EA040269B1 - ANTIBODIES TO CD38 FOR THE TREATMENT OF LIGHT CHAIN AMYLOIDOSIS AND OTHER CD38-POSITIVE HEMATOLOGICAL MALIGNANT TUMORS - Google Patents

Description

Scope of the invention

The present invention relates to methods for the treatment of light chain amyloidosis and other CD38 positive hematological malignancies.

Prerequisites for the creation of the invention

Malignant B-cell tumors include chronic lymphocytic B-cell leukemia, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse large cell lymphoma, multiple myeloma, Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma, and AIDS-associated non-Hodgkin's lymphoma. Malignant B-cell tumors account for more than 85% of diagnosed lymphomas.

Multiple myeloma (MM) is characterized by a latent accumulation of secretory plasma cells in the bone marrow with a low proliferative index and an extended life cycle. The disease ultimately affects 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 can be classified as multiple myeloma. The likelihood of developing multiple myeloma increases in older people, with the average age at diagnosis being about 61 years.

Light chain amyloidosis (ALC) (also called systemic amyloidosis) is a clonal plasma cell dysfunction in which fragments of misfolded immunoglobulin light chains are deposited in tissues. Monoclonal plasma cells in the bone marrow produce misfolded immunoglobulin light chains that accumulate in tissues and cause toxicity in vital organs leading to organ failure and death (Comenzo et al., Leukemia 26: 2317-25, 2012). Clinical manifestations depend on the affected organs; amyloidosis often occurs in the kidneys, heart, skin, nervous system, and soft tissues such as the tongue (Merlini and Belotti, NEJM, 349: 583-596, 2003), leading to albuminuria and renal failure, heart failure, arrhythmias , risk of sudden cardiac death, hepatomegaly, bloating, early satiety, paresthesias, dysesthesias, orthostatic hypotension, constipation or diarrhea (Chaulagain and Comenzo; Curr Hematol Malig Rep 8: 291-8, 2013).

CD38 is a type II membrane protein whose function is receptor-mediated adhesion and signaling, as well as mediating calcium mobilization through ectoenzymatic activity, catalyzing the formation of cyclic ADP-ribose (cADPR) from NAD ⁺ , and hydrolysis of cADFR to ADP-ribose (ADPR) . CD38 mediates cytokine secretion as well as lymphocyte activation and proliferation (Funaro et al., J Immunolog 145:2390-6, 1990; Guse et al., Nature 398:70-3, 1999) and regulates levels through its NAD-glycohydrolase activity extracellular NAD ⁺ , which is involved in the modulation of the regulatory T cell compartment (Adriouch et al., 14:1284-92, 2012; Chiarugi et al., Nature Reviews 12:741-52, 2012).

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

Current treatments for light chain amyloidosis and multiple myeloma include various chemotherapy drugs with or without autologous stem cell transplantation. However, both diseases remain largely incurable. Thus, there is a need for additional therapeutic agents for the treatment of multiple myeloma and light chain amyloidosis.

Statement of the Invention

The present invention provides a method of treating a patient with a CD38-positive hematological malignancy, comprising administering an anti-CD38 antibody to a patient requiring it for a time period sufficient to cure the CD38-positive hematological malignancy, wherein the patient is undergoing hematopoietic stem cell transplantation (HSCT).

The present invention also provides a method of treating a patient with light chain amyloidosis (ALC) comprising administering an anti-CD38 antibody to a patient requiring it for a time sufficient to cure the ALC.

The present invention also provides a method of treating a patient with light chain amyloidosis (ALC) comprising administering an anti-CD38 antibody to a patient requiring it for a time sufficient to cure the ALC, the patient undergoing hematopoietic stem cell transplantation (HSCT).

Brief description of the drawings

In FIG. 1 shows the relative expression of CD38, CD32B, IL6R, gp130, and CD16 in transcriptional profiles of CD138+ clonal plasma cells in patients with newly diagnosed ALC (n=16 patients with ALC, GEO GSE24128). LnQLE: natural logarithm of quantitative expression level.

In FIG. 2 shows the percentage (%) of NK cells in mononuclear cells in the peripheral blood of ALC patients 3 weeks after stem cell transplantation (SCT).

In FIG. 3 shows that CD38 is expressed in CD34+ hematopoietic progenitor cells.

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In FIG. 4A shows that DARZALEX™ (daratumumab) has no effect on the proliferation of unselected mobilized blood progenitors thawed after cryopreservation, as assessed by progenitor cells' ability to form a similar number of colonies in the presence of 500 ng/mL or 1000 ng/mL DARZALEX™ (daratumumab) versus isotype control. In this assay, granulocyte-macrophage colony-forming units (CFU-GM) were assessed after 14 days of methylcellulose culture in the presence of DARZALEX™ (daratumumab), with isotype control or antibody-free control, and plotted % colonies formed versus antibody-free control. *p<0.05; paired t-test. All analyzes were performed in triplicate, and 3 independent experiments were performed using progenitor cell samples from 3 different patients. Data show mean ±SD.

In FIG. 4B shows that DARZALEX™ (daratumumab) has no effect on the proliferation of unselected mobilized blood progenitor cells thawed after cryopreservation, as assessed by progenitor cells' ability to form a similar number of colonies in the presence of 500 ng/mL or 1000 ng/mL DARZALEX™ (daratumumab) versus isotype control. In this assay, erythroid blasting units (BFU-E) were assessed after 14 days of growth in methylcellulose in the presence of DARZALEX™ (daratumumab), with isotype control or no antibody control, and plotted % colonies formed vs. no antibody control. All analyzes were performed in triplicate, and 3 independent experiments were performed using progenitor cell samples from 3 different patients. Data show mean ±SD.

In FIG. 5A shows that DARZALEX™ (daratumumab) has no effect on the proliferation of fresh, unselected mobilized blood progenitor cells, as assessed by their ability to form a similar number of colonies in methylcellulose in the presence of 500 ng/ml or 1000 ng/ml DARZALEX™ (daratumumab ) compared to isotype control. Colony formation was measured on day 14 as CFU-GM, and % colony formation was plotted against control for the absence of antibodies. All analyzes were performed in triplicate, and 3 independent experiments were performed using progenitor cell samples from 3 different patients. Data show mean ±SD.

In FIG. 5B shows that DARZALEX™ (daratumumab) has no effect on the proliferation of fresh, unselected mobilized blood progenitor cells, as assessed by their ability to form a similar number of colonies in methylcellulose in the presence of 500 ng/ml or 1000 ng/ml DARZALEX™ (daratumumab ) compared to isotype control. Colony formation was measured on day 14 as BFU-E and plotted as % colony formation vs. no antibody control. All analyzes were performed in triplicate, and 3 independent experiments were performed using progenitor cell samples from 3 different patients. Data show mean ±SD.

In FIG. 6A shows that DARZALEX™ (daratumumab) does not kill CD34-selected hematopoietic progenitor cells by CDC despite high expression of CD38 in these cells. CD34+ cells were incubated for 1 hour in high-complement antibody-free (CTL) serum, 500 ng/ml DARZALEX™ (daratumumab) (Dara) or 500 ng/ml isotype control (Iso), after which the cells were plated on semi-solid medium. Colony formation was measured on day 14 as CFU-GM per 500 CD34 selected cells. Results are from a triplicate assay with cells from 1 MM patient and 2 ALC patients.

In FIG. 6B shows that DARZALEX™ (daratumumab) does not kill CD34-selected hematopoietic progenitor cells by CDC despite high expression of CD38 in these cells. CD34+ cells were incubated for 1 hour in high-complement antibody-free (CTL) serum, 500 ng/ml DARZALEX™ (daratumumab) (Dara) or 500 ng/ml isotype control (Iso), after which the cells were plated on semi-solid medium. Colony formation was measured on day 14 as BFU-E per 500 CD34 selected cells. DARZALEX™ (daratumumab) plates had more BFU-E reaching statistical significance. Results are from a triplicate assay with cells from 1 MM patient and 2 ALC patients (*p<0.01).

In FIG. 7A shows that DARZALEX™ (daratumumab) does not adversely affect fresh CD34-selected granulocyte-monocyte progenitor cells. Isolated CD34+ cells were incubated in media free of antibodies (CTL; control), DARZALEX™ (daratumumab) (Dara) or isotype control (Iso) - 500 ng/mL or 1000 ng/mL as indicated. Cells were placed in methylcellulose and colony formation was measured on day 14 as CFU-GM per 500 cells selected for CD34. Results are from a triplicate assay with cells from 1 MM patient and 2 ALC patients.

In FIG. 7B shows that DARZALEX™ (daratumumab) does not adversely affect fresh CD34-selected erythroid progenitor cells. Isolated CD34 cells were incubated in antibody-free medium (CTL; control), daratumumab, or isotype control (Iso) - 2 040269

500 ng/ml or 1000 ng/ml. Cells were placed in methylcellulose and colony formation was measured on day 14 as BFU-E per 500 CD34-selected cells. Results are from a triplicate assay with cells from 2 MM patients and one ALC patient, *p<0.02.

In FIG. 8 shows that FCyRIIIa-158aa polymorphisms affected ADCC mediated by DARZALEX™ (daratumumab). Patients with genotypes 158V/V (V/V) and 158F/V (F/V) showed an increased response rate compared with patients with genotype 158F/F (F/F) (the vertical line is the median for each group, P<0 .05, two-tailed Mann-Whitney test).

Detailed description of the invention

The term CD38 refers to the human CD38 protein (synonyms: ADP-ribosylcyclase 1, cADPH-hydrolase 1, cyclic ADP-ribose hydrolase 1). Human CD38 has an amino acid sequence as shown in GenBank in order of entry NP_001766 and in SEQ ID NO: 1. It is well known that CD38 is a type II single-pass membrane protein with amino acid residues 1-21 representing the cytosolic domain, amino acid residues 22-42 representing the transmembrane domain, and residues 43-300 representing the extracellular domain of CD38.

SEQ ID NO: 1

MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRF

PETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKIL LWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSV FWKTVSRRFAEAACDWHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSR DLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI

The term antibodies is intended to be used herein in a broad sense to include immunoglobulin molecules, including monoclonal antibodies, including murine, human, human-adapted, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, as well as single-chain antibodies.

Immunoglobulins can fall into five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further classified into IgA _b IgA ₂ , IgG _b IgG ₂ , IgG ₃ and IgG4 isotypes. The light chains of antibodies of any vertebrate species can be assigned, depending on the amino acid sequences of their constant domains, to one of two distinct types, namely kappa (k) and lambda (λ).

The term antibody fragments refers to the portion of an immunoglobulin molecule that retains the heavy chain and/or light chain antigen-binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining region (LCDR) 1, 2 and 3 , a heavy chain variable region (VH) or a light chain variable region (VL). Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, HV, CL, and CHI domains; F(ab) ₂ fragment - bivalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region; Fd fragment consisting of VH and CHI domains; an Fv fragment consisting of the VL and VH domains of one antibody arm; an antibody domain fragment (dAb) (Ward et al., Nature 341:544-6, 1989), which consists of a VH domain. The VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody configurations in which the VH/VL domains are paired intramolecularly or intermolecularly in cases where the VH and VL domains are expressed by separate single chain antibody constructs to form a monovalent an antigen-binding site such as a single chain Fv (scFv) or diabody; they are described, for example, in international patent publications WO 1998/44001, WO 1988/01649, WO 1994/13804 and WO 1992/01047. These antibody fragments are prepared using techniques known to those skilled in the art, and the fragments are screened for suitability in the same manner as for full length antibodies.

The term isolated antibody means an antibody or antibody fragment essentially free of other antibodies having different antigen specificity values (e.g., an isolated antibody that specifically binds to CD38 is essentially free of antibodies that specifically bind to antigens other than human CD38). However, an isolated antibody that specifically binds to CD38 may be cross-reactive with other antigens, such as human CD38 orthologues, eg Macaca fascicularis (cynomolgus monkey) CD38. Moreover, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The variable region of an antibody consists of a framework region separated by three antigen-binding sites. Antigen binding sites are defined using different terms: complementarity determining regions (CDRs), three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3), 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 term hypervariable regions, HVR or HV, three in VH

- 3 040269 (H1, H2, H3) and three in VL (L1, L2, L3), refers to antibody variable domain regions 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 use of specificity determining residues (SDRUs) (Almagro Mol Recognit 17:132-43, 2004). The international database ImMunoGeneTics (IMGT) (http://www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between CDR, HV and IMGT delimitations is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003.

As used herein, the term Chothia residues refers to Al-Lazikani-numbered VL and VH antibody residues (Al-Lazikani et al., J Mol Biol 273:927-48, 1997).

The terms framework or framework sequences are residual variable region sequences that differ from those defined as antigen-binding sites. Since antigen-binding sites, as described above, may be defined by various terms, the exact amino acid sequence of the framework depends on the definition of antigen-binding site.

The term humanized antibody refers to an antibody in which the antigen-binding sites are derived from a non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions, whereby the framework may not be an exact copy of the expressed human immunoglobulin or germline gene sequences.

The term human-adapted antibodies or human framework-adapted (HFA) antibodies refers to humanized antibodies adapted according to the methods described in US Patent Publication No. US 2009/0118127. Human-adapted antibodies are humanized by selecting human acceptor scaffolds based on maximum CDR and FR similarities, length and sequence similarity of CDR1 and CDR2 loops, and part of the light chain CDR3 loops.

The term human antibody refers to an antibody having heavy and light chain variable regions in which both the framework and antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region is also derived from sequences of human origin.

