HK1250928B

HK1250928B - Combination therapies for heme malignancies with anti-cd38 antibodies and survivin inhibitors

Info

Publication number: HK1250928B
Application number: HK18110389.8A
Authority: HK
Inventors: Parul Doshi; Henk M. LOKHORST; Tuna MUTIS
Original assignee: Janssen Biotech, Inc.
Priority date: 2015-06-22
Filing date: 2016-06-22
Publication date: 2022-09-16

Description

Combination therapy of heme malignancies with anti-CD 38 antibodies and survivin inhibitors

Technical Field

The present invention relates to the combination therapy of heme malignancies.

Background

Multiple Myeloma (MM) is a B-cell malignancy characterized by the potential accumulation of secretory plasma cells in the bone marrow with a low proliferative index and an extended lifespan. The disease eventually invades the bone and bone marrow, resulting in multiple tumors and lesions throughout the skeletal system. Approximately 1% of all cancers and slightly more than 10% of all hematological malignancies are attributable to MM. MM increased in incidence in the elderly population, with a median age at diagnosis of about 61 years.

Currently available MM treatment methods include chemotherapy regimens, stem cell transplantation, and transplantation,(thalidomide),(lenalidomide),(pomalidomide),(bortezomib),(carfilzomib),(panobinostat),(pamidronic acid) and(zoledronic acid). Current treatment regimens (including combinations of chemotherapeutic agents such as vincristine, BCNU, melphalan, cyclophosphamide, doxorubicin, and prednisone or dexamethasone) produce complete remission rates of only about 5%, and median survival from diagnosis is about 36-48 months. Recent advances have used high dose chemotherapy followed by autologous bone marrow or peripheral blood mononuclear cell transplantation to increase the rate of complete remission and extend the duration of remission. However, this only slightly extends the overall survival and no evidence of a cure is obtained. Finally, all MM patientsRelapse occurs even in the case of maintenance therapy using interferon-alpha (IFN- α) alone or in combination with steroids.

The efficacy of available treatment regimens for MM drugs is limited by: in up to 90% of patients, the cell proliferation rate is low and resistance develops. Chromosomal translocations, oncogene mutations, deregulation of signaling pathways (such as anti-apoptotic pathways and survival pathways), and the bone marrow microenvironment are thought to be associated with drug resistance in MM (see review, Abdi et al, Oncotarget 4: 2186-. The Bone Marrow (BM) microenvironment is involved in proliferation, survival, differentiation, migration and resistance of malignant plasma cells (Manier et al, J Biomed Biotechnol 2012; published online at 10.3.2012, doi: 10.1155/_2012/_ 157496).

CD38 is a type II transmembrane glycoprotein and an attractive target for antibody therapy of various heme malignancies, including multiple myeloma. anti-CD 38 antibodies are described, for example, in international patent publication No. wo2008/037257, international patent publication No. wo2008/047242, and international patent publication No. wo2007/042309, and are being evaluated for their efficacy in multiple myeloma and other heme malignancies in a clinical setting.

Disclosure of Invention

The invention provides a method of treating a subject having a CD38 positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD 38 antibody and a survivin inhibitor for a time sufficient to treat the CD38 positive hematological malignancy.

Drawings

Figure 1a protection of Bone Marrow Stromal Cells (BMSCs) in MM cell lines against Multiple Myeloma (MM) cell killing by ADCC induced by anti-CD 38 antibody, namely, darunavir. Luciferase-transduced CD38 in the Presence or absence of healthy donor BMSC (HD-BMSC)⁺UM9 MM cells were cultured for 16 hours and then incubated with consecutive concentrations of daratumab and HD-PBMC at a PBMC: MM cell ratio of 30: 1. MM cell viability was determined by bioluminescence imaging (BLI) after 4 hours. ADCC percent calculation relative to cell viability without daratumabRatio (%). Error bars represent the standard error of the mean (SEM) of three measurements. Data are representative of 3 independent experiments. The unpaired t-test was used to test for differences between cultures with or without BMSC (═ p < 0.05).

Figure 1b protection of Bone Marrow Stromal Cells (BMSCs) in MM cell lines by mediating pin killing of Multiple Myeloma (MM) cells by ADCC induced by anti-CD 38 antibody, namely, darunavir. Luciferase-transduced CD38 in the Presence or absence of HD-BMSC⁺RPMI8226MM cells were cultured for 16 hours, and then incubated with consecutive concentrations of daratumab and HD-PBMC at a PBMC: MM cell ratio of 30: 1. After 4 hours MM cell viability was determined by BLI. ADCC% was calculated relative to cell viability without daratumab. Error bars represent SEM of three measurements. Data are representative of 3 independent experiments. The unpaired t-test was used to test for differences between cultures with or without BMSC (═ p < 0.05).

Bmscs mediate protection against MM cell killing by ADCC induced by anti-CD 38 antibody, namely, darunavir, in primary MM patient samples. Whole bone marrow aspirate obtained from MM patient 1 was cultured in the presence (white bars) or absence (black bars) of autologous bone marrow stromal cells and then treated with the indicated concentration of darunavir. Autologous cells present in the aspirate are used as effector cells. Since BM-MNC already contain NK cells as effector cells, no effector cells are added. After 24 hours, CD138 in culture was determined by flow cytometry⁺Viability of MM cells. Error bars represent SEM of three measurements. The unpaired t-test was used to test for differences between cultures with or without BMSC (═ p < 0.05). The upper diagram: patient #1, the following figure: patient # 2. BMSC: bone marrow stromal cells. ADCC: antibody-dependent cellular cytotoxicity.

Bmscs mediate protection against MM cell killing by ADCC induced by anti-CD 38 antibody, namely, darunavir, in primary MM patient samples. Whole bone marrow aspirate obtained from MM patient 2 was cultured in the presence (white bars) or absence (black bars) of autologous bone marrow stromal cells and then spiked with the indicated concentration of darunavirAnd (5) line processing. Autologous cells present in the aspirate are used as effector cells. Since BM-MNC already contain NK cells as effector cells, no effector cells are added. After 24 hours, CD138 in culture was determined by flow cytometry⁺Viability of MM cells. Error bars represent SEM of three measurements. The unpaired t-test was used to test for differences between cultures with or without BMSC (═ p < 0.05). The upper diagram: patient #1, the following figure: patient # 2. BMSC: bone marrow stromal cells. ADCC: antibody-dependent cellular cytotoxicity.

FIG. 3 YM155 does not induce NK cell lysis. HD-PBMC and patient bone marrow mononuclear cells (BMMNC) were incubated with YM155 at the indicated concentration for 24 hours. Determination of viable CD3 by flow cytometry^-CD56⁺Number of NK cells and percentage lysis was calculated using untreated samples as negative controls.

Figure 4a. daratumab in combination with YM155 provides a synergistic effect on ADCC-induced killing of MM cells in the presence of stromal cells. Luciferase-transduced RPMI8226MM cells were cultured in the presence or absence of HD-BMSC. Daratumab and YM155 were added at the indicated concentrations. HD-PBMC were added to all wells as a source of NK cells at a PBMC: MM ratio of 40: 1 to induce ADCC. RPMI8226 cell viability was determined by BLI after 4 hours. The% lysis was calculated relative to the survival of RPMI8226 cells not receiving any treatment. In the case of YM155 and darunavir combined, the expected lysis values were derived from treatment with darunavir and YM155 alone, assuming that the cumulative effect is additive rather than synergistic. Statistical differences between the expected and observed results were determined in paired t-tests (═ P < 0.005, ═ P < 0.05). DARA: daratumab.

Fig. 4b. daratumab in combination with YM155 provides a synergistic effect on ADCC-induced killing of primary MM cells in the presence of stromal cells. Whole BM aspirates of MM patient 1 were cultured in the presence or absence of autologous MM-BMSCs. Daratumab and YM155 were added at the indicated concentrations. Since BM-MNC contain sufficient NK cells (in both cases, the ratio of NK to MM cells is approximately 30: 1), no effector cells are added. 24 hoursThereafter, live CD138 was treated by flow cytometry under each condition⁺MM cells were counted. % lysis was calculated relative to the viability of MM cells in BM-MNC cultured under the same conditions but not receiving any treatment. The statistical difference between the expected and observed results was determined in paired t-tests (═ P < 0.05).

Fig. 4c. daratumab in combination with YM155 provides a synergistic effect on ADCC-induced killing of primary MM cells in the presence of stromal cells. Whole BM aspirates from MM patient 2 were cultured in the presence or absence of autologous MM-BMSCs. Daratumab and YM155 were added at the indicated concentrations. Since BM-MNC contain sufficient NK cells (in both cases, the ratio of NK to MM cells is approximately 30: 1), no effector cells are added. After 24 hours, live CD138 was treated by flow cytometry under each condition⁺MM cells were counted. % lysis was calculated relative to the viability of MM cells in BM-MNC cultured under the same conditions but not receiving any treatment. The statistical difference between the expected and observed results was determined in paired t-tests (═ P < 0.05).

Fig. 4d. daratumab in combination with YM155 provides a synergistic effect on ADCC-induced killing of primary MM cells in the presence of stromal cells. Culture from cells containing CD138 in the Presence or absence of autologous MM-BMSC⁺Bone marrow mononuclear cells (BM-MNC) from 4 patients (patients 1-4 45%, 5.5%, 10.2%, 21.6%, respectively) of MM cells. Daramumab (1ng/ml) and appropriate sub-maximal concentrations of YM155 (125, 62, 75 and 50ng/ml for patients 1-4, respectively) were added. Since BM-MNCs contained enough NK cells (7.9%, 10.3% and 9.5%, respectively), no effector cells were added. After 24 hours, live CD138 was treated by flow cytometry under each condition⁺MM cells were counted. % lysis was calculated relative to the viability of MM cells in BM-MNC cultured under the same conditions but not receiving any treatment. Statistical differences between the expected and observed results were determined in paired t-tests (═ P < 0.05, ns: not significant). DARA: daratumab.

FIG. 5 anti-tumor effect of daratumab and YM155 combination therapy. In thatRAG2 implanted with UM9 cells^-/-γc^-/-Tumor burden was analyzed in mice for each treatment group. A composite scaffold coated with human MSCs and loaded with the luciferase-transduced MM cell line UM9 was subcutaneously implanted with RAG2^-/-γc^-/-The back of the mouse (4 scaffolds per mouse). Ten days after implantation, the growing tumors were visualized and quantified by BLI. Different groups of mice (n-4) were then treated with vehicle controls (controls) or with daratumab, YM155, or daratumab + YM155 as specified. YM155 (or its vehicle, i.e., PBS) was delivered at a rate of 1mg YM 155/kg/day for 10 days using a subcutaneous infusion pump. Each mouse (including mice in the control group) received T cell depleted HD-PBMC (5X 10)⁶Individual cells) as a source of human NK cells to induce ADCC. Mice were monitored weekly by BLI. Results are expressed as the average tumor load in each stent. Error bars represent SEM. Statistical differences between mice treated with darunavir and mice treated with darunavir + YM155(× P < 0.001) were calculated using the Mann-Whitney U test.

