml, 30 ng / ml, or 10 ng / ml 50 ADCC-inducing antibodies and / or - an EC of less than 5 μg / ml, for example less than 1 μg / ml, on daudi-luc or CD38-CHO cells by the method described in Example 6 herein; 50 antibodies that induce CDC in the presence of complement, such as and / or - Antibodies that inhibit cGDPR synthesis and / or - Antibodies that inhibit the synthesis of cADPR and / or 10 as determined by surface plasmon resonance as described in Example 20 herein -8 Less than M, e.g. 10 -8 M~10 -11 Within the range of M, e.g., 7 x 10 -9 M~10 -10 Affinity (K D ) an antibody that binds to human CD38 and / or an antibody that inhibits the synthesis of cGDPR by at least 25%, such as at least 30%, after 90 minutes at a concentration of 3 μg / ml, as determined by the spectrophotometric method described in Example 24 herein; and / or - Munshi et al., J. Biol. Chem. 275 an antibody that inhibits the synthesis of cADPR by at least 25%, e.g., at least 30%, after 90 minutes at a concentration of 3 μg / ml, as determined by the HPLC method described in J. Immunol., 21566-21571 (2000). is.

[0072] In one embodiment, the non-agonistic CD38 antibody used in the present invention is antibody -003. -003 has the V L region and V consisting of the sequence of SEQ ID NO: 7 H It is a human monoclonal IgG1 antibody with a nucleotide sequence.

[0073] In another embodiment, the non-agonistic CD38 antibody used in the present invention is antibody -005. -005 has the V L region and V consisting of the sequence of SEQ ID NO: 17 H It is a human monoclonal IgG1 antibody with a nucleotide sequence.

[0074] In a further embodiment, the non-agonistic CD38 antibody used in the present invention is antibody -024. -024 has the V sequence of SEQ ID NO:22. L region and V consisting of the sequence of SEQ ID NO: 27 H It is a human monoclonal IgG1 antibody with a nucleotide sequence.

[0075] In one embodiment, the non-agonistic CD38 antibodies used in the present invention are antibodies that bind to human CD38 encoded by human light chain and human heavy chain nucleic acids comprising the nucleotide sequences in their variable regions set forth in SEQ ID NO:1 and SEQ ID NO:6, respectively.

[0076] In one embodiment, the non-agonistic CD38 antibodies used in the present invention are antibodies that bind to human CD38 encoded by human light chain and human heavy chain nucleic acids comprising the nucleotide sequences in their variable regions set forth in SEQ ID NO:11 and SEQ ID NO:16, respectively.

[0077] In one embodiment, the non-agonistic CD38 antibodies used in the present invention are antibodies that bind to human CD38 encoded by human light chain and human heavy chain nucleic acids comprising the nucleotide sequences in their variable regions set forth in SEQ ID NO:21 and SEQ ID NO:26, respectively.

[0078] In still further embodiments, the non-agonistic CD38 antibody used in the present invention is one of the antibodies described in WO 2005 / 103083 (Morphosys), in particular an antibody comprising one or more of the sequences shown in Figure 1b and / or Figure 2B of WO 2005 / 103083.

[0079] Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse among individual antibodies than sequences outside the CDRs. Because the CDR sequences are responsible for the majority of antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular natural antibody by constructing an expression vector containing the CDR sequences from that particular natural antibody spliced ​​onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., Nature 1999, 14, 146-152). 332 , 323-327(1998), Jones, P. et al., Nature 321 , 522-525 (1986), and Queen, C. et al., PNAS USA 86 , 10029-10033(1989)).

[0080] It is well known in the art that the antibody heavy chain CDR3 domain plays a particularly important role in the binding specificity / affinity of an antibody to an antigen (Ditzel HJ, et al., J Immunol. 157 (2), 739-49 (1996), Barbas SM et al., J. Am. Chem. Soc. 116 , 2161-2162 (1994), and Barbas SM et al., Proc Natl Acad Sci USA 92 (7), 2529-33 (1995)), the antibody used in the present invention may comprise the heavy chain CDR3 of -003, -005, or -024. The antibody used in the present invention may also comprise the heavy and light chain CDR3s of -003, -005, or -024.

[0081] Thus, in a further embodiment of the method of the present invention, the antibody comprises a V H An antibody that comprises the CDR3 or an antibody that competes with the antibody for CD38 binding, for example, by binding to the same epitope as the antibody.

[0082] In one embodiment, competition is determined by use of an ELISA as described in the Examples section.

[0083] In another embodiment, competition is determined by use of FACS as described in the Examples section.

[0084] In another embodiment, the antibody has the V L CDR3 and V having the sequence shown in SEQ ID NO: 10 H It is an antibody containing CDR3.

[0085] In another embodiment, the antibody is an antibody comprising a human light chain and a human heavy chain variable region, wherein the light chain variable region has the sequence set forth in SEQ ID NO:3. L CDR1, V having the sequence shown in SEQ ID NO:4 L CDR2, and V having the sequence shown in SEQ ID NO:5 L The heavy chain variable region comprises a V CDR3 having the sequence shown in SEQ ID NO: 8. H CDR1, V having the sequence shown in SEQ ID NO:9 H CDR2, and V having the sequence shown in SEQ ID NO: 10 H Includes CDR3.

[0086] In another embodiment, the antibody has the amino acid sequence shown in SEQ ID NO:2. L a region or V having at least about 90%, e.g., at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO:2; L It is an antibody containing the region.

[0087] In another embodiment, the antibody has the amino acid sequence set forth in SEQ ID NO:7. H region, or a V having at least about 90%, e.g., at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO:7. H region, or V having 1 to 5, for example 1 to 3 amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO: 7. H It is an antibody containing the region.

[0088] In another embodiment, the antibody has the V H An antibody that comprises the CDR3, or an antibody that competes with the antibody for CD38 binding, for example, by binding to the same epitope as the antibody.

[0089] In another embodiment, the antibody has the V L CDR3 and V having the sequence shown in SEQ ID NO:20 H It is an antibody containing CDR3.

[0090] In another embodiment, the antibody is an antibody comprising a human light chain and a human heavy chain variable region, wherein the light chain variable region has the sequence set forth in SEQ ID NO:13. L CDR1, V having the sequence shown in SEQ ID NO: 14 L CDR2, and V having the sequence shown in SEQ ID NO: 15 L The heavy chain variable region comprises a V CDR3 having the sequence set forth in SEQ ID NO: 18. H CDR1, V having the sequence shown in SEQ ID NO: 19 H CDR2, and V having the sequence shown in SEQ ID NO:20 H Includes CDR3.

[0091] In another embodiment, the antibody has the amino acid sequence set forth in SEQ ID NO:12. L a region, or a V region having at least about 90%, for example at least about 95%, amino acid sequence identity with a sequence according to SEQ ID NO: 12 L It is an antibody containing the region.

[0092] In another embodiment, the antibody has the amino acid sequence set forth in SEQ ID NO:17. H a region or V having at least about 90%, for example at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO: 17; H region, or V having 1 to 5, for example 1 to 3 amino acid substitutions, deletions, or additions compared to the sequence set forth in SEQ ID NO: 17.H The antibody comprises a region.

[0093] In another embodiment, the antibody has the V H An antibody that comprises the CDR3, or an antibody that competes with the antibody for CD38 binding, for example, by binding to the same epitope as the antibody.

[0094] In another embodiment, the antibody has the V L CDR3 and V having the sequence shown in SEQ ID NO: 30 H It is an antibody containing CDR3.

[0095] In another embodiment, the antibody is an antibody comprising a human light chain and a human heavy chain variable region, wherein the light chain variable region has the sequence set forth in SEQ ID NO:23. L CDR1, V having the sequence shown in SEQ ID NO:24 L CDR2, and V having the sequence shown in SEQ ID NO: 25 L The heavy chain variable region comprises a V CDR3 having the sequence set forth in SEQ ID NO: 28. H CDR1, V having the sequence shown in SEQ ID NO:29 H CDR2, and V having the sequence shown in SEQ ID NO: 30 H Includes CDR3.

[0096] In another embodiment, the antibody has the amino acid sequence set forth in SEQ ID NO:22. L a region or V having at least about 90%, for example at least about 95%, amino acid sequence identity with a sequence according to SEQ ID NO: 22; L It is an antibody containing the region.

[0097] In another embodiment, the antibody has the amino acid sequence set forth in SEQ ID NO:27. H a region or V having at least about 90%, for example at least about 95%, amino acid sequence identity with a sequence according to SEQ ID NO: 27; Hregion, or V having 1 to 5, for example 1 to 3 amino acid substitutions, deletions, or additions compared to the sequence set forth in SEQ ID NO: 27. H It is an antibody containing the region.

[0098] In certain embodiments of the methods of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises one or more agents selected from the group consisting of melphalan, mechlorethamine, thioepa, chlorambucil, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin; and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0099] In certain embodiments of the methods of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises melphalan, and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0100] In another embodiment, the at least one non-corticosteroid chemotherapeutic agent comprises a glutamic acid derivative, such as thalidomide (Thalomid®) or a thalidomide analog, e.g., CC-5013 (lenalidomide, Revlimid™) or CC4047 (Actimid™), and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0101] In another embodiment, the at least one non-corticosteroid chemotherapeutic agent comprises thalidomide (Thalomid®) or a thalidomide analog, e.g., a glutamic acid derivative, such as CC-5013 (lenalidomide, Revlimid™) or CC4047 (Actimid™), and the antibody comprises a V antibody consisting of the sequence of SEQ ID NO: 12. L region and V consisting of the sequence of SEQ ID NO: 17 H It is a human monoclonal IgG1 antibody with a nucleotide sequence.

[0102] In another embodiment, the at least one non-corticosteroid chemotherapeutic agent comprises a proteasome inhibitor, such as bortezomib (Velcade®), and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0103] In another embodiment, the at least one corticosteroid comprises dexamethasone; at least one non-corticosteroid chemotherapeutic agent comprises a proteasome inhibitor, such as bortezomib (Velcade®); and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0104] In another embodiment, the at least one non-corticosteroid chemotherapeutic agent comprises a vinca alkaloid, such as vincristine, and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0105] In another embodiment, the at least one non-corticosteroid chemotherapeutic agent comprises an anthracycline, such as doxorubicin, and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0106] In another embodiment, the at least one corticosteroid comprises a glucocorticoid, and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0107] In another embodiment, the at least one corticosteroid comprises prednisone, and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0108] In another embodiment, the at least one corticosteroid comprises prednisone, and at least one non-corticosteroid chemotherapy agent comprises melphalan; and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 Ha human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0109] In another embodiment, the at least one corticosteroid comprises prednisone, and at least one non-corticosteroid chemotherapy agent comprises thalidomide, and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0110] In another embodiment, the at least one corticosteroid comprises prednisone, and at least one non-corticosteroid chemotherapy agent comprises melphalan and thalidomide, and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0111] In another embodiment, the at least one corticosteroid comprises dexamethasone, and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0112] In another embodiment, the at least one corticosteroid comprises dexamethasone, and at least one non-corticosteroid chemotherapy agent comprises thalidomide and / or lenalidomide, and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0113] In another embodiment, the at least one corticosteroid comprises dexamethasone, and at least one non-corticosteroid chemotherapeutic agent comprises vincristine and / or doxorubicin, and The antibody is - V consisting of the sequence of SEQ ID NO: 2 L region and V consisting of the sequence of SEQ ID NO: 7 H a human monoclonal IgG1 antibody having a region V consisting of the sequence of SEQ ID NO: 12 L region and V consisting of the sequence of SEQ ID NO: 17 H a human monoclonal IgG1 antibody having a region, V consisting of the sequence of SEQ ID NO: 22 L region and V consisting of the sequence of SEQ ID NO: 27 H Human monoclonal IgG1 antibody with a region is selected from the group consisting of:

[0114] Antibodies suitable for use in the present invention also include variants of the antibodies of the Examples. V as used in the context of CD38 antibodies L , V H , or functional variants of the CDRs still 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 antibodies may be associated with greater affinity, selectivity, and / or specificity than the parent antibody.

[0115] "Mutant" anti-CD38 antibodies may be derived from CDRs or other V H and / or V L An antibody that differs from a parent antibody (typically produced by immunization) by one or more suitable amino acid residue modifications in sequence, be they substitutions, deletions, insertions, or terminal sequence additions (provided that such changes retain, if not improve, at least a substantial amount of the epitope-binding characteristics of the parent antibody).

[0116] Thus, for example, in an antibody variant, one or more amino acid residues may be introduced or inserted into or near one or more hypervariable regions of the parent antibody, such as one or more CDRs. The anti-CD38 antibody variant may contain any number of inserted amino acid residues, again provided that at least a substantial amount of the epitope-binding characteristics of the parent antibody are retained. The anti-CD38 antibody variant of the invention may contain, for example, about 1 to 30 inserted amino acid residues, e.g., about 1 to 10, e.g., about 2 to 10, e.g., 2 to 5, or e.g., about 1 to 5 inserted amino acid residues. Similarly, the anti-CD38 antibody variant of the invention may contain, for example, about 1 to 30 deleted amino acid residues, e.g., about 1 to 10, e.g., about 2 to 10, e.g., 2 to 5, or e.g., about 1 to 5 deleted amino acid residues. Similarly, anti-CD38 antibody variants of the invention may comprise, for example, about 1 to 30 substituted amino acid residues, e.g., about 1 to 10, e.g., about 2 to 10, e.g., about 2 to 5, or e.g., about 1 to 5 substituted amino acid residues. Similarly, anti-CD38 antibody variants useful for the invention may comprise, for example, about 1 to 30 terminal sequence amino acid residue additions, e.g., about 1 to 10, e.g., about 2 to 10, e.g., 2 to 5, or e.g., about 1 to 5 terminal sequence amino acid residue additions. Antibody variants of the invention may also comprise a combination of two or more of such insertions, deletions, substitutions, and terminal sequence amino acid residue additions, provided that the variant retains at least a substantial proportion of the affinity, specificity, and / or selectivity of the parent antibody with respect to one or more CD38 epitopes.

[0117] In one embodiment, the antibody used in the invention comprises a variant V that essentially consists of a sequence having at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95% sequence identity with a sequence according to any one of SEQ ID NO: 10 or SEQ ID NO: 20 or SEQ ID NO: 30. H and the antibody comprises a variant V CDR3 of SEQ ID NO: 10 or SEQ ID NO: 20 or SEQ ID NO: 30, respectively.H and having at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or more) of the epitope binding characteristics of an antibody having the CDR3 sequence of SEQ ID NO:7 or SEQ ID NO:17 or SEQ ID NO:27, respectively. H Antibodies having the sequences, for example, V and V of SEQ ID NO:7, respectively. H Sequence and V of SEQ ID NO:2 L an antibody having the sequence V of SEQ ID NO: 17; H Sequence and V of SEQ ID NO: 12 L an antibody having the sequence V of SEQ ID NO: 27 H Sequence and V of SEQ ID NO: 22 L It is an antibody having the sequence

[0118] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100) and takes into account the number of gaps, and the length of each gap, that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm, such as that described in the non-limiting examples below.

[0119] Percent identity between two nucleotide sequences may be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using a NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. Percent identity may also be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The GAP program in the GCG software package (available at http: / / www.gcg.com) as incorporated into the ALIGN program (2nd ed.) by E. Meyers and W. Miller, Comput. Appl. Biosci. 4The percent identity between two nucleotide or amino acid sequences may be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol., 11-17 (1988), as 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 gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. 48 The algorithm of J. Am. Chem. Soc., 444-453 (1970) may be used to determine percent identity between two amino acid sequences.

[0120] 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, about 95% or more (e.g., about 65-99%) of the substitutions in the variant are conservative amino acid residue exchanges. 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: Classes of amino acid residues for conservative substitutions TIFF2025143484000001.tif42137 Classes of alternative conservative amino acid residue substitutions TIFF2025143484000002.tif43137 Physical and functional classification of alternative amino acid residues TIFF2025143484000003.tif122139

[0121] More conservative substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Additional amino acid groups may also be formulated using the principles described, for example, in Creighton (1984) Proteins: Structure and Molecular Properties (2nd ed., 1993), W. H. Freeman and Company.

[0122] When making hypervariable region insertions to generate variant antibodies, the typical range of lengths of the hypervariable region in question in known antibodies should be taken into consideration. For example, for the first hypervariable region of the light chain variable domain, the insertion should be substantially similar to, and therefore longer than, the V length of the parent antibody while retaining the expected approximate size. L It may be introduced into the CDR1 sequence, which, according to Kabat et al., supra, typically has about 9 to 20 (e.g., about 10 to 17) residues overall. L CDR2 typically has an overall length of about 5-10 residues; L CDR3 typically has a length of about 7 to 20 residues; H CDR1 typically has a length of about 10 to 15 residues; H CDR2 typically has a length of about 15-20 residues; and V H CDR3 typically has a length of about 6 to 30 residues (e.g., 3 to 25 residues). H Insertions in the region are typically H CDR3 and typically the parent V, using alignment and numbering as described in Kabat H around residues 97-102 of CDR3 (e.g., parent V H Adjacent to residue 100 of the CDR3 sequence or the parent V HThe insertion of an amino acid residue in the hypervariable region of the antibody may be made near the C-terminus of the domain (i.e., C-terminal to residue 100 of the CDR3 sequence). Antibody variants with inserted amino acid residues in their hypervariable region may be prepared randomly, particularly if the initial binding affinity of the parent antibody for the target antigen is such that randomly generated antibody variants can be readily screened. Phage display, for example, provides a convenient method for screening such random variants.

[0123] In a further embodiment, the non-agonistic CD38 antibody used in the invention has the V L Sequence and V of SEQ ID NO:7 H an antibody having the sequence (such as antibody-003), or V of SEQ ID NO: 12 L Sequence and V of SEQ ID NO: 17 H an antibody having the sequence V of SEQ ID NO:22 (such as antibody-005); L Sequence and V of SEQ ID NO: 27 H An antibody having the sequence V of SEQ ID NO:2 is an antibody that has been characterized for its ability to compete (competitively inhibit) or cross-compete (i.e., relatively partially inhibit epitope binding) with an antibody having the sequence V of SEQ ID NO:2 (e.g., antibody -024). L Sequence and V of SEQ ID NO:7 H an antibody having the sequence V of SEQ ID NO: 12 L Sequence and V of SEQ ID NO: 17 H an antibody having the sequence V of SEQ ID NO: 22 L Sequence and V of SEQ ID NO: 27 H The antibody may be a Fab fragment derived from an antibody that binds to an epitope that is identical to or overlaps with the epitope bound by an antibody having the sequence. Competition for binding to CD38 or a portion of CD38 by two or more antibodies may be determined by any suitable technique. In one embodiment, competition is determined, for example, as described in Examples 7, 8, and 9.

[0124] Competition, as determined by ELISA and / or FACS analysis, often represents significantly greater than about 5% relative inhibition. It may be desirable to set a higher threshold of relative inhibition as a criterion / determinant of a suitable level of competition in certain contexts (e.g., when using competition analysis to select or screen new antibodies designed with the intended function of blocking binding of another peptide or molecule that binds to CD38 (e.g., the CD31 antigen, EndoCAM, GPIIA', PECAM-1, platelet / endothelial cell adhesion molecule, or a natural binding partner of CD38, such as CD31, also referred to as a natural anti-CD38 antibody)). Thus, for example, it is possible to set a criterion for competitiveness such that at least about 10% relative inhibition is detected; at least about 15% relative inhibition is detected; 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 be characterized 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).

[0125] In a further embodiment, the non-agonistic CD38 antibody used in the present invention comprises the V L Sequence and V of SEQ ID NO:7 H an antibody having the sequence (such as antibody-003), or V of SEQ ID NO: 12 L Sequence and V of SEQ ID NO: 17 H an antibody having the sequence V of SEQ ID NO:22 (such as antibody-005); L Sequence and V of SEQ ID NO: 27 H It is an antibody that specifically binds to a CD38 epitope that is also specifically bound by an antibody having the sequence (such as antibody -024).

[0126] V of SEQ ID NO:2 L Sequence and V of SEQ ID NO:7 H an antibody having the sequence (such as antibody-003), or V of SEQ ID NO: 12 L Sequence and V of SEQ ID NO: 17 H an antibody having the sequence (such as antibody-005), or V of SEQ ID NO:22 L Sequence and V of SEQ ID NO: 27 H The CD38 epitope bound by an antibody having the sequence (such as antibody-024) may be identified by standard mapping and characterization techniques, and further refined by any suitable technique, numerous examples of which are available to those skilled in the art. These techniques may also be used to generally identify and / or characterize the epitopes of anti-CD38 antibodies. As one example of such a mapping / characterization method, the epitope of an anti-CD38 antibody may be determined by epitope "footprinting" using 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 amide protons in receptor and ligand proteins occur, and the backbone amide groups involved in protein binding are thought to be protected from back-exchange and therefore remain undeuterated. At this point, relevant regions may be identified by proteolysis with pepsin, fast 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 epitope mapping (NMR), which compares the position of signals in two-dimensional NMR spectra of a free antigen and an antigen complexed with an antigen-binding peptide, such as an antibody. An antigen is typically 15 The antigen is selectively radioisotope-labeled with N, so that only signals corresponding to the antigen are seen in the NMR spectrum, and signals from the antigen-binding peptide are not seen. Antigen signals arising from amino acids involved in the interaction with the antigen-binding peptide are typically thought to shift their position in the spectrum of the complex compared to the spectrum of the free antigen, and amino acids involved in binding may be identified in this way. 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).

[0127] Epitope mapping / characterization may also be performed using mass spectrometry methods. See, e.g., Downward, J Mass Spectrom. 35 (4), 493-503(2000) and Kiselar and Downard, Anal Chem. 71 (9), 1792-801 (1999).