The human antibody contains heavy and/or light chain variable regions that are derived from sequences of human origin, wherein the antibody variable regions are derived from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include phage-displayed human immunoglobulin gene libraries and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci as described herein. The human antibody may contain amino acid differences from the human germline or rearranged immunoglobulin sequences due, for example, to naturally occurring somatic mutations or intentional substitutions at scaffold or antigen binding sites. Typically, a human antibody in amino acid sequence 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 to the amino acid sequence encoded by the human germline gene or the rearranged immunoglobulin gene. In some instances, a human antibody may contain consensus framework sequences derived from human framework sequence assays, such as those described in Knappik et al., J Mol Biol 296:57-86, 2000, or synthetic HCDR3s included in immunoglobulin gene libraries human displayed on phage, for example, as described in Shi et al., J Mol Biol 397:385-96, 2010 and international patent publication WO 2009/085462). Antibodies in which the antigen-binding sites are derived from a non-human species do not fit the definition of a human antibody.

The isolated humanized antibodies may be synthetic. Human antibodies, although derived from human immunoglobulin sequences, can be generated using systems such as phage display, including synthetic CDRs and/or synthetic scaffolds, or can be subjected to in vitro mutagenesis to improve the properties of the antibodies, resulting in the production of antibodies, in natural conditions not included in the set of human germline antibodies in vivo.

The term recombinant antibody includes all antibodies produced, expressed, generated or isolated by recombinant methods, such as antibodies isolated from an animal (e.g. mouse or rat) that is transgenic or transchromosomal for human immunoglobulin genes, or derived from it hybridomas, antibodies, isolated from a host cell transformed to express an antibody, antibodies isolated from a recombinant combinatorial antibody library, and antibodies produced, expressed, generated, or isolated by any other means that involve splicing human immunoglobulin gene sequences with other DNA sequences, or antibodies generated in vitro by exchanging Fab arms, such as bispecific antibodies.

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The term monoclonal antibody refers to a preparation of antibody molecules of a single molecule composition. A monoclonal antibody composition exhibits single binding specificity and affinity for a particular epitope or, in the case of a bispecific monoclonal antibody, dual binding specificity for two separate epitopes. Therefore, a monoclonal antibody is a population of antibodies with the same amino acid composition in each heavy and each light chain, except for possible well-known changes, such as the removal of the C-terminal lysine from the heavy chain of the antibody. Monoclonal antibodies may have heterogeneous glycosylation within an antibody population. A monoclonal antibody may be monospecific or multispecific or monovalent, bivalent or multivalent.

A bispecific antibody is included in the term monoclonal antibody.

The term epitope refers to the portion of an antigen to which an antibody specifically binds. Typically, epitopes are composed of reactive (such as polar, nonpolar, or hydrophobic) surface groupings of moieties, such as amino acid or polysaccharide side chains, and they may have specific three-dimensional structure characteristics as well as specific charge characteristics. An epitope may be formed from contiguous and/or non-contiguous amino acids forming a conformational space block. In the case of a non-contiguous epitope, amino acids from different regions of the linear sequence of the antigen are in close proximity to each other in three-dimensional space due to the folding of the protein molecule.

The term variant refers to a polypeptide or polynucleotide that differs from a control polypeptide or control polynucleotide by one or more modifications, such as substitutions, insertions, or deletions.

The expression in combination with means that two or more therapeutic agents together can be administered to a subject in admixture, simultaneously as separate agents, or sequentially as separate agents in any order.

The terms "treat" or "treatment" refer to therapeutic treatment, the purpose of which is to slow down (reduce) an undesired physiological change or disease, or to achieve a favorable or desired clinical outcome during treatment. Beneficial or desirable clinical outcomes include relief of symptoms, reduction in disease severity, stabilization (i.e., no worsening) of the disease state, delay or retardation of disease progression, alleviation or temporary amelioration of the disease state, and remission (partial or complete) as detectable, so undetectable. The term "treatment" can also mean prolongation of survival compared to that expected in the absence of treatment of a subject. Those in need of treatment include subjects who already have an undesired physiological change or disease, as well as subjects prone to physiological changes or disease.

The expression "growth inhibiting" (for example, in relation to cells such as tumor cells) refers to a measurable reduction in cell growth in vitro or in vivo when cells are brought into contact with a therapeutic agent or a combination of therapeutic or drugs compared to the growth of the same cells growing in appropriate control conditions well known to those skilled in the art. The inhibition of cell growth in vitro or in vivo may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Inhibition of cell growth can occur according to a variety of mechanisms, for example, through antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, necrosis, or inhibition of cell proliferation.

The term therapeutically effective amount refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex and body weight of the subject, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the subject. Examples of indicators of an effective amount of a therapeutic agent or combination of therapeutic agents include, for example, improvement in the well-being of the patient, reduction in tumor burden, cessation or slowing of tumor growth, and/or the absence of cancer cell metastasis to other areas of the body.

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

The present invention provides a method of treating patients with a CD38-positive hematological malignancy, wherein the patient is transplanted with a hematopoietic stem cell transplant with an anti-CD38 antibody that does not kill (e.g., does not mediate killing) CD34+ hematopoietic stem cells within a transplant that is also CD38 positive. The present invention also provides a method for treating patients with light chain amyloidosis. The present invention is based, at least in part, on the discovery that an antibody to

CD38, DARZALEX™ (daratumumab), is effective in killing ALC plasma cells but does not kill CD38+CD34+ hematopoietic stem cells isolated from patients with light chain amyloidosis or multiple myeloma, allowing combination treatment with injection

DARZALEX™ (daratumumab) and hematopoietic stem cell transplantation.

The methods of the invention may be used to treat an animal patient within any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats, and farm animals.

The present invention provides a method of treating a patient with a CD38-positive hematological malignancy, comprising administering an anti-CD38 antibody to a patient requiring it for a time period sufficient to cure the CD38-positive hematological malignancy, wherein the patient is undergoing hematopoietic stem cell transplantation (HSCT).

The term CD38-positive hematologic malignancy refers to a hematologic malignancy that is characterized by the presence of CD38-expressing tumor cells, including leukemias, lymphomas, myeloma, and abnormal plasma cell function. Examples of CD38-positive hematologic malignancies include 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 lymphocyte lymphoma (LML), B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including well-differentiated, moderately differentiated and poorly differentiated PL, cutaneous cell lymphoma center of the follicle, marginal zone B-cell lymphoma (MALT type, nodular and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (LB), plasmacytoma, multiple myeloma (MM), plasmacytic leukemia, post-transplantation lymphoproliferative disorder, Waldenström's macroglobulinemia, plasma tarry leukemias, anaplastic large cell lymphoma (ALCL), and light chain amyloidosis (ALC).

The term plasma cell dysfunction as used herein means disorders that are characterized by clonal plasma cells and include multiple myeloma, light chain amyloidosis, and Waldenström's macroglobulinemia. Light chain amyloidosis and Waldenström's macroglobulinemia occur independently of multiple myeloma. They can also present at the same time as multiple myeloma and develop either before or after the development of multiple myeloma.

Thus, the definitions of CD38-positive hematologic malignancy and abnormal plasma cell function may overlap.

In some embodiments, the CD38 positive hematologic cancer is light chain amyloidosis (ALC).

In some embodiments, the CD38 positive hematologic cancer is multiple myeloma (MM).

In some embodiments, the CD38-positive hematologic malignancy is Waldenström's macroglobulinemia.

In some embodiments, the CD38 positive hematologic cancer is diffuse large B cell lymphoma (DLBCL).

In some embodiments, the CD38 positive hematologic cancer is non-Hodgkin's lymphoma.

In some embodiments, the CD38 positive hematologic malignancy is acute lymphoblastic leukemia (ALL).

In some embodiments, the CD38 positive hematologic cancer is follicular lymphoma (FL).

In some embodiments, the CD38-positive hematologic malignancy is Burkitt's lymphoma (BL).

In some embodiments, the CD38-positive hematologic malignancy is mantle cell lymphoma (MCL).

In some embodiments, the CD38 positive hematologic cancer is a plasma cell dysfunction.

In some embodiments, the CD38-positive hematologic malignancy is light chain amyloidosis (ALC), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (LB), follicular lymphoma (FL) or mantle cell lymphoma (MCL).

Examples of B-cell non-Hodgkin's lymphomas are lymphogranulomatosis, primary effusion lymphoma, intravascular large B-cell lymphoma, mediastinal large-cell lymphoma, heavy chain diseases (including γ-, μ-, and α-chains), lymphomas induced by immunosuppressive therapy. such as ciclosporin-induced lymphoma and metatrexate-induced lymphoma.

In some embodiments, the dysfunction of cells expressing CD38 is Hodgkin's lymphoma.

Other examples of disorders associated with CD38-expressing cells include T-cell and NK-cell malignancies, including mature T-cell and NK-cell neoplasms, including T-cell prolymphocytic leukemia, large granular lymphocyte T-cell leukemia, aggressive NK cell leukemia, adult T-cell leukemia/lymphoma, extranodal nasal-type NK/T-cell lymphoma, 78 enteropathic T-cell lymphoma, T-cell liver and spleen lymphoma, subcutaneous panniculitis-like T-cell lymphoma, blast NK cell lymphoma, fungoid granuloma/Sezari syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disorders (primary cutaneous anaplastic large cell lymphoma K-ALCL, lymphomatoid papulosis, borderline lesions), angioimmunoblastic T-cell lymphoma, non-specific T-cell lymphoma, and anaplastic large cell lymphoma.

Examples of malignant diseases associated with myeloid cells include acute myeloid leukemia, including acute promyelocytic leukemia, and chronic myeloproliferative diseases, including chronic myeloid leukemia.

The present invention also provides a method of treating a patient with light chain amyloidosis (ALC) comprising administering an anti-CD38 antibody to a patient requiring it for a time sufficient to cure the ALC.

The present invention also provides a method of treating a patient with light chain amyloidosis, comprising administering an anti-CD38 antibody to a patient requiring it for a time sufficient to cure the light chain amyloidosis, the patient undergoing hematopoietic stem cell transplantation (HSCT).

The present invention also provides a method of treating a patient with multiple myeloma, comprising administering an anti-CD38 antibody to a patient requiring it for a time sufficient to cure the multiple myeloma, the patient undergoing a hematopoietic stem cell transplant (HSCT).

In some embodiments, an 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.

In some embodiments, the anti-CD38 antibody binds to at least the SKRNIQFSCKNIYR region (SEQ ID NO: 2) and the

EKVQTLEAWVIHGG (SEQ ID NO: 3)

Human CD38 (SEQ ID NO: 1).

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

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

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.

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 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 SEQ ID NO: 5.

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

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

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.

An antibody epitope includes some or all of the residues having the sequences shown in SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, an antibody epitope contains at least one amino acid in the region

SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least one amino acid in the region

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EKVQTLEAWVIHGG (SEQ ID NO: 3)

Human CD38 (SEQ ID NO: 1).

In some embodiments, the antibody epitope contains at least two amino acids in the region

SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least two amino acids in the region

EKVQTLEAWVIHGG (SEQ ID NO: 3)

Human CD38 (SEQ ID NO: 1).

In some embodiments, the antibody epitope contains at least three amino acids in the region

SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least three amino acids in the region

EKVQTLEAWVIHGG (SEQ ID NO: 3)

Human CD38 (SEQ ID NO: 1).

An example of an antibody that binds to a region

SKRNIQFSCKNIYR (SEQ ID NO: 2) and region

EKVQTLEAWVIHGG (SEQ ID NO: 3)

Human CD38 (SEQ ID NO: 1) is DARZALEX™ (daratumumab).

An example of an anti-CD38 antibody that is useful in the methods of the present invention is DARZALEX™ (daratumumab). DARZALEX™ (daratumumab) contains the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) shown in SEQ ID NO: 4 and 5 respectively, the heavy chain CDRs of HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 6, 7 and 8, respectively, and the LCDR1, LCDR2, and LCDR3 light chain CDRs of SEQ ID NOs: 9, 10, and 11, respectively, have the IgG1/K subtype and are described in US Patent No. 7,829,693. ID NO: 12 and the amino acid sequence of the light chain is shown in SEQ ID NO: 13.

Antibodies can be assessed for their competition with a control antibody, such as DARZALEX™ (DARZALEX™ (daratumumab)) having VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for binding to CD38 using well known in vitro methods. In an exemplary method, CHO cells recombinantly expressing CD38 can be incubated with unlabeled control antibody for 15 minutes at 4°C followed by incubation with excess fluorescently labeled test antibody for 45 minutes at 4°C. After washing in phosphate buffered saline with bovine serum albumin (PBS/BSA), fluorescence can be measured by flow cytometry using standard methods. In another exemplary method, the CD38 extracellular domain can be coated onto the surface of an ELISA plate. An excess of unlabeled control antibody can be added over about 15 minutes, and biotinylated test antibodies can be added afterwards. After washes in PBS/Tween, binding of the biotinylated antibody of interest can be detected with horseradish peroxidase (HRP) conjugated streptavidin and signal detected using standard methods. Obviously, in competitive assays, the control antibody may be labeled and the test antibody unlabeled. Test antibody competes with control antibody if test antibody inhibits test antibody binding, or test antibody inhibits control antibody binding to CD38 by at least 80%, e.g. 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. The epitope of the antibody of interest can be further determined, for example by peptide mapping or hydrogen/deuterium shielding assays by known methods, or by crystal structure determination.

Antibodies binding to the area

SKRNIQFSCKNIYR (SEQ ID NO: 2) and region

EKVQTLEAWVIHGG (SEQ ID NO: 3)

Human CD38 (SEQ ID NO: 1) can be obtained, for example, by immunizing mice with peptides having the amino acid sequences shown in SEQ ID NO: 2 and 3 using standard methods and as described herein below and characterizing the resulting antibodies by binding to peptides, for example, using an ELISA assay or a mutagenesis assay.