Detailed Description

"CD 38" refers to human CD38 protein (synonyms: ADP-ribosyl cyclase 1, cADPr hydrolase 1, cyclic ADP-ribosyl hydrolase 1). Human CD38 has the amino acid sequence shown in SEQ ID NO: 1. It is well known that CD38 is a single-pass transmembrane type II membrane protein with amino acid residues 1-21 representing the cytoplasmic domain, amino acid residues 22-42 representing the transmembrane domain, and residues 43-300 representing the extracellular domain of CD 38.

As used herein, the term "antibody" is used in a broad sense to include immunoglobulin molecules, including monoclonal antibodies, including murine, human adapted, humanized and chimeric monoclonal antibodies; an antibody fragment; bispecific or multispecific antibodies; dimeric, tetrameric or multimeric antibodies; and single chain antibodies.

Depending on the heavy chain constant domain amino acid sequence, immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM. IgA and IgG are further sub-classified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG 4. Antibody light chains of any vertebrate species can be assigned to one of two completely different types, κ and λ, based on the amino acid sequence of the constant domains.

The term "antibody fragment" refers to a portion of an immunoglobulin molecule that retains heavy and/or light chain antigen binding sites, such as heavy chain complementarity determining regions (HCDR)1, 2, and 3, light chain complementarity determining regions (LCDR)1, 2, and 3, a heavy chain variable region (VH) or a light chain variable region (VL). Antibody fragments include: a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CHI domains; f (ab)₂A fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; a Fd fragment consisting of VH and CHI domains; fv fragments consisting of the VL and VH domains of a one-armed antibody; domain antibody (dAb) fragments (Ward et al, Nature 341: 544-546, 1989), which consist of VH domains. The VH and VL domains can be engineered and can be linked together via synthetic linkers to form various types of single chain antibody designs, wherein in the case where the VH and VL domains are expressed from separate single chain antibody constructs, the VH/VL domains pair intramolecularly or intermolecularly to form a monovalent antigen binding site, such as single chain fv (scfv) or diabodies, as described, for example, in international patent publication nos. wo1998/44001, WO1988/01649, WO1994/13804, and WO 1992/01047. These antibody fragments are obtained using techniques well known to those skilled in the art, and the fragments are screened for use in the same manner as full-length antibodies.

The phrase "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds CD38 is substantially free of antibodies that specifically bind antigens other than human CD 38). However, isolated antibodies that specifically bind CD38 may be cross-reactive to other antigens, such as the orthologous antigen of human CD38, such as cynomolgus monkey (Macaca fascicularis) CD 38. Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The antibody variable region consists of a "framework" region interrupted by three "antigen binding sites". Different terms are used to define antigen binding sites: (i) three Complementarity Determining Regions (CDRs) in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat, J Exp Med 132: 211-50, 1970; Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, national institutes of Health, Bethesda, Md., 1991). (ii) Three "hypervariable regions", "HVRs" or "HV" in VH (H1, H2, H3) and three in VL (L1, L2, L3) refer to regions of antibody variable domains which are hypervariable structurally, as defined by Chothia and Lesk (Chothia and Lesk, Mol Biol 196: 901-17, 1987). Other terms include "IMGT-CDR" (Lefranc et al, Dev company Immunol 27: 55-77, 2003) and "specificity determining residue usage" (SDRU) (Almagro Mol Recognit 17: 132-43, 2004). The International Immunogenetics (IMGT) database (http:// www _ IMGT _ org) provides a standardized numbering and definition of antigen binding sites. The corresponding relationship between CDR, HV and IMGT descriptions is described in Lefranc et al, Dev company Immunol 27: 55-77, 2003.

As used herein, "Chothia residues" are antibody VL and VH residues, which are numbered according to Al-Lazikani (Al-Lazikani et Al, J Mol Biol 273: 927-48, 1997).

"framework" or "framework sequence" is the remaining sequence of the variable region except for those sequences defined as antigen binding sites. Because the antigen binding site can be defined by various terms as described above, the exact amino acid sequence of the framework depends on how the antigen binding site is defined.

"humanized antibody" refers to an antibody in which the antigen binding site is derived from a non-human species and the variable region framework is derived from human immunoglobulin sequences. Humanized antibodies may comprise substitutions in the framework regions such that the framework may not be an exact copy of the expressed human immunoglobulin or germline gene sequence.

"human antibody" refers to an antibody having a heavy chain variable region and a light chain variable region, wherein both the framework and the antigen-binding site are derived from sequences of human origin. If the antibody comprises a constant region, the constant region is also derived from a sequence of human origin.

If the variable region of an antibody is obtained from a system using human germline immunoglobulins or rearranged immunoglobulin genes, the human antibody comprises a heavy chain variable region or a light chain variable region that is "derived" from a sequence of human origin. Such systems include human immunoglobulin gene libraries displayed on phage, as well as transgenic non-human animals, such as mice bearing human immunoglobulin loci as described herein. A "human antibody" may comprise amino acid differences resulting from, for example, naturally occurring somatic mutations or deliberate introduction of substitutions in the framework or antigen-binding site when compared to human germline or rearranged immunoglobulin sequences. Typically, the amino acid sequence of a "human antibody" has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, a "human antibody" can comprise a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al, JMol Biol 296: 57-86, 2000); or synthetic HCDR3 incorporated into a phage-displayed human immunoglobulin gene library (e.g., as described in Shi et al, J Mol Biol 397: 385-96, 2010 and international patent publication No. wo 2009/085462). Antibodies whose antigen-binding sites are derived from non-human species are not included in the definition of "human antibodies".

The isolated humanized antibody may be synthetic. Although human antibodies are derived from human immunoglobulin sequences, they can be generated using systems such as phage display in conjunction with synthetic CDRs and/or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally occur within the full complement of human antibody germline in vivo.

"recombinant antibodies" include all antibodies prepared, expressed, formed or isolated by recombinant methods, such as antibodies isolated from animals (e.g., mice), i.e., transgenic or transchromosomes of human immunoglobulin genes, or antibodies isolated from hybridomas prepared therefrom (described further below); an antibody isolated from a host cell transformed to express the antibody; antibodies isolated from a recombinant combinatorial antibody library; and antibodies prepared, expressed, created or isolated by any other method involving the splicing of human immunoglobulin gene sequences with other DNA sequences, or antibodies generated in vitro using Fab arm swapping.

"monoclonal antibody" refers to a preparation of antibody molecules in a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope or, in the case of bispecific monoclonal antibodies, exhibit dual binding specificities for two different epitopes. Thus, a "monoclonal antibody" refers to a population of antibodies having a single amino acid composition in each heavy chain and each light chain, except for possible well-known changes, such as the removal of the C-terminal lysine from the antibody heavy chain. Monoclonal antibodies can have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, or monovalent, bivalent, or multivalent. Included within the term "monoclonal antibody" are bispecific antibodies.

An "epitope" refers to the portion of an antigen that specifically binds to an antibody. Epitopes are typically composed of chemically active (such as polar, non-polar or hydrophobic) surface groups of moieties (such as amino acids or polysaccharide side chains) and can have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be composed of contiguous and/or noncontiguous amino acids that form conformational space units. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are close in three dimensions due to the folding of the protein molecule.

A "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications (e.g., substitutions, insertions, or deletions).

Use in combination "means that two or more therapeutic agents may be administered to a subject together in admixture, simultaneously as a single agent, or sequentially in any order as a single agent.

"treatment" or "treating" refers to both therapeutic treatment as well as prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disease, such as the development or spread of a tumor or tumor cells. Beneficial or desired clinical results include alleviation of symptoms, diminishment of extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission, whether detectable or undetectable (whether partial or complete), and extended survival compared to expected survival in the absence of treatment. Individuals in need of treatment include individuals already suffering from a disorder or disease as well as individuals susceptible to a disorder or disease or individuals in whom a disorder or disease is to be prevented.

"therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on the following factors: such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents that may result in a decrease or attenuation of the associated resistance include, for example, an improvement in the patient's health condition, a reduction in tumor burden, a suppression or slowing of tumor growth, and/or the absence of metastasis of cancer cells to other locations in the body.

By "synergistic", "synergistic" or "synergistic" is meant an additive effect beyond that expected from the combination.

"inhibiting growth" (e.g., in relation to a cell, such as a tumor cell) refers to a measurable decrease in cell growth in vitro or in vivo upon contact with a therapeutic agent or combination of therapeutic agents or drugs, as compared to the growth of the same cell grown under appropriate control conditions well known to those skilled in the art. The inhibition of cell growth in vitro or in vivo may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100%. Inhibition of cell growth can occur by a variety of mechanisms, such as by ADCC, apoptosis, necrosis, or by inhibiting cell proliferation.

"subject" includes any human or non-human animal. "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like. The terms "subject" and "patient" are used interchangeably herein.

As used herein, "survivin" refers to a polypeptide having the sequence of SEQ ID NO: 22, or a pharmaceutically acceptable salt thereof. Survivin is a member of the apoptosis Inhibitor (IAP) family. Survivin is a bifunctional protein that acts as an inhibitor of apoptosis and a regulator of the cell cycle. Overexpression of survivin was observed in human malignancies and was positively correlated with poor prognosis, tumor recurrence and treatment resistance (Liu et al, Cancer biol. Ther., 7: 1053-5001060, 2008; Mita et al, Clin Cancer Res., 14: 5000-5005, 2008).

SEQ ID NO：22

MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAEKVRRAIEQLAAMD

"survivin inhibitor" refers to a molecule that inhibits, antagonizes, decreases, or suppresses the activity of survivin protein; such as molecules that inhibit the anti-apoptotic activity of survivin in cells. Survivin inhibitors may inhibit the anti-apoptotic activity of survivin by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100%. The survivin inhibitor may be a small molecule, peptide, vaccine, polynucleotide, DNA or RNA molecule.

The present invention is based, at least in part, on the following findings: bone Marrow Stromal Cells (BMSCs) present in the BM microenvironment protect MM cells from antibody-induced ADCC at least in part by upregulating survivin, and survivin inhibitors may improve antibody-mediated ADCC of MM cells and abrogate BMSC-induced ADCC resistance. BMSCs have been shown to protect MM cells from Cytotoxic T Lymphocyte (CTL) dependent cell lysis through cell adhesion-mediated immune resistance, and survivin has been found to be upregulated in anti-cytolytic MM cells (de Haart et al, Clin cancer Res 19: 5591-.

The invention also provides a method of inhibiting growth or proliferation of multiple myeloma cells in a subject, comprising administering to a subject in need thereof an anti-CD 38 antibody and a survivin inhibitor for a time sufficient to inhibit growth or proliferation of multiple myeloma cells.

"CD 38-positive hematologic malignancy" refers to hematologic malignancies characterized by the presence of tumor cells expressing CD38, and includes leukemias, lymphomas, and myelomas. Exemplary such CD 38-positive hematological malignancies are precursor B-cell lymphoblastic leukemia/lymphoma and B-cell non-hodgkin's lymphoma, acute promyelocytic leukemia, acute lymphocytic leukemia and mature B-cell tumors, such as 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), Follicular Lymphoma (FL) (including low, medium and high grade FLs), cutaneous follicular central lymphoma, marginal zone B-cell lymphoma (MALT type, lymphoid node and spleen type), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt Lymphoma (BL), plasmacytoma, Multiple Myeloma (MM), Plasma cell leukemia, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, plasma cell leukemia, and Anaplastic Large Cell Lymphoma (ALCL).

