Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
  • A61K31/203—Retinoic acids ; Salts thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
  • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
  • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/734—Complement-dependent cytotoxicity [CDC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the present invention relates to combination therapies with anti-CD38 antibodies and all-trans retinoic acid.
• B-cell malignancies include B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, multiple myeloma, Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma and AIDS-related Non-Hodgkin's Lymphoma.
• B-cell malignancies comprise more than 85% of diagnosed lymphomas.
• MM Multiple myeloma
• MM is a B cell malignancy characterized by the latent accumulation of secretory plasma cells in bone marrow with a low proliferative index and an extended life span. The disease ultimately attacks 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 hematologic malignancies, can be attributed to MM. Incidence of MM increases in the aging population, with the median age at time of diagnosis being about 61 years.
• CD38 is a type II membrane protein having function in receptor-mediated adhesion and signaling as well as mediating calcium mobilization via its ecto-enzymatic activity, catalyzing formation of cyclic ADP-ribose (cADPR) from NAD + and also hydrolyzing cADPR into ADP-ribose (ADPR).
• cADPR cyclic ADP-ribose
• CD38 mediates cytokine secretion and activation and proliferation of lymphocytes (Funaro et al, J Immunology 145:2390-6, 1990; Terhorst et al, Cell 771-80, 1981; Guse et al, Nature 398:70-3, 1999), and via its NAD glycohydrolase activity regulates extracellular NAD + levels which have been implicated in modulating 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 MM malignant plasma cells, and is implicated in various hematological malignancies.
• One embodiment of the invention is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to a patient in need thereof an anti-CD38 antibody in combination with all-trans retinoic acid (ATRA), wherein the anti-CD38 antibody induces killing of CD38-expressing cells in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
• ADCC antibody-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• Figure 1A shows that all-trans retinoic acid (ATRA) enhances CD38 expression on multiple myeloma (MM) cell lines in a dose dependent manner.
• MM cell lines all-trans retinoic acid (ATRA) enhances CD38 expression on multiple myeloma (MM) cell lines in a dose dependent manner.
• RPMI8226, UM9 and XG1 were incubated with RPMI-1640 medium alone or with 0-25 nM ATRA for 48 hours and then harvested to determine CD38 expression by flow cytometry.
• the graph shows results of one representative experiment.
• the Y axis shows the fold increase of mean fluorescent intensity (MFI) of CD38 surface expression.
• Figure IB shows that ATRA enhances CD38 expression on MM cell lines in a time dependent manner.
• MM cell lines RPMI8226, UM9 and XG1 were incubated with RPMI- 1640 medium alone or with 10 nM ATRA for 24, 48, 72 or 96 hours and then harvested to determine CD38 expression by flow cytometry.
• the graph shows results of one representative experiment.
• the Y axis shows the fold increase of mean fluorescent intensity (MFI) of CD38 surface expression.
• FIG. 2 shows that ATRA enhances CD38 expression on bone marrow mononuclear cells (BM-MNCs) from MM patients ex vivo.
• BM-MNCs bone marrow mononuclear cells
• BM-MNCs from 26 MM patients were incubated with RPMI-1640 medium alone or with 10 nM ATRA for 48 hours and then harvested to determine CD38 expression by flow cytometry.
• the Y axis shows the MFI of CD38 surface expression.
• Medium medium at 0 hours
• ns not
• Figure 3A shows daratumumab-induced complement-dependent cytotoxicity (CDC) (top panel) and antibody-dependent cell mediated cytotoxicity (ADCC) (bottom panel) in MM XG1 cell line pretreated with or without 10 nM ATRA for 48 hours prior to performing CDC or ADCC in the presence of 10 ⁇ g/ml daratumumab.
• the Y axis shows the percent (%) CDC or ADCC.
• Data show the mean and SEM of at least three experiments, p- values between the indicated groups were calculated using a paired student's t test.
• Dara daratumumab; * p ⁇ 0.05; ** p ⁇ 0.01.
• Figure 3B shows daratumumab-induced CDC (top panel) and ADCC (bottom panel) in MM RPMI8226 cell line pretreated with or without 10 nM ATRA for 48 hours prior to performing CDC or ADCC in the presence of 10 ⁇ g/ml daratumumab.
• the Y axis shows the percent (%) CDC or ADCC.
• Data show the mean and SEM of at least three experiments. P-values between the indicated groups were calculated using a paired student's t test.
• Dara daratumumab; ns: not significant.
• Figure 3C shows daratumumab-induced CDC (top panel) and ADCC (bottom panel) in MM UM9 cell line pretreated with or without 10 nM ATRA for 48 hours prior to performing CDC or ADCC in the presence of 10 ⁇ g/ml daratumumab.
• the Y axis shows the percent (%) CDC or ADCC.
• Data show the mean and SEM of at least three experiments. P-values between the indicated groups were calculated using a paired student's t test.
• Dara daratumumab; * p ⁇ 0.05; ns: not significant.
• Figure 4A shows that pretreatment of primary MM cells for 48 hours with 10 nM ATRA potentiates daratumumab-mediated CDC of the primary MM cells.
• MM cells were pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 0-10 ⁇ g/ml.
• the graph shows pooled results of 16 patient samples. *** p ⁇ 0.001, **** p ⁇ 0.0001.
• DARA daratumumab.
• Figure 4B shows that pretreatment of primary MM cells for 48 hours with 10 nM ATRA potentiates daratumumab-mediated ADCC of the primary MM cells.
• MM cells were pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 0-10 ⁇ g/ml.
• the graph shows pooled results of 13 patient samples. * p ⁇ 0.05.
• DARA daratumumab.
• Figure 5A shows the results of in vitro CDC of primary MM cells isolated from patient 1 and patient 2 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 5B shows the results of in vitro CDC of primary MM cells isolated from patient 3 and patient 4 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 5C shows the results of in vitro CDC of primary MM cells isolated from patient 5 and patient 6 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 5D shows the results of in vitro CDC of primary MM cells isolated from patient 7 and patient 8 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 5E shows the results of in vitro CDC of primary MM cells isolated from patient 9 and patient 10 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 5F shows the results of in vitro CDC of primary MM cells isolated from patient 11 and patient 12 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 5G shows the results of in vitro CDC of primary MM cells isolated from patient 13 and patient 14 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 5H shows the results of in vitro CDC of primary MM cells isolated from patient 15 and patient 16 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 6A shows the results of in vitro ADCC of primary MM cells isolated from patient 3 and patient 4 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 6B shows the results of in vitro ADCC of primary MM cells isolated from patient 7 and patient 8 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 6C shows the results of in vitro ADCC of primary MM cells isolated from patient 9 and patient 10 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 6D shows the results of in vitro ADCC of primary MM cells isolated from patient 14 and patient 15 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 6E shows the results of in vitro ADCC of primary MM cells isolated from patient 16 and patient 17 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ / ⁇ 1.
• Figure 6F shows the results of in vitro ADCC of primary MM cells isolated from patient 18 pretreated for 48 hours with or without 10 nM ATRA as indicated in the Figure at daratumumab concentrations ranging from 1-10 ⁇ g/ml.
• Figure 7 shows CD38 expression levels in BM-MNCs isolated from MM patients before and after incubation of cells with (black bars) or without (white bars) in the presence of 10 nM ATRA.
• the same patient samples were used in ADCC and CDC assays as shown in Figures 4A, 4B, 5 and 6.
• Figure 8A shows ATRA-induced reduction of CD55, CD59 and CD46 expression on RPMI8226 cells after 48 hour incubation of cells with 0- 25 nM ATRA.
• MFI mean fluorescent intensity.
• Expression of CD55, CD59 and CD46 were assessed using flow cytometry.
• Figure 8B shows ATRA-induced reduction of CD55, CD59 and CD46 expression on UM9 cells after 48 hour incubation of cells with 0- 25 nM ATRA.
• MFI mean fluorescent intensity.
• Expression of CD55, CD59 and CD46 were assessed using flow cytometry.
• Figure 8C shows ATRA-induced reduction of CD55, CD59 and CD46 expression on XGl cells after 48 hour incubation of cells with 0- 25 nM ATRA.
• MFI mean fluorescent intensity.
• Expression of CD55, CD59 and CD46 were assessed using flow cytometry.
• Figure 9C shows effect of ATRA on CD46 expression on primary MM cells after 48 hour incubation of cells with (grey bars) or without (black bars) in 10 nM ATRA as indicated, ns: not significant.
• FigurelOA shows CD55 expression on primary MM cells isolated from 16 MM patients after 48 hour incubation of cells with (black bars) or without (white bars) 10 nM ATRA. The same patient samples were used in CDC assays as shown in Figure 5.
• Figure 10B shows CD59 expression on primary MM cells isolated from 16 MM patients after 48 hour incubation of cells with (black bars) or without (white bars) 10 nM ATRA. The same patient samples were used in CDC assays as shown in Figure 5.
• Figure IOC shows CD46 expression on primary MM cells isolated from 16 MM patients after 48 hour incubation of cells with (black bars) or without (white bars) 10 nM ATRA. The same patient samples were used in CDC assays as shown in Figure 5.
• FIG 11 shows that ATRA improves response to daratumumab in a humanized multiple myeloma mouse model.
• Rag2 ⁇ ⁇ Y c ⁇ ⁇ mice carrying mesenchymal stem cell (MSC)-coated scaffolds were inoculated with luciferase-transduced XGl cells.
• Mice were treated with control, ATRA plus T-cell depleted PBMCs as effector cells (PBMC-T), daratumumab plus PBMC-T, or daratumumab plus ATRA plus PBMC-T, and monitored weekly by bioluminescent imaging (BLI) for growth of the transduced XGl cells.