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SEQ ID NO: 2

SKRNIQFSCKNIYR

SEQ ID NO: 3

EKVQTLEAWVIHGG

SEQ ID NO: 4

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSA ISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDK ILWFGEPVFDYWGQGTLVTVS S SEQ ID NO: 5 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQ GTKVEIK

SEQ ID NO: 6

SEAMS

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

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK

SEQ ID NO: 13

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSS

Other examples of anti-CD38 antibodies that are useful in the methods of the present invention are mAb003 containing the sequences VH and VL of SEQ ID NOs: 14 and 15, respectively, and described in US Pat. No. 7,829,693. VH and VL mAb003 can be expressed as IgG1/K.

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SEQ ID NO: 14

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIAN

SAQKFQGRVTITADKS T S TAY

MDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSSAS

SEQ ID NO: 15

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVP

SRFSGSGSGTDFTLTISSLQP

EDFATYYCQQYNSYPRT FGQGTKVEIK;

mAb024 containing the sequences VH and VL with SEQ ID NO: 16 and 17, respectively, described in US patent No. 7,829,693. VH and VL mAb024 can be expressed as IgG1/K.

SEQ ID NO: 16

EVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMPGKGLEWMGIIYPHDSDAR

YS PS FQGQVT FSADKSIS TAY

LQWSSLKASDTAMYYCARHVGWGSRYWYFDLWGRGTLVTVSS

SEQ ID NO: 17

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP

ARFSGSGSGTDFTLTISSLEP

EDFAVYYCQQRSNWPPTFGQGTKVEIK;

MOR-202 (MOR-03087) containing the VH and VL sequences of SEQ ID NOs: 18 and 19, respectively, described in US Pat. No. 8,088,896. The VH and VL of MOR-202 can be expressed as IgG1/K.

SEQ ID NO: 18

QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTY

YADSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS

SEQ ID NO: 19

DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPE

RFSGSNSGNTATLTISGTQAE

DEADYYCQTYTGGASLVFGGGTKLTVLGQ;

Isatuximab containing the sequences VH and VL with SEQ ID NO: X and X, respectively, described in US patent No. 8153765. VH and VL of isatuximab can be expressed as IgG1/K.

SEQ ID NO: 20:

QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGT

IYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGD

YYGSNSLDYWGQGTSVTVSS

SEQ ID NO: 21:

DIVMTQSHLSMSTSLGDPVSITCKASQDVSTWAWYQQKPGQSPRRLIYS

ASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGG

GTKLEIK

Other examples of anti-CD38 antibodies that can be used in the methods of the invention include those described in International Patent Publication WO 05/103083, International Patent Publication WO 06/125640, International Patent Publication WO 07/042309, International Patent Publication WO 08/ 047242 or international patent publication WO 14/178820.

In some embodiments, ALC is characterized by cardiac stage I, cardiac stage II, or cardiac stage III.

In some embodiments, the ALC is recurrent or refractory.

ALC is diagnosed by a physician according to guidelines presented, for example, in

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National Comprehensive Cancer Network (http://www.nccn.org/professionals/physician_gls/f_guidelines_asp#site). Abnormalities in patients with ALC manifest themselves in various organ systems due to the accumulation of light chains and their misfolded intermediate forms in the form of amyloid fibers in vital organs, which causes organ dysfunction and death. Patients may have multiple organ systems affected at the time of diagnosis; about one third of patients have more than 3 affected organs at the time of diagnosis (Chaulagain and Comenzo, Curr Hematol Malig Rep 8: 291-8, 2013). Cardiac staging is necessary to predict the course of ALC, since cardiac abnormalities appear to be present in all patients with ALC at diagnosis, even if the patient is asymptomatic (Palladini et al., Blood 116: 3426-30, 2010; Kristen et al., Blood 116:2455-61, 2010). Based on the presence of one, two, or both of the cardiac biomarkers N-terminal brain natriuretic peptide prohormone (NTproBNP) and troponin T, ALC patients can be classified into cardiac stages I, II, or III, see e.g. Comenzo et al., Leukemia 26 : 2317-25, 2012.

The current choice of treatment options for ALC is directed at the elimination of immunoglobulin light chain-secreting plasma cells and includes 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 doses of Alkeran® (melphalan) can be used in conjunction with stem cell transplantation (see, for example, Anderson et al., Systemic light chain amyloidosis, NCCN Clinical Practice Guidelines in Oncology, Version I. 2015, NCCN. org. 2014). For the treatment of ALC, NINLARO® (ixazomib), a proteasome inhibitor, is currently being evaluated. For the treatment of ALC, NEOD001, a monoclonal antibody that targets the amyloid protein ALC, is currently being evaluated.

In some embodiments, MM is recurrent or refractory.

Currently available treatment options for MM include chemotherapy, stem cell transplant, Thalomid® (thalidomide), Revlimid® (lenalidomide), Velcade® (bortezomib), Kyprolis® (carfilzomib), Farydak® (panobinostat), Aredia® (pamidronate ) and Zometa® (zoledronic acid). Current treatment protocols, which include a combination of chemotherapy drugs such as Oncovin® (vincristine), BiCNU® (BCNU, carmustine), Alkeran® (melphalan), cyclophosphamide, Adriamycin® (doxorubicin) and prednisone or dexamethasone, provide a complete remission rate of only about 5%, and the median survival time from diagnosis is approximately 36-48 months. Additionally, positive results have been achieved with the agent ixazomib in a pivotal clinical trial in patients with recurrent multiple myeloma. Recent advances in high-dose chemotherapy followed by transplantation of autologous bone marrow or peripheral blood mononuclear cells have increased the rate of complete remission and duration of remission, but overall survival has not been much improved, and no cure has been reported. Ultimately, all MM patients relapsed even with interferon-alpha (IFN-α) maintenance therapy, either alone or in combination with steroids.

Various qualitative and/or quantitative methods can be used to determine relapsed or refractory forms of the disease. Symptoms that may be associated with recurrence or resistance of the disease are, for example, worsening or no improvement in the patient's condition, or return or worsening of various symptoms of hematological malignancy, and/or spread of cancer cells in the body from one location to other organs, tissues, or cells. Symptoms associated with hematologic malignancies may vary depending on the type of cancer. For example, symptoms associated with ALC may include fatigue, purpura, enlarged tongue, diarrhea or edema, proteinuria, or elevated plasma levels of free light chains.

In some embodiments, the HSCT is allogeneic, autologous, or syngeneic, i. from a twin donor. Autologous HSCT involves extracting the HSC from a subject and freezing the resulting HSC. Following myeloablation, the subject's stored HSCs are transplanted back into the subject. Allogeneic HSCT uses HSCs obtained from an allogeneic HSC donor whose HLA type matches the subject.

As used herein, the term hematopoietic stem cell transplantation means transplantation of blood stem cells derived from bone marrow (here known as bone marrow transplantation), blood (eg, peripheral blood and umbilical cord blood), or amniotic fluid.

In this document, the term hematopoietic stem cell transplantation means that the patient has already undergone, is undergoing or will undergo HSCT.

In some embodiments, the patient has received chemotherapy and/or radiation therapy prior to the HSCT.

Patients may receive chemotherapy and/or radiation therapy before surgery

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HSCT (so-called transplant preparation) to kill some or all of the patient's hematopoietic cells before transplantation. In the case of allogeneic HSCT, the patient may also be treated with immunosuppressants. An example of pre-transplant therapy is the use of high doses of 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, which can be used for treatment before transplantation, can be carried out in accordance with well-known protocols for this field. Radiation therapy may also be administered simultaneously, sequentially, or separately from anti-CD38 antibody therapy.

DARZALEX™ (daratumumab) may not mediate the destruction of CD38+CD34+ stem cells within the graft and is therefore an acceptable therapeutic agent in combination with HSCT. Antibodies that compete with daratumumab and/or antibodies that bind the same epitope as DARZALEX™ (daratumumab) may also fail to kill CD38+CD34+ stem cells.

Other examples of antibodies that may be useful in the methods of the invention described herein may not kill CD38+CD34+ stem cells within a graft and are therefore acceptable therapeutic agents in combination with HSCT. The inability of these antibodies to kill CD38+CD34+ stem cells within the graft is assessed using the methods described herein.

The anti-CD38 antibodies used in the methods of the invention can also be selected de novo from, for example, a phage display library where the phage is 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 from, for example, phage display libraries expressing antibody heavy and light chain variable regions as fusion proteins fused to the bacteriophage coat protein pIX, as described in Shi et al., J. Mol. Biol. 397:385-96, 2010 and International Patent Publication WO 09/085462). It is possible to screen antibody libraries for binding to the extracellular domain of human CD38, further characterize the resulting positive clones, isolate Fab from clone lysates and subsequently clone as full-length antibodies. Such use of phage display methods for the isolation of human antibodies is well known in the art. See, for example, US patent No. 5223409; US patent No. 5403484; and also US patent No. 5571698, US patent No. 5427908, US patent No. 5580717, US patent No. 5969108, US patent No. 6172197, US patent No. 5885793; US patent No. 6521404; US patent No. 6544731; US patent No. 6555313; US patent No. 6582915; and US Pat. No. 6,593,081.

In some embodiments described herein, the anti-CD38 antibody does not mediate the destruction of CD34-positive hematopoietic progenitor cells by complement-dependent cytotoxicity (CDC).

The expression does not kill or 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 if the measured cell killing in the presence of DARZALEX™ (daratumumab) is statistically insignificant compared to the cell killing in the presence of the isotype control. Isotype control is a well known term.

Destruction of CD34-positive hematopoietic progenitor cells by CDC can be measured on isolated fresh or frozen CD34+ cells by incubating the cells in 10% complement serum and 500 ng/mL of anti-CD38 antibody and then assaying the degree of colony formation of the cells, plated on a semi-solid medium according to known methods. ways. For example, BFU-E and CFU-GM production can be assessed after 14 days of culture using commercial reagents such as MethoCult™ from Stem Cell Technologies.

The Fc region of an antibody can mediate antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). Such function may be mediated by binding of the Fc effector domain(s) to an Fc receptor on an immune cell with phagocytic or lytic activity, or by binding of the Fc effector domain(s) to components of the complement system. Typically, the effect(s) mediated by Fc-binding cells or complement components results in inhibition and/or depletion of target cells, eg CD38 expressing cells. Human IgG isotypes IgG1, IgG2, IgG3 and IgG4 show different ability to perform effector functions. ADCC can be mediated by IgG1 and IgG3, ADCP can be mediated by IgG1, IgG2, IgG3 and IgG4, and CDC can be mediated by IgG1 and IgG3.

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

In some embodiments, the anti-CD38 antibody induces the killing of plasma cells,

- 12 040269 expressing CD38, in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or modulation of CD38 enzymatic activity in vitro.

In some embodiments, the anti-CD38 antibody induces in vitro killing of CD38 expressing cells by ADCC, ADCP, or CDC.

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

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

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

The terms antibody-dependent cellular cytotoxicity, antibody-dependent cell-mediated cytotoxicity, or ADCC is a mechanism for inducing cell death that depends on the interaction of antibody-coated target cells with effector cells with lytic activity, such as natural killer cells, monocytes, macrophages, and neutrophils, via Fc gamma receptors. (FcyR) expressed on effector cells. For example, NK cells express FcyRIIIa while monocytes express FcyRI, FcyRII and FcyRIIIa. The death of antibody-coated target cells, such as CD38 expressing cells, occurs as a result of the activity of effector cells through the secretion of membrane pore-forming proteins and proteases. To evaluate the ADCC activity of an anti-CD38 antibody, the antibody can be added to CD38-expressing cells in combination with immune system effector cells that can be activated by antigen-antibody complexes, resulting in cytolysis of the target cell. Cytolysis is essentially detected by the release of labels (eg, radioactive substrates, fluorescent dyes, or natural intracellular proteins) from lysed cells. Examples of effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Examples of target cells include Dowdy cells (ATCC® CCL-213™) or B-cell leukemia or lymphoma tumor cells expressing CD38. In an example assay, target cells are labeled with 20 µCi ⁵¹ Cr for 2 hours and washed thoroughly. The target cell concentration can be adjusted to 1 x 10 ⁶ cells/ml and anti-CD38 antibodies are added at various concentrations. The assay is started by adding Daudi cells at a ratio of effector cell:target cell equal to 40:1. After incubation for 3 hours at 37° C., the assay is terminated by centrifugation and the release of ⁵¹ Cr from the lysed cells is measured on a scintillation counter. The percentage of cellular cytotoxicity can be calculated as % of the maximum lysis that can be induced by adding 3% perchloric acid to target cells. Anti-CD38 antibodies used in the methods of the invention can induce ADCC at levels of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. compared with control (cell lysis induced by 3% perchloric acid).

