Anti-cd38 antibodies for treatment of light chain amyloidosis and other cd38-positive hematological malignancies

Abstract

This invention relates to a method for treating light chain amyloidosis and other CD38-positive hematologic malignancies with anti-CD38 antibodies.

Description

Technical Field

[0001] [

Cross-reference to related applications

[0002] This application claims priority to U.S. patent application Ser. No. 15 / 160,476, filed May 20, 2016, and International Patent Application No. PCT / US16 / 33544, filed May 20, 2016, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to methods for treating light-chain amyloidosis and other CD38-positive hematological malignancies. Prior Art

[0004] B-cell malignancies include B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Birch's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, multiple myeloma, Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma, and AIDS-related non-Hodgkin's lymphoma. B-cell malignancies account for over 85% of diagnosed lymphomas.

[0005] Multiple myeloma (MM) is characterized by the latent accumulation of secretory plasma cells in the bone marrow, which have a low proliferation rate and a prolonged lifespan. The disease ultimately attacks the bones 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. The incidence of MM increases in the aging population, with the median age at diagnosis being approximately 61 years.

[0006] Light-chain amyloidosis (AL), also known as systemic amyloidosis, is a clonal plasma cell disorder in which misfolded immunoglobulin light chain fragments accumulate in tissues. Single plasma cells in the bone marrow produce misfolded immunoglobulin light chains, which accumulate in tissues and cause toxicity in vital organs, leading to organ failure and death (Comenzo et al., Leukemia 26:2317-25, 2012). Clinical features depend on the organs involved; amyloidosis often manifests in the kidneys, heart, skin, nervous system, and soft tissues (e.g., tongue) (Merlini and Belotti, NEJM, 349:583-596, 2003), leading to albuminuria and renal failure, heart failure, arrhythmias, sudden cardiac death, hepatomegaly, abdominal distension, early satiety, paresthesias, dysesthesias, orthostatic hypotension, constipation, or diarrhea (Chaulagain and Comenzo; Curr Hematol Malig Rep 8:291-8, 2013).

[0007] CD38 is a type II membrane protein that plays a role in receptor-mediated adhesion and signaling. It also mediates calcium mobilization through its extracellular enzymatic activity, catalyzing the formation of cyclic ADP-ribose (cADPR) from NAD+ and hydrolyzing cADPR to ADP-ribose (ADPR). CD38 mediates interleukin secretion and lymphocyte activation and proliferation (Funaro et al., J Immunology 145:2390-6, 1990; Guse et al., Nature 398:70-3, 1999). Its NAD glycohydrolase activity regulates extracellular NAD+ levels, which are involved in regulating the regulatory T cell compartment (Adriouch et al., J Immunology 14:1284-92, 2012; Chiarugi et al., Nature Reviews 12:741-52, 2012).

[0008] CD38 is expressed on malignant plasma cells in multiple myeloma and is implicated in various hematological malignancies.

[0009] Current treatments for light-chain amyloidosis and multiple myeloma include various chemotherapies with or without autologous stem cell transplantation. However, both diseases remain largely incurable. Therefore, additional therapeutic agents for multiple myeloma and light-chain amyloidosis are needed. Summary of the Invention

[0010] The present invention provides a method for treating a patient suffering from a CD38-positive hematological malignancy, comprising administering to the patient in need thereof an anti-CD38 antibody for a period of time sufficient to treat the CD38-positive hematological malignancy, wherein the patient is undergoing hematopoietic stem cell transplantation (HSCT).

[0011] The present invention also provides a method for treating a patient suffering from light-chain amyloidosis (AL), comprising administering an anti-CD38 antibody to the patient in need thereof for a period of time sufficient to treat AL.

[0012] The present invention also provides a method for treating a patient suffering from light-chain amyloidosis (AL), comprising administering to the patient in need thereof an anti-CD38 antibody for a period of time sufficient to treat AL, wherein the patient is undergoing hematopoietic stem cell transplantation (HSCT). Simple diagram description

[0013] Figure 1 shows the relative expression of CD38, CD32B, IL6R, gp130, and CD16 in the transcriptional profile of CD138+ clonal plasma cells from newly diagnosed AL patients (n = 16 patients with AL, GEO GSE24128). LnQLE: natural logarithm of the quantitative level of expression. [Figure 2] shows the percentage (%) of NK cells in peripheral blood mononuclear cells of AL patients 3 weeks after stem cell transplantation (SCT). [Figure 3] shows that CD38 is expressed on CD34+ hematopoietic progenitor cells. Figure 4A shows that DARZALEX™ (daratumumab) has no effect on the proliferation of thawed, cryopreserved, unselected mobilized blood progenitor cells. This was assessed by the ability of progenitor cells to form similar numbers of colonies in the presence of 500 ng or 1000 ng / ml DARZALEX™ (daratumumab) compared to isotype controls. In this assay, colony-forming units (CFU-GM) were assessed after 14 days of culture in methylcellulose in the presence of DARZALEX™ (daratumumab), isotype controls, or a no-antibody control, and the percentage of colonies formed was plotted against that of the no-antibody control. *p<0.05, paired t-test. All assays were performed in triplicate, and three independent experiments were performed using progenitor cell samples from three different patients. Data are shown as mean ± SD. Figure 4B shows that DARZALEX™ (daratumumab) has no effect on the proliferation of thawed, cryopreserved, unselected mobilized blood progenitor cells. This was assessed by the ability of progenitor cells to form similar numbers of colonies in the presence of 500 ng or 1000 ng / ml DARZALEX™ (daratumumab) compared to an isotype control. In this assay, blast-forming unit erythroid (BFU-E) was assessed after 14 days of growth in methylcellulose in the presence of DARZALEX™ (daratumumab), an isotype control, or a no-antibody control, and the percentage of colonies formed was plotted against that of the no-antibody control. All assays were performed in triplicate, and three independent experiments were performed using progenitor cell samples from three different patients. Data are shown as mean ± SD. Figure 5A shows that DARZALEX™ (daratumumab) has no effect on the proliferation of fresh, unselected mobilized blood progenitor cells, assessed by their ability to form similar numbers of colonies in methylcellulose in the presence of 500 ng or 1000 ng / ml DARZALEX™ (daratumumab) compared to isotype controls. Colony formation was measured as CFU-GM on day 14, and the percentage of colonies formed was plotted against the no-antibody control. All assays were performed in triplicate, and three independent experiments were performed using progenitor cell samples from three different patients. Data are shown as mean ± SD. Figure 5B shows that DARZALEX™ (daratumumab) has no effect on the proliferation of fresh, unselected mobilized blood progenitor cells, assessed by their ability to form similar numbers of colonies in methylcellulose in the presence of 500 ng or 1000 ng / ml DARZALEX™ (daratumumab) compared to isotype controls. Colony formation was measured as BFU-E on day 14, and the percentage of colonies formed was plotted against the no-antibody control. All assays were performed in triplicate, and three independent experiments were performed using progenitor cell samples from three different patients. Data show mean ± SD. Figure 6A shows that DARZALEX™ (daratumumab) does not kill CD34-selected hematopoietic progenitor cells by CDC, despite high CD38 expression on the cells. CD34+ cells were cultured for 1 hour with high-complement serum containing either no antibody (CTL), 500 ng / ml DARZALEX™ (daratumumab) (Dara), or 500 ng / ml isotype control (Iso), after which the cells were plated on semisolid culture medium. Colony formation was measured as CFU-GM per 500 CD34-selected cells on day 14. Results are from triplicate assays of cells from one patient with MM and two patients with AL. Figure 6B shows that DARZALEX™ (daratumumab) does not kill CD34-selected hematopoietic progenitor cells by CDC, despite high CD38 expression on the cells. CD34+ cells were cultured for 1 hour with high-complement serum containing either no antibody (CTL), 500 ng / ml DARZALEX™ (daratumumab) (Dara), or 500 ng / ml isotype control (Iso), after which the cells were plated on semisolid culture medium. Colony formation was measured as BFU-E per 500 CD34-selected cells on day 14. There were significantly more BFU-E in the wells treated with DARZALEX™ (daratumumab), achieving statistical significance. Results are from triplicate assays of cells from one patient with MM and two patients with AL. (*p<0.01). Figure 7A shows that DARZALEX™ (daratumumab) has no adverse effects on fresh CD34-selected granulocyte-monocyte precursor cells. Isolated CD34+ cells were cultured in medium lacking the following: antibody (CTL; ​​control), DARZALEX™ (daratumumab) (Dara), or isotype control (Iso) at 500 ng / ml or 1000 ng / ml, as indicated. Cells were plated in methylcellulose, and colony formation as CFU-GM per 500 CD34-selected cells was measured on day 14. Results are from triplicate assays of cells from one patient with MM and two patients with AL. Figure 7B shows that DARZALEX™ (daratumumab) has no adverse effects on fresh CD34-selected erythroid progenitor cells. Isolated CD34- cells were cultured in medium without antibody (CTL; ​​control), with 500 ng / ml or 1000 ng / ml of daratumumab, or an isotype control (Iso). Cells were plated in methylcellulose, and colony formation as BFU-E was measured per 500 CD34-selected cells on day 14. Results are from triplicate assays of cells from two patients with MM and one patient with AL. *p<0.02. Figure 8 shows that DARZALEX™ (daratumumab)-mediated ADCC is affected by the FcγRIIIa-158aa polymorphism. Patients with the 158V / V (V / V) and 158F / V (F / V) genotypes showed improved response rates compared with those with the 158F / F (F / F) genotype (vertical lines represent the median of each group, P < 0.05, Mann Whitney, two-tailed). Implementation Method

[0014] "CD38" refers to the human CD38 protein (synonyms: ADP-ribosyl cyclase 1, cADP-hydrolase 1, cyclo-ADP-ribose hydrolase 1). Human CD38 has GenBank accession number NP_001766 and the amino acid sequence shown in SEQ ID NO: 1. It is well known that CD38 is a single-pass type II membrane protein with amino acid residues 1 to 21 representing the cytoplasmic domain, amino acid residues 22 to 42 representing the transmembrane domain, and residues 43 to 300 representing the CD38 extracellular domain. SEQ ID NO: 1

[0015] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulin molecules, including monoclonal antibodies (including murine, human, human-adapted, humanized, and chimeric monoclonal antibodies), antibody fragments, bispecific or multispecific antibodies, dimers, tetramers, or multimeric antibodies, and single-chain antibodies.

[0016] Immunoglobulins can be divided into five major classes: IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of their heavy chain constant domains. IgA and IgG are further subdivided into the following isotypes: IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains from any vertebrate species can be assigned to one of two distinct types: kappa (κ) and lambda (λ), depending on the amino acid sequence of their constant domains.

[0017] An "antibody fragment" refers to a portion of an immunoglobulin molecule that retains the antigen-binding sites of the heavy and / or light chains, such as the heavy chain complementarity-determining regions (HCDRs) 1, 2, and 3, the light chain complementarity-determining regions (LCDRs) 1, 2, and 3, the heavy chain variable region (VH), or the light chain variable region (VL). Antibody fragments include Fab fragments, which are monovalent fragments consisting of the VL, VH, CL, and CHI domains; F(ab)2 fragments, which are bivalent fragments consisting of two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments, which consist of the VH and CHI domains; Fv fragments, which consist of the VL and VH domains of a single antibody arm; and domain antibody (dAb) fragments (Ward et al., Nature 341:544-6, 1989), which consist of the VH domain. VH and VL domains can be engineered and linked together via synthetic linkers to form various types of single-chain antibody designs, in which the VH / VL domains pair intramolecularly, or intermolecularly, when the VH and VL domains are expressed by separate single-chain antibody constructs, to form monovalent antigen-binding sites, such as single-chain Fvs (scFvs) or diabodies, as described, for example, in International Patent Publication Nos. WO1998 / 44001, WO1988 / 01649, WO1994 / 13804, and WO1992 / 01047. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as full-length antibodies.

[0018] An "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds CD38 is substantially free of antibodies that specifically bind antigens other than human CD38). However, an isolated antibody that specifically binds CD38 may cross-react with other antigens, such as heterologs of human CD38, such as cynomolgus monkey (Macaca fascicularis) CD38. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0019] The variable region of an antibody consists of a "framework" region interrupted by three "antigen binding sites." Various terms are used to define these antigen-binding sites: the complementarity determining regions (CDRs) (three in VH (HCDR1, HCDR2, HCDR3) and three in 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, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991); the "hypervariable region," "HVR," or "HV" (three in VH (H1, H2, H3) and three in VL (L1, L2, L3)) refers to regions of the antibody variable domain that are structurally hypervariable as defined by Chothia and Lesk (Chothia and Lesk, Mol Biol. 196:901-17, 1987). Other terms include "IMGT-CDR" (Lefranc et al., Dev Comparat 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 standardized numbering and definitions of antigen-binding sites. The correspondence between CDR, HV, and IMGT delineations is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003.

[0020] As used herein, "Chothia residues" are VL and VH residues numbered according to Al-Lazikani (Al-Lazikani et al., J Mol Biol 273:927-48, 1997).

[0021] The term "framework" or "framework sequence" refers to the remaining sequence in the variable region outside of the antigen-binding site. Because the antigen-binding site can be defined using various terms, the exact amino acid sequence of the framework depends on how the antigen-binding site is defined.

[0022] A "humanized antibody" is an antibody whose antigen-binding site is derived from a non-human species and whose variable region framework is derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions, so the framework may not be an exact replica of the expressed human immunoglobulin or germline gene sequence.

[0023] "Human-adapted" or "human framework adapted" (HFA) antibodies are humanized antibodies adapted according to the methods described in US Pat. Publ. No. US2009 / 0118127. Human-adapted antibodies are humanized by selecting an acceptor human framework based on maximum CDR and FR similarity, length compatibility, and sequence similarity between the CDR1 and CDR2 loops and a portion of the light chain CDR3 loop.

[0024] A "human antibody" refers to an antibody that has heavy and light chain variable regions in which both the framework and antigen-binding site are derived from human sequences. If the antibody contains a constant region, the constant region is also derived from human sequences.