[0128] Protease digestion techniques may also be useful in the context of epitope mapping and identification techniques. Antigenic determinant-associated regions / sequences may be determined by protease digestion, for example, by using about a 1:50 ratio of trypsin to CD38 in an overnight (O / N) digestion at 37°C and pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification. Peptides protected from trypsin cleavage by the antibody may then be identified by comparing the sample subjected to trypsin digestion with a sample incubated with the antibody and then subjected to, for example, trypsin digestion (thereby revealing a binding footprint). Other enzymes, such as chymotrypsin, pepsin, and the like, may also or alternatively be used in similar epitope characterization methods. In these measurements, the V of SEQ ID NO:2 may be compared with the V of SEQ ID NO:2. L Sequence and V of SEQ ID NO:7 H an antibody having the sequence (such as antibody-003), or V of SEQ ID NO: 12 L Sequence and V of SEQ ID NO: 17 H an antibody having the sequence V of SEQ ID NO:22 (such as antibody-005); L Sequence and V of SEQ ID NO: 27 H Antibodies that give significantly the same results as antibodies having the sequences (such as antibody-024) are designated V of SEQ ID NO: 2, respectively. L Sequence and V of SEQ ID NO:7 H an antibody having the sequence (such as antibody-003), or V of SEQ ID NO: 12 L Sequence and V of SEQ ID NO: 17 H an antibody having the sequence V of SEQ ID NO:22 (such as antibody-005); L Sequence and V of SEQ ID NO: 27 H The antibody is considered to bind to the same epitope as an antibody having the sequence (such as antibody-024). For a discussion of similar techniques, see, for example, Manca, Ann Ist Super Sanita. 27 (1), 15-9 (1991).

[0129] Other methods potentially useful in mapping epitopes include crystallography, X-ray diffraction (such as the X-ray diffraction / sequence study technique developed by Poljak and others in the 1970s and 1980s), and the application of multipin peptide synthesis technology. Computer-based methods, such as sequence analysis and three-dimensional structural analysis and docking, may also be used to identify antigenic determinants. Epitopes may also be determined by molecular modeling, for example, using the structure of CD38 with docking of the structures of Fab fragments of individual monoclonal antibodies. These and other mapping methods are discussed in Epitope Mapping: A Practical Approach (Westwood and Hay, eds.), 2001, Oxford University Press.

[0130] Antibodies used in the present invention may have any suitable affinity and / or avidity for one or more epitopes contained at least in part in CD38. Affinity refers to the strength of binding of an antibody to such epitopes. Typically, affinity is measured by the dissociation constant K, defined as [Ab] x [Ag] / [Ab-Ag]. d where [Ab-Ag] is the molar concentration of the antibody-antigen complex (or antibody-antigen complex), [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant K a is 1 / K dSuitable methods for determining specificity and affinity 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).

[0131] The anti-CD38 antibody used in the present invention has a specificity of about 10 to at least one epitope that is at least partially contained in CD38. 4 ~about 10 10 M -1 Such antibodies may have an affinity for CD38 that is at least as great as -003, -005, and -024, and in some embodiments, at least about as great as -003, -005, and -024. Affinity may be determined by any of the methods described elsewhere herein, or their known equivalents in the art. One example of a method that may be used to determine affinity is described in Munson & Pollard, Anal. Biochem., 107 , 220 (1980) Scatchard analysis. Binding affinity may also be determined by equilibrium assays (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic assays (e.g., BIACORE™ analysis).

[0132] Typically, the dissociation constant of an anti-CD38 antibody used in the invention is less than about 100 nM, less than about 50 nM, less than about 10 nM, about 5 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.1 nM or less, about 0.01 nM or less, or even about 0.001 nM or less.

[0133] Non-limiting examples of anti-CD38 antibodies suitable for use in the present invention include: (a) a complete, functional immunoglobulin molecule comprising (i) two identical chimeric heavy chains comprising a variable region with human B-cell surface antigen specificity and a human constant region, and (ii) two identical, whole (i.e., non-chimeric) human light chains; (b) a complete, functional immunoglobulin molecule comprising (i) two identical chimeric heavy chains comprising a variable region and a human constant region as shown, and (ii) two identical, whole (i.e., non-chimeric) non-human light chains; and (c) a monovalent antibody, i.e., a complete, functional immunoglobulin molecule comprising (i) two identical chimeric heavy chains comprising a variable region as shown, and a human constant region, and (ii) two different light chains, only one of which has the same specificity as the variable region of the heavy chain. The resulting antibody molecule binds only at one end and is therefore incapable of bivalent binding. As another example, the immunoglobulin-related peptides provided by the present invention may be said to include: (a) whole immunoglobulin molecules; (b) scFvs; (c) monoclonal antibodies; (d) human antibodies; (e) chimeric antibodies; (f) humanized antibodies; (g) Fab fragments; (h) Fab' fragments; (i) F(ab')2 fragments; (j) Fv molecules; and (k) disulfide-linked Fv molecules.

[0134] In one embodiment, the antibody used in the present invention is a polyclonal antibody. In one embodiment, the antibody used in the present invention is a monoclonal antibody. In a further embodiment, the antibody used in the present invention is a human monoclonal antibody. In another further embodiment, the antibody used in the present invention is a humanized antibody. In another further embodiment, the antibody used in the present invention is a chimeric antibody. In another further embodiment, the antibody used in the present invention is a monoclonal antibody originating entirely from a mammalian species other than humans. In a further embodiment, the antibody used in the present invention is a fully murine monoclonal antibody.

[0135] In one embodiment, the antibody used in the present invention is glycosylated in a eukaryotic cell. In another embodiment, the antibody used in the present invention further comprises a chelator linker for attaching a radioisotope. In a further embodiment, the antibody used in the present invention is in a substantially isolated form.

[0136] A monoclonal antibody refers to a composition comprising a homogeneous antibody population with uniform structure and specificity. Typically, a monoclonal antibody is an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprise a population that is identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, and each monoclonal antibody is typically directed against a single epitope, in contrast to polyclonal antibody preparations that typically contain different antibodies directed against different epitopes. The fact that an antibody is monoclonal should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies of the present invention can be prepared by first preparing the monoclonal antibodies described in Kohler et al., Nature 256 , 495 (1975), or may be made by recombinant DNA methods. Monoclonal antibodies may also be produced using the methods described, for example, by Clackson et al., Nature 352 , 624-628(1991) and Marks et al., J. Mol. Biol. 222The antibodies may also be isolated from phage antibody libraries using the techniques described in J. Immunol., 581-597 (1991).

[0137] Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from mouse splenic B cells obtained from a mouse 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. Monoclonal antibodies may also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rats, dogs, primates, etc.

[0138] Alternatively, cloned antibody genes can be expressed in other expression systems, including prokaryotic cells such as microorganisms, e.g., E. coli for the production of single-chain Fv antibodies, algae, and insect cells. Furthermore, antibodies can be produced in transgenic non-human animals, such as in milk from sheep and rabbits, or in eggs from hens, or in transgenic plants. See, e.g., Verma, R., et al., J. Immunol. Meth. 216 , 165-181(1998);Pollock, et al., J. Immunol. Meth. 231 , 147-157(1999); and Fischer, R., et al., Biol. Chem. 380 , 825-839(1999).

[0139] In some embodiments, human monoclonal antibodies against CD38 may be generated using transgenic or transchromosomal mice that carry parts of the human immune system rather than the mouse system. Such transgenic and transchromosomic mice include those referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice." Human monoclonal antibodies generated in such mice may be abbreviated as HuMabs.

[0140] HuMAb mice contain human immunoglobulin gene minilocuses encoding unrearranged human heavy (μ and γ) chain 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 and 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 (Reviewed in Taylor, L. et al., Nucleic Acids Research, 536-546 (1995)). Preparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research, 536-546 (1995). 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), Fishwild, D. et al., Nature Biotechnology 14 , 845-851 (1996). See also U.S. Patent No. 5,545,806, U.S. Patent No. 5,569,825, U.S. Patent No. 5,625,126, U.S. Patent No. 5,633,425, U.S. Patent No. 5,789,650, U.S. Patent No. 5,877,397, U.S. Patent No. 5,661,016, U.S. Patent No. 5,814,318, U.S. Patent No. 5,874,299, U.S. Patent No. 5,770,429, U.S. Patent No. 5,545,807, WO 98 / 24884, WO 94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918, and WO 01 / 09187.

[0141] HCo7 mice have a JKD disruption in their endogenous light chain (kappa) gene (Chen et al., ENBO J. 12 , 821-830 (1993)), a CMD disruption in their endogenous heavy chain gene (as described in Example 1 of WO 01 / 14424), a KCo5 human kappa light chain transgene (Fishwild et al., Nature Biotechnology 14 , 845-851 (1996)), and the HCo7 human heavy chain transgene (as described in US Pat. No. 5,770,429).

[0142] HCo12 mice have a JKD disruption in their endogenous light chain (kappa) gene (Chen et al., ENBO J. 12, 821-830 (1993)), a CMD disruption in their endogenous heavy chain gene (as described in Example 1 of WO 01 / 14424), a KCo5 human kappa light chain transgene (Fishwild et al., Nature Biotechnology 14 , 845-851 (1996)), and the HCo12 human heavy chain transgene (as described in Example 2 of WO 01 / 14424). In the KM mouse strain, the endogenous mouse kappa light chain gene is expressed as described in Chen et al., ENBO J. 12 The mouse strain is homozygously disrupted as described in Fishwild et al., Nature Biotechnology 1993, 811-820, and the endogenous mouse heavy chain gene is homozygously disrupted as described in Example 1 of WO 01 / 01987. 14 , 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF(SC20) as described in WO 02 / 43478.

[0143] KM mice contain a human heavy chain transchromosome and a human kappa light chain transgene. The endogenous mouse heavy and light chain genes have also been disrupted in KM mice so that immunization of the mice results in the production of human immunoglobulins rather than mouse immunoglobulins. The construction of KM mice and their use to produce human immunoglobulins is described in detail in WO 02 / 43478. Spleen cells from these transgenic mice can be used to produce hybridomas secreting human monoclonal antibodies using well-known techniques.

[0144] Human monoclonal or polyclonal antibodies, or antibodies originating from other species, used in the present invention may also be produced transgenically by creating another non-human mammal or plant transgenic for the immunoglobulin heavy and light chain sequences of interest and producing the antibody in a recoverable form therefrom. In connection with transgenic production in mammals, antibodies may be produced in and recovered from the milk of goats, cows, or other mammals. See, e.g., U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957.

[0145] Additionally, human antibodies for use in the present invention or antibodies from other species for use in the present invention may be generated through 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 may be subjected to further maturation, such as affinity maturation, as such techniques are well known in the art (see, 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 U.S. Patent No. 5,733,743. When display techniques are used to produce non-human antibodies, such antibodies may be humanized, e.g., as described elsewhere herein.

[0146] Examples of how to make humanized antibodies can be found, for example, in U.S. Patent No. 6,054,297, U.S. Patent No. 5,886,152, and U.S. Patent No. 5,877,293. The use of Ig cDNA for the construction of chimeric immunoglobulin genes is also known in the art (see, for example, Liu et al., PNAS USA 84, 3439 (1987) and J. Immunol. 139, 3521 (1987)).

[0147] The anti-CD38 antibody was synthesized using human V-cells prepared from mRNA derived from human lymphocytes. L and V H Antibody display libraries may also be recovered from recombinant combinatorial antibody libraries, such as scFv phage display libraries, which may be made from cDNA. Methods for preparing and screening such libraries are known in the art. Many commercially available kits are available for making phage display libraries. There are also other methods and reagents that can be used in making and screening antibody display libraries (e.g., U.S. Pat. No. 5,223,409; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; Fuchs et al., Bio / Technology 9, 1370-1372(1991), Hay et al., Hum. Antibod. Hybridomas 3 , 81-85(1992), Huse et al., Science 246 , 1275-1281(1989), McCafferty et al., Nature 348 , 552-554(1990), Griffiths et al., EMBO J 12 , 725-734(1993), Hawkins et al., J. Mol. Biol. 226 , 889-896(1992), Clackson et al., Nature 352 , 624-628(1991), Gram et al., PNAS USA 89 , 3576-3580(1992), Garrad et al., Bio / Technology 9 , 1373-1377(1991), Hoogenboom et al., Nuc Acid Res 19 , 4133-4137(1991), and Barbas et al., PNAS USA 88 , 7978-7982 (1991). L and V H The nucleic acid sequence of V may be selected using any suitable method. L and V H Nucleic acids of the present invention may be selected by utilizing the epitope imprinting method described in WO 93 / 06213. Antibody libraries, such as scFv libraries, may be selected by utilizing the epitope imprinting method described in WO 92 / 01047, McCafferty et al., Nature 348 , 552-554(1990), and Griffiths et al., EMBO J 12Such antibody libraries may be prepared and screened using known and suitable methods (with human CD38-containing peptides as antigens), such as those described in J. Immunol., 725-734 (1993). Such antibody libraries are a feature of the present invention that may be used therapeutically to provide a more comprehensive immune response; as tools in screening methods (e.g., by competitive assays) for immunogenic peptides, small molecules, other anti-CD38 antibodies, etc.; and / or in diagnostic methods and compositions (e.g., immunoassay chips containing panels of such antibodies, optionally in conjunction with other antibodies, may be prepared by standard techniques). Once the first human V L and V H Once a segment is selected, the first selected V L and V H Perform a "mix-and-match" experiment to screen different pairs of segments for binding to CD38-containing peptides and identify the desired V L / V H Pair combinations may be selected. For example, the reactivity of peptides may be determined by ELISA or other suitable epitope analysis methods (see, e.g., Scott, JK and Smith, GP Science, for a review of such techniques and principles). 249 , 386-390(1990), Cwirla et al., PNAS USA 87 , 6378-6382(1990), Felici et al., J. Mol. Biol. 222 , 301-310(1991), and Kuwabara et al., Nature Biotechnology 15 , 74-78 (1997)). Antibodies may be selected by their affinity for the antigen and / or by their kinetics of dissociation from the antigen (off-rate) (see, e.g., Hawkins et al., J. Mol. Biol. 226 , 889-896 (1992).

[0148] High affinity antibody peptides, such as human single-chain Fv (scFv) and Fab antibody fragments, may also be isolated from such libraries using panning techniques in which the antigen of interest is immobilized on a solid surface, such as a microtiter plate or beads (see, e.g., Barbas and Burton, Trends. Biotechnol. 14 , 230-234(1996) and Aujame et al., Hum. Antibodies 8 , 155-68 (1997)). Phage display of large naive libraries also makes it possible to directly isolate human antibodies without immunization (see, e.g., Haard et al., J. Biol. Chem. 274 (26), 18218-18230 (1999)).

[0149] Antibodies suitable for use in the present invention may be selected based on their ability to provide or not provide complement fixation. There are many antibody isotypes capable of complement fixation and CDC, including, but not limited to: mouse IgM, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgM, human IgG1, and human IgG3. Isotypes not included include, but are not limited to, human IgG2 and human IgG4. Isotyping and other methods for modifying the functional characteristics of antibody complement fixation and CDC are known in the art.

[0150] The anti-CD38 antibodies used in the present invention may be prepared by recombinant expression in any suitable type of cell or animal. Suitable methods for antibody production are known in the art and include, for example, those described in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988); Harlow and Lane: Using Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press (1999)); U.S. Patent No. 4,376,110; and Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley InterScience NY, (1987, 1992). The anti-CD38 antibodies originally described by Kohler et al., Nature 256Monoclonal antibodies may be made using the hybridoma method described by Goding, J., 495 (1975), or by other well-known, later-developed methods (see, e.g., Goding, Monoclonal Antibodies: Principle and Practice, pp. 59-103 (Academic Press, 1986)). Hybridomas useful for producing the anti-CD38 antibodies of the present invention are also provided by the present invention. Any suitable type of myeloma, heteromyeloma, phoblastoid cell, plasmacytoma, or other equivalent, and any suitable type of antibody-expressing cell may be used to form such hybridomas by chemical fusion, electrical fusion, or any other suitable technique. Transformed, immortalized B cells may also be used to efficiently produce the antibodies of the present invention and are also provided by the present invention. Such cells may be produced by standard techniques, such as transformation with Epstein-Barr virus or a transforming gene. (See, e.g., "Continuously Proliferating Human Cell Lines Synthesizing Antibodies of Predetermined Specificity," Zurawaki, VR et al., in Monoclonal Antibodies, edited by Kennett, RH et al., Plenum Press, NY 1980, pp. 19-33.)

[0151] Recombinant cells containing exogenous nucleic acid encoding an anti-CD38 antibody may be prepared by any suitable technique (e.g., transfection / transformation with naked DNA plasmid vectors, viral vectors, invasive bacterial cell vectors, or other whole cell vectors containing antibody-encoding sequences delivered into cells by calcium phosphate precipitation-facilitated transfection, receptor-mediated targeting and transfection, biolistic delivery, electroporation, dextran-mediated transfection, liposome-mediated transformation, protoplast fusion, direct microinjection, etc.). Methods for transforming / transfecting cells are well known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (2nd ed., 1989 and 3rd ed., 2001) and F. Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987)). Such recombinant cells are a feature of the present invention.

[0152] Cell lines available as hosts for recombinant protein expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and many other cell lines. Other cell lines that may be used are insect cell lines, such as Sf9 cells. When a nucleic acid (or a vector containing the nucleic acid) encoding a protein, such as an anti-CD38 antibody, is introduced into mammalian host cells, the protein may be produced by culturing the host cells for a period of time sufficient to allow expression of the protein in the host cells or by secretion of the protein into the culture medium in which the host cells are grown. The antibody may be recovered from the culture medium using standard protein purification methods. Alternatively, if directly expressed without a secretory signal, the antibody may be recovered from host cell lysates.

[0153] When a recombinant expression vector encoding an anti-CD38 antibody gene is introduced into a mammalian host, the antibody is produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells and secretion of the antibody into the culture medium that the host cells grow in. Purification of antibodies from cell cultures, cell lysates, and animals (e.g., from the ascites of transgenic animals producing anti-CD38 antibodies) may be achieved by application of any number of suitable techniques known in the art, including, for example, immunoaffinity column purification; sulfuric acid precipitation; chromatofocusing; preparative SDS-PAGE, etc.

[0154] Human monoclonal antibodies of the present invention can also be produced using a variety of other techniques, including conventional monoclonal antibody methodologies, e.g., Kohler and Milstein, Nature 256Antibodies may also be produced by standard somatic cell hybridization techniques, such as those described in J. Immunol., 495 (1975). Other techniques for producing monoclonal antibodies may also be utilized, such as phage display techniques using libraries of human antibody genes. In certain embodiments, the anti-CD38 antibodies of the present invention are produced by the use of hybridomas generated in a mouse system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0155] To generate fully human monoclonal antibodies against CD38, transgenic or transchromosomal mice containing human immunoglobulin genes (e.g., HCo12, HCo7, or KM mice) can be immunized with enriched preparations of CD38 antigen and / or cells expressing CD38, as described, for example, by Lonberg et al. (1994), supra, Fishwild et al. (1996), supra, and WO 98 / 24884. Alternatively, mice can be immunized with DNA encoding human CD38. At the time of the first injection, mice can be 6 to 16 weeks old. For example, HuMAb mice can be immunized intraperitoneally with an enriched preparation of CD38 antigen (5 to 50 μg). Additionally, in the event that immunization with a purified or enriched CD38 antigen preparation does not result in antibodies, immunization with cells expressing CD38, such as a cell line, can be used to stimulate the immune response.

[0156] Accumulating experience with various antigens has shown that HuMAb transgenic mice respond best when initially immunized intraperitoneally (ip) or subcutaneously (sc) with CD38-expressing cells in complete Freund's adjuvant, followed by biweekly ip immunizations (up to a total of 10 times) with CD38-expressing cells in PBS. Throughout the immunization protocol, immune responses can be monitored with plasma samples obtained by retroorbital bleeding. Plasma can be screened by FACS analysis, and mice with sufficient titers of anti-CD38 human immunoglobulin can be used for fusions. Mice can be boosted intravenously with CD38-expressing cells for Example 4, 4 and 3 days before sacrifice and removal of the spleen.

[0157] To generate hybridomas producing human monoclonal antibodies against human CD38, spleen cells and lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. For example, a single-cell suspension of splenic lymphocytes from immunized mice can be fused to SP2 / 0 non-secretory mouse myeloma cells (ATCC, CRL 1581) in 50% PEG (w / v). Cells can be plated at approximately 1 x 10 per well in flat-bottom microtiter plates and then incubated for 2 weeks in selection medium containing, in addition to the usual reagents, 10% fetal bovine serum, 5-10% origen hybridoma cloning factor (IGEN), and 1 x HAT (Sigma). After approximately 2 weeks, cells can be cultured in medium in which the HAT is replaced with HT. Individual wells may then be screened by ELISA for antibodies containing human kappa light chains and by FACS analysis using CD38-expressing cells for CD38 specificity. Once extensive hybridoma growth occurs, usually after 10-14 days, medium may be observed. Antibody-secreting hybridomas may be replated and screened again, and if still positive for human IgG, anti-CD38 monoclonal antibodies may be subcloned at least twice by limiting dilution. Stable subclones may then be cultured in vitro to generate antibody in tissue culture medium for characterization.

[0158] Human antibodies of the invention can also be produced in host cell transfectomas using a combination of recombinant DNA technology and gene transfection methods as are well known in the art, for example, see Morrison, S., Science 229 , 1202(1985).

[0159] For example, to express an antibody, or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains may be obtained by standard molecular biology techniques (e.g., PCR amplification, site-directed mutagenesis) and inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, or more typically, both genes may be inserted into the same expression vector. The antibody gene may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). V H segment is operably linked to a CH segment in a vector and V L Full-length antibody genes of any antibody isotype may be created by inserting the antibody light and heavy chain variable regions described herein into an expression vector already encoding the heavy and light chain constant regions of the desired isotype, such that the segments are operably linked to a CL segment in the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0160] In addition to the antibody chain genes, recombinant expression vectors carry regulatory sequences that enable and control the expression of the antibody chain genes in a host cell. Furthermore, recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically, the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cells into which the vector has been introduced. Examples of selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0161] For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The host cell may be a prokaryotic or eukaryotic host cell, such as a mammalian host cell. For example, antigen-binding fragments may be expressed in prokaryotic host cells, and full-length antibodies may be expressed in eukaryotic host cells.

[0162] In some embodiments, the antibodies are expressed in eukaryotic cells, such as mammalian host cells. Exemplary mammalian host cells for expressing the recombinant antibodies of the invention include CHO cells (see, e.g., RJ Kaufman and PA Sharp, Mol. Biol. 159 , 601-621 (1982), used in conjunction with a DHFR selectable marker, such as that described by Urlaub and Chasin, PNAS USA 77, 4216-4220 (1980)), NS / 0 myeloma cells, COS cells, HEK293 cells, and SP2.0 cells. Specifically, for use with NS / 0 myeloma cells, another example of an expression system is the GS (glutamine synthetase) gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841.

