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
  • A61K31/33—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
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
• • 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
• • 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
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal 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
  • C07K2317/56—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
  • C07K2317/70—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
• • 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]

Definitions

• the present invention relates to methods of treatment of acute lymphoblastic leukemia with anti-CD38 antibodies.
• CDS 8 is a multifunctional 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) and ADPR.
• CDS 8 mediates cytokine secretion and activation and proliferation of lymphocytes (Funaro et al, J Immunol 145:2390-6, 1990; Terhorst et al., Cell 771-80, 1981; Guse et al, Nature 398:70-3, 1999).
• CD38 via its NAD glycohydrolase activity, also 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 12:743 -52, 2012).
• CD38 signaling occurs via cross-talk with antigen-receptor complexes on T and B cells or other types of receptor complexes, e.g ., MHC molecules, involving CD38 in several cellular responses, but also in switching and secretion of IgGl.
• CD38 is a type II transmembrane glycoprotein expressed on hemopoietic cells such as medullai thymocytes, activated T- and B-cells, resting NK cells and monocytes, lymph node germinal center iymphoblasts, plasnsa B cells, intrafollicular vas and dendritic cells. A portion of normal bone marrow cells, particular precursor cells as well as umbilical cord cells are CD38-positive. In addition to lymphoid precursor cells, CD38 is expressed on erythrocyies and on platelets, and expression is also found in some solid tissues such as gut, brain, prostate, bone, and pancreas. Mature resting T- and B-cells express limited to no surface CD38.
• CD38 is also 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/myeiocyiic leukemia, acute myeloid leukemia, chronic myeloid leukemia, follicular lymphoma, Buxkitt'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
• CD38 has been described on epithelial/endothelial cells of different origin, including glandular epithelium in prostate, islet cells in pancreas, ductal epitheliuin in glands, including parotid gland, bronchial epithelial cells, cells in testis and ovary and tumor epitheliuin 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 CDS 8.
• Acute lymphoblastic leukemia is characterized by impaired early lymphoid development and is classified as either B-cell or T-cell ALL.
• Buriktt's lymphoma (“Mature B ceil lymphoma”) is also classified as ALL.
• incidence of ALL is about 6000 new cases per year, or approximately ! in 50,000.
• Both genetic and environmental factors contribute to ALL, with several chromosomal rearrangement and subrnicroscopic genetic alterations identified (inaba el at, Lancet 381 : 1943-55, 2013).
• Overall response rates to therapy in children having ALL is about 80%, and about 45%-60% in adults with ALL.
• prognosis in relapsed ALL is poor.
• One embodiment of the invention is a method of treating a subject having acute lymphoblastic leukemia (ALL), comprising administering to a patient in need thereof an anti-CD38 antibody that competes for binding to CDS 8 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VI.) of SEQ ID NO: 5, wherein the anti-CD38 antibody induces in vitro killing of ALL cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody- dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), apoptosis, or in vitro modulation of CDS 8 enzymatic activity.
• ADCC antibody-dependent cell-mediated cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• CDC complement dependent cytotoxicity
• apoptosis or in vitro modulation of CDS 8 enzymatic activity.
• Figure 1 shows the efficacy of daratumumab in ALL patient tumor model of disease (ALL 7015 model).
• Graph was plotted by Mean + SEM; Mean + SEM plotted only when there were 80% or more animals (at least 8 animals per each cohort) on the study for each time point (initially there were 10 mice per each cohort).
• Y- axis shows the percentage of tumor burden measured as % of human CD4.T cells divided by live cells.
• Figure 2 shows the efficacy of daratumumab in ALL patient tumor model of disease (ALL 7043 model).
• Graph was plotted by Mean ⁇ SEM; Mean ⁇ SEM plotted only when there were 80% or more animals (at least 8 animals per each cohort) on the study for each time point (initially there were 10 mice per each cohort).
