Subcutaneous formulations of anti-CD38 antibodies and uses thereof

Abstract

The present invention relates to subcutaneous formulations of anti-CD38 antibodies and uses thereof.

Description

[0001] This application is a divisional application of an invention application filed on November 1, 2016, with Chinese national application number 201680077740.2 and invention name “Subcutaneous preparation of anti-CD38 antibodies and their uses”. Technical Field

[0002] The present invention relates to a subcutaneous preparation of an anti-CD38 antibody and uses thereof. Background Art

[0003] CD38 is a multifunctional protein whose functions include receptor-mediated adhesion and signal transduction, as well as calcium mobilization via its extracellular enzyme activity, catalyzing the formation of cyclic ADP ribose (cADPR) and ADPR. CD38 mediates the secretion of cytokines and the activation and proliferation of lymphocytes (Funaro et al., J Immunol 145:2390-6, 1990; Terhorst et al., Cell 771-80, 1981; Guse et al., Nature 398:70-3, 1999). CD38 also regulates extracellular NAD via its NAD glycohydrolase activity. + levels, which is involved in regulating the regulatory T cell compartment (Adriouch et al., 14:1284-92, 2012; Chiarugi et al., Nature Reviews 12:741-52, 2012). 2+ In addition to signal transduction by , CD38 signaling also occurs through cross-responses with antigen-receptor complexes or other types of receptor complexes (such as MHC molecules) on T cells and B cells, so that CD38 is not only involved in several cellular responses, but also in the conversion and secretion of IgG1. CD38 is expressed on various malignant cells.

[0004] Anti-CD38 antibodies for the treatment of multiple myeloma and other heme malignancies are under development. The antibodies can be injected or infused via the intravenous (IV) route. The amount of antibody that can be administered via the intravenous route is limited by the physicochemical properties of the antibody, especially its solubility and stability in a suitable liquid formulation and the volume of the infusion fluid.

[0005] Therefore, there is a need for additional anti-CD38 antibody preparations and pharmaceutical compositions. Summary of the invention

[0006] The present invention provides a pharmaceutical composition comprising an anti-CD38 antibody and hyaluronidase.

[0007] The present invention also provides a pharmaceutical composition comprising an anti-CD38 antibody and a hyaluronidase having the amino acid sequence of SEQ ID NO:22.

[0008] The present invention also provides a method of treating cancer in a subject, comprising subcutaneously administering to a subject in need thereof a pharmaceutical composition comprising an anti-CD38 antibody and a hyaluronidase for a period of time sufficient to treat the cancer.

[0009] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising subcutaneously administering the pharmaceutical composition of the present invention to a subject in need thereof for a period of time sufficient to treat the CD38-positive hematological malignancies.

[0010] The present invention also provides a method for treating multiple myeloma, comprising subcutaneously administering the pharmaceutical composition of the present invention to a subject in need thereof for a period of time sufficient to treat the multiple myeloma.

[0011] The present invention also provides a unit dosage form comprising:

[0012] an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in an amount of about 1200 mg to about 5000 mg;

[0013] A hyaluronidase in an amount of about 30,000 U to about 45,000 U;

[0014] Histidine at a concentration of about 5 mM to about 15 mM;

[0015] sorbitol at a concentration of about 100 mM to about 300 mM;

[0016] PS-20 at a concentration of about 0.01% w / v to about 0.04% w / v; and

[0017] The concentration is about 1 mg / mL to about 2 mg / mL of methionine, and the pH is about 5.5.

[0018] The present invention also provides a unit dosage form according to claim 74, comprising:

[0019] an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in an amount of about 1800 mg;

[0020] An amount of about 30,000 U of hyaluronidase;

[0021] Histidine at a concentration of about 10 mM;

[0022] Sorbitol at a concentration of about 300 mM;

[0023] PS-20 at a concentration of about 0.04% w / v; and

[0024] The concentration was about 1 mg / mL of methionine and the pH was about 5.5.

[0025] The invention also provides a container comprising a unit dosage form of the invention.

[0026] The present invention also provides a container comprising the pharmaceutical composition of the present invention. DETAILED DESCRIPTION

[0027] "CD38" refers to human CD38 protein (synonyms: ADP-ribosyl cyclase 1, cADPr hydrolase 1, cyclic ADP-ribose hydrolase 1). Human CD38 has GenBank accession number NP_001766 and the amino acid sequence shown in SEQ ID NO: 1. As is well known, CD38 is a single-pass type II membrane protein, which has amino acid residues 1-21 representing the cytoplasmic domain, amino acid residues 22-42 representing the transmembrane domain, and residues 43-300 representing the extracellular domain of CD38.

[0028] SEQ ID NO:1

[0029] MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLL GYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI

[0030] "Antibody" refers broadly to and includes immunoglobulin molecules, specifically monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies and chimeric monoclonal antibodies), antigen-binding fragments, bispecific or multispecific antibodies, dimers, tetramers or multimers, single-chain antibodies, domain antibodies, and any other modified configurations of immunoglobulin molecules that contain antigen-binding sites with desired specificity. "Full-length antibodies" are composed of two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and their multimers (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of domains CH1, hinge, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL is composed of three CDR and four FR segments, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0031] "Complementarity determining regions (CDRs)" are "antigen binding sites" in antibodies. Different terms are used to define CDRs: (i) three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) three in VH (H1, H2, H3) and three in VL (L1, L2, L3) "hypervariable region", "HVR" or "HV" refers to the structurally hypervariable region of the antibody variable domain, as defined by Chothia and Lesk (Chothia and Lesk, Mol Biol 196:901-17, 1987). The International Immunogenetics (IMGT) database (http: / / www_imgt_org) provides standardized numbering and definitions of antigen binding sites. The correspondence between CDR, HV and IMGT descriptions is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003. Unless otherwise expressly stated, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" used herein include CDRs defined by any of the above methods (Kabat, Chothia or IMGT).

[0032] Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct types, kappa and lambda, based on the amino acid sequence of their constant domains.

[0033] "Antigen binding fragment" refers to a portion of an immunoglobulin molecule that retains the antigen binding properties of a parent full-length antibody. Exemplary antigen binding fragments are heavy chain complementary determining regions (HCDR) 1, 2 and / or 3, light chain complementary determining regions (LCDR) 1, 2 and / or 3, heavy chain variable region (VH), or light chain variable region (VL), Fab, F(ab')2, Fd and Fv fragments, and domain antibodies (dAbs) consisting of one VH domain or one VL domain. The VH and VL domains can be linked together via synthetic linkers to form various types of single-chain antibody designs, in which the VH and VL domains are expressed by separate chains and the VH / VL domains are paired intramolecularly or intermolecularly to form a monovalent antigen-binding site, such as a single-chain Fv (scFv) or a diabody; for example, as described in International Patent Publication No. Wo1998 / 44001, International Patent Publication No. WO1988 / 01649; International Patent Publication No. WO1994 / 13804; International Patent Publication No. WO1992 / 01047.

[0034] "Monoclonal antibody" refers to an antibody population with a single amino acid composition in each heavy chain and each light chain, except for possible well-known changes, such as removal of the C-terminal lysine from the antibody heavy chain. Monoclonal antibodies usually bind to one antigenic epitope, but multispecific monoclonal antibodies bind to two or more different antigens or epitopes. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, or monovalent, divalent or multivalent. Multispecific antibodies, such as bispecific antibodies or trispecific antibodies are included in the term monoclonal antibody.

[0035] An "isolated antibody" refers to an antibody or antigen-binding fragment thereof that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to human CD38 is substantially free of antibodies that specifically bind to antigens other than human CD38). In the case of a bispecific antibody, the bispecific antibody specifically binds to two antigens of interest and is substantially free of antibodies that specifically bind to antigens other than the two antigens of interest. An "isolated antibody" encompasses antibodies that have been isolated to a higher degree of purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0036] "Humanized antibody" refers to an antibody in which the antigen binding site is derived from a non-human species and the variable region framework is derived from a human immunoglobulin sequence. Humanized antibodies may contain mutations deliberately introduced into the framework region so that the framework may not be an exact copy of the expressed human immunoglobulin sequence or germline gene sequence.

[0037] "Human antibody" refers to an antibody having a heavy chain variable region and a light chain variable region, wherein the framework and antigen binding site are derived from sequences of human origin. If the antibody contains a constant region or a portion of a constant region, the constant region is also derived from sequences of human origin.

[0038] If the variable region of the antibody is obtained by a system using human germline immunoglobulins or rearranged immunoglobulin genes, then the human antibody comprises a heavy chain variable region or a light chain variable region derived from a sequence of human origin. Such exemplary systems are human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals, such as mice or rats carrying human immunoglobulin loci as described herein. When compared with human germline immunoglobulin sequences or rearranged immunoglobulin sequences, human antibodies generally comprise amino acid differences caused by amino acid changes introduced during cloning and VDJ recombination in non-human animals, such as naturally occurring somatic mutations, displacements deliberately introduced into frameworks or antigen binding sites, and amino acid differences caused by VDJ recombination. Typically, the amino acid sequence of 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 to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, a human antibody may include a consensus framework sequence obtained by human framework sequence analysis (e.g., Knappik et al., J Mol Biol 296:57-86, described in 2000); or a synthetic HCDR3 bound to a human immunoglobulin gene library displayed on a phage (e.g., Shi et al., J Mol Biol 397:385-96, described in 2010 and International Patent Publication No. WO2009 / 085462).

[0039] The definition of "human antibody" does not include antibodies whose antigen binding sites are derived from non-human species.

[0040] "Recombinant" includes antibodies and other proteins that are prepared, expressed, created or isolated by recombinant means.

[0041] "Epitope" refers to the part of an antigen that specifically binds to an antibody. An epitope is usually composed of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups of a portion such as an amino acid or a polysaccharide side chain, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope may be composed of continuous and / or discontinuous amino acids that form a conformational space unit. For a discontinuous epitope, amino acids from different parts of the linear sequence of an antigen are close in three-dimensional space due to the folding of the protein molecule.

[0042] "Multispecific" refers to an antibody that specifically binds to at least two different antigens or two different epitopes within an antigen (eg, three, four, or five different antigens or epitopes).

[0043] "Bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific antibodies may have cross-reactivity to other related antigens, or may bind to epitopes shared between two or more different antigens.

[0044] A "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications (eg, substitutions, insertions, or deletions).

[0045] "In combination with" means that two or more therapeutic agents are administered to the subject together as a mixture, simultaneously as single agents, or sequentially in any order as single agents.

[0046] "Pharmaceutical composition" refers to the product obtained by mixing an anti-CD38 antibody with a hyaluronidase, and includes both fixed and non-fixed combinations. Pharmaceutical compositions generally contain a pharmaceutically acceptable carrier. "Fixed combination" refers to a single pharmaceutical composition comprising an anti-CD38 antibody and a hyaluronidase that is administered simultaneously in a single entity or dosage form. "Non-fixed combination" refers to separate pharmaceutical compositions or unit dosage forms of an anti-CD38 antibody and a hyaluronidase that are administered simultaneously, concurrently or sequentially as separate entities without specific time interval constraints, wherein such administration provides effective levels of the two compounds in the subject.

[0047] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0048] "Treatment" or "treatment" refers to treatment with a therapeutic agent, wherein the subject will slow (mitigate) the development or spread of an undesirable physiological change or disease such as a tumor or tumor cell, or is used to provide a beneficial or desired clinical outcome during treatment. Beneficial or desired clinical outcomes include relief of symptoms, attenuation of disease severity, stabilization of the disease state (i.e., no worsening), delayed or slowed disease progression, no metastasis, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonging survival compared to the expected survival of the subject if he or she is not receiving treatment. Those subjects in need of treatment include those subjects who already have an undesirable physiological change or disease, and those subjects who are prone to having a physiological change or disease.

[0049] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improvement in the patient's health status, reduction in tumor burden, arrest or slowing of tumor growth, and / or the absence of metastasis of cancer cells to other parts of the body.

[0050] "Inhibition of growth" (e.g., with respect to tumor cells) refers to a measurable decrease in the growth of a tumor cell or tumor tissue in vitro or in vivo when contacted with a therapeutic drug or combination of therapeutic drugs, as compared to the growth of the same tumor cell or tumor tissue in the absence of the therapeutic drug or combination of therapeutic drugs. The inhibition of tumor cell or tumor tissue growth in vitro or in vivo can be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

[0051] "CD38-positive hematological malignancies" refers to hematological malignancies characterized by the presence of tumor cells expressing CD38, including leukemias, lymphomas, and myelomas. Examples of such CD38-positive hematological malignancies include precursor B-cell lymphoblastic leukemia / lymphoma and B-cell non-Hodgkin's lymphoma, acute promyelocytic leukemia, acute lymphoblastic leukemia, and mature B-cell tumors, 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, lymphadenopathy and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), plasma cell neoplasm, multiple myeloma, plasma cell leukemia, post-transplant lymphoproliferative disorder, light-chain amyloidosis, Waldenstrom's macroglobulinemia, plasma cell leukemia and anaplastic large cell lymphoma (ALCL).

[0052] "About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless otherwise expressly stated in the examples or elsewhere in the specification, "about" means within one standard deviation, or up to a range of 5%, whichever is greater, according to customary practice in the art.

[0053] Pharmaceutical composition

[0054] The present invention provides a pharmaceutical composition comprising an anti-CD38 antibody and hyaluronidase.

[0055] Hyaluronidase is an enzyme that degrades hyaluronic acid (EC 3.2.1.35) and reduces the viscosity of hyaluronic acid (hyaluronan) in the extracellular matrix to increase tissue permeability. The enzymatic activity of hyaluronidase (including rHuPH20) can be defined by units / mL (U / mL) or by the total enzyme activity (U) in a specific preparation, as further explained below.

[0056] rHuPH20 is a recombinant hyaluronidase ( Recombinant) is described in International Patent Publication No. WO2004 / 078140.

[0057] The standard definition of a unit of enzyme activity (U) is the amount of enzyme per unit time that catalyzes the reaction of a substrate of a defined amount, such as one mole or one nanomole of substrate per minute. The technology used to measure the activity of hyaluronidase preparations is known in the art, and the activity of hyaluronidase preparations is usually expressed in USP units or units (hereinafter "units"). Exemplary methods for measuring activity can be found in U.S. Patent No. 7,767,429.

[0058] Hyaluronidase activity refers to the ability of enzyme catalysis hyaluronic acid cracking.American Pharmacopeia (USP) XXII provides the assay method of hyaluronidase, wherein hyaluronidase activity is indirectly determined in the following manner: after making enzyme and HA react at 37 DEG C for 30 minutes, measure the amount of remaining higher molecular weight hyaluronic acid or hyaluronan (HA) substrate (USP XXII-NFXVII (1990) 644-645United States Pharmacopeia Convention, Inc, Rockville, Md.). Reference standard solution can be used in determination to determine the relative activity (in units) of any hyaluronidase. The in vitro assay for measuring the hyaluronidase activity of hyaluronidase (such as soluble rHuPH20) is known in the art, and is described herein. Exemplary assay method includes micro-turbidity assay method (see, e.g., Example 3) described below, which indirectly measures the cracking of hyaluronidase to hyaluronic acid by the insoluble precipitation formed when detecting uncracked hyaluronic acid and serum albumin. Reference standards can be used, for example, to generate a standard curve to determine the activity in units of the hyaluronidase being tested.

[0059] The pharmaceutical composition can be used to subcutaneously administer an anti-CD38 antibody to a subject in need of anti-CD38 antibody treatment, such as a subject with cancer (e.g., a CD38-positive hematological malignancy). Without wishing to be bound by any particular theory, subcutaneous administration of an anti-CD38 antibody may have reduced infusion-related reactions and achieve improved response rates when compared to intravenous administration of the anti-CD38 antibody.