The term antibody-dependent cellular phagocytosis (ADCP) refers to a mechanism for killing antibody-coated target cells by internalization by phagocytic cells such as macrophages or dendritic cells. ADCP can be assessed using monocytic macrophages as effector cells and Dowdy cells (ATCC® CCL-213™) or B-cell leukemia or lymphoma tumor cells expressing CD38 as target cells engineered to express green fluorescent protein (GFP ) or another labeled molecule. The effector cell:target cell ratio can be, for example, 4:1. Effector cells can be incubated with target cells for 4 hours, with or without anti-CD38 antibody. After incubation, the cells can be separated using accutase. Identification of macrophages can be done with anti-CD11b and anti-CD14 antibodies associated with a fluorescent label, and percentage phagocytosis can be determined based on % fluorescent GFP in CD11+CD14+ macrophages using standard methods. Anti-CD38 antibodies used in the methods of the invention can induce ADCP at levels of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

The term complement-dependent cytotoxicity, or CDC, refers to a cell death induction mechanism in which the Fc effector domain of a target-bound antibody binds and activates the C1q complement component, which in turn activates the complement cascade leading to death of the target cell. Complement activation can also lead to the deposition of complement components on the surface of target cells, which promotes the manifestation of ADCC by binding to complement receptors (eg, CR3) on leukocytes. The CDC for cells expressing CD38 can be measured, for example, by seeding Daudi cells at 1 x 10 ⁵ cells/well (50 µl/well) in RPMI-B (RPMI supplemented with 1% BSA), adding 50 µl of anti-CD38 antibody to the wells until final concentration in the range of 0-100 μg/ml, incubating the reaction mixture for 15 min at room temperature, adding 11 μl of pooled human serum to the wells and incubating the reaction mixture for 45 min at 37°C. Percentage (%) Lysed Cells

- 13 040269 can be defined as % of cells stained with propidium iodide when analyzed by FACS using standard methods. Anti-CD38 antibodies used in the methods of the invention can induce a CDC of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

The ability of monoclonal antibodies to induce ADCC can be enhanced by designing their oligosaccharide component. Human IgG1 or IgG3 are N-glycosylated at Asn297 by most glycans in the well-known 2-antennary forms G0, G0F, G1, G1F, G2 or G2F. Antibodies produced by unengineered CHO cells typically have a fucose content of glycans of about at least 85%. Removal of central fucose from type 2-anthenary complex oligosaccharides attached to Fc regions enhances antibody ADCC through improved FcyRIIIa binding without altering antigen binding or CDC activity. Such mAbs can be generated by a variety of methods that have been reported to result in successful expression of relatively highly defucosylated antibodies bearing type 2-anthenary complex Fco-oligosaccharides, such as controlling the osmolality of the culture medium (Konno et al., Cytotechnology 64: 249-65, 2012), use as a host cell line of the variant CHO line Lec13 (Shields et al., J Biol Chem 277:26733-40, 2002), use as a host cell line of the variant CHO line EB66 (Olivier et al., MAbs; 2(4), 2010; online to print; PMID: 20562582), using the YB2/0 rat hybridoma cell line as a host cell line (Shinkawa et al., J Biol Chem 278:3466- 73, 2003), introduction of small interfering RNA specific for the α 1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-8, 2004), or co-expression of β-1,4-N-acetylglucosaminyltransferase III and α-mannosidases II of the Golgi complex, or the use of α-mannosidase I inhibitor, kyfunesin (Ferrara et al., J Biol Chem 281:50326, 2006; Ferrara et al., Biotechnol Bioeng 93:851-61, 2006; Xhou et al., Biotechnol Bioeng 99:652-65, 2008). ADCC caused by antibodies to CD38, which are used in the methods of the invention and in some embodiments of all of the following numbered embodiments without exception, can also be enhanced by certain substitutions in the Fc of the antibody. Examples of substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 (residue numbering follows the EC suffix) as described in US Patent No. 6,737,056. antibodies to CD38, which are used in the methods of the invention and in some embodiments of all of the numbered embodiments listed below, without exception, can also be enhanced by certain substitutions in the Fc of the antibody. Examples of substitutions are, for example, substitutions at amino acid positions 423, 268, 267 and/or 113 (residue numbering according to the EC index) as described in Moore et al., Mabs 2:181-9, 2010.

In some embodiments, the anti-CD38 antibody comprises a substitution in the Fc of the antibody.

In some embodiments, the anti-CD38 antibody comprises a substitution 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 follows the EC index).

In some embodiments, the anti-CD38 antibody comprises a substitution in the Fc of the antibody at amino acid positions 113, 267, 268, and/or 423 (EC residue numbering).

In some embodiments, the anti-CD38 antibody comprises a 2-antennary glycan structure with about 0% to about 15% fucose, e.g., 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0%.

In some embodiments, the anti-CD38 antibody comprises a 2-antennary glycan structure with a fucose content of about 50, 40, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7 , 6, 5, 4, 3, 2, 1 or 0%.

Fc substitutions and reduced fucose can enhance anti-CD38 ADCC activity.

The term fucose content refers to the amount of fucose monosaccharide within the saccharide chain at position Asn297. The relative amount of fucose is the percentage of fucose-containing structures related to all glycostructures. They can be characterized and quantified in a variety of ways, e.g. 1) using time-of-flight matrix-assisted laser desorption/ionization (MALDI-TOF) of a sample treated with N-glycosidase F (e.g. complex, hybrid, oligomannose and high-mannose structures), as described in International Patent Publication No. WO 2008/077546; 2) by enzymatic release of Asn297 glycans followed by derivatization and detection/quantitation by HPLC (UHPLC) with detection by fluorescence and/or HPLC-MS (UHPLC-MS); 3) analysis of the intact protein of the native or reconstituted mAb with or without Asn297 glycan treatment with Endo S or another enzyme that cleaves the bond between the first and second GlcNAc monosaccharides, keeping the fucose attached to the first GlcNAc; 4) cleavage of the mAb into its constituent peptides by enzymatic cleavage (eg, trypsin or Lys-C endopeptidase) and subsequent separation, detection and quantification by HPLC-MS (UHPLC-MS); or 5) separating the mAb oligosaccharides from the Asn 297 mAb protein by specific enzymatic deglycosylation with PNGase F. -activated laser desorption/ionization (MALDI) by comparing experimental masses with theoretical masses, determine the degree of sialylation by ion-exchange HPLC (GlycoSep C), separate and quantify oligosaccharide forms by hydrophilicity criterion by ordinary phase HPLC (GlycoSep N) and separate and quantify oligosaccharides by high-performance laser-induced fluorescence capillary electrophoresis (HPCE-LIF).

The expressions low-fucose or low-fucose used in the application refer to antibodies with a fucose content of about 0-15%.

The term normofucose or normal fucose as used herein refers to antibodies with a fucose content greater than about 50%, typically greater than about 60%, 70%, 80%, or greater than 85%.

An anti-CD38 antibody used in the methods of the invention can induce the killing of cells expressing CD38 by apoptosis in vitro. Methods for assessing apoptosis are well known and include, for example, staining with annexin IV using standard methods. Anti-CD38 antibodies of the methods of the invention can induce apoptosis at about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of cells.

An anti-CD38 antibody used in the methods of the invention can induce the killing of cells expressing CD38 in vitro by modulating CD38 enzymatic activity. CD38 is a multifunctional ectoenzyme with the activity of ADP-ribosylcyclase 1, catalyzing the formation of cyclic ADP-ribose (cADFR) and ADFR from NAD ⁺ , which also has the function of hydrolyzing NAD ⁺ and cADFR into ADFR. CD38 also catalyzes the exchange of the nicotinamide group of NADP ⁺ for nicotinic acid under acidic conditions to produce NADP+ (nicotinic acid adenine dinucleotide phosphate). Modulation of human CD38 enzymatic activity by anti-CD38 antibodies used in the methods of the invention can be measured in the assay described in Graeff et al., J. Biol. Chem. 269:30260-7, 1994). For example, an NGD ⁺ substrate can be incubated with CD38, and modulation of cyclic GDP-ribose (cGDFR) production can be monitored spectrophotometrically by excitation at 340 nm and emission at 410 nm at various time points after the addition of antibody at various concentrations. Inhibition of cADFR synthesis can be determined according to the HPLC method described in Munshi et al., J. Biol. Chem. 275:21566-71, 2000. Anti-CD38 antibodies used in the methods of the 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%.

Antibodies that are substantially identical to an antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 can be used in the methods of the invention. The term substantially identical means that the compared amino acid sequences of the VH or VL antibodies are identical or have minor differences. Minor differences are substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in the VH and/or VL of the antibody that do not adversely affect the properties of the antibody . Percent identity can be determined, for example, by pairwise alignment using the default settings in the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, CA, USA). The protein sequences of the present invention can be used as a search sequence when performing searches in public or proprietary databases, for example, to identify related sequences. Examples of programs used to perform such searches are the XBLAST or BLASTP programs (http_//www_ncbi_nlm/nih_gov) or the GenomeQuest™ package (GenomeQuest, Westboro, Massachusetts, USA) using default settings. Examples of substitutions that can be made for antibodies that specifically bind to CD38 used in the methods of the invention are, for example, conservative substitutions with amino acids having similar charge, hydrophobic properties, or stereochemical characteristics. Conservative substitutions can also be made to improve the properties of the antibody, such as stability or affinity, or to improve the effector functions of the antibody. You can make 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid substitutions, for example in VL and/or VH antibodies to CD38. Moreover, any native heavy or light chain residue can also be replaced with alanine, as previously described in relation to alanine scanning mutagenesis (MacLennan et al., Acta Physiol Scand Suppl 643:55-67, 1998; Sasaki et al., Adv Biophys 35 :1-24, 1998). Those skilled in the art can determine the desired amino acid substitutions when such substitutions are needed. Amino acid substitutions can be made, for example, using PCR mutagenesis (US patent No. 4683195). Libraries of variants can be generated using well-known methods, for example, by using random (NNK) or non-random codons, such as DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp ), and screening libraries for variants with desired properties. Generated variants can be tested for their binding to CD38, their ability to induce ADCC, ADCP or apoptosis, or to modulate CD38 enzymatic activity in vitro using the methods described herein.

The term conservative modifications means amino acid modifications that do not significantly alter or affect the binding characteristics of an antibody containing amino acid sequences. Conservative modifications include substitutions, additions and deletions of amino acids. Conservative substitutions are those in which an amino acid is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are well defined and include amino acids with acidic side chains (eg, aspartic acid, glutamic acid), basic side chains (eg, lysine, arginine, histidine), non-polar side chains (eg, alanine, valine, leucine , isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g. glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g. phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (eg, glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (eg, asparagine, glutamine), beta-branched side chains (eg, threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Moreover, any native residue in the polypeptide can be replaced by alanine, according to the method previously described as alanine-scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; Sasaki et al., (1988) Adv Biophys 35: 1-24). Amino acids in the antibodies of the present invention can be replaced by known methods, for example by PCR mutagenesis (US patent No. 4683195). Alternatively, variant libraries can be generated, for example, by using random (NNK) or non-random codons, such as DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Tip). The characteristics of the resulting antibody variants can be tested using the assays described herein.

In some embodiments, the antibody can bind CD38 with a dissociation constant (KD) of less than about 1x10' ⁷ M, 1x10'8 M, 1x10'9 M, 1x10'1° M, 1x10'11 M, 1x10' ¹² M, 1x10' ¹³ M, 1x10' ¹⁴ M or 1x10' ¹⁵ M as determined by the surface plasmon resonance or KinExA method known to those skilled in the art. In some embodiments, the antibody binds human CD38 with a KD of less than about 1x10'8 M. In some embodiments, the antibody binds human CD38 with a KD of less than about 1x10'9 M.

KinExA instrumentation, ELISA or competitive binding assays are known to those skilled in the art. The measured affinity of the interaction in a particular antibody/CD38 pair may vary when measured under different conditions (eg, osmolarity, pH). Thus, measurements of affinity and other binding parameters (eg, KD, K _on , Koff) are typically performed under standardized conditions and using a standardized buffer solution, such as the buffer solution described herein. Those skilled in the art will appreciate that the inherent error in affinity measurements, such as using Biacore 3000 or ProteOn equipment (measured as standard deviation, SD), can typically be 5-33% for measurements within typical detection limits. Therefore, the term about in the context of KD characterizes the typical standard deviation in the analysis. For example, a typical SD for a KD value of 1x10'9 M is up to ±0.33x10'9 M.

In some embodiments, the anti-CD38 antibody is a bispecific antibody. The VL and/or VH regions of existing anti-CD38 antibodies, or the VL and VH regions identified de novo as described above, can be constructed to generate bispecific full length antibodies. Such bispecific antibodies can be made by modulating CH3 interactions between the heavy chains of monospecific antibodies to form bispecific antibodies using technologies such as those described in US Pat. No. 7,695,936; international patent publication WO 04/111233; US Patent Publication No. US 2010/0015133; US Patent Publication No. US 2007/0287170; international patent publication WO 2008/119353; US Patent Publication No. US 2009/0182127; US Patent Publication No. US 2010/0286374; US Patent Publication No. US 2011/0123532; international patent publication WO 2011/131746; international patent publication WO 2011/143545; or US Patent Publication No. US 2012/0149876. Additional bispecific structures into which the VL and/or VH regions of the antibodies of the present invention can be inserted are, for example, dual variable domain immunoglobulins (International Patent Publication WO 2009/134776) or structures comprising different dimerization domains to connect the two arms of antibodies with different specificities, such as leucine zipper or collagen dimerization domains (international patent publication WO 2012/022811, US patent No. 5932448; US patent No. 6833441).

For example, bispecific antibodies can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and generating a bispecific heterodimeric antibody from the two original monospecific homodimeric antibodies under reducing conditions, which promotes isomerization of the disulfide bond, according to

- 16 040269 methods described in international patent publication WO 2011/131746. In these methods, a first monospecific divalent antibody (eg, an anti-CD38 antibody) and a second monospecific divalent antibody are designed to have specific substitutions in the CH3 domain that provide heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to cause the cysteines in the hinge region to undergo disulfide bond isomerization; thus forming a bispecific antibody by exchanging Fab arms. The incubation conditions can be optimally returned to non-reducing conditions. Examples of suitable reducing agents include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol, preferably reducing the agent is selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. For example, an incubation for at least 90 minutes 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 can be used, e.g. at pH=7.0 or at pH=7.4.

Possible exemplary CH3 mutations in the first heavy chain and second heavy chain of the bispecific antibody include K409R and/or F405L.

In some embodiments, the anti-CD38 antibody is conjugated to a toxin. Methods of conjugation and acceptable toxins are well known.

In some embodiments, the subject with ALC is homozygous for phenylalanine at position 158 in CD16 (genotype FcyRIIIa-158F/F) or heterozygous for valine and phenylalanine at position 158 in CD16 (genotype FcyRHIa-158F/V). CD16 is also known as the Fc gamma receptor IIIa (FcyRHIa) or low affinity receptor for the Fc region of immunoglobulin gamma, isoform III-A.

