EA042589B1 - COMPOSITIONS OF ANTIBODIES TO CD38 FOR SUBCUTANEOUS ADMINISTRATION AND THEIR APPLICATION - Google Patents

Description

Sequence listing

This application contains a sequence listing submitted via EFS-Web, the entire content of which is incorporated herein by reference in its entirety. The ASCII text file created on October 28, 2016 is named JBI5070USNP_ST25.txt and is 26 kilobytes in size.

Scope of the invention

The present invention relates to subcutaneous anti-CD38 antibody formulations and their uses.

Prerequisites for the creation of the invention

CD38 is a multifunctional protein whose function is receptor-mediated adhesion and signaling, as well as mediating calcium mobilization through its exoenzymatic activity, catalyzing the formation of cyclic ADP-ribose (cADFR) and ADFR. CD38 mediates cytokine secretion as well as lymphocyte activation and proliferation (Funaro et al., J Immunol 145:2390-6, 1990; Terhorst et al., Cell 771-80, 1981; Guse et al., Nature 398:70-3 , 1999). Through its NAD-glycohydrolase activity, CD38 also regulates levels of extracellular NAD+, which is involved in modulating the regulatory T cell compartment (Adriouch et al., 14:1284-92, 2012; Chiarugi et al., Nature Reviews 12:741-52, 2012). In addition to Ca2+ signaling, CD38 signaling occurs through cross-exchange with antigen-receptor complexes on T and B cells or other types of receptor complexes, such as major histocompatibility complex (MHC) molecules, involving CD38 in several cellular responses, as well as switching and secretion of IgG1. CD38 is expressed on various cancer cells.

Anti-CD38 antibodies are being investigated as a treatment for multiple myeloma and other hematological malignancies. Antibodies are administered or infused by the intravenous (IV) route. The amount of antibody that can be administered intravenously is limited by the physicochemical properties of the antibody, in particular its solubility and stability in an acceptable liquid formulation, as well as the volume of the infusion fluid.

Thus, there is a need for additional formulations and pharmaceutical compositions containing an anti-CD38 antibody.

Statement of the Invention

The invention provides a pharmaceutical composition containing an anti-CD38 antibody and hyaluronidase.

The invention also provides a pharmaceutical composition containing an anti-CD38 antibody and rHuPH20 hyaluronidase having the amino acid sequence of SEQ ID NO: 22.

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

The invention also provides a method for treating a CD38-positive hematologic cancer, comprising subcutaneously administering to a subject in need thereof a pharmaceutical composition of the invention for a time period sufficient to treat the CD38-positive hematologic cancer.

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

The invention also provides a unit dosage form containing an antibody to CD38 containing VH with SEQ ID NO: 4 and VL with SEQ ID NO: 5, in an amount of from about 1200 to about 5000 mg;

hyaluronidase in an amount of from about 30,000 to about 45,000 U;

histidine at a concentration of about 5 to about 15 mM;

sorbitol at a concentration of about 100 to about 300 mM;

PS-20 at a concentration of about 0.01 to about 0.04% w/v. and methionine at a concentration of about 1 to about 2 mg/ml, pH about 5.5.

The invention also provides a single dosage form according to item 74, containing an antibody to CD38 containing VH with SEQ ID NO: 4 and VL with SEQ ID NO: 5, in an amount of about 1800 mg;

hyaluronidase in an amount of about 30,000 U;

histidine at a concentration of about 10 mM;

sorbitol at a concentration of about 300 mm;

PS-20 at a concentration of about 0.04% w/v. and methionine at a concentration of about 1 mg/ml, pH about 5.5.

The invention also provides a container containing a unit dosage form of the invention.

The invention also provides a container containing the pharmaceutical composition of the invention.

- 1 042589

Detailed description of the invention

The term CD38 refers to the human CD38 protein (synonyms: ADP-ribosylcyclase 1, cADPH-hydrolase 1, cyclic ADP-ribose hydrolase 1). Human CD38 has an amino acid sequence which is shown in GenBank in order of entry NP 001766 and in SEQ ID NO: 1. It is well known that CD38 is a type II single-stranded membrane protein with amino acid residues 1-21 representing the cytosolic domain, amino acid residues 22- 42 representing the transmembrane domain and residues 43-300 representing the extracellular domain of CD38.

SEQ ID NO: 1

MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRF

PETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKIL LWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSV FWKTVSRRFAEAACDWHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSR DLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI

The term antibodies is intended in a broad sense and includes immunoglobulin molecules, including monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, antigen-binding fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single-chain antibodies, domain-specific antibodies, and any another modified configuration of the immunoglobulin molecule that contains an antigen-binding site of the desired specificity. Full-length antibodies consist of two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (consisting of CH1 domains, CH2 hinge region 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 regions of hypervariability, referred to as complementarity determining regions (CDRs), between which are frame regions (FRs). Each VH and VL region consists of three CDRs and four FRs arranged in the amino-terminal to carboxy-terminal direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

Complementarity determining regions (CDRs) represent antigen-binding sites in an antibody. CDRs can be defined using different terms: (i) complementarity determining regions (CDRs), three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3), based on sequence variability (Wu and Kabat, J Exp Med 132:211-50, 1970; Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991); (ii) hypervariable regions, HVR or HV, three in VH (H1, H2, H3) and three in VL (L1, L2, L3), refer to antibody variable domain regions that are structurally hypervariable, as defined by Chothia, and Lesk (Chothia and Lesk, Mol. Biol 196:901-17, 1987). The international database ImMunoGeneTics (IMGT) (http://www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between the CDR, HV, and IMGT delimitations is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003. As used herein, the designations CDR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include CDRs defined by any of methods described above, according to Kabat, Chothia or IMGT, unless otherwise expressly indicated in this description.

Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be classified into five major classes, IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further classified into IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4 isotypes. The light chains of antibodies of any vertebrate species can be assigned, depending on the amino acid sequences of their constant domains, to one of two distinct types, namely kappa (k) and lambda (λ).

The term antigen-binding fragment refers to the part of the immunoglobulin molecule that retains the antigen-binding properties of the parent full-length antibody. Exemplary antigen binding fragments are heavy chain complementarity determining regions (HCDR) 1, 2 and/or 3, light chain complementarity determining region (LCDR) 1, 2 and/or 3, heavy chain variable region (VH), or light chain variable region (VL). ), fragments of Fab, F(ab')2, Fd and Fv, as well as domain antibodies (dAb) consisting of either a single VH domain or a single VL domain. The VH and VL domains can be linked together via a synthetic linker to form various types of single chain antibody configurations, in which the VH/VL domains are paired intramolecularly or intermolecularly in cases where the VH and VL domains are expressed by separate antibody chains to form a monovalent antigen binding site, eg single chain Fv (scFv) or diabody; they are described, for example, in International Patent Publication No. WO 1998/44001, International Patent Publication No. WO 1988/01649; International Patent Publication No. WO 1994/13804; International Patent Publication No. WO 1992/01047.

A monoclonal antibody is a population of antibodies with the same amino acid

- 2 042589 becoming in each heavy and each light chain, with the exception of possible well-known changes, such as the removal of the C-terminal lysine from the heavy chain of the antibody. Monoclonal antibodies generally bind to a single antigenic epitope, with the exception of multispecific monoclonal antibodies that bind to two or more distinct antigens or epitopes. Bispecific monoclonal antibodies bind to two distinct antigenic epitopes. Within an antibody population, monoclonal antibodies may have heterogeneous glycosylation. The monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. The term monoclonal antibody includes a multispecific antibody, such as a bispecific antibody or a trispecific antibody.

The term isolated antibody means an antibody or antigen-binding fragment that is substantially free of other antibodies having different antigen specificity values (e.g., an isolated antibody that specifically binds to human CD38 is essentially free of antibodies that specifically bind to antigens other than human CD38) . In the case of a bispecific antibody, the 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. The term isolated antibody includes antibodies isolated to be of higher purity, such as antibodies that are 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 pure. , 94, 95, 96, 97, 98, 99 or 100%.

The term humanized antibodies refers to antibodies in which the antigen-binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences.

Humanized antibodies may include deliberately introduced mutations in the framework regions, whereby the framework region may not be an exact copy of the expressed human immunoglobulin or germline gene sequences.

The term human antibodies refers to antibodies having heavy and light chain variable regions in which both the framework regions and the antigen binding sites 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.

A human antibody contains heavy or light chain variable regions that are derived from sequences of human origin if the antibody variable regions are derived from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems are phage-displayed human immunoglobulin gene libraries and non-human transgenic animals such as mice or rats harboring human immunoglobulin loci as described herein. A human antibody typically contains amino acid differences from the human germline or rearranged immunoglobulin sequences due, for example, to naturally occurring somatic mutations or intentional substitutions at the scaffold or antigen binding site and amino acid changes introduced during VDJ cloning and recombination in animals other than person. Typically, a human antibody will have an amino acid sequence of at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99 or 100% identical to the amino acid sequence encoded by the human germline gene or the rearranged immunoglobulin gene. In some cases, the human antibody may contain consensus framework sequences derived from human framework sequence assays, such as those described in Knappik et al., J Mol Biol 296:57-86, 2000, or synthetic HCDR3s included in human immunoglobulin gene libraries displayed on phage, for example, as described in Shi et al., J Mol Biol 397:385-96, 2010 and International Patent Publication No. WO 2009/085462.

Antibodies in which the antigen-binding sites are derived from a non-human species do not fit the definition of a human antibody.

The term recombinant refers to antibodies and other proteins that are produced, expressed, created or isolated by recombinant methods.

The term epitope refers to the portion of an antigen to which an antibody specifically binds. Typically, epitopes are composed of reactive (such as polar, nonpolar, or hydrophobic) surface groupings of components such as amino acid or polysaccharide side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope may be formed from continuous and/or discontinuous amino acids forming a conformational spatial unit. In the case of a discontinuous epitope, amino acids from different parts of the linear sequence of the antigen come close to each other in three-dimensional space by folding the protein molecule.

The term multispecific refers to an antibody that specifically binds to at least two different antigens or two different epitopes within the same antigen,

- 3 042589 for example three, four or five different antigens or epitopes.

The term bispecific refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen. A bispecific antibody may be cross-reactive with other related antigens, or may bind to an epitope that is common to two or more different antigens.

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

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

The term "pharmaceutical composition" refers to a product that is formed by combining an anti-CD38 antibody and a hyaluronidase and includes both bound and unbound combinations. The pharmaceutical composition typically includes a pharmaceutically acceptable carrier. The expression related combinations refers to a single pharmaceutical composition containing an anti-CD38 antibody and hyaluronidase administered simultaneously in the form of a single unit or dose. The expression unrelated combination refers to separate pharmaceutical compositions containing an anti-CD38 antibody and hyaluronidase, or unit dosage forms, administered as separate elements simultaneously, in parallel or sequentially without specific limiting time frames, and such administration provides effective concentrations of the two compounds in the body of the subject.

The expression pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, excluding the active ingredient, which is non-toxic to the subject. A pharmaceutically acceptable carrier may include, without limitation, a buffer, excipient, stabilizer, or preservative.

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

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

The term "growth inhibiting" (e.g., in relation to tumor cells) refers to a measurable reduction in the growth of tumor cells or tumor tissue in vitro or in vivo when brought into contact with a therapeutic agent or combination of therapeutics or drugs compared to the growth of the same tumor cells or tumor tissue. tissue in the absence of a therapeutic agent or a combination of therapeutic drugs. Inhibition of tumor cell or tumor tissue growth in vitro or in vivo may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

The term CD38-positive hematologic malignancy refers to a hematologic malignancy that is characterized by the presence of CD38-expressing tumor cells, including leukemias, lymphomas, and myeloma. Examples of such CD38-positive hematologic malignancies include B-cell lymphoblastic leukemia/progenitor lymphoma and B-cell non-Hodgkin's lymphoma, acute promyelocytic leukemia, acute lymphoblastic leukemia, and mature B-cell neoplasms such as B-cell chronic lymphocytic leukemia (CLL). ) / small lymphocyte lymphoma (LML), B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including well-differentiated, moderately differentiated and poorly differentiated PL, cutaneous lymphoma from follicle center cells, marginal zone B-cell lymphoma (MALT type, nodular and splenic), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (LB), plasma

- 4 042589 cytoma, multiple myeloma, plasmacytic leukemia, post-transplant lymphoproliferative disorder, light chain amyloidosis, Waldenström's macroglobulinemia, plasmacytic leukemia and anaplastic large cell lymphoma (ALCL).

The term about means within an acceptable range of error for a particular value as determined by one skilled in the art, the error depending in part on how that value is measured or determined, ie, the limitations of the measurement system. Unless explicitly stated otherwise in the examples or elsewhere in the description in the context of a particular assay, result, or embodiment, the term about means within one standard deviation, in accordance with practice accepted in the art, or up to 5%, depending on that which is more.

Pharmaceutical compositions

The invention provides a pharmaceutical composition containing an anti-CD38 antibody and hyaluronidase.

Hyaluronidase is an enzyme that degrades hyaluronic acid (EC 3.2.1.35) and reduces the viscosity of hyaluronan in the extracellular matrix, thus increasing tissue permeability. Enzymatic activity of hyaluronidase, including rHuPH20, can be determined in units of activity per ml (U/ml) or total enzymatic activity in a particular formulation (U), as further described below.

rHuPH20 is a recombinant hyaluronidase (recombinant HYLENEX®) and is described in International Patent Publication No. WO 2004/078140.