[0060] In some embodiments, the pharmaceutical composition is a fixed combination.

[0061] In some embodiments, the pharmaceutical composition is a non-fixed combination.

[0062] In some embodiments, the pharmaceutical composition comprises about 1 mg / mL to about 180 mg / mL of the anti-CD38 antibody.

[0063] In some embodiments, the pharmaceutical composition comprises about 10 mg / mL to about 180 mg / mL of the anti-CD38 antibody.

[0064] In some embodiments, the pharmaceutical composition comprises about 20 mg / mL to about 160 mg / mL of the anti-CD38 antibody.

[0065] In some embodiments, the pharmaceutical composition comprises about 20 mg / mL to about 140 mg / mL of the anti-CD38 antibody.

[0066] In some embodiments, the pharmaceutical composition comprises about 20 mg / mL to about 120 mg / mL of the anti-CD38 antibody.

[0067] In some embodiments, the pharmaceutical composition comprises about 40 mg / mL to about 120 mg / mL of the anti-CD38 antibody.

[0068] In some embodiments, the pharmaceutical composition comprises about 60 mg / mL to about 120 mg / mL of the anti-CD38 antibody.

[0069] In some embodiments, the pharmaceutical composition comprises about 80 mg / mL to about 120 mg / mL of the anti-CD38 antibody.

[0070] In some embodiments, the pharmaceutical composition comprises about 100 mg / mL to about 120 mg / mL of the anti-CD38 antibody.

[0071] In some embodiments, the pharmaceutical composition comprises about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, or about 180 mg / mL of an anti-CD38 antibody.

[0072] In some embodiments, the pharmaceutical composition comprises about 20 mg / mL of the anti-CD38 antibody.

[0073] In some embodiments, the pharmaceutical composition comprises about 100 mg / mL of the anti-CD38 antibody.

[0074] In some embodiments, the pharmaceutical composition comprises about 120 mg / mL of the anti-CD38 antibody.

[0075] In some embodiments, the pharmaceutical composition comprises about 50 U / mL to about 5000 U / mL of hyaluronidase.

[0076] In some embodiments, the pharmaceutical composition comprises about 500 U / mL to about 5000 U / mL of hyaluronidase.

[0077] In some embodiments, the pharmaceutical composition comprises about 1000 U / mL to about 5000 U / mL of hyaluronidase.

[0078] In some embodiments, the pharmaceutical composition comprises about 2000 U / mL to about 5000 U / mL of hyaluronidase.

[0079] In some embodiments, the pharmaceutical composition comprises about 50 U / mL to about 2000 U / mL of hyaluronidase.

[0080] In some embodiments, the pharmaceutical composition comprises about 500 U / mL to about 2000 U / mL of hyaluronidase.

[0081] In some embodiments, the pharmaceutical composition comprises about 1000 U / mL to about 2000 U / mL of hyaluronidase.

[0082] In some embodiments, the pharmaceutical composition comprises about 500 U / mL, about 600 U / mL, about 700 U / mL, about 800 U / mL, about 900 U / mL, about 1000 U / mL, about 1100 U / mL, about 1200 U / mL, about 1300 U / mL, about 1400 U / mL, about 1500 U / mL, about 1600 U / mL, about 1700 U / mL, about 1800 U / mL, about 1900 U / mL, about 2000 U / mL, about 2100 U / mL, about 2200 U / mL, about 2300 U / mL, about 2400 U / mL, about 2500 U / mL, about 2600 U / mL, about 2700 U / mL, In some embodiments, the present invention relates to a hyaluronidase of about 400 U / mL, about 2800 U / mL, about 2900 U / mL, about 3000 U / mL, about 3100 U / mL, about 3200 U / mL, about 3300 U / mL, about 3400 U / mL, about 3500 U / mL, about 3600 U / mL, about 3700 U / mL, about 3800 U / mL, about 3900 U / mL, about 4000 U / mL, about 4100 U / mL, about 4200 U / mL, about 4300 U / mL, about 4400 U / mL, about 4500 U / mL, about 4600 U / mL, about 4700 U / mL, about 4800 U / mL, about 4900 U / mL or about 5000 U / mL of hyaluronidase.

[0083] In some embodiments, the pharmaceutical composition comprises about 500 U / mL of hyaluronidase.

[0084] In some embodiments, the pharmaceutical composition comprises about 2000 U / mL of hyaluronidase.

[0085] In some embodiments, the pharmaceutical composition comprises about 5000 U / mL of hyaluronidase.

[0086] In some embodiments, the pharmaceutical composition comprises about 1200 mg to about 5000 mg of the anti-CD38 antibody.

[0087] In some embodiments, the pharmaceutical composition comprises about 1200 mg to about 2400 mg of the anti-CD38 antibody.

[0088] In some embodiments, the pharmaceutical composition comprises about 1200 mg to about 1800 mg of the anti-CD38 antibody.

[0089] In some embodiments, the pharmaceutical composition comprises about 1200 mg of the anti-CD38 antibody.

[0090] In some embodiments, the pharmaceutical composition comprises about 1400 mg of the anti-CD38 antibody.

[0091] In some embodiments, the pharmaceutical composition comprises about 1600 mg of the anti-CD38 antibody.

[0092] In some embodiments, the pharmaceutical composition comprises about 1800 mg of the anti-CD38 antibody.

[0093] In some embodiments, the pharmaceutical composition comprises about 2000 mg of the anti-CD38 antibody.

[0094] In some embodiments, the pharmaceutical composition comprises about 2200 mg of the anti-CD38 antibody.

[0095] In some embodiments, the pharmaceutical composition comprises about 2400 mg of the anti-CD38 antibody.

[0096] In some embodiments, the pharmaceutical composition comprises about 2600 mg of the anti-CD38 antibody.

[0097] In some embodiments, the pharmaceutical composition comprises about 2800 mg of the anti-CD38 antibody.

[0098] In some embodiments, the pharmaceutical composition comprises about 3000 mg of the anti-CD38 antibody.

[0099] In some embodiments, the pharmaceutical composition comprises about 3500 mg of anti-CD38 antibody.

[0100] In some embodiments, the pharmaceutical composition comprises about 4000 mg of the anti-CD38 antibody.

[0101] In some embodiments, the pharmaceutical composition comprises about 4500 mg of the anti-CD38 antibody.

[0102] In some embodiments, the pharmaceutical composition comprises about 5000 mg of the anti-CD38 antibody.

[0103] In some embodiments, the pharmaceutical composition comprises about 750 U to about 75,000 U of hyaluronidase.

[0104] In some embodiments, the pharmaceutical composition comprises about 7500 U to about 45000 U of hyaluronidase.

[0105] In some embodiments, the pharmaceutical composition comprises about 30,000 U to about 45,000 U of hyaluronidase.

[0106] In some embodiments, the pharmaceutical composition comprises about 7500U, about 8000U, about 8500U, about 9000U, about 10000U, about 15000U, about 20000U, about 21000U, about 22000U, about 23000U, about 24000U, about 25000U, about 26000U, about 27000U, about 28000U, about 29000U, about 30000U, about 31000U, about 32000U, about 33000U, about 34000U In some embodiments, the present invention relates to a hyaluronidase in an amount of about 50,000 U, about 55,000 U, about 60,000 U, about 65,000 U, about 70,000 U, or about 75,000 U of hyaluronidase.

[0107] In some embodiments, the pharmaceutical composition comprises about 5000 mg of anti-CD38 antibody and about 30000 U of hyaluronidase.

[0108] In some embodiments, the pharmaceutical composition comprises about 5000 mg of anti-CD38 antibody and about 45000 U of hyaluronidase.

[0109] In some embodiments, the pharmaceutical composition comprises about 3000 mg of anti-CD38 antibody and about 30000 U of hyaluronidase.

[0110] In some embodiments, the pharmaceutical composition comprises about 3000 mg of anti-CD38 antibody and about 45000 U of hyaluronidase.

[0111] In some embodiments, the pharmaceutical composition comprises about 2800 mg of anti-CD38 antibody and about 30000 U of hyaluronidase.

[0112] In some embodiments, the pharmaceutical composition comprises about 2800 mg of anti-CD38 antibody and about 45000 U of hyaluronidase.

[0113] In some embodiments, the pharmaceutical composition comprises about 2600 mg of anti-CD38 antibody and about 30,000 U of hyaluronidase.

[0114] In some embodiments, the pharmaceutical composition comprises about 2600 mg of anti-CD38 antibody and about 45000 U of hyaluronidase.

[0115] In some embodiments, the pharmaceutical composition comprises about 2400 mg of anti-CD38 antibody and about 30000 U of hyaluronidase.

[0116] In some embodiments, the pharmaceutical composition comprises about 2400 mg of anti-CD38 antibody and about 45000 U of hyaluronidase.

[0117] In some embodiments, the pharmaceutical composition comprises about 2200 mg of anti-CD38 antibody and about 30,000 U of hyaluronidase.

[0118] In some embodiments, the pharmaceutical composition comprises about 2200 mg of anti-CD38 antibody and about 45000 U of hyaluronidase.

[0119] In some embodiments, the pharmaceutical composition comprises about 2000 mg of anti-CD38 antibody and about 30000 U of hyaluronidase.

[0120] In some embodiments, the pharmaceutical composition comprises about 2000 mg of anti-CD38 antibody and about 45000 U of hyaluronidase.

[0121] In some embodiments, the pharmaceutical composition comprises about 1800 mg of an anti-CD38 antibody and about 30,000 U of hyaluronidase.

[0122] In some embodiments, the pharmaceutical composition comprises about 1800 mg of an anti-CD38 antibody and about 45,000 U of hyaluronidase.

[0123] In some embodiments, the pharmaceutical composition comprises about 1600 mg of anti-CD38 antibody and about 30,000 U of hyaluronidase.

[0124] In some embodiments, the pharmaceutical composition comprises about 1600 mg of anti-CD38 antibody and about 45,000 U of hyaluronidase.

[0125] In some embodiments, the hyaluronidase is rHuPH20 having the amino acid sequence of SEQ ID NO:22.

[0126] In some embodiments, the anti-CD38 antibody in the pharmaceutical composition competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO:4 and a light chain variable region (VL) of SEQ ID NO:5.

[0127] In some embodiments, the anti-CD38 antibody in the pharmaceutical composition binds to at least region SKRNIQFSCKNIYR (SEQ ID NO: 2) and region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).

[0128] In some embodiments, the anti-CD38 antibody in the pharmaceutical composition comprises heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 of SEQ ID NOs 6, 7 and 8, 9, 10 and 11, respectively.

[0129] In some embodiments, the anti-CD38 antibody in the pharmaceutical composition comprises a heavy chain variable region (VH) that is 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4, and a light chain variable region (VL) that is 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:5.

[0130] In some embodiments, the anti-CD38 antibody in the pharmaceutical composition comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:5.

[0131] In some embodiments, the anti-CD38 antibody in the pharmaceutical composition comprises the heavy chain of SEQ ID NO:12 and the light chain of SEQ ID NO:13.

[0132] SEQ ID NO:2

[0133] SKRNIQFSCKNIYR

[0134] SEQ ID NO:3

[0135] EKVQTLEAWVIHGG

[0136] SEQ ID NO:4

[0137] <h2 style=";text-align:left;direction:ltr">EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0138] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0139] <h2 style=";text-align:left;direction:ltr"> EIVLTQSPATLSLSPGERATLLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0140] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0141] <h2 style=";text-align:left;direction:ltr"> SFAMS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0142] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0143] <h2 style=";text-align:left;direction:ltr"> AISGSGGGTYYADSVKG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0144] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0145] <h2 style=";text-align:left;direction:ltr"> DKILWFGEPVFDY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0146] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0147] <h2 style=";text-align:left;direction:ltr"> RASQSVSSYLA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0148] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0149] <h2 style=";text-align:left;direction:ltr"> DASNRAT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0150] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0151] <h2 style=";text-align:left;direction:ltr"> QQRSNWPPTF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0152] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:12<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0153] EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0154] SEQ ID NO:13

[0155] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0156] Other exemplary anti-CD38 antibodies useful in the pharmaceutical compositions and methods of the invention are:

[0157] mAb003 comprises the VH and VL sequences of SEQ ID NOs 14 and 15, respectively, and is described in U.S. Patent 7,829,693. The VH and VL of mAb003 may be expressed as IgG1 / κ;

[0158] mAb024 comprises the VH and VL sequences of SEQ ID NOs 16 and 17, respectively, and is described in U.S. Patent 7,829,693. The VH and VL of mAb024 can be expressed as IgG1 / κ;

[0159] MOR-202 (MOR-03087) comprising the VH and VL sequences of SEQ ID NOs 18 and 19, respectively, and described in U.S. Patent No. 8,088,896. The VH and VL of MOR-202 may be expressed as IgG1 / κ; or

[0160] Isatuximab; comprising the VH and VL sequences of SEQ ID NOs 20 and 21, respectively, described in U.S. Patent No. 8,153,765. The VH and VL of Isatuximab can be expressed as IgG1 / κ.

[0161] SEQ ID NO:14

[0162] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSSAS

[0163] SEQ ID NO:15

[0164] DIQMTQSPSSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK

[0165] SEQ ID NO:16

[0166] EVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMPGKGLEWMGIIYPHDSDARYSPSFQGQVTFSADKSISTAYLQWSSLKASDTAMYYCARHVGWGSRYWYFDLWGRGTLVTVSS

[0167] SEQ ID NO:17

[0168] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPGLLIYDASNRASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK

[0169] SEQ ID NO:18

[0170] QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS

[0171] SEQ ID NO:19

[0172] DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVLGQ

[0173] SEQ ID NO 20:

[0174] QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGTIYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDYYGSNSLDYWGQGTSVTVSS

[0175] SEQ ID NO:21:

[0176] DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKLEIK

[0177] Other exemplary anti-CD38 antibodies useful in the pharmaceutical compositions of the invention are those described in International Patent Publication No. WO05 / 103083, International Patent Publication No. WO06 / 125640, International Patent Publication No. WO07 / 042309, International Patent Publication No. WO08 / 047242, or International Patent Publication No. WO14 / 178820.

[0178] An exemplary anti-CD38 antibody that can be used in the pharmaceutical composition of the present invention is Daratumumab. Daratumumab comprises heavy chain variable region (VH) and light chain variable region (VL) amino acid sequences shown in SEQ ID NOs: 4 and 5, respectively, HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 6, 7 and 8, respectively, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 9, 10 and 11, respectively, and is of IgG1 / κ subtype, described in U.S. Pat. No. 7,829,693. The heavy chain amino acid sequence of Daratumumab is shown in SEQ ID NO: 12, and the light chain amino acid sequence is shown in SEQ ID NO: 13.