[0025] Human antibodies comprise heavy and / or light chain variable regions that are "derived from" human sequences, where the variable regions of the antibodies are obtained using systems that utilize human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage and transgenic non-human animals (such as mice or rats carrying human immunoglobulin gene loci), as described herein. "Human antibodies" may contain amino acid differences when compared to human germline or rearranged immunoglobulin sequences due, for example, to naturally occurring somatic mutations or to deliberate substitutions in the structure or antigen-binding site. Generally, a human antibody is 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% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, a "human antibody" may comprise a consensus framework sequence derived from human framework sequence analysis, such as described by Knappik et al. (J Mol Biol 296:57-86, 2000), or incorporated into a synthetic HCDR3 of a human immunoglobulin gene repertoire displayed on phage, such as described by Shi et al. (J Mol Biol 397:385-96, 2010) and International Patent Publication No. WO2009 / 085462. Antibodies whose antigen-binding sites are derived from non-human species are not included in the definition of a human antibody.

[0026] Isolated humanized antibodies can be synthetic. Human antibodies derived from human immunoglobulin sequences can be produced using systems such as phage display incorporating synthetic CDRs and / or incorporating synthetic frameworks, or can be genetically modified in vitro to improve antibody properties, thereby obtaining antibodies that do not naturally exist in the in vivo human antibody germline repertoire.

[0027] "Recombinant antibodies" include all antibodies produced, expressed, created, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice or rats) genetically or chromosomally transfected with human immunoglobulin genes or from hybridomas produced therefrom; antibodies isolated from host cells transformed to express the antibodies; antibodies isolated from recombinant, combinatorial antibody libraries; and antibodies produced, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other immunoglobulin gene sequences; or bispecific antibodies generated in vitro using, for example, Fab arm exchange.

[0028] A "monoclonal antibody" refers to a preparation of antibody molecules composed of a single molecule. A monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope, or, in the case of a bispecific monoclonal antibody, dual binding specificities for two different epitopes. "Monoclonal antibody" therefore refers to an antibody population with a single amino acid composition in each heavy chain and each light chain, except for possible well-known alterations, such as removal of the C-terminal lysine from the antibody heavy chain. Within an antibody population, monoclonal antibodies may have heterogeneous glycosylation. Monoclonal antibodies may be monospecific or multispecific, and monovalent, bivalent, or multivalent. Bispecific antibodies are also encompassed by the term monoclonal antibody.

[0029] An "epitope" refers to the portion of an antigen that specifically binds to an antibody. Epitopes typically consist of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groupings of molecular moieties (such as amino acids or polysaccharide side chains) and may possess specific three-dimensional structural and charge characteristics. Epitopes can include contiguous and / or noncontiguous amino acids that form a conformational unit. In noncontiguous epitopes, amino acids from different parts of the antigen's linear sequence are brought into close proximity in three-dimensional space by the folding of the protein molecule.

[0030] A "variant" is a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications (eg, substitutions, insertions, or deletions).

[0031] "In combination with" means that two or more therapeutic agents can be administered to a subject together as a mixture, simultaneously as a single dose, or sequentially in any order as a single dose.

[0032] "Treatment" or "treatment" refers to therapeutic management in which the object is to slow down (lessen) an undesired physiological change or disease, or to provide a beneficial or desired clinical result during treatment. Beneficial or desired clinical results include alleviation of symptoms, reduction in severity of disease, stabilization of the disease state (i.e., non-worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonging survival as compared to expected survival in a subject not receiving treatment. Those in need of treatment include those already with the undesired physiological change or disease, as well as those susceptible to such change or disease.

[0033] "Inhibit growth" (e.g., with reference to cells, such as tumor cells) means that when the cells are contacted with a therapeutic agent or combination of therapeutic agents or drugs, there is a measurable decrease in the growth of the cells in vitro or in vivo, as compared to the growth of the same cells grown under appropriately controlled conditions known to those of ordinary skill in the art. Inhibition of cell growth in vitro or in vivo can be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Inhibition of cell growth can occur through various mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, necrosis, or through inhibition of cell proliferation.

[0034] A "therapeutically effective amount" refers to an amount effective at the dosage and for the amount of time necessary to achieve the desired therapeutic outcome. A therapeutically effective amount may vary depending on factors such as the subject's disease state, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the subject. Exemplary indicators of an effective amount of a therapeutic agent or combination of therapeutic agents include, for example, an improvement in patient well-being, a reduction in tumor burden, a slowing or cessation of tumor growth, and / or the absence of metastasis of cancer cells to other locations in the body.

[0035] "Patient" includes any human or nonhuman animal. "Non-human animals" include all vertebrates, such as mammals and non-mammals, for example, non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "patient" and "subject" are used interchangeably herein.

[0036] The present invention provides methods for treating patients with CD38-positive hematological malignancies undergoing hematopoietic stem cell transplantation with anti-CD38 antibodies that do not kill (e.g., mediate killing) CD34+ hematopoietic stem cells within the transplant, which are also CD38-positive. The present invention also provides methods for treating patients with light-chain amyloidosis. The present invention is based, at least in part, on the discovery that the anti-CD38 antibody DARZALEX™ (daratumumab) effectively kills AL plasma cells but does not kill CD38+CD34+ hematopoietic stem cells isolated from patients with light-chain amyloidosis or multiple myeloma, thereby enabling the combined treatment of DARZALEX™ (daratumumab) and hematopoietic stem cell transplantation.

[0037] The methods of the present invention can be used to treat animal patients belonging to any category. Examples of such animals include mammals, such as humans, mice, dogs, cats, and farm animals.

[0038] The present invention provides a method for treating a patient suffering from a CD38-positive hematological malignancy, comprising administering to the patient in need thereof an anti-CD38 antibody for a period of time sufficient to treat the CD38-positive hematological malignancy, wherein the patient is undergoing hematopoietic stem cell transplantation (HSCT).

[0039] "CD38-positive hematological malignancies" refers to a hematological malignancy characterized by the presence of tumor cells expressing CD38, including leukemias, lymphomas, myelomas, and plasma cell disorders. Examples of CD38-positive hematological malignancies include precursor B-cell lymphoblastic leukemia / lymphoma and B-cell non-Hodgkin's lymphoma, acute promyelocytic leukemia, acute lymphoblastic leukemia and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL) (including low-grade, intermediate-grade, and high-grade FL), cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT-type, nodal-type, and splenic-type), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BCL), and leukemia. lymphoma (BL), plasma cell neoplasms, multiple myeloma (MM), plasma cell leukemia, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, plasma cell leukemia, anaplastic large cell lymphoma (ALCL), and light-chain amyloidosis (AL).

[0040] As used herein, "plasma cell disorders" refer to disorders characterized by pure plasma cell lines, including multiple myeloma, light-chain amyloidosis, and Waldenstrom's macroglobulinemia. Light-chain amyloidosis and Waldenstrom's macroglobulinemia can occur independently of multiple myeloma. They can also coexist with multiple myeloma and develop before or after the development of multiple myeloma.

[0041] Therefore, the definitions of "CD-38 positive hematological malignancies" and "plasma cell disorders" may partially overlap.

[0042] In some embodiments, the CD38-positive hematological malignancy is light-chain amyloidosis (AL).

[0043] In some embodiments, the CD38-positive hematological malignancy is multiple myeloma (MM).

[0044] In some embodiments, the CD38-positive hematological malignancy is Waldenstrom's macroglobulinemia.

[0045] In some embodiments, the CD38-positive hematological malignancy is diffuse large B-cell lymphoma (DLBCL).

[0046] In some embodiments, the CD38-positive hematological malignancy is non-Hodgkin's lymphoma.

[0047] In some embodiments, the CD38-positive hematological malignancy is acute lymphoblastic leukemia (ALL).

[0048] In some embodiments, the CD38-positive hematological malignancy is follicular lymphoma (FL).

[0049] In some embodiments, the CD38-positive hematological malignancy is Birch's lymphoma (BL).

[0050] In some embodiments, the CD38-positive hematological malignancy is mantle cell lymphoma (MCL).

[0051] In some embodiments, the CD38-positive hematological malignancy is a plasma cell disorder.

[0052] In some embodiments, the CD38-positive hematological malignancy is light-chain amyloidosis (AL), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), Birch's lymphoma (BL), follicular lymphoma (FL), or mantle cell lymphoma (MCL).

[0053] Exemplary B-cell non-Hodgkin's lymphomas are lymphomatoid granulomatosis, primary effusion lymphoma, intravascular large B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain diseases (including gamma, mu, and alpha diseases), lymphomas induced by immunosuppressive therapy (such as cyclosporine-induced lymphoma and methotrexate-induced lymphoma).

[0054] In some embodiments, the disorder involving cells expressing CD38 is Hodgkin's lymphoma.

[0055] Other examples of disorders involving cells expressing CD38 include malignancies derived from T cells and NK cells, including mature T-cell and NK-cell neoplasms, including T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma (nasal type), enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, embryoblastic NK-cell lymphoma, mycosis fungoides / Sézary syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disorders (primary cutaneous anaplastic large cell lymphoma (C-ALCL), lymphomatoid papulosis, borderline lesions), angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma not otherwise specified, and anaplastic large cell lymphoma.

[0056] Examples of malignant diseases derived from myeloid cells include acute myeloid leukemia (including acute promyelocytic leukemia), and chronic myeloproliferative diseases (including chronic myeloid leukemia).

[0057] The present invention also provides a method for treating a patient suffering from light-chain amyloidosis (AL), comprising administering an anti-CD38 antibody to the patient in need thereof for a period of time sufficient to treat AL.

[0058] The present invention also provides a method of treating a patient suffering from light-chain amyloidosis, comprising administering an anti-CD38 antibody to the patient in need thereof for a period of time sufficient to treat the light-chain amyloidosis, wherein the patient is undergoing hematopoietic stem cell transplantation (HSCT).

[0059] The present invention also provides a method of treating a patient suffering from multiple myeloma, comprising administering an anti-CD38 antibody to the patient in need thereof for a period of time sufficient to treat the multiple myeloma, wherein the patient is undergoing hematopoietic stem cell transplantation (HSCT).

[0060] In some embodiments, the anti-CD38 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.

[0061] In some embodiments, the anti-CD38 antibody binds to at least the SKRNIQFSCKNIYR region (SEQ ID NO: 2) and the EKVQTLEAWVIHGG region (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).

[0062] In some embodiments, the anti-CD38 antibody comprises the heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively.

[0063] In some embodiments, the anti-CD38 antibody comprises light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively.

[0064] In some embodiments, the anti-CD38 antibody comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively.

[0065] In some embodiments, the anti-CD38 antibody comprises a heavy chain variable region (VH) amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4 and a light chain variable region (VL) amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5.

[0066] In some embodiments, the anti-CD38 antibody comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:5.

[0067] In some embodiments, the anti-CD38 antibody contains a heavy chain comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12 and a light chain comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13.

[0068] In some embodiments, the anti-CD38 antibody comprises a heavy chain of SEQ ID NO: 12 and a light chain of SEQ ID NO: 13.

[0069] The epitope of the antibody includes some or all of the residues having the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the antibody epitope comprises at least one amino acid in the SKRNIQFSCKNIYR (SEQ ID NO: 2) region of human CD38 (SEQ ID NO: 1) and at least one amino acid in the EKVQTLEAWVIHGG (SEQ ID NO: 3) region. In some embodiments, the antibody epitope comprises at least two amino acids in the SKRNIQFSCKNIYR (SEQ ID NO: 2) region of human CD38 (SEQ ID NO: 1) and at least two amino acids in the EKVQTLEAWVIHGG (SEQ ID NO: 3) region. In some embodiments, the antibody epitope comprises at least three amino acids in the SKRNIQFSCKNIYR (SEQ ID NO: 2) region of human CD38 (SEQ ID NO: 1) and at least three amino acids in the EKVQTLEAWVIHGG (SEQ ID NO: 3) region.

[0070] An exemplary antibody that binds to the SKRNIQFSCKNIYR (SEQ ID NO: 2) and EKVQTLEAWVIHGG (SEQ ID NO: 3) regions of human CD38 (SEQ ID NO: 1) is DARZALEX™ (daratumab).

[0071] An exemplary anti-CD38 antibody that can be used in the methods of the present invention is DARZALEX™ (daratumumab). DARZALEX™ (daratumumab) comprises the heavy chain variable region (VH) and light chain variable region (VL) amino acid sequences shown in SEQ ID NOs: 4 and 5, respectively, the heavy chain CDRs (HCDR1, HCDR2, and HCDR3) of SEQ ID NOs: 6, 7, and 8, respectively, and the light chain CDRs (LCDR1, LCDR2, and LCDR3) of SEQ ID NOs: 9, 10, and 11, respectively. It is of the IgG1 / κ subtype and is described in U.S. Patent No. 7,829,693. The heavy chain amino acid sequence of DARZALEX™ (daratumumab) is shown in SEQ ID NO: 12, and the light chain amino acid sequence is shown in SEQ ID NO: 13.

[0072] Conventional in vitro methods can be used to assess competition for CD38 binding between an antibody and DARZALEX™ (daratumumab), which has a VH of SEQ ID NO:4 and a VL of SEQ ID NO:5. In one exemplary method, recombinant CD38-expressing CHO cells can be incubated with an unlabeled reference antibody at 4°C for 15 minutes, followed by incubation with an excess of a fluorescently labeled test antibody at 4°C for 45 minutes. After washing in PBS / BSA, fluorescence can be measured by flow cytometry using standard methods. In another exemplary method, the extracellular domain of CD38 can be coated on the surface of an ELISA plate. An excess of the unlabeled reference antibody can be added for approximately 15 minutes, followed by the addition of the biotinylated test antibody. After washing in PBS / Tween, binding of the biotinylated test antibody can be detected using horseradish peroxidase (HRP)-conjugated streptavidine and signal detected using standard methods. In such competition assays, it will be apparent that the reference antibody can be labeled and the test antibody can be unlabeled. A test antibody competes with a reference antibody when the reference antibody inhibits binding of the test antibody to CD38 by at least 80%, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The epitope of the test antibody can be further defined using conventional methods, such as peptide mapping or hydrogen / deuterium protection assays, or by crystal structure determination.

