Patent Application
20180117150
Disclosed are combination therapies comprising an anti-CD38 antibody and cyclophosphamide for the treatment of CD38-positive hematological malignancies.
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.
Currently available therapies for MM include chemotherapy regimens, stem cell transplantation, THALOMID® (thalidomide), REVLIMID® (lenalidomide), POMALYST® (pomalidomide), VELCADE® (bortezomib), NINLARO (ixazomib), KYPROLIS® (carfilzomib), FARADYK® (panobinostat), AREDIA® (pamidronate), ZOMETA® (zoledronic acid) and DARZALEX® (daratumumab). Current treatment protocols, which include a combination of chemotherapeutic agents such as vincristine, carmustine (BCNU), melphalan (Alkeran®), cyclophosphamide, doxorubicin (Adriamycin), and prednisone or dexamethasone, yield a complete remission rate of only about 5%, and median survival is approximately 36-48 months from the time of diagnosis. Recent advances using high dose chemotherapy followed by autologous bone marrow or peripheral blood mononuclear cell transplantation have increased the complete remission rate and remission duration. Nevertheless, overall survival has only been slightly prolonged, and no evidence for a cure has been obtained as yet. Ultimately, it it thought that all MM patients will relapse, even under maintenance therapy with interferon-alpha (IFN-α) alone or in combination with steroids.
Efficacy of the available drug treatment regimens for MM is limited by the low cell proliferation rate and development of drug resistance in up to 90% of patients. Chromosomal translocations, oncogene mutations, dysregulated signaling pathways such as anti-apoptotic and survival pathways, and bone marrow (BM) niche have been implicated to contribute to drug resistance in MM (for review, see Abdi et al., Oncotarget 4: 2186-2207, 2013). The BM niche is implicated in proliferation, survival, differentiation, migration, and drug resistance of the malignant plasma cells (Manier et al., J Biomed Biotechnol 2012; published online 2012 Oct. 3, doi:_10.1155/_2012/_157496).
Provided herein are methods of treating a subject having a CD38-positive hematological malignancy, the methods comprising administering to the subject a therapeutically effective amount of an anti-CD38 antibody and cyclophosphamide for a time sufficient to treat the CD38-positive hematological malignancy. In some aspects of the methods, a therapeutically effective amount of dexamethasone, lenalidomide, bortezomib, or any combination thereof may also be administered to the subject.
The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed methods, there are shown in the drawings exemplary embodiments of the methods; however, the methods are not limited to the specific embodiments disclosed. In the drawings:
The disclosed methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods are not limited to the specific methods described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods.
Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed methods are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.
Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the invention as described herein. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the invention be limited to the specific values recited when defining a range. All ranges are inclusive and combinable.
When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.
It is to be appreciated that certain features of the disclosed methods which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.
As used herein, the singular forms “a,” “an,” and “the” include the plural.
Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.
“About” when used in reference to numerical ranges, cutoffs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. As many of the numerical values used herein are experimentally determined, it should be understood by those skilled in the art that such determinations can, and often times will, vary among different experiments. The values used herein should not be considered unduly limiting by virtue of this inherent variation. Thus, the term “about” is used to encompass variations of±10% or less, variations of±5% or less, variations of±1% or less, variations of±0.5% or less, or variations of±0.1% or less from the specified value.
“Comprising” is intended to include examples encompassed by the terms “consisting essentially of” and “consisting of”; similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.” Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.
“CD38” refers to the human CD38 protein (UniProtKB accession no. P28907) (synonyms: ADP-ribosyl cyclase 1, cADPr hydrolase 1, cyclic ADP-ribose hydrolase 1). Human CD38 has an amino acid sequence as shown in SEQ ID NO: 1. CD38 is a single pass type II transmembrane protein with amino acid residues 1-21 representing the cytosolic domain, amino acid residues 22-42 representing the transmembrane domain, and residues 43-300 representing the extracellular domain. Anti-CD38 antibodies are described, for example, in Int'l Pat. Pub. No. WO2008/037257, Int'l Pat. Pub. No. WO2008/047242 and Int'l Pat. Pub. No. WO2007/042309, and are being evaluated in clinical settings for their efficacy in multiple myeloma and other hematological malignancies.
“Antibody,” is meant in a broad sense and includes immunoglobulin molecules including, monoclonal antibodies (such as 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 IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.
“Antibody fragment” refers to a portion of an immunoglobulin molecule that retains the antigen binding properties of the parental full length antibody. Exemplary antibody fragments are heavy chain complementarity determining regions (HCDR) 1, 2, and 3, light chain complementarity determining regions (LCDR) 1, 2, and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include: a Fab fragment, a monovalent fragment consisting of the VL, VH, constant light (CL), and (constant heavy 1) CH1 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; and a domain antibody (dAb) fragment (Ward et al., Nature 341: 544- 546, 1989), which consists of a VH domain or a VL 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 intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int'l Pat. Pub. Nos. WO1998/44001, WO1988/01649, WO1994/13804, and WO1992/01047. These antibody fragments are obtained using techniques well known to those of skill in the art, and the fragments are screened for utility in the same manner as are full length antibodies.
“Isolated antibody” refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated anti-CD38 antibody is substantially free of antibodies that specifically bind antigens other than human CD38) or to a homogenous population of antibodies which have been substantially separated and/or purified away from other components of the system the antibody is produced in, such as a recombinant cell, as well as antibodies that have been subjected to at least one purification or isolation step An isolated anti-CD38 antibody, can have cross-reactivity to other antigens, such as orthologs of human CD38, such as Macaca fascicularis (cynomolgus) CD38.
An antibody variable region consists of a “framework” region interrupted by three “antigen binding sites.” The antigen binding sites are defined using various terms: (i) 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 132: 211-50, 1970; Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991); and (ii) “Hypervariable regions” (“HVR” or “HV”), three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3) refer to the regions of the 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). The International ImMunoGeneTics (IMGT) database (http://www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations is described in Lefranc et al., Dev Comparat Immunol 27: 55-77, 2003.
“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 framework regions 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. If the antibody contains a constant region, the constant region is also derived from sequences of human origin. “Derived from,” as used in the context of humanized antibodies, means that the region in question is at least 80% homologous in sequence to the corresponding region of the immunoglobulin from the species in which it is based.
“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 if 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. “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. Typically, a “human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, a “human antibody” may 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, as described in, for example, Shi et al., J Mol Biol 397: 385-96, 2010 and Int'l Pat. Pub. No. WO2009/085462. Antibodies in which antigen binding sites are derived from a non-human species are not included in the definition of “human antibody”.
“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 of human immunoglobulin gene sequences to other DNA sequences, or antibodies that are generated in vitro using Fab arm exchange.
“Monoclonal antibody” refers to a preparation of antibody molecules of a 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. Monoclonal antibody therefore refers to an antibody population with single amino acid composition in each heavy and each light chain, except for possible well known alterations such as removal of C-terminal lysine from the antibody heavy chain Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. A bispecific antibody is included in the term monoclonal antibody.
“Epitope” refers to 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.
“In combination with” 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,” “treatment,” and like terms refer to both therapeutic treatment and prophylactic or preventative measures, and includes reducing the severity and/or frequency of symptoms, eliminating symptoms and/or the underlying cause of the symptoms, reducing the frequency or likelihood of symptoms and/or their underlying cause, improving or remediating damage caused, directly or indirectly, by the CD38-positive hematological malignancy. Treatment also includes prolonging survival as compared to the expected survival of a subject not receiving treatment. Subjects to be treated include those that have 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.
“Therapeutically effective amount” refers to an amount of the disclosed combination therapy effective, at dosages and for periods of time necessary, to achieve a desired treatment. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the combination therapy to elicit a desired response in the subject. Exemplary indicators of a therapeutically effect amount 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.