[0179] The present invention also provides a pharmaceutical composition comprising an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 and hyaluronidase rHuPH20 of SEQ ID NO: 22, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0180] The present invention also provides a pharmaceutical composition comprising an anti-CD38 antibody and hyaluronidase rHuPH20 of SEQ ID NO: 22, wherein the anti-CD38 antibody comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs 6, 7 and 8, respectively, and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs 9, 10 and 11, respectively, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0181] The present invention also provides a pharmaceutical composition comprising between about 1200 mg and 1800 mg of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and between about 30,000 U and 45,000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0182] The present invention also provides a pharmaceutical composition comprising about 1800 mg of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and about 30,000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0183] The present invention also provides a pharmaceutical composition comprising about 1800 mg of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and about 45000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0184] The present invention also provides a pharmaceutical composition comprising about 1600 mg of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and about 30,000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0185] The present invention also provides a pharmaceutical composition comprising about 1600 mg of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and about 45000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0186] The present invention also provides a pharmaceutical composition comprising about 1200 mg of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and about 30,000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0187] The present invention also provides a pharmaceutical composition comprising about 1200 mg of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and about 45,000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0188] SEQ ID NO:22

[0189] MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL

[0190] Anti-CD38 antibodies useful in the pharmaceutical compositions of the invention can also be selected de novo, for example, from phage display libraries, in which phage are engineered to express human immunoglobulins or portions thereof, such as Fab, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., J Mol Biol 296:57-86, 2000; Krebs et al., J Immunol Meth 254:67-84, 2001; Vaughan et al., Nature Biotechnology 14:309-314, 1996; Sheets et al., PITAS (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J Mol Biol 227:381, 1991; Marks et al., J Mol Biol 222:581, 1991). CD38 binding variable domains can be isolated, for example, from phage display libraries that express antibody heavy chain variable regions and light chain variable regions as fusion proteins with phage pIX coat protein, as described in Shi et al., J. Mol. Biol. 397:385-96, 2010 and International Patent Publication WO09 / 085462. Antibody libraries can be screened for those that bind to the extracellular domain of human CD38, the resulting positive clones further characterized, Fab isolated from 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. Patent 5,223,409, U.S. Patent 5,403,484, U.S. Patent 5,571,698, U.S. Patent 5,427,908, U.S. Patent 5,580,717, U.S. Patent 5,969,108, U.S. Patent 6,172,197, U.S. Patent 5,885,793, U.S. Patent 6,521,404, U.S. Patent 6,544,731, U.S. Patent 6,555,313, U.S. Patent 6,582,915, and U.S. Patent 6,593,081.

[0191] Known in vitro methods can be used to assess the competition of antibodies with reference antibodies (such as, anti-CD38 antibodies comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5) for binding to CD38. In an exemplary method, CHO cells recombinantly expressing CD38 can be incubated with unlabeled reference antibodies for 15 minutes at 4°C and then incubated with excess fluorescently labeled test antibodies for 45 minutes at 4°C. After washing in PBS / BSA, fluorescence can be measured by flow cytometry using standard methods. In another exemplary method, the extracellular portion of human CD38 can be coated on the surface of an ELISA plate. An excess of unlabeled reference antibodies can be added within about 15 minutes, followed by the addition of biotinylated test antibodies. After washing in PBS / Tween, the binding of the biotinylated test antibody can be detected using horseradish peroxidase (HRP)-conjugated streptavidin, and the signal can be detected using standard methods. Obviously, in a competition assay, the reference antibody can be labeled, while the test antibody is not labeled. When the reference antibody inhibits binding of the test antibody, the test antibody competes with the reference antibody, or the test antibody inhibits binding of the reference antibody by at least 80%, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The epitope of the test antibody can be further defined using known methods, e.g., by peptide mapping or hydrogen / deuterium protection assays, or by crystal structure determination.

[0192] Antibodies that bind to region SKRNIQFSCKNIYR (SEQ ID NO:2) and region EKVQTLEAWVIHGG (SEQ ID NO:3) of human CD38 (SEQ ID NO:1) can be produced, for example, by immunizing mice with peptides having the amino acid sequences shown in SEQ ID NOs:2 and 3 using standard methods and those described herein, and characterizing the binding of the obtained antibodies to the peptides using, for example, ELISA or mutagenesis studies.

[0193] The present invention also provides a pharmaceutical composition comprising: an anti-CD38 antibody comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the following sequences:

[0194] a. VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15;

[0195] b. VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17;

[0196] c. VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or

[0197] d. VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21; and hyaluronidase rHuPH20 of SEQ ID NO: 22.

[0198] The present invention also provides a pharmaceutical composition, comprising: an anti-CD38 antibody containing:

[0199] a. VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15;

[0200] b. VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17;

[0201] c. VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or

[0202] d. VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21; and hyaluronidase rHuPH20 of SEQ ID NO: 22.

[0203] The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., such as salts, buffers, antioxidants, sugars, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifiers, or combinations thereof.

[0204] Exemplary buffers that may be used are acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffer, HEPPSO, and HEPES.

[0205] Exemplary antioxidants that may be used are ascorbic acid, methionine, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, lecithin, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, and tartaric acid.

[0206] Exemplary amino acids that may be used are histidine, isoleucine, methionine, glycine, arginine, lysine, L-leucine, trileucine, alanine, glutamic acid, L-threonine, and 2-phenylalanine.

[0207] Exemplary surfactants that can be used are polysorbates (e.g., polysorbate-20 or polysorbate-80); polyoxamers (e.g., poloxamer 188); Triton; sodium octylglucoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocoamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitopropyl-, or isostearamidopropyl-betaine (e.g., lauramidopropyl); myristamidopropyl-, palmitopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and MONAQUA TM series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., PLURONICS TM , PF68, etc.).

[0208] Exemplary preservatives that can be used are phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), alkylbenzyldimethylammonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof.

[0209] Exemplary sugars that can be used are monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars such as glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol and isomaltulose.

[0210] Exemplary salts that can be used are acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc., and those derived from non-toxic organic acids, such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, etc., and those derived from non-toxic organic amines, such as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc. An exemplary salt is sodium chloride.

[0211] The amount of pharmaceutically acceptable carrier in a pharmaceutical composition can be determined experimentally based on the activity of the carrier and the desired formulation characteristics (eg, stability and / or minimal oxidation).

[0212] In some embodiments, the pharmaceutical composition comprises acetic acid.

[0213] In some embodiments, the pharmaceutical composition comprises acetic acid at a concentration of about 1 mM to about 50 mM.

[0214] In some embodiments, the pharmaceutical composition comprises acetic acid at a concentration of about 10 mM to about 40 mM.

[0215] In some embodiments, the pharmaceutical composition comprises acetic acid at a concentration of about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0216] In some embodiments, the pharmaceutical composition comprises acetic acid at a concentration of about 25 mM.

[0217] In some embodiments, the pharmaceutical composition comprises sodium chloride (NaCl).

[0218] In some embodiments, the pharmaceutical composition comprises NaCl at a concentration of about 20 mM to about 100 mM.

[0219] In some embodiments, the pharmaceutical composition comprises NaCl at a concentration of about 40 mM to about 80 mM.

[0220] In some embodiments, the pharmaceutical composition comprises NaCl at a concentration of about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.

[0221] In some embodiments, the pharmaceutical composition comprises NaCl at a concentration of about 60 mM.

[0222] In some embodiments, the pharmaceutical composition comprises a carbohydrate.

[0223] In some embodiments, the sugar is sucrose.

[0224] In some embodiments, the sugar is sorbitol.

[0225] In some embodiments, the carbohydrate is mannitol.

[0226] In some embodiments, the pharmaceutical composition comprises a saccharide at a concentration of about 50 mM to about 500 mM.

[0227] In some embodiments, the pharmaceutical composition comprises a saccharide at a concentration of about 50 mM to about 450 mM.

[0228] In some embodiments, the pharmaceutical composition comprises a saccharide at a concentration of about 50 mM to about 400 mM.

[0229] In some embodiments, the pharmaceutical composition comprises a saccharide at a concentration of about 50 mM to about 350 mM.

[0230] In some embodiments, the pharmaceutical composition comprises a saccharide at a concentration of about 100 mM to about 350 mM.

[0231] In some embodiments, the pharmaceutical composition comprises a saccharide at a concentration of about 100 mM to about 300 mM.

[0232] In some embodiments, the pharmaceutical composition comprises a concentration of about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM, about 500 mM, about 510 mM, about 520 mM, about 530 mM, about 540 mM, about 550 mM, about 560 mM, about 570 mM, about 580 mM, about 590 mM, about 600 mM, about 610 mM, about 620 mM, about 630 mM, about 640 mM, about 650 mM, about 660 mM, about 670 mM, about 680 mM, about 690 mM, about 710 mM, about 720 mM, about 73 about 450mM, about 460mM, about 470mM, about 480mM, about 490mM, or about 500mM of a carbohydrate.

[0233] In some embodiments, the pharmaceutical composition comprises mannitol.

[0234] In some embodiments, the pharmaceutical composition comprises mannitol at a concentration of about 100 mM to about 180 mM.

[0235] In some embodiments, the pharmaceutical composition comprises mannitol at a concentration of about 120 mM to about 160 mM.

[0236] In some embodiments, the pharmaceutical composition comprises mannitol at a concentration of about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, or about 180 mM.

[0237] In some embodiments, the pharmaceutical composition comprises mannitol at a concentration of about 140 mM.

[0238] In some embodiments, the pharmaceutical composition comprises polysorbate.

[0239] In some embodiments, the pharmaceutical composition comprises polysorbate-20 (PS-20).

[0240] In some embodiments, the pharmaceutical composition comprises polysorbate-20 (PS-20) at a concentration of about 0.01% w / v to about 0.1% w / v.

[0241] In some embodiments, the pharmaceutical composition comprises polysorbate-20 (PS-20) at a concentration of about 0.01% w / v to about 0.08% w / v.

[0242] In some embodiments, the pharmaceutical composition comprises polysorbate-20 (PS-20) at a concentration of about 0.01% w / v to about 0.04% w / v.

[0243] In some embodiments, the pharmaceutical composition comprises polysorbate 20 (PS-20) at a concentration of about 0.01% w / v, 0.02% w / v, 0.03% w / v, 0.04% w / v, 0.05% w / v, 0.06% w / v, 0.07% w / v, 0.08% w / v, 0.09% w / v, or 0.1% w / v.

[0244] The present invention also provides a pharmaceutical composition comprising:

[0245] About 20 mg / mL to about 120 mg / mL of an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 dissolved in about 25 mM acetic acid, about 60 mM sodium chloride, about 140 mM mannitol, and about 0.04% w / v polysorbate-20 (PS-20)

[0246] medium; a pH of about 5.5; and

[0247] About 30,000 U to about 45,000 U of hyaluronidase dissolved in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate 80, pH 6.5.

[0249] In some embodiments, the hyaluronidase is rHuPH20 (SEQ ID NO: 22).

[0250] In some embodiments, the pharmaceutical composition is a non-fixed combination.

[0251] The present invention also provides a pharmaceutical composition comprising:

[0252] about 20 mg / mL of an anti-CD38 antibody comprising the VH of SEQ ID NO:4 and the VL of SEQ ID NO:5 dissolved in about 25 mM acetic acid, about 60 mM sodium chloride, about 140 mM mannitol, and about 0.04% w / v polysorbate-20 (PS-20); pH about 5.5; and

[0253] About 30,000 U of hyaluronidase, dissolved in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate 80, pH 6.5.

[0254] In some embodiments, the hyaluronidase is rHuPH20 (SEQ ID NO: 22).

[0255] In some embodiments, the pharmaceutical composition is a non-fixed combination.

[0256] The present invention also provides a pharmaceutical composition comprising:

[0257] about 20 mg / mL of an anti-CD38 antibody comprising the VH of SEQ ID NO:4 and the VL of SEQ ID NO:5 dissolved in about 25 mM acetic acid, about 60 mM sodium chloride, about 140 mM mannitol, and about 0.04% w / v polysorbate-20 (PS-20); pH about 5.5; and

[0258] About 45,000 U of hyaluronidase, dissolved in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate 80, pH 6.5.

[0259] In some embodiments, the hyaluronidase is rHuPH20 (SEQ ID NO: 22).

[0260] In some embodiments, the pharmaceutical composition is a non-fixed combination.

[0261] In some embodiments, the pharmaceutical composition comprises histidine.

[0262] In some embodiments, the pharmaceutical composition comprises histidine at a concentration of about 1 mM to about 50 mM.

[0263] In some embodiments, the pharmaceutical composition comprises histidine at a concentration of about 5 mM to about 50 mM.

[0264] In some embodiments, the pharmaceutical composition comprises histidine at a concentration of about 5 mM to about 30 mM.

[0265] In some embodiments, the pharmaceutical composition comprises histidine at a concentration of about 5 mM to about 20 mM.

[0266] In some embodiments, the pharmaceutical composition comprises histidine at a concentration of about 5 mM to about 15 mM.

[0267] In some embodiments, the pharmaceutical composition comprises histidine at a concentration of about 5 mM to about 10 mM.

[0268] In some embodiments, the pharmaceutical composition comprises a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM 47mM, about 48mM, about 49mM, or about 50mM of histidine.

[0269] In some embodiments, the pharmaceutical composition comprises histidine at a concentration of about 5 mM.

[0270] In some embodiments, the pharmaceutical composition comprises histidine at a concentration of about 10 mM.

[0271] In some embodiments, the pharmaceutical composition comprises histidine at a concentration of about 15 mM.

[0272] In some embodiments, the pharmaceutical composition comprises histidine at a concentration of about 20 mM.

[0273] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 50 mM to about 500 mM.

[0274] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 50 mM to about 450 mM.

[0275] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 50 mM to about 400 mM.

[0276] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 50 mM to about 350 mM.

[0277] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 100 mM to about 350 mM.

[0278] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 100 mM to about 300 mM.

[0279] In some embodiments, the pharmaceutical composition comprises a concentration of about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM, about 500 mM, about 510 mM, about 520 mM, about 530 mM, about 540 mM, about 550 mM, about 560 mM, about 570 mM, about 580 mM, about 590 mM, about 600 mM, about 610 mM, about 620 mM, about 630 mM, about 640 mM, about 650 mM, about 660 mM, about 670 mM, about 680 mM, about 690 mM, about 710 mM, about 720 mM, about 73 470mM, about 480mM, about 490mM or about 500mM sorbitol.

[0280] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 50 mM.

[0281] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 100 mM.

[0282] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 150 mM.

[0283] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 200 mM.

[0284] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 250 mM.

[0285] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 300 mM.

[0286] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 350 mM.

[0287] In some embodiments, the pharmaceutical composition comprises sorbitol at a concentration of about 400 mM.

[0288] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 50 mM to about 500 mM.

[0289] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 50 mM to about 450 mM.

[0290] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 50 mM to about 400 mM.

[0291] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 50 mM to about 350 mM.

[0292] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 100 mM to about 350 mM.

[0293] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 100 mM to about 200 mM.

[0294] In some embodiments, the pharmaceutical composition comprises a concentration of about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM, about 500 mM, about 510 mM, about 520 mM, about 530 mM, about 540 mM, about 550 mM, about 560 mM, about 570 mM, about 580 mM, about 590 mM, about 600 mM, about 610 mM, about 620 mM, about 630 mM, about 640 mM, about 650 mM, about 660 mM, about 670 mM, about 680 mM, about 690 mM, about 710 mM, about 720 mM, about 73 about 470mM, about 480mM, about 490mM, or about 500mM of sucrose.

[0295] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 50 mM.

[0296] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 100 mM.

[0297] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 150 mM.

[0298] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 200 mM.

[0299] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 250 mM.

[0300] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 300 mM.

[0301] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 350 mM.

[0302] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 400 mM.

[0303] In some embodiments, the pharmaceutical composition comprises methionine.

[0304] In some embodiments, the pharmaceutical composition comprises methionine at a concentration of about 0.1 mg / mL to about 5 mg / mL.

[0305] In some embodiments, the pharmaceutical composition comprises methionine at a concentration of about 0.1 mg / mL to about 2.5 mg / mL.

[0306] In some embodiments, the pharmaceutical composition comprises methionine at a concentration of about 1 mg / mL to about 2 mg / mL.

[0307] In some embodiments, the pharmaceutical composition comprises methionine at a concentration of about 0.5 mg / mL, about 1 mg / mL, about 1.1 mg / mL, about 1.2 mg / mL, about 1.3 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1 / 7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, about 2.0 mg / mL, about 2.1 mg / mL, about 2.2 mg / mL, about 2 / 3 mg / mL, about 2.4 mg / mL, about 2.5 mg / mL, about 2.6 mg / mL, about 2.7 mg / mL, about 2.8 mg / mL, about 2.9 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, or about 5 mg / mL.