CD38 is expressed in a variety of hematologic malignancies, including multiple myeloma, leukemias and lymphomas such as B-cell chronic lymphocytic leukemia, T-cell and B-cell acute lymphocytic leukemia, fahrenheit macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, prolymphocytic/myelocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, follicular lymphoma, burkitt's lymphoma, Large Granular Lymphocytic (LGL) leukemia, NK cell leukemia, and plasma cell leukemia. Expression of CD38 on epithelial/endothelial cells of different origins has been described, including glandular epithelial cells in the prostate, islet cells in the pancreas, ductal epithelial cells in glands (including parotid), bronchial epithelial cells, cells in the testis and ovary, and tumor epithelial cells in colorectal adenocarcinoma. Other diseases in which CD38 expression may be implicated include, for example, lung bronchial epithelial cancers, breast cancers (evolved from epithelial lining malignant proliferation in breast ducts and leaflets), pancreatic tumors evolved from beta cells (insulinomas), tumors evolved from intestinal epithelial cells (e.g., adenocarcinomas and squamous cell carcinomas), cancers in the prostate, and seminomas and ovarian cancers in the testis. In the central nervous system, neuroblastoma expresses CD 38.

In some embodiments, the CD 38-positive hematological malignancy is Multiple Myeloma (MM), Acute Lymphoblastic Leukemia (ALL), non-hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt's Lymphoma (BL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), Acute Myeloid Leukemia (AML), or Chronic Lymphocytic Leukemia (CLL).

In some embodiments, the CD 38-positive hematological malignancy is MM.

In some embodiments, the CD 38-positive hematologic malignancy is ALL.

In some embodiments, the CD 38-positive hematologic malignancy is NHL.

In some embodiments, the CD 38-positive hematologic malignancy is DLBCL.

In some embodiments, the CD 38-positive hematological malignancy is BL.

In some embodiments, the CD 38-positive hematologic malignancy is FL.

In some embodiments, the CD 38-positive hematologic malignancy is MCL.

In some embodiments, the CD 38-positive hematological malignancy is AML.

In some embodiments, the CD 38-positive hematological malignancy is CLL.

In some embodiments, the CD 38-positive hematologic malignancy is a plasma cell disease.

In some embodiments, the plasma cell disease is light chain Amyloidosis (AL), Multiple Myeloma (MM), or fahrenheit macroglobulinemia.

In some embodiments, the plasma cell disease is AL.

In some embodiments, the plasma cell disease is MM.

In some embodiments, the plasma cell disease is fahrenheit macroglobulinemia.

Examples of B-cell non-hodgkin lymphomas are lymphomatoid granulomatosis, primary effusion lymphoma, intravascular large B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain diseases (including lower heavy chain disease, μ heavy chain disease, and α heavy chain disease), lymphomas induced by treatment with immunosuppressive agents (such as cyclosporine-induced lymphomas and methotrexate-induced lymphomas).

In one embodiment, the disease involving cells expressing CD38 is hodgkin's lymphoma.

Other examples of diseases involving cells expressing CD38 include malignancies derived from T cells and NK cells, including: mature T cell and NK cell tumors including T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, rhinotypic, 78 enteropathic 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 disease (primary cutaneous anaplastic large cell lymphoma C-ALCL, lymphomatoid papulosis, junctional lesions), angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma non-specific and anaplastic large cell lymphoma.

Examples of malignancies derived from bone marrow cells include acute myelogenous leukemia (including acute promyelocytic leukemia) and chronic myeloproliferative disease (including chronic myelogenous leukemia).

Any anti-CD 38 antibody can be used in the methods of the invention. The variable regions of the anti-CD 38 antibody can be obtained from an existing anti-CD 38 antibody and optionally cloned as a full-length antibody using standard methods. Exemplary antibody variable regions that bind CD38 that can be used are described, for example, in International patent publication Nos. WO05/103083, WO06/125640, WO07/042309, WO08/047242, WO12/092612, WO06/099875, and WO11/154453A 1.

An exemplary anti-CD 38 antibody that can be used is DARZALEX^TM(daratumab). DARZALEX^TM(daratumab) comprises the amino acid sequences as set forth in SEQ ID NOs: 4 and 5, and the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) shown as SEQ ID NOs: 6. 7 and 8 and the heavy chain complementarity determining region (HCDR)1, HCDR2 and HCDR3 amino acid sequences shown in SEQ ID NOs: 9. 10 and 11, and is of the IgG1/κ subtype, LCDR2 and LCDR3 amino acid sequences. DARZALEX^TM(daratumab) heavy chain amino acid sequence is set forth in SEQ ID NO: 12, the light chain amino acid sequence is set forth in SEQ ID NO: shown in fig. 13.

SEQ ID NO：1

MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI

SEQ ID NO：2

SKRNIQFSCKNIYR

SEQ ID NO：3

EKVQTLEAWVIHGG

SEQ ID NO：4

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSA

ISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDK

ILWFGEPVFDYWGQGTLVTVSS

SEQ ID NO：5

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

SEQ ID NO：6

SFAMS

SEQ ID NO：7

AISGSGGGTYYADSVKG

SEQ ID NO：8

DKILWFGEPVFDY

SEQ ID NO：9

RASQSVSSYLA

SEQ ID NO：10

DASNRAT

SEQ ID NO：11

QQRSNWPPTF

SEQ ID NO：12

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：13

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Another exemplary anti-CD 38 antibody that can be used is mAb003, which comprises the amino acid sequences set forth in SEQ ID NOs: 14 and 15, which antibodies are described in U.S. patent No.7,829,693.

SEQ ID NO：14

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQGRVTITADKSTSTAY

MDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSSAS

SEQ ID NO：15

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP

EDFATYYCQQYNSYPRTFGQGTKVEIK

Another exemplary anti-CD 38 antibody that can be used is mAb024 comprising the amino acid sequences of SEQ ID NOs: 16 and 17, which antibodies are described in U.S. patent No.7,829,693.

SEQ ID NO：16

EVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMPGKGLEWMGIIYPHDSDARYSPSFQGQVTFSADKSISTAY

LQWSSLKASDTAMYYCARHVGWGSRYWYFDLWGRGTLVTVSS

SEQ ID NO：17

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP

EDFAVYYCQQRSNWPPTFGQGTKVEIK

Another exemplary anti-CD 38 antibody that may be used is MOR-202(MOR-03087) which comprises the amino acid sequences of SEQ ID NOs: 18 and 19, which antibodies are described in U.S. patent No.8,088,896.

SEQ ID NO：18

QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS

SEQ ID NO：19

DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAE

DEADYYCQTYTGGASLVFGGGTKLTVLGQ

Another exemplary anti-CD 38 antibody that can be used is isatuximab, which comprises the amino acid sequences set forth in SEQ ID NOs: 20 and 21, which antibodies are described in U.S. patent No.8,153,765. The VH and VL of isatuximab may be expressed as IgG1/κ.

SEQ ID NO：20

QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGT

IYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGD

YYGSNSLDYWGQGTSVTVSS

SEQ ID NO：21

DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKLEIK

anti-CD 38 antibodies useful in the methods of the invention may also be selected de novo from, for example, phage display libraries in which phages are engineered to express human immunoglobulins or portions thereof, such as Fab, single chain antibody (scFv) or unpaired or paired antibody variable regions (Knappik et al, J Mol Biol 296: 57-86, 2000; Krebs et al, J Immunol Meth 254: 67-84, 2001; Vaughan et al, Nature Biotechnology 14: 309-314, 1996; Sheets et al, PITAS (USA) 95: 6157-6162, 1998; Hoogboenbo and Winter, J Mol Biol 227: 381, 1991; Marks et al, J Mol 222: 581, 1991). CD38 binding variable domains can be isolated, for example, from phage display libraries that express antibody heavy and light chain variable regions as fusion proteins with phage pIX coat proteins, as described in Shi et al, (2010) j.mol.biol.397: 385-96 and international patent publication No. wo 09/085462. Antibodies that bind to the extracellular domain of human CD38 can be screened from antibody libraries and the positive clones obtained can be further characterized, Fab isolated from clone lysates and then cloned as full length antibodies. Such phage display methods for isolating human antibodies are well established in the art. See, for example, U.S. Pat. No.5,223,409, U.S. Pat. No.5,403,484, U.S. Pat. No.5,571,698, U.S. Pat. No.5,427,908, U.S. Pat. No.5,580,717, U.S. Pat. No.5,969,108, U.S. Pat. No.6,172,197, U.S. Pat. No.5,885,793, U.S. Pat. No.6,521,404, U.S. Pat. No.6,544,731, U.S. Pat. No.6,555,313, U.S. Pat. No.6,582,915, and U.S. Pat. No.6,593,081.

The present invention also provides a method of treating a subject having a CD38 positive hematological malignancy, comprising administering to a subject in need thereof an antibody that binds to a polypeptide comprising SEQ ID NO: 4 and the VH of SEQ ID NO: antibody to VL of 5 competes for binding to anti-CD 38 antibody to CD38 and the survivin inhibitor for a time sufficient to treat a CD38 positive hematological malignancy.

The invention also provides a method of treating a subject having multiple myeloma comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 4 and the VH of SEQ ID NO: antibody to VL of 5 competes for binding to anti-CD 38 antibody to CD38 and the survivin inhibitor for a time sufficient to treat multiple myeloma.

The invention also provides a method of treating a subject having a CD38 positive hematological malignancy, comprising administering to a subject in need thereof an anti-CD 38 antibody that binds to region SKRNIQFSCKNIYR (SEQ ID NO: 2) and region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38(SEQ ID NO: 1) and a survivin inhibitor for a time sufficient to treat the CD38 positive hematological malignancy.

The invention also provides a method of treating a subject having multiple myeloma comprising administering to a subject in need thereof an anti-CD 38 antibody that binds to region SKRNIQFSCKNIYR (SEQ ID NO: 2) and region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38(SEQ ID NO: 1) and a survivin inhibitor for a time sufficient to treat multiple myeloma.

In some embodiments, the anti-CD 38 antibody comprises SEQ ID NOs: 6. HCDR1, HCDR2 and HCDR3 of 7 and 8.

In some embodiments, the anti-CD 38 antibody comprises SEQ ID NOs: 9. LCDR1, LCDR2 and LCDR3 of 10 and 11.

In some embodiments, the anti-CD 38 antibody comprises SEQ ID NOs: 6. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of 7,8, 9,10 and 11.

In some embodiments, the anti-CD 38 antibody comprises a heavy chain variable region comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 4, and a VH comprising an amino acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 5, or a VL of an amino acid sequence having 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of seq id No. 5.

In some embodiments, the anti-CD 38 antibody comprises SEQ ID NO: 4 and the VH of SEQ ID NO: 5 VL.

In some embodiments, the anti-CD 38 antibody comprises SEQ ID NO: 14 and SEQ ID NO: 15 VL.

In some embodiments, the anti-CD 38 antibody comprises SEQ ID NO: 16 and the VH of SEQ ID NO: 17 VL.

In some embodiments, the anti-CD 38 antibody comprises SEQ ID NO: 18 and SEQ ID NO: 19 VL of (d).