“Inhibit growth” (e.g., referring to cells, such as tumor cells) refers to a measurable decrease in in vitro or in vivo cell growth upon contact with a therapeutic or the combination therapy when compared to the growth of the same cells in the absence of the combination therapy. Inhibition of growth of a cell in vitro or in vivo may be at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or about 100%. Inhibition of cell growth can occur by a variety of mechanisms, for example by ADCC, apoptosis, necrosis, or by inhibition of cell proliferation.
“Subject” includes any human or nonhuman animal “Nonhuman animal” includes all vertebrates, e g , mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms “subject” and “patient” can be used interchangeably herein.
The following abbreviations are used throughout the disclosure: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), bortezomib (Bort); Burkitt's lymphoma (BL), chronic lymphocytic leukemia (CLL), complement-dependent cytotoxicity (CDC), complementarity determining region (CDR), constant light (CL), (constant heavy 1) CH1 domains, cyclophosphamide (CTX); daratumumab (DARA); diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), heavy chain CDR (HCDR), heavy chain variable region (VH), Hodgkin's lymphoma (HL), lenalidomide (LEN); light chain CDR (LCDR), light chain variable region (VL), multiple myeloma (MM), mantle-cell lymphoma (MCL); non-Hodgkin's lymphoma (NHL); bone marrow (BM); peripheral blood (PB); mononuclear cell (MC).
Provided here are methods of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject a therapeutically effective amount of an anti-CD38 antibody and cyclophosphamide for a time sufficient to treat the CD38-positive hematological malignancy.
“CD38-positive hematological malignancy” refers to a hematological malignancy characterized by the presence of tumor cells expressing CD38, including leukemias, lymphomas, and myeloma. Exemplary CD38-positive hematological malignancies are precursor B-cell lymphoblastic leukemia/lymphoma, B-cell non-Hodgkin's lymphoma, acute promyelocytic leukemia, acute lymphoblastic leukemia and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia(CLL)/small lymphocytic lymphoma (SLL), B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL) (including low-grade, intermediate- grade and high-grade FL), cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), plasmacytoma, multiple myeloma (MM), plasma cell leukemia, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, plasma cell leukemias, anaplastic large-cell lymphoma (ALCL), T-cell acute lymphocytic leukemia, primary systemic amyloidosis, mantle-cell lymphoma, pro-lymphocytic/myelocytic leukemia, acute myeloid leukemia (AML) (including acute promyelocytic leukemia), chronic myeloid leukemia (CML), large granular lymphocytic (LGL) leukemia, NK-cell leukemia, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T-cell leukemia/lymphoma, nasal type extranodal NK/T cell lymphoma, 78 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 CD38 positive T-cell lymphoproliferative disorders (primary cutaneous anaplastic large cell lymphoma C-ALCL, lymphomatoid papulosis, borderline lesions), angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma unspecified, and anaplastic large cell lymphoma.
Examples of B-cell non-Hodgkin's lymphomas include 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.
In some embodiments, the CD38-positive hematological malignancy is multiple myeloma (MM). In some embodiments, the CD38-positive hematological malignancy is acute lymphoblastic leukemia (ALL). In some embodiments, the CD38-positive hematological malignancy is non-Hodgkin's lymphoma (NHL). In some embodiments, the CD38-positive hematological malignancy is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the CD38-positive hematological malignancy is Burkitt's lymphoma (BL). In some embodiments, the CD38-positive hematological malignancy is follicular lymphoma (FL). In some embodiments, the CD38-positive hematological malignancy is mantle-cell lymphoma (MCL). In some embodiments, the CD38-positive hematological malignancy is acute myeloid leukemia (AML). In some embodiments, the CD38-positive hematological malignancy is chronic lymphocytic leukemia (CLL). In some embodiments, the CD38-positive hematological malignancy is any combination of the above CD38-positive hematological malignancies. Thus, disclosed herein are methods of treating a subject having multiple myeloma, comprising administering to the subject a therapeutically effective amount of an anti-CD38 antibody and cyclophosphamide for a time sufficient to treat the multiple myeloma.
In some embodiments, the CD38-positive hematological malignancy is not light chain amyloidosis (AL). In such embodiments, the methods of treating a subject having a CD38-positive hematological malignancy comprise administering to the subject a therapeutically effective amount of an anti-CD38 antibody and cyclophosphamide for a time sufficient to treat the CD38-positive hematological malignancy, wherein the CD38-positive hematological malignancy is not light chain amyloidosis.
Any anti-CD38 antibody may be used in the disclosed methods. For example, the variable regions of the anti-CD38 antibodies may be obtained from existing anti-CD38 antibodies and optionally cloned as full length antibodies using standard methods. Exemplary antibody variable regions that bind CD38 that may be used are described in Int'l Pat. Pub. Nos. WO2005/103083, WO2006/125640, WO2007/042309, WO2008/047242, WO2012/092612, WO2006/099875, and WO2011/154453A1.
The anti-CD38 antibody can bind to a region of human CD38 comprising SKRNIQFSCKNIYR (SEQ ID NO: 2) and a region of human CD38 comprising EKVQTLEAWVIHGG (SEQ ID NO: 3). An anti-CD38 antibody binds to a region of human CD38 comprising SEQ ID NO: 2 and a region of human CD38 comprising SEQ ID NO: 3 when the antibody binds at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residues within SEQ ID NO: 2 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residues within SEQ ID NO: 3. In some embodiments, the anti-CD38 antibody binds at least one amino acid in a region of human CD38 comprising SEQ ID NO: 2 and at least one amino acid in a region of human CD38 comprising SEQ ID NO: 3. In some embodiments, the anti-CD38 antibody binds at least two amino acids in a region of human CD38 comprising SEQ ID NO: 2 and at least two amino acids in a region of human CD38 comprising SEQ ID NO: 3. In some embodiments, the anti-CD38 antibody binds at least three amino acids in a region of human CD38 comprising SEQ ID NO: 2 and at least three amino acids in a region of human CD38 comprising SEQ ID NO: 3. Antibodies binding to a region of human CD38 comprising SEQ ID NO: 2 and a region of human CD38 comprising SEQ ID NO: 3 may be generated, for example, by immunizing mice with peptides having amino acid sequences comprising SEQ ID NOs: 2 and 3 using standard methods and as described herein, and characterizing the obtained antibodies for binding to the peptides using, for example, ELISA or mutagenesis studies.
An exemplary anti-CD38 antibody that binds to a region of human CD38 comprising SEQ ID NO: 2 and a region of human CD38 comprising SEQ ID NO: 3 is DARZALEXTM (daratumumab), which comprises: a heavy chain complementarity determining region (CDR) 1, a heavy chain CDR2, and a heavy chain CDR3 of SEQ ID NOs: 6, 7, and 8, respectively, and a light chain complementarity determining region (CDR) 1, a light chain CDR2, and a light chain CDR3 of SEQ ID NOs: 9, 10, and 11, respectively, a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5; and a heavy chain amino acid sequence of SEQ ID NO: 12 and light chain amino acid sequence of SEQ ID NO: 13.
The anti-CD38 antibody can comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11. The anti-CD38 antibody can comprise a VH comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 4 and a VL comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 5. In some embodiments, the anti-CD38 antibody can comprise a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 5. The anti-CD38 antibody can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a light chain comprising the amino acid sequence of SEQ ID NO: 13.
The anti-CD38 antibody can comprise a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a VL comprising the amino acid sequence of SEQ ID NOs:15. The anti-CD38 antibody can comprise a VH comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 15 and a VL comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 15. In some embodiments, the anti-CD38 antibody can comprise a VH comprising the amino acid sequence of SEQ ID NO: 14 and a VL comprising the amino acid sequence of SEQ ID NOs:15. In some embodiments, for example, the anti-CD38 antibody can comprise mAb003 (described in U.S. Pat. No. 7,829,693, incorporated herein by reference).
The anti-CD38 antibody can comprise a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 16, and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 17. The anti-CD38 antibody can comprise a VH comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 16 and a VL comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 17. In some embodiments, the anti-CD38 antibody can comprise a VH comprising the amino acid sequence of SEQ ID NO: 16 and a VL comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, for example, the anti-CD38 antibody can comprise mAb024 (described in U.S. Pat. No. 7,829,693, incorporated herein by reference).