• the Figure shows tumor load per treatment group with 4 mice per group and each mouse with 4 scaffolds.
• Statistical differences between mice treated with daratumumab and mice treated with daratumumab plus ATRA were calculated using the Mann- Whitney U-test. * P ⁇ 0.05, ** PO.01, *** PO.001 ;
• CD38 refers to the human CD38 protein (synonyms: ADP-ribosyl cyclase 1, cADPr hydrolase 1, Cyclic ADP-ribose hydrolase 1). Human CD38 has the amino acid sequence shown in SEQ ID NO: 1
• Antibodies as used herein is meant in a broad sense and includes
• immunoglobulin molecules including, monoclonal antibodies including murine, human, human-adapted, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, and single chain antibodies.
• Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
• IgA and IgG are further sub-classified as the isotypes IgAi, IgA 2 , IgGi, IgG 2 , IgG 3 and IgG 4 .
• Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
• Antibody fragments refers to a portion of an immunoglobulin molecule that retains the heavy chain and/or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL).
• HCDR heavy chain complementarity determining regions
• LCDR light chain complementarity determining regions
• VH heavy chain variable region
• VL light chain variable region
• Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, a F(ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a domain antibody (dAb) (Ward et ah, Nature 341 :544- 546, 1989), which consists of a VH domain.
• VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH/VL domains pair intramolecularly, or
• isolated antibody refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an antibody that specifically binds CD38).
• An isolated antibody that specifically binds CD38 may have cross-reactivity to other antigens, such as orthologs of human CD38 such as Macaca fascicularis (cynomolgus) CD38.
• an isolated antibody may be substantially free of other cellular material and/or chemicals.
• An antibody variable region consists of a "framework" region interrupted by three "antigen binding sites".
• the antigen binding sites are defined using various terms:
• Complementarity Determining Regions three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat J Exp Med 132:211-50, 1970; Kabat et al Sequences of Proteins of Immunological Interest, 5th Ed.
• HVR HVR
• HV three in the VH (HI, H2, H3) and three in the VL (LI, L2, L3) refer to the regions of an antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk Mol Biol 196:901-17, 1987).
• Other terms include “IMGT-CDRs” (Lefranc et al, Dev Comparat Immunol 27:55-77, 2003) and “Specificity Determining Residue Usage” (SDRU) (Almagro, Mol Recognit 17:132-43, 2004).
• IMGT International ImMunoGeneTics
• Chothia residues as used herein are the antibody VL and VH residues numbered according to Al-Lazikani (Al-Lazikani et al, J Mol Biol 273:927-48, 1997).
• Framework or “framework sequences” are the remaining sequences of a variable region other than those defined to be antigen binding sites.
• Humanized antibody refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework so that the framework may not be an exact copy of expressed human immunoglobulin or germline gene sequences.
• Human-adapted antibodies or “human framework adapted (HFA)” antibodies refers to humanized antibodies adapted according to methods described in U.S. Pat. Publ. No. US2009/0118127. Human-adapted antibodies are humanized by selecting the acceptor human frameworks based on the maximum CDR and FR similarities, length compatibilities and sequence similarities of CDR1 and CDR2 loops and a portion of light chain CDR3 loops.
• Human antibody refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin.
• a human antibody comprises heavy or light chain variable regions that are "derived from" sequences of human origin where the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin loci as described herein.
• a human antibody may contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to for example naturally occurring somatic mutations or intentional introduction of substitutions in the framework or antigen binding sites.
• a human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene.
• a human antibody may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al, J Mol Biol 296:57-86, 2000), or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al, J Mol Biol 397:385-96, 2010 and Intl. Pat. Publ. No.
• Antibodies in which antigen binding sites are derived from a non- human species are not included in the definition of human antibody.
• Isolated humanized antibodies may be synthetic. Human antibodies may be generated using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties.
• Recombinant antibody includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse or a rat) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, or antibodies that are generated in vitro using Fab arm exchange such as bispecific antibodies.
• Monoclonal antibody refers to a preparation of antibody molecules of single molecular composition.
• a monoclonal antibody composition displays a single binding specificity via its VH, VL and/or VH/VL pair and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes.
• Epitope as used herein means a portion of an antigen to which an antibody specifically binds.
• Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
• An epitope may be composed of contiguous and/or noncontiguous amino acids that form a conformational spatial unit. For a noncontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule.
• Variant refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitution, insertion or deletion.
• “In combination with” as used herein means that two or more therapeutics maybe administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order.
• treat refers to therapeutic treatment wherein the object is to slow down (lessen) an undesired physiological change or disease, or provide a beneficial or desired clinical outcome during treatment, such as the development, growth or spread of tumor or tumor cells.
• beneficial or desired clinical outcomes include alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
• Treatment can also mean prolonging survival as compared to expected survival if a subject was not receiving treatment.
• Those in need of treatment include those subjects already with the undesired physiological change or diseaseas well as those subjects prone to have the physiological change or disease.
• “Inhibits growth” refers to a measurable decrease in the cell growth in vitro or in vivo when contacted with a therapeutic or a combination of therapeutics or drugs when compared to the growth of the same cells grown in appropriate control conditions well known to the skilled in the art. Inhibition of growth of a cell 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 may occur by a variety of mechanisms, for example by antibody-dependent cell-mediated toxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), apoptosis, necrosis, or inhibition of cell proliferation.
• a "therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
• a therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual.
• Exemplary indicators of an effective therapeutic or combination of therapeutics that include, for example, improved well-being of the patient, reduction of a tumor burden, arrested or slowed growth of a tumor, and/or absence of metastasis of cancer cells to other locations in the body.
• the invention provides methods for treating patients having CD38-positive hematological malignancy with the combination of a CD38 antibody and all-trans retinoic acid (ATRA).
• ATRA all-trans retinoic acid
• the invention is based, at least in part, on the discovery that ATRA augments anti-CD38 antibody daratumumab-mediated lysis by ADCC and/or CDC of primary MM cells expressing low, intermediate or high levels of CD38 by enhancing CD38 expression on MM cells.
• ATRA is also able to induce daratumumab-mediated ADCC and/or CDC in primary MM samples which were resistant to daratumumab- mediated CDC and/or ADCC in vitro or were obtained from heavily pretreated multiple myeloma patients having double-refractory (lenalidomide- and bortezomib-refractory) disease.
• ATRA augmented daratumumab-mediated CDC to a higher extent than ADCC, which may be explained by the findings that ATRA also down-regulates complement- inhibitory proteins CD55 and CD59.
• ATRA (CAS 302-79-4) has a well-known molecular structure.
• One embodiment of the invention disclosed herein is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject in need thereof an anti- CD38 antibody in combination with all-trans retinoic acid (ATRA).
• ATRA all-trans retinoic acid
• One embodiment of the invention disclosed herein is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject in need thereof an anti- CD38 antibody in combination with all-trans retinoic acid (ATRA), wherein the anti- CD38 antibody induces killing of CD38-expressing cells in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
• ADCC antibody-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• the methods of the invention may be used to treat an animal subject belonging to any classification.
• animals include mammals such as humans, rodents, dogs, cats and farm animals.
• the anti-CD38 antibody induces killing of the CD38-expressing cells by CDC in vitro.
• CD38-positive hematological malignancy refers to a hematological malignancy characterized by the presence of tumor cells expressing CD38 including leukemias, lymphomas and myeloma.
• Examples of such CD38-positive hematological malignancies are precursor B-cell lymphoblastic leukemia/lymphoma and B-cell non-Hodgkin's lymphoma, acute promyelocytic leukemia, acute lymphoblastic leukemia and mature B- cell neoplasms, such as B-cell chronic lymphocytic leukemia(CLL)/small lymphocytic lymphoma (SLL), B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate- grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B
• lymphoproliferative disorder Waldenstrom's macroglobulinemia, plasma cell leukemias and anaplastic large-cell lymphoma (ALCL).
• ACL anaplastic large-cell lymphoma