In some embodiments, the anti-CD 38 antibody comprises SEQ ID NO: 20 and SEQ ID NO: 21 VL of.

The antibodies can be evaluated against a reference antibody (e.g., DARZALEX) using well-known in vitro methods^TMI.e. having SEQ ID NO: 4 and the VH of SEQ ID NO: daratumab for VL of 5) competes for binding to CD 38. In an exemplary method, CHO cells recombinantly expressing CD38 may be incubated with unlabeled reference antibody at 4 ℃ for 15 minutes, and then incubated with excess fluorescently labeled test antibody at 4 ℃ for 45 minutes. After washing in PBS/BSA, fluorescence can be measured by flow cytometry using standard methods. In another exemplary method, the extracellular portion of human CD38 may be coated on the surface of an ELISA plate. An excess of unlabeled reference antibody can be added over about 15 minutes, followed by the addition of biotinylated test antibody. After washing in PBS/tween, binding of the biotinylated test antibody can be detected using horseradish peroxidase (HRP) conjugated streptavidin and the signal detected using standard methods. It will be apparent that in a competition assay, the reference antibody may be labelled and the test antibody unlabelled. The test antibody competes with the reference antibody when the reference antibody inhibits binding of the test antibody or the test antibody inhibits binding of the reference antibody by at least 80%, 85%, 90%, 95%, or 100%. The epitope of the test antibody can be further defined using known methods, by, for example, peptide mapping or hydrogen/deuterium protection assays, or by crystal structure determination.

When the anti-CD 38 antibody binds to at least 1,2, 3,4, 5, 6,7, 8, 9,10, 11, 12, 13, or 14 residues within region SKRNIQFSCKNIYR (SEQ ID NO: 2) and region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38(SEQ ID NO: 1), the antibody binds to the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 3. in some embodiments, the anti-CD 38 antibody binds to at least one amino acid in region SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least one amino acid in region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38(SEQ ID NO: 1). In some embodiments, the anti-CD 38 antibody binds to at least two amino acids in region SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least two amino acids in region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38(SEQ ID NO: 1). In some embodiments, the anti-CD 38 antibody binds to at least three amino acids in region SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least three amino acids in region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38(SEQ ID NO: 1).

An exemplary antibody that binds to region SKRNIQFSCKNIYR (SEQ ID NO: 2) and region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38(SEQ ID NO: 1) is DARZALEX^TM(daratumab).

An antibody that binds to region SKRNIQFSCKNIYR (SEQ ID NO: 2) and region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38(SEQ ID NO: 1) can be produced, for example, by the following process: using standard methods and as described herein with a peptide having SEQ ID NO: 2 and 3 and characterization of the binding of the obtained antibodies to the peptides using, for example, ELISA or mutagenesis studies.

The Fc portion of an antibody can mediate antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC), as described in more detail below. Such functions may be mediated by: the Fc effector domain binds to an Fc receptor on an immune cell with phagocytic or lytic activity, or the Fc effector domain binds to a component of the complement system. Typically, Fc binding cells or effects mediated by complement components result in the inhibition and/or depletion of target cells (e.g., cells expressing CD 38). Human IgG isotypes IgG1, IgG2, IgG3 and IgG4 exhibit differential capabilities in effector function. ADCC may be mediated by IgG1 and IgG3, ADCP may be mediated by IgG1, IgG2, IgG3 and IgG4, and CDC may be mediated by IgG1 and IgG 3.

In some embodiments, the anti-CD 38 antibody is an IgG1, IgG2, IgG3, or IgG4 isotype.

In some embodiments, the anti-CD 38 antibody is an IgG1 isotype.

In some embodiments, the anti-CD 38 antibody is an IgG2 isotype.

In some embodiments, the anti-CD 38 antibody is an IgG3 isotype.

In some embodiments, the anti-CD 38 antibody is an IgG4 isotype.

In some embodiments, the anti-CD 38 antibody induces killing of CD 38-expressing cells by antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or apoptosis.

In some embodiments, the anti-CD 38 antibody induces killing of CD38 expressing cells by ADCC.

In some embodiments, the anti-CD 38 antibody induces killing of CD38 expressing cells by ADCP.

In some embodiments, the anti-CD 38 antibody induces killing of CD38 expressing cells by CDC.

In some embodiments, the anti-CD 38 antibody kills cells expressing CD38 by induction of apoptosis.

"antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a mechanism for inducing cell death that relies on the interaction of antibody-coated target cells with effector cells having lytic activity, such as natural killer cells, monocytes, macrophages and neutrophils, via Fc γ receptors (Fc γ R) expressed on the effector cells. For example, NK cells express Fc γ RIIIa, while monocytes express Fc γ RI, Fc γ RII, and Fc γ RIIIa. The activity of effector cells by secreting pore-forming proteins and proteases leads to death of antibody-coated target cells such as MM cells expressing CD 38. To assess ADCC activity of an anti-CD 38 antibody, the antibody can be added to cells expressing CD38 in combination with immune effector cells that can be activated by the antigen-antibody complex, thereby lysing the target cells. Cell lysis is detected based on the release of a label (e.g., radioactive substrate, fluorescent dye, or native intracellular protein) from the lysed cells. Exemplary effector cells for use in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and NK cells. A multiple myeloma cell line or primary MM cell expressing CD38 may be used as the target cell. In an exemplary assay, MM cell lines engineered to express luciferase enzymes were incubated with anti-CD 38 antibodies. Freshly isolated PBMC effector cells were added at a target to effector cell ratio of 40: 1. 4 hours after PBMC addition, fluorescein was added, the resulting bioluminescent signal emitted by viable MM cells was measured using a luminometer (SpectraMax, Molecular Devices) over 20 minutes, and the ADCC percentage of MM cells was calculated using the following formula: ADCC% × 1- (mean bioluminescence signal in the absence of PBMC/mean bioluminescence signal in the presence of PBMC) × 100%. An anti-CD 38 antibody "induces ADCC in vitro" when the% ADCC is at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, or 100% in an in vitro assay (such as one of the assays described above).

"complement-dependent cytotoxicity" or "CDC" refers to a mechanism of inducing cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q, and C1q in turn activates the complement cascade, resulting in the death of the target cell. Activation of complement may also result in the deposition of complement components on the surface of target cells that promote ADCC by binding to complement receptors on leukocytes (e.g., CR 3). In an exemplary assay, primary BM-MNC cells isolated from patients with B cell malignancies can be treated with anti-CD 38 antibody derived from 10% pooled human serum and complement at a concentration of 0.3-10 μ g/mL for 1 hour, and can be treated using van der Veer et al, haemotologica 96: 284 + 290, 2011, van der Veer et al, Blood Cancer J1 (10): e41, 2011 by flow cytometry⁺Viability of MM cells. Percent MM cell lysis can be determined relative to an isotype control as described herein. anti-CD 38 antibodies used in the methods of the invention can induce CDC by about 20%, 25%, 30%, 35%, 400%, 45%, 50%, 55%, 60%, 65%, 700%, 75%, 80%, 85%, 90%, 95%, or 100%.

"antibody-dependent cellular phagocytosis" ("ADCP") refers to a mechanism by which antibody-coated target cells are eliminated by internalization of phagocytic cells, such as macrophages or dendritic cells. ADCP can be evaluated as follows: (ii) use of monocyte-derived macrophages as effector cells and use of Daudi cells expressing CD 38: (CCL-213^TM) Or B-cell leukemia or lymphoma tumor cells as target cells engineered to express GFP or other marker molecules. The effector to target cell ratio may be, for example, 4: 1. Effector cells can be incubated with target cells for 4 hours with or without anti-CD 38 antibody. After incubation, the cells can be isolated using a cell digest. Macrophages can be identified using anti-CD 11b and anti-CD 14 antibodies conjugated to fluorescent markers, and can be based on CD11 using standard methods⁺CD14⁺Percent phagocytosis was determined by% GFP fluorescence in macrophages. anti-CD 38 antibodies used in the methods of the invention can induce ADCP by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

ADCC by anti-CD 38 antibodies may be enhanced by making certain substitutions in the antibody Fc. Exemplary substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (numbering of residues according to the EU index), as described in U.S. Pat. No.6,737,056.

In some embodiments, the anti-CD 38 antibody comprises an amino acid substitution in the antibody Fc.

In some embodiments, the anti-CD 38 antibody comprises a substitution at amino acid position 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (numbering residues according to the EU index) in the antibody Fc.

ADCC by anti-CD 38 antibodies may also be enhanced by engineering the oligosaccharide component of the antibody. Human IgG1 or IgG3 were N-glycosylated with most glycans in the form of double-branched G0, G0F, G1, G1F, G2, or G2F at Asn 297. Antibodies produced by CHO cells that are not engineered typically have a glycan fucose content of about at least 85%. Removal of core fucose from a double-branched complex-type oligosaccharide linked to an Fc region can enhance ADCC of an antibody via improved Fc γ RIIIa binding without altering antigen binding or CDC activity. Such modified antibodies can be achieved using different methods reported to result in the successful expression of relatively high defucosylated antibodies with double-branched complex-type Fc oligosaccharides, such as control of the osmolality of the culture (Konno et al, Cytotechnology 64: 249-65, 2012), the use of the variant CHO cell line Lec13 as host cell line (Shields et al, J Biol Chem 277: 26733-26740, 2002), the use of the variant CHO cell line EB66 as host cell line (Olivier et al, MAbs; 2 (2010), pre-printing electronic edition; PMID: 20562582), the use of the large mouse hybridoma cell line YB2/0 as host cell line (Shinkawa et al, J tecl Chem: 3466-3473, 2003), the introduction of small interfering RNA specific for the α 1, 6-fucosyltransferase (T8) gene (Moriol et al, Biohng 908: 88-85901) or 85901, 4-N-acetylglucosaminyltransferase III and golgi alpha-mannosidase II or the potent alpha-mannosidase I inhibitor kifansine (Ferrara et al, J Biol Chem 281: 5032-.

In some embodiments, the anti-CD 38 antibody has a bimolecular glycan structure with a fucose content of between about 0% to about 15% (e.g., 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%).

In some embodiments, the anti-CD 38 antibody has a bimolecular glycan structure with a fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

Substitutions in the Fc and reduced fucose content can enhance ADCC activity of the anti-CD 38 antibody.

"fucose content" means the amount of fucose monosaccharide within the sugar chain at Asn 297. The relative amount of fucose is the percentage of fucose-containing structures relative to all sugar structures. These can be characterized and quantified by a variety of methods, such as: 1) MALDI-TOF using N-glycosidase F treated samples (e.g., complex structures, mixed structures, and oligomeric and high mannose structures), as described in international patent publication No. wo 2008/0775462; 2) by enzymatic release of Asn297 glycans, followed by derivatization and detection/quantification by HPLC (UPLC) and/or HPLC-MS with fluorescence detection (UPLC-MS); 3) performing an intact protein analysis on the native or reduced mAb with or without treatment of Asn297 glycan with Endo S or other enzymes that cleave between a first GlcNAc monosaccharide and a second GlcNAc monosaccharide, leaving a fucose attached to the first GlcNAc; 4) digestion of the antibody into component peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), followed by separation, detection and quantification by HPLC-MS (UPLC-MS); 5) antibody oligosaccharides were separated from antibody proteins by specific enzymatic deglycosylation with PNGase F at Asn 297. The oligosaccharides thus released can be fluorescently labelled, isolated and identified by various complementary techniques which allow: performing fine characterization on the glycan structure by comparing experimental mass with theoretical mass by using matrix-assisted laser desorption ionization (MALDI) mass spectrometry; determination of the degree of sialylation by ion exchange hplc (glycosep c); isolating and quantifying the oligosaccharide form by normal phase hplc (glycocep n) according to hydrophilic standards; and separating and quantifying oligosaccharide by high performance capillary electrophoresis laser induced fluorescence (HPCE-LIF).