The anti-CD38 antibody can comprise a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 18, and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 19. The anti-CD38 antibody can comprise a VH comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 18 and a VL comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 19. In some embodiments, the anti-CD38 antibody can comprise a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, for example, the anti-CD38 antibody can comprise MOR-202 (MOR-03087) (described in US. Pat. No. 8,088,896, incorporated herein by reference).
The anti-CD38 antibody can comprise a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 20, and a light chain CDR1, a light chain CDR2, and a light chain CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 21. The anti-CD38 antibody can comprise a VH comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 20 and a VL comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 21. In some embodiments, the anti-CD38 antibody can comprise a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, for example, the anti-CD38 antibody can comprise isatuximab (described in U.S. Pat. No. 8,153,765, incorporated herein by reference). In some aspects, the VH and the VL of isatuximab may be expressed as IgG1/κ.
Antibodies that are substantially identical to those disclosed herein may be used in the disclosed methods. The term “substantially identical” means that the antibody heavy chain or light chain amino acid sequences are identical, or have “insubstantial differences,” compared to the antibody disclosed herein. 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. Antibody sequences can be compared, for example, by pairwise alignments using the default settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, Calif.). The protein sequences of the disclosed antibodies 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 GenomeQuest™ (GenomeQuest, Westborough, Mass.) suite using the default settings. Antibodies that are substantially identical to the disclosed antibodies can be generated, for example, by making conservative modifications to the amino acid sequences of the disclosed antibodies. “Conservative modifications” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequences. Conservative modifications include amino acid substitutions, additions, and deletions. “Conservative substitutions” are those in which the amino acid is replaced with an amino acid residue having a similar side chain The families of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), an amide (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643: 55-67; Sasaki et al., (1988) Adv Biophys 35: 1-24). Exemplary substitutions that can be made to the anti-CD38 antibodies used in the disclosed methods include, 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, including 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 and/or the light chain of the anti-CD38 antibody. Furthermore, any native residue in the heavy and/or light chain may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., Acta Physiol Scand Suppl 643: 55-67, 1998; Sasaki et al., Adv Biophys 35: 1-24, 1998). Suitable amino acid substitutions may be determined by those skilled in the art at the time such substitutions are desired Amino acid substitutions may be performed, for example, by PCR mutagenesis (as disclosed in 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) 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 and their ability to induce ADCC using methods described herein.
The anti-CD38 antibody can be of the IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the anti-CD38 antibody is of the IgG1 isotype. In some embodiments, the anti-CD38 antibody is of the IgG2 isotype. In some embodiments, the anti-CD38 antibody is of the IgG3 isotype. In some embodiments, the anti-CD38 antibody is of the IgG4 isotype.
Anti-CD38 antibodies used in the disclosed methods may also be selected de novo from, for example, a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., J Mol Biol 296: 57-86, 2000; Krebs et al., J Immunol Meth 254: 67-84, 2001; Vaughan et al., Nature Biotechnology 14: 309-314, 1996; Sheets et al., PITAS (USA) 95: 6157-6162, 1998; Hoogenboom and Winter, J Mol Biol 227: 381, 1991; Marks et al., J Mol Biol 222: 581, 1991). CD38 binding variable domains may be isolated, for example, from phage display libraries expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al (2010) J. Mol. Biol. 397: 385-96 and Int'l Pat. Pub. No. WO2009/085462. The antibody libraries may be screened for binding to human CD38 extracellular domain and the obtained positive clones may be further characterized and the Fabs isolated from the clone lysates, and subsequently cloned as full length antibodies. Such phage display methods for isolating human antibodies are established in the art. See for example: U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,571,698; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,580,717; U.S. Pat. No. 5,969,108; U.S. Pat. No. 6,172,197; U.S. Pat. No. 5,885,793; U.S. Pat. No. 6,521,404; U.S. Pat. No. 6,544,731; U.S. Pat. No. 6,555,313; U.S. Pat. No. 6,582,915; and U.S. Pat. No. 6,593,081.
Also disclosed are methods of treating a subject having a CD38-positive hematological malignancy, comprising administering to the subject a therapeutically effective amount of: an anti-CD38 antibody that competes for binding to CD38 with a reference antibody and cyclophosphamide,
wherein the reference antibody comprises:
In some embodiments, the CD38-positive hematological malignancy is multiple myeloma, and the methods comprise administering to the subject a therapeutically effective amount of:
an anti-CD38 antibody that competes for binding to CD38 with a reference antibody and cyclophosphamide,
wherein the reference antibody comprises any one of a) to q) above.
Antibodies may be evaluated for their competition with a reference antibody (such as references antibodies a) to q) above) for binding to CD38 using well known in vitro methods. In an exemplary method, CHO cells recombinantly expressing CD38 may be incubated with unlabeled reference antibody 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. In another exemplary method, an extracellular portion of human CD38 may be coated on the surface of an ELISA plate. Excess unlabeled reference antibody 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 antibody may be detected using horseradish peroxidase (HRP)-conjugated streptavidin and the signal detected using standard methods. In the competition assays, the reference antibody may be labeled and the test antibody may be unlabeled. The test antibody competes with the reference antibody when the reference antibody inhibits binding of the test antibody, or the test antibody inhibits binding of the reference antibody, by at least about 90%, 95%, or 100%. The epitope of the test antibody may further be defined, for example, by peptide mapping or hydrogen/deuterium protection assays using known methods, or by crystal structure determination.
The anti-CD38 antibody can induce killing of CD38-expressing cells by antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or apoptosis. In some embodiments, the anti-CD38 antibody induces killing of CD38-expressing cells by ADCC. In some embodiments, the anti-CD38 antibody induces killing of CD38-expressing cells by ADCP. In some embodiments, the anti-CD38 antibody induces killing of CD38-expressing cells by CDC. In some embodiments, the anti-CD38 antibody induces killing of CD38-expressing cells by apoptosis. In some embodiments, the anti-CD38 antibody induces killing of CD38-expressing cells by any combination of ADCC, ADCP, CDC, and apoptosis.
“Antibody-dependent cellular cytotoxicity,” “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 (NK) cells, monocytes, macrophages, and neutrophils via Fc gamma receptors (FcγR) expressed on effector cells. For example, NK cells express FcγRllla, whereas monocytes express FcγRI, FcγRII, and FcγRllla. Death of the antibody-coated target cell, such as CD38-expressing MM cell, occurs as a result of effector cell activity through the secretion of membrane pore-forming proteins and proteases. To assess ADCC activity of an anti-CD38 antibody, 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 may be detected by the release of a label (e.g., radioactive substrates, fluorescent dyes, or natural intracellular proteins) from the lysed cells. Exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Multiple myeloma cell lines or primary MM cells that express CD38 may be used as target cells. In an exemplary assay, MM cell lines engineered to express luciferase are incubated with anti-CD38 antibodies. Freshly isolated PBMC effector cells are added at a target:effector cell ratio of 40:1. 4 hours after addition of PBMC, luciferin is added and the resulting bioluminescent signal emitted from surviving MM cells can be determined within 20 minutes using a luminometer (SpectraMax, Molecular Devices), and the percentage ADCC of MM cells can calculated using the formula: % ADCC=1−(mean bioluminescent signal in the absence of PBMCs/mean bioluminescent signal in the presence of PBMCs)×100%. Anti-CD38 antibodies used in the disclosed methods can induce ADCC by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 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 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. In an exemplary assay, 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 μg/ml, and the survival of primary CD38+ MM cells may be determined by flow cytometry using techniques described in van der Veer et al., Haematologica 96: 284-290, 2011; van der Veer et al., Blood Cancer J 1(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 disclosed methods may induce CDC by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
“Antibody-dependent cellular phagocytosis,” or “ADCP,” refers to a mechanism of elimination of antibody-coated target cells by internalization by 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 molecules. Effector:target 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 may be identified with anti-CD11b and anti-CD14 antibodies coupled to a fluorescent label, and percent phagocytosis may be determined based on % GFP fluorescence in the CD11+CD14+ macrophages using standard methods. Anti-CD38 antibodies used in the disclosed methods may induce ADCP by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% .