• CD38 is expressed in a variety of malignant hematological diseases, including multiple myeloma, leukemias and lymphomas, such as B-cell chronic lymphocytic leukemia, T- and B-cell acute lymphocytic leukemia, Waldenstrom macroglobulinemia, primary systemic amyloidosis, mantle-cell lymphoma, pro-lymphocytic/myelocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, follicular lymphoma, Burkitt's lymphoma, large granular lymphocytic (LGL) leukemia, NK-cell leukemia and plasma-cell leukemia.
• multiple myeloma such as B-cell chronic lymphocytic leukemia, T- and B-cell acute lymphocytic leukemia, Waldenstrom macroglobulinemia, primary systemic amyloidosis, mantle-cell lymphoma, pro-lymphocy
• CD38 has been described on epithelial/endothelial cells of different origin, including glandular epithelium in prostate, islet cells in pancreas, ductal epithelium in glands, including parotid gland, bronchial epithelial cells, cells in testis and ovary and tumor epithelium in colorectal adenocarcinoma.
• CD38 expression could be involved, include, e.g., broncho-epithelial carcinomas of the lung, breast cancer (evolving from malignant proliferation of epithelial lining in ducts and lobules of the breast), pancreatic tumors, evolving from the ⁇ -cells (insulinomas), tumors evolving from epithelium in the gut (e.g. adenocarcinoma and squamous cell carcinoma), carcinoma in the prostate gland, and seminomas in testis and ovarian cancers.
• neuroblastomas express CD38.
• the CD38-positive hematological malignancy is multiple myeloma.
• the CD38-positive hematological malignancy is diffuse large B-cell lymphoma (DLBCL).
• DLBCL diffuse large B-cell lymphoma
• the CD38-positive hematological malignancy is non-Hodgkin's lymphoma.
• the CD38-positive hematological malignancy is acute lymphoblastic leukemia (ALL).
• ALL acute lymphoblastic leukemia
• the CD38-positive hematological malignancy is follicular lymphoma (FL).
• the CD38-positive hematological malignancy is Burkitt's lymphoma (BL).
• the CD38-positive hematological malignancy is mantle cell lymphoma (MCL).
• the CD38-positive hematological malignancy is multiple myeloma, acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular lymphoma (FL) or mantle-cell lymphoma (MCL).
• ALL acute lymphoblastic leukemia
• NHL non-Hodgkin's lymphoma
• DLBCL diffuse large B-cell lymphoma
• BL Burkitt's lymphoma
• FL follicular lymphoma
• MCL mantle-cell lymphoma
• B-cell non-Hodgkin's lymphomas are lymphomatoid granulomatosis, primary effusion lymphoma, intravascular large B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain diseases (including ⁇ , ⁇ , and a disease), lymphomas induced by therapy with immunosuppressive agents, such as cyclosporine-induced lymphoma, and methotrexate-induced lymphoma.
• the disorder involving cells expressing CD38 is Hodgkin's lymphoma.
• disorders involving cells expressing CD38 include malignancies derived from T and NK cells including mature T cell and NK cell neoplasms including T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T-cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, blastic NK cell lymphoma, Mycosis Fungoides/Sezary Syndrome, primary cutaneous CD30 positive T-cell lymphoproliferative disorders (primary cutaneous anaplastic large cell lymphoma C- ALCL, lymphomatoid papulosis,
• malignancies derived from myeloid cells include acute myeloid leukemia, including acute promyelocytic leukemia, and chronic myeloproliferative diseases, including chronic myeloid leukemia.
• Any anti-CD38 antibody may be used in the methods of the invention as disclosed herein, including in the numbered embodiments listed below.
• the anti-CD38 antibody induces in vitro killing of CD38- expressing cells by antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC).
• ADCC antibody-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• variable regions of the anti-CD38 antibodies may be obtained from existing anti-CD38 antibodies, and cloned as full length antibodies or into various antibody formats and fragments using standard methods. Exemplary variable regions binding CD38 that may be used are described in Intl. Pat. Publ. Nos. WO05/103083, WO06/125640, WO07/042309, WO08/047242, WO12/092612, WO06/099875 and WOl 1/154453A1.
• An exemplary anti-CD38 antibody that may be used is daratumumab.
• Daratumumab comprises the heavy chain variable region (VH) and the light chain variable region (VL) amino acid sequences shown in SEQ ID NO: 4 and 5, respectively, heavy chain CDRs HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 6, 7 and 8, respectively, and light chain CDRs LCDR1 , LCDR2 and LCDR3 of SEQ ID NOs: 9, 10 and 11 , respectively, and is of IgGl/ ⁇ subtype and described in U.S. Pat. No. 7,829,693.
• Daratumumab heavy chain amino acid sequence is shown in SEQ ID NO: 12 and light chain amino acid sequence shown in SEQ ID NO: 13.
• Another exemplary anti-CD38 antibody that may be used is mAb003 comprising the VH and VL sequences of SEQ ID NOs: 14 and 15, respectively and described in U.S. Pat. No. 7,829,693.
• the VH and the VL of mAb003 may be expressed as IgGl/ ⁇ .
• mAb024 comprising the VH and VL sequences of SEQ ID NOs: 16 and 17, respectively, described in U.S. Pat. No. 7,829,693.
• the VH and the VL of mAb024 may be expressed as IgGl/ ⁇ .
• MOR-202 (MOR- 03087) comprising the VH and VL sequences of SEQ ID NOs: 18 and 19, respectively, described in US. Pat. No. 8,088,896.
• the VH and the VL of MOR-202 may be expressed as IgGl/ ⁇ .
• Isatuximab comprising the VH and VL sequences of SEQ ID NOs: 20 and 21, respectively, described in U.S. Pat. No. 8,153,765.
• the VH and the VL of Isatuximab may be expressed as IgGl/ ⁇ .
• anti-CD38 antibodies that may be used in the methods of the invention include those described in Int. Pat. Publ. No. WO05/103083, Intl. Pat. Publ. No. WO06/125640, Intl. Pat. Publ. No. WO07/042309, Intl. Pat. Publ. No. WO08/047242 or Intl. Pat. Publ. No. WO14/178820.
• Anti-CD38 antibodies used in the methods of the invention disclosed herein, including in the numbered embodiments listed below, may also be selected de novo from a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et ah, J Mol Biol 296:57-86, 2000; Krebs et ah, J Immunol Meth 254:67-84, 2001 ; Vaughan et ah, Nature Biotechnology 14:309-314, 1996; Sheets et ah, PITAS (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J Mol Biol 227:381, 1991 ; Marks et ah, J Mol Biol 222:581, 1991).
• human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody
• CD38 binding variable domains may be isolated from for example phage display libraries expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et ah, J. Mol. Biol. 397:385-96, 2010 and PCT Intl. Publ. No.
• the antibody libraries may be screened for binding to human CD38 extracellular domain, obtained positive clones further characterized, Fabs isolated from the clone lysates, and subsequently cloned as full length antibodies.
• phage display methods for isolating human antibodies are established in the art. See for example: US Pat. No. 5,223,409; US Pat. No. 5,403,484; and US Pat. No. 5,571,698, US Pat. No. 5,427,908, US Pat. No. 5,580,717, US Pat. No. 5,969,108, US Pat. No. 6,172,197, US Pat. No. 5,885,793; US Pat. No. 6,521,404; US Pat. No. 6,544,731 ; US Pat. No. 6,555,313; US Pat. No. 6,582,915; and US Pat. No. 6,593,081.
• the Fc portion of the antibody may mediate antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement dependent cytotoxicity (CDC).
• ADCC antibody-dependent cell-mediated cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• CDC complement dependent cytotoxicity
• Such functions may be mediated by binding of an Fc effector domain(s) to an Fc receptor on an immune cell with phagocytic or lytic activity or by binding of an Fc effector domain(s) to components of the complement system.
• the effect(s) mediated by the Fc-binding cells or complement components result in inhibition and/or depletion of target cells, e.g., CD38- expressing cells.
• ADCC may be mediated by IgGl and IgG3
• ADCP may be mediated by IgGl, IgG2, IgG3 and IgG4
• CDC may be mediated by IgGl and IgG3.
• the anti-CD38 antibody is of IgGl, IgG2, IgG3 or IgG4 isotype.
• the anti-CD38 antibody is of IgGl or IgG3 isotype.
• the anti-CD38 antibody induces in vitro killing of CD38 -expressing cells by ADCC.
• the anti-CD38 antibody induces in vitro killing of CD38-expressing cells by CDC.
• the anti-CD38 antibody induces killing of CD38-expressing cells by ADCC and CDC in vitro.
• Antibody-dependent cellular cytotoxicity or “antibody-dependent cell-mediated cytotoxicity” or “ADCC” is a mechanism for inducing cell death that depends upon the interaction of antibody-coated target cells with effector cells possessing lytic activity, such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (FcyR) expressed on effector cells.
• effector cells possessing lytic activity such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (FcyR) expressed on effector cells.
• FcyR Fc gamma receptors
• NK cells express FcyRIIIa
• monocytes express FcyRI, FcyRII and FcyRIIIa.
• Death of the antibody-coated target cell such as CD38-expressing cells, occurs as a result of effector cell activity through the secretion of membrane pore-forming proteins and proteases.
• the antibody may be added to CD38-expressing cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis is generally detected by the release of label (e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells.
• label e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins
• primary BM-MNC cells isolated from a patient with a B-cell malignancy such as MM may be used for the assay.
• BM-MNCs may be treated with an anti-CD38 antibody for 1 hour at a concentration of 0.3-10 ⁇ g/ml, and the survival of primary CD138 + MM cells may be determined by flow cytometry using techniques described in van der Veer et ah, Haematologica 96:284- 290, 2001 or in van der Veer et ah, Blood Cancer J l(10):e41, 2011. The percentage of MM cell lysis may be determined relative to an isotype control as described herein.
• Anti- CD38 antibodies used in the methods of the invention may induce ADCC by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of control.
• “Complement-dependent cytotoxicity”, or “CDC” refers to a mechanism for inducing cell death in which an Fc effector domain of a target-bound antibody binds and activates complement component Clq which in turn activates the complement cascade leading to target cell death. Activation of complement may also result in deposition of complement components on the target cell surface that facilitate ADCC by binding complement receptors (e.g., CR3) on leukocytes.