[0073] Antibodies that bind to the SKRNIQFSCKNIYR (SEQ ID NO: 2) and EKVQTLEAWVIHGG (SEQ ID NO: 3) regions of human CD38 (SEQ ID NO: 1) can be generated, for example, by immunizing mice with peptides having the amino acid sequences shown in SEQ ID NOs: 2 and 3 as described herein using standard methods, and characterizing the resulting antibodies for binding to the peptides using, for example, ELISA or mutagenesis studies. SEQ ID NO:2 SKRNIQFSCKNIYR SEQ ID NO:3 EKVQTLEAWVIHGG SEQ ID NO:4 SEQ ID NO:5 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 SEQ ID NO:13

[0074] Another exemplary anti-CD38 antibody that can be used in the methods of the present invention is mAb003, which comprises the VH and VL sequences of SEQ ID NOs: 14 and 15, respectively, and is described in U.S. Patent No. 7,829,693. The VH and VL of mAb003 can be expressed as IgG1 / κ. SEQ ID NO:14 SEQ ID NO:15 mAb024, comprising the VH and VL sequences of SEQ ID NOs: 16 and 17, respectively, is described in U.S. Patent No. 7,829,693. The VH and VL sequences of mAb024 can be denoted as IgG1 / κ. SEQ ID NO:16 SEQ ID NO: 17

[0075] MOR-202 (MOR-03087), comprising the VH and VL sequences of SEQ ID NOs: 18 and 19, respectively, and described in U.S. Patent No. 8,088,896, can be expressed as IgG1 / κ. SEQ ID NO: 18 SEQ ID NO: 19 Isatuximab; which comprises the VH and VL sequences of SEQ ID NOs: X and X, respectively, and is described in U.S. Patent No. 8,153,765. The VH and VL of isatuximab can be expressed as IgG1 / κ. SEQ ID NO 20: SEQ ID NO:21:

[0076] Other exemplary anti-CD38 antibodies that can be used in the methods of the invention include those described in International Patent Publication No. WO05 / 103083, International Patent Publication No. WO06 / 125640, International Patent Publication No. WO07 / 042309, International Patent Publication No. WO08 / 047242, or International Patent Publication No. WO14 / 178820.

[0077] In some embodiments, the AL is cardiac stage I, cardiac stage II, or cardiac stage III.

[0078] In some embodiments, AL is relapsed or refractory.

[0079] AL is diagnosed by physicians according to guidelines available, for example, from the National Comprehensive Cancer Network (http: / / www.nccn.org / professionals / physician_gls / f_guidelines_asp#site). AL patients experience damage in various organ systems due to the accumulation of light chains and misfolded intermediates of these light chains in the form of amyloid fibrils in vital organs, leading to organ dysfunction and death. Patients may have multiple organ systems affected at diagnosis; approximately one-third of patients have more than three organs affected at diagnosis (Chaulagain and Comenzo Curr Hematol Malig Rep 8:291-8, 2013). AL prognosis is influenced by cardiac staging, as cardiac damage appears to be present in all patients diagnosed with AL, even if they are asymptomatic (Palladini et al., Blood 116:3426-30, 2010; Kristen et al., Blood 116:2455-61, 2010). AL patients can be classified as cardiac stage I, II, or III based on the presence of one, two, or both of the cardiac biomarkers N-terminal prohormone of brain natriuretic peptide (NT-proBNP) and troponin T; see, e.g., Comenzo et al., Leukemia 26:2317-25, 2012.

[0080] Current treatment options for AL involve killing light chain immunoglobulin-secreting plasma cells and include a combination of agents such as Velcade® (bortezomib), cyclophosphamides such as Cytoxan® or Neosar®, Alkeran® (melphalan), Thalomid® (thalidomide), Revlimid® (lenalidomide), or Pomalyst® (pomalidomide), as well as steroids (dexamethasone), interferon alpha (IFN-α), and stem cell transplantation. High-dose Alkeran® (melphalan) can be used with stem cell transplantation (see, for example, Anderson et al., Systemic light chain amyloidosis, NCCN Clinical Practice Guidelines in Oncology Version 1.2015, NCCN.org.2014). NINLARO® (ixazomib), a proteasome inhibitor, is being evaluated for the treatment of AL. NEOD001, a monoclonal antibody targeting AL amyloid protein, is also being evaluated for the treatment of AL.

[0081] In some embodiments, the MM is relapsed or refractory.

[0082] Currently available MM treatments include chemotherapy, stem cell transplantation, Thalomid® (thalidomide), Revlimid® (lenalidomide), Velcade® (bortezomib), Kyprolis® (carfilzomib), Farydak® (panobinostat), Aredia® (pamidronate), and Zometa® (zoledronic acid). Current treatment regimens, including combinations of chemotherapy agents such as Oncovin® (vincristine), BiCNU® (BCNU, carmustine), Alkeran® (melphalan), cyclophosphamide, Adriamycin® (doxorubicin), and prednisone or dexamethasone, result in only approximately 5% complete remission rates, and median survival is approximately 36 to 48 months from the time of diagnosis. Furthermore, the investigational drug ixazomib has demonstrated positive results in pivotal clinical trials for patients with relapsed multiple myeloma. Recent advances in high-dose chemotherapy followed by autologous bone marrow or peripheral blood mononuclear cell transplantation have increased complete remission rates and duration of remission, but overall survival has only been modestly prolonged, and no evidence of a cure has been achieved. Ultimately, all patients with MM will relapse, even with maintenance therapy using interferon alpha (IFN-α) alone or in combination with steroids.

[0083] Various qualitative and / or quantitative methods can be used to determine the relapse or refractory nature of a disease. Symptoms that may be associated with relapse or resistance include, for example, a decline or stagnation in the patient's health, the return or worsening of various symptoms associated with a hematological malignancy, and / or the spread of cancer cells from one location in the body to other organs, tissues, or cells. Symptoms associated with a hematological malignancy may vary depending on the type of cancer. For example, symptoms associated with AL may include increased fatigue, purpura, enlarged tongue, diarrhea or edema, proteinuria, or increased plasma free light chains.

[0084] In some embodiments, HSCT is allogeneic, autologous, or syngeneic, i.e., the donor is a twin. Autologous HSCT involves extracting HSCs from the subject and freezing the resulting HSCs. Following myeloablation, the stored HSCs from the subject are transplanted into the subject. Allogeneic HSCT involves HSCs from an allogeneic HSC donor whose HLA typing matches that of the subject.

[0085] As used herein, "hematopoietic stem cell transplantation" is the transplantation of blood stem cells derived from bone marrow (in which case it is called a bone marrow transplant), blood (such as peripheral blood and umbilical cord blood), or amniotic fluid.

[0086] As used herein, the term "undergoing hematopoietic stem cell transplantation" means that the patient has received, is currently receiving, or will receive HSCT.

[0087] In some embodiments, the patient has completed chemotherapy and / or radiation therapy prior to HSCT.

[0088] Patients can be treated with chemotherapy and / or radiation therapy before HSCT (so-called pre-transplant preparation) to eliminate some or all of the patient's hematopoietic cells before transplantation. In the case of allogeneic HSCT, patients can also be treated with immunosuppressants. An exemplary pre-transplant preparation treatment is high-dose melphalan (see, for example, Skinner et al., Ann Intern Med 140:85-93, 2004; Gertz et al., Bone Marrow Transplant 34:1025-31, 2004; Perfetti et al., Haematologica 91:1635-43, 2006). Radiation therapy that can be used in pre-transplant treatment can be performed according to commonly known protocols in the field. Radiation therapy can also be provided simultaneously, sequentially, or separately with the anti-CD38 antibody.

[0089] DARZALEX™ (daratumumab) does not mediate the killing of CD38+CD34+ stem cells within the transplant, making it a suitable therapy for combination with HSCT. Antibodies that compete with (daratumumab) and / or bind to the same epitope as DARZALEX™ (daratumumab) also do not kill CD38+CD34+ stem cells.

[0090] Other exemplary antibodies that can be used in the methods of the invention disclosed herein are unable to kill CD38+CD34+ stem cells within the transplant and are therefore suitable therapies in combination with HSCT. The methods described herein can be used to assess whether an antibody has no ability to kill CD38+CD34+ stem cells within the transplant.

[0091] Anti-CD38 antibodies used in the methods of the invention can also be reselected, for example, from phage display libraries in which phage are engineered to express human immunoglobulins or portions thereof, such as Fab, single-chain antibodies (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; Hoogenboom and Winter, J Mol Biol 227:381, 1991; Marks et al., J Mol Biol 222:581, 1991). CD38-binding variable domains can be isolated, for example, from a phage display library expressing antibody heavy and light chain variable regions as fusion proteins with the phage pIX coat protein, as described in Shi et al., J. Mol. Biol., 397:385-96, 2010, and International Patent Publication No. WO09 / 085462. The antibody library can be screened for binding to the extracellular domain of human CD38. Positive clones obtained are further characterized, and Fabs are isolated from clone lysates and subsequently cloned as full-length antibodies. This phage display method for isolating human antibodies is well established in the art. See, e.g., U.S. Patent No. 5,223,409; U.S. Patent No. 5,403,484; and U.S. Patent No. 5,571,698, U.S. Patent No. 5,427,908, U.S. Patent No. 5,580,717, U.S. Patent No. 5,969,108, U.S. Patent No. 6,172,197, U.S. Patent No. 5,885,793; U.S. Patent No. 6,521,404; U.S. Patent No. 6,544,731; U.S. Patent No. 6,555,313; U.S. Patent No. 6,582,915; and U.S. Patent No. 6,593,081.

[0092] In some embodiments described herein, the anti-CD38 antibodies do not mediate killing of CD34-positive hematopoietic progenitor cells by complement-dependent cytotoxicity (CDC).

[0093] "Does not kill" or "does not mediate killing" refers to the inability of an anti-CD38 antibody to induce cell killing when compared to an appropriate control, such as an isotype control. An anti-CD38 antibody "does not kill" when cell killing measured in the presence of DARZALEX™ (daratumumab) is not statistically significant compared to cell killing in the presence of an isotype control. Isotype control is a well-known term.

[0094] Killing of CD34-positive hematopoietic progenitor cells by CDC can be measured in fresh or frozen isolated CD34+ cells by culturing the cells in 10% serum with complement and 500 ng / ml anti-CD38 antibody, followed by assaying the extent of colony formation of cells plated in semisolid culture medium according to known methods. For example, BFU-E and CFU-GM formation can be assessed after 14 days of culture using commercial reagents such as MethoCult™ from Stem Cell Technologies.

[0095] The Fc portion of an antibody mediates its effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). These functions can be mediated by binding of the Fc effector domain to Fc receptors on immune cells with phagocytic or lytic activity, or by binding of the Fc effector domain to components of the complement system. Generally, effects mediated by Fc-bound cells or complement components result in inhibition or depletion of target cells (e.g., CD38-expressing cells). Human IgG isotypes IgG1, IgG2, IgG3, and IgG4 exhibit distinct effects on their effector functions. ADCC can be mediated by IgG1 and IgG3, ADCP by IgG1, IgG2, IgG3, and IgG4, and CDC by IgG1 and IgG3.

[0096] In some embodiments, the anti-CD38 antibody is of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0097] In some embodiments, the anti-CD38 antibodies induce killing of CD38-expressing plasma cells in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or in vitro modulation of CD38 enzymatic activity.

[0098] In some embodiments, the anti-CD38 antibodies induce killing of cells expressing CD38 in vitro by ADCC, ADCP, or CDC.

[0099] In some embodiments, the anti-CD38 antibodies induce killing of cells expressing CD38 by ADCC in vitro.

[0100] In some embodiments, the anti-CD38 antibodies induce killing of CD38-expressing cells in vitro by ADCP.

[0101] In some embodiments, anti-CD38 antibodies induce killing of CD38-expressing cells by CDC in vitro.

[0102] Antibody-dependent cellular cytotoxicity, antibody-dependent cell-mediated cytotoxicity, or ADCC is a mechanism of cell death that relies on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer cells, monocytes, macrophages, and neutrophils) through Fcγ receptors (FcγRs) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. Death of antibody-coated target cells (such as CD38-expressing cells) occurs due to the activity of membrane pore-forming proteins and proteases secreted by the effector cells. To assess the ADCC activity of anti-CD38 antibodies, the antibody can be added to a combination of CD38-expressing cells and immune effector cells. The effector cells are activated by the antigen-antibody complex and cause cytolysis of the target cells. Cell lysis is typically detected by the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a native intracellular protein) from the lysed cells. Exemplary effector cells used in these assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Exemplary target cells include Daudi cells (ATCC® CCL-213™) or CD38-expressing B-cell leukemia or lymphoma tumor cells. In an exemplary assay, target cells are labeled with 20 μCi of 51Cr for 2 hours and washed thoroughly. The target cell concentration is adjusted to 1×106 cells / ml, and various concentrations of anti-CD38 antibody are added. The assay is initiated by adding Daudi cells at a 40:1 effector:target cell ratio. After incubation at 37°C for 3 hours, the assay is terminated by centrifugation, and 51Cr released from lysed cells is measured in a scintillation counter. Percent cytotoxicity is calculated as a percentage of the maximum lysis induced by the addition of 3% perchloric acid to the target cells. The anti-CD38 antibodies used in the methods of the present invention may induce ADCC of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the control group (cell lysis induced by 3% perchloric acid).

[0103] Antibody-dependent cellular phagocytosis (ADCP) refers to a mechanism of destruction of antibody-coated target cells through internalization by phagocytes, such as macrophages or dendritic cells. ADCP can be assessed using monocyte-derived macrophages as effector cells and Daudi cells (ATCC® CCL-213™) or CD38-expressing B-cell leukemia or lymphoma tumor cells engineered to express GFP or other marker molecules as target cells. The effector:target cell ratio can be, for example, 4:1. Effector and target cells can be cultured for 4 hours in the presence or absence of anti-CD38 antibodies. Following incubation, cells can be separated using accutase. Macrophages can be identified using fluorescently labeled anti-CD11b and anti-CD14 antibodies, and the percentage of phagocytosis can be determined based on the percentage of GFP fluorescence among these CD11+CD14+ macrophages using standard methods. The anti-CD38 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%.