As used herein, "low fucose" or "low fucose content" refers to antibodies having a fucose content of from about 0% to about 15%.

As used herein, "normal fucose" or "normal fucose content" refers to a fucose content of an antibody that is about greater than 50%, typically about greater than 80% or greater than 85%.

And a polypeptide comprising SEQ ID NO: 12 and SEQ ID NO: 13 can be used in the methods of the invention. As used herein, the term "substantially identical" means that the two antibody heavy or light chain amino acid sequences being compared are identical or have "insignificant differences. Non-significant differences refer to substitutions of 1,2, 3,4, 5, 6,7, 8, 9,10, 11, 12, 13, 14, or 15 amino acids in the heavy or light chain of an antibody that do not adversely affect the properties of the antibody. Percent identity can be determined, for example, by performing a two-sequence alignment using the default settings of the AlignX module of Vector ntiv.9.0.0(Invitrogen, Carlsbad, CA). The protein sequences of the invention may be used as query sequences to search public or patent databases, for example, to identify related sequences. Exemplary programs for performing such searches are XBLAST or BLASTP programs (http _/www _ ncbi _ nlm/nih _ gov) or GenomeQuest using default settings^TM(GenomeQuest, Westborough, MA) software package. Exemplary substitutions that can be made to the anti-CD 38 antibodies used in the methods of the invention are, for example, conservative substitutions with amino acids having similar charge, hydrophobicity, or stereochemical characteristics. Conservative substitutions may also be made to improve antibody properties, such as stability or affinity, or to improve antibody effector function. The heavy or light chain of the anti-CD 38 antibody may, for example, be subjected to 1,2, 3,4, 5, 6,7, 8, 9,10, 11, 12, 13, 14, or 15 amino acid substitutions. In addition, any natural residues in the heavy or light chain may also be replaced with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al, Acta Physiol Scan and Suppl 643: 55-67, 1998; Sasaki et al, Adv Biophys 35: 1-24, 1998). The desired amino acid substitutions can be determined by those skilled in the art when such substitutions are desired. Amino acid substitutions can be made, for example, by PCR mutagenesis (U.S. Pat. No.4,683,195). Libraries of variants can be generated using well-known methods, e.g., using random (NNK) or non-random codons (e.g., DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, T)yr, Trp)), and screening the library for variants having the desired properties. The resulting variants can be tested for binding to CD38 and their ability to induce ADCC using the methods described herein.

"conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Conservative modifications include amino acid substitutions, additions and deletions. Conservative substitutions are those in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well defined and include amino acids with side chains as follows: acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). In addition, any natural residues in the polypeptide may also be replaced with alanine as previously described for alanine scanning mutagenesis (MacLennan et al, (1988) Acta Physiol Scan and Suppl 643: 55-67; Sasaki et al, (1988) Adv Biophys 35: 1-24). Amino acid substitutions of the antibodies of the invention can be made by known methods, for example, by PCR mutagenesis (U.S. Pat. No.4,683,195). Alternatively, a library of variants can be generated, e.g., using random (NNK) or non-random codons, e.g., DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants can be tested for characteristics using the assays described herein.

In some embodiments, the anti-CD 38 antibody may have a range of affinities (K)_D) Binds to human CD 38. In one embodimentanti-CD 38 antibody at or below about 1X 10^-8M (e.g., 5X 10)^-9M、1×10^-9M、5×10^-10M、1×10^- ¹⁰M、5×10^-11M、1×10^-11M、5×10^-12M、1×10^-12M、5×10^-13M、1×10^-13M、5×10^-14M、1×10^-14M or 5X 10^-15M) or any range or value therein_DBinding to CD38, as measured by surface plasmon resonance or Kinexa methods as practiced by those skilled in the art. Exemplary affinity is equal to or less than 1 × 10^-8And M. Another exemplary affinity is equal to or less than 1 × 10^-9M。

KinExA instruments, ELISA or competitive binding assays are known to those skilled in the art. The measured affinity of a particular antibody/CD 38 interaction may vary if measured under different conditions (e.g., osmolarity, pH). Thus, affinity and other binding parameters (e.g., K)_D、K_on、K_off) The measurements of (a) are typically performed using standardized conditions and standardized buffers, such as the buffers described herein. Those skilled in the art will appreciate that the internal error (measured as standard deviation, SD) of affinity measurements using, for example, Biacore 3000 or ProteOn may typically be within 5% -33% of the measurements within typical detection limits. Thus, K_DThe term "about" in the background reflects the typical standard deviation in the assay. For example, 1X 10^-9K of M_DTypical SD of at most. + -. 0.33X 10^-9M。

In some embodiments, the anti-CD 38 antibody is a bispecific antibody. The VL and/or VH regions of existing anti-CD 38 antibodies, or VL and VH regions identified de novo as described herein, can be engineered into bispecific full length antibodies. Such bispecific antibodies can be prepared by modulating the CH3 interaction in the antibody Fc to form a bispecific antibody using techniques such as those described in the following patents: U.S. patent No.7,695,936, international patent publication No. wo04/111233, U.S. patent publication No. us2010/0015133, U.S. patent publication No. us2007/0287170, international patent publication No. wo2008/119353, U.S. patent publication No. us2009/0182127, U.S. patent publication No. us2010/0286374, U.S. patent publication No. us2011/0123532, international patent publication No. wo2011/131746, international patent publication No. wo2011/143545, or U.S. patent publication No. us 2012/0149876.

For example, bispecific antibodies of the invention can be produced in vitro in a cell-free environment by: according to the method described in international patent publication No. wo2011/131746, asymmetric mutations are introduced in the CH3 region of two monospecific homodimeric antibodies and bispecific heterodimeric antibodies are formed from the two parent monospecific homodimeric antibodies under reducing conditions that allow disulfide isomerization. In the methods, a first monospecific bivalent antibody (e.g., an anti-CD 38 antibody) and a second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; incubating the antibodies together under reducing conditions sufficient to disulfide isomerization of cysteines in the hinge region; thereby generating bispecific antibodies by Fab arm exchange. Optimally, the incubation conditions can be returned to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), Dithiothreitol (DTT), Dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine and β -mercaptoethanol, preferably a reducing agent selected from 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine. For example, incubation may be carried out for at least 90 minutes at a pH of 5-8, e.g., at a pH of 7.0 or at a pH of 7.4 and a temperature of at least 20 ℃, in the presence of at least 25mM 2-MEA or in the presence of at least 0.5mM dithiothreitol.

Exemplary CH3 mutations that can be used for the first and second heavy chains of a bispecific antibody are K409R and/or F405L.

Further bispecific structures into which the VL region and/or VH region of an antibody of the invention may be incorporated are, for example, Double Variable Domain (DVD) immunoglobulins (international patent publication No. wo2009/134776) or structures comprising multiple dimerization domains to connect two antibody arms with different specificities, such as leucine zippers or collagen dimerization domains (international patent publication No. wo2012/022811, U.S. patent No.5,932,448, U.S. patent No.6,833,441). DVD is a full length antibody comprising a heavy chain having the structure VH 1-linker-VH 2-CH and a light chain having the structure VL 1-linker-VL 2-CL; the linker is optional.

In some embodiments, the anti-CD 38 antibody is conjugated to a toxin. Conjugation methods and suitable toxins are well known.

In some embodiments, a subject with MM is homozygous for phenylalanine at position 158 of CD16 (Fc γ RIIIa-158F/F genotype), or heterozygous for valine and phenylalanine at position 158 of CD16 (Fc γ RIIIa-158F/V genotype). CD16 is also known as Fc γ receptor IIIa (Fc γ RIIIa) or low affinity immunoglobulin γ Fc region receptor III-a isoform. The valine/phenylalanine (V/F) polymorphism at residue 158 of Fc γ RIIIa protein has been shown to affect the affinity of Fc γ RIIIa for human IgG. Receptors with Fc γ RIIIa-158F/F or Fc γ RIIIa-158F/V polymorphisms exhibit reduced Fc binding and therefore reduced ADCC as compared to Fc γ RIIIa-158V/V. The absence or low amount of fucose on the human N-linked oligosaccharides increases the ability of the antibody to induce ADCC due to improved binding of the antibody to human Fc γ RIIIa (CD16) (Shields et al, J Biol Chem 277: 26733-40, 2002). The patient can be analyzed for Fc γ RIIIa polymorphisms using routine methods.

In some embodiments, the survivin inhibitor is a small molecule.

In some embodiments, the survivin inhibitor is a polynucleotide.

Survivin inhibitors may inhibit survivin-induced apoptosis by any mechanism, such as inhibiting survivin gene transcription or protein expression, inhibiting survivin protein dimerization, enhancing destabilization or inducing degradation thereof, and the like.

An exemplary small molecule survivin inhibitor is YM 155. YM155 binds to the survivin promoter and inhibits its transcription. Other exemplary small molecule survivin inhibitors are nordihydroguaiaretic acid derivatives, for example, as described in U.S. patent No.6,608,108 and molecules described in U.S. patent publication No. us 2012/0122910. Other survivin polynucleotide inhibitors are described, for example, in U.S. Pat. No.6,838,283, International patent publication No. WO01/057059, WO09/114476, and WO 09/044793. Polynucleotide inhibitors include micrornas (mirnas), small interfering rnas (sirnas), allele-specific oligonucleotides (ASOs), and other polynucleotide inhibitors known in the art.

Administration/pharmaceutical composition

In the methods of the invention, the anti-CD 38 antibody can be provided in a suitable pharmaceutical composition comprising the anti-CD 38 antibody and a pharmaceutically acceptable carrier. The carrier may be a diluent, adjuvant, excipient, or vehicle with which the anti-CD 38 antibody is administered. Such vehicles may be liquids, such as water and oils, including those derived from petroleum, animal, vegetable or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% physiological saline solution and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by well-known conventional sterilization techniques, such as filtration. The composition may contain pharmaceutically acceptable auxiliary substances as necessary to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickening agents, lubricants, and coloring agents, and the like. The concentration of the molecules or antibodies of the invention in such pharmaceutical formulations can vary widely, i.e., from less than about 0.5% by weight, typically to at least about 1% by weight up to 15% by weight or 20% by weight, and will be selected based primarily on the desired dosage, fluid volume, viscosity, etc., depending on the particular mode of administration selected. Suitable vehicles and formulations containing other human proteins (e.g., human serum albumin) are described in, for example, Remington: the Science and Practice of Pharmacy, 21 st edition, Troy, D.B. eds, Lipincott Williams and Wilkins, Philadelphia, PA2006, part 5, Pharmaceutical Manufacturing, pages 691-.