The Fc portion of the anti-CD38 antibody may mediate antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement dependent cytotoxicity (CDC). 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. Typically, 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. Human IgG isotypes IgG1, IgG2, IgG3, and IgG4 exhibit differential capacity for effector functions. ADCC may be mediated by IgG1 and IgG3, ADCP may be mediated by IgG1, IgG2, IgG3, and IgG4, and CDC may be mediated by IgG1 and IgG3.
ADCC elicited by the anti-CD38 antibodies may be enhanced by certain substitutions in the antibody Fc region. In some embodiments, the anti-CD38 antibodies comprise a substitution in the Fc region at amino acid position 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, 430, or any combination thereof, wherein the residue numbering is according to the EU index (substitutions described in U.S. Pat. No. 6,737,056).
ADCC elicited by the anti-CD38 antibodies can also be enhanced by engineering an antibody oligosaccharide component. Human IgG1 or IgG3 are N-glycosylated at Asn297 with the majority of the glycans in the biantennary G0, G0F, G1, G1F, G2, or G2F forms. Antibodies produced by non-engineered CHO cells typically have a glycan fucose content (i.e. the amount of the fucose monosaccharide within the sugar chain at Asn297) 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 FcyRllla binding without altering antigen binding or CDC activity. Such modified antibodies 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 Lec13 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 ?1,6-fucosyltrasferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88: 901-908, 2004); or coexpression of β-1,4-N-acetylglucosaminyltransferase III and Golgi a-mannosidase II or a potent alpha-mannosidase I inhibitor, such as kifunensine (Ferrara et al., J Biol Chem 281: 5032-5036, 2006, Ferrara et al., Biotechnol Bioeng 93: 851-861, 2006; Xhou et al., Biotechnol Bioeng 99: 652-65, 2008).
In some embodiments, the anti-CD38 antibody can have a biantennary glycan structure with fucose content between about 0% to about 15%, for example 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%. In some embodiments, the anti-CD38 antibody can have a biantennary glycan structure with fucose content of about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%. Substitutions in the Fc region and reduced fucose content may enhance the ADCC activity of the anti-CD38 antibody.
Fucose content 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'l Pat. Pub. No. WO2008/0775462); 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 antibody to constituent peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), and subsequent separation, detection, and quantitation by HPLC-MS (UPLC-MS); and 5) separation of the antibody oligosaccharides from the antibody protein by specific enzymatic deglycosylation with PNGase F at Asn297. The oligosaccharides thus 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 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).
The anti-CD38 antibody may bind human CD38 with a range of affinities (KD). For example, the anti-CD38 antibody can bind CD38 with a KD equal to or less than about 1×10−8 M, for example 5×10−9 M, 1×10−9 M, 5×10−10 M, 1×10−10 M, 5×10−11 M, 1×10−11 M, 5×10−12 M, 1×10−12 M, 5×10−13 M, 1×10−13 M, 5×10−14 M, 1×10−14 M, 5×10−15 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. In some embodiments, the anti-CD38 antibody can bind to CD38 with an affinity of equal to or less than 1×10−8 M. In some embodiments, the anti-CD38 antibody can bind to CD38 with an affinity of equal to or less than 1×10−9 M.
Antibody affinity can be measured using KinExA instrumentation, ELISA, or competitive binding assays known to those skilled in the art. The measured affinity of a particular antibody/CD38 interaction may vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other binding parameters (e.g., KD, Kon, Koff) are typically made with standardized conditions and a standardized buffer. Those skilled in the art will appreciate that the internal error for affinity measurements for example using Biacore 3000 or ProteOn (measured as standard deviation, SD) may typically be within 5-33% for measurements within the typical limits of detection. Therefore the term “about” in the context of KD reflects the typical standard deviation in the assay. For example, the typical SD for a KD of 1×10−9 M is up to+0.33×10−9 M.
In some embodiments, the antibody that specifically binds CD38 is a non-agonistic antibody. A non-agonistic antibody that specifically binds CD38 refers to an antibody which upon binding to CD38 does not induce significant proliferation of a sample of peripheral blood mononuclear cells in vitro when compared to the proliferation induced by an isotype control antibody or medium alone.
In some embodiments, the non-agonistic antibody that specifically binds CD38 induces proliferation of peripheral blood mononuclear cells (PBMCs) in a statistically insignificant manner PBMC proliferation may be assessed by isolating PBMCs from healthy donors and culturing the cells at 1×105 cells/well in flat bottom 96-well plates in the presence or absence of a test antibody in 200 μl RPMI. After four day incubation at 37° C., 30 μl 3H-thymidine (16.7 μCi/ml) may be added, and culture may be continued overnight. 3H-thymidine incorporation may be assessed using a Packard Cobra gamma counter (Packard Instruments, Meriden, DT, USA), according to the manufacturer's instructions. Data may be calculated as the mean cpm (±SEM) of PBMCs obtained from several donors. Statistical significance or insignificance between samples cultured in the presence or absence of the test antibody is calculated using standard methods.
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. Such 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. They 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 agents, etc. The 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, 21st Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.
Suitable routes of administering the anti-CD38 antibody include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, oral, intranasal, intravaginal, rectal, or other means known to those skilled in the art. In some embodiments, for example, the anti-CD38 antibody is administered intravenously. The anti-CD38 antibody may be administered parentally by intravenous (IV) infusion or bolus injection. IV infusion can 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 of anti-CD38 antibody given to a patient having CD38-positive hematological malignancy is that sufficient to alleviate or at least partially arrest the disease being treated (“therapeutically effective amount”) and includes from about 0.005 mg to about 100 mg/kg, e.g. about 0.05 mg to about 30 mg/kg or about 5 mg to about 25 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg, or about 24 mg/kg. Suitable doses include, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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 of the anti-CD38 antibody 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/m2. Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) may be administered to treat MM, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses may be given.
The administration of the anti-CD38 antibody 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. For example, the anti-CD38 antibody may be administered at 8 mg/kg or at 16 mg/kg at weekly intervals 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 by maintenance therapy, such as, e.g., once a week for a period of 6 months or more. For example, the anti-CD38 antibodies may be provided as a daily dosage in an amount of about 0.1 mg/kg to about 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.
The anti-CD38 antibodies 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 and cyclophosphamide may be administered over any convenient timeframe. In some embodiments, the anti-CD38 antibody and the cyclophosphamide are administered simultaneously. In some embodiments, the anti-CD38 antibody and the cyclophosphamide are administered sequentially in any order. In some aspects, the cyclophosphamide is administered prior the anti-CD38 antibody.
In some embodiments, cyclophoshpamide is administered 300 mg/m2 weekly. In some embodiments, cyclophosphamide is administered 150 mg/m2 weekly. In some embociments, cyclophoshpamide is administered between 150 mg/m2 and 300 mg/m2 weekly. In some embodiments, cyclophsophamide is administered orally. In some embodiments, cyclophsophamide is administered for a period of 1, 2, 3, 4, 5 or 6 months. In some embodiments, cyclophosphamide si administered 40 mg/kg to 50 mg/kg intravenously divided into doses over 2-5 days. In some embodiments. In some embodiments, cyclophsophamide is administered 10-15 mg/kg orally every 7-10 days. In some embodiments, cyclophosphamide is administered 3 mg/kg-5 mg/kg orally twice weekly. In some embodiments, cyclophosphamide is administered 1 mg/kg per day to 5 mg/kg per day orally.