• Figure 3 shows the efficacy of daratumumab and daratumumab in combination with vincristine in ALL cell line tumor xenograft model (NALM-6 model). Animals were divided in four treatment groups and were administered, 10 mg kg daratumumab, 0.5 mg/kg vincristine, or daratumumab in combination with vincristine. Median survival time was plotted against days after tumor inoculation,
• CD38 refers to the human CD38 protein (synonyms: ADP-ribosyl cyclase 1, cADPr hydrolase 1 , cyclic ADP-ribose hydrolase 1 ). Human CD38 has an amino acid sequence shown in SEQ ID NO: 1
• antibodies as used herein is meant in a broad sense and includes immunoglobulin molecules including polyclonal antibodies, monoclonal antibodies including murine, human, human-adapted, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or mu!tispecific antibodies, dimeric, letrarneiic or multimeric antibodies, and single chain antibodies.
• Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, lgG and IgM, depending on the heavy chain constant domain amino acid sequence.
• IgA and lgG are further sub-classified as the isotypes IgA,, IgA 2 , IgGi, ig 3 ⁇ 4, IgG : . and Ig ⁇ 3 ⁇ 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, suc 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(ab3 ⁇ 4; 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 fragmen t consisting of the VL and VH domains of a single ami of an antibody; a domain antibody (dAb) fragment (Ward et al (1989) Nature 341 :544- 546), 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 intramoleculariy, or
• scFv single chain Fv
• diabody diabody
• isolated antibody refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding CD38 is substantially free of antibodies that specifically bind antigens other than hurnan CD38).
• An isolated antibody that specifically binds CD38 can have cross-reactivity to other antigens, such as orthologs of human CD38, such as Macaca fascic laris (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 such as Complementarity Determining Regions (CDRs), 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 332:21 1-50, 1970; Kabat et al Sequences of Proteins of immunological interest, 5th Ed.
• CDRs Complementarity Determining Regions
• HVR HVR
• HV HV
• VH three in the VH (HI, H2, H3) and three in the VL (LI, 12, L3)
• HVR HVR
• HV three in the VH
• VL VL
• Other terms include “lMGT-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 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. Because the antigen binding sites can be defined by various terms as described above, the exact amino acid sequence of a framework depends on how the antigen-binding site was defined.
• 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 regions 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. US2009/01 18127. 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 wherein 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, in some cases, "human antibody” may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al, i 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. Pubt. 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, while derived from human immunoglobulin sequences, 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, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.
• 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) 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 ofhuman 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 and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes.
• epitope 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 can be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous 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, substitutions, insertions or deletions,
• “Synergy”, “synergism” or “synergistic” mean more than the expected additive effect of a combination.
• the term “in combination with” as used herein means that two or more therapeutics can be administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order.
• treat refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of tumor or tumor cells.
• beneficial or desired clinical results 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 tliose already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
• 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%.
• ADCC antibody-dependent cell-mediated cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• CDC complement dependent cytotoxicity
• apoptosis necrosis, or by 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 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 .
• One embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, is a method of treating a subject having acute lymphoblastic leukemia (ALL), comprising administering to a patient in need thereof an anti-CD38 antibody that competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5, wherein the anti-CD38 antibody induces in vitro killing of ALL cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), apoptosis, or in vitro modulation of CD38 enzymatic activity.
• ADCC antibody-dependent cell-mediated cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• CDC complement dependent cytotoxicity
• Another embodiment of the invention described herein, and in some embodiments of each and ever ⁇ ' one of the numbered embodiments listed below, is a method of treating a subject having acute lymphoblastic leukemia (ALL), comprising administering to a patient in need thereof an anti-CD38 antibody that binds to the region
• SKRNIQFSCKNIYR SEQ ID NO: 2
• EKVQTLEAWV1HGG SEQ ID NO: 3
• the anti-CD38 antibody induces in vitro killing of ALL cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), apoptosis, or in vitro modulation of CD38 enzymatic activity.
• the epitope of the antibody includes some or all of the residues having the sequences shown in SEQ ID NO: 2 or SEQ ID NO: 3.
• the antibody epitope comprises at least one amino acid in the region SKRNIQF SCKNIYR (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 EKVQTLEAWV1HGG (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: 3 ).
• 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: 14) in the region EKVQTLEA WVIHGG (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: 14) as shown above is daratumumab (see Intl. Pat. Publ. No. WO2006/0998647).