[0308] In some embodiments, the pharmaceutical composition is at a pH of 5.0 to 6.0.

[0309] In some embodiments, the pharmaceutical composition is at a pH of 5.3 to 5.8.

[0310] In some embodiments, the pharmaceutical composition is at pH 5.5.

[0311] In some embodiments, the pharmaceutical composition is at pH 5.6.

[0312] The present invention also provides a pharmaceutical composition comprising:

[0313] about 1 mg / mL to about 180 mg / mL of an anti-CD38 antibody;

[0314] About 50 U / mL to about 5000 U / mL of hyaluronidase

[0315] About 5 mM to about 50 mM histidine; and

[0316] From about 50 mM to about 400 mM sorbitol.

[0317] In some embodiments, the hyaluronidase is rHuPH20.

[0318] The present invention also provides a pharmaceutical composition comprising:

[0319] about 1 mg / mL to about 180 mg / mL of an anti-CD38 antibody;

[0320] About 50 U / mL to about 5000 U / mL of hyaluronidase

[0321] about 5 mM to about 50 mM histidine;

[0322] about 50 mM to about 400 mM sorbitol;

[0323] From about 0.01% w / v to about 0.1% PS-20; and

[0324] About 0.1 mg / mL to about 2.5 mg / mL of methionine.

[0325] In some embodiments, the hyaluronidase is rHuPH20.

[0326] The present invention also provides a pharmaceutical composition comprising:

[0327] about 100 mg / mL to about 120 mg / mL of an anti-CD38 antibody;

[0328] about 50 U / mL to about 5000 U / mL of hyaluronidase;

[0329] About 10 mM histidine; and

[0330] From about 100 mM to about 300 mM sorbitol.

[0331] In some embodiments, the hyaluronidase is rHuPH20.

[0332] In some embodiments, the pharmaceutical composition further comprises:

[0333] From about 0.01% w / v to about 0.04% w / v PS-20.

[0334] In some embodiments, the pharmaceutical composition further comprises about 1 mg / mL to about 2 mg / mL methionine.

[0335] In some embodiments, the pharmaceutical composition further comprises about 100 mM to about 200 mM sucrose.

[0336] In some embodiments, the anti-CD38 antibody comprises:

[0337] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 6, 7, 8, 9, 10 and 11, respectively;

[0338] VH and VL of SEQ ID NOs 4 and 5, respectively; and / or

[0339] The heavy and light chains of SEQ ID NOs are 12 and 13, respectively.

[0340] In some embodiments, the anti-CD38 antibody comprises:

[0341] VH and VL of SEQ ID NOs 14 and 15, respectively;

[0342] VH and VL of SEQ ID NOs 16 and 17, respectively;

[0343] VH and VL of SEQ ID NOs: 18 and 19, respectively; or

[0344] VH and VL of SEQ ID NOs 20 and 21, respectively;

[0345] In some embodiments, the hyaluronidase comprises rHuPH20 (SEQ ID NO: 22). The present invention also provides a pharmaceutical composition comprising:

[0346] about 1 mg / mL to about 180 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0347] About 50 U / mL to about 5000 U / mL of hyaluronidase

[0348] About 5 mM to about 50 mM histidine; and

[0349] From about 50 mM to about 400 mM sorbitol.

[0350] In some embodiments, the hyaluronidase is rHuPH20.

[0351] The present invention also provides a pharmaceutical composition comprising:

[0352] about 1 mg / mL to about 180 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0353] about 50 U / mL to about 5000 U / mL of hyaluronidase;

[0354] about 5 mM to about 50 mM histidine;

[0355] about 50 mM to about 400 mM sorbitol;

[0356] From about 0.01% w / v to about 0.1% PS-20; and

[0357] About 0.1 mg / mL to about 2.5 mg / mL of methionine.

[0358] In some embodiments, the hyaluronidase is rHuPH20.

[0359] The present invention also provides a pharmaceutical composition comprising:

[0360] about 100 mg / mL to about 120 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0361] About 50 U / mL to about 5000 U / mL of rHuPH20;

[0362] About 10 mM histidine;

[0363] about 100 mM to about 300 mM sorbitol;

[0364] From about 0.01% w / v to about 0.04% w / v PS-20; and

[0365] About 1 mg / mL to about 2 mg / mL methionine.

[0366] The present invention also provides a pharmaceutical composition comprising:

[0367] about 100 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0368] About 500 U / mL of rHuPH20;

[0369] About 10 mM histidine;

[0370] About 300 mM sorbitol;

[0371] About 0.04% w / v PS-20; and

[0372] About 2 mg / mL of methionine; pH about 5.5.

[0373] The present invention also provides a pharmaceutical composition comprising:

[0374] about 120 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0375] About 2000 U / mL of rHuPH20;

[0376] About 10 mM histidine;

[0377] About 300 mM sorbitol;

[0378] About 0.04% w / v PS-20; and

[0379] About 1 mg / mL of methionine; pH about 5.6.

[0380] The present invention also provides a pharmaceutical composition comprising:

[0381] about 100 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0382] About 500 U / mL of rHuPH20;

[0383] About 10 mM histidine;

[0384] about 300 mM sorbitol; and

[0385] About 2 mg / mL of methionine; pH about 5.5.

[0386] The present invention also provides a pharmaceutical composition comprising:

[0387] about 100 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0388] About 500 U / mL of rHuPH20;

[0389] About 10 mM histidine;

[0390] About 300 mM sorbitol;

[0391] About 0.01% w / v PS-20; and

[0392] About 2 mg / mL of methionine; pH about 5.5.

[0393] The present invention also provides a pharmaceutical composition comprising:

[0394] about 100 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0395] About 500 U / mL of rHuPH20;

[0396] About 10 mM histidine;

[0397] About 300 mM sorbitol;

[0398] About 0.02% w / v PS-20; and

[0399] About 2 mg / mL of methionine; pH about 5.5.

[0400] The present invention also provides a pharmaceutical composition comprising:

[0401] about 100 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0402] About 500 U / mL of rHuPH20;

[0403] About 10 mM histidine;

[0404] About 300 mM sorbitol;

[0405] about 0.06% w / v PS-20; and

[0406] About 2 mg / mL of methionine; pH about 5.5.

[0407] The present invention also provides a pharmaceutical composition comprising:

[0408] about 100 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0409] About 50 U / mL of rHuPH20;

[0410] About 10 mM histidine;

[0411] About 300 mM sorbitol;

[0412] About 0.04% w / v PS-20; and

[0413] About 1 mg / mL of methionine; pH about 5.5.

[0414] The present invention also provides a pharmaceutical composition comprising:

[0415] about 100 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0416] About 500 U / mL of rHuPH20;

[0417] About 10 mM histidine;

[0418] About 300 mM sorbitol;

[0419] About 0.04% w / v PS-20; and

[0420] About 1 mg / mL of methionine; pH about 5.5.

[0421] The present invention also provides a pharmaceutical composition comprising:

[0422] about 100 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0423] About 2000 U / mL of rHuPH20;

[0424] About 10 mM histidine;

[0425] About 300 mM sorbitol;

[0426] About 0.04% w / v PS-20; and

[0427] About 1 mg / mL of methionine; pH about 5.5.

[0428] The present invention also provides a pharmaceutical composition comprising:

[0429] about 100 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0430] About 5000U / mL of rHuPH20;

[0431] About 10 mM histidine;

[0432] About 300 mM sorbitol;

[0433] About 0.04% w / v PS-20; and

[0434] About 1 mg / mL of methionine; pH about 5.5.

[0435] In some embodiments, the pharmaceutical composition is a fixed combination.

[0436] Preparations to be used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

[0437] The pharmaceutical composition of the present invention can be prepared by known methods. For example, the pharmaceutical composition can be prepared by dissolving, suspending or emulsifying the anti-CD38 antibody in a sterile aqueous medium or oily medium conventionally used for injection.

[0438] Drug administration

[0439] The pharmaceutical composition of the present invention may be administered as a non-fixed combination.

[0440] The pharmaceutical compositions of the invention may also be administered as a fixed combination, for example, in a unit dosage form (or dosage unit form). Fixed combinations may have the advantage of ease of administration and uniformity of dosage.

[0441] The present invention also provides a unit dosage form comprising: an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 in an amount of about 1200 mg to about 5000 mg; and rHuPH20 in an amount of about 30000 U to about 75000 U.

[0442] The present invention also provides a unit dosage form comprising: an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 in an amount of about 1200 mg to about 4000 mg; and rHuPH20 in an amount of about 30000 U to about 75000 U.

[0443] The present invention also provides a unit dosage form comprising: an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 in an amount of about 1200 mg to about 2400 mg; and rHuPH20 in an amount of about 30000 U to about 45000 U.

[0444] The present invention also provides a unit dosage form comprising: an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 in an amount of about 1200 mg to about 1800 mg; and rHuPH20 in an amount of about 30000 U to about 45000 U.

[0445] The present invention also provides a unit dosage form comprising:

[0446] an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in an amount of about 1200 mg to about 5000 mg;

[0447] An amount of about 30,000 U to about 75,000 U of rHuPH20;

[0448] Histidine at a concentration of about 5 mM to about 15 mM;

[0449] sorbitol at a concentration of about 100 mM to about 300 mM;

[0450] PS-20 at a concentration of about 0.01% w / v to about 0.04% w / v; and

[0451] The concentration is about 1 mg / mL to about 2 mg / mL of methionine, and the pH is about 5.5.

[0452] The present invention also provides a unit dosage form comprising:

[0453] an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in an amount of about 1200 mg to about 2400 mg;

[0454] An amount of about 30,000 U to about 45,000 U of rHuPH20;

[0455] Histidine at a concentration of about 10 mM;

[0456] Sorbitol at a concentration of about 300 mM;

[0457] PS-20 at a concentration of about 0.04% w / v; and

[0458] The concentration was about 1 mg / mL of methionine; the pH was about 5.5.

[0459] The present invention also provides a unit dosage form comprising:

[0460] an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in an amount of about 1200 mg to about 1800 mg;

[0461] An amount of about 30,000 U to about 45,000 U of rHuPH20;

[0462] Histidine at a concentration of about 10 mM;

[0463] Sorbitol at a concentration of about 300 mM;

[0464] PS-20 at a concentration of about 0.04% w / v; and

[0465] The concentration was about 1 mg / mL of methionine; the pH was about 5.5.

[0466] The present invention also provides a unit dosage form comprising:

[0467] an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in an amount of about 1200 mg to about 1800 mg;

[0468] An amount of about 30,000 U to about 45,000 U of rHuPH20;

[0469] Histidine at a concentration of about 5 mM to about 15 mM;

[0470] sorbitol at a concentration of about 100 mM to about 300 mM;

[0471] PS-20 at a concentration of about 0.01% w / v to about 0.04% w / v; and

[0472] The concentration is about 1 mg / mL to about 2 mg / mL of methionine, and the pH is about 5.5.

[0473] The present invention also provides a unit dosage form comprising:

[0474] an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in an amount of about 1800 mg;

[0475] An amount of about 30,000 U of rHuPH20;

[0476] Histidine at a concentration of about 10 mM;

[0477] Sorbitol at a concentration of about 300 mM;

[0478] PS-20 at a concentration of about 0.04% w / v; and

[0479] The concentration was about 1 mg / mL of methionine; the pH was about 5.5.

[0480] The present invention also provides a unit dosage form comprising:

[0481] an anti-CD38 antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in an amount of about 1800 mg;

[0482] An amount of about 45,000 U of rHuPH20;

[0483] Histidine at a concentration of about 10 mM;

[0484] Sorbitol at a concentration of about 300 mM;

[0485] PS-20 at a concentration of about 0.04% w / v; and

[0486] The concentration was about 1 mg / mL of methionine; the pH was about 5.5.

[0487] The pharmaceutical compositions of the present invention may be administered in a total volume of about 80 mL, 90 mL, 100 mL, 110 mL, or 120 mL.

[0488] The pharmaceutical compositions of the present invention can be administered in a total volume of about 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL, 100 mL, 105 mL, 110 mL, 115 mL or 120 mL.

[0489] The pharmaceutical compositions of the present invention may be administered in a total volume of about 10 mL.

[0490] The pharmaceutical compositions of the present invention may be administered in a total volume of about 15 mL.

[0491] The pharmaceutical compositions of the present invention may be administered in a total volume of about 20 mL.

[0492] The total volume administered may generally be smaller for fixed combinations when compared to non-fixed combinations.

[0493] The present invention also provides a container comprising the pharmaceutical composition of the present invention.

[0494] The invention also provides a container comprising a unit dosage form of the invention.

[0495] The container may be a vial, a cartridge, a syringe, a prefilled syringe or a disposable pen.

[0496] The pharmaceutical composition of the present invention can be repeatedly administered 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 more. The course of treatment can also be repeated, as in chronic administration. Repeated administration can be the same dose or different doses. For example, the pharmaceutical composition of the present invention can be administered once a week for eight weeks, then biweekly for 16 weeks, and then once every four weeks.

[0497] The pharmaceutical compositions of the present invention may be administered subcutaneously.

[0498] The pharmaceutical compositions of the present invention may be administered subcutaneously to the abdominal area.

[0499] Subcutaneous administration can be achieved using a device. The device can be a syringe, a prefilled syringe, an autoinjector (disposable or reusable), a pen injector, a patch injector, a wearable injector, or a walking syringe infusion pump with a subcutaneous infusion set.

[0500] For non-fixed combinations, 20 mg / mL anti-CD38 antibody in 25 mM sodium acetate, 60 mM sodium chloride, 140 mM D-mannitol, 0.04% polysorbate 20 (pH 5.5) can be mixed with 1 mg / mL (75-150 kU / mL) rHuPH20 in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate-80 (pH 6.5) and the mixture can be administered to the subject.

[0501] The pharmaceutical compositions of the present invention may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events in cancer progression, and / or reduce the risk of recurrence after cancer remission. This may be particularly useful for patients whose tumors are difficult to locate but are known to have tumors due to other biological factors.

[0502] Treatment

[0503] The present invention also provides a method for treating cancer, comprising administering the pharmaceutical composition of the present invention to a subject in need thereof for a period of time sufficient to treat the cancer.

[0504] In some embodiments, the cancer is a CD38-positive hematological malignancy.

[0505] In some embodiments, the CD38-positive hematological malignancy is multiple myeloma.

[0506] In some embodiments, the CD38-positive hematological malignancy is diffuse large B-cell lymphoma (DLBCL).

[0507] In some embodiments, the CD38-positive hematological malignancy is non-Hodgkin's lymphoma.

[0508] In some embodiments, the CD38-positive hematological malignancy is acute lymphoblastic leukemia (ALL).

[0509] In some embodiments, the CD38-positive hematological malignancy is follicular lymphoma (FL).

[0510] In some embodiments, the CD38-positive hematological malignancy is Burkitt's lymphoma (BL).

[0511] In some embodiments, the CD38-positive hematological malignancy is mantle cell lymphoma (MCL).

[0512] In some embodiments, the CD38-positive hematological malignancy is light chain amyloidosis (AL).

[0513] In some embodiments, the CD38-positive hematological malignancy is multiple myeloma, acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), follicular lymphoma (FL), or mantle cell lymphoma (MCL).

[0514] Examples of B-cell non-Hodgkin's lymphomas are lymphomatoid granulomatosis, primary effusion lymphoma, intravascular large B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain disease (including gamma heavy chain disease, mu heavy chain disease and alpha heavy chain disease), lymphomas induced by treatment with immunosuppressants (such as cyclosporine-induced lymphoma and methotrexate-induced lymphoma).