The anti-CD38 antibody and cyclophosphamide may be administered to a patient on the same day. Alternatively, the anti-CD38 antibody and cyclophosphamide may be administered on alternating days or alternating weeks or months, and so on. In some methods, the anti-CD38 antibody and cyclophosphamide 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. In some methods, 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 cyclophosphamide, consisting of a number of doses. A recovery period of 1, 2, or several days or weeks may be used between administration of the anti-CD38 antibody and cyclophosphamide.
The anti-CD38 antibody in combination with cyclophosphamide 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.
The anti-CD38 antibody may be administered as a pharmaceutical composition comprising the anti-CD38 antibody and a hyaluronidase. In some embodiments, the pharmaceutical composition comprising the anti-CD38 antibody and a hyaluronidase is administered subcutaneously. The concentration of the anti-CD38 antibody in the pharmaceutical composition may be about 20 mg/ml. The pharmaceutical composition may comprise between about 1,200 mg to about 1,800 mg of the anti-CD38 antibody. In some embodiments, the pharmaceutical composition may comprise about 1,200 mg of the anti-CD38 antibody. In some embodiments, the pharmaceutical composition may comprise about 1,600 mg of the anti-CD38 antibody. In some embodiments, the pharmaceutical composition may comprise about 1,800 mg of the anti-CD38 antibody. The pharmaceutical composition may comprise between about 30,000 U to about 45,000 U of the hyaluronidase. In some embodiments, the pharmaceutical composition may comprise about 1,200 mg of the anti-CD38 antibody and about 30,000 U of the hyaluronidase. In some embodiments, the pharmaceutical composition may comprise about 1,800 mg of the anti-CD38 antibody and about 45,000 U of the hyaluronidase. In some embodiments, the pharmaceutical composition may comprise about 1,600 mg of the anti-CD38 antibody and about 30,000 U of the hyaluronidase. In some embodiments, the pharmaceutical composition may comprise about 1,600 mg of the anti-CD38 antibody and about 45,000 U of the hyaluronidase.
The pharmaceutical composition may comprise the hyaluronidase rHuPH20 having the amino acid sequence of SEQ ID NO: 22, which is a recombinant hyaluronidase (HYLENEX® recombinant) described in Int'l Pat. Pub. No. WO2004/078140.
The administration of the pharmaceutical composition comprising the anti-CD38 antibody and the hyaluronidase may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose. For example, the pharmaceutical composition comprising the anti-CD38 antibody and the hyaluronidase may be administered once weekly for eight weeks, followed by once in two weeks for 16 weeks, followed by once in four weeks. The pharmaceutical compositions to be administered may comprise about 1,200 mg of the anti-CD38 antibody and about 30,000 U of hyaluronidase, wherein the concentration of the antibody that specifically binds CD38 in the pharmaceutical composition is about 20 mg/ml. The pharmaceutical composition comprising the anti-CD38 antibody and the hyaluronidase may be administered subcutaneously to the abdominal region.
The pharmaceutical composition comprising the anti-CD38 antibody and the hyaluronidase may be administered in a total volume of about 80 ml, 90 ml, 100 ml, 110 ml, or 120 ml. For administration, 20 mg/ml of the anti-CD38 antibody in 25 mM sodium acetate, 60 mM sodium chloride, 140 mM D-mannitol, 0.04% polysorbate 20, pH 5.5 may be mixed with rHuPH20, 1.0 mg/mL (75-150 kU/mL) in 10 mM L-Histidine, 130 mM NaCl, 10 mM L-Methionine, 0.02% Polysorbate 80, pH 6.5 prior to administration of the mixture to a subject.
The methods can further comprise administering a therapeutically effective amount of a corticosteroid. Exemplary corticosteroids include, for example, a glucocorticoid (cortisol, for example), prednisone, or dexamethasone. In some embodiments, the corticosteroid is dexamethasone. Thus, the methods can comprise administering to the subject a therapeutically effective amount of an anti-CD38 antibody, cyclophosphamide, and dexamethasone for a time sufficient to treat the CD38-positive hematological malignancy.
In some embodiments, corticosteroid is administered 80 mg weekly. In some embodiments, corticosteroid is administered 40 mg weekly. In some embodiments, corticosteroid is administered twice a week. In some embodiments, corticosteroid is administered once a week. In some embodiments, corticosteriod is administered orally. In some embodiments, corticosteroid is administered intravenously.
The methods can further comprise administering a therapeutically effective amount of a non-corticosteroid chemotherapeutic agent. Exemplary non-corticosteroid chemotherapeutic agents include glutamic acid derivatives or proteasome inhibitors. Exemplary glutamic acid derivatives include thalidomide (Thalomid®) or a thalidomide analog, e.g. CC-5013 (lenalidomide, Revlimid™), pomalidomide or CC4047 (Actimid™). In some embodiments, the glutamic acid derivative is lenalidomide. Thus, the methods can comprise administering to the subject a therapeutically effective amount of an anti-CD38 antibody, cyclophosphamide, and lenalidomide for a time sufficient to treat the CD38-positive hematological malignancy.
Exemplary proteasome inhibitors include bortezomib (Velcade®), carfilzomib, ixazomib, or vinca alkaloid, such as vincristine, or an anthracycline, such as doxorubicin. In some embodiments, the proteasome inhibitor is bortezomib. Thus, the methods can comprise administering to the subject a therapeutically effective amount of an anti-CD38 antibody, cyclophosphamide, and bortezomib for a time sufficient to treat the CD38-positive hematological malignancy.
In some embodiments, bortezomib is administered 1.5 mg/m2 once a week. In some embodiments, bortezomib is administered 1.3 mg/m2 once a week. In some embodiments, bortezomib is administered at about 1.3 mg/m2 to about 1.5 mg/m2 once a week. In some embodiments, bortezomib is administered 1.3 mg/m2 twice a week. In some embodiments, bortezomib is administered by subcutaneous injection.
In some embodiments, the methods can comprise administering to the subject a therapeutically effective amount of an anti-CD38 antibody, cyclophosphamide, dexamethasone, and lenalidomide for a time sufficient to treat the CD38-positive hematological malignancy. In some embodiments, the methods can comprise administering to the subject a therapeutically effective amount of an anti-CD38 antibody, cyclophosphamide, dexamethasone, and bortezomib for a time sufficient to treat the CD38-positive hematological malignancy. In some embodiments, the methods can comprise administering to the subject a therapeutically effective amount of an anti-CD38 antibody, cyclophosphamide, dexamethasone, lenalidomide, and bortezomib for a time sufficient to treat the CD38-positive hematological malignancy.
In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of an anti-CD38 antibody comprising a a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 11 and cyclophosphamide for a time sufficient to treat the CD38-positive hematological malignancy, wherein
In some embodiments,
In some embodiments, cyclophosphamide is administered orally.
In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of an anti-CD38 antibody comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 11, cyclophosphamide, bortezomib and dexamethasone for a time sufficient to treat the CD38-positive hematological malignancy, wherein
In some embodiments,
In some embodiments, the anti-CD38 antiboyd is administered intravenously.
In some embodiments, cyclophosphamide is administered orally.
In some embodiments, bortezomib is administered subcutaneously.
In some embodiments, dexamethasone is administered orally.
In some embodiments, the anti-CD38 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-CD38 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a light chain comprising the amino acid sequence of SEQ ID NO: 13.
In some embodiments, the methods can comprise administering to the subject a therapeutically effective amount of an anti-CD38 antibody, cyclophosphamide, dexamethasone, and bortezomib for a time sufficient to treat the CD38-positive hematological malignancy.
In some embodiments, the subject has one or more of the following chromosomal abnormalities:
The subject can have naive multiple myeloma, relapsed multiple myeloma, or refractory multiple myeloma. In some embodiments, the subject has high risk refractory and/or relapsed multiple myeloma.
The methods of treatment can improve one or more outcome measurements of the subject compared to a subject receiving cyclophosphamide, bortezomib or dexamethasone, of combinations thereof. Exemplary outcome measurements comprise progression-free survival, overall response rate, very good partial response or better, complete response or better, or any combination thereof.