• Daratumumab comprises VH and a 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, and light chain CDRs LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 9, 10 and 11 , respectively, and is of TgGl/ ⁇ subtype.
• 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.
• ⁇ ⁇ ⁇ IX is ! ⁇ ⁇ i ' KPRiil QYSSiA kVVS ⁇ I . iA ! . ⁇ IQi >VU NGK!A ⁇ ⁇ S
• Antibodies can be evaluated for their competition with daraturnumab having VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 for binding to CD38 using well known in vitro methods.
• CHO cells recombinant! ⁇ -' expressing CDS 8 may be incubated with unlabeled daraiumuniab for 15 min at 4°C, followed by incubation with an excess of fluorescently labeled lest antibody for 45 ruin at 4°C. After washing in PBS/BSA, fluorescence may be measured by flow cytometry using standard methods.
• extracellular portion of human CDS 8 may be coated on the surface of an ELISA plate.
• test antibody competes with daratumumab when daratumumab inhibits binding of the test antibody, or the test antibody inhibits binding of daratumumab by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% , 95% or 100%.
• the epitope of the test antibody can further be defined for example by peptide mapping or hydrogen/deuterium protection assays using known methods, or by crystal structure determination.
• Antibodies binding to the same region on CD38 as daratumumab can 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 can be further evaluated for example by assaying competition between daratumumab and a test antibody for binding to CDS 8 using well known in vitro methods and as described above.
• 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 function may ⁇ be mediated by binding of an Fc effector domain(s) to an Fc receptor on an immune ceil 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, for example CD38-expressing cells.
• Human IgG isotypes IgGl, IgG2, IgGS and IgG4 exhibit differential capacity for effector functions.
• ADCC may be mediated by IgGl and IgGS
• ADCP may be mediated by IgGl
• CDC may be mediated by IgGl and IgGS.
• the airti-CD38 antibody is of IgGl, IgG2, igG3 or IgG4 isotype.
• the anti-CD38 induces in vitro killing of ALL cells that express CD38 by ADCC.
• the anti-CD38 induces in vitro killing of ALL cells that express CD38 by CDC.
• the anti-CD38 antibody induces in vitro killing of ALL cells that express CD38 by ADCP.
• the aoti-CD38 antibody induces in vitro killing of ALL cells that express CD38 by apoptosis.
• the anti-CD38 antibody induces in vitro killing of ALL cells that express CD38 by ADCC and CDC
• the anti-CD38 antibody of the invention will induce in vivo killing of ALL cells that express CDS 8 by ADCC, CDC, ADCP, apoptosis or in vivo modulation of CDS 8 enzymatic activity.
• Antibody-dependent cellular cytotoxicity 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 ceils.
• effector cells possessing lytic activity such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (FcyR) expressed on effector ceils.
• FcyR Fc gamma receptors
• UK cells express FcyRIHa
• monocytes express FcyRi, FcyRII and FcvRIIIa.
• 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 ceils in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cyiolysis 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
• exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMC) and K cells.
• Exemplary target cells include Daudi cells (A ICC CCL-213 " ) or B cell leukemia or lymphoma tumor ceils expressing CD38. in an exemplary assay, target cells are labeled with 20 ⁇ of j l Cr for 2 hours and washed extensively.
• Cell concentration of the target cells can be adjusted to 1x 10" cells/ml, and anti-CD38 antibodies at various concentrations are added.
• Assays are started by adding Daudi cells at an effeetontarget cell ratio of 40: 1. After incubation for 3 hr at 37°C assays are stopped by centrifugation, and " "Cr release from lysed cells are measured in a scintillation counter. Percentage of cellular cytotoxicity may be calculated as % maximal lysis which may be induced by adding 3% perchloric acid to target cells.
• 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 (cell lysis induced by 3% perchloric acid).
• ADCP Antibody-dependent cellular phagocytosis
• phagocytic cells such as macrophages or dendritic cells.
• ADCP may be evaluated by using monocyte -derived macrophages as effector cells and Daudi cells (ATCC * CCL -213 ) or B cell leukemia or lymphoma tumor cells expressing CD38 as target cells engineered to express GFP or other labeled molecule.