• complement receptors e.g., CR3
• primary BM- MNC cells isolated from a patient with a B-cell malignancy may be treated with an anti- CD38 antibody and complement derived from 10% pooled human serum for 1 hour at a concentration of 0.3-10 ⁇ */ ⁇ 1, and the survival of primary CD138 + MM cells may be determined by flow cytometry using techniques described in van der Veer et ah, Haematologica 96:284-290, 2011; van der Veer et ah, Blood Cancer J l(10):e41, 2011. The percentage of MM cell lysis may be determined relative to an isotype control as described herein.
• Anti-CD38 antibodies used in the methods of the invention may induce CDC by 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 may be enhanced by engineering their oligosaccharide component.
• Human IgGl or IgG3 are N-glycosylated at Asn297 with the majority of the glycans in the well-known biantennary GO, G0F, Gl, GIF, G2 or G2F forms.
• Antibodies produced by non-engineered CHO cells typically have a glycan fucose content of about at least 85%. The removal of the core fucose from the biantennary complex-type oligosaccharides attached to the Fc regions enhances the ADCC of antibodies via improved FcyRIIIa binding without altering antigen binding or CDC activity.
• Such antibodies may be achieved using different methods reported to lead to the expression of relatively high defucosylated antibodies bearing the biantennary complex- type of Fc oligosaccharides such as control of culture osmolality (Konno et ah,
• ADCC elicited by anti-CD38 antibodies used in the methods of the invention may also be enhanced by certain substitutions in the antibody Fc.
• Exemplary substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residue numbering according to the EU index) as described in U.S. Pat. No.
• CDC elicited by anti-CD38 antibodies used in the methods of the invention may also be enhanced by certain substitutions in the antibody Fc.
• Exemplary substitutions are, for example, substitutions at amino acid positions 423, 268, 267 and/or 113 (residue numbering according to the EU index) as described in Moore et al., Mabs 2: 181-189, 2010.
• the anti-CD38 antibodies comprise a substitution in the antibody Fc.
• the anti-CD38 antibodies comprise a substitution in the antibody Fc at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 and/or 430 (residue numbering according to the EU index).
• the anti-CD38 antibodies comprise a substitution in the antibody Fc at amino acid position 1 13, 267, 268 and/or 423 (residue numbering according to the EU index).
• Another embodiment of the invention is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 antibody in combination with all-trans retinoic acid (ATRA), wherein the anti-CD38 antibody competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5 (daratumumab).
• ATRA all-trans retinoic acid
• Another embodiment of the invention is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 antibody in combination with all-trans retinoic acid (ATRA), wherein the anti-CD38 antibody induces killing of CD38-expressing cells in vitro by antibody-dependent cell- mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), wherein the anti-CD38 antibody competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5 (daratumumab).
• ATRA all-trans retinoic acid
• ADCC antibody-dependent cell- mediated cytotoxicity
• CDC complement dependent cytotoxicity
• Antibodies may be evaluated for their competition with daratumumab having the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for binding to CD38 using well known in vitro methods.
• CHO cells recombinantly expressing CD38 may be incubated with unlabeled daratumumab for 15 min at 4°C, followed by incubation with an excess of fluorescently labeled test antibody for 45 min at 4°C. After washing in PBS/BSA, fluorescence may be measured by flow cytometry using standard methods.
• extracellular portion of human CD38 may be coated on the surface of an ELISA plate.
• Excess of unlabeled daratumumab may be added for about 15 minutes and subsequently biotinylated test antibodies may be added. After washes in PBS/Tween, binding of the test biotinylated antibodies may be detected using horseradish peroxidase (HRP)-conjugated streptavidine and the signal detected using standard methods. It is readily apparent that in the competition assays, daratumumab may be labelled and the test antibody unlabeled. The test antibody competes with daratumumab when daratumumab inhibits binding of the test antibody, or the test antibody inhibits binding of daratumumab by 80%, 85%, 90% , 95% or 100%.
• HRP horseradish peroxidase
• the epitope of the test antibody can further be defined, for example, by peptide mapping or hydrogen/deuterium protection assays using known methods.
• Another embodiment of the invention disclosed herein, including in the numbered embodiments listed below, is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject in need thereof an anti- CD38 antibody that binds to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1) in combination with all-trans retinoic acid (ATRA).
• Another embodiment of the invention disclosed herein is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject in need thereof an anti- CD38 antibody that binds to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1) in combination with all-trans retinoic acid (ATRA), wherein the anti-CD38 antibody induces killing of CD38-expressing cells in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
• ADCC antibody-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• the antibody "binds to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID NO: 3)" when the antibody binds at least one amino acid residue within each region.
• the antibody may bind for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid residues within each region of SEQ ID NO:2 and SEQ ID NO: 3.
• the antibody may also optionally bind one or more residues outside of the regions of SEQ ID NO: 2 and SEQ ID NO: 3. Binding may be assessed by known methods such as mutagenesis studies or by resolving the crystal structure of CD38 in complex with the antibody.
• the antibody epitope comprises at least one amino acid in the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least one amino acid in the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). In some embodiments disclosed herein, including in the numbered embodiments listed below, the antibody epitope comprises 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) of human CD38 (SEQ ID NO: 1).
• the antibody epitope comprises 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) of human CD38 (SEQ ID NO: 1).
• the anti-CD38 antibody binds to an epitope comprising at least KRN in the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and comprising at least VQLT (SEQ ID NO: 22) in the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
• the anti- CD38 antibody binds to an epitope comprising at least KRN in the region
• SKRNIQFSCKNIYR (SEQ ID NO: 2) and comprising at least VQLT (SEQ ID NO: 22) in the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
• An exemplary antibody that binds to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1) or minimally to residues KRN and VQLT (SEQ ID NO: 22) as shown above is daratumumab having certain VH, VL and CDR sequences as described above.
• EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1) may be generated, for example, by immunizing mice with peptides having the amino acid sequences shown in SEQ ID NOs: 2 and 3 using standard methods and as described herein. Antibodies may be further evaluated, for example, by assaying competition between daratumumab and a test antibody for binding to CD38 as described above.
• the anti-CD38 antibody may bind human CD38 with a range of affinities (K D ).
• K D affinities
• the anti-CD38 antibody binds to CD38 with high affinity, for example, with a K D equal to or less than about 10 ⁇ 7 M, such as about 1, 2, 3, 4, 5, 6, 7, 8, or 9 x 10 "8 M, lxl 0 "9 M, about lxlO "10 M, about lxlO "11 M, about lxlO "12 M, about lxlO "13 M, about lxlO "14 M, about lxlO "15 M or any range or value therein, as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art.
• One exemplary affinity is equal to or less than lxlO "8 M
• the anti-CD38 antibody has a biantennary glycan structure with fucose content of about between 0% to about 15%, for example 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%.
• the anti-CD38 antibody has a biantennary glycan structure with 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%
• “Fucose content” refers to the amount of the fucose monosaccharide within the sugar chain at Asn297.
• the relative amount of fucose is the percentage of fucose- containing structures related to all glycostructures.
• Glycostructures may be characterized and quantified by multiple methods, for example: 1) using MALDI-TOF of N-glycosidase F treated sample (e.g. complex, hybrid and oligo- and high-mannose structures) as described in Int. Pat. Publ. No.
• WO2008/077546 2) by enzymatic release of the Asn297 glycans with subsequent derivatization and detection/ quantitation by HPLC (UPLC) with fluorescence detection and/or HPLC -MS (UPLC -MS); 3) intact protein analysis of the native or reduced mAb, with or without treatment of the Asn297 glycans with Endo S or other enzyme that cleaves between the first and the second GlcNAc monosaccharides, leaving the fucose attached to the first GlcNAc; 4) digestion of the mAb to constituent peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), and subsequent separation, detection and quantitation by HPLC -MS (UPLC-MS); or 5) separation of the mAb oligosaccharides from the mAb protein by specific enzymatic deglycosylation with PNGase F at Asn 297.
• UPLC enzy
• the oligosaccharides released may be labeled with a fluorophore, separated and identified by various complementary techniques which allow fine characterization of the glycan structures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry by comparison of the experimental masses with the theoretical masses, determination of the degree of sialylation by ion exchange HPLC (GlycoSep C), separation and quantification of the oligosacharride forms according to hydrophilicity criteria by normal-phase HPLC (GlycoSep N), and separation and quantification of the oligosaccharides by high performance capillary electrophoresis-laser induced fluorescence (HPCE-LIF).