[0163] Antibody genes may also be expressed in other expression systems, including prokaryotic cells such as microorganisms, e.g., E. coli for the production of scFv antibodies, algae, and insect cells. Furthermore, antibodies may be produced in transgenic non-human animals, such as in milk from sheep and rabbits or in eggs from hens, or in transgenic plants. See, e.g., Verma, R. et al., J. Immunol. Meth. 216 , 165-181(1998), Pollock et al., J. Immunol. Meth. 231 , 147-157(1999), and Fischer, R. et al., Biol. Chem. 380 , 825-839(1999).

[0164] Bispecific and multispecific antibodies In certain embodiments of the invention, the antibodies used may be derivatized or linked to another functional molecule, such as another peptide (such as a Fab' fragment) or protein, to create a bispecific or multispecific molecule that binds to multiple binding sites or target epitopes. For example, an antibody used in the invention may be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other binding molecules, such as another antibody, peptide, or binding mimetic.

[0165] Thus, the present invention includes bispecific and multispecific molecules comprising at least one first binding specificity for CD38 and a second binding specificity for a second target epitope. In certain embodiments of the present invention, the second target epitope is an Fc receptor, e.g., human FcγRI (CD64) or human Fcα receptor (CD89), or a T cell receptor, e.g., CD3. In certain embodiments, the present invention provides bispecific and multispecific molecules that can bind to both FcγR-, FcαR-, or FcεR-expressing effector cells (e.g., monocytes, macrophages, or polymorphonuclear cells (PMNs)) and CD38-expressing target cells. These bispecific and multispecific molecules target CD38-expressing cells to effector cells and induce Fc receptor-mediated effector cell activity, such as phagocytosis of CD38-expressing cells, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or superoxide anion generation.

[0166] In certain embodiments, the bispecific and multispecific molecules used in the present invention comprise at least one additional antibody as a binding specificity, including, for example, Fab, Fab', F(ab')2, Fv, or scFv. The additional antibody may also be a light or heavy chain dimer or any minimal fragment thereof, such as an Fv or single-chain construct as described by Ladner et al. in U.S. Pat. No. 4,946,778. The antibody may also be a binding domain immunoglobulin fusion protein as disclosed in U.S. Pat. Nos. 2003 / 0118592 and 2003 / 0133939.

[0167] In certain embodiments, the binding specificity for an Fc receptor is conferred by a human monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term "IgG receptor" refers to any of the eight gamma chain genes located on chromosome 1. These genes are part of the three Fc receptors. *The receptor classes encode a total of 12 transmembrane or soluble receptor isoforms, grouped into FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). In one embodiment, the Fcγ receptor is a human high-affinity FcγRI. The production and characterization of these monoclonal antibodies are described by Fanger et al. in WO 88 / 00052 and U.S. Pat. No. 4,954,617. These antibodies bind to epitopes of FcγRI, FcγRII, or FcγRIII at sites distinct from the Fcγ binding site of the receptor, and therefore, their binding is not substantially blocked by physiological levels of IgG. Specific anti-FcγRI antibodies useful in the present invention are mAb 22, mAb 32, mAb 44, mAb 62, and mAb 197. In another embodiment, the anti-Fcγ receptor antibody is a humanized form of mAb 22 (H22). The production and characterization of the H22 antibody is described in Graziano, RF et al., J. Immunol. 155 (10), 4996-5002 (1995) and WO 94 / 10332. The H22 antibody-producing cell line was deposited with the American Type Culture Collection on November 4, 1992 under the designation HA022CL1 and has the accession number CRL 11177.

[0168] In certain embodiments, binding specificity for an Fc receptor is conferred by an antibody that binds to a human IgA receptor, e.g., an Fcα receptor (FcαI (CD89)), the binding of which, in certain embodiments, is not blocked by human immunoglobulin A (IgA). The term "IgA receptor" is intended to include the gene product of a single α-gene (FcαRI) located on chromosome 19. This gene is known to encode several alternatively spliced ​​55- to 110-kDa transmembrane isoforms. FcαRI (CD89) is constitutively expressed on monocytes / macrophages, eosinophilic, and neutrophilic granulocytes, but not on non-effector cell populations. FcαRI has intermediate affinity for both IgA1 and IgA2, and affinity increases upon exposure to cytokines such as G-CSF or GM-CSF (Morton HC et al., Critical Reviews in Immunology 16 , 423-440 (1996)). Four FcαRI-specific monoclonal antibodies, identified as A3, A59, A62, and A77, which bind to FcαRI outside the IgA ligand-binding domain have been described (Monteiro, RC et al., J. Immunol. 148 , 1764(1992)).

[0169] FcαRI, FcγRI, FcγRII, and FcγRIII, particularly FcγRII and FcγRIII, are examples of triggering receptors for use in the present invention because they (1) are expressed primarily on immune effector cells, e.g., monocytes, PMNs, macrophages, and dendritic cells; (2) are expressed at high levels (e.g., 5,000-100,000 per cell); (3) are mediators of cytotoxic activity (e.g., ADCC, phagocytosis); and (4) mediate enhanced antigen presentation of antigens, including self-antigens, targeted by them.

[0170] Exemplary bispecific antibody molecules include (i) two antibodies conjugated together, one with specificity for CD38 and the other for a second target, (ii) one 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. Typically, the second target / second molecule is a molecule other than CD38. In some embodiments, the second molecule is a cancer antigen / tumor-associated antigen such as carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), RAGE (renal antigen), alpha-fetoprotein, CAMEL (antigen recognized by CTL on melanoma), CT antigen (e.g., MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE, etc.), mucin antigen (e.g., MUC1, mucin-CA125, etc.), ganglioside antigen, tyrosinase, gp75, C-myc, Mart1, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, and Ep-CAM. In some embodiments, the second molecule is a cancer-associated integrin, such as alpha5beta3 integrin. In some embodiments, 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 receptors, particularly those associated with cancer progression (e.g., one of the HER1-HER4 receptors). Other cancer progression-associated proteins discussed herein may also be suitable second molecules. In some embodiments, the second molecule is a molecule expressed on the surface of multiple myeloma cells, such as CD138.

[0171] In one embodiment, a bispecific antibody for use in the present invention is a diabody.

[0172] Bispecific and multispecific antibodies for use in the present invention can be produced using chemical techniques (see, e.g., D. M. Kranz et al., PNAS USA 78, 5807 (1981)), "polydoma" technology (see U.S. Pat. No. 4,474,893), or recombinant DNA technology.

[0173] Conjugates In certain embodiments, the present invention utilizes a CD38 antibody conjugated to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioisotope. Such conjugates are referred to herein as "immunoconjugates." Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins."

[0174] Cytotoxins or cytotoxic agents include 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, 1990. Further techniques related to the preparation of antibody immunotoxins can be found, for example, in Vitetta, Immunol. Today 14 , 252 (1993) and U.S. Pat. No. 5,194,594.

[0175] Suitable therapeutic agents for forming immunoconjugates useful in the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine, etc.), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine ... other platinum derivatives such as carbazine (DTIC), procarbazine, mitomycin C, cisplatin, and carboplatin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, calicheamicin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC), etc.), diphtheria toxin and related molecules (e.g., diphtheria Ricin A chain and active fragments and hybrid molecules thereof), 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, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordicacharantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, xeronine, mitogenin, restrictocin, phenomycin, and enomycin toxin. Therapeutic agents that may be administered in combination with antibodies, as described elsewhere herein, may also be useful candidates for therapeutic moieties for conjugation to antibodies used in the present invention. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, enzymatically active toxins, or active fragments thereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), or other growth factors and apoptosis-inducing proteins isolated from mitochondria.

[0176] Conjugates of antibodies and such cytotoxic moieties may be prepared using various bifunctional protein coupling agents. Examples of such agents include bifunctional derivatives of imidoesters such as SPDP, IT, dimethyl adipimidate HCl, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis(p-azidobenzoyl)hexanediamine, bis-diazonium derivatives such as bis-(p-diazoniumbenzoyl)ethylenediamine, diisocyanates such as tolylene 2,6-diisocyanate, and bis-active fluorine compounds such as 1,5-difluoro-2,4-dinitrobenzene, and antimitotic drugs (e.g., vincristine, vinblastine, docetaxel, paclitaxel, and vinorelbine).

[0177] In some embodiments, the present invention provides anti-CD38 antibodies conjugated to an immunomodulatory factor, such as an immunomodulatory cytokine, stem cell growth factor, lymphotoxin (such as a TNF, such as TNFα), or hematopoietic factor. Examples of such molecules that may be useful as conjugates include IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21, colony-stimulating factors (such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF)), interferons (such as IFNα, IFNβ, and IFNγ), stem cell growth factor designated "S1 factor," erythropoietin, and thrombopoietin, active fragments thereof, derivatives thereof, variants thereof, or any combination thereof.

[0178] Techniques for conjugating such therapeutic moieties to antibodies are well known, and include, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al., (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al., (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., (eds.), pp. 475-506 (1985); Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al., (eds.), pp. 303-16 (Academic Press 1985), "Analysis, Results, And Future Prospect of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy," and Thorpe et al., "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates," Immunol. Rev. 62 , 119-58 (1982).

[0179] Further useful conjugate substituents include anticancer retinoids, taxane conjugates (see, e.g., Jaime et al., Anticancer Res. 21 (2A), 1119-28 (2001)), cisplatin conjugates, thapsigargin conjugates, linoleic acid conjugates, calicheamicin conjugates (see, e.g., Damle et al., Curr Opin Pharmacol. 3 (4), 386-90 (2003)), doxorubicin conjugates, geldanamycin conjugates, and the like may also be useful in facilitating cancer treatment (see generally Trail et al., Cancer Immunol Immunother. 52 (5), 328-37 (2003).

[0180] Formulation and Mode of Administration The agents used in the present invention may be formulated with pharmaceutically acceptable carriers or diluents, as well as any 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.

[0181] Pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, should be suitable for the selected compound and the selected mode of administration used in the present invention. Suitability of carriers and other components of pharmaceutical compositions is determined based on the lack of a significant negative effect on the desired biological properties of the selected compound or pharmaceutical composition (e.g., no substantial effect on antigen binding (10% or less relative inhibition, 5% or less relative inhibition, etc.)).

[0182] Pharmaceutical compositions for use in the present invention may also include diluents, fillers, salts, buffers, surfactants (e.g., non-ionic surfactants such as Tween-80), stabilizers, stabilizers (e.g., sugars or non-protein amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in pharmaceutical compositions.

[0183] The actual dosage level of the active ingredient in the pharmaceutical composition may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition or its ester, salt, or amide employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0184] The pharmaceutical compositions may be administered by any suitable route and manner. Suitable routes of 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.

[0185] The compounds used in the present invention may be administered via any suitable route, such as oral, nasal, inhalable, topical (including buccal, transdermal, and sublingual), rectal, vaginal, and / or parenteral routes.

[0186] In some embodiments, one or more pharmaceutical compositions used in the present invention are orally administered, for example, using inert diluents or absorbable edible carriers.Active ingredient can be encapsulated in hard or soft gelatin capsules, compressed into tablets, or directly incorporated into the diet of subjects.Pharmaceutical compositions suitable for oral administration include ingestible tablets, oral tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., with carriers known to be suitable in the art.To enable oral administration, compound may need to be coated with a material that prevents its inactivation, or compound may need to be administered simultaneously with said material.

[0187] In certain embodiments, one or more of the compounds used in the present invention are administered parenterally.

[0188] The phrases "parenteral administration" and "administering parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include epithelial, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion.

[0189] In some embodiments, the compound is administered by intravenous or subcutaneous injection or infusion.

[0190] In some embodiments, the compounds used in the present invention are administered in crystalline form by subcutaneous injection. Yang et al., PNAS USA, 100 (12), 6934-6939 (2003).

[0191] The pharmaceutical compositions used in the present invention may be formulated for a particular route of administration, such as oral, nasal, topical (including buccal, transdermal, and sublingual), rectal, vaginal, and / or parenteral administration. Pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any method known in the art of pharmacy. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will usually be that amount of the composition that produces a therapeutic effect. Out of one hundred percent, this amount will usually range from about 0.01% to about 99% of the active ingredient, for example, from about 0.1% to about 70%, for example, from about 1% to about 30%.

[0192] Regardless of the route of administration selected, the compounds used in the present invention, which may be used in the form of a pharmaceutically acceptable salt or a suitable hydrated form, and / or pharmaceutical compositions may be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM et al., J. Pharm. Sci. 66 , 1-19 (1977). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, as well as salts derived from non-toxic organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as salts derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0193] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants, and absorption delaying agents, etc. that are physiologically compatible with the compounds used in the present invention.

[0194] Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions include water, saline, phosphate-buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solution, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical arts.

[0195] Pharmaceutically acceptable carriers include sterile injectable aqueous solution or dispersion and sterile powder for the immediate preparation of sterile injectable solution or dispersion.The use of such media and agents for pharmaceutical active substances is known in the art.Except when any conventional media or agent is incompatible with active compound, its use in pharmaceutical compositions is included.

[0196] Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0197] Pharmaceutical compositions containing the agents used in the present invention may also contain pharmaceutically acceptable antioxidants, for example: (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, alpha-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0198] The pharmaceutical compositions may also contain isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0199] Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0200] Pharmaceutical compositions used in the present invention may also contain one or more adjuvants appropriate to the chosen route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives, and buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds of the present invention may be blended with, for example, lactose, sucrose, powder (e.g., starch powder), cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphates and sulfates, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and / or polyvinyl alcohol. Other examples of adjuvants are QS21, GM-CSF, SRL-172, histamine dihydrochloride, thymocartin, Tio-TEPA, monophosphoryl lipid A / mycobacterial composition, alum, incomplete Freund's adjuvant, Montanide ISA, Livia adjuvant system, TiterMax adjuvant, Syntex adjuvant formulation, immune stimulating complexes (ISCOMs), Gelb adjuvant, CpG oligodeoxynucleotides, lipopolysaccharide, and polyinosinic:polycytidylic acid.

[0201] Prevention of presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, etc. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[0202] Therefore, the pharmaceutical composition of the present invention containing the compound of the present invention may also contain a suitable salt.Any suitable salt, such as any suitable form of alkaline earth metal salt (e.g., buffer salt), may be used to stabilize the compound used in the present invention.Suitable salts typically include sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride.In some embodiments, aluminum salts are used to stabilize the compound used in the present invention in pharmaceutical compositions, and these aluminum salts may also serve as adjuvants when such compositions are administered to patients.

[0203] The compound used in the present invention can be prepared with carriers that are expected to prevent compound from rapid release, such as controlled release preparations, including implants, transdermal patches and microencapsulated delivery systems.Such carriers include biodegradable and biocompatible polymers, such as gelatin, glyceryl monostearate, glyceryl distearate, ethylene vinyl acetate alone or with wax, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, or other materials well known in the art.The method for preparing such preparations is generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker Co., New York, 1978.

[0204] To administer the composition via certain routes of administration, it may be necessary to coat the compound with a material to prevent its inactivation or to administer the compound simultaneously with the material. For example, the compound used in the methods of the present invention may be administered to a subject in an appropriate carrier, such as a liposome, or a diluent. Liposomes include not only conventional liposomes but also water-in-oil-in-water CGF emulsions (Strejan et al., J. Neuroimmunol. 7 , 27(1984)).

[0205] Depending on the route of administration, the active compound may be coated in a material that protects the compound from the action of acids and other natural conditions that may inactivate the compound. For example, the compound may be administered to a subject in a suitable carrier, such as a liposome. Liposomes include not only traditional liposomes but also water-in-oil-in-water CGF emulsions (Strejan et al., J. Neuroimmunol. 7 , 27(1984)).

[0206] In some embodiments of the present invention, the compounds of the present invention may be formulated in liposomes. In a further embodiment, the liposomes contain a targeting moiety. In a further embodiment, the compounds in the liposomes are delivered by bolus injection to a site close to the desired area, for example, the site of inflammation or infection, or the site of a tumor. The composition must be fluid enough to allow easy syringability. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0207] In some embodiments, the compounds used in the present invention may be formulated to prevent or reduce their transport across the placenta. This may be done by methods known in the art, for example, by PEGylation of the compound or by using F(ab')2 fragments. Cunningham-Rundles C et al., J Immunol Methods. 152 , 177-190(1992), and Landor M., Ann Allergy Asthma Immunol 74 , 279-283 (1995).

[0208] Pharmaceutically acceptable carriers for parenteral administration include sterile injectable aqueous solution or dispersion and sterile powder for the immediate preparation of sterile injectable solution or dispersion.The use of such media and agents for pharmaceutically active substances is known in the art.Except when any conventional media or agent is incompatible with active compound, its use in pharmaceutical compositions is included.In addition, auxiliary active compounds can be incorporated into the composition.

[0209] Injectable pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other designated structures suitable for high drug concentrations. The carrier may be an aqueous or non-aqueous solvent or dispersion medium, including, for example, water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, glycerol, polyalcohols such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable composition may be achieved, for example, by including an agent that delays absorption, such as monostearate salts and gelatin, in the composition. Sterile injectable solution can be prepared by incorporating the active compound in the required amount in a suitable solvent with, for example, one or a combination of the ingredients listed above, and then optionally by sterile microfiltration.Usually, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other required ingredients, for example, from the ingredients listed above.For the preparation of sterile powder for sterile injectable solution, examples of the method of preparation are vacuum drying and freeze-drying (lyophilization), which produces a powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile filtered.

[0210] Sterile injectable solution can be prepared by incorporating the active compound in the required amount with one or a combination of the above-listed ingredients into a suitable solvent, and then, if necessary, by sterile microfiltration. Usually, dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients from the above-listed ingredients. For the preparation of sterile powder for sterile injectable solution, examples of the preparation method are vacuum drying and freeze-drying (lyophilization), which produces a powder of the active ingredient and any additional desired ingredients from the solution previously sterile-filtered.

[0211] In certain embodiments of the methods of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises melphalan, administered intravenously or orally.

[0212] In another embodiment of the method of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises an orally administered glutamic acid derivative, such as thalidomide (Thalomid®) or a thalidomide analog, e.g., CC-5013 (lenalidomide, Revlimid®) or CC4047 (Actimid®).

[0213] In another embodiment of the methods of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises a proteasome inhibitor, such as bortezomib (Velcade®), administered intravenously.

[0214] In another embodiment of the methods of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises a vinca alkaloid, such as vincristine, administered intravenously.

[0215] In another embodiment of the method of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises an anthracycline, such as doxorubicin, administered intravenously.

[0216] In another embodiment of the method of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises prednisone, administered orally.

[0217] In another embodiment of the method of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises prednisone, administered orally.

[0218] Patients and diseases to be treated Individuals who may be treated with the combination therapy of the present invention include human patients having disorders that may be corrected or ameliorated by inhibiting CD38 function, such as, for example, enzymatic activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation, and / or induction of lysis, and / or deleting / reducing the number of CD38-expressing cells.

[0219] For example, anti-CD38 antibodies may be used to elicit one or more of the following biological activities in vivo or in vitro: inhibiting CD38 function (such as enzymatic activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation, and / or induction of lysis), mediating phagocytosis or ADCC of CD38-expressing cells in the presence of human effector cells, and killing CD38-expressing cells by mediating CDC of CD38-expressing cells in the presence of complement or by killing CD38-expressing cells by apoptosis.

[0220] In certain embodiments, the immunoconjugates described herein may be used to target compounds (e.g., therapeutic agents, labels, cytotoxins, immunosuppressants, etc.) to cells that have CD38 bound to their surface by using targeting compounds such as therapeutic moieties in the immunoconjugates of the invention.

[0221] In certain embodiments, the present invention provides methods for killing cells having CD38 bound to their surface by administering an immunoconjugate of the invention.

[0222] The present invention provides a method for treating a disorder involving cells expressing CD38 in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of i) a non-agonistic antibody that binds to CD38; ii) at least one corticosteroid, and iii) at least one non-corticosteroid chemotherapy agent The present invention provides methods for inhibiting CD38-induced activity or reducing or decreasing the number of cells expressing CD38, which are associated with certain disorders, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody.

[0223] In certain embodiments of the invention, the disorder involving cells expressing CD38 is a tumorigenic disorder, such as a disorder characterized by the presence of tumor cells expressing CD38, including, for example, B-cell lymphomas, plasma cell malignancies, T / NK-cell lymphomas, and myeloid malignancies.

[0224] Examples of such tumorigenic diseases include B-cell lymphomas / leukemias, including precursor B-cell lymphoblastic leukemia / lymphoma and B-cell non-Hodgkin's lymphoma; mature B-cell neoplasms, such as acute promyelocytic leukemia, acute lymphoblastic leukemia, and B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL); B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL); These include follicular lymphoma (FL), including low-, intermediate-, and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal and splenic type), 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).

[0225] In certain embodiments, the disorder involving cells expressing CD38 is multiple myeloma.

[0226] Examples of B-cell non-Hodgkin's lymphomas are lymphomas induced by treatment with immunosuppressive drugs, such as lymphomatoid granulomatosis, primary effusion lymphoma, intravascular large B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain disease (including gamma, mu, and alpha disease), cyclosporine-induced lymphoma, and methotrexate-induced lymphoma.

[0227] In one embodiment of the invention, the disorder involving cells expressing CD38 may be Hodgkin's lymphoma.

[0228] Examples of disorders involving cells expressing CD38 may be malignancies derived from T cells and NK cells, including mature T cell and NK cell neoplasms, such as 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, enteropathic type 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.

[0229] Examples of malignancies derived from myeloid cells include acute myeloid leukemia, including acute promyelocytic leukemia, and chronic myeloproliferative disorders, including chronic myelogenous leukemia.

[0230] Medication and Treatment Regimen Treatments according to the present invention include the use of a "therapeutically effective amount" of a pharmaceutical agent. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount can vary depending on the disease state, age, sex, and weight of the individual, as well as the ability of the pharmaceutical agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. In the context of this combination therapy, a therapeutic amount includes an amount that is therapeutically effective only in combination with other compounds, e.g., an amount that is too small to be effective in monotherapy.

[0231] The "therapeutically effective amount" for tumor treatment may also be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer may be evaluated in an animal model system that is predictive of its effectiveness in human tumors. Alternatively, this property of a composition may be evaluated by examining the ability of a compound to inhibit cell growth or induce apoptosis in an in vitro assay known to those skilled in the art. A therapeutically effective amount of a therapeutic compound may reduce tumor size or otherwise improve symptoms in patients. Those skilled in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0232] The dosage regimen is adapted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. Parenteral compositions may be formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to a physically discrete unit suitable as a single dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage form of the present invention are determined by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the technical field of formulating such active compounds for the treatment of susceptibility in individuals.