[0515] In some embodiments, the cancer is a solid tumor.

[0516] The present invention also provides a method for treating CD38-positive malignant hematological tumors, comprising subcutaneously administering a pharmaceutical composition comprising an anti-CD38 antibody and a hyaluronidase to a subject in need thereof for a period of time sufficient to treat the CD38-positive malignant hematological tumors, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0517] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising subcutaneously administering to a subject in need thereof a pharmaceutical composition comprising an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 and hyaluronidase rHuPH20 of SEQ ID NO: 22 for a period of time sufficient to treat the CD38-positive hematological malignancies, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0518] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising between about 1200 mg-1800 mg of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 and between about 30,000 U-45,000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22 for a period of time sufficient to treat the CD38-positive hematological malignancies, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0519] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising between about 1200 mg and about 1800 mg of an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 and about 30,000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22 for a time sufficient to treat the CD38-positive hematological malignancies, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0520] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising between about 1200 mg and about 1800 mg of an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 and about 45,000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22 for a time sufficient to treat the CD38-positive hematological malignancies, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0521] The present invention also provides a method for treating CD38-positive malignant hematological tumors, comprising administering to a subject in need thereof about 1,600 mg of an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 and about 30,000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22 for a time sufficient to treat the CD38-positive malignant hematological tumors, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0522] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof about 1600 mg of an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 and about 45,000 U of hyaluronidase rHuPH20 of SEQ ID NO: 22 for a time sufficient to treat the CD38-positive hematological malignancies, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg / mL.

[0523] The present invention also provides a method for treating CD38-positive malignant hematological tumors, comprising administering a pharmaceutical composition comprising an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 and hyaluronidase to a subject in need thereof, wherein the pharmaceutical composition is a non-fixed combination.

[0524] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising:

[0525] about 20 mg / mL to about 120 mg / mL of an anti-CD38 antibody comprising the VH of SEQ ID NO:4 and the VL of SEQ ID NO:5 dissolved in about 25 mM acetic acid, about 60 mM sodium chloride, about 140 mannitol, and about 0.04% w / v polysorbate-20 (PS-20);

[0526] A pH of about 5.5; and

[0527] About 30,000 U to about 45,000 U of hyaluronidase dissolved in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate-80, pH 6.5.

[0529] In some embodiments, the hyaluronidase is rHuPH20.

[0530] In some embodiments, the pharmaceutical composition is a non-fixed combination.

[0531] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising:

[0532] about 20 mg / mL of an anti-CD38 antibody comprising the VH of SEQ ID NO:4 and the VL of SEQ ID NO:5 dissolved in about 25 mM acetic acid, about 60 mM sodium chloride, about 140 mannitol, and about 0.04% w / v polysorbate-20 (PS-20); pH is about 5.5; and

[0533] About 30,000 U of hyaluronidase, dissolved in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate-80, pH 6.5.

[0534] In some embodiments, the hyaluronidase is rHuPH20.

[0535] In some embodiments, the pharmaceutical composition is a non-fixed combination.

[0536] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising:

[0537] about 20 mg / mL of an anti-CD38 antibody comprising the VH of SEQ ID NO:4 and the VL of SEQ ID NO:5 dissolved in about 25 mM acetic acid, about 60 mM sodium chloride, about 140 mannitol, and about 0.04% w / v polysorbate-20 (PS-20); pH is about 5.5; and

[0538] About 45,000 U of hyaluronidase, dissolved in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate-80, pH 6.5.

[0539] In some embodiments, the hyaluronidase is rHuPH20.

[0540] In some embodiments, the pharmaceutical composition is a non-fixed combination.

[0541] The present invention also provides a method for treating CD38-positive malignant hematological tumors, comprising administering a pharmaceutical composition comprising an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 and hyaluronidase to a subject in need thereof, wherein the pharmaceutical composition is a fixed combination.

[0542] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising:

[0543] about 1 mg / mL to about 180 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0544] About 50 U / mL to about 5000 U / mL of hyaluronidase

[0545] About 5 mM to about 50 mM histidine; and

[0546] From about 50 mM to about 400 mM sorbitol.

[0547] In some embodiments, the hyaluronidase is rHuPH20.

[0548] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising:

[0549] about 1 mg / mL to about 180 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0550] About 50 U / mL to about 5000 U / mL of hyaluronidase

[0551] about 5 mM to about 50 mM histidine;

[0552] about 50 mM to about 400 mM sorbitol;

[0553] From about 0.01% w / v to about 0.1% PS-20; and

[0554] About 0.1 mg / mL to about 2.5 mg / mL of methionine.

[0555] In some embodiments, the hyaluronidase is rHuPH20.

[0556] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising:

[0557] about 100 mg / mL to about 120 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0558] about 50 U / mL to about 5000 U / mL of hyaluronidase;

[0559] About 10 mM histidine;

[0560] about 100 mM to about 300 mM sorbitol;

[0561] From about 0.01% w / v to about 0.04% w / v PS-20; and

[0562] About 1 mg / mL to about 2 mg / mL methionine.

[0563] In some embodiments, the hyaluronidase is rHuPH20.

[0564] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising:

[0565] about 100 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0566] About 500 U / mL of hyaluronidase;

[0567] About 10 mM histidine;

[0568] About 300 mM sorbitol;

[0569] About 0.04% w / v PS-20; and

[0570] About 2 mg / mL of methionine, pH about 5.5.

[0571] In some embodiments, the hyaluronidase is rHuPH20.

[0572] The present invention also provides a method for treating CD38-positive hematological malignancies, comprising administering to a subject in need thereof a pharmaceutical composition comprising:

[0573] about 120 mg / mL of a CD38 antibody comprising VH and VL of SEQ ID NOs 4 and 5, respectively;

[0574] About 2000 U / mL of rHuPH20;

[0575] About 10 mM histidine;

[0576] About 300 mM sorbitol;

[0577] About 0.04% w / v PS-20; and

[0578] About 1 mg / mL of methionine; pH about 5.6.

[0579] In some embodiments, the hyaluronidase is rHuPH20.

[0580] The anti-CD38 antibody in the pharmaceutical composition of the present invention can induce killing of tumor cells expressing CD38 through antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis or in vitro regulation of CD38 enzyme activity. The anti-CD38 antibody in the pharmaceutical composition of the present invention can also mediate anti-tumor efficacy through its immunomodulatory effect, which can be achieved by inducing CD4 + and CD8 + cell proliferation, and / or alleviate the suppression of inflammatory responses mediated by myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg).

[0581] "Antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with effector cells with lytic activity (such as natural killer cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγRs) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcvRIIIa. The activity of effector cells through the secretion of membrane pore-forming proteins and proteases can cause the death of antibody-coated target cells, such as cells expressing CD38. To assess the ADCC activity of antibodies that specifically bind to CD38, the antibodies can be added to cells expressing CD38 in combination with immune effector cells, which can be activated by the antigen-antibody complex, resulting in cytolysis of the target cells. Cytolysis is usually detected based on the release of markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from the lysed cells. Exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Exemplary target cells include Tregs or MDSCs expressing CD38. In an exemplary assay, 20 μCi of 51 Cr labeled target cells for 2 hours and then washed thoroughly. The cell concentration of target cells can be adjusted to 1×10 6 cells / ml and various concentrations of anti-CD38 antibody were added. The assay was initiated by adding target cells at a 40:1 effector cell:target cell ratio. After incubation for 3 hours at 37°C, the assay was terminated by centrifugation and the release of CD38 from the lysed cells was measured in a scintillation counter. 51 The percentage of cellular cytotoxicity can be calculated as the % of maximum lysis induced by the addition of 3% perchloric acid to target cells.

[0582] "Antibody-dependent cellular phagocytosis" ("ADCP") refers to a mechanism by which antibody-coated target cells are eliminated by internalization by phagocytes such as macrophages or dendritic cells. ADCP can be assessed by using Tregs or MDSCs expressing CD38 as target cells engineered to express GFP or other marker molecules. The effector cell: target cell ratio can be, for example, 4:1. The effector cells can be incubated with the target cells for 4 hours with or without anti-CD38 antibodies. After incubation, the cells can be separated using accutase. Anti-CD11b antibodies and anti-CD14 antibodies coupled to fluorescent markers can be used to identify macrophages, and standard methods can be used to identify CD11 based on the expression of CD11 + CD14 + The % GFP fluorescence in macrophages determined the percentage of phagocytosis.

[0583] "Complement-dependent cytotoxicity" or "CDC" refers to a mechanism of inducing cell death in which the Fc effector domain of a target-bound antibody binds and activates the complement component C1q, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0584] The ability of monoclonal antibodies to induce ADCC can be enhanced by engineering their oligosaccharide components. Most of the glycans of human IgG1 or IgG3 are N-glycosylated at Asn297 in the form of well-known bibranched G0, G0F, G1, G1F, G2 or G2F. Antibodies produced by unengineered CHO cells typically have a glycan fucose content of about at least 85%. Removing core fucose from bibranched complex-type oligosaccharides attached to the Fc region can enhance the ADCC of antibodies via improved FcγRIIIa binding without changing antigen binding or CDC activity. Such mAbs can be achieved using different methods that have been reported to result in the successful expression of relatively highly defucosylated antibodies with bibranched complex-type Fc oligosaccharides, such as controlling the culture osmotic pressure (Konno et al., Cytotechnology 64:249-65, 2012), using the variant CHO cell line Lec13 as a host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002), using the variant CHO cell line EB66 as a host cell line (Olivier et al., MAbs; 2(4), 2010; Epub ahead of print; PMID: 20562582), using the rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa et al., J Biol Chem 277:26733-26740, 2002), and using the variant CHO cell line EB66 as a host cell line (Olivier et al., MAbs; 2(4), 2010; Epub ahead of print; PMID: 20562582). 278:3466-3473, 2003), introduction of small interfering RNA specific for the α1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 2004), or co-expression of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or a potent α-glycosidase I inhibitor, kifnosine (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). ADCC elicited by the anti-CD38 antibodies used in the methods of the invention and in some embodiments of each of all numbered embodiments listed below can also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 (residue numbering according to the EU index) as described in U.S. Pat. No. 6,737,056.

[0585] In some embodiments, the anti-CD38 antibody comprises a substitution in the antibody Fc.

[0586] In some embodiments, the anti-CD38 antibody comprises a substitution at amino acid position 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 (residue numbering according to the EU index) in antibody Fc.

[0587] In some embodiments, the anti-CD38 antibody has a bibranched glycan structure with a fucose content of about 0% to about 15%, e.g., 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%.

[0588] In some embodiments, the anti-CD38 antibody has a bibranched glycan structure with a fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%.

[0589] Substitutions and reduced fucose content in Fc can enhance the ADCC activity of antibodies that specifically bind to CD38.

[0590] "Fucose content" means the amount of fucose monosaccharide within the sugar chain at Asn 297. The relative amount of fucose is the percentage of fucose-containing structures relative to all sugar structures. These can be characterized and quantified by a variety of methods, such as: 1) MALDI-TOF using samples treated with N-Glycosidase F (e.g., complex, mixed, and oligo- and high-mannose structures), as described in International Patent Publication No. WO2008 / 077546; 2) enzymatic release of the Asn297 glycan followed by derivatization and detection / quantification by HPLC (UPLC) and / or HPLC-MS (UPLC-MS) with fluorescence detection; 3) intact protein analysis of the mAb, either native or reduced, with or without treatment of the Asn297 glycan with Endo S or other enzymes that cleave between the first and second GlcNAc monosaccharides, leaving fucose attached to the first GlcNAc; 4) digestion of the mAb into component peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C) followed by separation, detection, and quantification by HPLC-MS (UPLC-MS); or 5) PNGase F at Asn297 mAb oligosaccharides are separated from mAb protein by specific enzymatic deglycosylation at 297. The released oligosaccharides can be fluorophore-labeled, separated, and identified by various complementary techniques that allow for detailed characterization of glycan structures by comparison of experimental and theoretical masses using matrix-assisted laser desorption ionization (MALDI) mass spectrometry, determination of the degree of sialylation by ion-exchange HPLC (GlycoSep C), separation and quantification of oligosaccharide forms based on hydrophilicity criteria by normal phase HPLC (GlycoSep N), and separation and quantification of oligosaccharides by high-performance capillary electrophoresis laser-induced fluorescence (HPCE-LIF).

[0591] As used herein, "low fucose" or "low fucose content" refers to an antibody having a fucose content of about 0% to 15%.

[0592] As used herein, "normal fucose" or "normal fucose content" refers to an antibody having a fucose content of greater than about 50%, typically greater than about 60%, 70%, 80% or greater than 85%.

[0593] In some embodiments of the methods described herein, and in all numbered embodiments listed below, the anti-CD38 antibody is of an IgG1, IgG2, IgG3, or IgG4 isotype.

[0594] Antibodies that are substantially identical to an antibody comprising a VH of SEQ ID NO: 4 and a VL of SEQ ID NO: 5 can be used in the methods of the present invention. As used herein, the term "substantially identical" means that the two antibody VH or VL amino acid sequences being compared are identical or have "non-significant differences". Non-significant differences refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids being substituted in the heavy or light chain of the antibody, and these substitutions do not adversely affect the properties of the antibody. Percent identity can be determined, for example, by pairwise alignment using the default settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, CA). The protein sequences of the present invention can be used as query sequences to search against public or patent databases, for example to identify related sequences. Exemplary programs for performing such searches are XBLAST or BLASTP programs (http_ / / www_ncbi_nlm / nih_gov) or GenomeQuest using default settings. TM (GenomeQuest, Westborough, MA) software package. Exemplary substitutions that can be made to the anti-CD38 antibodies used in the methods of the invention are, for example, conservative substitutions with amino acids of similar charge, hydrophobicity, or stereochemical properties. Conservative substitutions can also be made to improve antibody properties, such as stability or affinity, or to improve antibody effector function. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions can be made to the heavy chain or light chain of the anti-CD38 antibody. In addition, any native residue in the heavy chain or light chain can also be replaced with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., Acta Physiol Scand Suppl 643:55-67, 1998; Sasaki et al., Adv Biophys 35:1-24, 1998). The desired amino acid substitutions can be determined by those skilled in the art when such substitutions are needed. Amino acid substitutions can be performed, for example, by PCR mutagenesis (U.S. Pat. No. 4,683,195). Variant libraries can be generated using well-known methods, such as using random (NNK) or non-random codons (e.g., DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp)), and the library can be screened for variants with desired properties. The generated variants can be tested for binding to CD38, and their ability to induce ADCC, ADCP or apoptosis in vitro, or their ability to modulate CD38 enzymatic activity using the methods described herein.

[0595] In some embodiments, anti-CD38 antibodies can be used with a range of affinities (K D ) binds to human CD38. In one embodiment according to the invention, and in some embodiments of each of the following numbered embodiments, the anti-CD38 antibody binds to CD38 with high affinity, e.g., as determined by surface plasmon resonance or Kinexa methods, K D Equal to or less than about 10 -7 M, such as but not limited to 1-9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9)×10 - 8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 M or any range or value thereof, as practiced by those skilled in the art. An exemplary affinity is equal to or less than 1×10 -8 M. Another exemplary affinity is equal to or less than 1×10 -9 M.