According to the standard definition, one unit (U) of enzymatic activity is the amount of enzyme that catalyzes the reaction of a given amount of substrate per unit of time, such as one µmol or one nmol of substrate per minute. Methods for determining the activity of hyaluronidase preparations are known in the art, and the activity of hyaluronidase preparations is generally expressed in USP units or Units (hereinafter units). An example of a method for determining activity can be found in US Pat. No. 7,767,429.

Hyaluronidase activity reflects the ability to enzymatically catalyze the breakdown of hyaluronic acid. The United States Pharmacopeia (USP) XXII edition provides a hyaluronidase assay in which hyaluronidase activity is indirectly determined by measuring the amount of higher molecular weight hyaluronic acid (or hyaluronan, HA) substrate remaining after the enzyme reacts with HA for 30 min. at 37°C. (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention, Inc, Rockville, Maryland). The assay can use a reference standard solution to establish the relative activity (in units) of a particular hyaluronidase. In vitro assays for determining the activity of hyaluronidases, such as soluble rHuPH20, are known in the art and are described herein. Examples of assays include the microturbidimetric assay described below (see, for example, Example 3), which measures the degradation of hyaluronic acid by hyaluronidase indirectly by detecting the insoluble precipitate formed when undegraded hyaluronic acid is bound to serum albumin.

Reference standards can be used, for example, to construct a standard curve in order to determine the activity of the test hyaluronidase in units.

The pharmaceutical composition is used for subcutaneously administering an anti-CD38 antibody to a subject in need of anti-CD38 antibody therapy, such as a subject having a cancer, eg a CD38 positive hematologic malignancy. Without wishing to be bound by any theory, subcutaneous administration of anti-CD38 antibody may reduce infusion-related response and provide higher treatment response rates compared to intravenous administration of anti-CD38 antibody.

In some embodiments, the pharmaceutical composition is a linked combination.

In some embodiments, the implementation of the pharmaceutical composition is an unrelated combination.

In some embodiments, the implementation of the pharmaceutical composition contains from about 1 to about 180 mg/ml of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains from about 10 to about 180 mg/ml of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains from about 20 to about 160 mg/ml of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains from about 20 to about 140 mg/ml of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains from about 20 to about 120 mg/ml of antibodies to CD38.

In some embodiments, the pharmaceutical composition contains from about 40 to

- 5 042589 about 120 mg/ml anti-CD38 antibody.

In some embodiments, the implementation of the pharmaceutical composition contains from about 60 to about 120 mg/ml of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains from about 80 to about 120 mg/ml of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains from about 100 to about 120 mg/ml of antibodies to CD38.

In some embodiments, the pharmaceutical composition contains 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 anti-CD38 antibody.

In some embodiments, the implementation of the pharmaceutical composition contains about 20 mg/ml of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 100 mg/ml of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 120 mg/ml antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains from about 50 to about 5000 U/ml of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains from about 500 to about 5000 U/ml of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains from about 1000 to about 5000 U/ml of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains from about 2000 to about 5000 U/ml of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains from about 50 to about 2000 U/ml of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains from about 500 to about 2000 U/ml of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains from about 1000 to about 2000 U/ml of hyaluronidase.

In some embodiments, the pharmaceutical composition contains 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, 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 hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains about 500 U/ml of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains about 2000 U/ml of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains about 5000 U/ml of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains from about 1200 to about 5000 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains from about 1200 to about 2400 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains from about 1200 to about 1800 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 1200 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 1400 mg of antibodies to CD38.

In some embodiments, the pharmaceutical composition contains about 1600 mg of an anti-CD38 antibody.

In some embodiments, the implementation of the pharmaceutical composition contains about 1800 mg of antibodies to CD38.

- 6 042589

In some embodiments, the implementation of the pharmaceutical composition contains about 2000 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 2200 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 2400 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 2600 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 2800 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 3000 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 3500 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 4000 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 4500 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains about 5000 mg of antibodies to CD38.

In some embodiments, the implementation of the pharmaceutical composition contains from about 750 to about 75,000 units of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains from about 7500 to about 45000 U of hyaluronidase.

In some embodiments, the implementation of the pharmaceutical composition contains from about 30,000 to about 45,000 units of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 7,500 U, about 8,000 U, about 8,500 U, about 9,000 U, about 10,000 U, about 15,000 U, about 20,000 U, about 21,000 U, about 22,000 U, about 23,000 U, about 24 000 U, about 25000 U, about 26000 U, about 27000 U, about 28000 U, about 29000 U, about 30000 U, about 31000 U, about 32000 U, about 33000 U, about 34000 U, about 35000 U, about 36000 U , about 37,000 U, about 38,000 U, about 39,000 U, about 40,000 U, about 41,000 U, about 42,000 U, about 43,000 U, about 44,000 U, about 45,000 U, about 48,000 U, about 49,000 U, 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.

In some embodiments, the pharmaceutical composition contains about 5,000 mg of an anti-CD38 antibody and about 30,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 5,000 mg of an anti-CD38 antibody and about 45,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 3,000 mg of an anti-CD38 antibody and about 30,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 3,000 mg of an anti-CD38 antibody and about 45,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 2800 mg of an anti-CD38 antibody and about 30,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 2800 mg of an anti-CD38 antibody and about 45,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 2600 mg of an anti-CD38 antibody and about 30,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 2600 mg of an anti-CD38 antibody and about 45,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 2400 mg of an anti-CD38 antibody and about 30,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 2400 mg of an anti-CD38 antibody and about 45,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 2200 mg of an anti-CD38 antibody and about 30,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 2200 mg of an anti-CD38 antibody and about 45,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 2000 mg of an anti-CD38 antibody and about 30,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 2000 mg of an anti-CD38 antibody and about 45,000 U of hyaluronidase.

- 7 042589

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

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

In some embodiments, the pharmaceutical composition contains about 1600 mg of an anti-CD38 antibody and about 30,000 U of hyaluronidase.

In some embodiments, the pharmaceutical composition contains about 1600 mg of an anti-CD38 antibody and about 45,000 U of hyaluronidase.

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

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

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

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

In some embodiments, an anti-CD38 antibody in a 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.

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

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

SEQ ID NO: 2

SKRNIQFSCKNIYR

SEQ ID NO: 3

EKVQTLEAWVIHGG

SEQ ID NO: 4

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSA

ISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDK

ILWFGEPVFDYWGQGTLVTVS S

SEQ ID NO: 5

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD

ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQ

- 8 042589

GTKVEIK

SEQ ID NO: 6

SEAMS

SEQ ID NO: 7

AISGSGGGTYYADSVKG

SEQ ID NO: 8

DKILWFGEPVFDY

SEQ ID NO: 9

RASQSVSSYLA

SEQ ID NO: 10

DASNRAT

SEQ ID NO: 11

QQRSNWPPTF

SEQ ID NO: 12

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK

SEQ ID NO: 13

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLS S

Other examples of anti-CD38 antibodies that can be used in pharmaceutical compositions and methods of the invention are mAb003 containing the sequences VH and VL of SEQ ID NO: 14 and 15, respectively, and described in US patent No. 7829693. VH and VL mAb003 can be expressed as IgG1 /K;

mAb024 containing the sequences VH and VL with SEQ ID NO: 16 and 17, respectively, described in US patent No. 7829693. VH and VL mAb024 can be expressed as IgG1/K;

MOR-202 (MOR-03087) containing the VH and VL sequences of SEQ ID NOs: 18 and 19, respectively, described in US Pat. No. 8,088,896. The VH and VL of MOR-202 can be expressed as IgG1/K; or isatuximab containing the VH and VL sequences of SEQ ID NOs: 20 and 21, respectively, as described in US Pat. No. 8,153,765. The VH and VL of isatuximab can be expressed as IgG1/K.

- 9 042589

SEQ ID NO: 14

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIAN

SAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSSAS

SEQ ID NO: 15

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVP

SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK

SEQ ID NO: 16

EVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMPGKGLEWMGIIYPHDSDAR

YS PS FQGQVT FSADKSIS TAYLQWS S LKAS DTAMYYCARHVGWGSRYWYFDLWGRGTLVTVSS

SEQ ID NO: 17

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPGLLIYDASNRASGIP

ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK

SEQ ID NO: 18

QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS

SEQ ID NO: 19

DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPE

RFSGSNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVLGQ

SEQ ID NO 20:

SEQ ID NO: 21:

DIVMTQSHLSMSTSLGDPVSITCKASQDVSTWAWYQQKPGQSPRRLIYS

ASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGG

GTKLEIK

Other examples of anti-CD38 antibodies that can be used in the pharmaceutical compositions of the invention are those described in International Patent Publication No. WO 05/103083, International Patent Publication No. WO 06/125640, International Patent Publication No. WO 07/042309, International Patent Publication No. No. WO 08/047242 or International Patent Publication No. WO 14/178820.

An example of an anti-CD38 antibody that can be used in the pharmaceutical compositions of the invention is daratumumab. Daratumumab contains the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) as 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 NO: 9, 10 and 11, respectively, has the IgG1/K subtype and is described in US Patent No. 7,829,693. ID NO: 13.

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

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

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

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

The invention also provides a pharmaceutical composition containing about 1800 mg of an anti-CD38 antibody containing VH with SEQ ID NO: 4 and VL with SEQ ID NO: 5, and about 45,000 U of rHuPH20 hyaluronidase with SEQ ID NO: 22, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg/ml.

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

- 10 042589

The invention also provides a pharmaceutical composition containing about 1600 mg of an anti-CD38 antibody containing VH with SEQ ID NO: 4 and VL with SEQ ID NO: 5, and about 45,000 U of rHuPH20 hyaluronidase with SEQ ID NO: 22, wherein the concentration of the anti-CD38 antibody in the pharmaceutical composition is about 20 mg/ml.

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

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

SEQ ID NO: 22

MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEF CLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDH LDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEK AKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSW LWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF LSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQV LCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVA SL

The anti-CD38 antibodies used in the pharmaceutical compositions of the invention can also be selected de novo, for example from a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof, such as Fab, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., J Mol Biol 296:57-86, 2000; Krebs et al., J Immunol Meth 254:67-84, 2001; Vaughan et al., Nature Biotechnology 14:309314, 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 expressing antibody heavy and light chain variable regions as fusion proteins fused to the bacteriophage coat protein pIX, as described in Shi et al., J. Mol. Biol. 397:385-96, 2010 and International Patent Publication No. WO 09/085462). It is possible to screen antibody libraries for binding to the extracellular domain of human CD38, further characterize the resulting positive clones, isolate Fab from clone lysates and subsequently clone as full-length antibodies. Such use of phage display techniques for the isolation of human antibodies is well known in the art. See, for example: US Patent No. 5223409, US Patent No. 5403484, US Patent No. 5571698, US Patent No. 5427908, US Patent No. 5580717, US Patent No. 5969108, US Patent No. 6172197, US Patent No. 5885793, US Patent No. 6521404, US Patent No. 6544731, US Patent No. 6555313, US Patent No. 6582915 and US Patent No. 6593081.

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

Antibodies binding to the SKRNIQFSCKNIYR region (SEQ ID NO: 2) and the EK region

- 11 042589

VQTLEAWVIHGG (SEQ ID NO: 3) human CD38 (SEQ ID NO: 1) can be obtained, for example, by immunizing mice with peptides having the amino acid sequences shown in SEQ ID NO: 2 and 3, using standard methods and described in this document methods and characterization of the resulting antibodies in relation to binding to peptides, for example, using an ELISA assay or a mutagenesis assay.

The invention also provides a pharmaceutical composition containing an anti-CD38 antibody containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3:

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

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

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

d) VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21 and hyaluronidase rHuPH20 of SEQ ID NO: 22.

The invention also proposes a pharmaceutical composition containing an anti-CD38 antibody containing:

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

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

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

d) VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21 and hyaluronidase rHuPH20 of SEQ ID NO: 22.

The pharmaceutical compositions of the invention further comprise a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers are solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible, such as salts, buffers, antioxidants, saccharides, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifying agents, or combinations thereof.

Examples of buffers that can be used are acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffers, HEPPSO and HEPES.

Examples of antioxidants that can be used are ascorbic acid, methionine, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, lecithin, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, and tartaric acid.

Examples of amino acids that can be used are histidine, isoleucine, methionine, glycine, arginine, lysine, L-leucine, tri-leucine, alanine, glutamic acid, L-threonine and 2-phenylamine.

Examples of surfactants that can be used are polysorbates (eg polysorbate-20 or polysorbate-80); polyoxamers (eg, polyoxamer 188); Triton; sodium octylglycoside; lauryl, myristil, linoleyl or stearylsulfobetaine; lauryl, myristil, linoleyl, or stearylsarcosine; linoleyl, myristil or cetyl betaine;

lauramidopropyl, cocamidopropyl, linoleamidopropyl, myristamidopropyl, palmidopropyl, or isostearamidopropyl betaine (eg, lauramidopropyl);

myristamidopropyl palmidopropyl or isostearamidopropyl dimethylamine; sodium methyl cocoyl or disodium methyl oleyl taurate; and MONAQUA™ series surfactants (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropylene glycol, and ethylene/propylene glycol copolymers (eg, PLURONICS™ PF68, etc.).