• Effctortarget cell ratio may be for example 4: 1. Effector cells may be incubated with target cells for 4 hours with or without anti-CD38 antibody. After incubation, cells may be detached using accutase.
• Macrophages can be identified with anti-CD 1 l b and anti-CD 14 antibodies coupled to a fluorescent label, and percent phagocytosis can be determined based on % GFP fluorescent in the CD11 * CD34 + macrophages using standard methods.
• Anti-CD38 antibodies used in the methods of the invention may induce ADCP by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% .
• “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 C ! q 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
• CDC of CDS 8-expressing ceils can be measured for example by plating Daudi cells at 1 > ; 10 "' cells/well (50 ⁇ /well) in RPMI-B (RPMI supplemented with 1% BSA), adding 50 ⁇ anti-CD38 antibodies to the wells at final concentration between 0-100 ⁇ &' ⁇ , incubating the reaction for 15 min at room temperature, adding 11 ⁇ of pooled human serum to She wells, and incubation the reaction for 45 min at 37° C. Percentage (%) lysed cells may be detected as % propidium iodide stained cells in FACS assay using standard methods.
• 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 ADC ' C can 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 io the Fc regions enhances the ADCC of antibodies via improved FcyRiiia binding without altering antigen binding or CDC activity.
• Such mAbs can be achieved using different methods reported to lead to the successful expression of relatively high defucosylated antibodies bearing the biantennary complex-type of Fc oligosaccharides such as control of culture osmolality (Konno et al, Cytotechnology 64:249-65, 2012), application of a variant CHO line LecB as the host cell line (Shields et al, J Biol Chem 277:26733-26740, 2002), application of a variant CHO line EB66 as the host cell line (Olivier et al, MAbs ;2(4), 2010; Epub ahead of print; PMiD:20562582), application of a rat hybridoma cell line YB2/0 as the host cell line (Shinkawa et al, J Biol Chem 278:3466-3473, 2003), introduction of small interfering RNA specifically against the a 1,6-fucosyltrasferase ( FUT8) gene (Mori e
• 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.
• the anti-CD38 antibodies comprise a substitution in the antibody Fc. In some methods described herein, and in some embodiments of each and e very one of the numbered embodiments listed below, 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 or 430 (residue numbering according to the EU index).
• 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%, 1 1% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%
• “Fucose content” means 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. These may be characterized and quantified by multiple methods, for example: ⁇ ) using MALDI-TOF of N-glycosidase F treated sample (e.g. compiex, hybrid and oligo- and Mgh-mannose structujes) as described in Intl. Pat. Publ. No.
• WO20Q8/077546 2) by enzymatic release of the Asn297 glycans with subsequent derivatization and detection 7 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 UPLC
• the oligosaccharides released can 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 siaiylation 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 quantifi cation of the oiigosaccbarides by " nigh 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 refers to antibodies with fucose content of about over 50%, typically about over 60%, 70%, 80% or over 85%.
• the anti-CD38 antibodies used in the methods described herein, and in some embodiments of each and every one of the numbered embodiments listed below, may induce in vitro killing of ALL cells by apoptosis.
• Methods for evaluating apopiosis are well known, and include for example annexin IV staining using standard methods.
• the anti-CD38 antibodies used in the methods of the inve ntion may induce apoptosis in about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of cells.
• CD38 is a multifunctional ectoenzrne with ADP-ribosyl cyclase 1 activity catalyzing the formation of cyclic ADP-ribose (cADPR) and ADPR from NAD + , and also functions to hydrolyze NAD + and cADPR to ADPR.
• cADPR cyclic ADP-ribose
• CD38 also catalyzes the exchange of the nicotinamide group of NADP + with nicotinic acid under acidic conditions, to yield NAADP + (nicotinic acid-adenine dinucleoiide phosphate).
• Modulation of the enzymatic activity of human CD38 with anti-CD38 antibodies used in the methods of the invention may be measured in an assay described in Graeff et ai, J . Biol. Chem. 269, 30260-30267 (1994).
• substrate NGD* may be incubated with CD38, and the modulation of the production of cyclic GDP-ribose (cGDPR) may be monitored spectropbotometrically at excitation at 340 nM and emission at 410 nM at different time points alter addition of the antibody at various concentrations.