[0104] "Complement-dependent cytotoxicity" or "CDC" refers to a mechanism of cell death in which the Fc effector domain of an antibody bound to a target binds to and activates the complement component C1q, which in turn reactivates the complement cascade, leading to target cell death. Complement activation can also result in the deposition of complement components on the target cell surface, promoting ADCC by binding to complement receptors (e.g., CR3) on leukocytes. CDC in CD38-expressing cells can be measured, for example, by the following steps: seed Daudi cells at 1×105 cells per well (50 μl / well) in RPMI-B (RPMI supplemented with 1% BSA), add 50 μl of anti-CD38 antibody to the wells to a final concentration between 0 and 100 μg / ml, incubate the reaction at room temperature for 15 minutes, add 11 μl of pooled human serum to the wells, and incubate the reaction at 37°C for 45 minutes. The percentage (%) of lysed cells can be detected as the percentage of cells stained with propidium iodide in a FACS assay using standard methods. The anti-CD38 antibodies used in the methods of the present invention can induce CDC by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0105] The ability of monoclonal antibodies to induce ADCC can be enhanced by engineering their oligosaccharide components. Human IgG1 or IgG3 are N-glycosylated at Asn297, and the majority of glycans are in the well-known biantennary G0, G0F, G1, G1F, G2, or G2F forms. Antibodies produced by unengineered CHO cells typically have a glycan fucose content of approximately at least 85%. Removing core fucose from the biantennary complex oligosaccharide attached to the Fc region enhances antibody ADCC by improving FcγRIIIa binding without altering antigen binding or CDC activity. Such mAbs can be produced using various methods that have been reported to result in the successful expression of relatively high amounts of defucosylated antibodies (with biantennary complexed Fc oligosaccharides), such as controlling culture osmotic pressure (Konno et al., Cytotechnology 64:249-65, 2012), using a variant CHO strain Lec13 as a host cell line (Shields et al., J Biol Chem 277:26733-40, 2002), using a variant CHO strain EB66 as a host cell line (Olivier et al., MAbs; 2(4), 2010; electronic publication before print; PMID: 20562582), using a rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa et al., J Biol Chem 277:26733-40, 2002), and using a rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa et al., J Biol Chem 277:26733-40, 2002). 278:3466-73, 2003), introduction of short interfering RNA specifically targeting the α1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-8, 2004), or co-expression of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or kifunensine (a potent α-mannosidase I inhibitor) (Ferrara et al., J Biol Chem 281:5032-6, 2006; Ferrara et al., Biotechnol Bioeng 93:851-61, 2006; Xhou et al., Biotechnol Bioeng ADCC elicited by the anti-CD38 antibodies used in the methods described herein, and in some embodiments of each of the following numbered examples, can also be enhanced by certain substitutions in the antibody Fc.Exemplary substitutions include, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 (residue numbering according to the EU index), as described in U.S. Patent No. 6,737,056. CDC induced by the anti-CD38 antibodies used in the methods of the present invention, and in some embodiments of each of the numbered embodiments below, can also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions include, for example, substitutions at amino acid positions 423, 268, 267, and / or 113 (residue numbering according to the EU index), as described in Moore et al., Mabs 2:181-9, 2010.

[0106] In some embodiments, the anti-CD38 antibody comprises a substitution in the antibody Fc.

[0107] In some embodiments, the anti-CD38 antibody comprises substitutions in the Fc of the antibody at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, and / or 430 (residue numbering is according to the EU index).

[0108] In some embodiments, the anti-CD38 antibody comprises substitutions in the Fc region of the antibody at amino acid positions 113, 267, 268, and / or 423 (residue numbering is according to the EU index).

[0109] In some embodiments, the anti-CD38 antibodies comprise biantennary glycan structures having a trehalose content of about 0% to about 15%, e.g., 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0110] In some embodiments, the anti-CD38 antibody comprises biantennary glycan structures having a trehalose 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%.

[0111] Substitutions in Fc and reduced trehalose content can enhance the ADCC activity of the anti-CD38 antibody.

[0112] "Fucose content" refers to the amount of fucose monosaccharide in the carbohydrate chain at Asn297. The relative amount of fucose is the percentage of fucose-containing structures relative to all carbohydrate structures. These can be qualitatively and quantitatively determined by various methods, such as: 1) MALDI-TOF using samples treated with N-glycosidase F (e.g., complex, hybrid, and oligo- and high-mannose structures), as described in International Patent Publication No. WO2008 / 077546; 2) enzymatic release of the Asn297 glycan followed by derivatization and detection / quantification by HPLC (UPLC) with fluorescence detection and / or HPLC-MS (UPLC-MS); 3) intact protein analysis of native or reduced mAbs, with or without treatment of the Asn297 glycan with Endo S or other enzymes that cleave between the first and second GlcNAc monosaccharides, leaving fucose attached to the first GlcNAc; 4) enzymatic digestion. 5) Specific enzymatic deglycosylation at Asn 297 with PNGase F is performed to separate mAb oligosaccharides from the mAb protein. These released oligosaccharides can be labeled with fluorophores and separated and identified by various complementary techniques, including matrix-assisted laser desorption ionization (MALDI) mass spectrometry to determine the precise characterization of glycan structure by comparing experimental and theoretical masses, determination of sialylation by ion-exchange HPLC (GlycoSep C), separation and quantification of oligosaccharide forms based on hydrophilicity criteria by normal-phase HPLC (GlycoSep N), and separation and quantification of oligosaccharides by high-performance capillary electrophoresis-laser-induced fluorescence (HPCE-LIF).

[0113] As used in this application, “low fucose” or “low fucose content” refers to an antibody having a fucose content of about 0% to 15%.

[0114] As used herein, "normal fucose" or "normal fucose content" refers to an antibody having a fucose content of more than about 50%, typically more than about 60%, 70%, 80%, or more than 85%.

[0115] The anti-CD38 antibodies used in the methods of the present invention can induce killing of CD38-expressing cells through apoptosis in vitro. Methods for assessing apoptosis are known and include, for example, annexin IV staining using standard methods. The anti-CD38 antibodies used in the methods of the present invention can induce apoptosis in approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of cells.

[0116] The anti-CD38 antibodies used in the methods of the present invention can induce in vitro killing of CD38-expressing cells by modulating CD38 enzymatic activity. CD38 is a multifunctional exonuclease with ADP-ribosyl cyclase 1 activity. It catalyzes the formation of cyclic ADP-ribose (cADPR) and ADPR from NAD+ and also hydrolyzes NAD+ and cADPR to ADPR. CD38 also catalyzes the exchange of the nicotinamide group of NADP+ with nicotinic acid under acidic conditions to produce NAADP+ (nicotinic acid adenine dinucleotide phosphate). Modulation of human CD38 enzymatic activity by the anti-CD38 antibodies used in the methods of the present invention can be measured in the assay described in Graeff et al. (J. Biol. Chem. 269:30260-7, 1994). For example, the substrate NGD+ can be incubated with CD38, and the modulation of cyclic GDP-ribose (cGDPR) production can be monitored spectrophotometrically (excitation wavelength 340 nM and emission wavelength 410 nM) at various time points after addition of various concentrations of the antibody. Inhibition of cGDPR synthesis can be determined according to the HPLC method described in Munshi et al., J. Biol. Chem. 275:21566-71, 2000. The anti-CD38 antibodies used in the methods of the present invention can inhibit CD38 enzymatic activity by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0117] Antibodies that are substantially identical to the antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 can be used in the methods of the present invention. The term "substantially identical" means that the amino acid sequences of the compared antibody VH or VL are identical or have "insubstantial differences." Insubstantial differences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions in the antibody VH and / or VL that do not adversely affect antibody performance. Percent identity can be determined, for example, by pairwise alignment using the default settings of the AlignX module of Vector NTI v. 9.0.0 (Invitrogen, Carlsbad, CA). The protein sequences of the present invention can be used as query sequences to perform searches against public or patent databases to, for example, identify related sequences. Exemplary programs for performing such searches are XBLAST or BLASTP (http: / / www.ncbi.nlm.nih.gov), or the GenomeQuest™ (GenomeQuest, Westborough, MA) suite using default settings. Exemplary substitutions that can be made in antibodies that specifically bind CD38 and are used in the methods of the present invention include conservative substitutions with amino acids having similar charge, hydrophobicity, or stereochemistry. Conservative substitutions can also be made to improve antibody properties (e.g., stability or affinity) or to improve effector functions of the antibody. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, 13, 14, or 15 amino acid substitutions can be made in the anti-CD38 VH and / or VL. In addition, any native residue in the heavy or light chain can be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., Acta Physiol Scand Suppl 643:55-67, 1998; Sasaki et al., Adv Biophys 35:1-24, 1998). The desired amino acid substitutions can be determined by one of ordinary skill in the art when such substitutions are desired. Amino acid substitutions can be performed, for example, by PCR mutagenesis (U.S. Patent No. 4,683,195).Variant libraries can be generated using conventional methods, for example, using random (NNK) or non-random codons (e.g., DVK codons) encoding the 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp), and then screened to identify variants with desired properties. The generated variants can be tested in vitro using the methods described herein for their ability to bind to CD38, induce ADCC, ADCP, or apoptosis, or modulate CD38 enzymatic activity.

[0118] "Conservative modification" refers to an amino acid modification that does 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. A conservative substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, aspartic acid, glutamic acid, cysteine), and hydroxyl groups. The present invention also includes amino acids (e.g., cystine, 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., aspartic acid, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). In addition, any native residue in the polypeptide can be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; ​​Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions for antibodies of the present invention can be performed using known methods, such as PCR mutagenesis (U.S. Patent No. 4,683,195). Alternatively, a library of variants can be generated, for example, 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 their properties using the assays described herein.

[0119] In some embodiments, the antibodies can bind CD38 with a dissociation constant (KD) of less than about 1×10-7 M, 1×10-8 M, 1×10-9 M, 1×10-10 M, 1×10-11 M, 1×10-12 M, 1×10-13 M, 1×10-14 M, or 1×10-15, as determined by surface plasmon resonance or Kinexa methods practiced by one of ordinary skill in the art. In some embodiments, the antibodies bind to human CD38 with a KD of less than about 1×10-8 M. In some embodiments, the antibodies bind to human CD38 with a KD of less than about 1×10-9 M.

[0120] KinExA instrumentation, ELISA, or other competitive binding assays known to those skilled in the art. The affinity measured for a particular antibody / CD38 interaction may vary when measured under different conditions (e.g., osmolarity, pH). Therefore, affinity and other binding parameters (e.g., KD, Kon, Koff) are typically measured using standardized conditions and buffers, such as those described herein. Those skilled in the art will understand that within typical detection limits, the internal error (measured as standard deviation, SD) of affinity measurements (e.g., performed using Biacore 3000 or ProteOn) is typically within 5 to 33% of the measured value. Therefore, in the context of KD, the term "about" reflects the typical standard deviation within the assay. For example, a KD of 1 × 10-9 M typically has an SD of up to +0.33 × 10-9 M.

[0121] In some embodiments, the anti-CD38 antibody is a bispecific antibody. The VL and / or VH regions of an existing anti-CD38 antibody or the newly identified VL and VH regions described above can be engineered into a bispecific full-length antibody. Such bispecific antibodies can be made by modulating CH3 interactions between monospecific antibody heavy chains to form bispecific antibodies using techniques such as those described in 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. US2012 / 0149876. Other bispecific structures that can incorporate the VL and / or VH regions of the antibodies of the present invention are, for example, dual variable domain immunoglobulins (International Patent Publication No. WO2009 / 134776), or structures comprising various 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).

[0122] For example, bispecific antibodies can be produced in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming the bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions (to allow disulfide bond isomerization), according to the methods described in International Patent Publication No. WO2011 / 131746. In these methods, a first monospecific bivalent antibody (e.g., an anti-CD38 antibody) and a second monospecific bivalent antibody are engineered to have certain substitutions in the CH3 domain that promote heterodimer stability. These antibodies are incubated together under reducing conditions sufficient to allow disulfide bond isomerization of cysteines in the hinge region, thereby generating the bispecific antibody by Fab arm exchange. The incubation conditions can optimally be returned to non-reducing conditions. Exemplary reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol. Preferably, the reducing agent is selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of 5 to 8 (e.g., at a pH of 7.0 or at a pH of 7.4) for at least 90 minutes can be used.

[0123] Exemplary CH3 mutations that can be used in the first heavy chain and in the second heavy chain of the bispecific antibody are K409R and / or F405L.

[0124] In some embodiments, the anti-CD38 antibody is conjugated to a toxin. Conjugation methods and suitable toxins are known in the art.

[0125] In some embodiments, subjects with AL are homozygous for phenylalanine at position 158 of CD16 (FcγRIIIa-158F / F genotype) or heterozygous for valine and phenylalanine at position 158 of CD16 (FcγRIIIa-158F / V genotype). CD16 is also known as Fcγ receptor IIIa (FcγRIIIa) or the low-affinity immunoglobulin gamma Fc region receptor III-A isoform. The valine / phenylalanine (V / F) polymorphism at residue position 158 of the FcγRIIIa protein has been shown to affect the affinity of FcγRIIIa for human IgG. Receptors with the FcγRIIIa-158F / F or FcγRIIIa-158F / V polymorphisms exhibit reduced Fc binding and, therefore, reduced ADCC when compared to FcγRIIIa-158V / V. Low levels or absence of the human N-linked oligosaccharide fucose enhances the ability of antibodies to induce ADCC, due to enhanced binding to human FcγRIIIa (CD16) (Shields et al., J Biol Chem 277:26733-40, 2002). Patients can be analyzed for FcγRIIIa polymorphism using conventional methods.

[0126] The present invention also provides a method of treating a subject having AL, comprising administering to a subject in need thereof an anti-CD38 antibody that binds to the SKRNIQFSCKNIYR (SEQ ID NO:2) and EKVQTLEAWVIHGG (SEQ ID NO:3) regions of human CD38 (SEQ ID NO:1), wherein the anti-CD38 antibody induces in vitro killing of pathogenic plasma cells expressing CD38 by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or in vitro modulation of CD38 enzymatic activity, wherein the subject is homozygous for the valine at position 158 of CD16.

[0127] The present invention also provides a method of treating a subject having AL, comprising administering to a subject in need thereof an anti-CD38 antibody that binds to the SKRNIQFSCKNIYR region (SEQ ID NO:2) and the EKVQTLEAWVIHGG region (SEQ ID NO:3) of human CD38 (SEQ ID NO:1), wherein the anti-CD38 antibody induces in vitro killing of pathogenic plasma cells expressing CD38 by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or in vitro modulation of CD38 enzymatic activity, wherein the subject is homozygous for phenylalanine at position 158 of CD16 or heterozygous for valine and phenylalanine at position 158 of CD16.