The valine/phenylalanine (V/F) polymorphism at residue 158 of the FcyRIIIa protein has been shown to affect the affinity of FcyRIIIa for human IgG. The receptor with the FcyRIIIa-158F/F or FcyRIIIa-158F/V polymorphisms shows reduced interaction with Fc and thus reduced ADCC compared to FcyRIIIa-158V/V. The absence or low amount of fucose in human N-linked oligosaccharides enhances the ability of antibodies to induce ADCC due to improved antibody binding to human FcyRIIIa (CD16) (Shields et al., J Biol Chem 277:26733-40, 2002). Using standard methods, it is possible to analyze the presence of FcyRIIIa polymorphism in patients.

The invention also provides a method of treating a subject with ALC, comprising administering to a patient in need thereof an anti-CD38 antibody that binds to a region

SKRNIQFSCKNIYR (SEQ ID NO: 2) and region

EKVQTLEAWVIHGG (SEQ ID NO: 3)

Human CD38 (SEQ ID NO: 1), wherein the anti-CD38 antibody induces in vitro killing of pathogenic CD38-expressing plasma cells via antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or modulation enzymatic activity of CD38 in vitro, while the subject is homozygous for valine at position 158 in CD16.

The invention also provides a method of treating a subject with ALC, comprising administering to a patient in need thereof an anti-CD38 antibody that binds to a region

SKRNIQFSCKNIYR (SEQ ID NO: 2) and region

EKVQTLEAWVIHGG (SEQ ID NO: 3)

Human CD38 (SEQ ID NO: 1), wherein the anti-CD38 antibody induces in vitro killing of pathogenic CD38-expressing plasma cells via antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or modulation CD38 enzymatic activity in vitro, wherein the subject is homozygous for phenylalanine at position 158 in CD16 or heterozygous for valine and phenylalanine at position 158 in CD16.

The invention also provides a method of treating a patient with ALC, comprising administering to a patient in need thereof an anti-CD38 antibody that competes for CD38 binding 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, wherein the anti-CD38 antibody induces in vitro killing of pathogenic CD38-expressing plasma cells via antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or modulation of enzyme activity CD38 in vitro, with the patient homozygous for the valine at position 158 in CD16.

The invention also provides a method of treating a patient with ALC comprising administering to a patient in need thereof an anti-CD38 antibody that competes for CD38 binding with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light variable region.

- 17 040269 chain (VL) with SEQ ID NO: 5, and the antibody to CD38 induces the destruction of pathogenic plasma cells expressing CD38, in vitro through antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity ( CDC), apoptosis, or modulation of CD38 enzymatic activity in vitro, wherein the patient is homozygous for phenylalanine at position 158 in CD16 or heterozygous for valine and phenylalanine at position 158 in CD16.

The present invention also provides a method of treating a patient with ALC, comprising determining whether the patient is homozygous or heterozygous for the valine at position 158 in CD16; and administering to the patient 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;

contains the sequences of complementarity determining regions of the heavy chain (HCDR) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) with SEQ ID NO: 6, 7 and 8, respectively, and the sequence of complementarity determining regions of the light chain (LCDR) 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) with SEQ ID NOs: 9, 10 and 11, respectively; or contains 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 time sufficient to cure the patient.

In some embodiments, the step of determining if a patient is homozygous or heterozygous for the valine at position 158 in CD16 is performed by polymerase chain reaction (PCR) and sequencing.

Introduction/pharmaceutical compositions

In the methods of the present invention, it is possible to provide the anti-CD38 antibody in acceptable pharmaceutical compositions comprising the anti-CD38 antibody and a pharmaceutically acceptable carrier. The carrier may be a diluent, adjuvant, excipient or carrier medium with which the anti-CD38 antibody is administered. Such carriers may be liquids such as water and oils, including oils derived from petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, a 0.4% saline solution and a 0.3% glycine solution can be used. These solutions are sterile and essentially free of particulate matter. They can be sterilized using well known standard sterilization techniques (eg filtration). The compositions may contain pharmaceutically acceptable adjuvants necessary to approximate physiological conditions, such as pH adjusting agents and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of anti-CD38 antibodies in such a pharmaceutical formulation can vary considerably, i. e. from less than about 0.5 wt.%, usually at least from about 1 and up to 15 or 20, 25, 30, 35, 40, 45 or 50 wt.%, and will be mainly selected based on the required dose, fluid volumes , viscosity values, etc. according to the specific route of administration chosen. Suitable carrier media 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, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006 , Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.

The route of administration of the anti-CD38 antibody may be any suitable route, such as parenteral administration, such as intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, inhalation, transmucosal (oral, intranasal, intravaginal, rectal), or other means known to those skilled in the art.

The anti-CD38 antibody in the methods of the present invention can be administered to a patient by any suitable route, for example parenterally via intravenous (iv) infusion or bolus injection, intramuscularly or subcutaneously, or intraperitoneally. IV infusion may be given, for example, over 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 h.

The dose of anti-CD38 antibody administered to a patient is sufficient to attenuate or at least partially delay the disease being treated (therapeutically effective amount), and may sometimes be from 0.005 mg to about 100 mg/kg, for example, from 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, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg, but may be even higher, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.

A fixed unit dose may also be administered, such as 50, 100, 200, 500, or 1000 mg, or the dose may be based on the patient's body surface area, such as 500, 400, 300, 250, 200, or 100 mg/m ² . For the treatment of ALC, 1 to 8 doses (eg, 1, 2, 3, 4, 5, 6, 7, or 8) can usually be given, but 9, 10, 11, 12, 13, 14, 15, 16 can also be given. , 17, 18, 19, 20 or more doses.

Administration of the anti-CD38 antibody in the methods of the present invention may be repeated after one day, two days, three days, four days, five days, 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 more. Repeated courses of treatment in the form of long-term administration are also possible.

- 18 040269

Repeated administration can be carried out at the same dose or at a different dose. For example, an anti-CD38 antibody can be administered at 8 or 16 mg/kg weekly intervals for 8 weeks, followed by 8 or 16 mg/kg every other week for an additional 16 weeks, followed by 8 or 16 mg/kg every four weeks by intravenous infusion.

In some embodiments, the anti-CD38 antibody is administered at a dose of 16 mg/kg once a week for 8 weeks followed by a dose of 16 mg/kg once every two weeks for 16 weeks and followed by a dose of 16 mg/kg once four weeks before discontinuation.

In some embodiments, the anti-CD38 antibody is administered at a dose of 8 mg/kg once a week for 8 weeks followed by a dose of 8 mg/kg once every other week for 16 weeks and followed by a dose of 8 mg/kg once four weeks before discontinuation.

In some embodiments, the anti-CD38 antibody is administered at a dose of 16 mg/kg once a week for 4 weeks followed by a dose of 16 mg/kg once every other week for 16 weeks and followed by a dose of 16 mg/kg once four weeks before discontinuation.

In some embodiments, the anti-CD38 antibody is administered at a dose of 8 mg/kg once a week for 4 weeks followed by a dose of 8 mg/kg once every two weeks for 16 weeks and followed by a dose of 8 mg/kg once four weeks before discontinuation.

The anti-CD38 antibody can be administered as maintenance therapy, eg once a week for a period of 6 months or more.

For example, the anti-CD38 antibody can be administered as a daily dose of about 0.1-100 mg/kg, such as 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 per day on 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 or alternatively 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 after the start of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

The anti-CD38 antibody can also be administered prophylactically to reduce the risk of developing cancer, slow the onset of events when the cancer progresses, and/or reduce the risk of recurrence when the cancer is in remission. This may be particularly useful in patients who have difficulty localizing a tumor that is identified based on other biological factors.

The anti-CD38 antibody may be lyophilized for storage and reconstituted in an appropriate vehicle prior to use. This technique has been shown to be effective for standard protein preparations and well known lyophilization and reconstitution techniques can be used.

Subcutaneous administration of pharmaceutical compositions containing an antibody that specifically binds to CD38 and to hyaluronidase

The anti-CD38 antibody can be administered subcutaneously as a pharmaceutical composition containing the anti-CD38 antibody and hyaluronidase.

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

The pharmaceutical composition for subcutaneous administration may contain from about 1200 mg to about 1800 mg of anti-CD38 antibody.

A pharmaceutical composition for subcutaneous administration may contain about 1200 mg of an anti-CD38 antibody.

A pharmaceutical composition for subcutaneous administration may contain about 1600 mg of an anti-CD38 antibody.

A pharmaceutical composition for subcutaneous administration may contain about 1800 mg of an anti-CD38 antibody.

A pharmaceutical composition for subcutaneous administration may contain from about 30,000 units. up to about 45000 units. hyaluronidase.

A pharmaceutical composition for subcutaneous administration may contain about 1200 mg of anti-CD38 antibody and about 30,000 units. hyaluronidase.

A pharmaceutical composition for subcutaneous administration may contain about 1800 mg of anti-CD38 antibody and about 45,000 units. hyaluronidase.

A pharmaceutical composition for subcutaneous administration may contain about 1600 mg of anti-CD38 antibody and about 30,000 units. hyaluronidase.

A pharmaceutical composition for subcutaneous administration may contain about 1600 mg of anti-CD38 antibody and about 45,000 units. hyaluronidase.

The pharmaceutical composition for subcutaneous administration may contain hyaluronidase rHuPH20 with the amino acid sequence of SEQ ID NO: 22.

rHuPH20 is a recombinant hyaluronidase (recombinant HYLENEX®) and is described in international patent publication WO 2004/078140.

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

- 19 040269

SEQ ID NO: 22

MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEF

CLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDH LDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEK AKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSW LWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF LSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQV LCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVA

SL

Administration of a pharmaceutical composition containing an anti-CD38 antibody and hyaluronidase may be repeated after one day, two days, three days, four days, five days, 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 in the form of long-term administration are also possible. Repeated administration can be carried out at the same dose or at a different dose. For example, a pharmaceutical composition containing an anti-CD38 antibody and hyaluronidase can be administered once a week for eight weeks followed by once every two weeks for 16 weeks followed by once every four weeks. Pharmaceutical compositions for administration may contain about 1200 mg of anti-CD38 antibody and about 30,000 units. hyaluronidase, and the concentration of the antibody that specifically binds CD38 in the pharmaceutical composition is about 20 mg/ml. Pharmaceutical compositions for administration may contain about 1800 mg of anti-CD38 antibody and about 45,000 units. hyaluronidase. Pharmaceutical compositions for administration may contain about 1600 mg of anti-CD38 antibody and about 30,000 units. hyaluronidase. Pharmaceutical compositions for administration may contain about 1600 mg of anti-CD38 antibody and about 45,000 units. hyaluronidase.

A pharmaceutical composition containing an anti-CD38 antibody and hyaluronidase can be administered subcutaneously in the abdominal region.

A pharmaceutical composition containing an anti-CD38 antibody and hyaluronidase may be administered in a total volume of about 80, 90, 100, 110, or 120 ml.

For administration, 20 mg/ml anti-CD38 antibody in 25 mM sodium acetate, 60 mM sodium chloride, 140 mM D-mannitol, 0.04% polysorbate-20, pH 5.5 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, prior to administration of the mixture to the subject.

Combination Therapies

The anti-CD38 antibody may be administered in combination with a second therapeutic agent.

The present invention also provides a method of treating light chain amyloidosis (ALC) comprising administering an anti-CD38 antibody in combination with a proteasome inhibitor to a patient in need thereof for a time sufficient to cure the ALC.

The present invention also provides a method for the treatment of light chain amyloidosis (ALC) comprising administering an anti-CD38 antibody in combination with a proteasome inhibitor and a corticosteroid to a patient in need thereof for a time sufficient to cure the ALC.

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

In some embodiments, the implementation of the second therapeutic agent is a proteasome inhibitor.

In some embodiments, the proteasome inhibitor is Velcade® (bortezomib), or vinca alkaloids, such as vincristine, or an anthracycline, such as doxorubicin.

In some embodiments, the implementation of the second therapeutic agent is a corticosteroid.

In some embodiments, the corticosteroid is dexamethasone.

In some embodiments, the corticosteroid is prednisone.

In some embodiments, the implementation of the second therapeutic agent is a cyclophosphamide.

In some embodiments, the implementation of the second therapeutic agent is a derivative of glutamic acid.

In some embodiments, the glutamic acid derivative is Thalomid® (thalidomide), Revlimid® (lenalidomide), Actimid® (CC4047).

- 20 040269

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), stem cell transplant, Ninlaro® (ixazomib), or NEOD001.

Bortezomib can be administered at 1.3 mg/m ² subcutaneously twice a week or once a week.

Cyclophosphamide can be administered intravenously (intermittent therapy): 40-50 mg/kg (400-1800 mg/m ² ) distributed over 2-5 days, may be repeated at intervals of 2-4 weeks;

intravenously (continuous daily therapy): 60-120 mg / m ² / day (1-2.5 mg / kg / day);

orally (intermittent therapy): 400-1000 mg/m ² distributed over 4-5 days; or orally (continuous daily therapy): 50-100 mg/m ² /day or 1-5 mg/kg/day.

Dexamethasone can be administered at 40 mg weekly or 20 mg before and after anti-CD38 administration.

Melphalan can be administered at a dose of 9 mg/m ² orally once a day from the 1st to the 4th day of each cycle up to the 9th cycle.

Thalidomide can be administered at a dose of 200 mg orally once a day.

Lenalidomide can be administered at a dose of 25 mg/day orally from days 1 to 21 of each cycle.

Pomalidomide can be administered at a dose of 4 mg orally from days 1 to 21 of a repeated 28-day cycle.