Examples of preservatives that can be used are phenol, m-cresol, p-cresol, ocresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkylparaben (methyl, ethyl, propyl, butyl, etc.). ), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thiomersal, or mixtures thereof.

Examples of saccharides 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, glycerin, arabitol. , silitol, sorbitol, mannitol, melibiose, melesitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucite, maltitol, lactitol or isomaltulose.

Examples of salts that can be used are addition salts and basic addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyl alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Basic addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., as well as from non-toxic organic amines such as N,N'-dibenzylethylenediamine, Nmethylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like. An example of a salt is sodium chloride.

The amounts of pharmaceutically acceptable carrier(s) in pharmaceutical compositions may

- 12 042589 can be determined experimentally based on the activities of the carrier(s) and the desired characteristics of the composition, such as stability and/or minimal oxidation.

In some embodiments, the implementation of the pharmaceutical composition contains acetic acid.

In some embodiments, the implementation of the pharmaceutical composition contains acetic acid in a concentration of from about 1 to about 50 mm.

In some embodiments, the implementation of the pharmaceutical composition contains acetic acid at a concentration of from about 10 to about 40 mm.

In some embodiments, the pharmaceutical composition contains 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.

In some embodiments, the implementation of the pharmaceutical composition contains acetic acid at a concentration of about 25 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sodium chloride (NaCl).

In some embodiments, the implementation of the pharmaceutical composition contains NaCl at a concentration of from about 20 to about 100 mm.

In some embodiments, the implementation of the pharmaceutical composition contains NaCl at a concentration of from about 40 to about 80 mm.

In some embodiments, the pharmaceutical composition contains 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.

In some embodiments, the implementation of the pharmaceutical composition contains NaCl at a concentration of about 60 mm.

In some embodiments, the implementation of the pharmaceutical composition contains a saccharide.

In some embodiments, the saccharide is sucrose.

In some embodiments, the saccharide is sorbitol.

In some embodiments, the saccharide is mannitol.

In some embodiments, the implementation of the pharmaceutical composition contains a saccharide at a concentration of from about 50 to about 500 mm.

In some embodiments, the implementation of the pharmaceutical composition contains saccharide at a concentration of from about 50 to about 450 mm.

In some embodiments, the implementation of the pharmaceutical composition contains saccharide at a concentration of from about 50 to about 400 mm.

In some embodiments, the implementation of the pharmaceutical composition contains saccharide at a concentration of from about 50 to about 350 mm.

In some embodiments, the implementation of the pharmaceutical composition contains saccharide at a concentration of from about 100 to about 350 mm.

In some embodiments, the implementation of the pharmaceutical composition contains saccharide at a concentration of from about 100 to about 300 mm.

In some embodiments, the pharmaceutical composition comprises a saccharide at 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, or about 500 mM.

In some embodiments, the implementation of the pharmaceutical composition contains mannitol.

In some embodiments, the pharmaceutical composition contains mannitol at a concentration of about 100 to about 180 mM.

In some embodiments, the pharmaceutical composition contains mannitol at a concentration of about 120 to about 160 mM.

In some embodiments, the composition contains 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.

In some embodiments, the implementation of the pharmaceutical composition contains mannitol at a concentration of about 140 mm.

In some embodiments, the implementation of the composition contains polysorbate.

In some embodiments, the implementation of the pharmaceutical composition contains polysorbate-20 (PS-20).

In some embodiments, the pharmaceutical composition contains polysorbate-20

- 13 042589 (PS-20) at a concentration of from about 0.01 to about 0.1% wt./about.

In some embodiments, the implementation of pharmaceutical (PS-20) at a concentration of from about 0.01 to about 0.08% wt./about.

In some embodiments, the implementation of pharmaceutical (PS-20) at a concentration of from about 0.01 to about 0.04% wt./about.

In some embodiments, the pharmaceutical composition contains polysorbate-20 the composition contains polysorbate-20 the composition contains 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% wt./about.

The invention also provides a pharmaceutical composition comprising about 20 to about 120 mg/mL of an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 in about 25 mM acetic acid, about 60 mM sodium chloride , about 140 mm mannitol and about 0.04% wt./about. polysorbate-20 (PS-20); at a pH of about 5.5 and from about 30,000 to about 45,000 U of hyaluronidase in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate-80, pH 6.5.

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

In some embodiments, the implementation of the pharmaceutical composition is an unrelated combination.

The invention also provides a pharmaceutical composition containing about 20 mg/ml of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in about 25 mM acetic acid, about 60 mM sodium chloride, about 140 mM mannitol and about 0.04% wt./about. polysorbate-20 (PS-20); at pH about 5.5 and about 30,000 units of hyaluronidase in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate-80, pH 6.5.

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

In some embodiments, the implementation of the pharmaceutical composition is an unrelated combination.

The invention also provides a pharmaceutical composition containing about 20 mg/ml of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in about 25 mM acetic acid, about 60 mM sodium chloride, about 140 mM mannitol and about 0.04% wt./about. polysorbate-20 (PS-20); at pH about 5.5 and about 45,000 units of hyaluronidase in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate-80, pH 6.5.

In some embodiments, the hyaluronidase is rHuPH20 (SEQ ID NO:

22).

In some embodiments, the implementation of the pharmaceutical composition is a specific combination.

In some embodiments, the implementation of the pharmaceutical composition contains histidine.

non-binding In some embodiments, pharmaceutical centration is from about 1 to about 50 mM.

In some embodiments, the pharmaceutical centration is from about 5 to about 50 mM.

In some embodiments, the pharmaceutical centration is from about 5 to about 30 mM.

In some embodiments, the pharmaceutical centration is from about 5 to about 20 mM.

In some embodiments, the pharmaceutical centration is from about 5 to about 15 mM.

In some embodiments, the pharmaceutical centration is from about 5 to about 10 mM.

In some embodiments, the pharmaceutical composition contains a histidine concomposition contains a histidine concomposition contains a histidine concomposition contains a histidine composition contains a histidine , about 7 mM to 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, or about 50 mM.

In some embodiments, the implementation of the pharmaceutical composition contains histidine at a concentration of about 5 mm.

In some embodiments, the implementation of the pharmaceutical composition contains histidine in con

- 14 042589 centration about 10 mM.

In some embodiments, the implementation of the pharmaceutical composition contains histidine at a concentration of about 15 mm.

In some embodiments, the implementation of the pharmaceutical composition contains histidine at a concentration of about 20 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of from about 50 to about 500 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of from about 50 to about 450 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of from about 50 to about 400 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of from about 50 to about 350 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of from about 100 to about 350 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of from about 100 to about 300 mm.

In some embodiments, the pharmaceutical composition contains sorbitol at 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, or about 500 mM.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of about 50 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of about 100 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of about 150 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of about 200 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of about 250 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of about 300 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of about 350 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sorbitol at a concentration of about 400 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose in a concentration of from about 50 to about 500 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose in a concentration of from about 50 to about 450 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose at a concentration of from about 50 to about 400 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose in a concentration of from about 50 to about 350 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose in a concentration of from about 100 to about 350 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose in a concentration of from about 100 to about 200 mm.

In some embodiments, the pharmaceutical composition contains sucrose at 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, or about 500 mM.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose at a concentration of about 50 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose in the end

- 15 042589 Traction about 100 mM.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose at a concentration of about 150 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose at a concentration of about 200 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose at a concentration of about 250 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose at a concentration of about 300 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose at a concentration of about 350 mm.

In some embodiments, the implementation of the pharmaceutical composition contains sucrose at a concentration of about 400 mm.

In some embodiments, the implementation of the composition contains methionine.

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

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

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

In some embodiments, the pharmaceutical composition contains 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 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.

In some embodiments, the implementation of the pH of the pharmaceutical composition is 5.0-6.0.

In some embodiments, the implementation of the pH of the pharmaceutical composition is 5.3-5.8.

In some embodiments, the implementation of the pH of the pharmaceutical composition is 5.5.

In some embodiments, the implementation of the pH of the pharmaceutical composition is 5.6.

The invention also provides a pharmaceutical composition comprising about 1 to about 180 mg/ml of an anti-CD38 antibody;

about 50 to about 5000 U/ml hyaluronidase, about 5 to about 50 mM histidine, and about 50 to about 400 mM sorbitol.

In some embodiments, the hyaluronidase is rHuPH20.

The invention also provides a pharmaceutical composition comprising about 1 to about 180 mg/ml of an anti-CD38 antibody;

about 50 to about 5000 U/ml hyaluronidase;

about 5 to about 50 mM histidine;

about 50 to about 400 mM sorbitol;

about 0.01 to about 0.1% PS-20; and about 0.1 to about 2.5 mg/ml methionine.

In some embodiments, the hyaluronidase is rHuPH20.

The invention also provides a pharmaceutical composition comprising about 100 to about 120 mg/ml of an anti-CD38 antibody;

about 50 to about 5000 U/ml hyaluronidase;

about 10 mM histidine; and about 100 to about 300 mM sorbitol.

In some embodiments, the hyaluronidase is rHuPH20.

In some embodiments, the implementation of the pharmaceutical composition further contains from about 0.01 to about 0.04% PS-20.

In some embodiments, the implementation of the pharmaceutical composition further contains from about 1 to about 2 mg/ml of methionine.

In some embodiments, the implementation of the pharmaceutical composition further contains from about 100 to about 200 mm sucrose.

In some embodiments, the anti-CD38 antibody comprises

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 6, 7, 8, 9, 10 and 11, respectively;

VH and VL with SEQ ID NOs: 4 and 5, respectively, and/or the heavy chain and light chain of SEQ ID NOs: 12 and 13, respectively.

In some embodiments, the anti-CD38 antibody comprises

VH and VL with SEQ ID NO: 14 and 15, respectively;

- 16 042589

VH and VL with SEQ ID NO: 16 and 17, respectively;

VH and VL with SEQ ID NO: 18 and 19, respectively, or

VH and VL with SEQ ID NO: 20 and 21, respectively;

In some embodiments, the hyaluronidase is rHuPH20 (SEQ ID NO: 22)

The invention also provides a pharmaceutical composition comprising about 1 to about 180 mg/ml of an anti-CD38 antibody comprising VH and VL of SEQ ID NOs: 4 and 5, respectively;

about 50 to about 5000 U/ml hyaluronidase;

about 5 to about 50 mM histidine; and about 50 to about 400 mM sorbitol.

In some embodiments, the hyaluronidase is rHuPH20.

The invention also provides a pharmaceutical composition comprising about 1 to about 180 mg/ml of an anti-CD38 antibody comprising VH and VL of SEQ ID NOs: 4 and 5, respectively;

about 50 to about 5000 U/ml hyaluronidase;

about 5 to about 50 mM histidine;

about 50 to about 400 mM sorbitol;

about 0.01 to about 0.1% PS-20; and about 0.1 to about 2.5 mg/ml methionine.

In some embodiments, the hyaluronidase is rHuPH20.

The invention also provides a pharmaceutical composition comprising about 100 to about 120 mg/ml of an anti-CD38 antibody comprising VH and VL of SEQ ID NOs: 4 and 5, respectively;

about 50 to about 5000 U/ml rHuPH20;

about 10 mM histidine;

about 100 to about 300 mM sorbitol;

from about 0.01 to about 0.04% w/v. PS-20 and about 1 mg/ml to about 2 mg/ml methionine.

The invention also provides a pharmaceutical composition containing about 100 mg/ml of an antibody to CD38 containing VH and VL with SEQ

ID NO: 4 and 5, respectively;

about 500 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol;

about 0.04% w/v PS-20 and about 2 mg/ml methionine at a pH of about 5.5.

The invention also provides a pharmaceutical composition containing about 120 mg/ml of an anti-CD38 antibody containing VH and VL with SEQ ID NOs: 4 and 5, respectively;

about 2000 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol;

about 0.04% w/v PS-20; and about 1 mg/ml methionine; at a pH of about 5.6.

The invention also provides a pharmaceutical composition containing about 100 mg/ml of an anti-CD38 antibody containing VH and VL with SEQ ID NOs: 4 and 5, respectively;

about 500 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol and about 2 mg/ml methionine at a pH of about 5.5.

The invention also provides a pharmaceutical composition containing about 100 mg/ml of an anti-CD38 antibody containing VH and VL with SEQ ID NOs: 4 and 5, respectively;

about 500 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol;

about 0.01% w/v PS-20 and about 2 mg/ml methionine at a pH of about 5.5.

The invention also provides a pharmaceutical composition containing about 100 mg/ml of an anti-CD38 antibody containing VH and VL with SEQ ID NOs: 4 and 5, respectively;

about 500 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol;

- 17 042589 about 0.02% w/v PS-20 and about 2 mg/ml methionine at a pH of about 5.5.

The invention also provides a pharmaceutical composition containing about 100 mg/ml of an anti-CD38 antibody containing VH and VL with SEQ ID NOs: 4 and 5, respectively;

about 500 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol;

about 0.06% wt./about. PS-20 and about 2 mg/ml methionine at a pH of about 5.5.

The invention also provides a pharmaceutical composition containing about 100 mg/ml of an anti-CD38 antibody containing VH and VL with SEQ ID NOs: 4 and 5, respectively;

about 50 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol;

about 0.04% w/v PS-20 and about 1 mg/ml methionine at a pH of about 5.5.