[0233] Effective dosages and dosing regimens for anti-CD38 antibodies used in the present invention depend on the disease or condition to be treated and may be determined by one of skill in the art. An exemplary, non-limiting range for a therapeutically effective amount of an anti-CD38 antibody used in the present invention is about 0.1-100 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 0.5 mg / kg, e.g., about 0.3, about 1, or about 3 mg / kg. In another embodiment, the antibody is administered at a dose of 1 mg / kg or more, e.g., 1-20 mg / kg, e.g., 5-20 mg / kg, e.g., 8 mg / kg.

[0234] A physician or veterinarian with ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician or veterinarian may start the dosage of the drug utilized in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Generally, a suitable daily dose of the composition of the present invention will be the amount of the compound that is the minimum effective dose to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Administration may be intravenous, intramuscular, intraperitoneal, or subcutaneous, for example, administered near the target site. If desired, an effective daily dose of the pharmaceutical composition may be administered as two, three, four, five, six, or more separate doses administered at appropriate intervals throughout the day, optionally in unit dosage form. While it is possible to administer the compounds of the present invention alone, it is preferable to administer the compounds as pharmaceutical compositions as described above.

[0235] In one embodiment, the anti-CD38 antibody is administered at a concentration 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, such as 3 to 5 times. Administration may be by continuous infusion over a period of 2 to 24 hours, such as 2 to 12 hours.

[0236] In certain embodiments, 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.

[0237] In some embodiments, the anti-CD38 antibody may be administered by infusion up to eight times, e.g., four to six times, at a weekly dosage of 250 mg to 2000 mg, e.g., 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg, or 2000 mg. Administration may be by continuous infusion over a period of 2 to 24 hours, e.g., 2 to 12 hours. Such a regimen may be repeated one or more times as needed, e.g., after six to twelve months. Dosage may be determined or adjusted by, for example, obtaining a biological sample and measuring the amount of a compound of the invention in the blood at the time of administration using an anti-idiotypic antibody targeting the antigen-binding region of the anti-CD38 antibody.

[0238] In a further embodiment, the anti-CD38 antibody may be administered once a week for 2 to 12 weeks, such as 3 to 10 weeks, for example 4 to 8 weeks.

[0239] In certain embodiments, the anti-CD38 antibody may be administered by maintenance therapy, such as, for example, once a week for six months or more.

[0240] In some embodiments, the anti-CD38 antibody may be administered by a regimen comprising a single infusion of the anti-CD38 antibody, followed by an infusion of an anti-CD38 antibody conjugated to a radioisotope, which may be repeated, for example, 7-9 days later.

[0241] As a non-limiting example, treatment according to the present invention may be administered using a single or divided dose every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, at a dose of about 0.1 to 100 mg / day for at least one of 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after initiation of treatment, or alternatively, for at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks, or any combination thereof. mg / kg, for example, 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.

[0242] In some embodiments of the methods of the present invention, at least one non-corticosteroid chemotherapeutic agent comprises melphalan, and at least one corticosteroid comprises prednisone. Typically, melphalan is administered intravenously (IV), but it can also be administered orally (PO), for example, in the range of 0.2-0.25 mg / kg per day, or, for example, 7-9 mg / m. Prednisone may be administered, for example, at 2 mg / kg for 4 days every 4-6 weeks (Alexanian et al., J Am Med Assoc 1969;208:1680). In other embodiments, melphalan can be used in high-dose regimens at single doses up to 140 mg / m2 (IV) or intermediate doses ranging from 25 to 75 mg / m2 (IV), one example being 40 mg / d administered on days 1-4, 9-12, and 17-20 in a 5-weekly cycle (Tsakanikas et al., Oncology 1991;48:369, Richardson PG Am J Oncol 2005;4:737).

[0243] In another embodiment of the method of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises thalidomide (Thalomid®), and the at least one corticosteroid comprises dexamethasone. Thalidomide can be used, for example, at a dose of 200 mg / d (PO), or, for example, in the range of 50-400 mg / d, with a dose of 40 mg / d of dexamethasone, either administered daily or sequentially, for example, on days 1-4, 9-12, 17-20 of each 28-day cycle (Rajkumar SV J Clin Oncol 2006;24:431).

[0244] In another embodiment of the method of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises lenalidomide, and the at least one corticosteroid comprises dexamethasone. Lenalidomide can be administered, for example, at a dose of 25 mg / d administered daily (PO), and dexamethasone can be administered, for example, in the range of 40 mg / d administered (PO), for example, on days 1-4, 9-12, 17-20 of a 28-day cycle, and optionally thereafter only on days 1-4 of each cycle (Rajkumar SV, ASH 2004).

[0245] In another embodiment of the method of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises bortezomib (Velcade®). Bortezomib can be used, for example, in combination with dexamethasone. This combination can be used in both induction and maintenance settings. One example is bortezomib 1.3 mg / m2 on days 1, 4, 8, and 11 in a 21-day cycle (induction phase, usually up to 8 cycles), followed by 5-weekly cycles on days 1, 8, 11, 15, and 22 for maintenance (Richardson PG N Engl J Med 2005;352:2487).

[0246] In another embodiment of the method of the present invention, the at least one non-corticosteroid chemotherapeutic agent comprises vincristine and doxorubicin, and the at least one corticosteroid comprises dexamethasone. Vincristine may be administered, for example, at 0.4 mg per day by continuous IV infusion (days 1-4 in a 4-week cycle), and doxorubicin may be administered, for example, at a dose of 9 mg / m² / d by continuous IV infusion on days 1-4 in a 4-week cycle. Dexamethasone may be administered, for example, at 40 mg on days 1-4, 9-12, and 17-21 in a 4-week cycle. Alternatively, pegylated liposomal doxorubicin may be used (at a dose of 40 mg / m² in a weekly cycle) (Rifkin Cancer 2006; 106:848).

[0247] Further combinations The combination therapy of the present invention may further be combined with other pharmaceutical agents, i.e., additional therapeutic agents related to the disease or condition to be treated. Such administration may be simultaneous, separate, or sequential. For simultaneous administration, the agents may be administered as one composition or as separate compositions, as appropriate.

[0248] Thus, the present invention provides methods for treating disorders involving cells expressing CD38, as described above, comprising the triple therapy of the present invention in combination with one or more additional therapeutic agents, as described below.

[0249] In certain embodiments, the combination therapy of the present invention may include administration of at least one chemotherapeutic agent, at least one anti-inflammatory agent, or at least one immunosuppressive and / or immunomodulatory agent.

[0250] In certain embodiments, 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 similar agents.

[0251] In certain embodiments, such chemotherapeutic agents may be selected from antibiotics such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC), and similar agents.

[0252] In certain embodiments, such chemotherapeutic agents may be selected from antimitotic agents such as taxanes, eg, docetaxel, and paclitaxel.

[0253] In some embodiments, such chemotherapeutic agents may be selected from topoisomerase inhibitors, such as topotecan.

[0254] In certain embodiments, such chemotherapeutic agents may be selected from growth factor inhibitors, such as inhibitors of ErbB1 (EGFR) (e.g., gefitinib (Iressa®), cetuximab (Erbitux®), erlotinib (Tarceva®), 2F8 (disclosed in WO 2002 / 100348), and similar agents), inhibitors of ErbB2 (Her2 / neu) (e.g., transtuzumab (Herceptin®) and similar agents), and similar agents. In certain embodiments, such growth factor inhibitors may be farnesyltransferase inhibitors, such as SCH-66336 and R115777. In certain embodiments, such growth factor inhibitors may be vascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab (Avastin®).

[0255] In certain embodiments, such chemotherapeutic agents may be tyrosine kinase inhibitors, such as imatinib (Gleevec, Gleevec STI571), lapatinib, PTK787 / ZK222584, and similar agents.

[0256] In certain embodiments, such chemotherapeutic agents may be histone decerase inhibitors. Examples of such histone decerase inhibitors include hydroxamic acid-based hybrid polar compounds, such as SAHA (suberoylanilide hydroxamic acid).

[0257] In certain embodiments, such a chemotherapeutic agent may be a P38a MAP kinase inhibitor, such as SCIO-469.

[0258] In a further embodiment, the combination therapy of the present invention further comprises administration to a subject in need thereof at least one inhibitor of angiogenesis, neovascularization, and / or other angiogenesis.

[0259] 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, Madison, NJ), R115777 (Janssen Pharmaceuticals, Titusville, NJ), and similar agents), antagonists of angiogenic growth factors (e.g., ZD6474, SU6668, antibodies against angiogenic agents and / or their receptors (e.g., VEGF, bFGF, and angiopoietin-1), Sugen 5416, SU5402, anti-angiogenic ribozymes (e.g., angiozymes), interferon alpha (e.g., interferon alpha 2a), suramin, and similar agents), VEGF-R kinase inhibitors and other anti-angiogenic tyrosine kinase inhibitors (e.g., SU011248), inhibitors of endothelial-specific integrin / survival signals (e.g., vitaxin and similar agents), copper antagonists / chelators (e.g., tetrathiomolybdate, captopril, and similar agents), carboxyamidotriazole (CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E, as well as nucleotide molecules that inhibit angiogenesis (e.g., antisense VEGF-cDNA, cDNA encoding angiostatin, cDNA encoding p53, and cDNA encoding defective VEGF receptor-2) and similar agents.

[0260] 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-alpha-v / beta-3 integrin and anti-kininostatin mAbs), and similar agents.

[0261] In further embodiments, the combination therapy of the invention further includes administration of an anti-cancer immunogen, such as a cancer antigen / tumor-associated antigen (e.g., epithelial cell adhesion molecule (EpCAM / TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7), a cancer-associated viral vaccine (e.g., a human papillomavirus vaccine), a tumor-derived heat shock protein, and similar agents. Many other suitable cancer antigens / tumor-associated antigens described elsewhere herein and similar molecules known in the art may also or instead 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 to the MG7 antibody, and other anti-cancer anti-idiotype antibodies (e.g., Birebent et al., Vaccine. 21 (15), 1601-12(2003), Li et al., Chin Med J.(Engl). 114 (9), 962-6(2001), Schmitt et al., Hybridoma. 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 Methods. 264 (1-2), 121-33 (2002)). Such anti-idiotypic Abs may optionally be conjugated to a carrier, which may be a synthetic (typically 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 cells (e.g., erythrocytes - see, e.g., Wi et al., J Immunol Methods. 122 (2), 227-34 (1989)).

[0262] In a further embodiment, the combination therapy of the invention further includes administration of a bisphosphonate. Examples of potentially suitable bisphosphonates are pamidronate (Aredia®), zoledronic acid (Zometa®), clodronate (Bonefos®), risedronate (Actonel®), ibandronate (Boniva®), etidronate (Didronel®), alendronate (Fosamax®), tiludronate (Skelid®), incadronate (Yamanouchi Pharmaceutical), and minodronate (YM529, Yamanouchi).

[0263] In a further embodiment, the combination therapy of the present invention further comprises the administration of 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®).

[0264] In a further embodiment, the combination therapy of the invention further comprises the administration of an erythropoiesis agent. Examples of suitable erythropoiesis agents are erythropoietin (EPO) and erythropoiesis-stimulating proteins (e.g., Aranesp®), such as epoetin alfa (e.g., Procrit®, Epogen®, and Eprex®) and epoetin beta (e.g., NeoRecormon®).

[0265] In a further embodiment, the combination therapy of the present invention further includes the administration of an anti-cancer cytokine, chemokine, or a combination 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 may include Glu-Leu-Arg (ELR)-negative chemokines such as IP-10, MCP-3, MIG, and SDF-1α from the human CXC and CC chemokine families. Suitable cytokines include cytokine derivatives, cytokine mutants, cytokine fragments, and cytokine fusion proteins.

[0266] In a further embodiment, the combination therapy of the invention further includes the administration of 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).

[0267] In a further embodiment, the combination therapy of the present invention further includes the administration of a cell cycle control / apoptosis regulator (or "regulatory agent"). Cell cycle control / apoptosis regulators may include (i) molecules that target and regulate cell cycle control / apoptosis regulators such as cdc-25 (e.g., NSC 663284, etc.), (ii) cyclin-dependent kinases that overstimulate the cell cycle (e.g., flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase regulators (e.g., BIBR 1532, SOT-095, GRN163, and compositions described in, for example, U.S. Patent No. 6,440,735 and U.S. Patent 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®).

[0268] In a further embodiment, the combination therapy of the present invention further includes the administration of hormone-modulating agents, such as agents useful in antiandrogen and antiestrogen therapy. Examples of such hormone-modulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / ethinyl, antiandrogens (such as flutamide / eulexin), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone / Provera, megestol acetate / Megase), adrenocorticosteroids (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin), aromatase inhibitors (such as anastrazole / arimidex, aminoglutethimide / citraden, exemestane), hormone inhibitors (such as octreotide / sandostatin), and similar agents.

[0269] In a further embodiment, the combination therapy of the present invention further includes the administration of an anti-anergy agent (e.g., a small molecule compound, protein, glycoprotein, or antibody that breaks tolerance to tumor and cancer antigens). An example of such a compound is MDX-010 (Phan et al., PNAS USA 100 , 8372(2003)) are molecules that block the activity of CTLA-4.

[0270] In a further embodiment, the combination therapy of the present invention further comprises the administration of a nucleic acid or vector containing 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 or deregulated gene, or an siRNA targeted to a mutated or deregulated gene. Examples of tumor suppressor gene targets include, for example, BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1, and DCC.

[0271] In a further embodiment, the combination therapy of the present invention further includes administration of an anti-cancer nucleic acid, such as Genasense (augmerosen / G3139), LY900003 (ISIS 3521), ISIS 2503, OGX-011 (ISIS 112989), LE-AON / LEraf-AON (liposomally 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.

[0272] In a further embodiment, the combination therapy of the present invention further includes administration of 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).

[0273] In further embodiments, the combination therapy of the present invention further includes the administration of viruses, viral proteins, etc. Replication-deficient viruses, which are typically capable of one or only a few rounds of replication in vivo and are targeted to tumor cells, may, for example, be useful components of such compositions and methods. Such viral agents may include or be associated with nucleic acids encoding immune stimulatory substances, such as GM-CSF and / or IL-2. Both natural oncolytic viruses and such recombinant oncolytic viruses (e.g., HSV-1 virus, reovirus, replication-deficient adenovirus, and replication-competent adenovirus, etc.) may be useful components of such methods and compositions (e.g., Shah et al., J Neurooncol. 65 (3), 203-26(2003), Stiles et al., Surgery. 134 (2), 357-64(2003), Sunarmura et al., Pancreas. 28 (3), 326-9(2004), Teshigahara et al., J Surg Oncol. 85 (1), 42-7(2004), Varghese et al., Cancer Gene Ther. 9 (12), 967-78(2002), Wildner et al., Cancer Res. 59 (2), 410-3(1999), Yamanaka, Int J Oncol. 24 (4), 919-23(2004), and Zwiebel et al., Semin Oncol. 28 (4), 336-43 (2001).

[0274] In further embodiments, the combination therapy of the present invention may further involve "whole cell" and "adoptive" immunotherapy methods. For example, such methods involve the use of immune system cells (e.g., CD4 + and / or CD8 +These methods and compositions may include the infusion or reinfusion of tumor-infiltrating lymphocytes (TILs), such as T cells (e.g., T cells expanded with tumor-specific antigens and / or gene augmentation), antibody-expressing B cells or other antibody-producing / presenting cells, dendritic cells (e.g., recombinant dendritic cells expressing anti-cytokines, dendritic cells cultured with DC-expanding 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 contexts include Canvaxin™, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and Melacine® cell lysates. Antigens released from cancer cells, optionally mixed with adjuvants such as alum, and mixtures thereof (see, e.g., Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001), may also be components in such methods and combination compositions.

[0275] In a further embodiment, the combination therapy of the present invention may further include the application of internal vaccination. Internal vaccination refers to the induced death of tumor or cancer cells in a patient, such as drug-induced or radiation-induced tumor cell death, which typically results in the induction 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 may be humoral (i.e., antibody-complement mediated) or cellular (e.g., the generation and / or expansion of endogenous cytotoxic T lymphocytes that recognize internally killed tumor cells or parts thereof).

[0276] In a further embodiment, the combination therapy of the present invention further comprises the administration of complement.Therefore, it is also within the scope of the present invention to use a composition comprising an anti-CD38 antibody together with serum or complement.In these compositions, the complement may be in close proximity to the anti-CD38 antibody, for example, by conjugation, or may be adapted for simultaneous administration.Alternatively, the anti-CD38 antibody and the complement or serum may be administered separately.

[0277] In further embodiments, the combination therapies of the invention further include administration of differentiation-inducing agents, retinoic acid and retinoic acid analogs (such as all-trans retinoic acid, 13-cis retinoic acid, and similar agents), vitamin D analogs (such as seocalcitol and similar agents), ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk2, Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC, c-met, Ron, Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7, and similar agents.

[0278] In further embodiments, the combination therapy of the invention further includes administration of a modulator of cathepsin B, cathepsin D dehydrogenase activity, glutathione-S-transferase (such as glutatylcysteine ​​synthase and lactate dehydrogenase), or similar agent.

[0279] In a further embodiment, the combination therapy of the invention further comprises the administration of estramustine and epirubicin.

[0280] In a further embodiment, the combination therapy of the present invention further includes the administration of an HSP90 inhibitor such as 17-allylaminogeldanamycin, an antibody against a tumor antigen such as PSA, CA125, KSA, etc., an inhibitor of an integrin such as integrin β1, VCAM, or similar agent.

[0281] In further embodiments, the combination therapy of the invention further includes the administration of agents having an effect on cell signaling (such as anti-LFAs), such as calcineurin inhibitors (such as, for example, valspodar, PSC 833, and other MDR-1 or p-glycoprotein inhibitors), TOR inhibitors (such as, for example, sirolimus, everolimus, and rapamycin), and inhibitors of the "lymphocyte homing" mechanism (such as, for example, FTY720), and adhesion molecule inhibitors.

[0282] In a further embodiment, the combination therapy of the present invention further includes radiation therapy.

[0283] Radiotherapy may involve radiation or provide the associated administration of radiopharmaceuticals to patients. The source of radiation may be either external or internal to the patient being treated (radiation treatment may be, for example, in the form of external beam radiation therapy (EBRT), brachytherapy (BT), or skeletal targeted radiation therapy). Radioactive elements that can be used in carrying out 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.

[0284] In a further embodiment, the combination therapy of the present invention further comprises autologous peripheral stem cell transplantation or bone marrow transplantation.

[0285] In a further embodiment, the combination therapy of the present invention further includes orthopedic intervention.

[0286] Orthopedic interventions may be used in the treatment of disorders involving cells expressing CD38, such as multiple myeloma, to help control pain or preserve function or mobility. Such interventions include physical therapy, bone fixation to prevent or treat fractures, or surgery (minor or major) to repair fractures.

[0287] In a further embodiment, the combination therapy of the present invention further includes the delivery of one or more agents that facilitate the access of the CD38 antibody or combination composition to the inside of the tumor. Such a method may be performed, for example, in conjunction with the delivery of relaxin, which can loosen tumor stiffness (see, for example, U.S. Patent No. 6,719,977). In some embodiments, the anti-CD38 antibody used in the present invention may be conjugated to a cell-penetrating peptide (CPP). Cell-penetrating peptides and related peptides (e.g., artificially produced cell-penetrating antibodies) are described, for example, in Zhao et al., J Immunol Methods. 254 (1-2), 137-45(2001), Hong et al., Cancer Res. 60 (23), 6551-6(2000), Lindgren et al., Biochem J. 377 (Pt 1), 69-76(2004), Buerger et al., J Cancer Res Clin Oncol. 129 (12), 669-75(2003), Pooga et al., FASEB J. 12 (1), 67-77(1998), and Tseng et al., Mol Pharmacol. 62 (4), 864-72(2002).

[0288] In a further embodiment, the combination therapy of the present invention further includes administration of at least one anti-inflammatory agent.

[0289] In certain embodiments, such anti-inflammatory agents may be selected from steroid drugs and NSAIDs (non-steroidal anti-inflammatory drugs).

[0290] In some embodiments, such anti-inflammatory agents are selected from the group consisting of aspirin and other salicylates, Cox-2 inhibitors (such as rofecoxib and celecoxib), NSAIDs (such as ibuprofen, fenoprofen, naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac, oxaprozin, and indomethacin), anti-IL6R antibodies, anti-IL8 antibodies (such as 10F8 described in WO 2004 / 058797), anti-IL15 antibodies, anti-IL15R antibodies, anti-CD4 antibodies, anti-CD11a antibodies (such as efalizumab), anti-alpha-4 / beta-1 integrin (V integr ...IL6R antibodies, anti-IL8 antibodies (such as 10F8 described in WO 2004 / 058797), anti-IL15 antibodies, anti-IL15R antibodies, anti-CD4 antibodies, anti-CD11a antibodies (such as efalizumab), anti-IL6R antibodies, anti-IL8 antibodies (such as 10F8 described in WO 2004 / 058797), anti-IL15 antibodies, anti-IL15R L A4) antibodies (e.g., natalizumab), CTLA4-Ig for the treatment of inflammatory diseases, disease modifying antirheumatic drugs (DMARDs) such as prednisolone, prednisone, methotrexate, hydroxychloroquine, sulfasalazine, pyrimidine synthesis inhibitors (e.g., leflunomide), IL-1 receptor blocking agents (e.g., anakinra), TNF-α blocking agents (e.g., etanercept, infliximab, and adalimumab), and similar agents.

[0291] In a further embodiment, the combination therapy of the present invention further comprises administration of at least one immunosuppressive and / or immunomodulatory agent to a subject in need thereof.

[0292] In certain embodiments, 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 similar agents.

[0293] In certain embodiments, such immunosuppressive and / or immunomodulatory agents may be selected from immunosuppressive antibodies, such as antibodies that bind to p75 of the IL-2 receptor, or antibodies that bind to, for example, 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.

[0294] In certain embodiments, 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 (such as antibodies against CTLA4), and similar agents.

[0295] In a further embodiment, the combination therapy of the invention further includes administration of an anti-C3b(i) antibody.

[0296] In further embodiments, the combination therapy of the invention further includes administration of a histone deacetylase inhibitor (eg, phenylbutyrate) and / or a DNA repair agent (eg, DNA repair enzymes and related compositions such as dimelysin).