[0128] The present invention also provides a method for treating a patient suffering from AL, comprising administering to the patient in need thereof an anti-CD38 antibody that 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, wherein the anti-CD38 antibody induces killing of CD38-positive pathogenic plasma cells in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or in vitro modulation of CD38 enzymatic activity, wherein the subject is homozygous for valine at position 158 of CD16.

[0129] The present invention also provides a method for treating a patient suffering from AL, comprising administering to a patient in need thereof an anti-CD38 antibody that 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, wherein the anti-CD38 antibody induces in vitro killing of CD38-positive pathogenic plasma cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or in vitro modulation of CD38 enzymatic activity, wherein the patient is homozygous for phenylalanine at position 158 of CD16 or heterozygous for valine and phenylalanine at positions 158 of CD16.

[0130] The present invention also provides a method for treating a patient suffering from AL, comprising: determining whether the patient is homozygous or heterozygous for valine at position 158 of CD16; and Patients were given anti-CD38 antibodies. It 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; comprising heavy chain complementary determining region (HCDR): 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) sequences of SEQ ID NOs: 6, 7, and 8, respectively, and light chain complementary determining region (LCDR): 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) sequences of SEQ ID NOs: 9, 10, and 11, respectively; or comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5, for a period of time sufficient to treat the patient.

[0131] In some embodiments, polymerase chain reaction (PCR) and sequencing are used to determine whether the patient is homozygous or heterozygous for the valine at position 158 of CD16. [Administration / Pharmaceutical Composition]

[0132] In the methods of the present invention, the anti-CD38 antibody can be provided in a suitable pharmaceutical composition comprising the anti-CD38 antibody and a pharmaceutically acceptable carrier. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the anti-CD38 antibody is administered. Such vehicles can be liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizing, thickening, lubricating, and coloring agents. The concentration of anti-CD38 antibody in such pharmaceutical formulations can vary widely, i.e., from less than about 0.5% by weight, typically up to at least about 1% by weight, to as much as 15 or 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight, and will be selected primarily based on the desired dose, fluid volume, viscosity, etc., depending on the particular mode of administration chosen. Suitable vehicles and formulations (including other human proteins, such as human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B. Ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092, see especially pp. 958-989.

[0133] The mode of administration of the anti-CD38 antibody can be any suitable route, such as parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, nasal, vaginal, rectal), or other means known to those skilled in the art.

[0134] In the methods of the present invention, the anti-CD38 antibody can be administered to the patient via any suitable route, such as parenterally (by intravenous (IV) infusion or bolus injection), intramuscularly, subcutaneously, or intraperitoneally. IV infusion can 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.

[0135] The dosage of the anti-CD38 antibody administered to a patient is sufficient to alleviate or at least partially arrest the disease to be treated (a "therapeutically effective amount") and can sometimes be 0.005 mg to about 100 mg / kg, e.g., about 0.05 mg to about 30 mg / kg, or about 5 mg to about 25 mg / kg, or about 4 mg / kg, about 8 mg / kg, about 16 mg / kg, or about 24 mg / kg, or, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg, and can be even higher, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg.

[0136] Fixed unit doses, such as 50, 100, 200, 500, or 1000 mg, may also be administered, or the dose may be based on the surface area of ​​the patient, such as 500, 400, 300, 250, 200, or 100 mg / m 2 . Typically, 1 to 8 doses (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) are administered to treat AL, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses may be administered.

[0137] Administration of the anti-CD38 antibody in the methods of the present invention can be repeated after one, two, three, four, five, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer. Repeated treatment courses are also possible, such as for chronic administration. Repeated administration can be at the same dose or at different doses. For example, the anti-CD38 antibody can be administered at 8 mg / kg or 16 mg / kg at weekly intervals for 8 weeks, followed by 8 mg / kg or 16 mg / kg every two weeks for an additional 16 weeks, followed by 8 mg / kg or 16 mg / kg administered by intravenous infusion every four weeks.

[0138] In some embodiments, the anti-CD38 antibody is administered at 16 mg / kg once weekly for 8 weeks, then at 16 mg / kg once every two weeks for 16 weeks, and then at 16 mg / kg once every four weeks until discontinuation.

[0139] In some embodiments, the anti-CD38 antibody is administered at 8 mg / kg once weekly for 8 weeks, then at 8 mg / kg once every two weeks for 16 weeks, and then at 8 mg / kg once every four weeks until discontinuation.

[0140] In some embodiments, the anti-CD38 antibody is administered at 16 mg / kg once weekly for 4 weeks, then at 16 mg / kg once every two weeks for 16 weeks, and then at 16 mg / kg once every four weeks until discontinuation.

[0141] In some embodiments, the anti-CD38 antibody is administered at 8 mg / kg once weekly for 4 weeks, then at 8 mg / kg once every two weeks for 16 weeks, and then at 8 mg / kg once every four weeks until discontinuation.

[0142] Anti-CD38 antibodies can be administered as maintenance therapy, for example, once a week for 6 months or longer.

[0143] For example, at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 after initiation of treatment, or at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, after initiation of treatment, the anti- The CD38 antibody can be provided at a daily dosage of about 0.1 to 100 mg / kg per day, for example, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg, in single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0144] Anti-CD38 antibodies can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of cancer progression events, and / or reduce the risk of recurrence while the cancer is in remission. This may be particularly useful in patients who are known to have a tumor but whose location is difficult due to other biological factors.

[0145] Anti-CD38 antibodies can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective for conventional protein formulations and well-known lyophilization and reconstitution techniques can be used. [Subcutaneous administration of a pharmaceutical composition comprising an antibody specifically binding to CD38 and hyaluronidase]

[0146] The anti-CD38 antibody can be administered subcutaneously in the form of a pharmaceutical composition comprising the anti-CD38 antibody and hyaluronidase.

[0147] The concentration of the anti-CD38 antibody in the pharmaceutical composition for subcutaneous administration may be about 20 mg / ml.

[0148] The pharmaceutical composition for subcutaneous administration may contain between about 1,200 mg and 1,800 mg of the anti-CD38 antibody.

[0149] The pharmaceutical composition for subcutaneous administration may contain about 1,200 mg of anti-CD38 antibody.

[0150] The pharmaceutical composition for subcutaneous administration may contain about 1,600 mg of anti-CD38 antibody.

[0151] The pharmaceutical composition for subcutaneous administration may contain about 1,800 mg of anti-CD38 antibody.

[0152] The pharmaceutical composition for subcutaneous administration may contain between about 30,000 U and 45,000 U of hyaluronidase.

[0153] The pharmaceutical composition for subcutaneous administration may contain about 1,200 mg of anti-CD38 antibody and about 30,000 U of hyaluronidase.

[0154] The pharmaceutical composition for subcutaneous administration may contain about 1,800 mg of anti-CD38 antibody and about 45,000 U of hyaluronidase.

[0155] The pharmaceutical composition for subcutaneous administration may contain about 1,600 mg of anti-CD38 antibody and about 30,000 U of hyaluronidase.

[0156] The pharmaceutical composition for subcutaneous administration may contain about 1,600 mg of anti-CD38 antibody and about 45,000 U of hyaluronidase.

[0157] The pharmaceutical composition for subcutaneous administration may comprise hyaluronidase rHuPH20 having the amino acid sequence of SEQ ID NO: 22.

[0158] rHuPH20 is a recombinant hyaluronidase (HYLENEX® recombinant) and is described in International Patent Publication No. WO 2004 / 078140.

[0159] 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: 22]

[0160] Administration of the pharmaceutical composition comprising an anti-CD38 antibody and hyaluronidase can be repeated after one, two, three, four, five, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer. Repeated courses of treatment are also possible, such as for chronic administration. Repeated administration can be at the same dose or at different doses. For example, the pharmaceutical composition comprising an anti-CD38 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 can comprise approximately 1,200 mg of anti-CD38 antibody and approximately 30,000 IU of hyaluronidase, wherein the concentration of the antibody that specifically binds to CD38 in the pharmaceutical composition is approximately 20 mg / ml. The pharmaceutical composition to be administered can comprise approximately 1,800 mg of anti-CD38 antibody and approximately 45,000 IU of hyaluronidase. The pharmaceutical composition to be administered may contain about 1,600 mg of an anti-CD38 antibody and about 30,000 U of hyaluronidase. The pharmaceutical composition to be administered may contain about 1,600 mg of an anti-CD38 antibody and about 45,000 U of hyaluronidase.

[0161] The pharmaceutical composition comprising the anti-CD38 antibody and hyaluronidase can be administered subcutaneously to the abdominal area.

[0162] The pharmaceutical composition comprising the anti-CD38 antibody and hyaluronidase can be administered in a total volume of about 80 ml, 90 ml, 100 ml, 110 ml, or 120 ml.

[0163] For administration, 20 mg / ml of anti-CD38 antibody (in 25 mM sodium acetate, 60 mM sodium chloride, 140 mM D-mannitol, 0.04% polysorbate 20, pH 5.5) can be mixed with rHuPH20 (1.0 mg / mL (75-150 kU / mL) in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate 80, pH 6.5) and the mixture can be administered to the subject. [Combination therapy]

[0164] The anti-CD38 antibody can be administered in combination with a second therapeutic agent.

[0165] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody and a proteasome inhibitor for a period of time sufficient to treat AL.

[0166] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody, a proteasome inhibitor, and a corticosteroid for a period of time sufficient to treat AL.

[0167] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody in combination with a proteasome inhibitor, a corticosteroid, and cyclophosphamide for a period of time sufficient to treat AL.

[0168] In some embodiments, the second therapeutic agent is a proteasome inhibitor.

[0169] In some embodiments, the proteasome inhibitor is Velcade® (bortezomib); or a vinca alkaloid, such as vincristine; or an anthracycline, such as doxorubicin.

[0170] In some embodiments, the second therapeutic agent is a corticosteroid.

[0171] In some embodiments, the corticosteroid is dexamethasone.

[0172] In some embodiments, the corticosteroid is prednisone.

[0173] In some embodiments, the second therapeutic agent is cyclophosphamide.

[0174] In some embodiments, the second therapeutic agent is a glutamate derivative.

[0175] In some embodiments, the glutamate derivative is Thalomid® (thalidomide), Revlimid® (lenalidomide), or Actimid® (CC4047).

[0176] In some embodiments, the second therapeutic agent is Velcade® (bortezomib), a cyclophosphamide such as Cytoxan® or Neosar®, Alkeran® (melphalan), Thalomid® (thalidomide), Revlimid® (lenalidomide), or Pomalyst® (pomalidomide), a corticosteroid (dexamethasone), interferon alpha (IFN-α), a stem cell transplant, Ninlaro® (ixazomib), or NEOD001.

[0177] Bortezomib can be administered twice a week or once a week at 1.3 mg / m2 SQ Cyclophosphamide can be administered IV (intermittent therapy) at 40 to 50 mg / kg (400 to 1800 mg / m2) divided over 2 to 5 days; may be repeated at 2 to 4 week intervals; IV (continuous daily therapy): 60 to 120 mg / m2 / day (1 to 2.5 mg / kg / day); PO (intermittent therapy): 400 to 1000 mg / m2 divided over 4 to 5 days or PO (continuous daily therapy): 50 to 100 mg / m2 / day or 1 to 5 mg / kg / day.

[0178] Dexamethasone can be administered at 40 mg / week or 20 mg before and after administration of anti-CD38 antibody.

[0179] Melphalan can be administered orally at 9 mg / m2 once daily on days 1 to 4 of each cycle until cycle 9.

[0180] Thalidomide can be administered orally at a dose of 200 mg once daily.

[0181] Lenalidomide can be administered orally at 25 mg / day on days 1 to 21 of each cycle.

[0182] Pomalidomide can be administered orally at a dose of 4 mg on days 1 to 21 of repeated 28-day cycles.

[0183] Ixazomib can be administered IV at 24 mg / kg every 28 days.

[0184] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, and bortezomib for a period of time sufficient to treat AL.

[0185] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib and cyclophosphamide for a period of time sufficient to treat AL.

[0186] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib, cyclophosphamide, and a corticosteroid for a period of time sufficient to treat AL.

[0187] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib, cyclophosphamide, and dexamethasone for a period of time sufficient to treat AL.

[0188] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib and a corticosteroid for a period of time sufficient to treat AL.

[0189] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib and dexamethasone for a period of time sufficient to treat AL.

[0190] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, and melphalan for a period of time sufficient to treat AL.

[0191] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib and melphalan for a period of time sufficient to treat AL.

[0192] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib, melphalan, and a corticosteroid for a period of time sufficient to treat AL.

[0193] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib, melphalan, and dexamethasone for a period of time sufficient to treat AL.

[0194] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, and IFN-α for a period of time sufficient to treat AL.

[0195] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with IFN-α and a corticosteroid for a period of time sufficient to treat AL.

[0196] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with IFN-α and dexamethasone for a period of time sufficient to treat AL.

[0197] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with lenalinomide for a period of time sufficient to treat AL.

[0198] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with lenalinomide and cyclophosphamide for a period of time sufficient to treat AL.

[0199] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with lenalinomide, cyclophosphamide, and a corticosteroid for a period of time sufficient to treat AL.

[0200] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with lenalidomide, cyclophosphamide, and dexamethasone for a period of time sufficient to treat AL.

[0201] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with pomalidomide for a period of time sufficient to treat AL.

[0202] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with pomalidomide and a corticosteroid for a period of time sufficient to treat AL.

[0203] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with pomalidomide and dexamethasone for a period of time sufficient to treat AL.

[0204] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with thalidomide for a period of time sufficient to treat AL.

[0205] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with thalidomide and a corticosteroid for a period of time sufficient to treat AL.

[0206] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with thalidomide and dexamethasone for a period of time sufficient to treat AL.

[0207] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, and ixazomib for a period of time sufficient to treat AL.

[0208] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 and bortezomib for a period of time sufficient to treat light-chain amyloidosis (AL).

[0209] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with bortezomib and cyclophosphamide for a period of time sufficient to treat AL.

[0210] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with bortezomib, cyclophosphamide, and a corticosteroid for a period of time sufficient to treat AL.

[0211] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with bortezomib, cyclophosphamide, and dexamethasone for a period of time sufficient to treat AL.

[0212] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5, bortezomib, and a corticosteroid for a period of time sufficient to treat AL.

[0213] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with bortezomib and dexamethasone for a period of time sufficient to treat AL.

[0214] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 and melphalan for a period of time sufficient to treat AL.