The invention also provides a pharmaceutical composition containing about 100 mg/ml of an anti-CD38 antibody containing VH and VL with SEQ ID NOs: 4 and 5, respectively;

about 500 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol;

about 0.04% w/v PS-20 and about 1 mg/ml methionine at a pH of about 5.5.

The invention also provides a pharmaceutical composition containing about 100 mg/ml of an anti-CD38 antibody containing VH and VL with SEQ ID NOs: 4 and 5, respectively;

about 2000 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol;

about 0.04% w/v PS-20 and about 1 mg/ml methionine at a pH of about 5.5.

The invention also provides a pharmaceutical composition containing about 100 mg/ml of an anti-CD38 antibody containing VH and VL with SEQ ID NOs: 4 and 5, respectively;

about 5000 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol;

about 0.04% w/v PS-20 and about 1 mg/ml methionine at a pH of about 5.5.

In some embodiments, the pharmaceutical composition is a linked combination.

Formulations for in vivo administration must be substantially sterile. Sterility can be achieved, for example, by filtration through sterile filter membranes.

Pharmaceutical compositions of the invention can be obtained by known methods. For example, pharmaceutical compositions can be prepared, for example, by dissolving, suspending, or emulsifying an anti-CD38 antibody in a sterile aqueous or oily medium conventionally used for injection.

Introduction

The pharmaceutical compositions of the invention may be administered as an unbound combination.

The pharmaceutical compositions of the invention may also be administered as a related combination, for example as a unit dosage form (or unit dosage form). Related combinations may be advantageous due to ease of administration and dose uniformity.

The invention also provides a unit dosage form containing an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 in an amount of from about 1200 to about 5000 mg, and rHuPH20 in an amount of from about 30,000 to about 75,000 Unit

The invention also provides a unit dosage form containing an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 in an amount of from about 1200 to about 4000 mg, and rHuPH20 in an amount of from about 30,000 to about 75,000 Unit

The invention also provides a unit dosage form containing an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 in an amount of from about 1200 to about 2400 mg, and rHuPH20 in an amount of from about 30,000 to about 45,000 Unit

The invention also provides a unit dosage form containing an anti-CD38 antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 in an amount of from about 1200 to about 1800 mg, and rHuPH20 in an amount of from about 30,000 to about 45,000 Unit

- 18 042589

The invention also provides a unit dosage form containing an antibody to CD38 containing VH with SEQ ID NO: 4 and VL with SEQ ID NO: 5, in an amount of from about 1200 to about 5000 mg;

rHuPH20 in an amount of from about 30,000 to about 75,000 U;

histidine at a concentration of about 5 to about 15 mM;

sorbitol at a concentration of about 100 to about 300 mM;

PS-20 at a concentration of about 0.01% to about 0.04% w/v. and methionine at a concentration of about 1 to about 2 mg/ml, pH about 5.5.

The invention also provides a unit dosage form containing an antibody to CD38 containing VH with SEQ ID NO: 4 and VL with SEQ ID NO: 5, in an amount of from about 1200 to about 2400 mg;

rHuPH20 in an amount of about 30,000 to about 45,000 U;

histidine at a concentration of about 10 mM;

sorbitol at a concentration of about 300 mm;

PS-20 at a concentration of about 0.04% w/v. and methionine at a concentration of about 1 mg/mL; pH around 5.5.

The invention also provides a unit dosage form containing an antibody to CD38 containing VH with SEQ ID NO: 4 and VL with SEQ ID NO: 5, in an amount of from about 1200 to about 1800 mg;

rHuPH20 in an amount of about 30,000 to about 45,000 U;

histidine at a concentration of about 10 mM;

sorbitol at a concentration of about 300 mm;

PS-20 at a concentration of about 0.04% w/v. and methionine at a concentration of about 1 mg/mL; pH around 5.5.

The invention also provides a unit dosage form containing an antibody to CD38 containing VH with SEQ ID NO: 4 and VL with SEQ ID NO: 5, in an amount of from about 1200 to about 1800 mg;

rHuPH20 in an amount of about 30,000 to about 45,000 U;

histidine at a concentration of about 5 to about 15 mM;

sorbitol at a concentration of about 100 to about 300 mM;

PS-20 at a concentration of about 0.01 to about 0.04% w/v; and methionine at a concentration of about 1 to about 2 mg/ml, pH about 5.5.

The invention also provides a unit dosage form containing an antibody to CD38 containing VH with SEQ ID NO: 4 and VL with SEQ ID NO: 5, in an amount of about 1800 mg;

rHuPH20 in the amount of about 30,000 U;

histidine at a concentration of about 10 mM;

sorbitol at a concentration of about 300 mm;

PS-20 at a concentration of about 0.04% w/v. and methionine at a concentration of about 1 mg/mL; pH around 5.5.

The invention also provides a unit dosage form containing an antibody to CD38 containing VH with SEQ ID NO: 4 and VL with SEQ ID NO: 5, in an amount of about 1800 mg;

rHuPH20 in an amount of about 45,000 U;

histidine at a concentration of about 10 mM;

sorbitol at a concentration of about 300 mm;

PS-20 at a concentration of about 0.04% w/v. and methionine at a concentration of about 1 mg/mL; pH around 5.5.

The pharmaceutical composition of the invention may be administered in a total volume of about 80 ml, 90 ml, 100 ml, 110 ml or 120 ml.

The pharmaceutical composition of the invention may 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.

The pharmaceutical composition of the invention may be administered in a total volume of about 10 ml.

The pharmaceutical composition of the invention may be administered in a total volume of about 15 ml.

The pharmaceutical composition of the invention may be administered in a total volume of about 20 ml.

The total volume of administration may generally be less for bound combinations than for unbound combinations.

The invention also provides a container containing the pharmaceutical composition of the invention.

The invention also provides a container containing a unit dosage form of the invention.

- 19 042589

The container may be a vial, cassette, syringe, pre-filled syringe, or disposable pen.

The administration of the pharmaceutical compositions of the invention can be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or more. In addition, repeated courses of treatment in the form of long-term administration are possible. Repeated administration can be carried out at the same dose or at a different dose. For example, pharmaceutical compositions of the invention may be administered once a week for eight weeks followed by once every two weeks for 16 weeks followed by once every four weeks.

The pharmaceutical composition of the invention may be administered subcutaneously.

The pharmaceutical composition of the invention can be administered subcutaneously in the abdomen.

Subcutaneous administration can be performed using a device. The device may be a syringe, a prefilled syringe, an autoinjector (single or reusable), a pen, a patch autoinjector, a wearable autoinjector, or an ambulatory syringe infusion pump with subcutaneous infusion systems.

For unbound combinations, 20 mg/mL anti-CD38 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, prior to administration of the mixture to the subject.

The pharmaceutical compositions of the invention may also be administered prophylactically to reduce the risk of developing cancer, slow the onset of events in cancer progression, and/or reduce the risk of recurrence in the event of cancer remission. This may be particularly useful in patients who have difficulty localizing a tumor that is identified based on other biological factors.

Methods of treatment

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

In some embodiments, the cancer is a CD38 positive hematologic malignancy.

In some embodiments, the CD38 positive hematologic tumor is multiple myeloma.

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

malignant malignant

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

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

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

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

In some embodiments, the CD38 positive tumor is mantle cell lymphoma (MCL).

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

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

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

In some embodiments, the cancer is a solid tumor.

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

The invention also provides a method for treating a CD38-positive hematologic malignancy comprising 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, subcutaneously for a time sufficient to treat CD38 positive hematological malignancy, wherein the anti-CD38 antibody concentration in the pharmaceutical composition is about 20 mg/mL.

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

The invention also provides a method of treating a CD38-positive hematologic malignancy comprising administering to a subject in need thereof a pharmaceutical composition comprising about 1200 to 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 rHuPH20 hyaluronidase of SEQ ID NO: 22 for a time period sufficient to treat a CD38-positive hematological malignancy, wherein the anti-CD38 antibody concentration in the pharmaceutical composition is about 20 mg/mL.

The invention also provides a method of treating a CD38-positive hematologic malignancy comprising administering to a subject in need thereof a pharmaceutical composition comprising about 1200 to 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 rHuPH20 hyaluronidase of SEQ ID NO: 22 for a time period sufficient to treat a CD38-positive hematological malignancy, wherein the anti-CD38 antibody concentration in the pharmaceutical composition is about 20 mg/mL.

The invention also provides a method for treating a CD38-positive hematological malignancy comprising administering to a subject in need about 1600 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 rHuPH20 hyaluronidase with SEQ ID NO: 22 for a time period sufficient to treat a CD38 positive hematologic malignancy, wherein the anti-CD38 antibody concentration in the pharmaceutical composition is about 20 mg/mL.

The invention also provides a method for treating a CD38-positive hematologic malignancy comprising administering to a subject in need 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 rHuPH20 hyaluronidase with SEQ ID NO: 22 for a time period sufficient to treat a CD38 positive hematologic malignancy, wherein the anti-CD38 antibody concentration in the pharmaceutical composition is about 20 mg/mL.

The invention also provides a method for treating a CD38-positive hematological malignant tumor, comprising 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, wherein the pharmaceutical composition is an unrelated combination.

The invention also provides a method of treating a CD38-positive hematologic malignancy comprising administering to a subject in need thereof a pharmaceutical composition comprising about 20 to about 120 mg/mL of an anti-CD38 antibody comprising a VH of SEQ ID NO: 4 and a VL of SEQ ID NO. : 5, in about 25 mm acetic acid, about 60 mm sodium chloride, about 140 mannitol and about 0.04% wt./about. polysorbate-20 (PS-20); at a pH of about 5.5 and from about 30,000 to about 45,000 U of hyaluronidase in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate-80, pH 6.5.

In some embodiments, the hyaluronidase is rHuPH20.

In some embodiments, the implementation of the pharmaceutical composition is an unrelated combination.

The invention also provides a method of treating a CD38-positive hematologic malignancy comprising administering to a subject in need thereof a pharmaceutical composition comprising about 20 mg/mL of an anti-CD38 antibody comprising a VH of SEQ ID NO: 4 and a VL of SEQ ID NO: 5, in about 25 mm acetic acid, about 60 mm sodium chloride, about 140 mannitol and about 0.04% wt./about. polysorbate-20 (PS-20); at a pH of about 5.5; and about 30,000 units of hyaluronidase in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate-80, pH 6.5.

In some embodiments, the hyaluronidase is rHuPH20.

In some embodiments, the implementation of the pharmaceutical composition is an unrelated combination.

The invention also provides a method for treating a CD38-positive hematological malignancy, comprising administering to a subject in need thereof a pharmaceutical composition comprising

- 21 042589 about 20 mg/ml of an anti-CD38 antibody containing VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, in about 25 mM acetic acid, about 60 mM sodium chloride, about 140 mannitol and about 0.04 % w/v

polysorbate-20 (PS-20); at a pH of about 5.5; and about 45,000 U of hyaluronidase in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate-80, pH 6.5.

In some embodiments, the hyaluronidase is rHuPH20.

In some embodiments, the implementation of the pharmaceutical composition is an unrelated combination.

The invention also provides a method for treating a CD38-positive hematological malignant tumor, comprising 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, wherein the pharmaceutical composition is a related combination.

The invention also provides a method of treating a CD38-positive hematological malignancy comprising administering to a subject in need thereof a pharmaceutical composition comprising about 1 mg/mL to about 180 mg/mL of an anti-CD38 antibody comprising VH and VL of SEQ ID NO: 4 and 5 respectively;

from about 50 U/ml to about 5000 U/ml hyaluronidase from about 5 mm to about 50 mm histidine; and about 50 mM to about 400 mM sorbitol.

In some embodiments, the hyaluronidase is rHuPH20.

The invention also provides a method of treating a CD38-positive hematological malignancy comprising administering to a subject in need thereof a pharmaceutical composition comprising about 1 mg/mL to about 180 mg/mL of an anti-CD38 antibody comprising VH and VL of SEQ ID NO: 4 and 5 respectively;

from about 50 U/ml to about 5000 U/ml hyaluronidase from about 5 mm to about 50 mm histidine;

about 50 mM to about 400 mM sorbitol;

from about 0.01% wt./about. up to about 0.1% PS-20; and from about 0.1 mg/ml to about 2.5 mg/ml methionine.

In some embodiments, the hyaluronidase is rHuPH20.

The invention also provides a method of treating a CD38-positive hematologic malignancy comprising administering to a subject in need thereof a pharmaceutical composition comprising about 100 mg/mL to about 120 mg/mL of an anti-CD38 antibody comprising VH and VL of SEQ ID NO: 4, and 5 respectively;

about 50 U/ml to about 5000 U/ml hyaluronidase;

about 10 mM histidine;

about 100 mM to about 300 mM sorbitol;

from about 0.01% wt./about. up to about 0.04% wt./about. PS-20; and about 1 mg/ml to about 2 mg/ml methionine.

In some embodiments, the hyaluronidase is rHuPH20.

The invention also provides a method of treating a CD38-positive hematological malignancy comprising administering to a subject in need thereof a pharmaceutical composition comprising about 100 mg/mL of an anti-CD38 antibody comprising VH and VL of SEQ ID NOs: 4 and 5, respectively;

about 500 U/ml hyaluronidase;

about 10 mM histidine;

about 300 mm sorbitol;

about 0.04% w/v PS-20; and about 2 mg/ml methionine, at a pH of about 5.5.

In some embodiments, the hyaluronidase is rHuPH20.