[0297] In a further embodiment, the combination therapy of the present invention includes anti-cancer directed photodynamic therapy (e.g., optionally performed with the use of a photosensitizing agent) - anti-cancer laser therapy, e.g., Zhang et al., J Control Release. 93 (2), 141-50 (2003)), anti-cancer sound and shock wave therapy (see, for example, Kambe et al., Hum Cell. 10 (1), 87-94 (1997)), and / or anticancer nutritional supplementation treatment (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)).

[0298] Unless otherwise indicated herein or otherwise clearly contradicted by context, all methods described herein can be performed in any suitable order.

[0299] All patents, pending patent applications, and other publications cited herein are incorporated by reference in their entirety.

[0300] This invention is further illustrated by the following examples which should not be construed as further limiting. [Example]

[0301] Example 1 Preparation of luciferase-transfected (Daudi-luc) cells Daudi cells (originating from Burkitt's lymphoma) were cultured in RPMI 1640 medium supplemented with 10% FCS (Optimum C241, Wisent, St. Bruno, QC, Canada), 2 mM L-glutamine, 100 IU / ml penicillin, 100 mg / ml streptomycin, and 1 mM sodium pyruvate (all obtained from Gibco BRL, Life Technologies, Paisley, Scotland). The medium was replenished twice a week. Prior to transfection, cells were split at 1–1.5 × 10 cells per well to ensure viability and optimal growth. 6 cells / ml.

[0302] Luciferase transfection 8.2×10 6 CD38 +Daudi cells were harvested in 350 μl RPMI (supplemented with 10% dFCS, Gibco BRL) and transferred to an electroporation cuvette (Biorad, Hemel Hempstead, Herts, UK). Then, 40 μg gWIZ luciferase from GTS (Aldevron, Fargo, ND, USA) and 10 μg pPur vector (BD Biosciences, Alphen a / d Rijn, The Netherlands), conferring puromycin resistance, were added. After the cells were placed on ice for 10 minutes, they were electroporated (250 V, 950 μF; Gene Pulser II, Biorad Laboratories GmbH, Munchen, Germany). The cells were again placed on ice and harvested in 40 ml RPMI (supplemented with 10% FCS). The cells were then plated in 96-well tissue culture plates (100 μl per well). After 48 hours, puromycin (final concentration: 1 μg / ml; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) was added, and puromycin-resistant clones were further grown in 24-well tissue culture plates.

[0303] Determination of luciferase activity The luciferase activity of the cells was determined using a luciferase assay system (#E4030, Promega, Madison, WI, USA). 5 The cells were centrifuged in an Eppendorf centrifuge (13,500 rpm, 1 min), and the pellet was washed in 100 μl PBS. After centrifugation (13,500 rpm, 1 min), the pellet was lysed in 20 μl reporter lysis buffer (Promega), frozen, and thawed. After centrifugation (13,500 rpm, 1 min), 20 μl of the supernatant was discarded, and 100 μl of luciferase assay reagent (in a special luminometer tube from Promega) was added. Luminescence was measured (10 s) in a luminometer (LB9507, Berthold, Vilvoorde, Belgium).

[0304] Example 2 Immunization of mice and generation of hybridomas Immunization Protocol for -003 HCo12 mice were immunized every two weeks with 20 μg of purified HA-CD38. The initial immunization was performed i.p. in the presence of 100 μl PBS mixed with 100 μl complete Freund's adjuvant (CFA). Following this initial immunization, subsequent boosts (13x) with purified HA-CD38 were performed alternately sc and i.p. in the presence of 100 μl PBS mixed with 100 μl incomplete Freund's adjuvant (IFA). After titer expansion, mice were boosted i.v. with 20 μg HA-CD38 in PBA.

[0305] Immunization Protocol for -005 and -024 HCo12 mice were immunized every two weeks with 20 μg of purified HA-CD38 and NIH-3T3-CD38 transfected cells, alternating between the two. The first immunization consisted of 5 × 10 cells in 100 μl PBS mixed with 100 μl CFA. 6 Immunizations with transfected cells were performed i.p., and second and subsequent immunizations with HA-CD38 were performed sc 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 expansion, mice were boosted i.v. with 20 μg HA-CD38 in PBS.

[0306] Generation of hybridomas producing human monoclonal antibodies against CD38 Mouse splenocytes were isolated from HCo12 mice and fused with a mouse myeloma cell line using PEG according to standard protocols. The resulting hybridomas were then screened for human antibody production by ELISA and for CD38 specificity by FACS analysis using human CD38-transfected NS / 0 cells and by ELISA using recombinant HA-CD38 protein binding. Three hybridoma cell lines expressing human monoclonal anti-CD38 antibodies, -003, -005, and -024, respectively, were selected.

[0307] Example 3 Transfection of NIH cells with CD38 The vector (pclpuroCD38) for generating NIH-3T3-CD38 cells was obtained from Professor M. Glennie (Tenovus Research Laboratory, Southampton General Hospital, Southampton, UK). NIH-3T3 cells (DSMZ, ACC 59; 150,000 cells / well; 0.5 ml; 96-well flat-bottom plates, Greiner) were cultured in DMEM (supplemented with glucose [4.5 g / L], 10% FCS, L-glutamine, and pyruvate-Na; BioWhittaker) for 24 hours. DNA (0.8 μg) and Lipofectamine (Invitrogen, Breda, The Netherlands) were then diluted in DMEM and mixed (20 minutes, room temperature). The mixture (100 μl) was then added to each well and incubated (overnight, 37°C).

[0308] Screening for CD38 expression NIH-3T3-CD38 cells were washed (in 1 ml PBS) and trypsinized (200 μl, trypsin-EDTA, BioWhittaker). Then, 1 ml of DMEM was added, and the mixture was pipetted into a FACS tube. After centrifugation (1200 rpm, 5 min), the cells were washed in FACS buffer (FB; PBS, 0.05% BSA, 0.02% NaN3) and resuspended in 1 ml FB. After centrifugation (1200 rpm, 5 min), the supernatant was removed, and mouse anti-human CD38-PE (1 / 50 dilution, Sanquin, Amsterdam, The Netherlands) was added. The cells were washed twice in FB and then resuspended in FB for flow cytometry.

[0309] Magnification and selection After trypsinization, cells were transferred to T25 flasks (Greiner) in DMEM (supplemented with 4.5 g / L glucose, 2 mM L-glutamine, and puromycin (2 μg / ml) BioWhittaker). After 2 weeks in puromycin-containing medium, puromycin-resistant cells were examined for stable CD38 expression by flow cytometry. NIH-3T3-CD38-selected cells were subcloned by limiting dilution. After expansion of these cells, all 15 NIH-3T3-CD38 clones were screened for CD38 expression. CD38high NIH-3T3-CD38 cells were frozen in liquid nitrogen (-80°C) until use.

[0310] Culturing NIH-3T3-CD38 cells Cells were cultured in DMEM (supplemented with glucose (4.5 g / l), 10% FCS, 2 mM L-glutamine, sodium pyruvate, penicillin, and streptomycin). Cells were passaged twice a week by the use of trypsin / EDTA and cultured at 1 × 10 6 The cells were seeded at a concentration of 100 cells / T75 flask. The CD38high NIH-3T3-CD38 cells were frozen in liquid nitrogen (-80°C) until use.

[0311] Purification of HA-CD38 antigen Sepharose 4B (Amersham Bioscience, Uppsala, Sweden) was coupled with anti-CD38 antibody (Serotec, Oxford, UK). The column (HR5 / 20 column tube packed to a bed height of 12 cm, column volume 2.4 ml; maximum flow rate 0.5 ml / min) was equilibrated with at least 5 column volumes (CV) of PBS. The sample was filtered and loaded onto the column. The column was washed with PBS until the signal returned to baseline (approximately 3 CV). Elution was performed with 0.1 M glycine, pH 2. The eluted fractions were neutralized with 1% (v / v) 2 M Tris-HCl, pH 9.

[0312] Purification of anti-CD38 antibody Human anti-CD38 antibodies were purified from tissue culture supernatants. First, the supernatant was filtered through a 0.20 μM dead-end filter. The supernatant was then loaded onto a 5 ml Protein A column (rProtein A FF, Amersham Bioscience) and eluted with 0.1 M citric acid-NaOH, pH 3. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9, and dialyzed overnight against 12.6 mM sodium phosphate, 140 mM NaCl, pH 7.4 (B. Braun, Oss, The Netherlands). After dialysis, the sample was sterile filtered through a 0.20 μM dead-end filter.

[0313] Purification of His-CD38 batches The protein is present in the cell culture supernatant of His-CD38-expressing cells carrying a DNA construct containing the sequence of the extracellular domain of CD38. An additional poly-His tag sequence is included in the construct and present at the N-terminus of the protein. This tag allows purification by solid-phase metal affinity chromatography. In this process, the chelator immobilized on the chromatography resin binds to Co. 2+ In fact, a sequence containing six histidine amino acids is 2+Therefore, it is thought that the His-tagged CD38 protein binds strongly to such a column, whereas other proteins present in the culture supernatant pass through or are washed away. The strongly bound His-tagged CD38 protein is then purified by the His-CoA method. 2+ Once sufficient His-CD38 has been purified, the eluate is removed from the protein by buffer exchange on a desalting column.

[0314] Example 4 Binding of -003, -005, and -024 to CD38-transfected CHO (CHO-CD38) cells, Daudi-luc cells, and fresh multiple myeloma (MM) tumor cells After harvesting and counting, Daudi-luc cells, CD38-transfected CHO cells, and control CHO cells were resuspended in PBS (1 × 10 6 The cells were then placed in a 96-well V-bottom plate (100 μl / well) and washed twice in PBS-BSA (PBS supplemented with 0.1% BSA and 0.02% azide-Na). Then, 50 μl of antibody solution in PBS-BSA was added to the cells (4°C, 30 min). After three washes in PBS-BSA, 50 μl (1:400 dilution) of rabbit anti-human IgG-FITC antibody solution in PBS-BSA was added (4°C, 30 min in the dark). The cells were washed three times, and specific binding of the CD38 antibody to CHO-CD38 cells and Daudi-luc cells was detected by flow cytometry. HuMab-KLH (a human monoclonal antibody against KLH (keyhole limpet hemocyanin) produced by Genmab BV, Utrecht, The Netherlands, using the immunization protocol described elsewhere herein) was used as a control. Figures 1 and 2 show the binding of different ECs to different ECs. 50 However, we show that -003, -005, and -024 bind to CHO-CD38 cells and Daudi-luc cells (Table 1). No binding was observed to control CHO cells (data not shown).

[0315] Fresh MM tumor cells were obtained from Dr. Lokhorst (University Medical Center Utrecht, Utrecht, The Netherlands). Tumor cells were isolated from the bone marrow of a multiple myeloma patient by Ficoll (BioWhittaker; Lymphocyte Separation Medium, Cat. 17-829E) gradient centrifugation. After harvesting and counting, MM cells (100,000 cells / well) were resuspended in 25 μl FITC-conjugated CD38-specific antibody and 25 μl CD138. After incubation (4°C, 30 min), cells were washed in PBS-BSA, and PE-conjugated goat anti-mouse IgG (1:200; Jackson ImmunoResearch Europe, Soham, UK) was added. After incubation (4°C, 30 min) and washing the cells in PBS-BSA, fluorescence was measured by flow cytometry.

[0316] Figure 3 shows that -003, -005, and -024 bind to MM cells.

[0317] Table 1. EC values ​​of anti-CD38 antibody binding to CHO-CD38 cells, Daudi-luc cells, and fresh MM tumor cells. 50 value TIFF2025143484000004.tif23128

[0318] Example 5 antibody-dependent cell-mediated cytotoxicity Daudi-luc cells, fresh multiple myeloma tumor cells, fresh plasma cell leukemia tumor cells, and JK6L and AMO-1 multiple myeloma cells (5 × 10 6 cells), 100 μCi 51 RPMI supplemented with Cr (chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, The Netherlands) ++The cells were harvested in RPMI 1640 culture medium supplemented with 10% cosmic bovine serum (HyClone, Logan, UT, USA), and the mixture was incubated in a water bath at 37°C for 1 hour. After washing the cells (twice in PBS, 1500 rpm, 5 min), the cells were resuspended in RPMI 1640 culture medium. ++ The cells were resuspended in 1×10 PBS and counted by trypan blue exclusion. 5 The concentration was adjusted to cells / ml.

[0319] Preparation of effector cells Fresh peripheral blood mononuclear cells (healthy volunteers, UMC Utrecht, Utrecht, The Netherlands) were isolated from 40 ml of heparinized blood with Ficoll (BioWhittaker; Lymphocyte Separation Medium, Cat. 17-829E) according to the manufacturer's instructions. ++ After resuspension of cells at 1 x 10, count cells by trypan blue exclusion and 7 The concentration was adjusted to cells / ml.

[0320] ADCC setup 50 μl 51 Cr-labeled target cells were pipetted into a 96-well plate, 50 μl of antibody was added, and RPMI ++ The cells were diluted in 0.1% Triton-X100 (final concentrations: 10, 1, 0.1, 0.01 μg / ml). The cells were incubated (room temperature, 15 min), and 50 μl effector cells were added, resulting in an effector-to-target ratio of 100:1 (for determination of maximum lysis, 100 μl 5% Triton-X100 was added instead of effector cells; for determination of spontaneous lysis, 50 μl target cells and 100 μl RPMI++ were used). The cells were spun down (500 rpm, 5 min) and incubated (37°C, 5% CO2, 4 h). After spinning down the cells (1500 rpm, 5 min), 100 μl of supernatant was collected in a micronic tube and counted in a gamma counter. The percentage specific lysis was calculated as follows: (cpm sample - cpm target cells only) / (cpm maximum lysis - cpm target cells only) where cpm is counts per minute.

[0321] In Daudi-luc cells (Fig. 4 and Table 2), -003, -005, and -024 induce lysis by ADCC, and -003 and -005 perform slightly better than rituximab (an anti-CD20 mAb). Interestingly, when fresh multiple myeloma tumor cells (obtained from Dr. H. Lokhorst, UMCU, The Netherlands) are used as target cells, ADCC is induced by -003, -005, and -024 (Fig. 5A and Table 2).

[0322] Table 2. EC of CD38-specific antibodies obtained by ADCC 50 value TIFF2025143484000005.tif22128

[0323] Enrichment of human peripheral blood mononuclear cells Erlangen Human blood from human volunteers (University Erlangen, Erlangen, Germany) was diluted twice in RPMI 1640, and blood cells were layered on Ficoll (Lymphocyte Separation Medium 1077 g / ml, 710 g, RT, 20 min; BioWhittaker, Cambrex Bio Science Vervier, Verviers, Belgium, Cat. 17-829E, Lot No. 0148 32). Peripheral blood mononuclear cells (MNCs) were collected from the interphase, washed, and resuspended in RPMI 1640 culture medium supplemented with 10% FCS, 2 mM L-glutamine, 5 U / ml penicillin, and 50 μg / ml streptomycin (all from BioWhittaker), to which 25 mM HEPES (BioWhittaker) was added.

[0324] ADCC Setup II Target B cells (fresh plasma cell leukemia tumor cells, JK6L and AMO-1 B cell lines, obtained from Dr. T. Valerius, University of Erlangen, Erlangen, Germany) were administered at 20 μCi 51 After extensive washing in RPMI-10, cells were plated at 1 × 10 5 MNCs (50 μl), sensitizing antibody (50 μl), and RPMI-10 (50 μl) were added to a round-bottom microtiter plate (Greiner Bio-One GmbH, Frickenhausen, Germany). The assay was initiated by adding fresh plasma cell leukemia tumor cells, JK6L, or AMO-1 cells (50 μl) to a final volume of 200 μl. An effector-to-target (E:T) ratio of 40:1 was used. After incubation (3 h, 37°C), the assay was stopped by centrifugation, and triplicate samples were collected. 51 Cr release was measured as counts per minute (cpm) in a scintillation counter. The percentage of cellular cytotoxicity was calculated using the following formula: % specific lysis = (experimental cpm - basal cpm) / (maximum cpm - basal cpm) x 100 The largest 51 Cr release was determined by adding perchloric acid (3% final concentration) to target cells, and basal release was measured in the absence of sensitizing antibody and effector cells.

[0325] In both multiple myeloma cell lines (i.e., JK6L and AMO-1), even when CD38 expression is low (AMO-1 cell line), lysis is induced by both -003 and -005 (Figs. 6 and 7).

[0326] -003, -005, and -024 induce ADCC of primary plasma cell leukemia tumor cells (Fig. 5B).

[0327] Example 6 Complement-dependent cytotoxicity After harvesting and counting Daudi-luc cells, cell viability should be ≥90%. After washing (PBS), cells were collected at 2.0 × 10 6 The cells are then resuspended in RPMI-B (RPMI supplemented with 1% BSA) at 1 x 10 cells / ml. The cells are then plated in a 96-well round-bottom plate at 1 x 10 5 Cells were plated at 50 μl per well. 50 μl of antibody was then added to the wells (final concentrations ranging from 0 to 100 μg / ml (3-fold dilution in RPMI-B)). After incubation (room temperature, 15 min), 11 μl of pooled human serum (pooled 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). 10 μl of propidium iodide (PI; Sigma-Aldrich Chemie BV) was then added to this suspension (10 μg / ml solution). Lysis was detected by flow cytometry (FACScalibur™, Becton Dickinson, San Diego, CA, USA) by measuring the percentage of dead cells (corresponding to PI-positive cells).

[0328] Figure 8 and Table 2 show that lysis of Daudi-luc cells is induced by -005 (~60% maximal lysis), and that lysis by -003 is only seen at very high antibody concentrations. -024 does not induce CDC in Daudi cells (data not shown). In CHO-CD38 cells, lysis is induced by both -003, -005, and -024 (Figure 9 and Table 3). Lysis by -003 is induced at higher concentrations. In tumor cells (all from Drs. Lokhorst and Bloem, University Medical Center Utrecht, The Netherlands) obtained from different MM patients (A: 3% refractory tumor cells, B: 9% refractory tumor cells, C: 30-40% tumor cells, and D: 70% tumor cells), CDC-mediated lysis is observed in the presence of -005 but not -003 (Figure 10). -024 also induced lysis of MM tumor cells (Fig. 10E).

[0329] Table 3. EC of CD38-specific antibodies obtained by CDC 50 value TIFF2025143484000006.tif22128

[0330] Example 7 Cross-blocking study using FACS CHO-CD38 cells were incubated with excess unlabeled CD38-specific antibody (4°C, 15 min). Then, the cells were incubated with FITC-labeled CD38-specific antibody (concentration EC 90 (near 4°C, 45 min). After washing the cells twice with PBS-BSA, fluorescence was measured by flow cytometry. Figure 11 shows that unlabeled -003 blocks the binding of FITC-labeled -003, but not FITC-labeled -005. Also, unlabeled -005 blocks the binding of FITC-labeled -005, but not FITC-labeled -003. Because they do not compete for binding, -003 and -005 bind to different epitopes.

[0331] Example 8 Cross-blocking study using ELISA Soluble human CD38 is coated onto the surface of an ELISA plate. The coated CD38 is incubated with an excess of unlabeled CD38-specific antibody for approximately 15 minutes, after which biotinylated CD38-specific antibody is added (at a concentration of EC 90 After washing three times with PBS / Tween, streptavidin conjugated with horseradish peroxidase (HRP) is added and the mixture is incubated for 1 hour at room temperature. The complex can be detected by adding ABTS solution, and HRP-mediated substrate conversion is measured at OD 405 nm using an ELISA reader.

[0332] Example 9 Cross-blocking study using sandwich ELISA CD38-specific antibodies are coated onto the surface of ELISA plates. The plate-bound antibodies are incubated with biotinylated soluble CD38 in the presence of excess CD38-specific antibodies in the fluid phase. After washing with PBS / Tween, bound biotinylated CD38 is detected with HRP-conjugated streptavidin for 1 hour at RT. The complex can be detected by adding ABTS solution (after washing with PBS / Tween), and HRP-mediated substrate conversion is measured at OD 405 nm using an ELISA reader.

[0333] Example 10 Immunohistochemical reactivity with a panel of human tissues and cross-reactivity with cynomolgus monkey tissues Sections from frozen human tissue (obtained from Dr. H. Niessen, Free University Medical Center, Amsterdam, The Netherlands) and monkey tissue (Inveresk Research, Glasgow, Scotland) were cut at 6 μm and air-dried overnight. These cryostat sections were fixed in acetone (RT, 10 min) and air-dried (approximately 5 min). The sections were then incubated with 1x citrate / phosphate buffer containing 0.1% H2O2 (pH 5.8; Sigma) to block endogenous peroxidase. After 20 min at RT, the sections were washed twice with PBS and 0.05% Tween-20 (PBST, RT, 5 min; Riedel de Haen, Germany). The sections were then incubated with avidin (RT, 15 min; DAKO, Glostrup, Denmark), washed twice with PBST, and incubated with biotin to block endogenous biotin (RT, 15 min; DAKO). The sections were washed twice with PBST and then resuspended in PBST. ++ The sections were preincubated (RT, 20 min) with PBST supplemented with 10% normal human serum (NHS, CLB, Amsterdam, Netherlands) and 10% normal goat serum (NGS; DAKO). After blotting, the sections were resuspended in 2% PBST. ++The sections were then incubated with the indicated concentrations of FITC-labeled primary antibodies diluted in 2% PBST (RT, 60 min). ++ The sections were incubated with rabbit anti-FITC (1:1000; DAKO) in PBST (RT, 30 min). After washing with PBST, the sections were resuspended in 2% PBST. ++ The sections were then incubated with goat anti-rabbit biotin (1:400; DAKO) in 2% PBST (RT, 30 min). ++ The sections were incubated with SABC-HRP (1:100; DAKO) in PBST for 30 min at room temperature. After washing the sections twice in PBST, they were incubated with amino-ethyl-carbazole (AEC) developing solution (50 mM acetate buffer, pH 4.9, 0.01% HO; Riedel-de-Haen) for 10 min at room temperature. Finally, the sections were washed in Millipore HO for 5 min and counterstained with hematoxylin (DAKO). The sections were mounted with a coverslip using glycerol gel at 37°C and examined under a light microscope (Axiovision-2; Zeiss, Thornwood, NY, USA).