• MALDI matrix-assisted laser desorption ionization
• Low fucose or “low fucose content” as used in the application refers to antibodies with fucose content of about 0%> - 15%.
• Normal fucose or 'normal fucose content
• fucose content refers to antibodies with fucose content of about over 50%o, typically about over 60%>, 70%o, 80%o or over 85%o.
• the anti-CD38 antibody comprises the heavy chain complementarity determining regions (HCDR) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) sequences of SEQ ID NOs: 6, 7 and 8, respectively.
• the anti-CD38 antibody comprises the light chain complementarity determining regions (LCDR) 1 (LCDRl), 2 (LCDR2) and 3 (LCDR3) sequences of SEQ ID NOs: 9, 10 and 11 , respectively.
• the anti-CD38 antibody comprises the heavy chain complementarity determining regions (HCDR) 1 (HCDRl), 2 (HCDR2) and 3 (HCDR3) sequences of SEQ ID NOs: 6, 7 and 8, respectively and the light chain complementarity determining regions (LCDR) 1 (LCDRl), 2 (LCDR2) and 3 (LCDR3) sequences of SEQ ID NOs: 9, 10 and 11, respectively.
• the anti-CD38 antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 4 and the light chain variable region (VL) of SEQ ID NO: 5.
• the anti-CD38 antibody comprises the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO: 13.
• the anti-CD38 antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 14 and the light chain variable region (VL) of SEQ ID NO: 15.
• the anti-CD38 antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO: 17.
• the anti-CD38 antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 18 and the light chain variable region (VL) of SEQ ID NO: 19.
• the anti-CD38 antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 20 and the light chain variable region (VL) of SEQ ID NO: 21.
• the anti-CD38 antibody comprises a heavy chain comprising an amino acid sequence that is 95%, 96%>, 97%>, 98%> or 99% identical to that of SEQ ID NO: 12 and a light chain comprising an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to that of SEQ ID NO: 13.
• Antibodies that are substantially identical to the antibody comprising the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO: 13 may be used in the methods of the invention, and in some embodiments of each and every one of the numbered embodiments listed below.
• the term "substantially identical” as used herein means that the two antibody heavy chain or light chain amino acid sequences being compared are identical or have "insubstantial differences.” Insubstantial differences are substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in an antibody heavy chain or light chain that do not adversely affect antibody properties. Percent identity can be determined for example by pairwise alignment using the default settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, CA).
• the protein sequences of the present invention can be used as a query sequence to perform a search against public or patent databases to, for example, identify related sequences.
• Exemplary programs used to perform such searches are the XBLAST or BLASTP programs (http_//www_ncbi_nlm/nih_gov), or the GenomeQuestTM (GenomeQuest, Westborough, MA) suite using the default settings.
• Exemplary substitutions that can be made to the anti- CD38 antibodies used in the methods of the invention are for example conservative substitutions with an amino acid having similar charge, hydrophobic, or stereochemical characteristics. Conservative substitutions may also be made to improve antibody properties, for example stability or affinity, or to improve antibody effector functions.
• 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions may be made for example to the heavy or the light chain of the anti-CD38 antibody.
• any native residue in the heavy or light chain may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et ah, Acta Physiol Scand Suppl 643:55-67, 1998; Sasaki et al, Adv Biophys 35:1-24, 1998). Desired amino acid substitutions may be determined by those skilled in the art at the time such substitutions are desired. Amino acid substitutions may be done for example by PCR mutagenesis (U.S. Pat. No. 4,683,195).
• variants may be generated using well known methods, for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp) and screening the libraries for variants with desired properties.
• the generated variants may be tested for their binding to CD38, their ability to induce ADCC, ADCP or apoptosis in vitro using methods described herein.
• the anti-CD38 antibody is a bispecific antibody.
• the VL and/or the VH regions of the existing anti-CD38 antibodies or the VL and VH regions identified de novo as described above may be engineered into bispecific full length antibodies.
• Such bispecific antibodies may be made by modulating the CH3 interactions between the two monospecific antibody heavy chains to form bispecific antibodies using technologies such as those described in U.S. Pat. No. 7,695,936; Int. Pat. Publ. No.
• WO2011/143545 or U.S. Pat. Publ. No. US2012/0149876.
• Additional bispecific structures into which the VL and/or the VH regions of the antibodies of the invention can be incorporated are for example Dual Variable Domain Immunoglobulins (Int. Pat. Publ. No. WO2009/134776), or structures that include various dimerization domains to connect the two antibody arms with different specificity, such as leucine zipper or collagen dimerization domains (Int. Pat. Publ. No. WO2012/022811, U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441).
• Another embodiment of the invention is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 antibody in combination with all-trans retinoic acid (ATRA), wherein the CD38-positive hematological malignancy is multiple myeloma (MM), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular lymphoma (FL) or mantle-cell lymphoma (MCL).
• MM multiple myeloma
• ALL acute lymphoblastic leukemia
• NHL non-Hodgkin's lymphoma
• DLBCL diffuse large B-cell lymphoma
• BL Burkitt's lymphoma
• FL follicular lymphoma
• MCL mantle-cell lymphoma
• Another embodiment of the invention is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 antibody in combination with all-trans retinoic acid (ATRA), wherein the anti-CD38 antibody induces killing of CD38-expressing cells in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), wherein the CD38 -positive hematological malignancy is multiple myeloma (MM), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular lymphoma (FL) or mantle-cell lymphoma (MCL).
• ATRA all-trans retinoic acid
• ADCC antibody-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• CD38 -positive hematological malignancy is multiple
• Another embodiment of the invention is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 antibody in combination with all-trans retinoic acid (ATRA), wherein the CD38-positive hematological malignancy is multiple myeloma (MM).
• ATRA all-trans retinoic acid
• Another embodiment of the invention is a method of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 antibody in combination with all-trans retinoic acid (ATRA), wherein the anti-CD38 antibody induces killing of CD38-expressing cells in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), wherein the CD38 -positive hematological malignancy is multiple myeloma (MM).
• ATRA all-trans retinoic acid
• ADCC antibody-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• the invention also provides for a method of treating a subject having a CD38- positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 in combination with all-trans retinoic acid (ATRA), wherein the subject is resistant to or has acquired resistance to treatment with the anti-CD38 antibody.
• ATRA all-trans retinoic acid
• the invention also provides for a method of treating a subject having a CD38- positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 in combination with all-trans retinoic acid (ATRA), wherein the anti-CD38 antibody induces killing of CD38-expressing cells in vitro by antibody- dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), wherein the subject is resistant to or has acquired resistance to treatment with the anti-CD38 antibody.
• ATRA all-trans retinoic acid
• the invention also provides for a method of treating a subject having a CD38- positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 in combination with all-trans retinoic acid (ATRA), wherein the subject is resistant to or has acquired resistance to treatment with at least one chemotherapeutic agent.
• ATRA all-trans retinoic acid
• the invention also provides for a method of treating a subject having a CD38- positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 in combination with all-trans retinoic acid (ATRA), wherein the anti-CD38 antibody induces killing of CD38-expressing cells in vitro by antibody- dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), wherein the subject is resistant to or has acquired resistance to treatment with at least one chemotherapeutic agent.
• ATRA all-trans retinoic acid
• the invention also provides for a method of treating a subject having multiple myeloma, comprising administering to the subject in need thereof an anti-CD38 in combination with all-trans retinoic acid (ATRA), wherein the subject is resistant to or has acquired resistance to treatment with at least one chemotherapeutic agent.
• ATRA all-trans retinoic acid
• the invention also provides for a method of treating a subject having multiple myeloma, comprising administering to the subject in need thereof an anti-CD38 in combination with all-trans retinoic acid (ATRA), wherein the anti-CD38 antibody induces killing of CD38-expressing cells in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), wherein the subject is resistant to or has acquired resistance to treatment with at least one chemotherapeutic agent.
• ATRA all-trans retinoic acid
• ADCC antibody-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• the subject is resistant to or has acquired resistance to treatment with at least one chemotherapeutic agent, wherein the at least one chemotherapeutic agent is lenalidomide, bortezomib, melphalan, dexamethasone or thalidomide.
• the subject is resistant to or has acquired resistance to treatment with at least one chemotherapeutic agent, wherein the at least one chemotherapeutic agent is lenalidomide, bortezomib, melphalan, dexamethasone, thalidomide, cyclophosphamide,