The invention also provides a method of treating a CD38-positive hematologic malignancy comprising administering to a subject in need thereof a pharmaceutical composition comprising about 120 mg/mL of an anti-CD38 antibody comprising VH and VL of SEQ ID NOs: 4 and 5, respectively;

about 2000 U/ml rHuPH20;

about 10 mM histidine;

about 300 mm sorbitol;

about 0.04% w/v PS-20; and about 1 mg/ml methionine; at a pH of about 5.6.

- 22 042589

In some embodiments, the hyaluronidase is rHuPH20.

Anti-CD38 antibodies in the pharmaceutical compositions of the invention can induce the killing of CD38-expressing tumor cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or modulation of CD38 enzymatic activity. The anti-CD38 antibodies in the pharmaceutical compositions of the invention may also mediate antitumor efficacy by immunomodulatory action by inducing the proliferation of CD4+ and CD8+ T cells and/or attenuating the inhibition of inflammatory responses mediated by myeloid suppressor cells (MDSC) and regulatory T cells (Treg).

The terms antibody-dependent cellular cytotoxicity, antibody-dependent cell-mediated cytotoxicity, or ADCC is a mechanism for inducing cell death that depends on the interaction of antibody-coated target cells with effector cells with lytic activity, such as natural killer cells, monocytes, macrophages, and neutrophils, via Fc gamma receptors. (FcyR) expressed on effector cells. For example, NK cells express FcyRIIIa while monocytes express FcyRI, FcyRII and FcvRIIIa. The death of antibody-coated target cells, such as CD38 expressing cells, occurs as a result of the activity of effector cells through the secretion of membrane pore-forming proteins and proteases. To assess the ADCC activity of an antibody that specifically binds to CD38, the antibody can be added to CD38 expressing cells in combination with immune effector cells that can be activated by antigen-antibody complexes, resulting in cytolysis of the target cell. Cytolysis is essentially detected by the release of labels (eg, radioactive substrates, fluorescent dyes, or naturally occurring intracellular proteins) from 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 example assay, target cells are labeled with 20 µCi of 51Cr for 2 hours and washed thoroughly. Target cell concentration can be adjusted to 1 x 10 ⁶ cells/ml and anti-CD38 antibodies are added at various concentrations. The analysis begins with the addition of target cells in the ratio of effector cells: target cells 40:1. After incubation for 3 hours at 37° C., the assay is terminated by centrifugation and the release of 51 Cr from lysed cells is measured on a scintillation counter. The percentage of cellular cytotoxicity can be calculated as % of the maximum lysis that can be induced by adding 3% perchloric acid to target cells.

Antibody-dependent cellular phagocytosis (ADCP) refers to a mechanism for killing antibody-coated target cells by internalization by phagocytic cells such as macrophages or dendritic cells. ADCP can be assessed using Tregs or MDSCs expressing CD38 as target cells engineered to express GFP or other labeled molecule. The ratio of effector cells: target cells can be, for example, 4:1. Effector cells can be incubated with target cells for 4 hours, with or without anti-CD38 antibody. After incubation, cells can be separated using accutase. Identification of macrophages can be done with anti-CD11b and anti-CD14 antibodies associated with a fluorescent label, and the percentage of phagocytosis can be determined based on the % fluorescent GFP in CD11+CD14+ macrophages using standard methods.

Complement-dependent cytotoxicity or CDC refers to a cell death induction mechanism in which the Fc effector domain of a target-bound antibody binds and activates the C1q complement component, which in turn activates the complement cascade leading to death of the target cell. Complement activation can also lead to the deposition of complement components on the surface of target cells, which facilitates ADCC by binding complement receptors (eg, CR3) on leukocytes.

The ability of monoclonal antibodies to induce ADCC can be enhanced by designing their oligosaccharide component. Human IgG1 or IgG3 undergo N-glycosylation at Asn297 by most glycans in the well-known 2-antennary forms GO, GOF, G1, G1F, G2 or G2F. The fucose content of antibody glycans produced by unengineered CHO cells is typically at least about 85%. Removal of the central fucose from type 2-anthenary complex oligosaccharides attached to Fc regions enhances antibody ADCC through improved FcyRIIIa binding without altering antigen binding or CDC activity. Such mAbs can be generated by a variety of methods that have been reported to result in successful expression of relatively highly defucosylated antibodies bearing Fc-oligosaccharide type 2-anthenary complex, such as controlling the osmolality of the culture medium (Konno et al., Cytotechnology 64:249 -65, 2012), use as a host cell line of the variant CHO cell line Lec13 (Shields et al., J Biol Chem 277:26733-26740, 2002), use as a host cell line of the variant CHO cell line EB66 (Olivier et al., MAbs;2(4), 2010; electronic edition to printed edition

- 23 042589 Denmark; PMID: 20562582), use of YB2/0 rat hybridoma cell line as host cell line (Shinkawa et al., J Biol Chem 278:3466-3473, 2003), administration of a-1,6 gene specific small interfering RNA -fucosyltransferase (FUT8) (Mori et al., Biotechnol Bioeng88:901-908, 2004), or co-expression of β-1,4-N-acetylglucosaminyltransferase III and α-mannosidase II of the Golgi complex, or the use of a strong inhibitor of alpha-mannosidase I, kifunzin (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 caused by antibodies to CD38, which are used in the methods of the invention and in some embodiments of all of the following numbered embodiments without exception, can also be enhanced by certain substitutions in the Fc of the antibody. Examples of substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 (residue numbering corresponds to the EU index), as described in US patent No. 6,737,056.

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

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

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

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

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

The term fucose content refers to the amount of fucose monosaccharide within the saccharide chain at position Asn297. The relative amount of fucose is the percentage of fucose-containing structures related to all glycostructures. They can be characterized and quantified in a variety of ways, for example: 1) using time-of-flight matrix-assisted laser desorption/ionization (MALDI-TOF) of a sample treated with N-glycosidase F (e.g., complex, hybrid, oligomannose and high-mannose structures) as described in international Patent Publication No. WO2008/077546; 2) by enzymatic release of Asn297 glycans followed by derivatization and detection/quantitation by HPLC (UHPLC) with fluorescence detection and/or HPLC-MS (UHPLC-MS); 3) analysis of intact protein of native or reconstituted mAb with or without treatment of Asn297 glycans with Endo S enzyme or other enzyme that cleaves the bond between the first and second GlcNAc monosaccharides, keeping the fucose attached to the first GlcNAc; 4) cleavage of the mAb into its constituent peptides by enzymatic cleavage (eg, trypsin or Lys-C endopeptidase) and subsequent separation, detection and quantification by HPLC-MS (UHPLC-MS); or 5) separating the mAb oligosaccharides from the Asn 297 mAb protein by specific enzymatic deglycosylation with PNGase F. The released oligosaccharides can be fluorophore-labeled, separated, and identified by various ancillary methods that allow: ionization (MALDI) by comparing experimental masses with theoretical masses, determine the degree of sialylation using ion-exchange HPLC (GlycoSep C), separate and quantify oligosaccharide forms by hydrophilicity criterion using ordinary phase HPLC (GlycoSep N) and separate and quantify oligosaccharide forms using high-performance laser-induced fluorescence capillary electrophoresis (HPCE-LIF).

As used herein, the terms "low fucose" or "low fucose" refer to antibodies with a fucose content of about 0-15%.

As used herein, the terms normofucose or normal fucose refer to antibodies with a fucose content greater than about 50%, typically greater than about 60%, 70%, 80%, or greater than 85%.

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

The methods of the invention can use antibodies that are substantially identical to an antibody comprising a VH of SEQ ID NO: 4 and a VL of SEQ ID NO: 5. As used herein, the term substantially identical means that the compared amino acid sequences of the VH or VL of the two antibodies are identical or have minor differences. Minor differences are substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in the heavy chain or light chain of an antibody that do not adversely affect the properties of the antibody . The percentage of identity can be

- 24 042589 to determine, for example, by pairwise alignment using the default settings in the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, CA). The protein sequences of the present invention can be used as a search sequence when performing searches in public or proprietary databases, for example, to identify related sequences. Examples of programs used to perform such searches are the XBLAST or BLASTP programs (http_//www_ncbi_nlm/nih_gov) or the GenomeQuest™ package (GenomeQuest, Westboro, Massachusetts) using default settings. Examples of substitutions that can be made for anti-CD38 antibodies used in the methods of the invention are, for example, conservative substitutions with amino acids having similar charge, hydrophobic properties, or stereochemical characteristics. Conservative substitutions may also be made to improve antibody properties, such as stability or affinity, or to improve antibody effector functions. You can make 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid substitutions, for example, in the heavy or light chain of an anti-CD38 antibody. Moreover, any native residue in the heavy or light chain can also be replaced with alanine, as previously described in relation to alanine scanning mutagenesis (MacLennan et al., Acta Physiol Scand Suppl 643:55-67, 1998; Sasaki et al., Adv Biophys 35:1-24, 1998). Those skilled in the art can determine the desired amino acid substitutions when such substitutions are needed. Amino acid substitutions can be carried out, for example, using PCR mutagenesis (US patent No. 4,683,195). Libraries of variants can be created using well-known methods, for example, by using random (NNK) or non-random codons, such as DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp), and screening libraries for variants with desired properties. Generated variants can be tested for their binding to CD38, their ability to induce ADCC, ADCP or apoptosis, or to modulate CD38 enzymatic activity in vitro using the methods described herein.

In some embodiments, an anti-CD38 antibody can bind to human CD38 with different affinities (KD). In one embodiment according to the invention, and in some embodiments of all of the numbered embodiments listed below without exception, an anti-CD38 antibody binds to CD38 with high affinity, for example with a KD value equal to or less than about 10-7 M, such as, without limitation, 1-9, 9 (or any range or value included therein, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) x 10 ^-8 M, 10 ^-9 M, ^10-10M , ^10-11M , ^10-12M , ^10-13M , ^10-14M , ^10-15M , or any range or value within it, as determined by Surface Plasmon Resonance or Kinexa as practiced experts in this field. In one example, the affinity value is 1x10 ^-8 M or less. In another example, the affinity value is 1x10 ^-9 M or less.

In some embodiments, the anti-CD38 antibody is a bispecific antibody. The VL and/or VH regions of existing anti-CD38 antibodies, or the VL and VH regions identified de novo as described herein, can be constructed to generate bispecific full length antibodies. Such bispecific antibodies can be produced by modulating CH3 interactions between the heavy chains of monospecific antibodies to form bispecific antibodies using techniques such as those described in US Pat. No. 7,695,936; International Patent Publication No. WO 04/111233; US Patent Publication No. US2010/0015133; US Patent Publication No. US2007/0287170; International Patent Publication No. WO 2008/119353; US Patent Publication No. US2009/0182127; US Patent Publication No. US2010/0286374; US Patent Publication No. US2011/0123532; International Patent Publication No. WO 2011/131746; International Patent Publication No. WO 2011/143545; or US Patent Publication No. US 2012/0149876. Additional bispecific structures into which the VL and/or VH regions of the antibodies of the invention can be inserted are, for example, dual variable domain immunoglobulins (International Patent Publication No. WO 2009/134776) or structures comprising different dimerization domains to connect two antibody arms with different specificity such as leucine zipper or collagen dimerization domains (International Patent Publication No. WO 2012/022811, US Patent No. 5,932,448; US Patent No. 6,833,441).

For example, bispecific antibodies can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and generating a bispecific heterodimeric antibody from the two parental monospecific homodimeric antibodies under reducing conditions, which promotes isomerization of the disulfide bond, according to the methods described in International Patent Publication No. WO 2011/131746. In the methods, a first monospecific divalent antibody (eg, an anti-CD38 antibody) and a second monospecific divalent antibody are designed to have specific substitutions in the CH3 domain that provide heterodimer stability; antibodies are incubated together under reducing conditions sufficient to ensure that cysteines in the hinge region undergo isomerization

- 25 042589 disulfide bond; thus, as a result of the exchange of Fab arms, a bispecific antibody is formed. The incubation conditions can be optimally returned to non-reducing conditions. Examples of reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and betamercaptoethanol, preferably a reducing agent. is selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. For example, an incubation of at least 90 minutes at a temperature of at least 20° C. in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at pH 5-8, such as pH 7.0 or pH 7.4.

Examples of CH3 mutations that can be used in the first heavy chain and in the second heavy chain of the bispecific antibody are K409R and/or F405L.

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

Combined therapies.

The pharmaceutical compositions of the invention may be administered in combination with a second therapeutic agent or combinations thereof.

The second therapeutic agent may be melphalan, mechlorethamine, thioepa, chlorambucil, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannite, streptozocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin, thalidomide or a thalidomide analog, lenalidomide or CC4047, a proteasome inhibitor such as bortezomib, or a vinca alkaloid such as vincristine, or an anthracycline such as doxorubicin.

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

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

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

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 streptozocin.

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

In some embodiments, the glutamic acid derivative is Revlimid® (lenalidomide), thalidomide, or Pomalyst® (pomalidomide).

In some embodiments, the subject is additionally administered a corticosteroid.

In some embodiments, the corticosteroid is dexamethasone or predison.

The second therapeutic agent, or combinations thereof, is typically administered at dosages recommended for said agent.

The pharmaceutical composition of the invention can be administered simultaneously or sequentially with the second therapeutic agent or combinations thereof.

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

Example 1 Subcutaneous delivery of 2% human immunoglobulin G (IgG) together with recombinant human hyaluronidase PH20 (rHuPH20) in a minipig model.