The mode of administration of the anti-CD 38 antibody in the methods of the invention may be by any suitable route, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, mucosal (oral, intranasal, intravaginal, rectal), or other means as will be appreciated by those of skill in the art.

The anti-CD 38 antibody in the methods of the invention can be administered to the patient by any suitable route, for example, parenterally by Intravenous (IV) infusion or bolus injection, intramuscularly or subcutaneously or intraperitoneally. Intravenous infusion may be administered, for example, over 15, 30, 60, 90, 120, 180, or 240 minutes, or over 1,2, 3,4, 5, 6,7, 8, 9,10, 11, or 12 hours.

The dose administered to a patient with a CD38 positive hematological malignancy is sufficient to alleviate or at least partially arrest the disease being treated ("therapeutically effective amount"), and sometimes may be 0.005mg/kg to about 100mg/kg, such as about 0.05mg/kg to about 30mg/kg or about 5mg/kg to about 25mg/kg, or about 4mg/kg, about 8mg/kg, about 16mg/kg or about 24mg/kg, or such as about 1,2, 3,4, 5, 6,7, 8, 9 or 10mg/kg, but may be even higher, such as about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.

Fixed unit doses, e.g., 50, 100, 200, 500, or 1000mg, may also be administered, or the dose may be based on the surface area of the patient, e.g., 500, 400, 300, 250, 200, or 100mg/m². Typically, between 1 and 8 doses (e.g., 1,2, 3,4, 5, 6,7, or 8 doses) may be administered to treat MM, but 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be administered.

The anti-CD 38 antibody in the methods of the invention can be administered repeatedly after one, two, three, four, five, six, one, two, three, one, five, six, seven, two, three, four, five, six or more days. The course of treatment may also be repeated as in chronic administration. The repeated administration may be at the same dose or at different doses. For example, the anti-CD 38 antibody in the methods of the invention can be administered by intravenous infusion at 8mg/kg or at 16mg/kg at weekly intervals for 8 weeks, followed by 8mg/kg or 16mg/kg once every two weeks for a further 16 weeks, followed by 8mg/kg or 16mg/kg once every four weeks.

The anti-CD 38 antibody can be administered in the methods of the invention by maintenance therapy, such as, for example, once a week for 6 months or longer.

For example, the anti-CD 38 antibody in the methods of the invention can be provided as a daily dose in an amount of about 0.1-100mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 1.0, 1.5, 2, 1.5, 2, 5,9, 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks for at least one day of day 1,2, 3,4, 5, 6, 14, 1,6, 1.1, 1.9, 2, 1.1, 6, 4, or 2 hours, or any combination thereof, at least one day after initiation of treatment, or alternatively for at least one week 1,2, 3, 30, 31, 32, or 20 weeks, or any combination thereof, 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/day.

The anti-CD 38 antibodies in the methods of the invention can also be administered prophylactically to reduce the risk of acquiring cancer, delay the onset of events in the progression of cancer, and/or reduce the risk of relapse after remission of cancer. This may be particularly useful for patients whose tumors are difficult to locate and are known to have due to other biological factors.

The anti-CD 38 antibodies in the methods of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has proven effective for conventional protein formulations and well known lyophilization and reconstitution techniques can be employed.

In the methods of the invention, the anti-CD 38 antibody is administered in combination with a survivin inhibitor.

In the methods of the invention, an anti-CD 38 antibody is administered in combination with the survivin inhibitor YM 155.

YM155 used in the method of the present invention is easily obtained according to the production process disclosed in International patent publication No. WO01/60803 and WO 2004/092160.

YM155 may be administered orally or parenterally or intravenously. In this regard, injectable formulations for intravenous administration include those containing sterile aqueous or nonaqueous solutions, suspensions and emulsions. The aqueous solvent includes, for example, distilled water for injection and physiological saline. Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, polysorbate 80, and the like. Such compositions may contain other tonicity adjusting agents, preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizing agents and solubilizing agents. For example, these compositions may be sterilized by bacterial filter filtration, sterilizer, or blending of radiation. Alternatively, sterile solid compositions may be prepared and dissolved or suspended in sterile water or sterile solvent for injection immediately prior to use.

In intravenous administration, YM155 may be, for example, at 0.1-20mg/m²Daily (e.g., at 1-10 mg/m)²Daily) in one or more doses per day or by infusion (continuous instillation). YM155 may be 3-10mg/m²Daily continuous infusion is for 4 to 20 days, e.g. 4 to 14 days, or 5,7, 10 or 14 and/or 7 days. When further administration is continued, the following drug cycles may be employed: which includes a drug holiday period of 1 to 2 months, 7 to 21 or 14 days after the end of the drug period described above. Alternatively, YM155 may be 3-8mg/m²The dose/day was administered continuously by infusion for 7 days, then entered into a drug holiday for 14 days; the cycle is repeated for one cycle depending on the conditions. The administration frequency, dose, number of infusions, drug cycle and the like can be suitably determined depending on the individual conditions concerning the kind of the anticancer agent, the state of the patient, age, sex and the like.

In the methods of the invention, the combination of the anti-CD 38 antibody and the survivin inhibitor may be administered within any convenient time frame. For example, the anti-CD 38 antibody and the survivin inhibitor may be administered to the patient on the same day. However, the anti-CD 38 antibody and the survivin inhibitor may also be administered on alternate days or weeks or months, etc. In some methods, the anti-CD 38 antibody and the survivin inhibitor may be administered close enough in time that they are simultaneously present at detectable levels in the patient being treated (e.g., in serum). In some methods, a course of the anti-CD 38 antibody consisting of multiple doses over a period of time is followed or preceded by a course of the survivin inhibitor consisting of multiple doses. A recovery period of 1,2 or several days or weeks may be used between administration of the anti-CD 38 antibody and the survivin inhibitor.

The anti-CD 38 antibody in combination with a survivin inhibitor may be administered in conjunction with any form of radiation therapy and/or surgery, including external irradiation, Intensity Modulated Radiation Therapy (IMRT) and/or any form of radiosurgery, including gamma knife, radio knife, linac, and interstitial radiation (e.g., implantation of radioactive seeds, GliaSite balloons).

Subcutaneous administration of a pharmaceutical composition comprising an antibody that specifically binds CD38 and hyaluronidase

The anti-CD 38 antibody can be administered subcutaneously as a pharmaceutical composition comprising an anti-CD 38 antibody and hyaluronidase.

The concentration of the anti-CD 38 antibody may be about 20mg/ml in a pharmaceutical composition administered subcutaneously.

A pharmaceutical composition for subcutaneous administration may comprise between about 1200mg to 1800mg of an anti-CD 38 antibody.

A pharmaceutical composition for subcutaneous administration may comprise about 1,200mg of an anti-CD 38 antibody.

A pharmaceutical composition for subcutaneous administration may comprise about 1,600mg of an anti-CD 38 antibody.

A pharmaceutical composition for subcutaneous administration may comprise about 1,800mg of the anti-CD 38 antibody.

A pharmaceutical composition for subcutaneous administration may comprise between about 30,000U to 45,000U hyaluronidase.

A pharmaceutical composition for subcutaneous administration may comprise about 1,200mg of an anti-CD 38 antibody and about 30,000U of hyaluronidase.

A pharmaceutical composition for subcutaneous administration may comprise about 1,800mg of an anti-CD 38 antibody and about 45,000U of hyaluronidase.

A pharmaceutical composition for subcutaneous administration may comprise about 1,600mg of an anti-CD 38 antibody and about 30,000U of hyaluronidase.

A pharmaceutical composition for subcutaneous administration may comprise about 1,600mg of an anti-CD 38 antibody and about 45,000U of hyaluronidase.

A pharmaceutical composition for subcutaneous administration may comprise a peptide having SEQ ID NO: 23, or a hyaluronidase rHuPH 20.

rHuPH20 is a recombinant hyaluronidase (rHuPH 20)Recombinant) described in international patent publication No. wo 2004/078140.

Hyaluronidase is an enzyme that degrades hyaluronic acid (EC 3.2.1.35) and reduces the viscosity of hyaluronic acid in the extracellular matrix, thereby increasing tissue permeability.

SEQ ID NO：23

MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL

The pharmaceutical composition comprising the anti-CD 38 antibody and hyaluronidase can be administered repeatedly after one, two, three, four, five, six, seven, two, three, four, five, six or more days. The course of treatment may also be repeated as in chronic administration. The repeated administration may be at the same dose or at different doses. For example, a pharmaceutical composition comprising an anti-CD 38 antibody and hyaluronidase can be administered once a week for eight weeks, then once every two weeks for 16 weeks, and then once every four weeks. The pharmaceutical composition to be administered may comprise about 1,200mg of an anti-CD 38 antibody and about 30,000U of hyaluronidase, wherein the concentration of antibody that specifically binds to CD38 in the pharmaceutical composition is about 20 mg/ml. The pharmaceutical composition to be administered may comprise about 1,800mg of the anti-CD 38 antibody and about 45,000U of hyaluronidase. The pharmaceutical composition to be administered may comprise about 1,600mg of anti-CD 38 antibody and about 30,000U of hyaluronidase. The pharmaceutical composition to be administered may comprise about 1,600mg of anti-CD 38 antibody and about 45,000U of hyaluronidase.

The pharmaceutical composition comprising the anti-CD 38 antibody and hyaluronidase can be administered subcutaneously to the abdominal region.

The pharmaceutical composition comprising the anti-CD 38 antibody and the hyaluronidase enzyme can be administered in a total volume of about 80ml, 90ml, 100ml, 110ml, or 120 ml.

For administration, 25mM sodium acetate, 60mM sodium chloride, 140mM D-mannitol, 20mg/mL anti-CD 38 antibody in 0.04% polysorbate 20(pH5.5) can be mixed with 10mM L-histidine, 130mM NaCl, 10mM L-methionine, 1.0mg/mL rHuPH20(75-150kU/mL) in 0.02% polysorbate 80(pH6.5), and the mixture can then be administered to a subject.

Other embodiments of the invention

1. An anti-CD 38 antibody for use in treating a subject having a CD38 positive hematological malignancy in combination with a survivin inhibitor.

2. A survivin inhibitor for use in combination with an anti-CD 38 antibody in treating a subject having a CD38 positive hematological malignancy.

3. A combination of an anti-CD 38 antibody and a survivin inhibitor for use in treating a subject having a CD38 positive hematological malignancy.

4. The anti-CD 38 antibody for use according to embodiment 1, the survivin inhibitor for use according to embodiment 2 or the combination according to embodiment 3, wherein the CD38 positive hematological malignancy is Multiple Myeloma (MM), Acute Lymphocytic Leukemia (ALL), non-hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt's Lymphoma (BL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), Acute Myeloid Leukemia (AML) or Chronic Lymphocytic Leukemia (CLL).

5. The anti-CD 38 antibody for use according to embodiment 1 or 4, the survivin inhibitor for use according to embodiment 2 or 4, or the combination according to embodiment 3 or 4, wherein the CD38 positive hematological malignancy is a plasma cell disease.

6. The anti-CD 38 antibody for use according to embodiment 1,4 or 5, the survivin inhibitor for use according to embodiment 2,4 or 5, or the combination according to embodiment 3,4 or 5, wherein the plasma cell disease is light chain Amyloidosis (AL), Multiple Myeloma (MM) or fahrenheit macroglobulinemia.