• doxorubicin hydroxydaunorubicin (doxorubicin), vincristine or prednisone.
• the subject is resistant to or has acquired resistance to treatment with at least one chemotherapeutic agent, wherein the at least one chemotherapeutic agent is lenalidomide and/or bortezomib.
• Symptoms that may be associated with resistance include, for example, a decline or plateau of the well-being of the patient, an increase in the size of a tumor, increase in the number of cancer cells, arrested or slowed decline in growth of a tumor or tumor cells, and/or the spread of cancerous cells in the body from one location to other organs, tissues or cells.
• Re-establishment or worsening of various symptoms associated with tumor may also be an indication that a subject has developed or is susceptible to developing resistance to an anti-CD38 antibody or other therapeutic agent.
• the symptoms associated with cancer may vary according to the type of cancer.
• symptoms associated with B-cell malignancies may include swollen lymph nodes in neck, groin or armpits, fever, night sweats, coughing, chest pain, unexplained weight loss, abdominal swelling or pain, or a feeling of fullness.
• Remission in malignant lymphomas is standardized using the Cheson criteria (Cheson et ah, J Clin Oncology 25:579-586, 2007), which guidelines can be used to determine if a subject has developed a resistance to an anti-CD38 antibody or other therapeutic agent.
• the subject having a CD38 -positive hematological malignancy is homozygous for phenylalanine at position 158 of CD 16 (FcyRIIIa-158F/F genotype) or heterozygous for valine and pheynylalanine at position 158 of CD16 (FcyRIIIa-158F/V genotype).
• CD16 is also known as the Fc gamma receptor Ilia (FcyRIIIa) or the low affinity immunoglobulin gamma Fc region receptor III -A isoform.
• Valine/phenylalanine (V/F) polymorphism at FcyRIIIa protein residue position 158 has been shown to affect FcyRIIIa affinity to human IgG.
• Receptor with FcyRIIIa-158F/F or FcyRIIIa-158F/V polymorphisms demonstrates reduced Fc engagement and therefore reduced ADCC when compared to the FcyRIIIa- 158V/V.
• the lack of or low amount of fucose on human N-linked oligosaccharides improves the ability of the antibodies to induce ADCC due to improved binding of the antibodies to human FcyRIIIa (CD16) (Shields et al, J Biol Chem 277:26733-40, 2002).
• Patients can be analyzed for their FcyRIIIa polymorphism using routine methods.
• the invention also provides for the method of treating a subject having a CD38- positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 in combination with all-trans retinoic acid (ATRA), wherein the subject is homozygous for phenylalanine at position 158 of CD 16 or heterozygous for valine and pheynylalanine at position 158 of CD 16.
• ATRA all-trans retinoic acid
• the invention also provides for the method of treating a subject having a CD38- positive hematological malignancy, comprising administering to the subject in need thereof an anti-CD38 in combination with all-trans retinoic acid (ATRA), wherein the anti-CD38 antibody induces killing of CD38-expressing cells in vitro by antibody- dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), wherein the subject is homozygous for phenylalanine at position 158 of CD 16 or heterozygous for valine and pheynylalanine at position 158 of CD 16.
• ATRA all-trans retinoic acid
• ADCC antibody- dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• the anti-CD38 antibodies may be provided in suitable pharmaceutical compositions comprising the anti-CD38 antibody and a pharmaceutically acceptable carrier.
• the carrier may be diluent, adjuvant, excipient, or vehicle with which the anti-CD38 antibody is administered.
• vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration).
• compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc.
• concentration of the molecules or antibodies of the invention in such pharmaceutical formulation may vary widely, i.e., from less than about 0.5%, usually to at least about 1 % to as much as 15 or 20%> by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected.
• Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.
• the mode of administration of the anti-CD38 antibody in the methods of the invention may be any suitable route such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal) or other means appreciated by the skilled artisan, as well known in the art.
• parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal) or other means appreciated by the skilled artisan, as well known in the art.
• the anti-CD38 antibody in the methods of the invention may be administered to a patient by any suitable route, for example parentally by intravenous (i.v.) infusion or bolus injection, intramuscularly or subcutaneously or intraperitoneally.
• i.v. infusion may be given over for, example, 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.
• the dose given to a patient having a CD38-positive hematological malignancy is sufficient to alleviate or at least partially arrest the disease being treated ("therapeutically effective amount") and may be sometimes 0.005 mg/kg to about 100 mg/kg, e.g.
• a fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 500, 400, 300, 250, 200, or 100 mg/m 2 .
• 1 and 8 doses e.g., 1, 2, 3, 4, 5, 6, 7 or 8
• the administration of the anti-CD38 antibody in the methods of the invention and in some embodiments of each and every one of the numbered embodiments listed below, 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 longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose.
• the anti-CD38 antibody in the methods of the invention may be administered at 8 mg/kg or at 16 mg/kg at weekly interval for 8 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every two weeks for an additional 16 weeks, followed by administration at 8 mg/ kg or at 16 mg/kg every four weeks by intravenous infusion.
• the anti-CD38 antibodies may be administered in the methods of the invention and in some embodiments of each and every one of the numbered embodiments listed below, by maintenance therapy, such as, e.g ., once a week for a period of 6 months or more.
• anti-CD38 antibodies in the methods of the invention and in some embodiments of each and every one of the numbered embodiments listed below may be provided as a daily dosage in an amount 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 day 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 week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
• Anti-CD38 antibodies in the methods of the invention and in some embodiments of each and every one of the numbered embodiments listed below, may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission. This may be especially useful in patients wherein it is difficult to locate a tumor that is known to be present due to other biological factors.
• the anti-CD38 antibody in the methods of the invention and in some
• the anti-CD38 antibody in the methods of the invention and in some
• embodiments of each and every one of the numbered embodiments listed below may be administered in combination with all-trans retinoic acid (ATRA).
• ATRA all-trans retinoic acid
• ATRA may be provided as a dosage of 45 mg/m 2 /day PO or 25 mg/m 2 /day PO.
• the anti-CD38 antibody in the methods of the invention and in some
• embodiments of each and every one of the numbered embodiments listed below may be administered in combination with all-trans retinoic acid (ATRA) and a third therapeutic agent.
• ATRA all-trans retinoic acid
• the third therapeutic agent may be melphalan, mechlorethamine, thioepa, chlorambucil, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin, thalidomide or a thalidomide analog, lenalidomide or CC4047, a proteasome inhibitor, such as bortezomib or vinca alkaloid, such as vincristine or an anthracycline, such as doxorubicin.
• An anti-CD38 antibody for use in treating a subject having a CD38-positive hematological malignancy, in combination with all-trans retinoic acid (ATRA).
• ATRA for use in treating a subject having a CD38-positive hematological
• ADCC antibody-dependent cell-mediated cytotoxicity
• MM multiple myeloma
• ALL acute lymphoblastic leukemia
• NHL non-Hodgkin's lymphoma
• DLBCL diffuse large B-cell lymphoma
• BL Burkitt's lymphoma
• FL follicular lymphoma
• MCL mantle-cell lymphoma
• anti-CD38 antibody for use according to embodiment 1, 4-9, ATRA for use according to embodiment 2, 4-9, or the combination for use according to embodiment 3-9, wherein the at least one chemotherapeutic agent is lenalidomide or bortezomib.
• anti-CD38 antibody for use according to embodiment 1, 4-10, ATRA for use according to embodiment 2, 4-10, or the combination for use according to embodiment 3-10, wherein
• the anti-CD38 antibody is of IgGl, IgG2, IgG3 or IgG4 isotype
• the anti-CD38 antibody competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5;
• the anti-CD38 antibody binds to the region SKRNIQF SCKNI YR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1);
• the anti-CD38 antibody comprises the heavy chain complementarity determining regions (HCDR) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) sequences of SEQ ID NOs: 6, 7 and 8, respectively;
• the anti-CD38 antibody comprises the light chain complementarity
• LCDR LCDR 1
• LCDR2 LCDR2
• LCDR3 LCDR3
• the anti-CD38 antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 4 and the light chain variable region (VL) of SEQ ID NO: 5;
• the anti-CD38 antibody comprises a heavy chain comprising an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to that of SEQ ID NO: 12 and a light chain comprising an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to that of SEQ ID NO: 13; h. the anti-CD38 antibody comprises the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO: 13 i. the anti-CD38 antibody comprises th VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15;.
• the anti-CD38 antibody comprises th VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17;
• the anti-CD38 antibody comprises th VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19; or
• the anti-CD38 antibody comprises th VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
• a human mAb against an innocuous antigen (HIV-1 gpl20) was used as an isotype control as described previously (van der Veers et al, Haematologica 96:284-290, 2011; van der Veers et al, Blood Cancer J l :e41, 2011). All-trans retinoic acid (ATRA) was purchased from Sigma- Aldrich and diluted in DMSO.