• Inhibition of the synthesis of cADPR can be determined according to the HPLC method described in Munsbi et al, J. Biol. Chem. 275, 21566-21571 (2000).
• anti-CD38 antibodies used in the methods of the invention described herei n, and in some embodime nts of each and every one of the numbered embodiments listed below, may inhibit CD38 enzymatic activity by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% .
• 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 anii-CD38 antibody comprises the light chain complementarity determining regions (LCDR) 1 (LCDR1), 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 a heavy chain of SEQ ID NO: 12 and a light chain of SEQ ID NO: 13.
• 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.
• 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, 30, 1 1, 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 NT! v.9.0.0 (Invitrogen, Carlsbad, CA).
• the protein sequences of the present invention ca 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
• 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, 1 1, 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 aianine scanning mutagenesis (MacLennan et al, 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 PGR mutagenesis (U.S. Pat. No. 4,683, 195).
• Libraries of variants may be generated using well known methods, for example using random (NNK) or non-random codons, for example DVK codons, whicli encode 1 1 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, or modulate CD38 enzymatic activity in vitro using methods described herein.
• the anti-CD38 antibody in one embodiment according to the invention, and in some embodiments of each and every one of the numbered embodiments listed below, the anti-CD38 antibody binds to CD38 with high affinity, for example, with a K D equal to or less than about 10 " ' M, such as but not limited to, 1-9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) X 10 "8 , 10 "9 , 10 ";0 , 10 "i; , 10 "12 , 30 " ' 3 , 10 " 14 , 10 “i5 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 I xlO "8 M.
• the anti-CD38 antibody is a bispecific antibody.
• the VL and/or the VH regions of the existing anti-CD38 antibodies or the VI. 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 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.
• the anti-CD38 antibody is conjugated to a toxin. Conjugation methods and suitable toxins are well known.
• the ALL is B-cell lineage ALL.
• i n some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the ALL is T-cell lineage ALL. in some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the ALL is adult ALL.
• the ALL is pediatric ALL.
• the anti-CD38 antibody is administered as a remission induction or as a postinduction therapy.
• the ALL is refractory or relapsed ALL.
• the subject has a white blood cell count of at least about 1 x 10 9 /L.
• the ALL cells have a Philadelphia chromosome.
• “Philadelphia chromosome” or “Ph” refers to a well-known chromosomal translocation between chromosomes 9 and 22, resulting in the oncogenic BCR-ABL gene fusion with constitutively active tyrosine kinase activity. The translocation results in a portion of the BCR gene from chromosome 22ql 1 becoming fused with a portion of the ABL gene from chromosome 9q34, and is designated as t(9;22)(q34;ql 3 ) under the international System for Human Cytogenetic ⁇ Nomenclature (ISCN). Depending on the precise location of the fusion, the molecular weight of the resulting fusion protein can range from 185 to 210 kDa. “Philadelphia chromosome” refers to all BCR-ABL fusion proteins formed due the (9;22)(q34;ql 1) translocation.
• Ph chromosome is present in about 20% of adults with ALL and a small percentage of children with ALL and is associated with poor prognosis.
• patients with Ph+ positive ALL may be on tyrosine kinase inhibitor (TKI) regimen and may have therefore become resistant to the TKI.
• TKI tyrosine kinase inhibitor
• the anti-CD38 antibodies may thus be administered to a subject who has become resistant to selective or partially selective BCR-ABL inhibitors.
• Exemplary BCR-ABL inhibitors are for example imatinib, dasattnib, nilotinib, bosutinib, ponatinib, bafetinib, saracatinib, tozasertib, danusertib or ibrutinib.
• chromosomal rearrangements identified in B-lineage ALL patients are t(v; l lq23) (MLL rearranged), t( ! ; !9)(q23;pl3.3); TCF3-PBX1 (E2A-PBX1), t(12;2 )(pl3;q22); ETV6-RUNX1 (TEL-AMLi) and t(5; 14)(q31 ;q32); IL3-IGH.
• the subject has ALL with t(v; 1 lq23) (MLL rearranged), t(l ; 19)(q23;pl3.3); TCF3-PBXl (E2A-PBX1), t( 12 :21 )( p 13 :q22 ) : ETV6-RUNX 1 (TEL- AMLi) or t(5; 14)(q31 ;q32); IL3-IGH chromosomal rearrangement.