Ixazomib can be administered at a dose of 24 mg/kg IV every 28 days.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with bortezomib for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib and cyclophosphamide, for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib, cyclophosphamide, and a corticosteroid, for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib, cyclophosphamide, and dexamethasone, for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib and a corticosteroid, for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib and dexamethasone, for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with melphalan for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with bortezomib and melphalan for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib, melphalan, and a corticosteroid, for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with bortezomib, melphalan, and dexamethasone, for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with IFN-α, for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with IFN-α and a corticosteroid, for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with IFN-α and dexamethasone, for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with lenalinomide, for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with lenalinomide and cyclophosphamide, for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with lenalinomide, cyclophosphamide, and a corticosteroid, for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with lenalinomide, cyclophosphamide, and dexamethasone, for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with pomalinomide, for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with pomalinomide and a corticosteroid, for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with pomalinomide and dexamethasone, for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with thalidomide, for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with thalidomide and a corticosteroid, for a time sufficient to cure ALC.

- 22 040269

The invention also provides a method for treating light chain amyloidosis (ALC) comprising administering to a patient in need thereof an anti-CD38 antibody comprising HCDR1, HCDR2 and HCDR3 with SEQ

ID NOs: 6, 7, and 8, respectively, and containing LCDR1, LCDR3, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, in combination with thalidomide and dexamethasone, for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) 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 comprising LCDR1, LCDR3 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, in combination with ixazomib for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with bortezomib, for a time period sufficient to cure ALC.

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

The invention also provides a method for the treatment of light chain amyloidosis (ALC), comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with bortezomib, cyclophosphamide, and a corticosteroid, over time. enough to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC), comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with bortezomib, cyclophosphamide, and dexamethasone, over time. enough to cure ALC.

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

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

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

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

The invention also provides a method for the treatment of light chain amyloidosis (ALC), comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with bortezomib, melphalan, and a corticosteroid, over time. enough to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC), comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with bortezomib, melphalan, and dexamethasone, over time. enough to cure ALC.

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

The invention also provides a method for the treatment of light chain amyloidosis (ALC), comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with IFN-α and a corticosteroid, over time. enough to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with IFN-α and dexamethasone, over time. enough to cure ALC.

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

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

- 23 040269

The invention also provides a method for treating light chain amyloidosis (ALC) comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID

NO: 5, in combination with lenalinomide, cyclophosphamide and corticosteroid for a time sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC) comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with lenalinomide, cyclophosphamide, and dexamethasone, over time. enough to cure ALC.

The invention also provides a method for treating light chain amyloidosis (ALC) comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with pomalinomide, for a time period sufficient to cure ALC.

The invention also provides a method for the treatment of light chain amyloidosis (ALC), comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with pomalinomide and a corticosteroid, for a period of time sufficient to cure ALC.

The invention also provides a method for treating light chain amyloidosis (ALC) comprising administering to a patient in need thereof an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in combination with pomalinomide and dexamethasone, for a period of time sufficient to cure ALC.

The invention also provides a method for treating light chain amyloidosis (ALC) 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 NOs: 4 and VL of SEQ ID NO: 5, in combination with thalidomide, for a time sufficient to cure ALC.

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

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

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

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

In some embodiments, the patient is resistant to cyclophosphamide treatment.

In some embodiments, the patient is resistant to corticosteroid treatment.

In some embodiments, the patient is resistant to treatment with a proteasome inhibitor, cyclophosphamide, and a corticosteroid.

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

In some embodiments, the combination of the anti-CD38 antibody and the second therapeutic agent may be administered at any convenient time interval. For example, the anti-CD38 antibody and the second therapeutic agent can be administered to a patient on the same day and even in the same intravenous infusion. However, the anti-CD38 antibody and the second therapeutic agent may also be administered on alternating days or alternating weeks or months, etc. In some embodiments, the anti-CD38 antibody and the second therapeutic agent can be administered close enough in time that they are simultaneously present at detectable levels (eg, in serum) in the patient being treated. In some embodiments, a complete course of treatment with an anti-CD38 antibody consists of a series of doses administered over a period of time followed or preceded by a course of treatment with a second therapeutic agent consisting of a series of doses. A recovery period of 1, 2, or several days or weeks can be used between administration of the anti-CD38 antibody and the second therapeutic agent.

An anti-CD38 antibody or a 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, Linac and interstitial irradiation (eg, implanted radioactive seeds, GliaSite balloon), and/or with surgical treatment.

Although the invention has been described in general terms, embodiments will be further described in the following examples, which should not be construed as limiting the scope of the claims.

Additional embodiments of the invention

The following are some additional embodiments of the invention in accordance with

- 24 040269 descriptions provided in other parts of this document. Elements from the embodiments of the invention set forth above, described as being related to the invention disclosed herein, also apply to any and all of these additionally numbered embodiments.

1. An anti-CD38 antibody that 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, for use in the treatment of a patient with CD38-positive hematological malignancy, and the patient is undergoing hematopoietic stem cell transplantation (HSCT).

2. An anti-CD38 antibody for use according to Embodiment 1, wherein the CD38 positive hematologic malignancy is

a) light chain amyloidosis (ALC), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular lymphoma (FL) or mantle cell lymphoma (MCL);

b) plasma cell disease;

c) light chain amyloidosis (ALC);

d) multiple myeloma (MM); or

e) Waldenström's macroglobulinemia.

3. An anti-CD38 antibody for use according to embodiment 2, wherein the ALC is characterized by cardiac stage I, cardiac stage II, cardiac stage III, and is relapsed or refractory.

4. An anti-CD38 antibody for use according to any one of embodiments 1-3, wherein the HSCT

a) is allogeneic, autologous or syngeneic; or

b) contains transplantation of blood stem cells derived from bone marrow, blood or amniotic fluid.

5. An anti-CD38 antibody for use according to any of embodiments 1-4, wherein the anti-CD38 antibody is administered before, during, or after the HSCT.

6. An anti-CD38 antibody for use according to any one of embodiments 1-5, wherein the patient has received chemotherapy and/or radiation therapy prior to HSCT.

7. An anti-CD38 antibody for use according to any one of embodiments 1-6, wherein the anti-CD38 antibody binds to the region

SKRNIQFSCKNIYR (SEQ ID NO: 2) and region

EKVQTLEAWVIHGG (SEQ ID NO: 3)

Human CD38 (SEQ ID NO: 1).

8. An anti-CD38 antibody for use according to any one of embodiments 1-7, wherein the anti-CD38 antibody comprises the sequences of heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and complementarity determining sequences light chain regions (LCDR) 1, LCDR2 and LCDR3 with SEQ ID NO: 9, 10 and 11, respectively.

9. An anti-CD38 antibody for use according to any one of embodiments 1-8, 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 according to any of embodiments 1-9, wherein the anti-CD38 antibody comprises a heavy chain containing 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 containing an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13.

11. An anti-CD38 antibody for use according to any one of embodiments 1-10, wherein the anti-CD38 antibody comprises a heavy chain, which is a heavy chain of SEQ ID NO: 12, and a light chain of SEQ ID NO: 13.

12. An anti-CD38 antibody that 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, for use in the treatment of a patient with light chain amyloidosis.

13. An anti-CD38 antibody for use according to embodiment 12, wherein the patient is resistant to treatment with a proteasome inhibitor, cyclophosphamide, and/or a corticosteroid.

14. Anti-CD38 antibody for use according to embodiment 13, wherein the proteosome inhibitor is Velcade® (bortezomib).

15. An anti-CD38 antibody for use according to any one of embodiments 13, 14, wherein the corticosteroid is dexamethasone.

16. An anti-CD38 antibody for use according to any of embodiments 12-15, wherein the anti-CD38 antibody is administered in combination with a second therapeutic agent.

17. An anti-CD38 antibody for use according to embodiment 16, wherein the second therapeutic

- 25 040269 The tic agent is a proteasome inhibitor, cyclophosphamide, or a corticosteroid.

18. An anti-CD38 antibody for use according to any of embodiments 16, 17, wherein the proteosome inhibitor is Velcade® (bortezomib) or NINLARO® (ixazomib).

19. An anti-CD38 antibody for use according to any one of embodiments 16-18, wherein the corticosteroid is dexamethasone.

20. Anti-CD38 antibody for use according to any of embodiments 16-19, 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 alpha interferon.

21. An anti-CD38 antibody for use according to any one of embodiments 12-20, wherein the anti-CD38 antibody is administered in combination with a proteasome inhibitor, cyclophosphamide, and a corticosteroid.

22. Anti-CD38 antibody for use according to embodiment 21, wherein the proteosome inhibitor is Velcade® (bortezomib).

23. An anti-CD38 antibody for use according to any of embodiments 21, 22, wherein the proteasome inhibitor is NINLARO® (ixazomib).

24. An anti-CD38 antibody for use according to any one of embodiments 21-23, wherein the corticosteroid is dexamethasone.

25. An anti-CD38 antibody for use according to any one of embodiments 21-24, wherein the proteosome inhibitor is Velcade® (bortezomib) and the corticosteroid is dexamethasone.

26. An anti-CD38 antibody for use according to any one of embodiments 21-25, wherein the proteosome inhibitor is NINLARO® (ixazomib) and the corticosteroid is dexamethasone.

27. An anti-CD38 antibody for use according to any of embodiments 16-26, wherein the anti-CD38 antibody and the second therapeutic agent are administered simultaneously, sequentially, or separately.

28. An anti-CD38 antibody for use according to any of embodiments 16-27, wherein the anti-CD38 antibody and the second therapeutic agent are administered simultaneously, sequentially, or separately.

29. An anti-CD38 antibody for use according to any one of embodiments 21-28, wherein the anti-CD38 antibody, proteasome inhibitor, cyclophosphamide, and corticosteroid are administered simultaneously, sequentially, or separately.

30. An anti-CD38 antibody for use according to any one of embodiments 12-29, wherein ALC is characterized by cardiac stage I, cardiac stage II, cardiac stage III, and is relapsed or refractory.

31. An anti-CD38 antibody for use according to any one of embodiments 12-30 wherein the patient is undergoing hematopoietic stem cell transplantation (HSCT).

32. An anti-CD38 antibody for use according to any one of embodiments 12-31, wherein the HSCT is allogeneic, autologous, or syngeneic.

33. An anti-CD38 antibody for use according to any one of embodiments 12-32, wherein the HSCT comprises transplantation of blood stem cells derived from bone marrow, blood, or amniotic fluid.

34. An anti-CD38 antibody for use according to any of embodiments 12-33, wherein the anti-CD38 antibody is administered before, during, or after the HSCT.

35. An anti-CD38 antibody for use according to any one of embodiments 12-34, wherein the patient has received chemotherapy and/or radiation therapy prior to HSCT.

36. An anti-CD38 antibody for use according to any one of embodiments 12-35, wherein the patient is additionally given radiotherapy.

37. An anti-CD38 antibody for use according to any of embodiments 12-36, wherein the anti-CD38 antibody does not mediate destruction of CD34-positive hematopoietic progenitor cells by complement-dependent cytotoxicity (CDC).

38. An anti-CD38 antibody for use according to any one of embodiments 12-37, wherein the anti-CD38 antibody induces the killing of CD38-positive plasma cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) , apoptosis, or modulation of CD38 enzymatic activity.

39. An anti-CD38 antibody for use according to any one of embodiments 12-38, 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) with SEQ ID NO: 5.

40. An anti-CD38 antibody for use according to any one of embodiments 12-39, wherein the anti-CD38 antibody binds to at least a region

SKRNIQFSCKNIYR (SEQ ID NO: 2) and region

EKVQTLEAWVIHGG (SEQ ID NO: 3)

Human CD38 (SEQ ID NO: 1).

- 26 040269

41. An anti-CD38 antibody for use according to any one of embodiments 12-40, wherein the anti-CD38 antibody comprises the amino acid sequences of the heavy chain complementarity determining regions 1 (HCDR), HCDR2, and HCDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and the amino acid sequences light chain complementarity determining regions 1 (LCDR1), LCDR2 and

LCDR3 with SEQ ID NO: 9, 10 and 11, respectively.

42. An anti-CD38 antibody for use according to any of embodiments 12-41, wherein the anti-CD38 antibody comprises a VH containing an amino acid sequence that is 95%, 96, 97, 98, 99, or 100% identical to the sequence of SEQ ID NO: 4, and VL containing an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 5.

43. An anti-CD38 antibody for use according to any of embodiments 12-42, wherein the anti-CD38 antibody comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5.

44. An anti-CD38 antibody for use according to any one of embodiments 12-43, wherein the anti-CD38 antibody comprises a heavy chain containing an amino acid sequence that is 95%, 96, 97, 98, 99, or 100% identical to the sequence of SEQ ID NO: 12 , and a light chain containing an amino acid sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 13.

45. An anti-CD38 antibody for use according to any of embodiments 12-44, wherein the anti-CD38 antibody comprises the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO: 13.

46. An anti-CD38 antibody for use according to any of embodiments 12-46, wherein the anti-CD38 antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3:

a) VH with SEQ ID NO: 14 and VL with SEQ ID NO: 15;

b) VH with SEQ ID NO: 16 and VL with SEQ ID NO: 17;

c) VH with SEQ ID NO: 18 and VL with SEQ ID NO: 19; or

d) VH with SEQ ID NO: 20 and VL with SEQ ID NO: 21, with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 defined by Kabat, Chothia or IMGT.

47. Anti-CD38 antibody for use according to embodiment 46, wherein the anti-CD38 antibody comprises

e) VH with SEQ ID NO: 14 and VL with SEQ ID NO: 15;

f) VH with SEQ ID NO: 16 and VL with SEQ ID NO: 17;

g) VH with SEQ ID NO: 18 and VL with SEQ ID NO: 19; or

h) VH with SEQ ID NO: 20 and VL with SEQ ID NO: 21.

48. An anti-CD38 antibody for use according to any of embodiments 12-47, wherein the anti-CD38 antibody is humanized or human.

49. An anti-CD38 antibody for use according to any one of embodiments 12-48, wherein the anti-CD38 antibody is of the isotype IgG1, IgG2, IgG3, or IgG4.