• LUC-transduced MM cell lines were co-cultured with effector cells (freshly isolated PBMCs from healthy donors) at an effector to target ratio of 1 :25 in white opaque 96-well flat bottom plates (Costar) in the presence of daratumumab (0.001, 0.01, 0.1, and 1.0 ⁇ g/mL) for four hours.
• BM-MNCs Freshly isolated BM-MNCs, containing 2-57% malignant plasma cells as determined by flow cytometry, were immediately used in ex vivo experiments.
• BM-MNCs containing the malignant plasma cells, as well as the patient's own effector cells, were incubated in RPMI + 10% fetal bovine serum with daratumumab (0.01-10 ⁇ g/mL) in 96-well flat-bottom plates in fully humidified incubators at 37°C, 5% C02-air mixture for 48 h. Sample viability at incubation was more than 98%, as assessed by using ToPro-3 (Invitrogen/Molecular Probes).
• BM-MNCs were treated with daratumumab (0.3-10 ⁇ g/mL) and complement for 1 hour prior to flow cytometric analysis. Pooled human serum (10%) was used as a source of complement. The survival of primary CD138 + MM cells in the BM-MNCs was determined by flow- cytometry as previously described (van der Veers et al., Haematologica 96:284-290, 201 1 ; van der Veers et al., Blood Cancer J 1 :e41, 201 1).
• MM cells were enumerated by single platform flow-cytometric analysis of CD138 + cells (with CD138-PE (Beckman Coulter, Miami, FL, USA)) in the presence of Flow-Count Fluorospheres (Beckman Coulter) to determine absolute numbers of cells.
• Anti- CD38, anti-CD138, and anti-CD56 were purchased from Beckman Coulter; anti-CD3, anti-CD16, anti-CD55, anti-CD59 from BD Biosciences; and anti-CD46 from Biolegend.
• Flow cytometry was done using a FACS-Calibur device (Becton Dickinson); the data were analyzed using the CellQuest software.
• Example 2 ATRA increases CD38 expression on MM cell lines and in primary MM cells
• An increase in CD38 expression levels may enhance the efficacy of daratumumab to kill MM cells via ADCC or CDC.
• Interaction of ATRA with nuclear retinoic acid receptors results in altered expression of target genes including induction of CD38 expression (Malavasi F. J Leukoc Biol 90:217-219, 2011 ; Drach et ah, Cancer Res 54:1746-1752, 1994). Therefore, effect of ATRA on MM cell lines RPMI8226, UM9, and XG1 was studied.
• MM cells were incubated with RPMI-1640 medium alone or with ATRA ranging from 0 - 25 nM for 48 hours ( Figure 1A) or were incubated with 10 nM ATRA for 24, 48, 72 or 96 hours ( Figure IB) and then harvested to determine CD38 expression by flow cytometry using a FACS-Calibur device (Becton Dickinson) and anti- CD38 antibody (Beckman Coulter). The data were analyzed using the CellQuest software.
• BM-MNCs from 26 MM patients were incubated with RPMI-1640 medium alone or with 10 nM ATRA for 48 hours, incubated at 4°C for 20 min with FITC-conjugated CD38 antibody (Beckman Coulter) and then harvested to determine CD38 expression by flow cytometry.
• Flow cytometric analyses were performed using a FACS-Calibur device (Becton Dickinson); the data were analyzed using the CellQuest software.
• ATRA induced CD38 expression (median increase 1.7-fold, range 1.0 - 26.5- fold) ( Figure 2).
• There was also a significant upregulation of CD138 expression levels (median increase: 2.0-fold), which is characteristic of MM cell differentiation.
• no significant changes in the expression of other plasma cell antigens, such as HLA A/B/C or CD56 were observed in response to ATRA.
• Example 3 ATRA-mediated upregulation of CD38 enhances both daratumumab- mediated ADCC and CDC against MM cells
• CDC and ADCC were assessed using bioluminescence imaging (BLI) based ADCC and CDC assays as described above.
• CDC and ADCC were assessed using Flow cytometry-based ex vivo ADCC and CDC assays in BM-MNC as described above.
• cells were pre-treated with 10 nM ATRA or solvent control for 48 hours, followed by incubation with or without daratumumab in the presence of PBMCs as effector cells for assessment of ADCC or in the presence of human serum as complement source for analysis of CDC. Isotype control was added at 10 ⁇ / ⁇ 1, and 10% heat-inactivated serum was used as control for CDC.
• Figure 3A, Figure 3B and Figure 3C show the results of daratumumab-induced CDC and ADCC in the XG1, RPMI8226 and UM9 cell lines, respectively.
• Example 4 ATRA-mediated upregulation of CD38 enhances both daratumumab- mediated ADCC and CDC against primary MM cells.
• Figure 4A and Figure 4B show results of daratumumab-induced CDC and ADCC, respectively, in primary MM cells pretreated for 48 hours with or without 10 nM ATRA.
• the graphs in Figure 4A and Figure 4B represent pooled results of 16 or 13 patient samples, respectively.
• Figure 5 shows results of daratumumab-induced CDC in primary MM cells from each patient.
• Figure 5A shows daratumumab-induced CDC in primary MM cells form patient 1 and patient 2.
• Figure 5B shows daratumumab-induced CDC in primary MM cells form patient 3 and patient 4.
• Figure 5C shows daratumumab-induced CDC in primary MM cells form patient 5 and patient 6.
• Figure 5D shows daratumumab-induced CDC in primary MM cells form patient 7 and patient 8.
• Figure 5E shows daratumumab- induced CDC in primary MM cells form patient 9 and patient 10.
• Figure 5F shows daratumumab-induced CDC in primary MM cells form patient 11 and patient 12.
• Figure 5G shows daratumumab-induced CDC in primary MM cells form patient 13 and patient 14.
• Figure 5h shows daratumumab-induced CDC in primary MM cells form patient 15 and patient 16.
• ATRA induced daratumumab-mediated CDC in primary MM cells that were not responsive to daratumumab alone in vitro (for example patients 1, 4, 8, 12, 13, 15 and 16). These primary MM cells were isolated from patients with refractory or double refractory disease as indicated in Table 1. In some patient primary MM cell samples, ATRA had no additional effect enhancing daratumumab-mediated CDC (for example see patients 6, 7 and 14).
• Figure 6 shows results of daratumumab-induced ADCC in primary MM cells from each patient.
• Figure 6A shows daratumumab-induced CDC in primary MM cells form patient 3 and patient 4.
• Figure 6B shows daratumumab-induced CDC in primary MM cells form patient 7 and patient 8.
• Figure 6C shows daratumumab-induced CDC in primary MM cells form patient 9 and patient 10.
• Figure 6D shows daratumumab-induced CDC in primary MM cells form patient 14 and patient 15.
• Figure 6E shows
• daratumumab-induced CDC in primary MM cells form patient 16 and patient 17.
• Figure 6f shows daratumumab-induced CDC in primary MM cells form patient 18.
• ATRA induced daratumumab-mediated ADCC most primary MM cells tested. These primary MM cells were isolated from patients with refractory or double refractory disease as indicated in Table 1.
• Table 1 shows the baseline characteristics of the BM-MNC of the tested 19 MM patients.
• * lenalidomide- and/or bortezomib-refractory disease is defined as progressive disease on lenalidomide - and bortezomib -therapy, no response (less than partial response) to lenalidomide - and bortezomib -therapy, or progressive disease within 60 days of stopping a lenalidomide - and bortezomib -containing regimen, according to the International Uniform Response Criteria for Multiple Myeloma.
• BM-MNCs bone marrow mononuclear cells. MM; multiple myeloma. M; male. F;
• Example 5 ATRA downregulates CD55 and CD59 expression in primary MM cells
• ATRA had no effect or minimal effect on the ability of PBMCs from healthy donors to induce ADCC on human MM cell lines L363-CD38, LME-1, RPMI8226 and UM9 (data not shown). On the contrary, ATRA reduced expression levels of complement-inhibitory proteins CD55, CD59 and CD46 on MM cell lines and primary MM cells. In RPMI8226 ( Figure 8A), L363 ( Figure 8B) and XG-1 ( Figure 8C) cells, ATRA reduced expression levels of CD55, CD59, and CD46.
• Hybrid scaffolds consisting of three 2-3 mm biphasic calcium phosphate particles were coated in vitro with human mesenchymal stromal cells (MSCs; 2xl0 5 cells/scaffold). After a week of in vitro culture in a osteogenic medium, humanized scaffolds were implanted subcutaneously into RAG2 " ⁇ yc ' ⁇ mice, as described previously (Groen et al, Blood. 19;120:e9-el6, 2012; de Haart et al, Clin.Cancer Res. 19:5591-5601, 2013).
• mice received a sublethal irradiation dose (3 Gy, 200 kV, 4 mA) and luciferase-transduced XG1 cells were injected directly into the scaffold (lxlO 6 cells/scaffold).
• a sublethal irradiation dose 3 Gy, 200 kV, 4 mA
• luciferase-transduced XG1 cells were injected directly into the scaffold (lxlO 6 cells/scaffold).
• three weeks after inoculation when there was visible tumor growth in the scaffolds by bioluminescent imaging (BLI), different groups of mice were treated with 1) vehicle, 2) ATRA plus T-cell depleted PBMC as effector cells (PBMC-T), 3) daratumumab plus PBMC-T, and 4) daratumumab plus ATRA plus PBMC- T.
• PBMC-T 8x l0 6 cells/mouse were given intravenously on days 24, 31, and 38; and ATRA (10 mg/kg) was given via intraperitoneal injection on days 21-24, 28-31, and 35-38.
• PBMC-T were prepared by Ficoll-Hypaque density-gradient centrifugation of buffy coats, and subsequent depletion of T cells by CD3 -beads using the EasySepTM-technology
• mice experiments The statistical differences between the different treatment groups in the mice experiments were calculated using a Mann-Whitney test. -values below 0.05 were considered significant.
• Luciferase-transduced XG1 multiple myeloma cells developed into aggressive tumors in immunodeficient RAG2 " ⁇ ⁇ c _ " mice in a humanized bone marrow
• mice were co-injected with NK cell-enriched (T cell-depleted) PBMCs of a healthy donor in combination with daratumumab and/or ATRA, as RAG2 " ⁇ y c ⁇ ' ⁇ mice are devoid of NK cells.
• BLI was performed weekly for 5 weeks.
• daratumumab markedly slowed tumor progression, whereas ATRA as single agent had no effect.
• ATRA also significantly enhanced the anti-MM effect of daratumumab in this model.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Immunology (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Engineering & Computer Science (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Molecular Biology (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Oncology (AREA)
• Biomedical Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Peptides Or Proteins (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Priority Applications (19)