• Chromosomal rearrangements can be identified using well known methods, for example fluorescent in situ hybridization, karyotyping, pulsed field gel electrophoresis, or sequencing.
• the subject is resistant or has acquired resistance to treatment with at least one BCR-ABL kinase inhibitor.
• the at least one BCR-ABL kinase inhibitor is imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, saracatinib, tozasertib, danusertib or ibrutinib.
• Various qualitative and/or quantitative methods may be used to determine if a subject is resistant, has developed or is susceptible to developing a resistance to treatment with at least one BCR-ABL kinase inhibitor.
• 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 de veloping resistance to at least one BCR-ABL kinase inhibitor.
• the symptoms associated with cancer may vary according to the type of cancer.
• symptoms associated with ALL 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.
• Other means to determine if a subject has developed a resistance to at least one BCR-ABL kinase inhibitor include analyses of tumor burden in a patient with ALL.
• the anti-CD38 antibody is administered in combination with vincristine.
• the subject having ALL is homozygous for phenylalanine at position 158 of CD16 (FcyRITIa- 158F/F genotype) or heterozygous for valine and pheynylalanine at position 158 of CD16 (FcyRITIa- 158F/V genotype).
• CD 16 is also known as the Fc gamma receptor ilia (FcyRIIia) or the low affinity immunoglobulin gamma Fc region receptor lil-A isoform.
• Valine/phenylalanine (V/F) polymorphism at FcyRIIia protein residue position 1 8 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 invention also provides for the method of treating a subject having ALL, comprising administering to a patient in need thereof an anii-CD38 antibody that binds to the region SKRNIQF SCKNIYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1), wherein the anti-CD38 antibody induces in vitro killing of CD38-expressing cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), apoptosis, or in vitro modulation of CD38 enzymatic activity, wherein the subject is homozygous for phenylalanine at position 158 of CD 16 or heterozygous for valine and pheynylalanine at position 158 of CD 16.
• ADCC antibody-dependent cell-mediated cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• CDC complement dependent cytotoxicity
• the invention also provides for the method of treating a subject having ALL, comprising administering to a patient in need thereof an anti-CD38 antibody that competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5, wherein the anti- CD38 antibody induces in vitro killing of ALL.
• an anti-CD38 antibody that competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5, wherein the anti- CD38 antibody induces in vitro killing of ALL.
• ADCC antibody-dependent cell-mediated cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• CDC complement dependent cytotoxicity
• apoptosis or in vitro modulation of CD38 enzymatic activity, wherein the subject is homozygous for phenylalanine at position 158 of CD16 or heterozygous for valine and pheynylalanine at position 158 of CD ! 6.
• 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 can be used. These solutions are sterile and generally free of particulate matter.
• compositions may be sterilized by conventional, well-known sterilization techniques (e.g., filtration).
• the 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 age nts, 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 described herein, and in some embodiments of each and every one of the numbered embodiments listed below, 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 described herein, and in some embodiments of each and every one of the numbered embodiments listed below, 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 intraperitoneaily.
• i.v. infusion may be given over for example 15, 30, 60, 90, 120, 180, or 240 minutes, or from i , 2, 3, 4, 5, 6, 7, 8, 9, 10, i 1 or 12 hours.
• the dose given to a patient having ALL is sufficient to alleviate or at least partially arrest the disease being treated ("therapeutically effective amount") and may be sometimes 0.005 mg to about 100 mg/kg, e.g. about 0,05 mg to about 30 rng/'kg or about 5 mg to about 25 mg/kg, or about 4 mg kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg , or for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.
• a fixed unit dose may also be 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/ii .
• 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 described herein, 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 possibie, 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 described herein, and in some ernbodiments 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 described herein, 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, 35, 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, 30, 3 1, 12, 13, 14, 15, 16, 3 7, 1 8, 19, 20, 23 , 22,
• Anti-CD38 antibodies in the methods of the invention described herein, 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 described herein, and in some embodiments of each and every one of the numbered embodiments listed below, may be lyophiiized for storage and reconstituted in a suitable carrier prior to use. This technique lias been shown to be effective with conventional protein preparations and well known lyophilization and reconstitution techniques can be employed.