50. An anti-CD38 antibody for use according to any one of embodiments 12-49, wherein the anti-CD38 antibody is of the IgG1 isotype.

51. An anti-CD38 antibody for use according to any one of embodiments 12-50, wherein the anti-CD38 antibody is administered intravenously.

52. An anti-CD38 antibody for use according to any one of embodiments 12-51, wherein the anti-CD38 antibody is administered subcutaneously in a pharmaceutical composition comprising an anti-CD38 antibody and hyaluronidase.

53. An anti-CD38 antibody for use according to embodiment 42, wherein the hyaluronidase is rHuPH20 of SEQ ID NO: 22.

Example 1. Materials and methods.

Patients with MM and ALC underwent mobilization and selection of blood stem cells in an IRB approved clinical trial (Tufts Medical Center IRB # 10680, DARZALEX™ (daratumumab) Preclinical Studies in Mobilized Stem Cells and Grafts for Patients with Clonal Plasma Cell Disease) requiring written informed consent . Mobilized blood stem and progenitor cells were used to investigate the effect of DARZALEX™ (daratumumab) on CD34+ cell growth in vitro. Both CD34-selected and non-selected mobilized blood progenitor cells from patients with MM and ALC were used. The effect of DARZALEX™ (daratumumab) or an isotype control antibody on progenitor colony growth in vitro was evaluated in semi-solid media assays as an indicator of the effect of DARZALEX™ (daratumumab) on human hematopoietic CD34+ progenitor cell proliferation.

Progenitor cell assays in methylcellulose containing recombinant cytokines (Stem Cell Technologies, Vancouver, CA, USA; Cat # 04435) were performed according to the manufacturer's instructions on CD34-selected and non-selected mobilized blood stem cells with 1- th day of leukapheresis products at different concentrations. CD34 selection was performed using a Miltenyi MiniMacs device. Cells not selected for CD34 were used at a concentration of 0.5x10 ⁴ /ml, and

- 27 040269 cells selected for CD34 - at a concentration of 500 cells / ml. Analyzes were performed with DARZALEX™ (daratumumab) or control antibody at varying concentrations; in some assays cells were plated in media containing DARZALEX™ (daratumumab) or a control antibody, and in other assays cells were plated after incubation at 37°C in 5% CO2 for 1 hour in complement-rich human serum with DARZALEX™ (daratumumab) or control antibody. Colonies were counted on day 14 (CFU-GM, BFU-E, CFU-Mix).

Example 2 CD38 is expressed on clonal ALC plasma cells.

Clonal plasma cells from ALC patients express high levels of mRNA for CD38 based on transcriptional profiles of bone marrow plasma cells sorted for CD138+ by FACS (n=16, GEO GSE24128; Zhou et al., Clin Lymphoma Myeloma Leuk 12: 49-58, 2012), which are obtained at the time of diagnosis before therapy (Fig. 1). In addition, in an immunophenotype analysis of bone marrow plasma cells for CD138+ from patients with newly diagnosed ALC, CD38 was consistently detected on the cell surface in all cases (Paiva et al., Blood 117: 3613-6, 2011).

Example 3 NK cells from ALC patients are functionalized and induce DARZALEX™ (daratumumab) mediated ADCC against CD38 expressing cells three weeks after stem cell transplantation (SCT).

According to the IRB-approved study protocol, which is described in Example 1 and requires written informed consent, NK cells (CD3-/CD56+/CD16+) were obtained from patients with ALC three weeks after SCT and evaluated by determining the percentage and number of NK cells in the samples peripheral blood, and the in vitro activity of these effector NK cells together with DARZALEX™ (daratumumab) was evaluated in ADCC assays on human plasma cells (MM1S cells) as targets. MM1S cells express high levels of CD38, making them suitable targets for DARA. Flow cytometry was performed using the FlowCellect™ human natural killer cell characterization kit (Millipore, Billerica, Massachusetts, USA; Cat # FCIM025164). Bioluminescent cytotoxicity assays (Cell Technology; Mountain View, CA, USA) were performed following the manufacturer's instructions. Target cell concentration was optimized to 5000 MM1S cells per well. Target cells were incubated with DARZALEX™ (daratumumab) or control antibody (human kappa IgG1, Sigma-Aldrich, St. Louis, MO, USA) at 100 ng/mL for 15 min at 37° C. incubator with 5% CO2. They were then added to wells with NK cells from patients at a concentration of effector:target cell=10:1. The number of NK cells in each sample was calculated based on the characterization of fresh samples from patients by flow cytometry. ADCC was determined by calculating the luminescence value for each reaction condition according to the manufacturer's instructions using standard controls. The amount of lysis per situation (% ADCC) was determined using the following calculations:

% ADCC=(sample) - (control 1) - (control 2)x100;

(control 3) - (control 1)

Sample: DARZALEX™ (daratumumab) sample.

Control 1: isotype control assay (targeted spontaneous release).

Control 2: no antibody, no effector cells.

Control 3: maximum lysis in the presence of lysis reagent.

The amount of specific lysis from DARZALEX™ (daratumumab) was calculated by subtracting % ADCC with isotype control from % ADCC with DARZALEX™ (daratumumab).

As shown in FIG. 2, NK cells accounted for almost one third of all mononuclear cells in peripheral blood from ALC patients (n=9) 3 weeks after SCT. In bioluminescent ADCC assays, NK cells from ALC patients (n=6) in combination with DARZALEX™ (daratumumab) had a 32% median specific lysis (range -3 to 73%).

Example 4 DARZALEX™ (daratumumab) does not induce the killing of hematopoietic CD34+ progenitor cells, even though the high level of CD38 expression seen in these cells.

CD38 expression on CD34+ cells was analyzed by flow cytometry using an APC-conjugated anti-human CD38 antibody (HIT2, Biolegend, San Diego, CA, USA). CD34+ cells had the appearance of myeloblasts and expressed high levels of CD38 (FIG. 3).

The effect of DARZALEX™ (daratumumab) on CD34+ cell growth in vitro was investigated using mobilized blood stem and progenitor cells from patients with MM and ALC who were mobilized and selected for blood stem cells in an IRB approved clinical trial requiring informed consent as described in Example 1. Both CD34-selected and unselected mobilized blood progenitor cells from MM and ALC patients were used, and the effect of daratumumab or an in vitro isotype control antibody on progenitor colony growth in semi-solid media assays was evaluated as an indicator effects of DARZALEX™ (daratumumab) on the proliferation of hematopoietic CD34+ human progenitor cells.

Progenitor cell assays in methylcellulose containing recombinant cytokines (Stem

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Cell Technologies, Vancouver, pc. California, USA; cat. no. 04435) was performed according to the manufacturer's instructions on CD34-selected or non-selected mobilized blood stem cells from day 1 of leukapheresis products at various concentrations. CD34 selection was performed using a Miltenyi MiniMacs device. Cells not selected for CD34 were used at a concentration of 0.5x10 ⁴ /ml, and cells selected for CD34 were used at a concentration of 500 cells/ml. Analyzes were performed with DARZALEX™ (daratumumab) or control antibody at varying concentrations; in some assays cells were plated in media containing DARZALEX™ (daratumumab) or a control antibody, and in other assays cells were plated after incubation at 37°C in 5% CO2 for 1 hour in complement-rich human serum with DARZALEX™ (daratumumab) or control antibody. Colonies were counted on day 14 (CFU-GM, BFU-E, CFU-Mix).

Thawed cryopreserved unselected mobilized blood progenitor cells grew in similar amounts in CFU-GM (Fig. 4A) and BFU-E (Fig. 4B) in cultures with DARZALEX™ (daratumumab) or in isotype control (500 or 1000 ng/ml ) compared to a control without antibodies. In these experiments, thawed, unselected, mobilized blood progenitor cells were applied at a concentration of 5x10 ⁴ cells/ml semi-solid medium (methylcellulose) in the presence of DARZALEX™ (daratumumab) or an isotype control (500 or 1000 ng/ml). CFU-GM and GFU-E were calculated after two weeks as the percentage of CFU-GM compared to a control without antibodies. It is worth noting that there was significantly more CFU-GM in the 1000 ng/mL plates of the isotype control antibody for an unclear reason.

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

The ability of DARZALEX™ (daratumumab) to induce CDC in fresh CD34-selected hematopoietic progenitors was evaluated. CD34+ cells were incubated in 10% complement-rich human serum without antibodies with 500 ng/ml DARZALEX™ (daratumumab) or 500 ng/ml isotype control for 1 hour and then applied directly to the semi-solid medium. Evaluated the formation of colonies on 500 selected for CD34 cells.

DARZALEX™ (daratumumab) did not reduce CFU-GM (FIG. 6A) or BFU-E (FIG. 6B), indicating that this antibody did not induce CDC on original CD34-selected progenitors despite expression CD38 on these cells. For unclear reasons, an increase in BFU-E was observed in samples treated with DARZALEX™ (daratumumab). DARZALEX™ (daratumumab) increased the amount of BFU-E produced.

The effect of DARZALEX™ (daratumumab) on fresh, CD34-selected progenitor hematopoietic cells was also tested by coating cells directly in methylcellulose containing 500 or 1000 ng/ml DARZALEX™ (daratumumab), or an isotype control, or a control without antibodies. Colony formation was assessed on day 14 for CFU-GM per 500 cells selected for CD34 (FIG. 7A) or for BFU-E per 500 cells selected for CD34 (FIG. 7B). No reduction in colony formation was observed at any concentration of DARZALEX™ (daratumumab), indicating that DARZALEX™ (daratumumab) was not toxic to colony formation of CD34+ cells in this assay system. In FIG. 6A, 6B and 7A show the results for mobilized blood progenitor cells from 1 MM patient and 2 ALC patients. In FIG. 7B shows results for mobilized blood progenitor cells from 2 MM patients and 1 ALC patient. BFU-E assays in the presence of 500 ng/mL DARZALEX™ (daratumumab) contained significantly more BFU-E for unclear reasons.

Example 5 Patient NK cell-mediated ADCC mediated by DARZALEX™ (daratumumab) is FcyRIIIA genotype dependent.

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

PCR-based analysis showed that of the ten patients examined in this study, six patients had a V/F or V/V polymorphism and had a 60% median lysis activity (31-98) and 4 had F/ alleles. F and 17% median lysis (0-32) (Fig. 8; P<0.05, two tailed Mann-Whitney test).

The baseline correlation between Fc receptor polymorphisms and ADCC under the influence of DARZALEX™ (daratumumab) may be considered in future clinical trials.

Example 6 Randomized phase 3 trial to evaluate the efficacy and safety of DARZALEX™ (daratumumab) in combination with cyclophosphamide, bortezomib and dexamethasone (CyBorD) versus CyBorD alone in cases of newly diagnosed systemic ALC amyloidosis.

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Conducted an open-label, phase 3, two-cohort study comparing therapy

DARZALEX™ (daratumumab) in combination with CyBorD (cyclophosphamide, bortezomib, and dexamethasone) and CyBorD alone in subjects with newly diagnosed ALC amyloidosis.

There are currently no approved drugs for the treatment of ALC amyloidosis. In the absence of an approved treatment, drugs developed for the treatment of multiple myeloma are prescribed. The CyBorD combination is most commonly used in the EU and US as the initial treatment for ALC amyloidosis (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).

the main objective

The main goal is to evaluate the complete hematological response in patients with ALC after treatment with DARZALEX™ (daratumumab) in combination with CyBorD compared with treatment with CyBorD alone.

Secondary Goals

Evaluation of progression-free survival based on mortality from any cause and disease progression (disease progression, including disease progression in hematology and disease progression in organs according to uniform recommendations);

assessment of organ response rates (OrRR) (Comenzo 2012): kidney, heart, liver;

assessment of indicators of the level of hematological response (ORR) and indicators of the level of hematological response VGPR or improved level (ie, complete response + VGPR);

assessment of indicators of progression in organs for the heart, kidneys, liver;

assessment of duration and time to hematologic CR and VGPR or improved response, respectively;

assessment of the duration and time to response in the organs;

evaluation of the safety and tolerability of DARZALEX™ (daratumumab) when administered in combination with CyBorD;

assessment of the pharmacokinetics of DARZALEX™ (daratumumab);

assessment of the immunogenicity of DARZALEX™ (daratumumab);

assessment of therapeutic effect by patient reported outcomes (PRO), including the SF-36 health questionnaire, the EuroQol-5 (EQ-5D-5L) and QLQ-C30 measurements of the European Organization for Research and Treatment of Cancer (EORTC).

Search targets

Evaluation of response biomarkers, including highly sensitive (HF) troponin T;

study of biomarkers to predict response or resistance to therapy;

study of minimal residual disease status in patients with amyloidosis.

Evaluation criteria

The main criterion for evaluation.

The main evaluation criterion is the indicators of a complete hematological response.

Secondary evaluation criteria.

Secondary efficacy measures include progression-free survival (PFS) based on all-cause mortality;

organ response rates (OrRR) for kidney, heart, liver;

overall survival (OS);

time to next treatment (TNT);

time to disease progression (TTP);

time to hematological progression of the disease;

overall hematological response;

indicators of hematological response VGPR or improved response;

time to complete hematologic response (or VGPR or improved response);

the duration of the complete hematological response (or VGPR, or improved response);

time to response in the body;

the duration of the response in the body;

assessment of the impact of treatment on reported patient outcomes, including the EORTC QLQ-C30, the Short Form 36 Health Survey (SF-36) and the European Five Dimensional Quality of Life Questionnaire (EQ-5D-5L).

Search endpoint

The exploratory endpoint is the assessment of minimal residual disease status in patients who have achieved a complete hematological response in bone marrow and blood.

Hypothesis

The main hypothesis of this study is that DARZALEX™ (daratumumab) in combination with CyBorD will improve overall hematologic response rates compared with CyBorD alone in subjects with newly diagnosed ALC amyloidosis.