[0596] In some embodiments, the anti-CD38 antibody is a bispecific antibody.The VL region and / or VH region of an existing anti-CD38 antibody or a VL region and VH region identified de novo as described herein can be engineered into a bispecific full-length antibody. Such bispecific antibodies can be prepared by modulating CH3 interactions between monospecific antibody heavy chains to form bispecific antibodies using techniques such as those described in the following patents: U.S. Patent No. 7,695,936; International Patent Publication No. WO04 / 111233; U.S. Patent Publication No. US2010 / 0015133, U.S. Patent Publication No. US2007 / 0287170; International Patent Publication No. WO2008 / 119353; U.S. Patent Publication No. US2009 / 0182127; U.S. Patent Publication No. US2010 / 0286374; U.S. Patent Publication No. US2011 / 0123532; International Patent Publication No. WO2011 / 131746; International Patent Publication No. WO2011 / 143545; or U.S. Patent Publication No. US2012 / 0149876. Other bispecific structures in which the VL region and / or VH region of the antibody of the present invention can be combined are, for example, dual variable domain immunoglobulins (International Patent Publication No. WO2009 / 134776), or structures including multiple dimerization domains to connect two antibody arms with different specificities, such as leucine zipper or collagen dimerization domains (International Patent Publication No. WO2012 / 022811, U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441).

[0597] For example, bispecific antibodies can be produced in vitro in a cell-free environment by the following process: according to the method described in International Patent Publication No. WO2011 / 131746, asymmetric mutations are introduced in the CH3 region of two monospecific homodimeric antibodies, and bispecific heterodimeric antibodies are formed from two parent monospecific homodimeric antibodies under reducing conditions that allow disulfide bond isomerization. In the method, a first monospecific bivalent antibody (e.g., an anti-CD38 antibody) and a second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; these antibodies are incubated together under reducing conditions sufficient to cause disulfide bond isomerization of cysteine ​​in the hinge region; thereby producing bispecific antibodies by Fab arm exchange. The incubation conditions are ideally restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine ​​and β-mercaptoethanol, preferably a reducing agent selected from 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine. For example, the following conditions can be used: in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a pH of 5-8, such as pH 7.0 or pH 7.4, at a temperature of at least 20° C., incubated for at least 90 minutes.

[0598] Exemplary CH3 mutations that can be used in the first and second heavy chains of the bispecific antibody are K409R and / or F405L.

[0599] The methods of the present invention can be used to treat animal patients belonging to any category. Examples of such animals include mammals, such as humans, rodents, dogs, cats, and farm animals.

[0600] Combination therapy

[0601] The pharmaceutical compositions of the present invention may be administered in conjunction with a second therapeutic agent or a combination thereof.

[0602] The second therapeutic agent can be melphalan, mechlorethamine, thiotepa, chlorambucil, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin, thalidomide or thalidomide analogs, lenalidomide or CC4047, a proteasome inhibitor (such as bortezomib), or a vinca alkaloid (such as vincristine) or an anthracycline (such as doxorubicin).

[0603] In some embodiments, the second therapeutic agent is a proteasome inhibitor.

[0604] In some embodiments, the proteasome inhibitor is bortezomib, carfilzomib, or ixazomib.

[0605] In some embodiments, the second therapeutic agent is an alkylating agent.

[0606] In some embodiments, the alkylating agent is busulfan, cyclophosphamide, bendamustine, chlorambucil, carboplatin, cisplatin, temozolomide, melphalan, busulfan, bendamustine, carmustine, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, thiotepa, trabectedin, or streptozotocin.

[0607] In some embodiments, the second therapeutic agent is a glutamate derivative.

[0608] In some embodiments, the glutamic acid derivative is (lenalidomide), thalidomide, or (Pomalidomide).

[0609] In some embodiments, the subject is further administered a corticosteroid.

[0610] In some embodiments, the corticosteroid is dexamethasone or prednisone.

[0611] The second therapeutic agent or combination thereof is generally administered at the recommended dose for that agent.

[0612] The pharmaceutical composition of the present invention may be administered simultaneously or sequentially with a second therapeutic agent or a combination thereof.

[0613]

[0066] While the present invention has been generally described, embodiments of the present invention will be further disclosed in the following examples, which should not be construed as limiting the scope of the claims.

[0614] Example 1: Subcutaneous delivery of 2% human immunoglobulin G (IgG) and recombinant human hyaluronidase in a minipig model PH20 (rHuPH20)

[0616] Minipigs are a clinical model suitable for evaluating subcutaneous (SC) administration conditions of biotherapeutics due to their anatomical similarity to human skin and clinical translatability (Mahj et al., Exp Toxicol Path 57:341-5, 2006). The purpose of this study was to evaluate and assess the conditions of administering 100 mL of a 20 mg / mL human IgG solution containing 200 U / mL, 500 U / mL or 800 U / mL of rHuPH20 at two different flow rates (2 mL / min and 4 mL / min). Endpoints included quantitative infusion pressure measurements and qualitative assessments of local infusion sites, such as swelling size and hardness.

[0617] Yucatan miniature pigs were subcutaneously infused with 100 mL of a solution containing 20 mg / mL immunoglobulin G (IgG) and 200 U / mL, 500 U / mL or 800 U / mL rHuPH20 at a flow rate of 2 mL / min or 4 mL / min. Real-time online pressure was measured during the infusion. After the infusion was completed, the visible swelling volume and area (if present) of the local infusion site were measured, and qualitative scoring was performed to the presence and severity of the erythema of the infusion site, the size and hardness of the swelling / blister and macroscopic observation. The infusion pressure was generally lower and in the range of about 40 mmHg to 60 mmHg (about 1 PSI) for both flows.

[0618] There were no statistical differences in pressure between the different concentrations of rHuPH20 and the two different flow rates, and the overall pressure was predictably slightly lower at a flow rate of 2 mL / min.

[0619] Unexpectedly, the number of infusions (10 out of 12) with visible and measurable swelling at the infusion site was found at the lower flow rate of 2 mL / min. This observation was observed at all three rHuPH20 concentrations. In contrast, at the higher flow rate of 4 mL / min, only 3 out of 12 infusions resulted in visible and measurable local swelling. Likewise, this was observed at each rHuPH20 concentration.

[0620] In all cases where local swelling was visible, the swelling subsided within one hour. In addition, the local swelling at the infusion site was usually soft to the touch and did not harden, as indicated by the swelling / hardening index (mean score <2). In the case of infusion with a 2mL / min flow rate, the incidence of erythema was more frequent; however, the severity of erythema was generally mild and completely subsided the next day. Other macroscopic observations of the infusion site were not noted in the study.

[0621] Three different concentrations of rHuPH20 (200 U / mL, 500 U / mL, or 800 U / mL) were evaluated in this study, and there were no statistical differences between the concentrations based on the study endpoints. Overall, the higher flow rate of 4 mL / min resulted in lower frequency of erythema, visible swelling, and local infusion site hardness compared to the 2 mL / min flow rate.

[0622] Test Articles and Methods

[0623] Test products

[0624] Materials in formulation buffer :

[0625] 25 mM sodium acetate (Spectrum; PN#S0104; Lot#1DI0271)

[0626] 60 mM sodium chloride (Spectrum; PN#S0155; Lot#1CE0421)

[0627] 140 mM D-mannitol (Spectrum; PN#MA165; Lot#1EB0316)

[0628] 0.04% Polysorbate 20 (JT Baker; PN#4116-04; Lot#0000017659)

[0629] pH 5.5 (glacial acetic acid adjusted to pH; Fisher Scientific; PN#A491-212;

[0630] Lot#080972)

[0631] Materials in drug substances :

[0632] Human gamma globulin (BioMed Supply; PN# HGG-1005;

[0633] Lot#BMS31309013)

[0634] · rHuPH20 [(Made by Cook Pharmica for Halozyme; Halozyme Lot #462-

[0635] ·021B(revialed,Cook Lot#104-001-HSTFIL-9054)]

[0636] preparation

[0637] Prior to the start of the study, 20 mg / mL IgG was mixed with 200 U / mL, 500 U / mL or 800 U / mL of rHuPH20 for 4 days. The solution was aliquoted into individual glass bottles, sealed with stoppers, and capped with crimps. All solutions were stored at 2-8°C until the start of the study, but were adapted to room temperature before infusion. In addition, samples were obtained from each formulation for rHuPH20 enzyme activity testing. The results of the enzyme activity assay confirmed that all dosing solutions were within 10% of the target concentration (data not shown).

[0638] Animal Description

[0639] Species: Pig (Sus scrofa domestica)

[0640] Breed: Yucatan Miniature Pig

[0641] Gender: Female

[0642] Age: > 3 months

[0643] Weight: Approx. 12kg

[0644] Quantity: 12 pieces

[0645] Source: S&S Farms (Ramona, CA)

[0646] Feeding

[0647] Animals were housed in steel stalls with running water available for use. Animals were fed twice daily (AM and PM) except for the study day (PM only). From the day of delivery to the day after the study was completed, the animals were weighed and recorded to assess the health status of the animals. All animals maintained their weight during this period (data not shown). The room environment was set to maintain a temperature of about 17-27°C and a relative humidity of 40-70%, and with a 12-hour lighting / 12-hour dark time cycle. Animals were adapted to the facility for 7 days before the start of the study.

[0648] Test Materials

[0649] High-pressure syringe pump (KD Scientific; Holliston, MA)

[0650] 23 ga x 3 / 4 inch winged infusion needle set with 12 inch tubing (Terumo Medical Corporation; Somerset, NJ)

[0651] 140-cc Luer lock syringe (Covidien; Mansfield, MA)

[0652] ·Additional device 7 inches (B / Braun; Bethlehem, PA)

[0653] ·PowerLab 4 / 30 (AD Instruments; Colorado Springs, CO)

[0654] Deltran-1 disposable pressure sensor (Utah Medical Products; Midvale, UT)

[0655] Digital calipers (Preisser Messtechnik; Gammertingen, Germany)

[0656] Isoflurane (Minrad International Company, Orchard Park, NY)

[0657] Isoflurane vaporizer (VetEquip; Pleasanton, CA)

[0658] Experimental design

[0659] The experimental design is summarized in the description of the group (Table 1) and the infusion description (Table 2) of each animal. In brief, 100mL of a solution comprising 20mg / mL IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20 was administered to the abdominal region of anesthetized Yucatan miniature pigs at a flow rate of 2mL / min or 4mL / min. The endpoints of the study included infusion pressure measurements using an online pressure sensor, local swelling (blister) volume and area (if possible) after infusion, and qualitative assessment of the infusion site (including photos).

[0660] Table 1 :

[0661]

[0662] Table 2 :

[0663]

[0664]

[0665] Research Process

[0666] Before starting the study, the general health of the animals was assessed and body weight was recorded. On the day of the study, the animals were anesthetized with isoflurane gas and placed on a heated operating table in a supine position (dorsal recumbence), and maintained under isoflurane gas throughout the duration of the process. The abdominal area was cleaned with isopropyl alcohol and wiped dry with clean gauze. The infusion site was located in the left and right abdominal area, starting from the cranial end of the inguinal fold and about 3-4 cm away from the cranial end of about 6 cm toward the midline. The infusion site was marked with a permanent marker and then photographed. Before infusion, the test article was adapted to room temperature. The test article was sucked into a 140cc syringe (>100mL to consider the volume required for starting the infusion line). A pressure sensor was attached to the syringe. Then a line extension device with a connected 23ga×3 / 4 inch winged infusion needle was attached to the sensor. Then the infusion hardware was prepared for the needle tip. The syringe was loaded into a syringe pump. Repeat this process, and each syringe contained a different test article. The needle was subcutaneously placed in the marked animal left and right abdominal infusion site. Make it online

[0667] The pressure sensor is reset to zero. Start online pressure recording, then make two syringe pumps start to infuse 100mL of test article with the flow rate of 2mL / min or 4mL / min simultaneously. After infusion is completed, stop online pressure data acquisition, remove pin, and insert pinhole with VetBond liquid adhesive seal to prevent any leakage. Use digital caliper to measure local infusion site swelling / blister area and volume. Use 5 point scoring system (respectively table 3, table 4 and table 5) to qualitatively assess the outward appearance (erythema), swelling / blister size and hardness (induration) of local infusion site in addition. Finally, take a picture of infusion site.

[0668] Table 3 :

[0669] Proportion illustrate 0 No erythema 1 Very mild erythema (barely noticeable) 2 Well-defined erythema 3 Moderate to severe erythema 4 Severe erythema (beet redness) to mild eschar formation

[0670] Table 4 :

[0671]

[0672] Table 5 :

[0673] Proportion illustrate 0 No noticeable difference in hardness after injection 1 Very slight hardening (almost unnoticeable) 2 Mild hardening 3 Moderate hardening 4 Very hard

[0674] Computational and statistical methods

[0675] Assessment of infusion pressure :

[0676] Infusion pressure (as measured by an in-line sensor) was recorded using LabChart 7, and the average pressure over the entire infusion period was calculated.

[0677] Assessment of local swelling volume and area :

[0678] The volume and area of ​​post-infusion swelling were measured using a digital caliper and recorded manually. Measurements were recorded as length, width, and height. Volume was calculated using the ellipsoid formula. Volume = 4 / 3πABC, where A = radius of length, B = radius of width, and C = radius of height. Area was calculated using a simple length × width formula.

[0679] Local Infusion Site Assessment :

[0680] After the infusion is completed, the local infusion site is independently evaluated by three independent assessors. Each assessor evaluates the presence of erythema in the skin at each infusion site, the size of local swelling and hardness. The scores according to the 0 to 4 rating scale are used to evaluate the three evaluation areas, with a score of 0 indicating no effect and 4 being severe. In addition, the erythema and swelling scores are used to calculate the initial irritation index (PII), which uses the formula PII = average value [(Σ erythema grade + Σ swelling grade) ÷ 2]. In addition, the swelling and hardness scores are used to calculate the swelling / hardening index (SII), which uses the formula SII = average value [(Σ swelling grade + Σ hardness grade) ÷ 2]. SII scores ≤ 2 are not considered hardening.

[0681] Statistical analysis :

[0682] Statistical comparisons between groups were performed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test for continuous variables and the nonparametric Krusal-Wallis test and Dunn's multiple comparison test for categorical variables. Statistical significance was determined at p < 0.05.

[0683] result

[0684] Assessment of infusion pressure :

[0685] 100mL of 20mg / mL IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20 was administered to the abdominal region of Yucatan miniature pigs at a flow rate of 2mL / min or 4mL / min. The IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20 was infused at a flow rate of 2mL / min resulting in an average infusion pressure of 40.5±0.1, 40.0±0.1 and 37.1±0.1 mmHg±SEM, respectively. The IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20 was infused at a flow rate of 4mL / min resulting in an average pressure of 49.9±0.1, 55.5±0.1 and 61.9±0.2 mmHg±SEM, respectively. There was no statistical difference in the infusion pressure at each flow rate between the various concentrations of rHuPH20, and there was no statistical difference in the infusion pressure between the two flow rates.

[0686] Assessment of local swelling volume and area :

[0687] After each infusion is completed, the swelling (if visible) of the local infusion site is marked and measured with a digital caliper. For the IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20, the flow infusion with 2mL / min causes 36.6 ± 14.4, 19.5 ± 6.5 and 31.4 ± 6.0cm respectively ± the average swelling volume of SEM, wherein 10 times are visible and measurable in 12 infusions. In contrast, under each rHuPH20 concentration, under the flow of 4mL / min, only 3 swelling volumes are detected in 12 infusions. The local swelling volume of every kind of flow is not statistically different between the rHuPH20 of various concentrations, and the local swelling volume is not statistically different between two kinds of flows.

[0688] Except volume calculation, also measured the local area of ​​visible swelling after infusion.For the IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20, the flow infusion with 2mL / min caused 78.2 ± 26.4, 59.7 ± 20.0 and 94.9 ± 9.7cm respectively ± the average swelling area of ​​SEM.Under each rHuPH20 concentration, for causing in 12 infusions, only 3 times have measurable swelling area with the flow infusion with 4mL / min.The local swelling area of ​​every kind of flow rate has no statistical difference between the rHuPH20 of various concentrations, and the local swelling area has no statistical difference between two kinds of flow rates.