[0334] Not only striated muscle (myocytes, Figures 12C and 13C), macrophages, lymphocytes, and plasma B cells (Figures 12A and 13A), but also tracheal epithelium stained with -003 and -005 (Figures 12B and 13B). -024 had similar, but less intense, staining of striated muscle and tracheal epithelium. Endothelial cell staining was not observed with either -003 (Figure 14D), -005 (Figure 14E), or -024 (data not shown), whereas clear staining was observed with positive control antibodies against the endothelial cell markers CD31 (Figure 14A) and vWF (Figure 14B). Anti-KLH was used as a negative control antibody (Figure 14C). -003 (Fig. 12D) and -024 (data not shown) cross-react with cynomolgus monkey lymphoid tissue, but -005 (Fig. 13D) does not.

[0335] Example 11 Cross-reactivity with cynomolgus or rhesus monkey peripheral blood mononuclear cells (PBMCs) by flow cytometry Five ml of cynomolgus monkey peripheral blood (Inveresk Research) was lysed by adding 4.5 ml shock buffer (1.7 mM NH4CL, 1 mM EDTA), 40 ml H2O, and 450 μl 10% KHCO3. After hemolysis, cells were centrifuged (1200 rpm, 10 min) and washed three times in PBS. After counting cells with trypan blue, cells were resuspended in PBS-BSA (1 × 10 6 cells / ml).

[0336] 17.5 ml of rhesus peripheral blood (BPRC, Rijswijk, The Netherlands) was diluted 1:1 with RPMI 1640 and layered on Ficoll (1.077 g / ml; BioWhittaker, Cat. 17-829E, Lot No. 0148 32). After centrifugation (710 g, RT, 20 min), the interphase was collected and washed twice in RPMI. After the final wash, cells were collected at 1 × 10 5 The cells were resuspended in RPMI 1640 at a concentration of cells / 50 μl.

[0337] Cells were transferred to a 96-well plate (100,000 PBMCs / well), washed in FACS buffer (PBS, 0.05% BSA, 0.02% NaN3), and incubated with primary antibodies (4°C, 30 min). After washing in PBS-BSA, 50 μl of FITC-labeled rb anti-hIgG (DAKO, Glostrup, Denmark) was added (4°C, 30 min). Finally, cells were collected in a total volume of 150 μl into FACS tubes. Samples were measured and analyzed using a FACScalibur™ (Becton Dickinson, San Diego, CA, USA).

[0338] Flow cytometry demonstrated cross-reactivity of -003, but not -005, with cynomolgus monkey lymphocytes (Figure 15A) and monocytes (Figure 15B). Cross-reactivity of -003, but not -005, was also observed with rhesus monkey mononuclear cells (Figure 15C).

[0339] Example 12 Internalization experiments CHO-CD38 cells were stained with a saturating concentration of FITC-conjugated CD38-specific antibody (on ice for 30 min). After washing the cells (in RPMI 1640 supplemented with 10% FCS), one cell pool was warmed to 37°C to allow for internalization, while the other pool was left on ice. At intervals of several minutes (0–120 min), cell aliquots were removed and transferred to ice-cold PBS-BSA to stop internalization. After washing the samples twice with PBS-BSA, EtBr (diluted in PBS-BSA, final concentration 2 mg / ml) was added to the samples to quench membrane-bound FITC. Fluorescence was measured by flow cytometry.

[0340] Figures 16A and 16B show that -003 and -005 are internalized by CHO-CD38 cells within 5 minutes at 37°C.

[0341] Example 13 In vivo SCID luciferase experiments In this model, tumor cells are transfected with firefly luciferase. Upon administration of luciferin (Molecular Probes, Leiden, The Netherlands) to mice, labeled cells can be detected in vivo by bioluminescence imaging using a sensitive CCD camera. Wetterwald et al., American Journal of Pathology 160 (3), 1143-1153 (2002).

[0342] Daudi cells were transfected with gWIZ luciferase from Gene Therapy Systems (San Diego, CA) and cultured in RPMI containing 10% FCS, Pen / Strep, sodium pyruvate, and 1 μg / ml puromycin (Sigma). Cells were analyzed by luminometer for luciferase expression (expressed as RLU / 1 × 10 cells) and by FACS for CD38 cells. 2.5 × 106 Luciferase-transfected Daudi cells / mouse were injected intravenously into SCID mice. Mice were treated with -003, -005, an isotype control antibody (HuMab-KLH), or rituximab (anti-CD20 antibody). Antibodies were injected intraperitoneally. Four treatment settings were used (see Table 4). In the preventive setting, antibody (100 μg / mouse) and cells were administered simultaneously. In treatment setting I, antibody (300 μg / mouse) was administered 7 days after cell administration. In treatment setting II, antibody (10 μg / mouse) was administered 14 days after cell administration. In treatment setting III, antibody (100 μg / mouse) was administered 7 days after cell administration. For imaging, mice were anesthetized with an i.p. injection of a mixture of ketamine, xylazine, and atropine. Synthetic D-luciferin (sodium salt, Molecular Probes) was administered intravenously at a dose of 25 mg / ml. Mice were then placed in a light, rigid box, and imaging began 3 minutes later using a liquid nitrogen-cooled CCD detector, VersArray 1300B (Roper Scientific). Luciferase photons were counted over a 5-minute exposure period. A black-and-white image was generated under illumination for reference. MetaVue software (Universal Imaging) was used for data collection and image analysis. One-way analysis of variance with Newman-Keuls post-hoc test was used with GraphPad PRISM version 3.02 (Graphpad Software) to confirm the statistical significance of differences between groups.

[0343] Table 4. Treatment settings for in vivo luciferase experiments TIFF2025143484000007.tif36128

[0344] Figures 17A and 17B show that -003 and -005 inhibit tumor cell growth in prophylactic and therapeutic settings I, similar to the inhibition observed with anti-CD20 antibodies. Both antibodies perform significantly better than isotype control antibodies. Also, in therapeutic setting II, CD38 antibodies slow the growth of Daudi-luc tumor cells (Figure 17C). In therapeutic setting III, -003 and -024 show clear inhibition of Daudi-luc tumor cell growth (Figure 17D).

[0345] Example 14 apoptosis Apoptosis assays were performed according to the manufacturer's instructions (Annexin-V Apoptosis Kit, BD Biosciences, Alphen ad Rijn, Netherlands). Briefly, CD38 mAb was added at a concentration of 5 μg / ml -003 or -005 or anti-CD20 antibody, alone or in the presence of cross-blocking rb-anti-hIgG (50 μg / ml), to 2.5 × 10 5 Cells (luciferase-transfected Daudi cells, 0.5 ml RPMI ++ (in a 24-well plate containing

[0346] After incubation (37°C, 5% CO2, 20 hours), the cells were carefully harvested and washed with binding buffer (1200 rpm, 4°C, 5 minutes, BD Biosciences). The pellet was resuspended in 100 μl binding buffer. 5 μl Annexin-V-FITC (BD Biosciences) and 10 μl PI (BD Biosciences) were then added to the suspension and incubated for 15 minutes at room temperature. 400 μl binding buffer was added, and the sample was measured (PI reading in FL2). For the analysis of apoptotic cells, all Annexin-V-positive cells were counted by flow cytometry using a FACScalibur flow cytometer with CellQuest Pro software (BD Biosciences). At least 10,000 events were collected for analysis. This population included both PI-positive and PI-negative cells.

[0347] Figure 18 shows that -003 and -005 do not induce apoptosis. However, apoptosis of target cells is observed after cross-linking. After cross-linking, -003 induced apoptosis similar to that induced by an anti-CD20 antibody (rituximab). After cross-linking, -005 was less able to induce apoptosis. Similar results were obtained when RAMOS cells were used as target cells (data not shown).

[0348] Example 15 Effect of -005 on tissue graft B cells in the RA-SCID mouse model Synovial tissue transplantation SCID mice, strain CB-17 / lcrCrI-SCID-bg, male / female, 4-12 weeks old, purchased from Charles River Laboratories Nederland (Maastricht, the Netherlands), were housed in IVC cages under standard temperature and lighting conditions and provided with laboratory chow and water ad libitum. Before transplantation, mice (three mice per experimental group, day 0) were anesthetized with an intraperitoneal injection of ketamine (NIMATEK, EuroVet) and xylazine (Rompun, Bayer) in a 1:1 ratio. A small incision was made in the skin using surgical scissors. Inflamed synovial tissue from a patient with rheumatoid arthritis undergoing joint replacement surgery was placed in six small pieces (2-3 mm in total) on each flank of the mice. 3 The mice were implanted subcutaneously as a mass of 1000 mg / kg of 10 ...

[0349] immunohistochemistry 5 μM frozen sections on SuperFrost (Menzel GmbH, Braunschweig) slides were prepared using a LEICA CM1900 cryostat and stored at -80°C. Thawed sections were fixed in acetone for 10 min, dried at room temperature, and washed in PBS for 3 × 5 min. All steps were performed at room temperature. Endogenous peroxidase activity was blocked by incubation with PBS supplemented with 0.3% hydrogen peroxide and 0.1% sodium azide for 20 min. Slides were washed 3 × 5 min in PBS and then incubated with 10% normal human serum (NHS) / 10% normal rabbit serum (NRbS) in PBS / 1% BSA for 30 min. Primary antibody (mouse mAb) diluted in PBS supplemented with 1% BSA / 10% NHS / 10% NRbS was then incubated for 60 min. After 3 × 2 min washes in PBS, HRP conjugate (goat anti-mouse Ig-HRP; DAKO P0447) diluted 1:50 in PBS (supplemented with 1% BSA / 10% NHS / 10% NRbS) was added for 30 min. The peroxidase signal was enhanced using the TSA™ Biotin System (Perkin Elmer Life Sciences, NEL700). Slides were washed 3 × 2 min in PBS and incubated with biotinyl tyramide diluted 1:1600 in amplification buffer for 30 min. After 3 × 2 min washes in PBS, streptavidin-HRP diluted 1:400 in PBS (supplemented with 1% BSA) was added for 30 min. Slides were washed 3 × 2 min in PBS and incubated with DAB solution (DAKO Cytomation K3465) for 5 min. The color reaction was stopped with distilled water. Finally, the slides were counterstained with hematoxylin (MERCK), washed in running water, and covered with Kaiser's glycerin and coverslips.

[0350] Scoring staining intensity Scoring of the stained synovial tissue xenografts was performed blindly by two trained individuals. The most intense section was first selected from a series of sections, and this reference section was given the maximum score of 8. The staining intensity of the other sections was then scored on a scale of 0 to 8 compared to the reference section.

[0351] statistical analysis Staining intensity scoring was analyzed by Kruskal-Wallis one-way ANOVA followed by Dunn's multiple comparison test using Graph Pad Prism version 4.01 (Graph Pad software, San Diego, CA, USA).

[0352] Figures 19 and 21 show that the number of anti-CD38 positive plasma cells is reduced after treatment with -005. Staining of plasma cells with anti-CD138 confirms that -005 results in a reduction in plasma cell numbers (Figures 20 and 22).

[0353] Example 16 Sequencing of the coding sequence of a human antibody against CD38 RNA preparation Total RNA was collected at 5 × 10 expressing monoclonal antibodies -003, -005, and -024, respectively, using an RNeasy kit (Qiagen, Westburg, Leusden, Netherlands) according to the manufacturer's protocol. 6 The cells were prepared from a hybridoma cell line.

[0354] cDNA preparation of -003, -005, and -024 5′ RACE of RNA Complementary DNA (cDNA) was prepared from 100 ng total RNA using the SMART RACE cDNA Amplification Kit (Clontech) according to the manufacturer's protocol.

[0355] Oligonucleotide primers were synthesized and quantified by Isogen Bioscience (Maarssen, The Netherlands). Primers were dissolved in HO to 100 pmol / μl and stored at -20°C. A summary of all PCR and sequencing primers is listed (Table 5). For PCR, PfuTurbo® Hotstart DNA polymerase (Stratagene, Amsterdam, The Netherlands; product #600322) was used according to the manufacturer's instructions. Each reaction mixture contained 200 μM mixed dNTPs (Roche Diagnostics, Almere, The Netherlands; product #1814362), 12 pmol of reverse primer (V) in a total volume of 30 μl containing PCR reaction buffer (supplied with the polymerase). H RACEG1A1, V for 3003-005 H RACEV for 3003-003 H ApaI and V L RACEV for 3003-003 and -005 L The PCR mixture contained 7.2 pmol of UPM-mixture (UPM-mixture: 2 μM short UPMH3 and 0.4 μM long UPMH3), 0.6 μl of 5' RACE cDNA template, and 1.5 units of PfuTurbo® Hotstart DNA polymerase. PCR reactions were performed in a TGradient Thermocycler 96 (Whatman Biometra, Goettingen, Germany; product # 050-801) using a 35-cycle program: denaturation at 95°C for 2 minutes; 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1.5 minutes; and a final extension at 72°C for 10 minutes. Where appropriate, the PCR mixture was stored at 4°C until further analysis or processing.

[0356] (Table 5) Primers TIFF2025143484000008.tif97150

[0357] pGEMT-Vector System II-003-2F5 VH and V L and -005 V L and -024 V H and V L Cloning The reaction products were separated by electrophoresis on a 1% TAE agarose gel and stained with ethidium bromide. Bands of the correct size were excised from the gel and DNA was isolated from the agarose using a Qiaex II gel extraction kit (Qiagen, catalog number 20021).

[0358] Gel-isolated PCR fragments were A-tailed by incubation with 200 μM dATP and 2.5 units of Amplitaq (Perkin Elmer) for 10 minutes at 72°C and purified using a minielute column (Qiagen). The A-tailed PCR fragments were cloned into the pGEMTeasy vector (Promega) using the pGEMT easy Vector System II kit and protocol (LJ270, page 3 / 4). 2 μl of the ligation mixture was transformed into OneShot DH5αT1R competent E. coli (Invitrogen) and plated on LB / Amp / IPTG / Xgal plates.

[0359] Sequence analysis 20(V H -003), 16(V L -003), 15(V L White colonies of 6 (VH and VL-024), 6 (VH and VL-005), and 6 (VH and VL-024) were picked, and the plasmids were isolated and sequenced with M13 reverse primers. The V regions of 6 (VH and VL-003) and 6 (VL-024) were then amplified by AGOWA (Berlin, Germany). L The region was sequenced. H The region was sequenced directly on the PCR product using primer HCseq5. Sequences were analyzed using the Vector NTI improved suite (Invitrogen).

[0360] Generation of expression vectors for antibodies -003, -005, -024, and Morphosys antibody 3079 -003 V H The coding region was cloned into VH3003-003 using primers VH3003-003for and RACEVHApaI, which introduced suitable restriction sites (HindIII and ApaI) and an ideal Kozak sequence (GCCGCCACC) for cloning into pConG1f0.4 (Lonza Biologics, Slough, UK). H The pConG1f0.4 vector contains the heavy chain constant region of human IgG1. H The PCR fragment was inserted in frame into the pConG1f0.4 vector using HindIII and ApaI. The construct was verified by sequence analysis.

[0361] -005 V H The coding region was cloned into the VH3005 vector using primers VH3003-5for and RACEVHApaI, which introduced suitable restriction sites (HindIII and ApaI) and an ideal Kozak sequence for cloning into pConG1f0.4. H The region was amplified by PCR from a pGemT plasmid clone containing the V H The PCR fragment was inserted in frame into the pConG1f0.4 vector using HindIII and ApaI. The construct was verified by sequence analysis.

[0362] -024 V H The coding region was cloned into the VH300324 vector using primers VH300324exfor and RACEVHApaI, which introduced suitable restriction sites (HindIII and ApaI) and an ideal Kozak sequence for cloning into pConG1f0.4. H The region was amplified by PCR from a pGemT plasmid clone containing the V H The PCR fragment was inserted in frame into the pConG1f0.4 vector using HindIII and ApaI. The construct was verified by sequence analysis.

[0363] Morphosys Antibody 3079 V H The coding region was synthesized by GeneArt (Regensburg, Germany) based on the data published in Patent Publication WO 2005 / 103083 A2. The coding region was codon-optimized for expression in HEK cells to improve expression levels, and suitable restriction sites (HindIII and ApaI) and an ideal Kozak sequence were introduced for cloning into pConG1f0.4. The plasmid containing the synthetic VH region was digested with ApaI and HindIII, and the VH fragment was inserted in frame into the pConG1f0.4 vector.

[0364] -005 V L The coding region was cloned into pConKappa0.4 (Lonza Biologics) using primers VL3003-5exfor and RACEVLBsiWI, which introduced suitable restriction sites (HindIII and Pfl23II) and an ideal Kozak sequence for cloning into VL3003-5exfor and RACEVLBsiWI. L The region was amplified by PCR from a pGemT plasmid clone containing the kappa light chain constant region. The pConKappa0.4 vector contains the kappa light chain constant region. L The PCR fragment was inserted in frame into the pConKappa0.4 vector using HindIII and Pfl23II. The construct was verified by sequence analysis.

[0365] -003 V L The coding region was cloned into pConKappa0.4 using primers VL3003-003for and RACEVLBsiWI, which introduced suitable restriction sites (HindIII and Pfl23II) and an ideal Kozak sequence for cloning into pConKappa0.4. L The region was amplified by PCR from a pGemT plasmid clone containing the V L The PCR fragment was inserted in frame into the pConKappa0.4 vector using HindIII and Pfl23II. The construct was verified by sequence analysis.

[0366] -024 V L The coding region was cloned into the V of -024 using primers VL3003-24-5exfor and RACEVLBsiWI, which introduced suitable restriction sites (HindIII and Pfl23II) and an ideal Kozak sequence for cloning into pConKappa0.4. L The region was amplified by PCR from a pGemT plasmid clone containing the V L The PCR fragment was inserted in frame into the pConKappa0.4 vector using HindIII and Pfl23II. The construct was verified by sequence analysis.

[0367] Based on the data published in patent application WO 2005 / 103083, GeneArt has determined that Morphosys antibody 3079 V L The coding region was synthesized. To improve expression levels, the coding region was codon-optimized for expression in HEK cells and included suitable restriction sites (HindIII and Pfl23II) and an ideal Kozak sequence for cloning into pConKappa0.4. Synthetic V L The plasmid containing the region was digested with Pfl23II and HindIII and the VH fragment was inserted in frame into the pConKappa0.4 vector.

[0368] The antibodies were transiently expressed in HEK-293F cells as described in Example 17 by co-transfecting their heavy and light chain vectors.

[0369] Generation of stable cell lines in CHO-K1SV cells For stable cell line generation, the -003 or -005 heavy and light chain vectors were combined into one two-gene vector by standard cloning techniques.

[0370] The -003 or -005 dual gene vector was linearized and transfected into CHO-K1SV (Lonza Biologics) cells essentially as described by the manufacturer. Stable cell lines were selected by selection with 25 μM L-methionine sulfoximine (MSX) as described by Lonza Biologics. The highest producing clones were selected and propagated in CD-CHO (Invitrogen) medium, and antibodies were purified from the cell culture medium as described in Example 3.

[0371] Example 17 Epitope mapping using site-directed mutagenesis Oligonucleotide primers were synthesized and quantified by Isogen Bioscience (Maarssen, The Netherlands). Primers were dissolved in HO to 100 pmol / μl and stored at -20°C. A summary of all PCR and sequencing primers is shown in Table 6. For PCR, PfuTurbo® Hotstart DNA polymerase (Stratagene, Amsterdam, The Netherlands) was used according to the manufacturer's instructions. Each reaction mixture contained 200 μM mixed dNTPs (Roche Diagnostics, Almere, The Netherlands), 10 pmol of both forward and reverse primers, 100 ng of genomic DNA or 1 ng of plasmid DNA, and 1 unit of PfuTurbo® Hotstart DNA polymerase in a total volume of 20 μl of PCR reaction buffer (supplemented with polymerase). PCR reactions were performed in a TGradient Thermocycler 96 (Whatman Biometra, Goettingen, Germany) using a 32-cycle program: denaturation at 95°C for 2 min; 30 cycles of 95°C for 30 s, a 60-70°C gradient (or another specified annealing temperature) for 30 s, and 72°C for 3 min; and a final extension at 72°C for 10 min. Where appropriate, PCR mixtures were stored at 4°C until further analysis or processing.

[0372] Agarose gel electrophoresis was performed using 50 ml of gel in 1x Tris-acetate-EDTA buffer according to Sambrook (Sambrook, Russell et al. 2000). DNA was visualized by including ethidium bromide in the gel and viewing under UV light. Gel images were recorded with a CCD camera and an image analysis system (GeneGnome; Syngene, via Westburg BV, Leusden, The Netherlands).

[0373] Purification of the desired PCR fragment was performed using the MinElute PCR Purification Kit (Qiagen, via Westburg, Leusden, The Netherlands; product # 28006) according to the manufacturer's instructions. The isolated DNA was quantified by UV spectroscopy (see below) and its quality was assessed by agarose gel electrophoresis.

[0374] Alternatively, PCR or digestion products (e.g., if multiple fragments were present) were separated by agarose electrophoresis using a 1% Tris-acetate-EDTA agarose gel. The desired fragments were excised from the gel and recovered using a QIAEX II Gel Extraction Kit (Qiagen; product # 20051) according to the manufacturer's instructions.

[0375] The optical density of nucleic acids was determined using a NanoDrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands) according to the manufacturer's instructions. DNA concentrations were determined using a 260 nm (1 OD 260 nm The concentration was measured by analyzing the optical density (OD) in units of 50 μg / ml. For all samples, the buffer in which the nucleic acids were dissolved was used as a reference.

[0376] Restriction enzymes and supplements were obtained from New England Biolabs (Beverly, MA, USA) or Fermetas (Vilnius, Lithuania) and used according to the manufacturer's instructions. DNA (100 ng) was digested with 5 units of enzyme in a final volume of 10 μl of the appropriate buffer (reaction volume was scaled up where appropriate). Digestions were incubated for a minimum of 60 min at the recommended temperature. For fragments requiring double digestion with restriction enzymes with incompatible buffer or temperature requirements, digestions were performed sequentially. When necessary, digestion products were purified by agarose gel electrophoresis and gel extraction.

[0377] Ligation of DNA fragments was performed using a Quick Ligation Kit (New England Biolabs) according to the manufacturer's instructions. For each ligation, vector DNA was mixed with approximately a three-fold molar excess of insert DNA.

[0378] Plasmid DNA (1–5 μl of DNA solution, typically 2 μl of DNA ligation mixture) was ligated into One Shot DH5α-T1 using the heat shock method according to the manufacturer's instructions. R The vector was transformed into Escherichia coli cells (Invitrogen, Breda, The Netherlands; product # 12297-016). Cells were then plated onto Luria-Bertani (LB) agar plates containing 50 μg / ml ampicillin. Plates were incubated at 37°C for 16-18 hours until bacterial colonies were evident.