[0215] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with bortezomib and melphalan for a period of time sufficient to treat AL.

[0216] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with bortezomib, melphalan, and a corticosteroid for a period of time sufficient to treat AL.

[0217] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with bortezomib, melphalan, and dexamethasone for a period of time sufficient to treat AL.

[0218] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 and IFN-α for a period of time sufficient to treat AL.

[0219] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with IFN-α and a corticosteroid for a period of time sufficient to treat AL.

[0220] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with IFN-α and dexamethasone for a period of time sufficient to treat AL.

[0221] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 and lenalinomide for a period of time sufficient to treat AL.

[0222] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with lenalinomide and cyclophosphamide for a period of time sufficient to treat AL.

[0223] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with lenalinomide, cyclophosphamide, and a corticosteroid for a period of time sufficient to treat AL.

[0224] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with lenalidomide, cyclophosphamide, and dexamethasone for a period of time sufficient to treat AL.

[0225] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 and pomalidomide for a period of time sufficient to treat AL.

[0226] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with pomalidomide and a corticosteroid for a period of time sufficient to treat AL.

[0227] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with pomalidomide and dexamethasone for a period of time sufficient to treat AL.

[0228] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with thalidomide for a period of time sufficient to treat AL.

[0229] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with thalidomide and a corticosteroid for a period of time sufficient to treat AL.

[0230] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with thalidomide and dexamethasone for a period of time sufficient to treat AL.

[0231] The present invention also provides a method for treating light-chain amyloidosis (AL), comprising administering to a patient in need thereof a combination of an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 and ixazomib for a period of time sufficient to treat AL.

[0232] In some embodiments, the patient is resistant to treatment with a proteasome inhibitor.

[0233] In some embodiments, the patient is resistant to treatment with cyclophosphamide.

[0234] In some embodiments, the patient is resistant to treatment with corticosteroids.

[0235] In some embodiments, the patient is resistant to treatment with a proteasome inhibitor, cyclophosphamide, and corticosteroids.

[0236] In some embodiments, the patient is resistant to treatment with Velcade® (bortezomib), cyclophosphamide, and dexamethasone.

[0237] In some embodiments, the combination of the anti-CD38 antibody and the second therapeutic agent can be administered within any convenient timeframe. For example, the anti-CD38 antibody and the second therapeutic agent can be administered to the patient on the same day, or even in the same intravenous infusion. However, the anti-CD38 antibody and the second therapeutic agent can also be administered on alternating days, weeks, months, etc. In some embodiments, the anti-CD38 antibody and the second therapeutic agent can be administered close enough in time so that they are simultaneously present at detectable levels in the treated patient (e.g., in the serum). In some embodiments, a course of treatment consisting of multiple doses of the second therapeutic agent is administered either before or after an entire course of treatment with the anti-CD38 antibody consisting of multiple doses over a period of time. A recovery period of one, two, several, or even several weeks can be used between administration of the anti-CD38 antibody and the second therapeutic agent.

[0238] The anti-CD38 antibody or the combination of an anti-CD38 antibody and a second therapeutic agent can be administered with any form of radiation therapy (including external beam radiation; intensity-modulated radiation therapy (IMRT); and any form of radiosurgery (including Gamma Knife, Cyberknife, linear accelerator (Linac), and interstitial radiation (e.g., implanted radioactive seeds, GliaSite balloons)) and / or with surgery.

[0239] While the present invention has been described in general terms, embodiments of the invention will be further disclosed in the following examples, which should not be construed as limiting the scope of the claims. [Further embodiments of the present invention]

[0240] Set forth below are certain further embodiments according to the invention disclosed elsewhere herein. Features from the embodiments of the invention set forth above that are described in relation to the invention disclosed herein also relate to each of these further numbered embodiments. [1.] An anti-CD38 antibody that competes with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5 for binding to CD38 and is used to treat a patient with a CD38-positive hematological malignancy, wherein the patient is undergoing hematopoietic stem cell transplantation (HSCT). [2.] An anti-CD38 antibody for use as described in Example 1, wherein the CD38-positive hematological malignancy is a. Light-chain amyloidosis (AL), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Birch's lymphoma (BL), follicular lymphoma (FL), or mantle cell lymphoma (MCL); b. Plasma cell diseases; c. Light chain amyloidosis (AL); d. Multiple myeloma (MM); or e. Waldenstrom macroglobulinemia. [3.] An anti-CD38 antibody for use as described in Example 2, wherein AL is cardiac stage I, cardiac stage II, cardiac stage III, recurrent, or refractory. [4.] An anti-CD38 antibody for use as described in any one of Examples 1 to 3, wherein the HSCT a. Allogeneic, autologous, or isogenic; or b. Includes transplantation of blood stem cells derived from bone marrow, blood, or amniotic fluid. [5.] An anti-CD38 antibody for use as described in any one of Examples 1 to 4, wherein the anti-CD38 antibody is administered before, during, or after HSCT. [6.] An anti-CD38 antibody for use as described in any one of Examples 1 to 5, wherein the patient has completed chemotherapy and / or radiotherapy before HSCT. [7.] An anti-CD38 antibody for use as described in any one of Examples 1 to 6, wherein the anti-CD38 antibody binds to the SKRNIQFSCKNIYR (SEQ ID NO: 2) region and the EKVQTLEAWVIHGG (SEQ ID NO: 3) region of human CD38 (SEQ ID NO: 1). [8.] An anti-CD38 antibody for use as described in any one of Examples 1 to 7, wherein the anti-CD38 antibody comprises heavy chain complementary determining region (HCDR) 1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 6, 7, and 8, respectively; and light chain complementary determining region (LCDR) 1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 9, 10, and 11, respectively. [9.] An anti-CD38 antibody for use as described in any one of Examples 1 to 8, wherein the anti-CD38 antibody comprises a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5. [10.] An anti-CD38 antibody for use as described in any one of Examples 1 to 9, wherein the anti-CD38 antibody comprises a heavy chain comprising an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12 and a light chain comprising an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13. [11.] An anti-CD38 antibody for use as described in any one of Examples 1 to 10, wherein the anti-CD38 antibody comprises a heavy chain of SEQ ID NO: 12 and a light chain of SEQ ID NO: 13. [12.] An anti-CD38 antibody that competes with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5 for binding to CD38 and is used to treat patients with light chain amyloidosis. [13.] An anti-CD38 antibody for use as described in Example 12, wherein the patient is resistant to treatment with a proteasome inhibitor, cyclophosphamide, and / or corticosteroids. [14.] An anti-CD38 antibody for use as described in Example 13, wherein the proteasome inhibitor is Velcade® (bortezomib). [15.] An anti-CD38 antibody for use as described in any one of Examples 13 to 14, wherein the corticosteroid is dexamethasone. [16.] An anti-CD38 antibody for use as described in any one of Examples 12 to 15, wherein the anti-CD38 antibody is administered in combination with a second therapeutic agent. [17.] An anti-CD38 antibody for use as described in Example 16, wherein the second therapeutic agent is a proteasome inhibitor, cyclophosphamide, or a corticosteroid. [18.] An anti-CD38 antibody for use as described in any one of Examples 16 to 17, wherein the proteasome inhibitor is Velcade® (bortezomib) or NINLARO® (ixazomib). [19.] An anti-CD38 antibody for use as described in any one of Examples 16 to 18, wherein the corticosteroid is dexamethasone. [20.] An anti-CD38 antibody for use as described in any one of Examples 16 to 19, wherein the second therapeutic agent is Velcade® (bortezomib), NINLARO® (ixazomib), Kyprolis® (carfilzomib), Farydak® (panobinostat), cyclophosphamide, Alkeran® (melphalan), Thalomid® (thalidomide), Revlimid® (lenalidomide), Pomalyst® (pomalidomide), dexamethasone, or interferon alpha. [21.] An anti-CD38 antibody for use as described in any one of Examples 12 to 20, wherein the anti-CD38 antibody is administered in combination with a proteasome inhibitor, cyclophosphamide, and a corticosteroid. [22.] An anti-CD38 antibody for use as described in Example 21, wherein the proteasome inhibitor is Velcade® (bortezomib). [23.] An anti-CD38 antibody for use as described in any one of Examples 21 to 22, wherein the proteasome inhibitor is NINLARO® (ixazomib). [24.] An anti-CD38 antibody for use as described in any one of Examples 21 to 23, wherein the corticosteroid is dexamethasone. [25.] An anti-CD38 antibody for use as described in any one of Examples 21 to 24, wherein the proteasome inhibitor is Velcade® (bortezomib) and the corticosteroid is dexamethasone. [26.] An anti-CD38 antibody for use as described in any one of Examples 21 to 25, wherein the proteasome inhibitor is NINLARO® (ixazomib) and the corticosteroid is dexamethasone. [27.] An anti-CD38 antibody for use as described in any one of Examples 16 to 26, wherein the anti-CD38 antibody and the second therapeutic agent are administered simultaneously, sequentially, or separately. [28.] An anti-CD38 antibody for use as described in any one of Examples 16 to 27, wherein the anti-CD38 antibody and the second therapeutic agent are administered simultaneously, sequentially, or separately. [29.] An anti-CD38 antibody for use as described in any one of Examples 21 to 28, wherein the anti-CD38 antibody, the proteasome inhibitor, cyclophosphamide, and the corticosteroid are administered simultaneously, sequentially, or separately. [30.] An anti-CD38 antibody for use as described in any one of Examples 12 to 29, wherein AL is cardiac stage I, cardiac stage II, cardiac stage III, recurrent, or refractory. [31.] An anti-CD38 antibody for use as described in any one of Examples 12 to 30, wherein the patient is undergoing hematopoietic stem cell transplantation (HSCT). [32.] An anti-CD38 antibody for use as described in any one of Examples 12 to 31, wherein the HSCT is allogeneic, autologous, or syngeneic. [33.] An anti-CD38 antibody for use as described in any one of Examples 12 to 32, wherein the HSCT comprises transplantation of blood stem cells derived from bone marrow, blood, or amniotic fluid. [34.] An anti-CD38 antibody for use as described in any one of Examples 12 to 33, wherein the anti-CD38 antibody is administered before, during, or after HSCT. [35.] An anti-CD38 antibody for use as described in any one of Examples 12 to 34, wherein the patient has completed chemotherapy and / or radiotherapy prior to HSCT. [36.] An anti-CD38 antibody for use as described in any one of Examples 12 to 35, wherein the patient is further treated with radiotherapy. [37.] An anti-CD38 antibody for use as described in any one of Examples 12 to 36, wherein the anti-CD38 antibody does not mediate the killing of CD34-positive hematopoietic progenitor cells by complement-dependent cytotoxicity (CDC). [38.] An anti-CD38 antibody for use as described in any one of Examples 12 to 37, wherein the anti-CD38 antibody induces killing of CD38-positive plasma cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular phagocytosis, complement-dependent cytotoxicity (CDC), apoptosis, or modulation of CD38 enzymatic activity. [39.] An anti-CD38 antibody for use as described in any one of Examples 12 to 38, wherein the anti-CD38 antibody competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5. [40.] An anti-CD38 antibody for use as described in any one of Examples 12 to 39, wherein the anti-CD38 antibody binds to at least the SKRNIQFSCKNIYR (SEQ ID NO: 2) region and the EKVQTLEAWVIHGG (SEQ ID NO: 3) region of human CD38 (SEQ ID NO: 1). [41.] An anti-CD38 antibody for use as described in any one of Examples 12 to 40, wherein the anti-CD38 antibody comprises the heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3 amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, and the light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3 amino acid sequences of SEQ ID NOs: 9, 10, and 11, respectively. [42.] An anti-CD38 antibody for use as described in any one of Examples 12 to 41, wherein the anti-CD38 antibody comprises a VH comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:4 and a VL comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5. [43.] An anti-CD38 antibody for use as described in any one of Examples 12 to 42, wherein the anti-CD38 antibody comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5. [44.] An anti-CD38 antibody for use as described in any one of Examples 12 to 43, wherein the anti-CD38 antibody comprises a heavy chain comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12 and a light chain comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13. [45.] An anti-CD38 antibody for use as described in any one of Examples 12 to 44, wherein the anti-CD38 antibody comprises a heavy chain of SEQ ID NO: 12 and a light chain of SEQ ID NO: 13. [46.] An anti-CD38 antibody for use as described in any one of Examples 12 to 46, wherein the anti-CD38 antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of: a. VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15; b. VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; c. VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or d. VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by Kabat, Chothia, or IMGT. [47.] An anti-CD38 antibody for use as described in Example 46, wherein the anti-CD38 antibody comprises e. VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15; f. VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; g. VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or h. VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21. [48.] An anti-CD38 antibody for use as described in any one of Examples 12 to 47, wherein the anti-CD38 antibody is a humanized antibody or a human antibody. [49.] An anti-CD38 antibody for use as described in any one of Examples 12 to 48, wherein the anti-CD38 antibody is of the IgG1, IgG2, IgG3, or IgG4 isotype. [50.] An anti-CD38 antibody for use as described in any one of Examples 12 to 49, wherein the anti-CD38 antibody is of the IgG1 isotype. [51.] An anti-CD38 antibody for use as described in any one of Examples 12 to 50, wherein the anti-CD38 antibody is administered intravenously. [52.] An anti-CD38 antibody for use as described in any one of Examples 12 to 51, wherein the anti-CD38 antibody is administered subcutaneously in the form of a pharmaceutical composition comprising the anti-CD38 antibody and hyaluronidase. [53.] An anti-CD38 antibody for use as described in Example 42, wherein the hyaluronidase is rHuPH20 of SEQ ID NO:22. [Example 1. Materials and Methods]

[0241] Mobilized blood stem cells and precursor cells from MM and AL patients who underwent blood stem cell mobilization and collection in an IRB-approved clinical study requiring written informed consent (Tufts Medical Center IRB #10680, Pre-Clinical Studies of DARZALEX™ (daratumumab) in Stem-Cell Mobilization and Transplant for Patients with Clonal Plasma cell Diseases) were used to study the effect of DARZALEX™ (daratumumab) on in vitro CD34+ cell growth. Unselected and CD34-selected mobilized blood precursor cells from patients with MM and AL were used. The effect of DARZALEX™ (daratumumab) or an isotype control antibody on the growth of in vitro pioneer cell colonies was evaluated in a semisolid assay as an indicator of the effect of DARZALEX™ (daratumumab) on the proliferation of CD34+ human hematopoietic precursor cells.