[0689] Local Infusion Site Assessment :

[0690] After infusion was completed, three independent assessors qualitatively scored the local infusion site for the presence and severity of erythema, the visible size of swelling, the physical firmness of the skin, the initial irritation index (PII) that incorporated the erythema and swelling scores, and the swelling / hardness index (SII) that incorporated the swelling and hardness scores to determine if induration was present.

[0691] The presence and severity of erythema were assessed. For IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20, the flow rate infusion at 2mL / min resulted in an average erythema score of 0.8±0.2, 0.4±0.1 and 1.0±0.3 respectively. The flow rate infusion at 4mL / min with IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20 resulted in an average erythema score of 0.3±0.1, 0.3±0.1. and 0.2±0.1 respectively. There was no statistical difference in the local swelling and erythema score of each flow rate between the rHuPH20 of various concentrations, and there was no statistical difference in the local swelling and erythema score between two kinds of flow rates. The size of visible local swelling was evaluated and resulted in mean swelling scores (± SEM) of 1.9 ± 0.4, 1.4 ± 0.3, and 2.0 ± 0.2 for IgG blended with 200 U / mL, 500 U / mL, or 800 U / mL rHuPH20, respectively, at a flow rate of 2 mL / min. IgG blended with 200 U / mL, 500 U / mL, or 800 U / mL rHuPH20, respectively, had less visible swelling with mean erythema scores (± SEM) of 0.6 ± 0.3, 0.9 ± 0.4, and 0.9 ± 0.4, respectively, at a flow rate of 4 mL / min. There was no statistical difference in the local swelling score between the various concentrations of rHuPH20 at each flow rate, except for IgG + 800 U / mL of rHuPH20 at 2 mL / min flow rate and IgG + 200 U / mL of rHuPH20 at 4 mL / min flow rate (p < 0.05), and there was no statistical difference in the local swelling score between the two flow rates. The physical hardness of the skin at the local infusion site was evaluated, and the infusion of IgG blended with 200 U / mL, 500 U / mL or 800 U / mL rHuPH20 at a flow rate of 2 mL / min resulted in an average hardness score of 1.5 ± 0.3, 1.0 ± 0.2 and 1.4 ± 0.2, respectively (± SEM). The IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20 had a mean hardness score (± SEM) of 0.5 ± 0.3, 0.7 ± 0.2 and 0.7 ± 0.3 respectively at a flow rate of 4mL / min. The local infusion site hardness score of each flow rate had no statistical difference between the rHuPH20 of various concentrations, and the local infusion site hardness score had no statistical difference between two flow rates. The initial stimulation index was calculated based on erythema and swelling scores. The IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20 had a PII score (± SEM) of 1.4 ± 0.3, 0.9 ± 0.2 and 1.5 ± 0.2 respectively at a flow rate of 2mL / min.The IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20 was infused at a flow rate of 4mL / min and had a mean score (± SEM) of 0.4 ± 0.2, 0.6 ± 0.3 and a lower PII score of 0.5 ± 0.3. Except the rHuPH20 of IgG+800U / mL under 2mL / min flow and the rHuPH20 of IgG200U / mL under 4mL / min flow (p < 0.05), the PII score of every kind of flow was not statistically different between the rHuPH20 of various concentrations, and the PII score was not statistically different between two kinds of flows. Swelling / hardening index was calculated based on swelling and hardness score. IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20 has 1.7 ± 0.3, 1.2 ± 0.2 and 1.7 ± 0.2 SII scores (± SEM) respectively with a flow rate infusion of 2mL / min. IgG blended with 200U / mL, 500U / mL or 800U / mL rHuPH20 has a mean score of 0.6 ± 0.3, 0.8 ± 0.3 and 0.8 ± 0.3 (± SEM) respectively with a flow rate infusion of 4mL / min. The SII score of each flow rate is not statistically different between the rHuPH20 of various concentrations, and the SII score is not statistically different between two kinds of flow rates. Based on average SII values ​​(score ≤ 2), the local infusion site is not considered as sclerosis. Finally, before and after each infusion is completed, photos are taken.

[0692] Example 2. Evaluation of subcutaneous delivery of daratumumab supplemented with recombinant human hyaluronidase (rHuPH20) for the treatment of An open-label, multicenter study to evaluate the safety and pharmacokinetics of sirolimus in subjects with relapsed or refractory multiple myeloma Phase 1b dose-escalation study

[0693] The purpose of this study is to evaluate the safety, pharmacokinetics and anti-tumor activity of subcutaneous (SC) or intravenous (IV) delivery of daratumumab to participants with relapsed or refractory multiple myeloma. This is an open-label multicenter 2-part 1b phase dose escalation / expansion study to evaluate the safety, pharmacokinetics and anti-tumor activity of daratumumab delivered SC or IV to participants with relapsed or refractory multiple myeloma. Up to about 48 participants in Part 1 and 80 participants in Part 2 will participate. The first part of the dose escalation phase is designed to determine the recommended Phase 2 dose (RP2D) based on the safety and pharmacokinetic (PK) data of daratumumab. Each part of the study will have 3 stages: screening phase, open-label treatment phase and post-treatment phase (from the final dose of the study drug to 8 weeks after treatment). In Part 1, participants will be assigned to continuous groups of about 8 participants in each group. Participants will be given DARA PH20 (daratumumab with recombinant human hyaluronidase [rHuPH20] added) via SC infusion, once a week in cycles 1 (28 days per cycle) and 2, once every 2 weeks in cycles 3-6, and once every 4 weeks in subsequent cycles in each group. After the last participant in each group completes cycle 3 day 1, the Safety Evaluation Team (SET) will evaluate safety and pharmacokinetic data according to protocol-specified criteria and make a decision whether to gradually increase the dose in the new group. The SET will review all safety and PK data from Part 1 to determine the RP2D before starting Part 2. In Part 2, participants will be randomized 1:1 to receive the recommended Phase 2 dose of DARA PH20 or IV delivery of 1200 mg DARA. The safety, pharmacokinetics, and antitumor activity of SC and IV delivery of daratumumab will be evaluated. The safety of participants will be monitored throughout the study.

[0694] Primary outcome indicators :

[0695] Serum trough concentration (Ctrough) of daratumumab (time range: up to day 1 of cycle 3 (28 days per cycle) in part 2). Ctrough: concentration before study drug administration.

[0696] Part 1 and Part 2: Number of participants with adverse events (AEs) and serious AEs (Time frame: Screening to Follow-up (30 days after last dose administration)).

[0697] An adverse event (AE) is any untoward medical occurrence in a participant receiving study drug without regard to the likelihood of causality. A serious adverse event (SAE) is an AE resulting in any of the following outcomes or considered significant for any other cause: death; initial or prolonged patient hospitalization; life-threatening experience (immediate risk of death); persistent or significant disability / asthenia; congenital anomaly.

[0698] Secondary outcome measures :

[0699] • Part 1 and Part 2: Serum concentrations of daratumumab and recombinant human hyaluronidase (rHuPH20) antibodies (plasma) (time frame: approximately 2 years). Serum levels of daratumumab and rHuPH20 antibodies were used to assess potential immunogenicity.

[0700] Part 1 and Part 2: Percentage of participants with a complete response (CR) (time frame: approximately 2 years). CR was defined as the proportion of participants who achieved CR (including sCR) according to the International Myeloma Working Group (IMWG) criteria.

[0701] Part 1 and Part 2: Percentage of participants with an overall response rate (ORR) (time frame: approximately 2 years). The overall response rate was defined as the percentage of participants who achieved a complete response or partial response according to the International Myeloma Working Group criteria during or after study treatment.

[0702] • Part 1 and Part 2: Duration of Response (DR) (Time Frame: approximately 2 years). DR is the time from the date of initial documented response (CR or PR) to the date of first documented PD as defined by the IMWG standard.

[0703] • Part 1 and Part 2: Time to response (time frame: approximately 2 years). Time to response is defined as the time from the date of the first dose of study treatment to the date of the first documented observed response (CR or PR).

[0704] Table 6 shows the study design. Table 7 shows the interventions.

[0705] Table 6 :

[0706]

[0707] Table 7 :

[0708]

[0709] Competence :

[0710] Participants were diagnosed with symptomatic (having symptoms) multiple myeloma (MM) according to the International Myeloma Working Group (IMWG)

[0711] standard :

[0712] - Measurable disease, as defined by any of the following: (a) immunoglobulin (Ig) G myeloma (serum monoclonal paraprotein [M-protein] level >= 1.0 gram / deciliter [g / dL] or urine M-protein level greater than or equal to (>=) 200 milligrams [mg] / 24 hours [h]); or (b)

[0713] IgA, IgD, or IgE multiple myeloma (serum M-protein level >= 0.5 g / dL or urine protein level >= 200 mg / 24 hours); or (c) light chain multiple myeloma (serum immunoglobulin free light chains >= 10 mg / dL and abnormal serum immunoglobulin kappa to lambda free light chain ratio)

[0714] - Participants must have an Eastern Cooperative Oncology Group (ECOG) performance status score of 0, 1, or 2

[0715] - Pretreatment clinical laboratory values ​​must meet protocol-specified parameters during screening

[0716] - Males who are sexually active with females of childbearing potential and do not have a vasectomy must agree to use appropriate contraception as determined by the investigator and must also agree not to donate sperm during the study and for 4 months after the last dose of daratumumab

[0717] Exclusion criteria :

[0718] - Participants have previously received daratumumab or other anti-cluster of differentiation 38 (anti-CD38) therapy

[0719] - Participants have received anti-myeloma therapy within 2 weeks prior to Day 1 of Cycle 1

[0720] - Participants have previously received allogeneic stem cell transplant; or participants have received autologous stem cell transplant (ASCT) within 12 weeks prior to Cycle 1 Day 1

[0721] - Participant has a history of malignancy within 5 years prior to Cycle 1 Day 1 (exceptions are squamous and basal cell carcinoma of the skin and carcinoma in situ of the cervix, or a malignancy deemed curable at minimal risk of recurrence by the investigator in conjunction with the sponsor's medical monitor)

[0722] - Participant shows clinical signs of multiple myeloma invading the meninges

[0723] Gender: Both

[0724] Age limit: 18 years old

[0725] Accept healthy volunteers: No

[0726] Interim readout for Part 1 (for safety / demographics / disease history, data cutoff July 21, 2016; For efficacy data, data cutoff is July 28, 2016)

[0727] method

[0728] Patients suffer from RRMM and have ≥2 previous treatments, including proteasome inhibitors (PI) and immunomodulatory drugs (IMiD). Part 1 of the Part 2 study added continuous groups of 1200mg and 1800mg DARA dosage levels to determine the recommended SC dose for Part 2. DARA-PH20 is administered in a 4-week treatment cycle: QW for 8 weeks, Q2W for 16 weeks, and then Q4W. DARA-PH20 is infused via a syringe pump at a dose of 1200mg in 60mL or 1800mg in 90mL within 20min at a rotation site in the abdominal cavity. Pre-infusion and / or post-infusion drugs include paracetamol, diphenhydramine, montelukast and methylprednisolone. In Part 2, participants are randomized to 1:1 to receive the recommended Phase 2 dose (RP2D) of SC DARA-PH20 or IV DARA (16mg / kg). The RP2D of DARA-PH20 will be selected based on the cumulative review of pharmacokinetic and safety data obtained from Part 1 and should achieve a maximum serum Ctrough during weekly dosing that is similar to or higher than that observed for the approved 16 mg / kg IV dose. The primary endpoints are Ctrough (up to Day 1 of Cycle 3) and safety of DARA. Secondary endpoints include overall response rate (ORR).

[0729] result

[0730] To date, 41 participants have been treated with SC DARA-PH20 at 1200 mg (n=8) and 1800 mg (n=33) dose levels in Part 1. Infusion-related reactions (IRRs) were reported by 9 / 41 participants (22%), and most were grade 1 / 2 in severity, including chills, fever, chills, vomiting, pruritus, tongue edema, non-cardiac chest pain, and asthma. After the first infusion, one participant developed grade 3 dyspnea, and one participant required hospitalization for fever and chills (both grade 2). All IRRs developed during or within 6 hours after the first SC infusion and were controlled with antihistamines, corticosteroids, antiemetics, or bronchodilator therapy. No IRRs were reported with subsequent infusions. Overall, the adverse event profile of DARA-PH20 was consistent with that of IV DARA. Grade 3 or higher drug-related adverse events were reported in 5 / 41 (12%) participants, including fatigue (2 participants), influenza, hypertension, dyspnea, and tumor lysis syndrome. SC administration of DARA-PH20 was well tolerated at the abdominal wall injection site, with 3 / 41 (7%) participants reporting grade 1 erythema, induration, or burning sensation. Analysis showed that the maximum Ctrough in the 1800 mg group was higher compared to the maximum Ctrough achieved after IV DARA (16 mg / kg).

[0731] In the 8 participants in the 1200 mg group (median of 5 prior therapies [range 2-10]; prior ASCT, 63%; refractory PI only, 0%; refractory IMiD only, 13%; dual refractory to PI and IMiD, 63%), an ORR of 25% was observed, including 2 partial responses (PR). The median duration of response was 14 (range 8-20) weeks. In the 1800 mg group, among the 17 response-evaluable participants assessed on Cycle 3 Day 1 (median of 4 prior therapies [range 2-7]; prior ASCT, 76%; refractory PI only, 6%; refractory IMiD only, 12%; dual refractory to PI and IMiD, 65%), an ORR of 41% was observed, including 3 excellent partial responses and 4 PRs. The median duration of response was 4 (range 4-8) weeks.

[0732] in conclusion :

[0733] SC DARA-PH20 was well tolerated and achieved serum trough concentrations similar to or higher than IV DARA, and the IRR rate was lower than IV DARA at a significantly shorter infusion time. Preliminary data suggest that SC DARA-PH20 may allow a similar response rate to IV DARA monotherapy in this participant population. The 1800 mg dose level of DARA-PH20 was selected as the RP2D for Part 2 of the study. These early data support further study of SC DARA in clinical trials.

[0734] Example 3: Development of co-formulation of daratumumab and hyaluronidase

[0735] Various co-formulations were evaluated to establish the overall physicochemical stability and delivery of daratumumab and rHuPH20 in the co-formulated products. The effects of active ingredient and / or excipient concentrations in the formulations were evaluated in some stability and / or animal studies (shelf stability, shake stability, and pig infusion studies). Table 8 provides a summary of the formulations used for various studies.

[0736] Table 8 :

[0737]

[0738] The ranges of excipients and active ingredients in the tested formulations are shown in Table 9.

[0739] Table 9 :

[0740] Preparation ingredients scope rHuPH20 0-2000U / mL Daratumumab 100-120 mg / mL Histidine 10mM Sorbitol 100-300mM sucrose 0-200mM Polysorbate 20 (PS20) 0.0-0.04%(w / v) Methionine 0-2mg / mL

[0741] The characteristics of the resulting formulations were tested in various assays, including evaluation of microscopic particles, microangiography imaging (MFI), size exclusion chromatography (SEC), capillary isoelectric focusing (cIEF), SDS-PAGE (non-reducing and reducing), peptide mapping, extractable volume, turbidity, osmolarity, and pH.

[0742] Sub-vis: Sub-visible particles of size ≥10 μm or ≥25 μm are usually aggregates of protein molecules and can be determined by the light obstruction HIAC method, whereby a solution is passed through a small hole and the light obstruction provides information on the size of the particles that pass through.

[0743] MFI: Orthogonal to light obscuration, microangiography (MFI) takes a snapshot image of particles flowing through and converts it back to the number of particles present in a specific volume of fluid. This method provides information on the presence of larger protein aggregates in solution.