7. The anti-CD 38 antibody for use according to any one of embodiments 1 or 4-6, the survivin inhibitor for use according to any one of embodiments 2 or 4-6, or the combination according to any one of embodiments 3 or 4-6, wherein the plasma cell disease is MM.

8. The anti-CD 38 antibody for use according to any one of embodiments 1 or 4-7, the survivin inhibitor for use according to any one of embodiments 2 or 4-7, or the combination of any one of embodiments 3 or 4-7, wherein the anti-CD 38 antibody induces CD 38-positive cell killing by antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or apoptosis.

9. The anti-CD 38 antibody for use according to any one of embodiments 1 or 4-8, the survivin inhibitor for use according to any one of embodiments 2 or 4-8, or the combination according to any one of embodiments 3 or 4-8, wherein the anti-CD 38 antibody is

IgG1, IgG2, IgG3, or IgG4 isotype; or

Igg1 isoforms.

10. The anti-CD 38 antibody for use according to any one of embodiments 1 or 4-9, the survivin inhibitor for use according to any one of embodiments 2 or 4-9, or the combination according to any one of embodiments 3 or 4-9, wherein the anti-CD 38 antibody is

a. And a polypeptide comprising SEQ ID NO: 4 and the heavy chain variable region (VH) of SEQ ID NO: 5 light chain variable region (VL) antibodies compete for binding to CD 38;

b. region SKRNIQFSCKNIYR (SEQ ID NO: 2) and region EKVQTLEAWVIHGG (SEQ ID NO: 3) that bind to human CD38(SEQ ID NO: 1);

c. comprises the amino acid sequences shown as SEQ ID NO: 6. 7 and 8 heavy chain complementarity determining region (HCDR)1(HCDR1), 2(HCDR2) and 3(HCDR3) sequences;

d. comprises the amino acid sequences shown as SEQ ID NO: 9. 10 and 11 light chain complementarity determining regions

(LCDR)1(LCDR1), 2(LCDR2) and 3(LCDR3) sequences;

e. comprises the amino acid sequences shown as SEQ ID NO: 6. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of 7,8, 9,10 and 11;

f. comprises a polypeptide comprising a nucleotide sequence substantially identical to SEQ ID NO: 4, and a heavy chain variable region (VH) comprising an amino acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 5 with 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of the light chain variable region (VL);

g. comprises the amino acid sequence of SEQ ID NO: 4 and the heavy chain variable region (VH) of SEQ ID NO: 5 light chain variable region (VL);

h. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising:

seq ID NO: 14 and SEQ ID NO: 15 VL;

seq ID NO: 16 and the VH of SEQ ID NO: 17 VL;

iii.SEQ ID NO: 18 and SEQ ID NO: 19 VL; or

Seq ID NO: 20 and SEQ ID NO: 21 VL; or

i. Comprises the following steps:

seq ID NO: 14 and SEQ ID NO: 15 VL;

seq ID NO: 16 and the VH of SEQ ID NO: 17 VL;

iii.SEQ ID NO: 18 and SEQ ID NO: 19 VL; or

Seq ID NO: 20 and SEQ ID NO: 21 VL of.

11. The anti-CD 38 antibody for use according to any one of embodiments 1 or 4-10, the survivin inhibitor for use according to any one of embodiments 2 or 4-10, or the combination according to any one of embodiments 3 or 4-10, wherein the anti-CD 38 antibody comprises the amino acid sequence of SEQ ID NO: 4 and the heavy chain variable region (VH) of SEQ ID NO: 5 (VL).

12. The anti-CD 38 antibody for use according to any one of embodiments 1 or 4-11, the survivin inhibitor for use according to any one of embodiments 2 or 4-11, or the combination according to any one of embodiments 3 or 4-11, wherein the survivin inhibitor is a small molecule or a polynucleotide or a vaccine.

13. The anti-CD 38 antibody for use according to any one of embodiments 1 or 4-12, the survivin inhibitor for use according to any one of embodiments 2 or 4-12, or the combination according to any one of embodiments 3 or 4-12, wherein the survivin inhibitor is YM 155.

14. The anti-CD 38 antibody for use according to any one of embodiments 1 or 4-13, the survivin inhibitor for use according to any one of embodiments 2 or 4-13, or the combination according to any one of embodiments 3 or 4-13, wherein the anti-CD 38 antibody and the survivin inhibitor are administered simultaneously, sequentially or separately.

15. An anti-CD 38 antibody for use in treating a subject having a CD38 positive hematological malignancy in combination with a survivin inhibitor, wherein the anti-CD 38 antibody comprises the amino acid sequences of SEQ ID NOs: 6. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of 7,8, 9,10 and 11.

16. The anti-CD 38 antibody for use according to embodiment 15, wherein the anti-CD 38 antibody comprises SEQ ID NO: 4 and the VH of SEQ ID NO: 5 VL.

17. The anti-CD 38 antibody for use according to embodiment 15 or 16, wherein the anti-CD 38 antibody is of the IgG1 isotype.

18. The anti-CD 38 antibody for use according to any one of embodiments 15-17, wherein the anti-CD 38 antibody comprises the amino acid sequence of SEQ ID NO: 12 and SEQ ID NO: 13, light chain.

19. The anti-CD 38 antibody for use according to any one of embodiments 15-18, wherein the anti-CD 38 antibody and the survivin inhibitor are administered simultaneously, sequentially or separately.

20. The anti-CD 38 antibody for use according to any one of embodiments 15-19, wherein the anti-CD 38 antibody is administered intravenously.

21. The anti-CD 38 antibody for use according to any one of embodiments 15-19, wherein the anti-CD 38 antibody is administered subcutaneously in a pharmaceutical composition comprising an anti-CD 38 antibody and hyaluronidase.

22. The anti-CD 38 antibody for use according to embodiment 21, wherein the hyaluronidase is SEQ ID NO: rHuPH20 of 23.

23. The anti-CD 38 antibody for use according to any one of embodiments 15-22, wherein the CD 38-positive hematological malignancy is multiple myeloma.

While the present invention has been generally described, embodiments of the invention are further disclosed in the following examples, which should not be construed as limiting the scope of the claims.

Materials and methods

Cells and cell culture

Bone marrow mononuclear cells (BM-MNC) and Peripheral Blood Mononuclear Cells (PBMC)

According to the declaration of helsinki, Bone Marrow (BM) aspirates were collected from MM patients or healthy individuals and Peripheral Blood (PB) was collected from healthy individuals using protocols and procedures approved by the institutional medical ethics committee. Healthy Donor (HD) -PBMC and BM-MNC were isolated from PB samples or BM aspirates, respectively, by Ficoll-Hypaque density gradient centrifugation. PBMC were used directly as effector cells in ADCC experiments; BM-MNC were cryopreserved until use.

Multiple Myeloma (MM) cell line

At 37 ℃ and with 5% CO₂Luciferase (Luc) -transduced human MM cell lines RPMI-8226 and UM9 were maintained in RPMI1640(Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and antibiotics (penicillin/streptomycin, Life Technologies) in a humid atmosphere of (r).

Bone Marrow Stromal Cells (BMSC)

Adherent stromal cells were isolated and cultured by plastic adhesion from BM-MNC of healthy individuals (hBMSC) or MM patients (pBMSC). Cells were cultured in optimem (invitrogen) containing 5% platelet lysate, heparin and antibiotics. Hbmscs were used in experiments up to passage 6, while pbmscs were used after passage 1 or passage 2.

Reagent

YM155(Sepantronium Bromide, 4, 9-dihydro-1- (2-methoxyethyl) -2-methyl-4, 9-dioxo-3- (2-pyrazinylmethyl) -1H-naphthalen [2, 3-d ] imidazolium Bromide, CAS 781661-94-7) (Selleck Chemicals) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1mM and stored in aliquots until use. YM155 was diluted in culture medium to the concentration specified in each experiment.

YM 155; formula I:

compartment-specific bioluminescence based antibody-dependent cell-mediated targeting multiple myeloma cell lines Cytotoxicity (ADCC) ("cell cytotoxicity assay based on compartment-specific BLI")

Mixing hBMSC at 1 × 10⁴The density of individual cells/well was seeded in white opaque flat bottom 96-well plates (Costar) containing 100. mu.l of medium. After six hours of adhesion, the luc transduced MM cell line was used at 1X 10⁴The density of individual cells/well was added to BMSC coated or uncoated wells. In the experiment for testing YM155, YM155 was added at a specified concentration together with MM cells. After 16 to 20 hours, daratumab was added at the indicated concentration and left at room temperature for 15 minutes. PBMCs freshly isolated from healthy individuals were then added as effector cells at the indicated effector target ratio. 4 hours after PBMC addition 125. mu.g/ml of Methoxyfluoralin (Promega) was added and the bioluminescent signal emitted by the surviving MM cells was measured using a luminometer (SpectraMax, Molecular Devices) over 20 minutes. The percent survival of MM cells was calculated using the formula: survival% (% mean bioluminescence signal in the absence of PBMC/mean bioluminescence signal in the presence of PBMC) × 100%. In these assays, viability of MM cells is a direct reflection of ADCC-mediated lysis and is associated with classical chromium release assays, such as McMillin et al, Nat Med 16: 483-.

FACS-based ADCC assay in multiple myeloma BM-MNC

Use of CD 138-derived antibodies in FACS-based ADCC assays⁺MM cell is 15% -35% of frozen BM-MNC of MM patients. Cells were thawed and cultured in RPMI containing 10% HS. After 16 to 20 hours, BM-MNC were counted by trypan blue exclusion and counted at 4X 10⁴The density of individual cells/well was seeded in 96-well round bottom plates. Daratumab and/or YM155 was added to the wells as specified for each experiment. After 24 hours, cells were stained with fluorescently conjugated anti-CD 138, anti-CD 38, anti-CD 56, and anti-CD 3 antibodies, and primary CD138 in BM-MNC was determined by FACS as described previously⁺Viability of MM cells (Groen et al, Blood 120: e9-e16, 2012). The percentage of lysis of MM cells was deduced using the formula: lysed cell% -1- (surviving CD138 in treated wells)⁺Surviving CD138 in cell count/control wells⁺Cell count) x 100%.

Flow cytometry

To determine the level of CD38 expression on MM cells, MM cells were cultured alone or with BMSCs and incubated with CD38 fluorescein-conjugated antibody. In addition, cells were stained with CD105 as a marker for BMSC. CD38 expression was determined on CD105 negative cells by the FACS described (de Haart et al, Clin Cancer Res 19: 5591-601, 2013).

In vivo tumor targeting experiments

Loaded with Luc in vitro⁺MM cell line UM9 (1X 10)⁶Individual cells/scaffold) was coated with a composite scaffold consisting of three 2 to 3mm biphasic calcium phosphate particles, which were then implanted into RAG2 as previously described^-/-γc^-/-In mice (Groen et al, Blood 120: e9-e16, 2012). Ten days after implantation, mice with tumors growing in the scaffolds were treated with vehicle control, darunavir + PBS or darunavir + YM 155. In addition, each mouse (including control group) also received T cell depleted HD-PBMC (5X 10)⁶Individual cells) as a source of human NK cells to induce ADCC. PBS and YM155 diluted in PBS were administered using a subcutaneous infusion pump (Alzet 1007D) delivering 1mg/kg/D of drug continuously. The pump was removed after 10 days. BLI was performed as described previously (Spaapen et al, C1in Cancer Res 16: 5481-88, 2010; Rozemuller et al, Haematologica 93: 1049-57，2008)。

Enzyme-linked immunosorbent assay (ELISA) for granzyme B

The cell-free supernatant was assayed for granzyme b (gzb) content using a commercial ELISA kit (Pelipair, Sanquin, Amsterdam, NL) according to the manufacturer's instructions.