[0379] Bacterial colonies were screened for the presence of vectors containing the desired sequences by colony PCR using ThermoStart PCR Master Mix (Abgene, via Wetsburg, Leusden, The Netherlands; product # AB-938-DC15 / b) and primers pConG1seq1 and pEE13.4seqrev2 (Table 6). Selected colonies were lightly touched with a 20 μl pipette tip and briefly introduced into 2 ml LB for small-scale culture, then resuspended in the PCR mixture. PCR was performed in a TGradient Thermocycler 96 using a 35-cycle program: denaturation at 95°C for 15 minutes; 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 2 minutes; followed by a final extension step of 72°C for 10 minutes. Where appropriate, PCR mixtures were stored at 4°C until analysis by agarose gel electrophoresis.

[0380] Plasmid DNA was isolated from E. coli cultures using the following kits from Qiagen (via Westburg, Leusden, The Netherlands) according to the manufacturer's instructions: For bulk plasmid preparations (50-150 ml cultures), either the HiSpeed ​​Plasmid Maxi Kit (product # 12663) or the HiSpeed ​​Plasmid Midi Kit (product # 12643) was used. For small-scale plasmid preparations (±2 ml cultures), the Qiaprep Spin Miniprep Kit (product # 27106) was used, and DNA was eluted in 50 μl elution buffer (supplied in the kit).

[0381] Construction of HA-CD38 expression vector pEE13.4HACD38 The extracellular domain of human CD38 was amplified from the plasmid pCIpuroCD38 (obtained from Professor M. Glennie, Tenovus Research Laboratory, Southampton General Hospital, Southampton, UK) using primers cd38forha and cd38exrev. This PCR reaction introduced an HA-tag. This PCR product was used as a template for a second PCR reaction using primers SPHMM38ex and cd38exrev. This PCR reaction introduced the signal peptide SPHMM, restriction sites, and an ideal Kozak sequence (GCCGCCACC) for optimal expression. After purification, this PCR fragment was cloned into the expression vector pEE13.4 (Lonza Biologics), and the complete coding sequence was confirmed by sequencing using primers pConKseq1, pEE13.4seqrev, cd38seq1for, and cd38seq2rev (Table 6). This construct was designated pEE13.4HACD38.

[0382] Site-directed mutagenesis Three single mutant proteins of huCD38 were constructed: a T to A mutation at position 237 (T237A, SEQ ID NO: 32), a Q to R mutation at position 272 (Q272R, SEQ ID NO: 33), or an S to F mutation at position 274 (S274F, SEQ ID NO: 34). Site-directed mutagenesis was performed using the QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, The Netherlands) according to the manufacturer's instructions. This method involves the introduction of a silent extra restriction site or the loss of a restriction site (an extra Xba1 site for the T237A mutant, an extra Bcg1 site for the Q272R mutant, and the loss of an Ssp1 site for the S274F mutant) to screen for successful mutagenesis. Briefly, 5 μl of 10× reaction buffer, 1 μl of oligonucleotide HACD38T237Afor2, HACD38Q272Rfor, or HACD38S274Ffor (100 pmol / μl), 1 μl of oligonucleotide HACD38T237Arev2, HACD38Q272Rrev, or HACD38S274Frev (100 pmol / μl), 1 μl of dNTP mix, 3 μl of Quick solution, 1 μl of plasmid pEE13.4HACD38 (50 ng / μl), and 1 μl of PfuUltra HF DNA polymerase were mixed in a total volume of 50 μl and subjected to an 18-cycle program in a TGradient Thermocycler 96 (Whatman Biometra, Goettingen, Germany; product # 050-801): 1 min denaturation at 95°C; 18 cycles of 95°C for 1 min; Amplification was performed using a PCR primer set at 37°C for 50 seconds, 60°C for 50 seconds, and 68°C for 10 minutes. The PCR mixture was stored at 4°C until further processing. The PCR mixture was then incubated with 1 μl DpnI at 37°C for 60 minutes to digest the pEE13.4HACD38 WT vector and stored at 4°C until further processing.The reaction mixture was precipitated with 5 μl of 3 M NaAc and 125 μl of ethanol, incubated at -20°C for 20 min, and spun down at 14,000 × g for 20 min at 4°C. The DNA pellet was washed with 70% ethanol, dried, and dissolved in 4 μl of water. A total reaction volume of 4 μl was prepared using One Shot Top 10DH5αT1 according to the manufacturer's instructions (Invitrogen). R The mutants were transformed into competent E. coli cells (Invitrogen, Breda, The Netherlands). Cells were then plated on Luria-Bertani (LB) agar plates containing 50 μg / ml ampicillin and incubated at 37°C for 16–18 hours until bacterial colonies were evident. Colonies were screened by colony PCR using primers pConG1seq1 and pEE13.4seqrev2 (Table 5) and digested with the relevant restriction enzymes to screen for incorporation of the mutagenic oligonucleotide. Two positive clones for each mutant were grown and plasmid DNA was isolated. The complete HACD38 coding sequence was determined using primers cd38seq1for, pConG1seq1, and pEE13.4seqrev2 to confirm the presence of the mutation and the absence of additional unwanted mutations.

[0383] DNA sequencing Plasmid DNA samples were sent to AGOWA (Berlin, Germany) for sequence analysis, and sequences were analyzed using VectorNTI modified software (Informax, Oxford, UK).

[0384] Transient expression in HEK-293F cells Freestyle™ 293-F (HEK-293 subclone adapted to growth in suspension and chemically defined Freestyle medium, (HEK-293F)) cells were obtained from Invitrogen and transfected with pEE13.4HACD38 and three constructs carrying the mutations T237A, Q272R, and S274F using 293fectin (Invitrogen) according to the manufacturer's protocol. Culture supernatants from transfected cells were used for anti-CD38 binding studies by ELISA.

[0385] Anti-CD38 antibody binding 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, applied to the ELISA plates, and incubated at RT for 1 h. After washing, serial dilutions of HuMab -003 and -005 were added and incubated at RT for 1 h. Bound antibodies were 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.

[0386] As can be seen from Figures 23A-23C, both -003 and -005 bind to wild-type human CD38. The binding of -003 was not affected by the introduction of the mutations T237A (Figure 23A), Q272R (Figure 23B), and S274F (Figure 23C). -005 was able to bind to CD38 with the mutation T237A (Figure 23A). The binding of -005 to CD38 with the mutation Q272R was significantly reduced by EC 50 Both the binding capacity and maximum binding capacity were significantly affected (Figure 23B). -005 was unable to bind to a mutant CD38 in which the serine at position 274 was replaced by phenylalanine (Figure 23C).

[0387] These data indicate that -003 and -005 bind to different epitopes. Furthermore, these studies revealed that binding of -005 to CD38 is sensitive to mutations at positions 272 and 274. In particular, S274 is essential for -005 binding to CD38.

[0388] Table 6 Primers TIFF2025143484000009.tif100149

[0389] Example 18 Induction of PBMC proliferation -003, -005, and -024 were synthesized essentially as described in Ausiello et al., Tissue antigens 56 , 538-547 (2000). Briefly, PBMCs from healthy donors were diluted in 200 μl RPMI ++ 1 × 10 cells / well in a flat-bottom 96-well plate in the presence of antibodies (final concentrations: 1.1-3.3-10-30 μg / ml) 5 Cells were cultured at 1000 x g / well. Stimulation of cells with IL-15 (333 ng / ml; Amgen, Thousand Oaks, CA, USA) was used as a positive control. After 4 days of incubation at 37°C, 30 μl of IL-15 was added to the wells. 3 H-thymidine (16.7 μCi / ml) was added and the culture was continued O / N. 3 H-thymidine incorporation was assessed using a Packard Cobra gamma counter (Packard Instruments, Meriden, DT, USA) according to the manufacturer's instructions. Data are presented as the mean cpm (±SEM) of PBMCs obtained from 10 donors. The results show that -003 and -005 did not induce significant PBMC proliferation (Figure 24A). Also, -024 did not induce significant PBMC proliferation (data not shown).

[0390] Example 19 IL-6 induction -003, -005, and -024 were compared with those described in Ausiello et al., Tissue antigens 56 , 538-547 (2000). Briefly, PBMCs were cultured in 500 μl RPMI ++ 1 × 10 cells in a 48-well plate in the presence of 20 μg / ml of antibody and 10 ng / ml LPS (Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands). 6 Cells / well were cultured. After O / N incubation at 37°C, supernatants were collected and stored at -20°C. IL-6 concentrations were assessed by ELISA (IL-6 ELISA kit, U-CyTech Biosciences, Utrecht, The Netherlands) according to the manufacturer's instructions. Data are shown as the mean concentration in pg / ml (±SEM) from seven donors. The results show that -003 and -005 did not induce significant IL-6 release (Figure 24B). Also, -024 did not induce significant IL-6 release (data not shown).

[0391] Example 20 Induction of IFN-γ release -003, -005, and -024 were compared with those described in Ausiello et al., Tissue antigens 56 , 538-547 (2000). Briefly, PBMCs were cultured in 500 μl RPMI ++ 1 × 10 cells in a 48-well plate in the presence of 20 μg / ml of antibody and 1 μg / ml OKT-3 (Sanquin, Amsterdam, The Netherlands). 6Cells / well were cultured. After O / N incubation at 37°C, supernatants were collected and stored at -20°C. IFN-γ concentrations were assessed by ELISA (IFN-γ ELISA kit, U-CyTech Biosciences, Utrecht, The Netherlands) according to the manufacturer's instructions. Data are presented as the mean concentration in pg / ml (±SEM) from nine donors. The results show that -003 and -005 did not induce the release of detectable IFN-γ levels (Figure 24C). Also, -024 did not induce significant IFN-γ release (data not shown).

[0392] Example 21 Binding affinity of -003 and -005 to recombinant CD38 The binding of -003 and -005 to CD38 was examined using surface plasmon resonance. Briefly, purified antibodies were immobilized onto a CM-5 sensor chip (Biacore, Uppsala, Sweden) via amine coupling. HA-tagged CD38 (see Example 3) was flown over the chip, and binding of the antigen to the mAb was detected by changes in refractive index at the surface of the chip using a Biacore 3000 (Biacore). The association and rate constants for -003 (Table 7) and -005 (Table 8) are summarized below as the mean ± SD of three experiments, indicating that both -003 and -005 have high affinity for CD38.

[0393] Table 7: Association constants and rate constants at 25°C TIFF2025143484000010.tif29128

[0394] Table 8: Association constants and rate constants at 25°C TIFF2025143484000011.tif29128

[0395] Example 22 Epitope mapping Epitope mapping using the PEPSCAN method Following published procedures (Geysen et al. 1984. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc Natl Acad Sci USA 81:3998; Slootstra et al. 1996. Structural aspects of antibody-antigen interaction revealed through small random peptide libraries. Mol Divers 1:87; Puijk et al. 2001. Segment synthesis. In PCT, The Netherlands, p.1), overlapping 20-mer linear peptides and 15-mer loop peptides covering the C-terminal 138 amino acids of human CD38 were synthesized. Furthermore, based on the C-terminal sequence, regions TIFF2025143484000012.tif5128 area TIFF2025143484000013.tif4128 and area We generated one loop peptide of different sizes covering TIFF2025143484000014.tif4128. Additionally, we designed an extra set of TIFF2025143484000015.tif4128 and The double loop region consisting of TIFF2025143484000016.tif5128 was reconstructed. The native cysteines were replaced with alanines. The peptides were screened by ELISA assay using the mini-PEPSCAN card in credit card format.

[0396] Peptide synthesis Peptides were synthesized using standard Fmoc chemistry and deprotected using TFA containing scavengers. The deprotected peptides were then reacted on the microarray with a 0.5 mM solution of 2,6-bis(bromomethyl)pyridine or 2,4,6-tris(bromomethyl)mesitylene in ammonium bicarbonate (20 mM, pH 7.9) supplemented with acetonitrile (1:1 [vol / vol]). The microarray was gently shaken in the solution while completely covered by the solution for 30–60 min. Finally, the microarray was thoroughly washed with excess Millipore HO and sonicated for 30 min at 70 °C in disruption buffer containing 1% sodium dodecyl sulfate and 0.1% β-mercaptoethanol in PBS (pH 7.2), followed by an additional 45 min in Millipore HO.

[0397] PEPSCAN ELISA-Assay 455-well credit-card format polyethylene cards containing covalently linked peptides were incubated (overnight at 4°C) with serum (e.g., diluted 1:1000 in blocking solution containing 5% horse serum [vol / vol] and 5% ovalbumin [wt / vol]). After washing, the peptides were incubated with rabbit anti-human Ig peroxidase (1:1000 dilution, 25°C, 1 h), and after washing, peroxidase substrate (2,2'-azino-di-3-ethylbenzthiazoline sulfonate and 2 μl / ml 3% HO) was added. After 1 h, color development was measured using a CCD camera and image processing system. The setup consisted of a CCD camera with a 55 mm lens (Sony CCD Video Camera XC-77RR, Nikon micro-nikkor 55 mm f / 2.8 lens), a camera adapter (Sony Camera adapter DC-77RR), and the Image Processing Software package Optimas, version 6.5 (Media Cybernetics, Silver Spring, MD 20910, USA; Optimas runs on a Pentium II computer system).

[0398] Methods for epitope display Individual amino acids were identified by dipeptide motifs, which represent the smallest unique unit in the human CD38 amino acid sequence. All dipeptide motifs present in each of the 1164 peptides tested were assigned the ELISA value obtained for the entire peptide. To rank dipeptide motifs from strongest to weakest binding, the relative signal was calculated by dividing the ELISA value obtained for each individual motif by the average ELISA value from all 1164 tested linear and loop peptides, and these were sorted for decreasing values. In this way, amino acid contributions to conformational epitopes were taken into account. For each mAb tested, dipeptide motifs with a score above 2.5 were selected (i.e., the ELISA value of peptides containing these motifs was at least 2.5 times the average ELISA value obtained for all 1164 peptides). The data were deconvoluted to obtain a single amino acid contribution that was displayed on the linear CD38 sequence by a scoring system. By walking along the linear CD38 sequence and using unique dipeptide units as reference points, one point was assigned each time a CD38 amino acid was present in this set of high-scoring peptides.

[0399] -003, -005, and -024 were all found to bind to the regions SKRNIQFSCKNIYR and EKVQTLEAWVIHGG of human CD38. -003 specifically recognized the motifs RNIQF and WVIH, while -005 specifically recognized the motifs KRN and VQTL.

[0400] Example 23 Enzyme activity The enzymatic activity of human CD38 was measured in an assay essentially as described by Graeff et al., J. Biol. Chem. 269, 30260-30267 (1994). Briefly, the substrate NGD +(80 μM) was incubated with CD38 (0.6 μg / ml of His-tagged human CD38 extracellular domain; see Example 3 for purification of His-CD38) in a buffer containing 20 mM Tris-HCl, pH 7.0. 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.

[0401] To examine the effects of -003, -005, and -024 on the enzymatic activity of CD38, we used the substrate NGD + Recombinant His-CD38 protein was preincubated with different antibodies at various concentrations (30, 3, 0.3, and 0.03 μg / ml) for 15 min at room temperature before the addition of antibodies. The production of cyclic GDP-ribose (cGDPR) was recorded at different time points (3, 6, 9, 12, 30, 45, 60, 75, and 90 min) after the addition of antibodies.

[0402] Figure 25B shows that -005 has a significant inhibitory effect on cGDPR production. After 90 minutes, addition of 30 and 3 μg / ml -005 resulted in a 32% and 34% decrease in cGDPR production (Table 9). Similar results were observed in independent experiments using different batches of -005.

[0403] No inhibitory effect on cGPDR production was observed after addition of -003 (Figure 25B, Table 9), -024 (Figure 25D, Table 9), or anti-KLH (Figure 25A, Table 9).

[0404] Based on these findings, -005 is a NAD + It is also expected to inhibit the synthesis of cyclic ADP-ribose (cADPR) from ATP. Inhibition of cADPR synthesis can be determined according to the HPLC method described in Munshi et al., J. Biol. Chem. 275, 21566-21571 (2000).

[0405] Table 9. cGDP-ribose production in the presence of CD38-specific antibodies or anti-KLH TIFF2025143484000017.tif29128

[0406] Example 24 Comparison of -003 and -005 with Morphosys antibody 3079 Antibodies -003 and -005 were functionally compared to Morphosys antibody 3079 (TH-3079). Methods for cloning and expression of Morphosys antibody TH-3079 are described in Example 16. Methods for CDC are described in Example 6. Methods for ADCC are described in Example 5. Figure 26A shows that -005, -003, and TH-3079 induce CDC-mediated lysis of CD38-transfected CHO cells, with similar maximal lysis. EC 50 When comparing values, the -005 antibody was better than TH3079 at inducing lysis of CHO-CD38 cells, with a 2-fold lower EC 50 (See Table 10)

[0407] Figure 26B shows that -005 is more potent than TH-3079 at inducing CDC-mediated lysis of Daudi-luciferase cells, with maximal lysis by -005 being 2-3 fold higher than that by TH3079. 50 When the values ​​were compared, the -005 antibody was similar to TH-3079 in inducing lysis of Daudi-luciferase cells (see Table 10). -003 did not induce significant CDC-mediated lysis of Daudi-luciferase cells.

[0408] Figure 26C shows that in this experiment, -005, -003, and TH-3079 mediate lysis of Daudi target cells via ADCC. 50 No differences were found in log lysis and maximum lysis (Table 11, n=5).

[0409] Table 10. Maximum lysis and EC50 values ​​of CD38-specific antibodies in CDC TIFF2025143484000018.tif42160

[0410] Table 11. Maximum lysis and EC of CD38-specific antibodies in ADCC 50 value TIFF2025143484000019.tif30155

[0411] Example 25 Inhibition of cell-expressed CD38 enzyme activity The enzymatic activity of human CD38 expressed in cells was measured in an assay essentially as described by Graeff et al., J. Biol. Chem. 269, 30260-30267 (1994). Briefly, the substrate NGD (80 μM) was added to 10 cells of human CD38-transfected cells in a buffer containing 20 mM Tris-HCl, pH 7.0, supplemented with 30 μg / ml IgG1. 5 The cGDPR was incubated with CHO cells (CHO-CD38 cells). The production of cGDPR 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.

[0412] To examine the effects of -005 and -003 on the enzymatic activity of cell-expressed CD38, CHO-CD38 cells were preincubated with different antibodies at various concentrations (30, 3, 0.3, and 0.03 μg / ml) for 15 min at room temperature before the addition of the substrate NGD. cGDPR production was recorded at different time points (3, 6, 9, 12, 30, 45, 60, 112, and 156 min) after the addition of the substrate NGD.

[0413] After 156 minutes, addition of 30 and 3 μg / ml of -005 resulted in a 21% and 18% reduction in cGDPR production. No inhibitory effect on cGDPR production was observed after addition of -003 or the IgG1 control antibody (Table 12).

[0414] Table 12. cGDP-ribose production in the presence of CD38-specific antibodies or IgG1 control TIFF2025143484000020.tif25128

[0415] Example 26 Binding of antibody -005 to EBV-transformed chimpanzee B cells After harvesting and counting, EBV-transformed chimpanzee B cells (received from the Biomedical Primate Research Centre, Department of Immunobiology, Rijswijk, The Netherlands) were resuspended (1 × 10 ) in PBS-BSA (PBS supplemented with 0.1% BSA and 0.02% azide-Na). 6 cells / ml). The cells were then placed in a 96-well V-bottom plate (100 μl / well) and washed twice in PBS-BSA. Then, 50 μl of FITC-labeled -005 antibody solution in PBS-BSA was added to the cells (4°C, 30 minutes). The cells were washed three times, and the specific binding of -005 to EBV-transformed chimpanzee B cells was detected by flow cytometry. FITC-labeled HuMab-KLH (a human monoclonal antibody against KLH (keyhole limpet hemocyanin) produced by Genmab BV, Utrecht, The Netherlands, using the immunization protocol described elsewhere herein) was used as a control. Figure 27 shows the dose-dependent binding of -005 to EBV-transformed chimpanzee B cells. Dose-dependent binding to EBV-transformed chimpanzee B cells was not observed with the control antibody HuMab-KLH.

[0416] Example 27 In vitro combination therapy of antibody-005 with dexamethasone and bortezomib Antibody -005 was tested for its ability to induce cell death of the multiple myeloma cell line UM6 in vitro in a triple combination setting with dexamethasone (Dex) and bortezomib (Bor; Velcade®). Results of the triple treatment were compared with single drug and double combination treatments.

[0417] 3×10 5 UM6 cells were incubated overnight at 37°C with medium alone, Dex (20 μM), Bor (15 pM), or a combination of Bor and Dex. After 23 hours, -005 (10 μg / ml) was added; and 15 minutes later, normal human serum was added, and the samples were incubated for an additional 45 minutes at 37°C. Finally, 10 μl of propidium iodide (PI; Sigma-Aldrich Chemie BV; 10 μg / ml) was added, and cell lysis was detected by flow cytometry using a FACS Calibur™ (Becton Dickinson) by measuring the percentage of PI-positive cells.

[0418] As can be seen in Figure 28, the triple treatment exceeded the lysis observed with either the single or double combination treatments. This effect was observed in two independent experiments.

[0419] Example 28 Patients with a clinical diagnosis of multiple myeloma are treated with a combination of anti-CD38 antibody -005, melphalan, and prednisone.

[0420] The compounds are administered to patients according to the following dosing schedule: - Antibody-005: 8 mg / kg IV once weekly for 4 weeks - Melphalan: 0.2 mg / kg IV daily for 4 days every 4 to 6 weeks -Prednisone: 2 mg / kg PO for 4 days every 4 to 6 weeks.

[0421] Response is determined by a decrease in M-protein in serum, a decrease in the number of plasma cells in bone marrow, and a decrease in Benze-Jones protein in urine, as well as a reduction / absence of new osteolytic bone lesions.

[0422] Example 29 Patients with a clinical diagnosis of multiple myeloma are treated with a combination of the anti-CD38 antibody -005, thalidomide, and dexamethasone.

[0423] The compounds are administered to patients according to the following dosing schedule: - Antibody-005: 8 mg / kg IV once weekly for 4 weeks - Thalidomide: 200 mg / day (PO) -Dexamethasone: 40 mg / day (PO) on days 1-4, 9-12, and 17-20 of each 28-day cycle.

[0424] Response is determined by a decrease in M-protein in serum, a decrease in the number of plasma cells in bone marrow, and a decrease in Benze-Jones protein in urine, as well as a reduction / absence of new osteolytic bone lesions.