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Study plan

This is a Phase 3, multicenter, open-label, two-cohort study comparing DARZALEX™ (daratumumab) in combination with CyBorD versus CyBorD alone in subjects with newly diagnosed ALC. Approximately 360 subjects in two randomized cohorts who initially received either CyBorD or DARZALEX™ (daratumumab) in combination with CyBorD were stratified by cardiac risk (stages I, II and IIIa). Each cycle lasted 4 weeks. DARZALEX™ (daratumumab) was administered at a dose of 16 mg/kg once a week for the first 2 cycles (8 weeks) of treatment, then once every 2 weeks for 4 cycles (16 weeks), and then every 4 weeks up to a maximum of 6 therapy cycles (24 weeks) along with CyBorD backbone (in both cohorts). Subjects randomized to the DARZALEX™ (daratumumab) group could continue DARZALEX™ (daratumumab) every 4 weeks after 6 cycles until disease progression for a maximum of 2 years. Subjects received weekly cyclophosphamide 300 mg/m ² orally or intravenously, bortezomib 1.3 mg/m ² subcutaneously, and dexamethasone 40 mg weekly. The cycles were repeated every 4 weeks. Maximum number of conducted cycles: 6.

Prior to randomization, 6 subjects treated with DARZALEX™ (daratumumab) plus CyBorD were pre-treated for at least 1 cycle to confirm the safety of the combination regimen. Dosages in these 6 subjects were spaced such that subjects received their first dose no earlier than 48 hours after the previous subject involved in the study. Safety assessment was performed by the organizer and external experts after completion of at least 1 cycle for 6 subjects before starting the randomized part of the protocol. Pre-treatment subjects completed all scheduled T&E evaluations and participated in the overall safety evaluation of the DARZALEX™ (daratumumab)+CyBorD administration regimen. However, these subjects are not included in the overall performance evaluation.

Subject Selection

Inclusion Criteria.

1. Subject must be at least 18 years of age.

2. Histopathological diagnosis of amyloidosis or light chain deposition disease based on the detection by polarization microscopy of green birefringent material in tissue samples stained with Congo red or a characteristic appearance in electron microscopy.

3. Indicators of light chain amyloidosis disease as defined by at least one of the following: serum monoclonal protein >=0.5 g/dL by protein electrophoresis, >200 mg of urinary monoclonal protein in 24 h by electrophoresis, serum free light chains >=5.0 mg/dL with abnormal kappa:lambda ratio or difference between folded and unfolded free light chains (dFLC) >5 mg/dL.

4. The patient must be diagnosed with ALC amyloidosis for the first time without prior systemic therapy. The only exception is that subjects may receive up to 4 weeks of therapy with bortezomib, cyclophosphamide, and/or dexamethasone (or an equivalent steroid) prior to emergency randomization if the subject is in urgent need of therapy.

5. General condition (PS) 0, 1 or 2 on the scale of the Eastern United Cancer Group (ECOG).

6. Prior to initiation of treatment, the subject must obtain clinical laboratory values that meet the following criteria in the screening phase:

i) absolute neutrophil count >1.0x10 ⁹ /l;

ii) hemoglobin level >7.5 g/dl (>5 mmol/l); blood transfusion is allowed to maintain Hb> 7.5;

iii) platelet count >50x10 ⁹ /l; transfusion of platelets is allowed;

iv) alanine aminotransferase (ALT) level <2.5 times upper limit of normal (ULN);

v) aspartate aminotransferase (AST) level <2.5 times the upper limit of normal (ULN);

vi) total bilirubin level <1.5xULN (with the exception of Gilbert's syndrome: direct bilirubin <2xULN);

vii) creatinine clearance >=20 ml/min; it should be noted that creatinine clearance can either be measured from a 24-hour urine test or calculated using an approved formula such as MDRD, CKD-epi or Cockcroft-Gault (see Appendix 3 for details);

viii) TSH and free T4 within normal limits.

Patients may be treated with thyroid hormones if necessary to correct concomitant hypothyroidism.

7. Women of childbearing age should use high-efficiency birth control continuously for 4 weeks prior to treatment, during therapy, during dosing intervals, and continue for 4 weeks after study drug is discontinued. Birth control must comply with local legal regulations regarding the methods of birth control used for subjects participating in clinical trials, on

- 31 040269 an example of the approved use of oral, injectable or implantable hormonal contraceptives; placement of intrauterine devices or intrauterine systems; barrier methods: using a condom with spermicidal foam/gel/film/cream/suppository or a barrier cap (membrane or cervical/vaginal cap) with spermicidal foam/gel/film/cream/suppository (if hormonal or intrauterine contraception is medically contraindicated, then you can use 2 or other effective or highly effective methods); sterilization of the male partner (this subject must have a single partner with a vasectomy); complete abstinence (if consistent with the subject's desired and habitual lifestyle) during and after the study (3 months after the last dose of DARZALEX™ (daratumumab) for women).

8. A man who has sex with a woman of childbearing age and has not undergone a vasectomy must consent to the use of a barrier method of birth control, such as either a condom with spermicidal foam/gel/film/cream/suppository or an obstruction cap (membrane or cervical/vaginal cap) with spermicidal foam/gel/film/cream/suppository at partner. All men are also excluded from sperm donation during the study and for 3 months after receiving the last dose of study drug.

9. A woman of childbearing potential at screening should have 2 negative urine or serum pregnancy tests, the first 10 to 14 days before dosing and the second within 24 hours before dosing.

10. Each subject must sign an Informed Consent Form (ICF) indicating that he/she understands the objectives and procedures required for the study and voluntarily participates in the study. Subjects must voluntarily comply with and be prepared to comply with the prohibitions and restrictions specified in this protocol in accordance with the provisions of the ICF.

exclusion criteria.

1. Previous therapy for ALC amyloidosis or multiple myeloma, except for one cycle (less than 4 weeks) with bortezomib, cyclophosphamide, and/or dexamethasone (or an equivalent steroid), prior to 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. Signs of serious cardiovascular disease as below:

a) NT-ProBNP >8500 ng/l;

b) heart failure class IIIB or IV according to the New York Heart Association (NYHA);

c) unstable angina or myocardial infarction within 6 months before the first dose;

d) 2nd or 3rd degree atrioventricular (AV) block or sick sinus syndrome if the subject does not have a pacemaker (Any degree of Mobitz type I AV block is acceptable);

e) a history of continuous ventricular tachycardia (>30 s) or cardiac syncope; a history of recurrent, intermittent ventricular tachycardia (>3 beats) despite antiarrhythmic therapy;

f) 12-lead screening ECG showing a corrected baseline QT interval (QTcF) >470 ms;

g) Lying systolic blood pressure <90 mmHg, or symptomatic orthostatic hypotension, or a decrease in standing systolic blood pressure >20 mmHg. Hg despite medical treatment (eg, midodrine, fludrocortisone);

h) left ventricular ejection fraction (LVEF) on transthoracic echocardiogram, MUGA scan, cardiac MRI or cardiac catheterization <40%. Evaluation required at screening.

4. Subjects scheduled for stem cell transplant during the first six cycles of protocol therapy are excluded. Stem cell sampling during the first six cycles of protocol therapy is allowed.

5. Diagnosis or treatment of other malignancies other than ALC, excluding

a) the therapy for the malignant disease was curative and there were no known active manifestations of the disease for >5 years before randomization;

b) sufficiently cured non-melanoma skin cancer or lentigo maligna without signs of disease;

c) well-treated carcinoma in situ (eg cervical, breast) without evidence of disease.

6. Subject with known chronic obstructive pulmonary disease (COPD), with a forced expiratory volume in 1 s (FEV1) <50% of normal prognosis. It should be noted that it is required

- 32 040269 FEV1 study for patients with suspected COPD, and subjects with FEV1<50% of normal prognosis should be excluded.

7. Subject has known moderate or severe persistent asthma within the past 2 years (see Appendix 5) or has current unmanaged asthma of any classification. (It should be noted that subjects with current managed intermittent asthma or managed moderate persistent asthma are eligible to participate in the study).

8. Subject known to be positive for human immunodeficiency virus (HIV) serology, known to be positive for hepatitis B surface antigen, or known to have a history of hepatitis C.

9. Sensitive (grade 3) or painful (grade 1) peripheral neuropathy.

10. Known hypersensitivity to bortezomib, boron, or mannitol.

11. The subject has a comorbid medical condition or disease (eg, an active systemic infection) that would interfere with procedures or interfere with study results, or, in the opinion of the investigator, would jeopardize participation in this study.

12. Any form of secondary or hereditary (ATTR) amyloidosis.

13. Subject has known allergies, hypersensitivity or intolerance to human monoclonal antibodies or proteins or excipients (see Investigator's Brochure), or known sensitivities to products derived from mammals.

14. The subject is known or suspected to be unable to comply with the study protocol (eg, due to alcohol, drug addiction, psychological distress), or the subject has any circumstances that would, in the investigator's opinion, make participation contrary to the subject's interests (eg, reduce well-being), or which may interfere with, limit or distort the assessments determined by the protocol.

15. Subject is pregnant, breastfeeding, or planning to become pregnant while participating in this study or within 6 months of last dose of study drug.

16. Subject took an experimental drug (including experimental vaccines) or used an invasive experimental medical device within 4 weeks prior to the start of Day 1 of Cycle 1 (except for experimental myeloma agents, which must not be taken within 2 weeks of the start of Cycle 1 day 1 of cycle 1, as described in exception #3).

17. Subject underwent major surgery within 2 weeks prior to the start of Day 1 of Cycle 1, or did not fully recover from surgery, or is scheduled for surgery at a time when the subject is expected to participate in the study, or within 2 weeks of administration of the latter dose of study drug.

Note. Subjects with planned surgical interventions that are performed under local anesthesia are eligible to participate.

Safety assessment

Safety will be assessed based on adverse events, laboratory test results, ECG, vital signs, physical examination data, and ECOG general condition. Any clinically important changes occurring during the study will be recorded in the adverse events section of the eCRF. Any clinically significant abnormality that persists at the end of the study/early termination will be observed by the investigator until corrected or clinically stable.

Efficiency

Categories by answer.

Disease assessment should be performed every 28 days on the scheduled assessment day (±3 days). If treatment is delayed for any reason, disease assessment should be performed as scheduled, regardless of any changes to the dosing regimen.

Disease assessment should be performed at a central laboratory (unless otherwise specified) according to time and event schedules prior to disease progression. This study will apply the consensus guidelines for criteria for response to ALC amyloidosis treatment (Comenzo et al., Leukemia 26: 2317-2325, 2012), which are presented below. When assessing free light chains, the amount of immunoglobulin, M-protein and determining immunofixation in serum and daily urine, the researcher will use the results provided by the central laboratory.

Subjects with a positive serum IFE and IFE-confirmed DARZALEX™ (daratumumab) interference who meet all other clinical criteria for a complete response (CR) will be considered to have a CR.

Agreed recommendations on international common response criteria

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Claims (4)

Category by answer Criteria Completed Normalization of levels and ratio of free light chains, negative immunofixation in blood serum and urine Very good partial Decrease in dFLC < 40 mg/l Partial More than 50% reduction in dFLC No answer Less PR Progression From CR, any detectable monoclonal protein or abnormal ratio of free light chains (light chains must be doubled) From PR, 50% increase in serum M-protein to > 0.5 g/dL, or 50% increase in urinary M-protein to > 200 mg/day (should show a visible peak) Increase free light chains by 50% to > 100 mg/L Abbreviations: CR, complete response; dFLC, difference between folded (iFLC) and unfolded (FLC) free light chains; FLC, free light chains; PR, partial response Example 7 Randomized phase 2 trial to evaluate the efficacy and safety of DARZALEX™ (daratumumab) as a single agent for the treatment of subjects with systemic ALC amyloidosis. A phase 2 open label study is underway to evaluate the safety and efficacy of DARZALEX™ (daratumumab) as a single agent in previously treated subjects with systemic ALC amyloidosis. Approximately 40 subjects were randomized into two cohorts, of which one received DARZALEX™ (daratumumab) and the other received placebo. Adult patients 18 years of age or older with biopsy-proven systemic ALC amyloidosis who have not achieved CR or VGPR after initial treatment, including Mayo Clinic stage I and II cardiac patients and including stage III patients with NT-proBNP<5000 ng alone /l (or BNP<1000 ng/l). Dosing schedule Two dosing regimens have been considered. Mode 1. Administer DARZALEX™ (daratumumab) in doses of 16 mg/kg IV, once a week x 8 doses, once every two weeks x 8 doses, and then every 4 weeks for up to 6 cycles. After 6 cycles, patients will continue DARZALEX™ (daratumumab) every 4 weeks until progression or selective discontinuation. Mode 2 Administer daratumumab in doses over six 28-day cycles of 16 mg/kg IV. In the first cycle, administer DARZALEX™ (daratumumab) every week on days 1, 8, 15, and 22. For cycles 2 and 3, administer DARZALEX™ (daratumumab) every other week on days 1 and 15. From cycles 4 to 6, administer DARZALEX™ (daratumumab) every 4 weeks on day 1. The main goal, secondary goals, main inclusion criteria and main exclusion criteria are the same as those described in example 6. CLAIM 1. A method of treating a patient with light chain amyloidosis (ALC), comprising administering an anti-CD38 antibody to the patient for a time period sufficient to cure the ALC, wherein the anti-CD38 antibody comprises a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region chain (VL) with SEQ ID NO: 5 and where the antibody against CD38 refers to the isotype IgG1. 2. The method of claim 1, wherein the patient is resistant to treatment with a proteasome inhibitor, cyclophosphamide, and/or a corticosteroid. 3. The method of claim 2 wherein the proteasome inhibitor is Velcade® (bortezomib). 4. The method of claim 2 wherein the corticosteroid is dexamethasone. -