Example 1 preservation of bone marrow stromal cells to confer antibody-dependent cellular cytotoxicity against multiple myeloma cells Protective action

Since stromal cells of the Bone Marrow (BM) microenvironment protect MM cells from CTL and NK-mediated cytotoxicity, it was evaluated whether similar protection against antibody-dependent cellular cytotoxicity (ADCC) induced by darunavir was developed.

Testing of healthy donor BMSCs on two CD38 with successive concentrations of Darandomu single antibody in the presence or absence of HD-PBMC as effector cells in a compartment-specific BLI-based cytotoxicity assay⁺ADCC induction of luciferase-transduced MM cell lines (i.e., UM9 and RPMI). In the absence of BMSC, daratumab mediates ADCC in a dose-dependent manner in two MM cell lines. Both cell lines were less sensitive to darunavir-induced ADCC in the presence of BMSC. Figure 1A shows the effect of BMSCs on darunavir-induced ADCC in UM9 cells, and figure 1B shows the effect of BMSCs on darunavir-induced ADCC in RPMI-8226 cells.

The ability of BMSCs to protect primary MM cells from darunavir-induced ADCC was also assessed in a FACS-based ADCC assay using the above method. In the assay, will contain at least 15% CD138⁺Malignant plasma cell BM-MNC and a sufficient number of autologous effector NK cells are incubated with Darandomia monoclonal antibody to induce ADCC. BM-MNCs were tested alone or in co-culture with autologous BMSCs to assess the effects of BMSCs. FACS-based viability assays to determine CD138 after 24 hours of culture⁺Cells were survived and lysis rates were calculated. In both donors tested, the primary MM cells induced less efficiently ADCC in the presence of autologous MM-BMSCs (fig. 2A and 2B), indicating a matrix of the tumor microenvironmentThe cells induced resistance to the darunavir monotherapy.

Example 2 BMSC-induced ADCC inhibition not caused by downregulation of CD38 or NK cell inhibition。

Possible changes in CD38 surface expression and NK cell activation were evaluated to understand the BMSC-mediated protection mechanism against ADCC.

MM cell lines UM9 and RPMI-8226 were cultured in the presence or absence of healthy donor BMSCs. Co-culture of MM cells with BMSCs did not down-regulate CD38 expression levels on either MM cell line (data not shown).

Since BMSCs are known to produce multiple immunosuppressive factors such as IDO, TGF- β or PGE-2, the protection of BMSCs against ADCC may be due to inhibition of NK cell activation, which in turn reduces their ability to degranulate and release granzyme B and perforin in the immune synapse, killing their targets. To this end, the effect of BMSC on NK cell activation by darunavir was determined using darunavir-mediated granzyme B secretion as a marker of NK cell activity. Levels of granzyme B in the supernatant were generally higher in the presence of BMSC (data not shown). Thus, BMSC-mediated protection against ADCC may not be due to NK cell inhibition.

Example 3 survivin inhibitor abrogated BMSC mediated protection against ADCC and with Daramose Together, the antibodies provide synergistic ADCC-mediated killing of multiple myeloma cells

BMSCs have been shown to protect MM cells from CTL lysis by upregulating survivin in MM cells. The modulation of survivin was evaluated using YM155, a small molecule inhibitor of survivin, according to the possible mechanism of BMSC-mediated protection against ADCC induced by darunavir.

The effect of YM155 on NK cell viability was first assessed. BM-MNC from MM patients were cultured for 24 hours in the presence of different doses of YM 155. MM cells (CD 138) by FACS assay⁺Cells) and NK cells (CD 3)^-CD138^-CD56⁺Cells) are detected. In already in rightNK cells were not affected at YM155 doses where MM cells showed some toxicity (fig. 3).

The effect of daratumab, YM155, or a combination of daratumab and YM155 was evaluated in RPMI-8226 cells and two MM patient samples using a concentration of YM155 that showed no toxicity to NK cells.

In the assay, for RPMI-8226 cells, daratumab and YM155 were used at concentrations of 0.3. mu.g/ml and 1nM, respectively; for MM patient samples, daratumab and YM155 were used at concentrations of 1. mu.g/ml and 120nM, respectively. Healthy donor PBMC were used as effector cells at an effector to target ratio of 40: 1 for RPMI-8226 cells and 30: 1 for MM patient samples.

In RPMI-8226 cells, daratumab alone or YM155 alone induced about 20% cell lysis in the absence of BMSC. In the presence of BMSC, darunavir induced about 10% cell lysis, while YM155 had no effect. The combination of daratumab and YM155 provided a synergistic effect, inducing about 50% cell lysis in the absence of BMSC and about 45% in the presence of BMSC (fig. 4A). The synergy in BMSC was about 5-fold for the combination of daratumab and YM 155. Similarly, the combination of daratumab and YM155 provided synergy in MM patient sample 1 (fig. 4B) with 120nM YM155, moderate synergy in MM patient sample 2 (fig. 4C) with lower amounts of YM155(64nM) and in the combination sample of MM cells derived from 4 patients (fig. 4D). Thus, YM155 abrogates BMSC protection from darunavir-mediated ADCC in MM cells and cell lines.

Thus, survivin upregulation may be an important mechanism to inhibit ADCC-mediated killing of MM cells, which can be prevented by drug-regulated survivin.

Example 4 in vivo anti-tumor Effect of Daramucimumab and YM155 combination therapy

In RAG2^-/-gc^-/-The expression was tested in a preclinical xenograft model in miceIn vivo correlation of the combination of ramucimumab and YM155, where MM tumors were grown in a humanized BM-like microenvironment created by subcutaneous implantation of a ceramic scaffold coated with human BMSCs. A composite scaffold coated with human MSCs and loaded with the luciferase-transduced MM cell line UM9 was subcutaneously implanted with RAG2^-/-γc^-/-The back of the mouse (4 scaffolds per mouse). Ten days after implantation, the growing tumors were visualized and quantified by BLI. Different groups of mice were then treated with vehicle controls (control) or with daraiumab, YM155, or daraiumab + YM155 (n ═ 4). YM155 or its vehicle, PBS, was delivered at a rate of 1mg YM 155/kg/day for 10 days using a subcutaneous infusion pump. Each mouse (including control group) received T cell depleted HD-PBMC (5X 10)⁶Individual cells) as a source of human NK cells to induce ADCC. Mice were monitored weekly by BLI. Fig. 5 shows the relative tumor growth for each group. Statistical differences between mice treated with darunavir single antibody and mice treated with darunavir + YM155 were calculated using the Mann-Whitney U test. Daratumab has marginal effects on tumor growth. YM155 has a more significant anti-MM effect, and in addition, exhibits a stronger synergistic effect with darunavir, and can significantly improve the anti-MM effect. These results indicate that clinical benefit can be expected from the combination of daratumab with the survivin inhibitor YM 155.

The presented results indicate that inhibition of survivin levels with small molecule YM155 not only improves darunavir-mediated ADCC in the absence of BMSC, but more importantly abolishes ADCC resistance induced by BMSC. Addition of YM155 to darunavir also showed enhanced anti-tumor effects in the absence of BMSC, suggesting that YM 155-darunavir combination therapy has potential benefits even if MM cells are not in direct contact with BMSC, such as in plasma cell leukemia. Furthermore, the significant improvement in ADCC (up to four-fold improvement in MM cell lysis) in the presence of BMSC indicates that, for MM cells present in BM, a greater benefit of combining darunavir therapy with YM155 can be achieved. It was also shown that YM155 treatment does not negatively interfere with NK cell function or viability, a prerequisite for considering the clinical application of this combination therapy.

Although efficacy of daratumab in combination with YM155 was demonstrated in multiple myeloma, it can be concluded that this combination therapy is also beneficial for other hematological tumors that express CD38, especially those that are predominantly present in BM. In this regard, AML is an outstanding candidate because AML cells express not only high levels of CD38, but also high levels of survivin, which is a predictor of poor clinical outcome. Another potential candidate for combination therapy may be CLL, since CLL cells have high survivin expression in BM and express CD38 in some patients. High CD38 and survivin expression in about 50% of non-hodgkin lymphomas, making this disease also relevant for efficacy assessment of the YM 155-darunavir monotherapy combination. In conclusion, the combination of daratumab and YM155 can be widely applied to various hematological tumors.

Claims

1. Use of an anti-CD 38 antibody and a survivin inhibitor in the manufacture of a medicament for treating a subject with Multiple Myeloma (MM), wherein the anti-CD 38 antibody comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NOs 6,7 and 8, respectively, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NOs 9,10 and 11, respectively, and is of the IgG1 isotype, and wherein the survivin inhibitor is YM 155.

2. The use of claim 1, wherein the anti-CD 38 antibody competes for binding to CD38 with an antibody comprising the heavy chain variable region (VH) of SEQ ID NO 4 and the light chain variable region (VL) of SEQ ID NO 5.

3. The use of claim 1, wherein the anti-CD 38 antibody binds to region SKRNIQFSCKNIYR and region EKVQTLEAWVIHGG of human CD38 of SEQ ID NO 1.

4. The use of claim 1, wherein the anti-CD 38 antibody comprises the VH of SEQ ID NO 4 and the VL of SEQ ID NO 5.

5. The use of claim 1, wherein the anti-CD 38 antibody comprises the heavy chain amino acid sequence of SEQ ID NO 12 and the light chain amino acid sequence of SEQ ID NO 13.

6. The use of any one of claims 1-5, wherein the anti-CD 38 antibody induces CD38 positive cell killing by Antibody Dependent Cellular Cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP), Complement Dependent Cytotoxicity (CDC), or apoptosis.

7. The use of any one of claims 1-5, wherein the anti-CD 38 antibody and the survivin inhibitor are to be administered simultaneously.

8. The use of claim 7, wherein the anti-CD 38 antibody is to be administered intravenously.

9. The use of claim 7, wherein the anti-CD 38 antibody is to be administered subcutaneously and wherein the medicament further comprises hyaluronidase.

10. The use of any one of claims 1-5, wherein the anti-CD 38 antibody and the survivin inhibitor are to be administered separately.

11. The use of claim 10, wherein the anti-CD 38 antibody and the survivin inhibitor are to be administered sequentially.

12. The use of claim 10, wherein the anti-CD 38 antibody is to be administered intravenously.

13. The use of claim 10, wherein the anti-CD 38 antibody is to be administered subcutaneously and wherein the medicament further comprises hyaluronidase.

14. The use of claim 11, wherein the anti-CD 38 antibody is to be administered intravenously.

15. The use of claim 11, wherein the anti-CD 38 antibody is to be administered subcutaneously and wherein the medicament further comprises hyaluronidase.