Applications Claiming Priority (4)

Publications (2)

Family

ID=55436472

Family Applications (1)

Country Status (25)

Cited By (30)

Families Citing this family (24)

Citations (37)

Family Cites Families (81)

• 2015
  • 2015-09-08 SG SG11201701867SA patent/SG11201701867SA/en unknown
  • 2015-09-08 EP EP15839752.1A patent/EP3191187B1/en active Active
  • 2015-09-08 MA MA040608A patent/MA40608A/fr unknown
  • 2015-09-08 AU AU2015315396A patent/AU2015315396B2/en active Active
  • 2015-09-08 WO PCT/US2015/048899 patent/WO2016040294A2/en not_active Ceased
  • 2015-09-08 BR BR112017004614-8A patent/BR112017004614A2/pt not_active Application Discontinuation
  • 2015-09-08 CA CA2960494A patent/CA2960494C/en active Active
  • 2015-09-08 MY MYPI2017700796A patent/MY192918A/en unknown
  • 2015-09-08 US US14/847,428 patent/US20160067205A1/en not_active Abandoned
  • 2015-09-08 CR CR20170086A patent/CR20170086A/es unknown
  • 2015-09-08 EA EA201790548A patent/EA035979B1/ru unknown
  • 2015-09-08 MX MX2017003115A patent/MX386886B/es unknown
  • 2015-09-08 CN CN201580060898.4A patent/CN108064182B/zh active Active
  • 2015-09-08 PH PH1/2017/500442A patent/PH12017500442B1/en unknown
  • 2015-09-08 JP JP2017513177A patent/JP6707531B2/ja active Active
  • 2015-09-08 PE PE2017000431A patent/PE20170676A1/es unknown
  • 2015-09-08 ES ES15839752T patent/ES2890669T3/es active Active
  • 2015-09-08 UA UAA201703276A patent/UA122212C2/uk unknown
  • 2015-09-08 KR KR1020177009199A patent/KR102586588B1/ko active Active
• 2016
  • 2016-12-21 US US15/386,391 patent/US10604580B2/en active Active
• 2017
  • 2017-03-08 IL IL251033A patent/IL251033B/en unknown
  • 2017-03-08 NI NI201700027A patent/NI201700027A/es unknown
  • 2017-03-08 CL CL2017000559A patent/CL2017000559A1/es unknown
  • 2017-03-09 GT GT201700048A patent/GT201700048A/es unknown
  • 2017-04-06 EC ECIEPI201721175A patent/ECSP17021175A/es unknown
  • 2017-04-07 ZA ZA2017/02483A patent/ZA201702483B/en unknown
• 2020
  • 2020-05-18 JP JP2020086765A patent/JP2020143108A/ja active Pending

Patent Citations (49)

Non-Patent Citations (39)

Also Published As

Similar Documents

Legal Events