The anti-CD38 antibody and cyclophosphamide may be administered together with autologous hematopoietic stem cell transplant (AHSC). The anti-CD38 antibody and cyclophosphamide may be administered after autologous hematopoietic stem cell transplant (AHSC). The anti-CD38 antibody, cyclophosphamide, dexamethasone and bortezomib may be administered together with autologous hematopoietic stem cell transplant (AHSC). The anti-CD38 antibody, cyclophosphamide, dexamethasone and bortezomib may be administered after autologous hematopoietic stem cell transplant (AHSC).
Provided here are methods of enhancing daratumumab-mediated antbody-dependent cellular phagocytosis (ADCP) in a subject, comprising administering to the subject daratumumab and cyclophosphamide.
In some embodiments, cyclophosphamide is administered at a dose of about 150-300 mg/m2
In some embodiments, daratumumab is administered at a dose of about 16 mg/kg.
“Enhancing” refers to measaruable increase in daratumumab-mediated ADCP.
The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.
Cells: The multiple myeloma cell line, MM1S, was sub-cultured in RPMI 1640 media supplemented with 10% FBS and 50 IU/ml penicillin and 50 μg/ml Streptomycin. For experiments, cells were plated at 2×105 cells/ml and treated with increasing doses of cyclophosphamide ranging from 0-10 μM. Additionally, cells were treated with combinations of cyclophosphamide and lenalidomide (10 μM, 1 μM) as well as bortezomib (10 μM, 1 nM). THP-1 cells were sub-cultured in RPMI 1640 media supplemented with 10% FBS and 50 IU/ml Penicillin and 50 μg/ml streptomycin. Cells were plated at a cell density of 2×105 cells/ml for experiments.
MM1 S conditioned media: MM 1 S conditioned media was generated by incubating MM1S cells for 24 hrs with cyclophosphamide, lenalidomide and/or bortezomib at concentrations indicated above, after which the media was removed and the cells washed thoroughly. Fresh media was added and the cells were further incubated for 24 hours after collecting the conditioned media. Flow Cytometry Analysis of cell surface expression of CD38 and CD47: Following treatment of MM1S cells with the indicated doses of cyclophosphamide, cells were harvested by pipetting into single cell suspensions in FACS Buffer (PBS/2%FBS) after 24 hours. All antibodies were purchased from BD Pharmingen™ unless otherwise stated. Cells were stained with anti-CD38 (APC-conjugated) and anti-CD47 (FITC-conjugated, eBiosciences) antibodies and the level of expression of CD38 and CD47 was assessed. Sytox advanced reagents were used to assess cell death. Cells were analysed by flow cytometry. Sytox positive cells were excluded from analysis. The mean fluorescent intensity was calculated by expressing the fluorescent intensity of each sample relative to the cell size.
Assessment of FcyRI and FcyRIIa/b expression on macrophages: The THP-1 human monocytic cell line was plated at a density of 2×105 cells/ml. Cells were incubated with conditioned media from untreated (CTX 0) or cyclophosphamide treated MM1S cells at the indicated concentrations. FcγRI and FcγRlla/b expression was assessed by flow cytometry 48 hours later using anti-FcγRI (APC-conjugated) and anti- FcγRIIa/b (PE-conjugated) antibodies. The mean fluorescent intensity was calculated by expressing the fluorescent intensity of each sample relative to the cell size and subtracting the values for the isotype control/unstained samples.
Antibody dependent cellular cytotoxicity or phagocytosis assays: THP-1 macrophages were seeded into 96-well plates and allowed to adhere. MM target cells (MM1S cells) were labelled with calcein-AM (Invitrogen, C-3100) and added to the control or conditioned macrophages at an effector:target (E:T) ratio of 2:1. MM cell were pre-incubated in the presence of 1 μg/ml IgG1 anti-CD38 antibody (daratumumab) or the relevant isotype control and then added to cultures. After 18h incubation, cells were imaged by fluorescent microscopy and then detached by pipetting to single cell suspensions. Antibody-dependent phagocytosis was assessed with flow cytometry (Accuri), and quantified as follows: Phagocytosis was quantified by frequency of remaining target cells (Calcein-AM positive) with and without Ab treatment and isotype control. Elimination of target cells was calculated using the following formula: % daratumumab Specific Cell Clearance=100-100*(% calcein labelled MM cells (DARA treated)/% calcein labelled MM cells (isotype control). To confirm that the clearance was mediated by macrophage mediated phagocytosis, experimental wells containing macrophages that were pre-incubated with 1μg/ml Cytochalasin D were included. Cytochalasin D inhibits actin polymerisation and has been shown to inhibit cell phagocytosis.
CD38 expression on multiple myeloma cells (MM1S) was induced by cyclophosphamide alone or cyclophosphamide in combination with lenalidomide or bortezomib.
CD47 expression on multiple myeloma cells (MM1S) was reduced by cyclophosphamide.
Conditioned media from cyclophosphamide-treated MM1S cells induced FcγRI and FcγRlla/b expression on macrophages.
Cyclophosphamide, lenalodomide and dexamethasone augmented daratumumab-mediated tumor cell killing by macrophages.
100−100*(The frequency of calcein stained MM1S cells in the presence of 1 μg/ml daratumumab/
The frequency of calcein stained MM1S cells in the presence of Isotype control). Thus, macrophage-mediated anti-myeloma activity of daratumumab was potentiated by cyclophosphamide, lenalidomide, bortezomib or cyclophosphamide in combination with lenalidomide or bortezomib.
Cytochalasin D reduced cyclophosphamide, lenalodomide and dexamethasone augmented daratumumab-mediated tumor cell killing by macrophages. Co-cultures of MM1S and THP-1 cells were incubated in the presence of cytochalasin to assess if daratumumab mediated MM1S killing by ADCP. Cytochalasin D inhibits actin polymerisation and has been shown to inhibit cell phagocytosis.
100−100*(The frequency of calcein stained MM1S cells in the presence of 1 μg/ml daratumumab/
The frequency of calcein stained MM1S cells in the presence of Isotype control). Potentiation of anti-myeloma activity of daratumumab with combination of cyclophosphamide or cyclophosphamide in combination with lenalidomide was therefore dependent on macrophage mediated ADCP.
A Phase 2 study evaluating the combination of daratumumab and oral cyclophosphamide, bortezomib, and dexamethasone (Dara-CyBorD) in subjects with previously untreated multiple myeloma, irrespective of eligibility for high-dose chemotherapy (HDT) and autologous stem cell transplant (ASCT), or relapsed multiple myeloma following one prior line of therapy is conducted. Clinical trials identificationi number NCT02951819.
The primary objective is to evaluate the complete response+ very good partial response (CR+VGPR) rate following 4 cycles of induction therapy with daratumumab plus CyBorD (Dara-CyBorD), in previously untreated subjects, and in relapsed subjects with multiple myeloma, as defined by the International Myeloma Working Group (IMWG) criteria.
The secondary objectives are to evaluate, in previously untreated subjects and in relapsed subjects with multiple myeloma:
An exploratory objective is to evaluate the clinical efficacy of Dara-CyBorD in molecular subgroups, including: dell7p, de113, t(4;14), t(11;14), t(14;16).
The primary endpoint is the proportion of subjects achieving CR+VGPR response following 4 cycles of induction therapy with Dara-CyBorD, in previously untreated subjects, and in relapsed subjects with multiple myeloma, as defined by the IMWG criteria.
The secondary efficacy endpoints include:
Clinical efficacy of Dara-CyBorD in high-risk molecular subgroups including: dell7p, del13, t(4;14), t(11;14), t(14;16).
The primary hypothesis of this study is that CR+VGPR rate following 4 cycles of induction therapy with Dara-CyBorD in previously untreated subjects is higher than 60%, and in relapsed subjects with multiple myeloma is higher than 30%, as defined by the IMWG criteria.