[0242] Methylcellulose precursor cell assays containing recombinant interleukins (Stem Cell Technologies, Vancouver, CA; catalog number 04435) were performed according to the manufacturer's instructions using several concentrations of unselected and CD34-selected mobilized blood stem cells from day 1 leukapheresis products. CD34 selection was performed using a Miltenyi MiniMacs apparatus. A concentration of 0.5 × 10⁴ / ml of CD34-unselected cells and 500 cells / ml of CD34-selected cells were used. Assays were performed with various concentrations of DARZALEX™ (daratumumab) or a control antibody; in some assays, cells were plated in medium containing DARZALEX™ (daratumumab) or a control antibody, while in other assays, cells were plated after one hour of incubation in complement-enriched human serum with DARZALEX™ (daratumumab) or a control antibody at 37°C in 5% CO₂. Colonies (CFU-GM, BFU-E, CFU-Mix) were counted on day 14. [Example 2. CD38 expression on cloned AL plasma cells]

[0243] Based on the transcriptional profile of FACS-sorted CD138+ bone marrow plasma cells, clonal plasma cells from patients with AL showed high levels of CD38 mRNA (n=16, GEO GSE24128; Zhou et al., Clin Lymphoma Myeloma Leuk 12:49-58, 2012) [(Figure 1)]. In addition, in an analysis of the immunophenotype of CD138+ bone marrow plasma cells from newly diagnosed AL patients, CD38 was found on the cell surface in all cases (Paiva et al., Blood 117:3613-6, 2011). [Example 3. NK cell lineage from a patient with AL is functional and induces DARZALEX against CD38-expressing cells 3 weeks after stem cell transplantation (SCT)] [TM] [Daratumumab-mediated ADCC]

[0244] Using the IRB-approved study protocol described in Example 1, which required written informed consent, we evaluated NK cells (CD3- / CD56+ / CD16+) obtained from AL patients three weeks after SCT. The percentage and number of NK cells in peripheral blood samples were assessed, as were the in vitro activity of these NK cell effectors in an ADCC assay targeting human plasma cells (MM1S cells) with DARZALEX™ (daratumumab). MM1S cells express high levels of CD38, making them a suitable target for DARA. Flow cytometry was performed using the FlowCellect™ Human Natural Killer Cell Characterization Kit (Millipore, Billerica, MA; Catalog No. FCIM025164). Bioluminescent cytotoxicity assays (Cell Technology; Mountain View, CA) were performed according to the manufacturer's instructions. The target cell concentration was optimized at 5000 MM1S cells per well. Target cells were incubated with DARZALEX™ (daratumumab) or a control antibody (human IgG1κ, Sigma-Aldrich, St. Louis, MO) at 100 ng / ml for 15 minutes in a 37°C, 5% CO2 incubator. These were then added to wells containing patient NK cells at a 10:1 effector:target cell concentration. The number of NK cells in each sample was calculated based on flow cytometric analysis of fresh patient samples. ADCC was determined by calculating the luminescence value for each reaction condition using a standard control according to the manufacturer's instructions. The amount of lysis (%ADCC) in each condition was determined using the following calculation: %ADCC = (sample) - (control 1) - (control 2) x 100 (control 3) - (control 1) Sample: Sample using DARZALEX™ (daratumumab) Control 1: Assay using isotype control (target spontaneous release) Control 2: no antibody, no effector cells Control 3: Maximum lysis in the presence of lysis reagent The amount of DARZALEX™ (daratumumab)-specific lysis was calculated by subtracting the % ADCC using the isotype control from the % ADCC using DARZALEX™ (daratumumab).

[0245] like As shown in Figure 2, NK cells comprised nearly one-third of all mononuclear cells in the peripheral blood of AL patients (n=9) 3 weeks after SCT. In a bioluminescent ADCC assay, NK cells from patients with AL (n=6) combined with DARZALEX™ (daratumumab) achieved a median specific lysis of 32% (range, -3 to 73%). [Example 4. Even though high CD38 expression was found on CD34+ hematopoietic progenitor cells, DARZALEX] [TM] [(Daratumumab) does not induce killing of CD34+ hematopoietic progenitor cells.]

[0246] CD38 expression on CD34+ cells was analyzed by flow cytometry using APC-conjugated anti-human CD38 (HIT2, Biolegend, San Diego CA). CD34+ cells had the appearance of myeloid blasts and expressed high levels of CD38 ( [Figure 3]).

[0247] As described in Example 1, the effect of DARZALEX™ (daratumumab) on in vitro CD34+ cell growth was studied using mobilized blood stem cells and progenitor cells from MM and AL patients who underwent blood stem cell mobilization and collection in an IRB-approved clinical study requiring informed consent. Unselected and CD34-selected mobilized blood progenitor cells from patients with MM and AL were used, and the effect of daratumumab or an isotype control antibody on in vitro progenitor cell colony growth was evaluated in a semisolid assay as an indicator of the effect of DARZALEX™ (daratumumab) on CD34+ human hematopoietic progenitor cell proliferation.

[0248] Methylcellulose precursor cell assays containing recombinant interleukins (Stem Cell Technologies, Vaneouver, CA; catalog number 04435) were performed according to the manufacturer's instructions using several concentrations of unselected or CD34-selected mobilized blood stem cells from day 1 leukapheresis products. CD34 selection was performed using a Miltenyi MiniMacs apparatus. A concentration of 0.5 × 10⁴ / ml of CD34-unselected cells and 500 cells / ml of CD34-selected cells were used. Assays were performed with various concentrations of DARZALEX™ (daratumumab) or a control antibody; in some assays, cells were plated in medium containing DARZALEX™ (daratumumab) or a control antibody, while in other assays, cells were plated after one hour of incubation in complement-enriched human serum with DARZALEX™ (daratumumab) or a control antibody at 37°C in 5% CO₂. Colonies (CFU-GM, BFU-E, CFU-Mix) were counted on day 14.

[0249] Thawed cryopreserved unselected mobilized blood progenitor cells grew similar numbers of CFU-GM (0.05 mmol / l) in culture with DARZALEX™ (daratumumab) or isotype control (500 or 1000 ng / ml) compared to no antibody controls. [Figure 4A]) and BFU-E ( [Figure 4B]). In these experiments, thawed, unselected mobilized human blood progenitor cells were plated at a concentration of 5 × 104 cells per ml of semisolid culture medium (methylcellulose) in the presence of 500 ng / ml or 1000 ng / ml of DARZALEX™ (daratumumab) or an isotype control. CFU-GM and GFU-E were counted after two weeks as a percentage of CFU-GM compared to a no-antibody control. Notably, for unknown reasons, significantly more CFU-GM were present in the wells containing 1000 ng / ml of the isotype control antibody.

[0250] The effect of DARZALEX™ (daratumumab) on fresh, unselected mobilized blood progenitor cells was also evaluated. DARZALEX™ (daratumumab) did not reduce CFU-GM ( [Figure 5A]) or BFU-E ( [Figure 5B]) DARZALEX™ (daratumumab) and the isotype control both increased CFU-GM formation at similar levels when compared to the no-antibody control.

[0251] The ability of DARZALEX™ (daratumumab) to induce CDC on fresh CD34+ selected hematopoietic progenitor cells was evaluated. CD34+ cells were cultured in 10% complement-enriched human serum without antibody, with 500 ng / ml DARZALEX™ (daratumumab), or with 500 ng / ml isotype control for 1 hour and then plated directly into semisolid culture medium. Colony formation was assessed per 500 CD34+ selected cells. DARZALEX™ (daratumumab) did not reduce CFU-GM ( [Figure 6A]) or BFU-E ( Figure 6B shows that the antibody does not induce CDC on naive CD34-selected progenitor cells, despite the presence of CD38 on these cells. For unknown reasons, more BFU-E were observed in samples treated with DARZALEX™ (daratumumab). DARZALEX™ (daratumumab) increased the number of BFU-E formed.

[0252] The effect of DARZALEX™ (daratumumab) on fresh CD34-selected hematopoietic progenitor cells was also tested by directly plating the cells into methylcellulose containing 500 ng / ml or 1000 ng / ml DARZALEX™ (daratumumab), or an isotype control, or no antibody. On day 14, for every 500 CFU-GM ( [Fig. 7A]) or BFU-E per 500 CD34 selected cells ( Colony formation was assessed using a 400-well plate (Figure 7B). DARZALEX™ (daratumumab) did not reduce colony formation at any concentration, indicating that DARZALEX™ (daratumumab) is not toxic to CD34+ cell colony formation in this assay system. The results in Figures 6A, 6B, and 7A show the results for mobilized blood progenitor cells from one patient with MM and two patients with AL. The results in Figure 7B show the results for mobilized blood progenitor cells from two patients with MM and one patient with AL. For unknown reasons, the BFU-E assay contained significantly more BFU-E in the presence of 500 ng / ml DARZALEX™ (daratumumab). [Example 5. DARZALEX via Patient NK Cells] [TM] [(Daratumumab)-mediated ADCC depends on the FcγRIIIA genotype.]

[0253] Genomic DNA from patient cells was used for PCR-based analysis using previously described methods and primers (Hatjiharissi et al., Blood 110:2561-2564, 2007). Amplicons were sequenced and analyzed for FcγRIIIA-158 polymorphism. DARZALEX™ (daratumumab)-specific cleavage in ADCC assays was compared in patients with V / F and V / V encoding FcγRIIIA-158aa alleles compared to F / F homozygotes.

[0254] PCR-based analysis showed that of the 10 patients analyzed in this study, 6 patients had V / F or V / V polymorphism with a median lytic activity of 60% (31 to 98), while 4 patients had F / F alleles with a median lytic activity of 17% (0 to 32) ( [Figure 8]; P < 0.05, Mann Whitney, two-tailed).

[0255] The initial correlation of Fc receptor polymorphism with DARZALEX™ (daratumumab) ADCC may be examined in future clinical trials. [Example 6. Evaluation of DARZALEX Compared to CyBorD Alone in Newly Diagnosed Systemic AL Amyloidosis] [TM] A randomized phase 3 study of the efficacy and safety of daratumumab in combination with cyclophosphamide, bortezomib, and dexamethasone (CyBorD).

[0256] A phase 3, two-group, open-label study was conducted comparing the combination of DARZALEX™ (daratumumab) and CyBorD (cyclophosphamide, bortezomib, and dexamethasone) with CyBorD alone in subjects with newly diagnosed AL amyloidosis.

[0257] There are currently no approved treatments for AL amyloidosis. In the absence of approved therapies, drugs developed for multiple myeloma are prescribed. CyBorD combinations are the most commonly used initial treatment for AL amyloidosis in the European Union and the United States (Venner et al., Blood 119:4387-4390, 2012; Mikhael et al., Blood 119:4391-4394, 2012; Jaggard et al., Hematologica 99:1479-1485, 2014; Palladini et al., Blood 126:612-615, 2015). [Main Purpose]

[0258] The primary objective was to evaluate the complete hematologic response following treatment with DARZALEX™ (daratumumab) in combination with CyBorD compared with CyBorD alone in patients with AL. [Secondary purpose] ● PFS was assessed based on all-cause mortality and progressive disease (PD, including hematologic PD and organ PD according to consensus guidelines). ●Assess the organ response rate (OrRR) (Comenzo 2012). Kidneys Heart Liver ●Assess the hematologic response rate (ORR) and the hematologic VGPR or better (i.e., CR+VGPR) response rate. ●Assess organ progression rates of the heart, kidneys, and liver. ●Evaluate the duration and time to achieve blood CR and VGPR or better response rates separately. ●Assess the duration and time of achievement of organ responses. • To evaluate the safety and tolerability of DARZALEX™ (daratumumab) when administered in combination with CyBorD. • To evaluate the pharmacokinetics of DARZALEX™ (daratumumab). • To evaluate the immunogenicity of DARZALEX™ (daratumumab). ● To assess treatment effects on patient-reported outcomes (PROs), including the SF-36 health questionnaire, EuroQol-5 dimensions (EQ-5D-5L), and the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30. [Purpose of Exploration] ●Assessment of biomarkers of response including high-sensitivity (HS) troponin T. • Discover biomarkers that predict response or resistance to treatment. ●Explore minimal residual disease status in patients with amyloidosis. [end] [Primary Endpoint]

[0259] The primary endpoint was complete hematological response rate. [Secondary End Points]

[0260] Secondary efficacy endpoints include: Progression-free survival (PFS) based on all-cause mortality. ●Organ response rate (OrRR) of kidney, heart, and liver. ●Overall survival (OS). ●Time to next treatment (TNT). ●Time to disease progression (TTP). ●Time to progression of hematologic disease. ●Overall hematologic response. Blood VGPR or better rate. ●Time to achieve complete hematologic response (or VGPR or better). Duration of complete hematologic response (or VGPR or better). ●Time required for organ response to occur Duration of organ response ● To assess treatment effects on patient-reported outcomes including the European Organization for Research and Treatment (EORTC) Quality of Life Questionnaire-C30, the 36-Item Short Form Health Survey (SF-36), and the European EQ-5D-5L questionnaire. [Exploration End]

[0261] The exploratory endpoint was minimal residual disease status in patients who achieved a complete hematologic response in the bone marrow and blood. [Assumption]

[0262] The primary hypothesis of this study was that the combination of DARZALEX™ (daratumumab) and CyBorD would improve complete hematologic response rates compared with CyBorD alone in subjects with newly diagnosed AL amyloidosis. Study Design:

[0263] This multicenter, Phase 3, two-cohort, open-label study compared the combination of DARZALEX™ (daratumumab) and CyBorD with CyBorD alone in subjects with newly diagnosed AL amyloidosis. Approximately 360 subjects, stratified by cardiac risk (stages I, II, and IIIa), were randomized into two cohorts to initially receive CyBorD or CyBorD plus DARZALEX™ (daratumumab). Each cycle was four weeks. DARZALEX™ (daratumumab) was administered at 16 mg / kg weekly for the first two treatment cycles (eight weeks), then every two weeks for four treatment cycles (16 weeks), and then every four weeks for up to six treatment cycles (24 weeks), based on CyBorD (in both cohorts). Patients randomized to the DARZALEX™ (daratumumab) group were allowed to continue DARZALEX™ (daratumumab) every four weeks after six cycles for up to two years after disease progression. Patients received weekly cyclophosphamide 300 mg / m² orally or IV, bortezomib 1.3 mg / m² quarterly, and dexamethasone 40 mg. Cycles were repeated every four weeks, with a maximum of six cycles administered.