Statement of essence

The miniature pig is a preclinical model suitable for the evaluation of biotherapeutic agents under subcutaneous (SC) administration due to its anatomical similarity to human skin and the possibility of clinical application of the results obtained (Mahj et al., Exp Toxicol Path 57: 341- 5, 2006). The aim of this study was to evaluate and analyze the conditions for injecting 100 ml of a 20 mg/ml human IgG solution containing 200, 500 or 800 U/ml rHuPH20 at two different flow rates (2 and 4 ml/min). Parameters assessed included quantitative measurements of the infusion pressure as well as quantitative assessments of the local infusion site, such as the size and density of the swelling.

Yucatan minipigs were infused subcutaneously with 100 ml of a solution containing 20 mg/ml immunoglobulin G (IgG) and 200, 500, or 800 U/ml rHuPH20 at a flow rate of 2 or 4 ml/min. During infusions, the pressure in the system was measured in real time. After the completion of infusions in the local areas of infusion, the volume and area of visible swelling (if any) were measured and performed

- 26 042589 a qualitative assessment of the infusion site for the presence and severity of erythema, the size and density of the swelling/blister, and macroscopic changes. The infusion pressure was generally low and ranged from ~5.3 to 8.0 kPa (40-60 mmHg (~1 psi)) at both flow rates.

There were no statistical differences in pressure between different concentrations of rHuPH20 and two different flow rates, with the overall pressure being slightly lower as expected at a flow rate of 2 ml/min.

An unexpected result was the number of infusions (10 out of 12) in which there was visible and measurable swelling at the infusion site at a lower flow rate of 2 ml/min. This phenomenon was observed at all three concentrations of rHuPH20. In contrast, only 3 out of 12 infusions at the higher flow rate of 4 ml/min resulted in visible and measurable local swelling. This phenomenon was also observed at each concentration of rHuPH20.

In all cases where local swelling was visible, it disappeared within an hour. In addition, local swelling at the infusion sites was essentially soft to the touch and loose, as seen in the swelling/induration score (mean score < 2). The incidence of erythema was greater with infusions at a flow rate of 2 ml/min; however, the overall severity of erythema was mild, and it completely disappeared by the next day. No other macroscopic changes were noted at the infusion sites in the study.

In the present study, three different concentrations of rHuPH20 (200, 500, or 800 U/mL) were evaluated, and as a result, no statistical differences were found between the concentrations based on the measurement of the evaluated study parameters. In general, at a higher flow rate of 4 ml/min, a lower incidence of erythema, visible swelling, and induration at the infusion site was observed than at a flow rate of 2 ml/min.

Test preparations and research methods.

Tested drugs.

Materials in the buffer solution for the composition.

Sodium acetate 25 mM (Spectrum; item no. S0104; batch no. 1DI0271).

Sodium chloride 60 mM (Spectrum; item no. S0155; serial no. 1CE0421).

D-mannitol 140 mM (Spectrum; item no. MA165; batch no. 1EB0316).

Polysorbate 200.04% (JT Baker; Item # 4116-04; Series # 0000017659).

pH 5.5 (glacial acetic acid for pH adjustment; Fisher Scientific; P/N A491-212; S/N 080972).

Materials in a medicinal substance.

Human gamma globulin (BioMed Supply; Item # HGG-1005; Series # BMS31309013).

rHuPH20 [ (manufactured by Cook Pharmica for Halozyme; Halozyme lot no. 462-021B (repackaged, Cook lot no. 104-001-HSTFIL-9054)].

The mg/ml IgG formulation was mixed with 200, 500, or 800 U/ml rHuPH20 4 days prior to study entry. The solutions were poured in portions into individual glass bottles, hermetically sealed with a stopper, and crimped with a cap. All solutions were stored at 2-8°C until the start of the study, but were left to warm to room temperature before infusions. In addition, a sample was taken from each composition to study the enzymatic activity of rHuPH20. The results of the enzyme activity analysis confirmed that the concentration of all dosing solutions was within 10% of the target concentration (data not shown).

Description of animals

Species: pig (Sus scrofa domestica).

Strain: Yucatan dwarf.

Gender: female.

Age: > 3 months.

Weight: ~ 12 kg.

Quantity: 12.

Source: S&S Farms (Ramona, California).

Animal care

Animals were kept in steel cages with an automatic water supply system in unlimited quantities. Animals were fed twice a day (before and after lunch), with the exception of the day of the study (only after lunch). The body weight of the animals was measured and recorded from the day of admission until one day after the completion of the study to assess the health status of the animals. During this period, all animals maintained body weight (data not shown). The room was maintained at a temperature of ~17-27°C and a relative humidity of 40-70%, as well as a 12-hour day/night cycle. Animals were allowed to acclimate to the room for 7 days prior to the start of the study.

Test materials

High pressure syringe pumps (KD Scientific; Holliston, Massachusetts).

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System with 23 ga x 1.9 cm (23 x 3/4 in.) butterfly infusion needle and 30 cm (12 in.) tubing (Terumo Medical Corporation; Somerset, NJ).

140 cm ³ syringe with Luer connector (Covidien; Mansfield, Massachusetts).

18 cm (7 in) extension system (B/Braun; Bethlehem, PA).

PowerLab 4/30 (AD Instruments; Colorado Springs, Colorado).

Deltran-1 disposable pressure transducer (Utah Medical Products; Midvale, Utah).

Digital caliper (Preisser Messtechnik; Hammertingen, Germany).

Isoflurane (Minrad International Company, Orchard Park, NY).

Isoflurane evaporator (VetEquip; Pleasanton, CA).

Experiment plan

The experimental plan is summarized in the description of the cohorts (Table 1) and the description of infusions for each animal (Table 2). Briefly, 100 ml of a solution containing 20 mg/ml IgG mixed with 200, 500, or 800 U/ml rHuPH20 was injected into the abdomen of anesthetized Yucatan minipigs at a flow rate of 2 or 4 ml/min. The study parameters evaluated included measurements of pressure with the built-in pressure sensor, volume and area (if possible) of swelling (blister) after infusion, and a qualitative assessment of the infusion site, including photographs.

Table 1

Cohort Test drug Flow rate (ml/min) 1 IgG+200 U/ml rHuPH20 2 ml/min 2 IgG+500 U/ml rHuPH20 3 IgG+800 U/ml rHuPH20 4 IgG+200 U/ml rHuPH20 4 ml/min 5 IgG+500 U/ml rHuPH20 6 IgG+800 U/ml rHuPH20

table 2

Identifier animal Flow rate (ml/min) Infusion in the left side Infusion in the right side 1 2 IgG+200 U/ml IgG+500 U/ml rHuPH20 rHuPH20 2 IgG+200 U/ml rHuPH20 IgG+800 U/ml rHuPH20 3 IgG+500 U/ml rHuPH20 IgG+200 U/ml rHuPH20 4 IgG+500 U/ml rHuPH20 IgG+800 U/ml rHuPH20 5 IgG+800 U/ml rHuPH20 IgG+200 U/ml rHuPH20 6 IgG+800 U/ml rHuPH20 IgG+500 U/ml rHuPH20 7 4 IgG+200 U/ml rHuPH20 IgG+500 U/ml rHuPH20 8 IgG+200 U/ml rHuPH20 IgG+800 U/ml rHuPH20 9 IgG+500 U/ml rHuPH20 IgG+200 U/ml rHuPH20 10 IgG+500 U/ml rHuPH20 IgG+800 U/ml rHuPH20 eleven IgG+800 U/ml rHuPH20 IgG+200 U/ml rHuPH20 12 IgG+800 U/ml rHuPH20 IgG+500 U/ml rHuPH20

n=4 infusions per cohort at one flow rate.

Each animal simultaneously received 2 different infusions (one at each opposite site).

rHuPH20 concentrations: 200 U/ml = 20,000 U total; 500 U/ml = 50,000 U total;

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800 U/ml = 80000 U total

The volume of infusion is 100 ml.

Criteria for evaluation.

Pressure in the system (infusion).

Measurement of the volume and area of the swelling/blister.

Qualitative assessment of erythema and induration, including photographs.

Research Procedure

Before the start of the study, the animals were assessed for general health and measured body weight. On the day of the study, animals were anesthetized with isoflurane gas, placed in a supine position on a heated surgical table, and maintained with isoflurane gas throughout the procedure. The abdomen was cleaned with isopropanol and wiped dry with clean gauze. The infusion sites were placed in the left and right abdomen, ~3-4 cm from the midline, starting from the anterior end of the inguinal fold, and after ~6 cm towards the anterior end. The infusion sites were marked with a permanent marker and subsequently photographed. Before infusion, the test preparations were warmed to room temperature. The test preparations were placed in a 140 cm ³ syringe (>100 ml to account for the volume required to fill the system). A pressure sensor was attached to the syringe. Thereafter, an extension set was attached to the transducer with a 23 da x 1.9 cm (23 da x 3/4 inch) butterfly infusion needle attached. Subsequently, the infusion set was filled to the tip of the needle. The syringe was placed in a syringe pump. This process was performed twice, with each syringe containing a different test drug. The needles were placed subcutaneously at the marked left and right infusion sites on the abdomen of the animal. The pressure sensor built into the system was reset. Pressure fixation in the system was started, and subsequently two syringe pumps were simultaneously started to inject 100 ml of the test preparations at a flow rate of 2 or 4 ml/min. After completion of the infusions, system pressure data collection was stopped, the needles were removed, and the needle hole was sealed with VetBond liquid adhesive to prevent leakage. The area and volume of the swelling/blister at the local infusion site was measured using a digital caliper. In addition, local infusion sites were qualitatively assessed for appearance (erythema), swelling/blister size, and density (induration) using a 5-point scoring system (Table 3, Tables 4 and 5, respectively). Finally, photographs were taken of the infusion sites.

Table 3

Meaning Description 0 No erythema 1 Very slight erythema (barely perceptible) 2 Well formed erythema 3 Moderate or severe erythema 4 Severe erythema (bright red) or formation of a small scab

Table 4

Meaning Description 0 No swelling 1 Very slight swelling 2 slight swelling 3 moderate swelling 4 Severe swelling Meaning 0 1 Table 5 Description No perceptible difference in density after injection Very slight induration (barely perceptible) 2 Light seal 3 Moderate compaction 4 Pronounced compaction

Calculations and statistical methods

Infusion pressure assessment

The values of the infusion pressure measured by the built-in sensor using LabChart 7 were recorded and the average pressure was calculated during the entire infusion period.

Assessment of the volume and area of local swelling

The volume and area of swelling after infusion was measured using a digital caliper and

- 29 042589 fixed manually. The measured values were recorded as length, width and height. The formula for an ellipsoid was used to calculate the volume. Volume=4/3πABC where A=length radius, B=width radius, C=height radius. To calculate the area, a simple formula length x width was used.

Evaluation of local infusion sites

After completion of the infusion, three different specialists independently evaluated the local infusion sites. Each specialist assessed the skin at each infusion site for the presence of erythema, the size of the local swelling and density. A scale from 0 to 4 points was used to evaluate the three study areas, with 0 points indicating no effect and 4 indicating a pronounced effect. In addition, the erythema and swelling scores were used to calculate the Primary Irritation Index (PII) by the formula PII=mean [(Σ erythema score+Σ swelling score)+2]. Moreover, swelling and induration scores were used to calculate the swelling/induration index (SII) by the formula SII=mean [(Σ swelling score+Σ density score) + 2]. If the SII was < 2, this was regarded as the absence of compaction.

Statistical analyzes

Statistical comparisons between cohorts were performed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test for continuous variables and a nonparametric Kruskal-Wallis test with Dunn's multiple comparison test for discrete variables. Statistical significance was defined as p < 0.05.

results

Infusion pressure assessment

100 ml of a 20 mg/ml IgG solution mixed with 200, 500, or 800 U/ml rHuPH20 was injected into the abdomen of Yucatan minipigs at a flow rate of 2 or 4 ml/min. When infused at a flow rate of 2 ml/min, the mean infusion pressures were 40.5 ± 0.1, 40.0 ± 0.1, and 37.1 ± 0.1 mmHg. ± standard error of the mean (SEM) for IgG when mixed with 200, 500 and 800 U/ml rHuPH20, respectively. With infusions at a flow rate of 4 ml/min, mean pressures were 49.9 ± 0.1, 55.5 ± 0.1, and 61.9 ± 0.2 mmHg. ± SEM for IgG when mixed with 200, 500, and 800 U/mL rHuPH20, respectively. The infusion pressure was not statistically different at different concentrations of rHuPH20 for each flow rate and was not statistically different at the two flow rates.

Assessment of the volume and area of local swelling

After completion of each infusion, swelling (if any) of the local infusion site was outlined and measured using a digital caliper. With infusions at a flow rate of 2 ml/min, the mean volume of swelling was 36.6 ± 14.4, 19.5 ± 6.5, and 31.4 ± 6.0 cm ³ ± SEM for IgG when mixed with 200, 500 and 800 U/ml rHuPH20, respectively, with 10 out of 12 infusions of swelling visible and measurable. In contrast, swelling volume was detected with only 3 of 12 infusions at a flow rate of 4 ml/min (one swelling at each concentration of rHuPH20). Local swelling volumes were not statistically different at different concentrations of rHuPH20 for each flow rate and were not statistically different at the two flow rates.