• anti-CD38 antibody in the methods of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, may be administered in combination with vincristine.
• Vincristine may be administered for example at about 0.1 to 2 mg/kg single dose i.p., for example 0.1 to 0.5 mg/kg single dose i.p, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0,6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mg/kg.
• Vincristine may be given as i.v. infusion.
• the combination of the anti-CD38 antibody and vincristine may be administered over any convenient timeframe.
• the anti-CD38 antibody and vincristine may be administered to a patient on the same day, and even in the same intravenous infusion.
• the anii-CD38 antibody and vincristine may also be administered on alternating days or alternating weeks or months, and so on.
• the anti-CD38 antibody and vincristine may be administered with sufficient proximity in time that they are simultaneously present (e.g., in the serum) at detectable levels in the patient being treated.
• an entire course of treatment with the anti-CD38 antibody consisting of a number of doses over a time period is followed or preceded by a course of treatment with vincristine, consisting of a number of doses.
• a recover ⁇ ' period of 1 , 2 or several days or weeks may be used between administration of the anti-CD38 antibody and vincristine.
• Aoti-CD38 antibody or a combination of anli-CD38 antibody and vincristine may be administered together with any form of radiation therapy including external beam radiation, intensity modulated radiation therapy (IMRT) and any form of radiosurgery' including Gamma Knife, Cyberknife, Linac, and interstitial radiation (e.g. implanted radioactive seeds, GliaSite balloon), and/or with surgery.
• IMRT intensity modulated radiation therapy
• radiosurgery' including Gamma Knife, Cyberknife, Linac, and interstitial radiation (e.g. implanted radioactive seeds, GliaSite balloon), and/or with surgery.
• An anti-CD38 antibody for use in treating a subject having acute lymphoblastic leukemia (ALL).
• An anti-CD38 antibody for use in treating a subject having ALL, in combination vincristine,
• Vincristine for use in treating a subject having ALL in combination with an anti-CD38 antibody.
• anti-CD38 antibody for use according to embodiment 1 or 2, vincristine for use according to embodiment 3, or the combination according to embodiment 4, 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.
• VH heavy chain variable region
• VL light chain variable region
• a.nti-CD38 antibody for use according to embodiment 1, 2 or 5, vincristine for use according to embodiment 3 or 5, or the combination according to embodiment 4 or 5, wherein the anti-CD38 antibody induces in vitro killing of ALL cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), apoptosis, or in vitro modulation of CDS 8 enzymatic activity, preferably wherein the anti-CEO 8 antibody induces in vitro killing of the ALL ceils by ADCC or CDC.
• ADCC antibody-dependent cell-mediated cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• CDC complement dependent cytotoxicity
• apoptosis or in vitro modulation of CDS 8 enzymatic activity, preferably wherein the anti-CEO 8 antibody induces in vitro killing of the ALL ceils by ADCC or CDC.
• anti-CD38 antibody for use according to embodiment 1, 2, 5 or 6, vincristine for use according to embodiment 3, 5 or 6, or the combination according to embodiment 4-6, wherein the anti-CD38 antibody binds to the region
• S RNIQFSCKNIYR SEQ ID NO: 2
• EKVQTLE AWVIHGG SEQ ID NO: 3
• aoti-CD38 antibody for use according to embodiment 1, 2, 5-7, vincristine for use according to embodiment 3, 5 -7, or the combination according to embodiment 4-7, wherein the aoti-CD38 antibody:
• a. is of IgGl, IgG2, IgG3 or IgG4 isotype
• b. 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%, 3 % or 0%; or
• c. comprise a substitution in the antibody Fc at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430, when residue numbering according to the Ell index,
• LCDR1 light chain complementarity determining regions
• LCDR2 light chain complementarity determining regions
• LCDR3 the light chain complementarity determining regions
• 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; or
• the atrti-CD38 antibody for use accordi ng to embodiment 1, 2, 5-9, vincristine for use accordi ng to embodiment 3, 5-9, or the combination according to embodiment 4-9, wherein ALL is B-cell lineage ALL, T-cell lineage ALL, adult ALL, pediatric ALL, refractory ALL or relapsed ALL.