[0264] Prior to randomization, a safety trial was conducted in six subjects treated with DARZALEX™ (daratumumab) plus CyBorD for at least one cycle to establish the safety of the combination regimen. Dosing for these six subjects was staggered so that subjects received their first dose no less than 48 hours after previously enrolled subjects. Safety assessments were conducted by the sponsor and external experts after the six subjects completed at least one cycle before the start of the randomized portion of the protocol. Subjects in the safety trial continued to undergo all planned assessments specified in the T&E and contributed to the overall safety assessment of the DARZALEX™ (daratumumab) + CyBorD regimen. However, these subjects were not included in the overall efficacy assessment. [Object selection] [Selection criteria] 1. The subject must be at least 18 years old. 2. The histopathological diagnosis of amyloidosis or light-chain degenerative diseases is based on the polarization microscopic detection or characteristic electron microscopic appearance of green birefringent material in Congo red-stained tissue samples. 3. Measurable amyloid light chain amyloidosis disease defined by at least one of the following: serum monoclonal protein >= 0.5 g / dL by protein electrophoresis, monoclonal protein > 200 mg / dL in urine after 24 hours of electrophoresis, serum free light chains >= 5.0 mg / dL, an abnormal kappa / lambda ratio, or a difference between involved and uninvolved free light chains (dFLC) 5mg / dL. 4. Patients must have newly diagnosed AL amyloidosis and have not received prior systemic therapy. The only exception is if the subject requires emergency treatment, in which case they may receive bortezomib, cyclophosphamide, and / or dexamethasone (or equivalent steroids) for up to 4 weeks prior to randomization. 5. Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0, 1, or 2 6. Subjects must have pre-treatment clinical laboratory values ​​that meet the following criteria during the screening phase: i) Absolute neutrophil count 1.0×109 / L; ii) Hemoglobin level 7.5 g / dL ( 5mmol / L); (blood transfusion to maintain Hb>7.5 is acceptable); iii) Platelet count > 50 × 109 / L; platelet transfusion is acceptable iv) Alanine aminotransferase level (ALT) 2.5 times the upper limit of normal (ULN); v) Aspartate aminotransferase (AST) 2.5 times the upper limit of normal (ULN); vi) Total bilirubin level 1.5×ULN, (except Gilbert syndrome: direct bilirubin <2×ULN); vii) Creatinine clearance 20 mL / min; Note that creatinine clearance can be measured by 24-hour urine studies or estimated using validated equations such as MDRD, CKD-epi, or Cockcroft Gault (see Appendix 3 for details) viii) TSH and free T4 are within normal limits. If necessary, patients can receive thyroid hormone therapy to correct underlying hypothyroidism. 7. Women of childbearing potential must use a highly effective method of birth control for 4 weeks before starting treatment, during treatment, during dose interruptions, and for 4 weeks after stopping the study drug. For subjects participating in clinical studies, birth control complies with local regulations regarding the use of birth control methods: for example, established use of oral, injectable, or implantable hormonal contraceptives; placement of an intrauterine device or intrauterine system; barrier methods: condoms with spermicidal foam / gel / film / cream / suppositories, or closure caps (diaphragms or cervical / dome caps) with spermicidal foam / gel / film / cream / suppositories (if hormonal or IUD contraception is medically contraindicated, two or other effective or highly effective methods may be used); sterilization of the male partner (the vasectomized partner should be the subject's only partner); and true abstinence during and after the study (for women, 3 months after the last dose of DARZALEX™ (daratumumab)) when consistent with the subject's preferred and usual lifestyle. 8. Men who are sexually active with women of childbearing age and have not undergone a vasectomy must agree to use a barrier method for birth control, such as condoms with spermicidal foam / gel / film / cream / suppositories, or their partner's use of a closure (diaphragm or cervical / dome cover) with spermicidal foam / gel / film / cream / suppositories. All men must also refrain from donating sperm during the study and for 3 months after receiving the last dose of study medication. 9. Women of childbearing age must undergo two negative serum or urine pregnancy tests at screening, the first within 10 to 14 days before administration and the second within 24 hours before administration. 10. Each subject must sign an Informed Consent Form (ICF), indicating that he or she understands the purpose of the study and the required procedures and is willing to participate in the study. The subject must be willing and able to comply with the prohibitions and restrictions set forth in this protocol, as mentioned in the ICF. [Exclusion criteria] 1. Previous treatment for AL amyloidosis or multiple myeloma, with the exception of a single cycle (up to 4 weeks) of bortezomib, cyclophosphamide, and / or dexamethasone (or equivalent steroids) before randomization. 2. Previous or current diagnosis of symptomatic multiple myeloma as defined by CRAB criteria, including the presence of lytic bone disease, plasmacytoma, and / or hypercalcemia. 3. Evidence of significant cardiovascular disease as follows: a.NT-ProBNP>8500ng / L. b. New York Heart Association (NYHA) classification IIIB or IV heart failure. c. Unstable angina or myocardial infarction within 6 months before the first dose. d. Grade 2 or 3 atrioventricular (AV) block or sick sinus syndrome, unless the subject has a pacemaker (any grade of Mobitz type I AV block is allowed). e. Known history of sustained (>30 seconds) ventricular tachycardia or cardiac syncope. Known history of recurrent non-sustained ventricular tachycardia (>3 times) despite antiarrhythmic therapy. f. Screening 12-lead ECG showing a corrected baseline QT interval (QTcF) >470 msec. g. Despite drug treatment (e.g., midodrine, fludrocortisone), the following symptoms persist: supine systolic blood pressure <90 mm Hg, or symptomatic orthostatic hypotension, or a decrease in resting systolic blood pressure >20 mm Hg. h. Left ventricular ejection fraction (LVEF) <40% as determined by transthoracic echocardiography, MUGA scan, cardiac MRI, or cardiac catheterization. This assessment is required during the screening period. 4. Subjects who are scheduled to undergo stem cell transplantation during the first six cycles of protocol treatment should be excluded. Stem cell collection during the first six cycles of protocol treatment is permitted. 5. Diagnosis or treatment of malignant tumors other than AL, except for the following: a. The malignancy has been treated with curative intent and there is no known active disease before randomization 5 years. b. Adequately treated non-melanoma skin cancer or lentigo maligna with no evidence of disease. c. Carcinoma in situ (e.g., cervical cancer, breast cancer) that has been adequately treated and has no evidence of disease 6. The subject is known to have chronic obstructive pulmonary disease (COPD), with a forced expiratory volume in 1 second (FEV1) < 50% of the predicted normal value. Note that an FEV1 test is required for patients suspected of having COPD; if the FEV1 is < 50% of the predicted normal value, the subject must be excluded. 7. Subjects who have been known to have moderate or severe persistent asthma (see Appendix 5) within the past 2 years, or currently have any category of uncontrolled asthma. (Note: Subjects currently with controlled intermittent asthma or controlled mild persistent asthma are permitted to participate in the study.) 8. Subjects are known to be seropositive for human immunodeficiency virus (HIV), known to be positive for hepatitis B surface antigen, or known to have a history of hepatitis C. 9. Grade 3 sensory or grade 1 painful peripheral neuropathy. 10. Known allergic reaction to bortezomib, boron, or mannitol 11. The subject has any concurrent medical condition or disease (e.g., active systemic infection) that may interfere with the study procedures or results, or that the investigator believes will pose a hazard to participation in this study. 12. Any form of secondary or familial (ATTR) amyloidosis. 13. Subjects with known allergy, anaphylaxis, or intolerance to the monoclonal antibody, or human protein, or its excipients (see Investigator's Brochure) or known sensitivity to mammalian-derived products. 14. The subject is known or suspected to be unable to comply with the study protocol (e.g., due to alcoholism, drug dependence, or psychiatric disorder), or suffers from any condition that, in the opinion of the investigator, would make participation in the study not in the best interests of the subject (e.g., impair their health), or that could prevent, limit, or confound protocol-mandated assessments. 15. The subjects are women who are pregnant, breastfeeding, or planning to become pregnant at the time of registration for this study or within 6 months after the last dose of study drug. 16. Subjects received investigational drugs (including investigational vaccines) or used invasive investigational medical devices within 4 weeks prior to Day 1 of Cycle 1 (except investigational anti-myeloma drugs, which cannot be taken within 2 weeks prior to Day 1 of Cycle 1, as described in Exclusion Criteria 3). 17. Subject has undergone major surgery within 2 weeks prior to Day 1 of Cycle 1, will not fully recover from surgery, or is scheduled to undergo surgery during the time of the subject's anticipated study participation or within 2 weeks after the last dose of study drug. Note: Subjects undergoing planned surgery under local anesthesia may participate. [Safety Assessment]

[0265] Safety will be measured by adverse events, laboratory test results, ECGs, vital sign measurements, physical examination findings, and ECOG performance status. Any clinically relevant changes occurring during the study will be recorded in the adverse events section of the eCRF. Any clinically significant abnormality persisting at study completion / early withdrawal will be followed by the investigator until resolution or clinical stabilization is achieved. [effect] [Reaction category]

[0266] Disease assessments will be performed every 28 days on the scheduled assessment day (± 3 days). If treatment is delayed for any reason, disease assessments will be performed according to the schedule, regardless of any changes in the dosing regimen.

[0267] Disease assessments will be performed by a central laboratory (unless otherwise specified) according to a time-based schedule until disease progression. This study will utilize the consensus recommendations for AL amyloidosis treatment response criteria (Comenzo et al., Leukema 26:2317-2325, 2012). Investigators will use results provided by the central laboratory for free light chain assessments, quantitative immunoglobulins, M-protein, and immunofixation measurements in serum and 24-hour urine. Subjects with positive serum IFE and confirmed IFE interference with DARZALEX™ (daratumumab) who meet all other clinical criteria for a complete response will be considered CRs. [International consensus recommendations for unified response standards] [Example 7. Evaluation of DARZALEX] [TM] A randomized phase 2 study of the efficacy and safety of daratumumab as a single agent in the treatment of patients with systemic AL amyloidosis.

[0268] A Phase 2, open-label study was conducted to evaluate the safety and efficacy of DARZALEX™ (daratumumab) as a single agent in previously treated subjects with systemic AL amyloidosis.

[0269] Approximately 40 subjects were randomized into two groups, one to receive DARZALEX™ (daratumumab) and the other to receive placebo.

[0270] Adult patients 18 years or older with biopsy-proven systemic AL amyloidosis who are not in CR or VGPR after initial treatment. Mayo Clinic Cardiac Stage I and II patients are included, while Stage III patients are only included if NT-proBNP is present. 5000ng / l (or BNP 1000ng / l). [Dosage regimen] Two dosing regimens are considered. Option 1: DARZALEX™ (daratumumab) is administered at 16 mg / kg IV weekly for 8 doses, every other week for 8 doses, and then every 4 weeks for 6 cycles. After 6 cycles, patients will continue to receive DARZALEX™ (daratumumab) every 4 weeks until progression or elective discontinuation. Option 2: Daratumumab was administered for six 28-day cycles at 16 mg / kg via IV route. For the first cycle, DARZALEX™ (daratumumab) was administered weekly on days 1, 8, 15, and 22. For cycles 2 and 3, administer DARZALEX™ (daratumumab) every other week on days 1 and 15. DARZALEX™ (daratumumab) administered every 4 weeks from cycle 4 to cycle 6, on day 1

[0271] The primary objective, secondary objectives, key inclusion criteria, and key exclusion criteria were similar to those described in Example 6.

Claims

1. The use of an anti-CD38 antibody in the manufacture of a medicament for treating patients newly diagnosed with light chain amyloidosis (AL), wherein the anti-CD38 antibody comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and wherein the anti-CD38 antibody is administered in combination with cyclophosphamide, bortezomib and dexamethasone (CyBorD), and wherein the patient does not have New York Heart Association (NYHA) classification IIIB or IV heart failure.

2. The use as described in claim 1, wherein the anti-CD38 anti-system is of IgG1, IgG2, IgG3 or IgG4 isotype.

3. The use as described in claim 1 or 2, wherein the anti-CD38 antibody is administered simultaneously with the CyBorD lineage.

4. The use as described in claim 1 or 2, wherein the anti-CD38 antibody and CyBorD are administered sequentially.

5. The use as described in claim 1 or 2, wherein the anti-CD38 antibody is administered separately from the CyBorD line.

6. The use as described in claim 1 or 2, wherein bortezomib, cyclophosphamide and corticosteroids are administered simultaneously.

7. The use as described in claim 1 or 2, wherein bortezomib, cyclophosphamide and dicortisol are administered in sequence.

8. The use as described in claim 1 or 2, wherein bortezomib, cyclophosphamide and corticosteroids are administered separately.

9. The use as described in claim 1 or 2, wherein the patient is undergoing a hematopoietic stem cell transplant (HSCT).

10. The use as described in claim 9, wherein the HSCT is an allogeneic variant.

11. The use as described in claim 9, wherein the HSCT is autologous or syngeneic.

12. The use as described in claim 9, wherein the HSCT comprises transplanting blood stem cells derived from bone marrow, blood, or amniotic fluid.

13. The use as described in claim 9, wherein the anti-CD38 anti-system is administered prior to HSCT.

14. The use as described in claim 9, wherein the anti-CD38 anti-system is administered during or after HSCT.

15. The use as described in claim 9, wherein the patient has completed chemotherapy and / or radiation therapy prior to HSCT.

16. The use as described in claim 1 or 2, wherein the patient is further treated with radiotherapy.

17. The use as described in claim 1 or 2, wherein the anti-CD38 antibody does not mediate the killing of CD34-positive hematopoietic precursor cells by complement-dependent cytotoxicity (CDC).

18. The use as described in claim 1 or 2, wherein the anti-CD38 antibody induces the killing of CD38-positive plasma cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or regulation of CD38 enzyme activity.

19. The use as described in claim 1, wherein the anti-CD38 antibody comprises the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO:

13.

20. The use as described in claim 1, wherein the anti-CD38 anti-system is a humanized antibody or human antibody.

21. The use as described in claim 1, wherein the anti-CD38 anti-system is of IgG1 isotype.

22. The use as described in claim 1, wherein the anti-CD38 anti-system is daratumumab.

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