This is a multicenter, single-arm, open-label, Phase 2 study evaluating the combination of daratumumab and oral cyclophosphamide, bortezomib, and dexamethasone (Dara-CyBorD) in subjects with previously untreated multiple myeloma, irrespective of eligibility for high-dose chemotherapy (HDT) and autologous stem cell transplant (ASCT), or relapsed multiple myeloma following one prior line of therapy. Relapsed multiple myeloma following one line of therapy is defined as having achieved at least a PR with first-line therapy before progression. Approximately 100 subjects will be enrolled into this study with at least 40 previously untreated multiple myeloma subjects and at least 40 subjects with relapsed multiple myeloma following one prior line of therapy.
Treatment phases consist of induction therapy with 4 to 8 cycles of Dara-CyBorD (this range allows for local standard of care and physician discretion), consolidation therapy with HDT/ASCT for eligible subjects, and maintenance therapy. Following induction and/or consolidation therapy with ASCT, subjects will receive maintenance therapy with daratumumab alone for 12 cycles (of 28 days each), or until disease progression (whichever occurs first). Maintenance therapy for subjects who have ASCT should begin approximately 90 days after ASCT. The follow-up period for each subject, after completion of maintenance therapy with daratumumab, will continue through 36 months following the start of induction therapy. Throughout the study, subjects will be monitored closely for adverse events, laboratory abnormalities, and clinical response. Disease response and progression will be based on assessments from IMWG Guidelines. Efficacy assessments include: M-protein measurements (serum and urine), immunofixation (serum and urine), serum free light chains, serum calcium corrected for albumin, serum immunoglobulins, examination of bone marrow aspirate or biopsy, and skeletal survey/documentation of extramedullary plasmacytomas. A Data Monitoring Committee will be commissioned for this study.
Screening for eligible subjects will be performed within 28 days before enrollment. The inclusion and exclusion criteria for enrolling subjects in this study are described below.
Each potential subject must satisfy all of the following criteria to be enrolled in the study:
Any potential subject who meets any of the following criteria will be excluded from participating in the study.
Approximately 100 subjects will be assigned to receive Dara-CyBorD as induction on a 28-day cycle length. All subjects will receive 4 to 8 cycles of oral cyclophosphamide 300 mg/m2 on Days 1, 8, 15, and 22; subcutaneous (SC) bortezomib 1.5 mg/m2 on Days 1, 8, and 15; and oral or intravenous (IV) dexamethasone 40 mg weekly. Subjects will concurrently receive daratumumab on 28-day cycles (Table 1). The initial dose of daratumumab will be given as a “split dose” of 8 mg/kg IV on C1D1 and C1D2. Starting Cycle 1 Day 8 through the completion of Cycle 2, daratumumab will be given weekly at 16 mg/kg IV. For Cycle 3 to Cycle 6, subjects will receive daratumumab 16 mg/kg IV once every 2 weeks. From Cycle 7 on, subjects will receive daratumumab 16 mg/kg IV once every 4 weeks, whether in the last induction cycles with CyBorD, or alone during the maintenance phase . Regardless of the number of induction cycles given, all eligible subjects are to receive 12 cycles of maintenance therapy with daratumumab 16 mg/kg IV monotherapy, given on 28-day cycles.
The induction regimen is the same dose, schedule and cycle length for all subjects in both study populations (previously untreated myeloma or relapsed myeloma with one prior line of therapy), although the number of induction cycles may vary from 4 to 8, based on local standard of care and physician discretion. Regardless of the number of induction cycles given, all subjects will receive maintenance therapy with daratumumab 16 mg/kg on Day 1 of a 28-day cycle for 12 cycles.
aSee dexamethasone dosing table
+None needed unless subject experiences an infusion reaction
Disease response and progression will be assessed based on IMWG Guidelines. Daratumumab detection on serum immunofixation (IFE) has been demonstrated in subjects treated with 16 mg/kg, and may interfere with the traditional IMWG criteria of negative serum IFE for CR or stringent CR (sCR). To mitigate this interference, the sponsor has developed a reflex assay that utilizes anti-idiotype antibody to bind daratumumab and confirm its interference on IFE. For all subjects with VGPR and a negative M protein by serum M-protein quantitation by electrophoresis (SPEP), reflex IFE testing will be performed to confirm the presence of daratumumab on IFE. In addition, for subjects who have an SPEP≤0.2 g/dL and detectable IgG kappa myeloma during the study, reflex testing will also be performed to determine whether the para-protein identified on SPEP/IFE is monoclonal daratumumab or the subject's endogenous myeloma protein.
Disease evaluations must be performed on Day 1 of each cycle during the induction phase with Dara-CyBorD. Disease evaluation should be performed following ASCT, on Day 1 of the first maintenance cycle dose, for subjects who undergo ASCT. For all subjects, disease evaluation will be done on Day 1 of the maintenance phase with daratumumab and then on Day 1 of every other cycle (approximately every 56 days) or, if there are concerns for relapse, sooner.
Safety evaluations will include adverse event monitoring, clinical laboratory testing (hematology and serum chemistry), pregnancy testing, electrocardiogram monitoring, vital sign measurements, physical examinations, and ECOG performance status.
Since daratumumab interferes with the indirect antiglobulin test (TAT), subjects will receive a subject identification wallet card for the study that includes the blood profile (ABO, Rh, and IAT) determined before the first infusion of daratumumab along with information on the IAT interference for healthcare providers/blood banks.
This study is a Phase Ib open label, single arm, adaptive multicentre trial. Patients with newly diagnosed Multiple Myeloma (MM) will be treated with Cyclophosphamide-Bortezomib-Dexamethasone (CyBorD) in combination with Daratumumab (DARA).
The study will consist of 2 phases: The Screening Phase will extend up to 28 days prior to Cycle 1, Day 1. The Treatment Phase will be conducted in 2 parts and will extend from Cycle 1 Day 1 until treatment discontinuation. Treatment Phase, Part 1:Induction/Transplantation/Consolidation Phase. The consolidation phase of treatment will begin approximately 30-60 days after Autologous Stem Cell Transplantation (ASCT), when the patient has recovered sufficiently and engraftment is complete. Treatment Phase, Part 2: Maintenance Phase treatment until a maximum duration of 2 years, documented disease progression, death, loss to follow-up, or withdrawal of consent, whichever occurs first.
Each patient must sign an Informed Consent Form (ICF) indicating that he or she understands the purpose of and procedures required for the study and is willing to participate in the study.
Screening, first within 10 to 14 days prior to first dose and the second within 24 hours prior to first dose.
Daratumumab [DARA] (16 mg/kg) will be administered by intravenous (IV) infusion once every week for 8 weeks (CyBorD Induction Cycle 1-2), then once every 2 weeks for 8 weeks (CyBorD Induction Cycle 3-4), and following transplantation once every 2 weeks for 8 weeks (CyBorD Consolidation Cycle 5-6). For the maintenance phase, patients with high risk disease will receive bortezomib as SC injection once every two weeks plus DARA (16 mg/kg IV) once every four weeks. The dose of bortezomib used for maintenance should be the maximum tolerated dose at the end of consolidation treatment but no more than 1.3 mg/m2. Patients with standard risk disease will receive DARA (16 mg/kg IV) once every four weeks. Patients will receive maintenance treatment for a maximum duration of 2 years, documented disease progression, death, loss to follow-up, or withdrawal of consent, whichever occurs first.
Cyclophosphamide will be administered 150-300 mg/m2 orally on days 1, 8, 15, 22 for four 28-day induction cycles (Cycles 1-4), and two consolidation cycles (Cycles 5-6).
Bortezomib will be administered at either a dose of 1.3 mg/m2(dose level 1 and 2) or 1.5 mg/m2 (dose level 3) as a SC injection once per week (Days 1, 8, 15 and 22) for four 28-day induction cycles (Cycles 1 to 4), and two consolidation cycles (Cycles 5 and 6).