[0425] Example 30 Patients with a clinical diagnosis of multiple myeloma are treated with a combination of the anti-CD38 antibody -005, lenalidomide, and dexamethasone.

[0426] The compounds are administered to patients according to the following dosing schedule: - Antibody-005: 8 mg / kg IV once weekly for 4 weeks - Lenalidomide: 25 mg / day (PO) -Dexamethasone: 40 mg / day (PO) on days 1-4, 9-12, and 17-20 of each 28-day cycle.

[0427] Response is determined by a decrease in M-protein in serum, a decrease in the number of plasma cells in bone marrow, and a decrease in Benze-Jones protein in urine, as well as a reduction / absence of new osteolytic bone lesions.

[0428] Example 31 Patients with a clinical diagnosis of multiple myeloma are treated with a combination of the anti-CD38 antibody -005, bortezomib, and dexamethasone.

[0429] The compounds are administered to patients according to the following dosing schedule: - Antibody-005: 8 mg / kg IV once weekly for 4 weeks - Bortezomib: 1.3 mg / m2 (IV) on days 1, 4, 6, and 11 of each 21-day cycle -Dexamethasone: 40 mg / day (PO) on days 1-4, 9-12, and 17-20 of each 28-day cycle.

[0430] Response is determined by a decrease in M-protein in serum, a decrease in the number of plasma cells in bone marrow, and a decrease in Benze-Jones protein in urine, as well as a reduction / absence of new osteolytic bone lesions.

[0431] Sequence information SEQUENCE LISTING <110> Genmab A / S <120> Combination treatment of CD38-expressing tumors <150> DK PA 2006 01232 <151> 2006-09-26 <150> US 60 / 847,329 <151> 2006-09-26 <160> 67 <170> PatentIn version 3.5 <210> 1 <211> 321 <212> DNA <213> homo sapiens <400> 1 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120 gagaaagccc ctaagtccct gatctatgct gcttccagtt tgcaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgccacag tataatagtt accctcggac gttcggccaa 300 321 - slowly slowly <210> 2 <211> 107 <212> PRT <213> homo sapiens <400> 2 Asp With Gln Met Thr Gln Serving Pro Serving Leu Serving Ala Serving Val Gly 1 5 10 15 Asp Arg Val Thr With Thr Cys Arg Is Served With Gln Gly And Served With Trp 20 25 30 Leu Wing Trp Tyr Gln Gln Lys Pro Glu Lys Wing Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Ser Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 11 <212> PRT <213> homo sapiens <400> 3 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 4 <211> 7 <212> PRT <213> homo sapiens <400> 4 Ala Ala Dear Dear Leu Gln Dear 1 5 <210> 5 <211> 9 <212> PRT <213> homo sapiens <400> 5 Gln Gln Tyr Asn Ser Tyr Pro Arg Thr 1 5 <210> 6 <211> 366 <212> DNA <213> homo sapiens <400> 6 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc agctatgctt tcagctgggt gcgacaggcc 120 cctggacaag gacttgagtg gatgggaagg gtcatccctt tccttggtat agcaaactcc 180 gcacagaaat tccaggggcag agtcacaatt accgcggaca aatccacgag cacagcctac 240 atggacctga gcagcctgag atctgaggac acggccgtat attactgtgc gagagatgat 300 atagcagcac ttggtccttt tgactactgg ggccaggaa cctgtcac cgtctcctca 360 gcctcc 366 <210> 7 <211> 122 <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 Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Val Ile Pro Phe Leu Gly Ile Ala Asn Ser Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Only Asp Lys Sert Thr Thr On Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Asp Ile Ala Ala Leu Gly Pro Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 <210> 8 <211> 5 <212> PRT <213> homo sapiens <400> 8 Ser Tyr Ala Phe Ser 1 5 <210> 9 <211> 17 <212> PRT <213> homo sapiens <400> 9 Arg Val Ile Pro Phe Leu Gly Ile Ala Asn Ser Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 10 <211> 11 <212> PRT <213> homo sapiens <400> 10 Asp Asp Ile Ala Ala Leu Gly Pro Phe Asp Tyr 1 5 10 <210> 11 <211> 321 <212> DNA <213> homo sapiens <400> 11 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120 ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag ctagagcct 240 gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctccgac gttcggccaa 300 gggaccaagg tggaaatcaa a 321 <210> 12 <211> 107 <212> PRT <213> homo sapiens <400> 12 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 13 <211> 11 <212> PRT <213> homo sapiens <400> 13 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 14 <211> 7 <212> PRT <213> homo sapiens <400> 14 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 15 <211> 10 <212> PRT <213> homo sapiens <400> 15 Gln Gln Arg Ser Asn Trp Pro Pro Thr Phe 1 5 10 <210> 16 <211> 372 <212> DNA <213> homo sapiens <400> 16 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggc cctgagactc 60 tcatgtgcag tctctggatt cacctttaac agctttgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtgg cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat atttctgtgc gaaagataag 300 attctctggt tcggggagcc cgtctttgac tactggggcc agggaaccct ggtcaccgtc 360 tcctcagcct cc 372 <210> 17 <211> 124 <212> PRT <213> homo sapiens <400> 17 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 <210> 18 <211> 5 <212> PRT <213> homo sapiens <400> 18 Ser Phe Ala Met Ser 1 5 <210> 19 <211> 17 <212> PRT <213> homo sapiens <400> 19 Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 20 <211> 13 <212> PRT <213> homo sapiens <400> 20 Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr 1 5 10 <210> 21 <211> 321 <212> DNA <213> homo sapiens <400> 21 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120 ggccaggctc ccgggctcct catctatgat gcttccaaca gggcctctgg catcccagcc 180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag ctagagcct 240 gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctctcac ttcggcgga 300 gggaccaagg tggagatcaa a 321 <210> 22 <211> 107 <212> PRT <213> homo sapiens <400> 22 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Gly Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 23 <211> 11 <212> PRT <213> homo sapiens <400> 23 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 24 <211> 7 <212> PRT <213> homo sapiens <400> 24 Asp Ala Ser Asn Arg Ala Ser 1 5 <210> 25 <211> 9 <212> PRT <213> homo sapiens <400> 25 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 26 <211> 366 <212> DNA <213> homo sapiens <400> 26 gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60 tcctgtaagg gttctggata cagctttttcc aactactgga tcggctgggt gcgccagatg 120 cccgggaaag gcctggagtg gatggggatc atctatcctc atgactctga tgccagatac 180 agcccgtcct tccaaggcca ggtcaccttc tcagccgaca agtccatcag caccgcctac 240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc gagacatgta 300 gggtggggat cgcggtactg gtacttcgat ctctggggcc gtggcaccct ggtcactgtc 360 tcctca 366 <210> 27 <211> 122 <212> PRT <213> homo sapiens <400> 27 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Asn Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro His Asp Ser Asp Ala Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Phe Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Val Gly Trp Gly Ser Arg Tyr Trp Tyr Phe Asp Leu Trp 100 105 110 Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 28 <211> 5 <212> PRT <213> homo sapiens <400> 28 Asn Tyr Trp Ile Gly 1 5 <210> 29 <211> 17 <212> PRT <213> homo sapiens <400> 29 Ile Ile Tyr Pro His Asp Ser Asp Ala Arg Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly <210> 30 <211> 13 <212> PRT <213> homo sapiens <400> 30 His Val Gly Trp Gly Ser Arg Tyr Trp Tyr Phe Asp Leu 1 5 10 <210> 31 <211> 300 <212> PRT <213> homo sapiens <400> 31 Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys 1 5 10 15 Arg Leu Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu Val 20 25 30 Leu Ile Leu Val Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln 35 40 45 Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu 50 55 60 Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val 65 70 75 80 Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser Lys 85 90 95 His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys Leu 100 105 110 Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp Ser Arg Ile 115 120 125 Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg Asp Met Phe Thr 130 135 140 Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp ...

Claims

1. for individuals i) a non-agonistic antibody that binds to CD38; ii) at least one corticosteroid, and iii) at least one non-corticosteroid chemotherapy agent including the administration of A method for inhibiting the growth and / or proliferation of tumor cells expressing CD38 in an individual in need thereof.

2. for individuals i) a non-agonistic antibody that binds to CD38; ii) optionally at least one corticosteroid, and iii) optionally at least one non-corticosteroid chemotherapy agent administration of followed by autologous peripheral stem cell transplantation or bone marrow transplantation, A method for treating cancer involving tumor cells that express CD38 in an individual in need thereof.

3. 3. The method of claim 1 or 2, wherein the at least one non-corticosteroid chemotherapeutic agent comprises a cytotoxic agent and / or an angiogenesis inhibitor.

4. 10. The method of any one of the preceding claims, wherein the at least one non-corticosteroid chemotherapeutic agent comprises an alkylating agent.

5. 10. The method of any one of the preceding claims, wherein the at least one non-corticosteroid chemotherapeutic agent comprises one or more agents selected from the group consisting of melphalan, mechlorethamine, thioepa, chlorambucil, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin.

6. 2. The method of any one of the preceding claims, wherein the at least one non-corticosteroid chemotherapeutic agent comprises a glutamic acid derivative such as thalidomide (Thalomid®) or a thalidomide analogue, e.g., CC-5013 (lenalidomide, Revlimid™) or CC4047 (Actimid™).

7. 10. The method of any one of the preceding claims, wherein the at least one non-corticosteroid chemotherapeutic agent comprises a proteasome inhibitor, such as bortezomib (Velcade®).

8. 10. The method of any one of the preceding claims, wherein the at least one non-corticosteroid chemotherapeutic agent comprises a vinca alkaloid, such as vincristine.

9. 10. The method of any one of the preceding claims, wherein the at least one non-corticosteroid chemotherapeutic agent comprises an anthracycline, such as doxorubicin.

10. 10. The method of any one of the preceding claims, wherein the at least one corticosteroid comprises a glucocorticoid.

11. 10. The method of any one of the preceding claims, wherein the at least one corticosteroid comprises prednisone.

12. 10. The method of any one of the preceding claims, wherein the at least one corticosteroid comprises prednisone and the at least one non-corticosteroid chemotherapeutic agent comprises melphalan.

13. 10. The method of any one of the preceding claims, wherein the at least one corticosteroid comprises prednisone and the at least one non-corticosteroid chemotherapeutic agent comprises thalidomide.

14. 10. The method of any one of the preceding claims, wherein the at least one corticosteroid comprises prednisone and the at least one non-corticosteroid chemotherapeutic agent comprises melphalan and thalidomide.

15. 10. The method of any one of the preceding claims, wherein the at least one corticosteroid comprises dexamethasone.

16. 10. The method of any one of the preceding claims, wherein the at least one corticosteroid comprises dexamethasone and the at least one non-corticosteroid chemotherapeutic agent comprises thalidomide and / or lenalidomide.

17. 10. The method of any one of the preceding claims, wherein the at least one corticosteroid comprises dexamethasone and the at least one non-corticosteroid chemotherapeutic agent comprises vincristine and / or doxorubicin.

18. 10. The method of any of the preceding claims, comprising the further administration of interferon-alpha.

19. The method of any of the preceding claims, wherein the antibody is a monoclonal antibody.

20. The method of any of the preceding claims, wherein the antibody is a human monoclonal antibody.

21. The method of any of the preceding claims, wherein the antibody is an antagonist of CD38.

22. The method of any one of the preceding claims, wherein the antibody does not induce significant IL-6 release by human monocytes or peripheral blood mononuclear cells, as determined by the method described in Example 19 of the present specification.

23. The method of any one of the preceding claims, wherein the antibody does not induce detectable release of IFN-γ by human T cells or peripheral blood mononuclear cells, as determined by the method described in Example 20 of the present specification.

24. The method of any one of the preceding claims, wherein the antibody is an antibody that is internalized by CD38-expressing cells, for example an antibody that is internalized by CHO-CD38 cells within 5 to 15 minutes at 37°C by the method described in Example 12 of the present specification.

25. The antibody is an antibody that induces ADCC, e.g., an EC200 of less than 15 ng / ml, e.g., less than 10 ng / ml, on Daudi-luc cells, as determined, e.g., by the method described in Example 5 herein. 50 values ​​and an EC of less than 75 ng / ml in MM cells, e.g., less than 50 ng / ml, 30 ng / ml, or 10 ng / ml 50 The method of any of the preceding claims, wherein the antibody is an antibody having a value.

26. The antibody is an antibody that induces CDC in the presence of complement and has an EC of less than 5 μg / ml, e.g., less than 1 μg / ml, on daudi-luc or CD38-CHO cells, e.g., by the method described in Example 6 herein. 50 The method of any of the preceding claims, wherein the antibody is an antibody having a value.

27. The method of any of the preceding claims, wherein the antibody is an antibody that inhibits the synthesis of cGDPR.

28. The method of any one of the preceding claims, wherein the antibody is an antibody that inhibits the synthesis of cADPR.

29. The antibody has a 10 -8 Less than M, e.g. 10 -8 M to 10 -11 Within the range of M, e.g., 7 x 10 -9 M to 10 -10 Affinity (K D The method of any one of the preceding claims, wherein the antibody binds to human CD38 at the

30. 10. The method of any one of the preceding claims, wherein the antibody inhibits the synthesis of cGDPR by at least 25%, such as at least 30%, after 90 minutes at a concentration of 3 μg / ml, as determined by the spectrophotometric method described in Example 24 herein.

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

32. The antibody comprises a V having the sequence shown in SEQ ID NO:

10. H The method of any one of the preceding claims, wherein the antibody is an antibody comprising a CDR3 or an antibody that competes with the antibody for CD38 binding, e.g., by binding to the same epitope as the antibody.

33. The antibody comprises a V having the sequence shown in SEQ ID NO:5 L CDR3 and V having the sequence shown in SEQ ID NO: 10 H The method of any one of the preceding claims, wherein the antibody comprises a CDR3.

34. the antibody comprises a human light chain and a human heavy chain variable region, The light chain variable region has the sequence shown in SEQ ID NO:

3. L CDR1, V having the sequence shown in SEQ ID NO: 4 L CDR2, and V having the sequence shown in SEQ ID NO: 5 L CDR3 and a heavy chain variable region having the sequence set forth in SEQ ID NO: 8 H CDR1, V having the sequence shown in SEQ ID NO: 9 H CDR2, and V having the sequence shown in SEQ ID NO: 10 H an antibody comprising CDR3, 10. The method of any one of the preceding claims.

35. The antibody has the amino acid sequence shown in SEQ ID NO:2 L region, or a V region having at least about 90%, e.g., at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO:

2. L The method of any one of claims 32 to 34, wherein the antibody comprises a region.

36. The antibody has the amino acid sequence shown in SEQ ID NO: 7 H a region, or a V region having at least about 90%, e.g., at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO: 7 H region, or V having 1 to 5, for example 1 to 3 amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO:

7. H The method of any one of claims 32 to 35, wherein the antibody comprises a region.

37. The antibody comprises a V having the sequence shown in SEQ ID NO: 20 H 32. The method of any one of claims 1 to 31, wherein the antibody is an antibody comprising a CDR3 or an antibody that competes with the antibody for CD38 binding, e.g., by binding to the same epitope as the antibody.

38. The antibody comprises a V having the sequence shown in SEQ ID NO:

15. L CDR3 and V having the sequence shown in SEQ ID NO: 20 H The method of any one of claims 1 to 31, wherein the antibody comprises a CDR3.

39. the antibody comprises a human light chain and a human heavy chain variable region, The light chain variable region has the sequence shown in SEQ ID NO:

13. L CDR1, V having the sequence shown in SEQ ID NO: 14 L CDR2, and V having the sequence shown in SEQ ID NO: 15 L CDR3 and a heavy chain variable region having the sequence set forth in SEQ ID NO: 18 H CDR1, V having the sequence shown in SEQ ID NO: 19 H CDR2, and V having the sequence shown in SEQ ID NO: 20 H an antibody comprising CDR3, 32. The method of any one of claims 1 to 31.

40. The antibody has the amino acid sequence shown in SEQ ID NO: 12 L region, or a V having at least about 90%, for example at least about 95%, amino acid sequence identity with a sequence according to SEQ ID NO:

12. L The method of any one of claims 37 to 39, wherein the antibody comprises a region.

41. The antibody has the amino acid sequence shown in SEQ ID NO:

17. H a region, or a V region having at least about 90%, e.g., at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO: 17 H region, or V having 1 to 5, for example 1 to 3 amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO:

17. H The method of any one of claims 37 to 40, wherein the antibody comprises a region.

42. The antibody comprises a V having the sequence shown in SEQ ID NO: 30 H 32. The method of any one of claims 1 to 31, wherein the antibody is an antibody comprising a CDR3 or an antibody that competes with the antibody for CD38 binding, e.g., by binding to the same epitope as the antibody.

43. The antibody comprises a V having the sequence shown in SEQ ID NO:

25. L CDR3 and V having the sequence shown in SEQ ID NO: 30 H The method of any one of claims 1 to 31, wherein the antibody comprises a CDR3.

44. the antibody comprises a human light chain and a human heavy chain variable region, The light chain variable region has the sequence shown in SEQ ID NO:

23. L CDR1, V having the sequence shown in SEQ ID NO: 24 L CDR2, and V having the sequence shown in SEQ ID NO: 25 L CDR3 and a heavy chain variable region having the sequence set forth in SEQ ID NO: 28 H CDR1, V having the sequence shown in SEQ ID NO: 29 H CDR2, and V having the sequence shown in SEQ ID NO: 30 H an antibody comprising CDR3, 32. The method of any one of claims 1 to 31.

45. The antibody has the amino acid sequence shown in SEQ ID NO: 22 L region, or a V having at least about 90%, for example at least about 95%, amino acid sequence identity with a sequence according to SEQ ID NO:

22. L The method of any one of claims 42 to 44, wherein the antibody comprises a region.

46. The antibody has the amino acid sequence shown in SEQ ID NO: 27 H region, or a V region having at least about 90%, e.g., at least about 95%, amino acid sequence identity with the sequence set forth in SEQ ID NO:

27. H region, or V having 1 to 5, for example 1 to 3 amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO:

27. H The method of any one of claims 42 to 45, wherein the antibody comprises a region.

47. 10. The method of any one of the preceding claims, wherein the antibody 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.

48. The antibody, (i) Human Hv1263 / 3M28(V H I) a heavy chain variable region amino acid sequence derived from a germline sequence and a light chain variable region amino acid sequence derived from a human L15 (VκI) germline sequence; or (ii) Human V H 3-DP-47 / V3-23(V H III) Heavy chain variable region amino acid sequence derived from germline sequence and light chain variable region amino acid sequence derived from human L6 (VκI) germline sequence is a human monoclonal antibody comprising 10. The method of any one of the preceding claims.

49. The method of any of the preceding claims, wherein the antibody is an antibody fragment or a single chain antibody.

50. The method of any one of the preceding claims, wherein the antibody is conjugated to a cytotoxic agent, a radioisotope, or a drug.

51. The antibody, An antibody as defined in any one of the preceding claims, and Binding specificity to human effector cells is a bispecific or multispecific molecule comprising 10. The method of any one of the preceding claims.

52. The antibody, An antibody as defined in any one of the preceding claims, and Binding specificity for CD3, CD4, CD138, IL-15R, membrane-bound or receptor-bound TNF-α, human Fc receptor, or membrane-bound or receptor-bound IL-15 is a bispecific or multispecific molecule comprising 10. The method of any one of the preceding claims.

53. The method of any of the preceding claims, wherein the tumor cells are multiple myeloma cells or chronic lymphocytic leukemia cells.

54. 10. The method of any of the preceding claims, wherein the tumor cells are relapsed or refractory tumor cells.

55. 10. The method of any of the preceding claims, wherein the individual is 65 years of age or older.

56. 55. The method of any one of claims 1 to 54, wherein the individual is under 65 years of age.

57. 10. The method of any of the preceding claims, wherein the individual has not received prior anti-cancer treatment for the same cancer.

58. 57. The method of any one of claims 1 to 56, wherein the individual has not responded to a prior anti-cancer treatment for the same cancer.

59. 60. The method of any one of claims 1-56 or 58, wherein the individual has previously undergone an autologous peripheral stem cell transplant or bone marrow transplant.

60. 60. The method of any one of claims 1 or 3-59, wherein the individual is enrolled to undergo a subsequent autologous peripheral stem cell transplant or bone marrow transplant.

61. 10. The method of any one of the preceding claims, wherein the antibody, the at least one corticosteroid, and the at least one non-corticosteroid chemotherapeutic agent are administered simultaneously.

62. 61. The method of any one of claims 1-60, wherein the antibody, the at least one corticosteroid, and the at least one non-corticosteroid chemotherapeutic agent are administered sequentially.

63. 10. The method of any one of the preceding claims, wherein the antibody, the at least one corticosteroid, and the at least one non-corticosteroid chemotherapeutic agent are all administered separately.

64. 61. The method of any one of claims 1-60, wherein the antibody, the at least one corticosteroid, and the at least one non-corticosteroid chemotherapeutic agent are co-administered as one or two pharmaceutical compositions.

65. 63. The method of claim 62, wherein the antibody is administered at least 1 day, such as at least 2 days, such as at least 1 week, before the administration of the at least one corticosteroid and the at least one non-corticosteroid chemotherapeutic agent.

66. 10. The method of any one of the preceding claims, wherein the antibody is administered at a dose of 1 mg / kg or more, such as a dose of 1 to 20 mg / kg, such as a dose of 5 to 20 mg / kg, such as a dose of 8 mg / kg.

67. 10. The method of any one of the preceding claims, wherein the antibody is administered once a week for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8 weeks.

68. 10. A method of treating cancer involving cells expressing CD38 in an individual, comprising the features of any one or more of the preceding claims.

69. 69. The method of claim 68, wherein the cancer is multiple myeloma or chronic lymphocytic leukemia.

70. 10. Use of an antibody that binds CD38 in the manufacture of a medicament for the treatment of cancer, which is or is to be administered in combination therapy with at least one corticosteroid and at least one non-corticosteroid chemotherapeutic agent.

71. 71. Use according to claim 70, comprising the features according to any one or more of claims 1 to 67.

72. Suitable for separate, sequential and / or simultaneous administration; i) a non-agonistic antibody that binds to CD38; ii) at least one corticosteroid, and iii) at least one non-corticosteroid chemotherapy agent 10. A therapeutic combination for inhibiting the growth and / or proliferation of tumor cells that express CD38, comprising:

73. 73. The therapeutic combination of claim 72, wherein the composition comprises the features of any one or more of claims 1 to 67.

Images