In addition to volume calculation, the local area of swelling visible after infusion was measured. When infused at a flow rate of 2 ml/min, the mean swelling area was 78.2 ± 26.4, 59.7 ± 20.0, and 94.9 ± 9.7 cm ² ± SEM for IgG when mixed with 200, 500 and 800 U/ml rHuPH20, respectively. With infusions at a flow rate of 4 ml/min, measurable areas of swelling occurred in only 3 out of 12 infusions (one swelling at each concentration of rHuPH20). Local swelling area values were not statistically different at different rHuPH20 concentrations for each flow rate and were not statistically different at the two flow rates.

Evaluation of local infusion sites

After completion of the infusions, three different specialists quantified the local infusion sites for the presence and severity of erythema, size of visible swelling, skin physical density, primary irritation score (PII), which included erythema and swelling scores, and swelling/induration score (SII), which included swelling and density scores to determine the presence of induration.

Evaluated the presence and severity of erythema

With infusions at a flow rate of 2 ml/min, the mean erythema score was (± SEM) 0.8 ± 0.2, 0.4 ± 0.1, and 1.0 ± 0.3 for IgG when mixed with 200, 500 and 800 U/ml rHuPH20, respectively. With infusions at a flow rate of 4 ml/min, the mean erythema score was (± SEM) 0.3 ± 0.1, 0.3 ± 0.1 and 0.2 ± 0.1 for IgG when mixed with 200, 500 and 800 U/ml rHuPH20, respectively. Local erythema scores were not statistically different at different rHuPH20 concentrations for each flow rate and were not statistically different at the two flow rates. The size of the visible local swelling was assessed, and when infused at a flow rate of 2 ml/min, the average swelling score was (± SEM) 1.9 ± 0.4, 1.4 ± 0.3 and 2.0 ± 0.2 in the case of IgG when mixed with 200, 500 and 800 U/ml rHuPH20, respectively. With infusions at a flow rate of 4 ml/min, visible swelling was less common, and the mean erythema score was (± SEM) 0.6 ± 0.3, 0.9 ± 0.4, and 0.9 ± 0.4 for IgG at mixing with 200, 500 and

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800 U/ml rHuPH20, respectively. Local swelling scores were not statistically different at different concentrations of rHuPH20 for each flow rate and were not statistically different at the two flow rates, except for IgG+800 U/mL rHuPH20 at a flow rate of 2 mL/min versus IgG+200 U/mL rHuPH20 at a flow rate of 4 ml/min (p < 0.05). Physical density of the skin was assessed at the local infusion site, and with infusions at a flow rate of 2 ml/min, the mean density score was (± SEM) 1.5 ± 0.3, 1.0 ± 0.2, and 1.4 ± 0.2 in the case of IgG when mixed with 200, 500 and 800 U/ml rHuPH20, respectively. With infusions at a flow rate of 4 ml/min, less density was observed at the local infusion site, and the mean density score was (± SEM) 0.5 ± 0.3, 0.7 ± 0.2 and 0.7 ± 0.3 in case of IgG when mixed with 200, 500 and 800 U/ml rHuPH20, respectively. Density scores at the local infusion site were not statistically different at different rHuPH20 concentrations for each flow rate and were not statistically different at the two flow rates. The primary irritation score was calculated based on the erythema and swelling scores. At 2 ml/min infusions, mean PII (± SEM) was 1.4 ± 0.3, 0.9 ± 0.2, and 1.5 ± 0.2 for IgG when mixed with 200, 500, and 800 U/ ml rHuPH20, respectively. With infusions at a flow rate of 4 ml/min, fewer PII scores were obtained, and the mean score was (± SEM) 0.4 ± 0.2, 0.6 ± 0.3, and 0.5 ± 0.3 for IgG at mixed with 200, 500 and 800 U/ml rHuPH20, respectively. PII scores were not statistically different at different concentrations of rHuPH20 for each flow rate and were not statistically different at the two flow rates, except for IgG+800 U/mL rHuPH20 at a flow rate of 2 mL/min compared to IgG+200 U/mL rHuPH20 at flow rate 4 ml/min (p < 0.05). The index of swelling/induration was calculated based on the scores of swelling and density. When infused at a flow rate of 2 ml/min, the mean SII was 1.7±0.3, 1.2±0.2, and 1.7±0.2 mmHg. Art. (± SEM) in the case of IgG when mixed with 200, 500 and 800 U/ml rHuPH20, respectively. With infusions at a flow rate of 4 ml/min, fewer SII scores were obtained, and the mean score was 0.6 ± 0.3, 0.8 ± 0.3, and 0.8 ± 0.3 (± SEM) for IgG at mixed with 200, 500 and 800 U/ml rHuPH20, respectively. SII scores were not statistically different at different rHuPH20 concentrations for each flow rate and were not statistically different at the two flow rates. If the mean value of SII was < 2, then this was regarded as the absence of compaction. Finally, photographs were taken before and after the completion of each infusion.

Example 2 An open-label, multicenter, phase 1b dose escalation study evaluating the safety and pharmacokinetics of daratumumab supplemented with recombinant human hyaluronidase (rHuPH20) administered subcutaneously for the treatment of subjects with relapsed or refractory multiple myeloma.

The aim of the study is to evaluate the safety, pharmacokinetics, and antitumor activity of daratumumab when administered subcutaneously (SC) or intravenously (IV) to participants with relapsed or refractory multiple myeloma. This is an open-label, multicenter, 2-part, phase 1b, dose/participant escalation study to evaluate the safety, pharmacokinetics, and antitumor activity of daratumumab when administered s.c. or i.v. to participants with relapsed or refractory multiple myeloma. The study will enroll up to approximately 48 participants in Part 1 and 80 participants in Part 2. The dose escalation phase (Part 1) is designed to determine the recommended dose for phase 2 (RP2D) based on the safety and pharmacokinetics (PK) of daratumumab. Each part of the study will have 3 phases: a screening phase, an open-label phase, and a post-treatment phase (from the last dose of study drug to week 8 after completion of treatment). In Part 1 participants will be placed into consecutive cohorts of approximately 8 participants each. Participants will receive DARA PH20 (daratumumab supplemented with recombinant human hyaluronidase [rHuPH20]) by SC infusion once a week on cycles 1 (each cycle of 28 days) and 2, every 2 weeks on cycles 3-6, and every 4 weeks on subsequent cycles of each cohort. After the last participant in each cohort completes Day 1 of Cycle 3, the Safety Assessment Panel (SET) will evaluate the pharmacokinetic and safety data against protocol-defined criteria and decide whether to increase the dose in the new cohort. The SET will evaluate all safety and PK data from Part 1 to determine 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 DARA 1200 mg. The safety, pharmacokinetics, and antitumor activity of s.c. and iv administration of daratumumab will be evaluated. The safety of participants will be monitored throughout the study.

Primary Performance Criteria

Residual serum concentrations (Ctrough) of daratumumab (time interval: up to day 1 of cycle 3 (each cycle 28 days) of part 2). Ctrough: concentration before drug administration.

Parts 1 and 2. Number of participants with adverse events (AEs) and serious AEs (time interval: from screening to follow-up 30 days after the last dose).

An adverse event (AE) is any adverse medical event in

- 31,042,589 participants who received study drug, regardless of the possibility of a causal relationship. A serious adverse event (SAE) is an AE that results in any of the following outcomes or is considered significant for any other reason: death; initial or prolonged hospitalization in a hospital; life-threatening condition (risk of immediate death); persistent or severe disability/incapacity; congenital anomaly.

Secondary performance criteria

Parts 1 and 2. Serum (plasma) concentrations of antibodies to daratumumab and recombinant human hyaluronidase.

(rHuPH20) (time interval: approximately 2 years).

Serum concentrations of antibodies to daratumumab and rHuPH20 to assess potential immunogenicity.

Parts 1 and 2. Percentage of participants with a complete response (CR) (time frame: approximately 2 years). CR is defined as the proportion of participants who achieve CR (including sCR) according to the International Myeloma Working Group (IMWG) criteria.

Parts 1 and 2. Percentage of participants with an overall response rate (ORR) (time frame: approximately 2 years). The overall response rate is defined as the percentage of participants who achieved a complete response or partial response according to the International Myeloma Task Force criteria during or after completion of study treatment.

Parts 1 and 2. Duration of response (DR) (time frame: approximately 2 years). DR is the time from the day of the initial documented response (CR or PR) to the day of the first documented case of disease progression (PD), as defined by the IMWG criteria.

Parts 1 and 2. Time to response (time frame: approximately 2 years). Time to response is defined as the time from the day of administration of the first dose of investigational treatment to the day of the first documented observed response (CR or PR).

In table. 6 shows the study plan. In table. 7 shows the interventions performed.

Table 6

Group number or name Type Description Part 1. Cohort 1 Obtained experimentally Participants will receive DARAPH20 1200 mg (daratumumab 1200 milligrams (mg) with recombinant human hyaluronidase [rHuPH20] 30,000 U) by subcutaneous (SC) infusion once a week in cycles 1 (each cycle lasts 2-8 days) and 2 , every 2 weeks in cycles 3-6, and then every 4 weeks in subsequent cycles until disease progression. Part 1. Cohort 2 Obtained experimentally Participants will receive DARAPH20 1800 mg (daratumumab 1800 milligrams (mg) with recombinant human hyaluronidase [rHuPH20] 45,000 U) by SC infusion once a week in cycles 1 and 2, every 2 weeks in cycles 3-6, and every other week thereafter 4 weeks in

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subsequent cycles until disease progression. Part 1. Cohort 3 Obtained experimentally Participants will receive DARAPH20 at a dose to be determined by the Study Evaluation Team (SET), once a week by SC infusion in cycles 1 and 2, every 2 weeks in cycles 3-6, and every 4 weeks thereafter in subsequent cycles until disease progression. In addition, up to three additional optional cohorts (cohorts 3b, 3c and 3d) can be recruited to repeat the dose level of daratumumab Part 2. Cohort 4 Obtained experimentally SC infusion of DARA-PH20 in RP2D (dose recommended for Phase 2) as determined in Part 1 will be administered to participants by SC infusion once a week in cycles 1 and 2, every 2 weeks in cycles 3-6 , and then every 4 weeks in subsequent cycles until disease progression. Part 2. Cohort 5 Obtained experimentally Daratumumab 1200 mg will be given to participants by intravenous (IV) infusion once a week in cycles 1 and 2, every 2 weeks in cycles 3-6, and every 4 weeks thereafter in subsequent cycles until disease progression.

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Table 7

Name of intervention Type Involved groups Description Daratumumab Subcutaneous (SC) infusion Medicinal means Part 1. Cohort 1 Part 1. Cohort 2 Part 1. Cohort 3 Part 2. Cohort 4 Participants will receive s/c infusion of daratumumab alone once a week in cycles 1 (each cycle lasts 28 days) and 2, every 2 weeks in cycles 3-6 and every 4 weeks in subsequent cycles. S / c infusion recombinant hyaluronidase [rHuPH20] Part 1. Cohort 1 Part 1. Cohort 2 Part 1. Cohort 3 Part 2. Cohort 4 Participants will receive s/c infusion of recombinant hyaluronidase [rHuPH20] together with daratumumab by subcutaneous infusion once a week in cycles 1 (each cycle lasts 28 days) and 2, every 2 weeks in cycles 3-6 and every 4 weeks in Daratumumab Intravenous (IV) infusion Part 2. Cohort 5 Participants will receive in / in infusion of daratumumab 1200 mg once a week in cycles 1 (each cycle 2 8 days) and 2, every 2 weeks in cycles 3-6 and every 4 weeks in subsequent cycles.

Selection criteria for participation

Participants who are confirmed to have symptomatic (with symptoms) multiple myeloma (MM) according to the International Myeloma Working Group (IMWG) diagnostic criteria.

Criteria:

measurable disease, defined as any of the following conditions:

(a) Myeloma immunoglobulin (Ig) G (serum concentration of monoclonal paraprotein [M-protein] > 1.0 gram/deciliter [g/dL] or urinary M-protein concentration equal to or greater than (>) 200 milligrams [mg] ]/24 hours [h]; or (b) multiple myeloma IgA, IgD IgE (serum M-protein >0.5 g/dl or urine M-protein > 200 mg/24 h); or (c) multiple myeloma of light chains (serum concentration of free light chains of immunoglobulin > 10 mg/dl and an abnormal ratio of free light chains of immunoglobulin kappa and lambda);

the participant must score 0, 1, or 2 on the Eastern Joint Oncology Group (ECOG) General Condition Scale;

the results of the assessment of clinical laboratory parameters before treatment should be consistent with the

- 34 042589 values set in the protocol during the screening phase;

A man who has sex with a woman of childbearing potential and who has not had a vasectomy must agree to use an appropriate method of contraception selected by the investigator and must agree not to donate sperm during the study and for 4 months after the last dose of daratumumab.

Exclusion Criteria:

the participant has previously received daratumumab or other drugs against differentiation cluster 38 (against CD38);

participant received anti-myeloma treatment for 2 weeks prior to day 1 of cycle 1;

the participant has previously had an allogeneic stem cell transplant; or the participant received an autologous stem cell transplant (ASCT) within 12 weeks prior to day 1 of cycle 1;

the participant was diagnosed with a malignant disease (excluding multiple myeloma) within 5 years prior to day 1 of cycle 1 (exceptions are squamous and basal cell carcinomas of the skin and carcinoma in situ of the cervix or a malignant disease that, in the opinion of the investigator and in agreement with the sponsor's designated specialist in considered cured in clinical studies with minimal risk of recurrence);

the participant has clinical signs of meningeal multiple myeloma.