Dexamethasone: Induction phase: Cycles 1-2: Dexamethasone will be administered orally on days 1-2, 8-9, 15-16, 22-23 at 40mg per day (i.e. total of 320mg per cycle). Cycles 3-4: Dexamethasone will be administered orally on days 1-2 at 40mg per day and subsequently 20 mg per day on days 8-9, 15-16, 22-23 (i.e. total of 200mg per cycle). Consolidation phase: Cycles 5-6: Dexamethasone will be administered orally on days 1-2, 8-9, 15-16, 22-23 at 20 mg per day (i.e. total of 160 mg per cycle). Maintenance phase: In the maintenance phase, dexamethasone at 20 mg or substitutions will be administered either intravenously or orally as pre-medication on DARA infusion days, 1 hour or less prior to the DARA infusion. Dexamethasone tablets are to be taken with or immediately after a meal or snack, preferably in the morning.
Ethics: Pre-screened newly diagnosed MM patients were enrolled into the sub-clinical study by informed consent according to predetermined protocols and standard operating procedures for sample draw and included extraction of relevant details from medical records. Follow up samples were also attained after 24 hours Cyclophosphamide (150-300 mg/m2) treatment as outlined in Cyclophosphamide-Bortezomib-Dexamethasone with Daratumumab (Dara-CyBorD) clinical trial (NCT02955810) protocol. The sub-study protocol, including the information leaflets and consent forms, the potential risks and measures to prevent incidences of risk was reviewed and approved by the Galway University Hospital Ethics Committee.
Samples Isolation: Isolation of mononuclear cells (MC) from PB and BM aspirates were performed by layering 3mL EDTA-anti-coagulated sample over 3mL endotoxin-free Ficoll Plaque density-gradient medium (GE Healthcare, Little Chalfont, United Kingdom) in 15-mL Falcon tubes (Sarstedt, North Rhine-Westphalia, Niimbrecht, Germany), and centrifuged at 420 relative centrifugal force (RCF) for 22 minutes at 4° C. without braking. The “buffy coat” layer was isolated and washed twice with Fluorescence-activated cell sorting (FACS buffer) [2% foetal calf serum (Lonza, Basel, Switzerland), PBS and 0.05% NaN3 (Sigma-Aldrich, St. Louis, Mo., USA)]. The MCs were pelleted by centrifugation at 300 RCF for 10 minutes at 4° C. with full acceleration and brake. Cells were counted using a haemocytometer and 5×105 cells per sample were stained for phenotypic characterisation by flow cytometry
Characterisation Studies: Plasma cells: Cell surface expression of CD38, CD47, CD319 (SLAMF7), B-cell maturation antigen (BCMA), MIC A/B, Programmed death-ligand (PDL) 1, PDL2, CD54, CD11a and CD48 were analysed on CD45-CD19-CD138+ Plasma cells by flow cytometry. Macrophages: Cell surface expression of CD32, CD64, SIRPla, PD-1 and CD163 were analysed on CD45+CD33+CX3CR1+−CD56-CD14+ macrophages by flow cytometry. Cells were stained with flourochrome-conjugated anti-human monoclonal antibodies for 20 minutes at 4° C. All antibodies were purchased from BD Biosciences unless otherwise stated. Cells were washed with FACS buffer and the level of surface expression was assessed for each cell population by flow cytometry. The mean fluorescent intensity (MFI) was calculated by expressing the fluorescent intensity of each sample relative to the cell size. Fluorescence compensation was set using single-stained controls, and matching median compensation algorithms were applied. Fluorescence minus one controls were used to set analysis gates controls and data were analyzed using Diva v8.0.1 acquisition software (BD Biosciences) or FlowJo® 7.6.5 software (TreeStar Inc., Olten, Switzerland).
Antibody Dependant cellular phagocytosis (ADCP): To determine the number of macrophages per sample, 50 μl of 5×106 cells/mL concentration of MC isolated from PB and BM were diluted using 150 μl FACS buffer. Based on forward and side scatter profiles, the total number of macrophages were determined by timed acquisition events per unit volume using an Accuri™ C6. PB and BM isolated macrophages were seeded into 96-well plates and allowed to adhere. MM target cells (MM1S cells) were labelled with CFSE (Thermofisher Scientific, Mass. USA) and pre-incubated in the presence of 1 μg/ml IgG1 anti-CD38 antibody (daratumumab) or the relevant isotype control. MM cells were then added to cultures at an effector:target (E:T) ratio of 2:1. After 12 h incubation, cells were imaged by fluorescent microscopy and then detached by pipetting to single cell suspensions. Cells were washed, incubated with mouse anti-human CD45-APC (BD Biosciences, San Jose, CA) and mouse anti-human CD14-PerCP-Cy5.5 (Miltenyi Biotech, Cologne, Germany) for 15 mins, then washed and re-suspended in 200 μl FACS buffer for sample acquisition. Macrophage and MM1S cells were gated using CFSE+ and CD45+CD14+ cells gating. Cell frequency was determined using the BD Canto IITM flow cytometer. Antibody-dependent phagocytosis was assessed and quanti?ed as follows: Phagocytosis was quanti?ed by frequency of remaining target cells (CFSE positive) with and without Ab treatment or isotype control. To confirm that the clearance was macrophage mediated phagocytosis, experimental wells containing macrophages that were pre-incubated with 1 μg/ml Cytochalasin D were included. Cytochalasin D inhibits actin polymerisation and has been shown to inhibit cell phagocytosis. ADCP mediated clearance was calculated using the following formula: 100-100*(Proportion of live Dara-treated MM1S cells in untreated macrophage co-culture/ Proportion of live Dara-treated MM1S cells in CytoD conditioned macrophage co-culture).
Patients enrolled in the clinical trial were treated with (1) cyclophosphamide 150 mg/m2 (first 3 patients only) then 300 mg/m2 weekly (remaining patients), (2) bortezomib 1.3 mg/m2 (first 6 patient only) or 1.5 mg/m2 (remaining patients) and weekly dexamethasone, as part of a 28 day cycle. Daratumumab was administered as described above. For Cycle 1 day 1, bortezomib was administered 24 hours after cyclophosphamide administration.
Plamsa cell and macrophage phenotypes in patient peripheral blood (PB) and bone marrow (BM) at baseline and after 24 hour treatment (patient had received cyclophosphamide, daratumumab and dexamethasone) were analyzed.
CD47 surface expression was evaluated on CD45−CD19−CD138+ plasma cells and found to be decreased on the plasma cells both in PB (
CD64 (FcyRI) surface expression was increased in macrophage precursors (CD45+CD56−CD33+CX3CR1+CD14+ cells) identified in patient PB (
CD32 (FcγRIIA) surface expression was not changed in the macrophage precursors (CD45+CD56−CD33+CX3CR1+CD14+ cells) in PB 24 hours post-treatment with cyclophosphamide, dexamethasone and daratumumab (
Cyclophosphamide also augmented tumour cytotoxicity by macrophages in co-culture of PB or BM-derived macrophages and multiple myeloma cells.
Co-cultures of MM1S and CD45+CD14+ macrophages from PB and BM were incubated in the presence of Cytochalasin D to assess if Daratumumab mediated MM1S killing occurs via ADCP. ADCP accounted for>15% of tumour cell clearance and was enhanced to>25% 24 hours after treatment with cyclophosphamide in BM (
To note, Daratumumab (16 mg/kg) infusion was administered to the patient 1 hour after cyclophosphamide. The minimal difference between Dara and Isotype within the ADCP assay (
Together, these data highlight that a detectable increase in CD64 surface expression by macrophages isolated from adults following cyclophosphamide treatment is coupled with an increased capability for ADCP-mediated cytotoxicity of MM cells. No changes in macrophage expression was observed in the “don't eat me receptor” inhibitory receptor SIRP1 however a decreased in the antigen for this receptor, CD47 was decreased on plasma cells in these patients. This data provides a strong rationale that cyclophosphamide can potentiate the anti-tumour effect of Dara by augmentation of macrophage-mediated ADCP.
Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.
The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62415749 | Nov 2016 | US |