[0744] SEC: Size Exclusion Chromatography whereby a column is used to distribute the molecules in a solution flowing through it according to their broad size range. Monomers, aggregates and fragments elute from the column at different times and therefore their relative proportions in the sample can be quantified using a standard UV detector.

[0745] cIEF: Capillary isoelectric focusing distributes molecules according to the charge on them and is a good indicator of overall chemical stability. For example, deamidation can result in a change in the charge of a molecule and will therefore be picked up via this method. This method provides an idea of ​​the overall acidity, basicity and % intact of the molecules present in solution.

[0746] SDS (reducing and non-reducing conditions): The SDS method provides information on the physical stability of the molecule. SDS provides a measure of the intact, aggregated, and fragmented species present in solution. Non-reducing SDS provides information on the intact, aggregated, and fragmented components of the antibody, while reduced SDS (after disulfide destruction) provides the same information for the heavy and light chains of the antibody.

[0747] Peptide mapping: Peptide mapping is an important technique for studying the primary structure of proteins. For recombinant protein drugs, peptide mapping is used for preliminary verification of structural characterization. Peptide mapping also provides information on post-translational modifications (e.g., deamidation, oxidation, etc.).

[0748] Extractable volume: This method provides information on the amount / volume of liquid that can be withdrawn from the vial after the corresponding time point.

[0749] Turbidity: A light scattering based method to assess the physical stability of a solution. An increase in the size of particles or aggregates results in an increase in the light scattering signal, thus obtained as the turbidity (opalescence) of the solution. Turbidity is measured in Nephelometric Turbidity Units (NTU).

[0750] Osmolarity: provides a measure of the total osmotic activity, which is dependent on the total true activity of the molecule (activity coefficient multiplied by concentration). The solution must approximate the osmolarity of the serum to be injected.

[0751] pH: provides an overall stability perspective and it is important that the pH remains constant throughout the storage life.

[0752] rHuPH20 enzyme activity: The determination of hyaluronidase activity is based on the precipitation formed when hyaluronic acid (HA) is combined with acidified serum. The activity is measured by incubating hyaluronidase with HA in a 96-well plate format at 37°C for 30 minutes and then adding acidified serum to precipitate the undigested HA. The resulting turbidity is measured at 640nm, and the decrease in turbidity caused by the enzymatic cleavage of the HA substrate is a measure of hyaluronidase activity.

[0753] The shelf stability of formulation 1 (100 mg / mL daratumumab, 10 mM histidine, 300 mM sorbitol, 0.04% PS20, 2 mg / mL methionine, 500 U / mL PrHuh20, pH 5.5) was evaluated using the assay. The samples were maintained in 25R vials (filled in 16 mL volume) at different temperatures (5°C, 25°C, and 40°C) and the vials were removed for analysis using various assays at different time points (0, 1, 2, 3, 4, 5, and / or 6 months). The data indicate that for both daratumumab and rHuPH20, the co-formulated product is stable under storage conditions, as indicated by various assays. The characteristics observed for particles, color, turbidity, sec, etc. are very similar to well-performing stable antibodies, and the data are equivalent to the stability data of some commercial mAb formulations.

[0754] Table 10 shows the number of particles in Formulation 1 over time as assessed using HIAC.

[0755] Table 11 shows the number of particles in Formulation 1 over time as assessed by MFI.

[0756] Table 12 shows the pH of Formulation 1 over time.

[0757] Table 13 shows the turbidity of Formulation 1 over time.

[0758] Table 14 shows the ratio of high molecular weight aggregates to low molecular weight fragments in Formulation 1 over time.

[0759] Table 15 shows the acidic and basic species in Formulation 1 over time as assessed by cIEF.

[0760] Table 16 shows the percent (%) purity of Formulation 1 over time as assessed by reducing SDS-PAGE.

[0761] Table 17 shows the percent (%) purity of Formulation 1 over time as assessed by non-reducing SDS-PAGE.

[0762] Table 18 shows the percent (%) biological activity of daratumumab in Formulation 1 and the enzyme activity of rhPH20 over time.

[0763] Table 10 :

[0764]

[0765] Table 11 :

[0766]

[0767] Table 12 :

[0768]

[0769] Table 13 :

[0770]

[0771]

[0772] Table 14 :

[0773]

[0774] Table 15 :

[0775]

[0776] Table 16

[0777]

[0778]

[0779] Table 17 :

[0780]

[0781] Table 18 :

[0782]

[0783] The agitation (shaking) stability of Formulation 1 was also evaluated using the above assays to characterize the formulations and to study the effect of PS concentration by varying the concentration of PS20 in the formulation only (Formulations 1, 3, 4, 5, and 6 in Table 8, where PS20 was varied to 0%, 0.01%, 0.02%, 0.04%, 0.06%). The data indicate that for both daratumumab and the enzyme, the co-formulations were stable under shaking conditions as indicated by the various assays. For all concentrations of PS except 0% (the 0% PS20 formulation had particles and was unstable), the characteristics observed, such as particles, color, turbidity, sec, etc., were very similar to those of well-performing stable antibodies, and the data were comparable to the stability data of some commercial mAb formulations (data not shown).

[0784] The shelf stability of formulation 2 was evaluated using the assay (120 mg / mL daratumumab, 10 mM histidine, 300 mM sorbitol, 0.04% PS20, 1 mg / mL methionine, 2000 U / mL rhuPH20, pH 5.5). The samples were maintained in 25R vials filled with 13.27 mL volume (1500 mg dose) at different temperatures, and the vials were removed for analysis using the following various assays. The collected data indicated that the co-formulated product was stable under storage conditions for both daratumumab and rHuPH20. The observed features, such as particles, color, turbidity, sec, etc., were very similar to well-performing stable antibodies, and the data were equivalent to the stability data of some commercial mAb formulations. rhuPH20 is extremely sensitive at higher temperatures and loses all activity very quickly when stored at 40°C. Table 19 shows the characteristics of the formulation.

[0785] Table 19 :

[0786]

[0787] Formulations 3-8 were tested for shelf stability or shake stability using some or all of the assays described. The data indicate that formulations 3-8 were stable under the conditions evaluated for both daratumumab and HuPH20 (formulations 7 and 8 did not have rHuPH20). Methionine was included in formulations 1-6 and 9-12 to provide additional oxidative stability. The characteristics observed for particles, color, turbidity, sec, etc. were very similar to those of well-performing stable antibodies, and the data are equivalent to stability data for some commercial mAb formulations (data not shown).

[0788] The agitation (shaking) stability of Formulation 1 was also evaluated using the above assays to characterize the formulations and to study the effect of PS20 concentration by varying the concentration of PS20 in the formulation alone (Formulations 1, 3, 4, 5, and 6 in Table 8, where PS20 was varied to 0%, 0.01%, 0.02%, 0.04%, 0.06%). The data indicate that for both daratumumab and rHuPH20, the co-formulations were stable under shaking conditions as indicated by the various assays. For all concentrations of PS except 0%, the characteristics observed, such as particles, color, turbidity, sec, etc., were very similar to those of well-performing stable antibodies, and the data were comparable to stability data for some commercial mAb formulations (data not shown).

[0789] For the evaluation of subcutaneous administration of daratumumab with different concentrations of enzyme in a porcine model (as described in Example 2), formulations 9-12 were also evaluated. These studies were conducted to determine the appropriate concentration of rhPH20 to deliver 16 mL of daratumumab. If the site can be measured and qualitatively assessed, the endpoint is the infusion pressure, swelling or blister area. A dose-dependent increase in infusion pressure was observed. All rhPH20 concentrations tested (50U / mL, 500U / mL, 2000U / mL, 5000U / mL) were sufficient to deliver 16 mL of daratumumab.

Claims

1. A pharmaceutical composition comprising about 1800 mg of an anti-CD38 antibody and about 30,000 U of a hyaluronidase, wherein the anti-CD38 antibody has an IgG1 isotype and comprises a heavy chain variable region sequence of SEQ ID NO: 4 and a light chain variable region sequence of SEQ ID NO: 5, and wherein the hyaluronidase is rHuPH20 recombinant hyaluronidase.

2. The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier. 3 . The pharmaceutical composition according to claim 2 , wherein the anti-CD38 antibody comprises a heavy chain sequence of SEQ ID NO: 12 and a light chain sequence of SEQ ID NO:

13.

4. The pharmaceutical composition according to claim 2, further comprising a) about 5 mM to about 50 mM histidine; and b) about 50 mM to about 400 mM sorbitol.

5. The pharmaceutical composition according to claim 4, further comprising a) about 0.01% w / v to about 0.1% w / v polysorbate-20 (PS-20); b) about 0.1 mg / mL to about 2.5 mg / mL methionine; or c) about 0.01% w / v to about 0.1% w / v PS-20 and about 0.1 mg / mL to about 2.5 mg / mL methionine.

6. The pharmaceutical composition according to claim 4, comprising a) about 10 mM histidine; and b) about 100 mM to about 300 mM sorbitol.

7. The pharmaceutical composition according to claim 6, further comprising a) from about 0.01% w / v to about 0.04% w / v PS-20; and b) about 1 mg / mL to about 2 mg / mL of methionine.

8. The pharmaceutical composition according to claim 5, comprising a) about 10 mM histidine; b) about 300 mM sorbitol; c) about 0.04% w / v PS-20; and d) about 1 mg / mL methionine; pH about 5.

6.

9. The pharmaceutical composition according to claim 5, comprising a) about 10 mM histidine; b) about 300 mM sorbitol; c) about 0.04% w / v PS-20; and d) about 2 mg / mL methionine; pH about 5.

5.

10. The pharmaceutical composition according to claim 5, comprising a) about 10 mM histidine; b) about 300 mM sorbitol; c) about 0.01% w / v PS-20; and d) about 2 mg / mL methionine; pH about 5.

5.

11. The pharmaceutical composition according to claim 5, comprising a) about 10 mM histidine; b) about 300 mM sorbitol; c) about 0.02% w / v PS-20; and d) about 2 mg / mL methionine; pH about 5.

5.

12. The pharmaceutical composition according to claim 5, comprising a) about 10 mM histidine; b) about 300 mM sorbitol; c) about 0.06% w / v PS-20; and d) about 2 mg / mL methionine; pH about 5.

5.

13. The pharmaceutical composition according to claim 5, comprising a) about 10 mM histidine; b) about 300 mM sorbitol; c) about 0.04% w / v PS-20; and d) about 1 mg / mL methionine; pH about 5.

5.

14. The pharmaceutical composition according to claim 2, which is in unit dosage form.

15. A method for treating CD38-positive multiple myeloma (MM) in a subject, comprising subcutaneously administering to a subject in need thereof a pharmaceutical composition comprising about 1800 mg of an anti-CD38 antibody and about 30,000 U of a hyaluronidase for a time sufficient to treat the CD38-positive MM, wherein the anti-CD38 antibody has an IgG1 isotype and comprises a heavy chain variable region sequence of SEQ ID NO: 4 and a light chain variable region sequence of SEQ ID NO: 5, and wherein the hyaluronidase is rHuPH20 recombinant hyaluronidase.

16. The method of claim 15, wherein the anti-CD38 antibody comprises a heavy chain sequence of SEQ ID NO: 12 and a light chain sequence of SEQ ID NO:

13.

17. The method of claim 15, wherein the pharmaceutical composition further comprises about 5 mM to about 50 mM histidine and about 50 mM to about 400 mM sorbitol, optionally further comprising about 0.01% w / v to about 0.1% w / v polysorbate-20 (PS-20), about 0.1 mg / mL to about 2.5 mg / mL methionine, or about 0.01% w / v to about 0.1% w / v PS-20 and about 0.1 mg / mL to about 2.5 mg / mL methionine.

18. The method of claim 17, wherein the pharmaceutical composition comprises about 10 mM histidine and about 100 mM to about 300 mM sorbitol, optionally further comprising about 0.01% w / v to about 0.04% w / v PS-20, about 1 mg / mL to about 2 mg / mL methionine, or about 0.01% w / v to about 0.04% w / v PS-20 and about 1 mg / mL to about 2 mg / mL methionine.

19. The method of claim 18, wherein the pharmaceutical composition comprises about 10 mM histidine, about 300 mM sorbitol, about 0.04% w / v PS-20, and about 1 mg / mL methionine; and the pH is about 5.

6.

20. The method of claim 18, wherein the pharmaceutical composition comprises about 10 mM histidine, about 300 mM sorbitol, about 0.04% w / v PS-20, and about 2 mg / mL methionine; and the pH is about 5.

5.

21. The method of claim 15, further comprising administering a second therapeutic agent.

22. The method of claim 21, wherein the second therapeutic agent is a proteasome inhibitor, an alkylating agent, or a glutamate derivative, or a combination thereof.

23. The method according to claim 22, wherein a) the proteasome inhibitor is bortezomib, carfilzomib or ixazomib; b) the alkylating agent is busulfan, cyclophosphamide, bendamustine, chloramphenicol, carboplatin, cisplatin, temozolomide, melphalan, carmustine, cyclohexyl lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, thiotepa, trabectedin or streptozotocin; and c) The glutamic acid derivative is lenalidomide, thalidomide or pomalidomide.

24. The method of claim 23, further comprising administering a corticosteroid.

25. The method of claim 24, wherein the corticosteroid is dexamethasone or prednisone.

26. The method of claim 25, wherein the corticosteroid is dexamethasone.

27. A unit dosage form comprising a) an anti-CD38 antibody of the IgG1 isotype in an amount of about 1800 mg, the anti-CD38 antibody comprising a heavy chain variable region sequence of SEQ ID NO: 4 and a light chain variable region sequence of SEQ ID NO: 5; b) a hyaluronidase in an amount of about 30,000 U, wherein the hyaluronidase is rHuPH20 recombinant hyaluronidase; c) histidine at a concentration of about 5 mM to about 15 mM; d) sorbitol at a concentration of about 100 mM to about 300 mM; e) polysorbate-20 (PS-20) at a concentration of about 0.01% w / v to about 0.04% w / v; and f) methionine at a concentration of about 1 mg / mL to about 2 mg / mL and a pH of about 5.

5.

28. The unit dosage form according to claim 27, wherein histidine is present at a concentration of about 10 mM.

29. The unit dosage form according to claim 28, wherein sorbitol is present at a concentration of about 300 mM.

30. The unit dosage form according to claim 29, wherein the polysorbate is present at a concentration of about 0.04% w / v.

31. The unit dosage form according to claim 30, wherein methionine is present at a concentration of about 1 mg / mL.

32. The unit dosage form according to claim 31, comprising sucrose at a concentration of about 100 mM to about 200 mM.

33. The unit dosage form of claim 32, wherein the anti-CD38 antibody comprises the heavy chain sequence of SEQ ID NO: 12 and the light chain sequence of SEQ ID NO:

13.

34. A container comprising the unit dosage form according to claim 33.

35. A unit dosage form comprising a) an anti-CD38 antibody of the IgG1 isotype in an amount of about 1800 mg, the anti-CD38 antibody comprising a heavy chain variable region sequence of SEQ ID NO: 4 and a light chain variable region sequence of SEQ ID NO: 5; b) a hyaluronidase in an amount of about 30,000 U, wherein the hyaluronidase is rHuPH20 recombinant hyaluronidase; c) histidine at a concentration of about 10 mM; d) sorbitol at a concentration of about 300 mM; e) polysorbate 20 (PS-20) at a concentration of about 0.04% w / v; and f) methionine at a concentration of about 1 mg / mL and a pH of about 5.

6.

36. A container comprising the unit dosage form according to claim 35.

37. The unit dosage form of claim 35, wherein the anti-CD38 antibody comprises the heavy chain sequence of SEQ ID NO: 12 and the light chain sequence of SEQ ID NO: 13.

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