• anti-CD38 antibody for use according to embodiment 1, 2, 5-10, vincristine for use according to embodiment 3, 5-10, or the combination according to embodiment 4-10, wherein the anti-CD38 antibody is administered as a remission induction or as postinduction therapy.
• anti-CD38 antibody for use according to embodiment 1, 2, 5-1 1, vincristine for use according to embodiment 3, 5-11 , or the combination according to embodiment 4-1 1, wherein the subject
• a. has a white blood cell count of at least about 1 x 10 * /L;
• b. has ALL cells with a Philadelphia chromosome.
• the subject is further treated or has been treated with radiotherapy; or b, the subject has received hematopoietic stem ceil transplantation.
• ALL 7015 model Turnor was resected from a 17 year old female having B ceil lineage ALL.
• White blood cell count (WBC) was 98 x 10 S /L, hemoglobin (HB) lOlg L and platelet count (pit) 24 x 10 9 /L.
• Philadelphia chromosome was evident in tumor cells with rearrangement BCR/ABL-P210 (i9;22)(q34:ql 1).
• Tumor ceils were negative for following rearrangements: TEL/AML1, E2A/PBX1, MLL related gene, SIL/TAL1 , IgH.
• Ratio of expression of Wilrn's tumor 1 gene (WTl ) to ABL gene (WT 1 /ABL) was 1 .2%.
• Grade 1 hyperplasia with 95% of primitive lymphocytes was evident in bone marrow, 92.8% of abnormal bone marrow cells expressed CD38.
• ALL 7473 model Tumor was resected from a 35 year old make having T cell lineage ALL, WBC was 7.4 x 10 9 /L, HB 1 12 g/L, and pit 73 x 10 9 /L. Tumor cells were negative for following chromosomal rearrangement: BCR/ABL. SIL/TAL, MLL related gene, TCR5. WTI/ABL was 2.0%. Grade Hi hyperplasia with 86% of primitive lymphocytes was evident in the bone marrow. 78'% of abnormal cells expressed CD38.
• NOD/SCID female, 3-4 weeks old were inoculated with 2x10 6 of ALL-7015 or ALL-7473 frozen DCis. The animals were evaluated every 3-4 days for the appearance of turnor DCis in the peripheral blood. Treatment was initiated when the tumor burden in the blood reached a specified level (ALL 7015: -4.2% and ALL 7473: -0.5%). The Tumor Burden (TB) was measured once a week by flow cytometry and measured as percentage of CD45 ' CD38 + cells in peripheral blood obtained from retro-orbital bleed. The animals were also monitored daily for morbidity and mortality. Death and observed clinical signs are recorded on the basis of the numbers of animals within each subset.
• Table 2 shows the tumor burden at different time points in the ALL 7015 model. Treatment of mice with daraturnumab showed significant tumor growth inhibition compared to mice treated with a control antibody.
• CB17 SCID mice were inoculated intravenously via the tail vein with the cell line NALM-6 tumor cells at i x 10 J in 300 uL PBS for tumor development.
• the date of tumor cell inoculation is denoted as Day 0.
• Animals were divided in four treatment groups and were administered daratumumab, vincristine, or daratuniumah in combination with vincristine at dosages as described in Table 3, NALM-6 cell line (ACC128, DZMZ) is established from the peripheral blood of a 10-year old man with ALL in relapse.
• Karyotype of ihe cell line is 46(43-47) ⁇ 2n>XY, t(5; 12)(q33.2;p l 3.2).
• n animal number
• Endpoint The major endpoint was animal survival. Each mouse was evaluated daily and mice that showed deteriorating and moribund condition (animals have lost significant body mass: body weight lost > 20%) and animals thai could not get to adequate food or water were euthanized with C ⁇ 3 ⁇ 4. The survival of all animals was followed and median survival time (MST) was calculated for each group. Body weights were measured twice per week. The surviving mice after a maximum of twice the median survival of the vehicle group were sacrificed, in addition, autopsy was performed at the termination to confirm the tumor progression.
• MST median survival time

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