Gender: both.

Age limit: 18 years old.

Participation of healthy volunteers: no.

Part 1 interim results (end of data collection: July 21, 2016 for safety/demographics/disease history and July 28, 2016 for efficacy data).

Ways

Patients had RRMM previously exposed to > 2 types of therapy, including a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD). Part 1 of the 2-part study used consecutive cohorts at dose levels of 1200 mg and 1800 mg of DARA to determine the recommended sc dose for Part 2. DARA-PH20 was administered in 4-week treatment cycles: once a week ( QW) for 8 weeks, once every two weeks (Q2W) for 16 weeks, and thereafter once every four weeks (Q4W). DARA-PH20 was infused at doses of 1200 mg in 60 ml over 20 min or 1800 mg in 90 ml over 30 min via a syringe pump into various sites on the abdomen. Pre- and/or post-infusion drugs included paracetamol, diphenhydramine, montelukast, and methylprednisolone. In Part 2, patients will be randomized 1:1 to receive the recommended phase 2 dose (RP2D) of DARA-PH20 sc or IV DARA (16 mg/kg). RP2D DARA-PH20 will be selected based on a cumulative review of the pharmacokinetic and safety data obtained in Part 1 and should provide a maximum serum Ctrough during weekly dosing that is equal to or greater than the Ctrough observed at the approved IV dose of 16 mg/kg . The main parameters evaluated were Ctrough DARA up to day 1 of cycle 3 and safety. Additional parameters assessed included overall response rate (ORR).

results

To date, 41 patients have been treated with DARA-PH20 Part 1 sc at doses of 1200 mg (n=8) and 1800 mg (n=33). Infusion-related reactions (IRRs) occurred in 9/41 patients (22%) and were most commonly grade 1/2, including chills, fever, tremors, vomiting, pruritus, swelling of the tongue, non-cardiac chest pain, and wheezing . One patient developed grade 3 dyspnea and one patient required hospitalization for fever and chills (both grade 2) after the first infusion. All IRRs developed within or within 6 hours of the first s.c. infusion and were treated with antihistamines, corticosteroids, antiemetics, or bronchodilators. There were no reports of IRR with subsequent infusions. Overall, the adverse event profile of DARA-PH20 was similar to that of IV DARA. Grade 3 or higher drug-related adverse events occurred in 5/41 (12%) patients and included fatigue (2 patients), colds, hypertension, dyspnea, and tumor disintegration syndrome. SC administration of DARA-PH20 was well tolerated at the site of administration, such as the abdominal wall, with 3/41 (7%) of the patient experiencing grade 1 erythema, induration, or burning sensation. The analysis showed a higher max. Ctrough in the 1800 mg cohort compared to max. Ctrough achieved after IV administration of DARA (16 mg/kg).

In a 1200 mg cohort of 8 patients (median 5 types of prior therapy [range 2–10]; prior ASCT, 63%; PI-only resistance, 0%; IMiD-only resistance, 13%; dual resistance to PI and IMiD, 63 %) saw an ORR of 25% including 2 partial responses (PR). The median time to response was 14 (range 8–20) weeks. Among 17 patients with the possibility of evaluation

- 35 042589 responses in the 1800 mg cohort assessed on day 1 of cycle 3 (median 4 prior treatments [range 2-7]; prior ASCT, 76%; PI-only resistance, 6%; IMiD-only resistance, 12 %; dual resistance to PI and IMiD, 65%) observed an ORR of 41%, consisting of 3 very good partial responses and 4 PRs. The median time to response was 4 (range 4–8) weeks.

conclusions

DARA-PH20 s.c. was well tolerated and produced serum residual concentrations equal to or greater than those of iv DARA, with a lower IRR compared to iv DARA over a significantly shorter infusion time. Preliminary data suggest that in this patient population, SC administration of DARA-PH20 may provide similar treatment response rates compared to IV DARA monotherapy. For part 2 of the study, the dose level of 1800 mg DARA-PH20 was chosen as the RP2D. These early data support the feasibility of further investigation of s.c. DARA in clinical trials.

Example 3 Development of combined formulations of daratumumab and hyaluronidase.

Various combination formulations were evaluated to determine the overall physicochemical stability and delivery of daratumumab and rHuPH20 in the combination formulation. The effect of active ingredient and/or excipient concentrations in formulations has been evaluated in several stability and/or animal studies (storage stability, shaking stability and infusion stability studies in pigs). In table. 8 is a summary of the formulations used in the various studies.

Table 8

Compound Daratumumab (mg/ml) rHuPH20 (U/ml) His (mM) Sorbitol / sucrose (mM) PS20 (% w/v) Met (mg/ml) pH 1 100 500 10 300 0.04 2 5, 5 2 120 2000 10 300 0.04 1 5, 6 3 100 500 10 300 0, 0 2 5, 5 4 100 500 10 300 0.01 2 5, 5 5 100 500 10 300 0.02 2 5, 5 6 100 500 10 300 0.06 2 5, 5 7 100 0 10 200/100 0.04 0 5, 5 8 100 0 10 100/200 0.04 0 5, 5 9 100 50 10 300 0.04 1 5, 5 10 100 500 10 300 0.04 1 5, 5 eleven 100 2000 10 300 0.04 1 5, 5 12 100 5000 10 300 0.04 1 5, 5

His: histidine

Met: methionine.

The ranges of excipients and active ingredients in the tested compositions are given in table. 9.

Table 9

Composition component Range rHuPH20 0-2000 U/ml Daratumumab 100-120 mg/ml Histidine 10 mmol Sorbitol 100-300 mM sucrose 0-200 mM Polysorbate-20 (PS20) 0.0-0.04% (w/v) Methionine 0-2 mg/ml

The generated formulations were examined in various assays to determine their characterization, including evaluation of invisible to the naked eye particles, microflow imaging (MFI), size exclusion chromatography (SEC), capillary isoelectric focusing (cIEF), polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (non-reducing and reducing), peptide mapping, extractable volume, turbidity, osmolality and pH.

Particles invisible to the naked eye (Sub-vis):

the collection of particles >10 µm or >25 µm, invisible to the naked eye, is usually an accumulation of protein molecules, and can be analyzed using the HIAC chiaroscuro method, in which the solution passes through a small hole, and blocking the light provides information about the size of the particle passing through it.

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MFI: Chiaroscuro Independent Microflow Imaging (MFI) captures snapshots of flowing particles and inversely converts to the number of particles present in a given volume of liquid. This method provides information about the large accumulations of proteins present in the solution.

SEC: A size exclusion chromatographic separation method in which a column is used to distribute molecules within a flowing solution according to a wide range of sizes. Monomers, clusters 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.

cIEF: capillary isoelectric focusing allows the distribution of molecules according to the charge on the molecule and is a good indicator of overall chemical stability. For example, deamidation can lead to a change in the charge of the molecule and thus can be detected using this method. The method gives an idea of the total % of acidic, basic and intact molecules present in the solution.

SDS (reducing and non-reducing conditions): The SDS method provides information about the physical stability of a molecule. SDS allows you to measure the intact, aggregated and fragmented components present in the solution. The non-reducing SDS provides information on the relevant intact, aggregated, and fragmented antibody constituents, and the reducing SDS (after breaking disulfide bonds) provides the same information for the heavy and light chains of the antibody.

Peptide mapping: Peptide mapping is the main way to study the primary structure of proteins. In the case of recombinant protein preparations, peptide mapping is used to initially confirm the described structure. Peptide mapping also provides information on post-translational modifications such as deamidation, oxidation, and the like.

Extractable Volume: This method provides information on the amount/volume of liquid that can be extracted from the vial after the appropriate timestamp.

Haze: based on light scattering method for assessing the physical stability of the solution. Increasing the size of particles or clusters leads to an increase in the light scattering signal and, therefore, can be used to determine the turbidity (opalescence) of the solution. Turbidity is measured in nephelometric turbidity units (NTU).

Osmolality: is a measure of the total osmotic activity, which depends on the total true activity of the molecules (activity factor times concentration).

The osmolality of the solution should be close to the osmolality of the injectable serum.

pH: gives an indication of overall stability and it is important that the pH remains constant throughout the shelf life.

Enzymatic activity of rHuPH20: The determination of hyaluronidase activity is based on the formation of a precipitate upon binding of hyaluronic acid (HA) to acidified serum. Activity is measured by incubating hyaluronidase with HA for 30 min in a 96-well plate at 37° C., followed by precipitation of uncleaved HA by adding acidified serum. The resulting turbidity is measured at 640 nm and the reduction in turbidity caused by enzymatic degradation of the HA substrate is a measure of hyaluronidase activity.

The storage 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 assessed using the assays described. Samples were stored in 25R vials (filled to a volume of 16 ml) at different temperatures (5, 25 and 40°C) and the vials were analyzed using different assays, at different time points (0, 1, 2, 3, 4, 5 and/or 6 months). The data show that the combination product is stable under the above storage conditions for both daratumumab and rHuPH20 as shown in various assays. The profile observed for particles, color, turbidity, sec, etc. was very similar to that of standard stable antibodies and the data is comparable to the stability data of some commercial mAb formulations.

In table. 10 shows the number of particles in Formulation 1 over time as assessed by HIAC.

In table. 11 shows the number of particles in Formulation 1 over time as assessed by MFI.

In table. 12 shows the pH value of formulation 1 over time.

In table. 13 shows the haze of Formulation 1 over time.

In table. 14 shows the percentage of high molecular weight clusters and low molecular weight fragments in formulation 1 over time.

In table. 15 shows the acidic and basic components in formulation 1 over time as assessed by cIEF.

In table. 16 shows the percentage (%) purity of formulation 1 over time as assessed by reconstituted SDS-PAGE.

In table. 17 shows the percentage (%) purity of formulation 1 over time as assessed by non-reducing SDS-PAGE.

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In table. 18 shows the bioactivity of daratumumab and the enzymatic activity of rhPH20 in percent (%) in formulation 1 over time.

Table 10

Storage temperature (°C) Mean total/mL of particles >10 µM or >25 µM 0 months 3 months 6 months > 10 µm > 25 µm > 10 µm > 25 µm > 10 µm > 25 µm 5 7.17 6, 33 6, 5 4.8 42.50 30, 83 25 7.17 6, 33 5, 5 1.8 110.50 81.83 40 7.17 6, 33 64.5 44.8

Table 11

0 months 3 months 6 months Temperature (°C) 5 5 25 40 5 25 Particles/ml > 2 - < 10 µm ECD 539 1155 3668 3371 686 3581 Particles/ml > 10 - < 2 5 µm ECD 9, 3 39 93 80 36 105 Particles/ml > 2 5 - < 7 0 µm ECD 1.4 2, 6 13 9.3 7.4 7.2 Particles/ml > 70 µm ECD 0.5 0.2 0.2 0.2 0, 0 0.5

Table 12

Storage temperature (°C) pH over storage time (months) 0 months 1 month 2 months 3 months 6 months 5 5, 7 n/a 5, 7 5, 7 5, 6 25 5, 7 5, 7 5, 7 5, 7 5, 6 40 5, 7 5, 7 5, 6 5, 7 n/a

month: month.

N/A=no data.

Table 13

Storage temperature (°C) Average number of NTUs during storage time (months) 0 months 1 month 2 months 3 months 6 months 5 3, 5 3, 5 3, 5 3, 4 25 3, 5 3.5 3, 6 3, 7 4, 6 40 3, 5 4 5, 5 8.2 n/a

month: month.

NTU; nephelometric units of turbidity.

N/A=no data.

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Table 14

Percentage (%) of components Storage time (months) Storage temperature (°C) hmw Monomer LMW 0 5 0.74 99, 25 0.01 25 0.74 99, 25 0.01 40 0.74 99.25 0.01 3 5 0.84 99, 16 0.02 25 1, 11 98.75 0.14 40 1, 87 94.50 3, 64 6 5 0.89 99, 10 n/a 25 1.30 98.38 n/a 40 n/a n/a n/a

HMW: high molecular weight components.

LMW: low molecular weight components.

N/A=no data.

Table 15

Storage time (months) Storage temperature (°C) Main peak, % % acidic peaks % of major peaks 0 5 69.2 27.8 3 25 69.2 27.8 3 40 69.2 27.8 3 3 5 68.2 28.9 2.9 25 63 32.8 4.2 40 31.3 60.2 8.5 6 5 68.1 28.7 3.2 25 55.4 38 6, 6 40 n/a n/a n/a

N/A=no data.

Table 16

Storage temperature (°C) % purity during storage time (in months) 0 months 1 month 2 months 3 months 6 months 5 98, 16 n/a 97.96 98 98.02 25 98, 16 97.87 97, 66 97.57 96, 75 40 98, 16 96.4 95 92, 88 n/a

month: month.

N/A=no data.

Table 17

Storage temperature (°C) % purity during storage time (in months) 0 1 2 3 6 5 97.56 97, 64 97, 64 97.5 25 97.56 97.27 96, 97 96, 62 95, 63 40 97.56 94.84 92, 61 89, 37 n/a

month: month.

N/A=no data.

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Table 18

Molecule Analysis Duration and storage temperature 0 months 6 months 6 months 5°C 25°C Daratumumab ADCC* 102 103 83 CDC* 95 101 93 РН2 0 Enzymatic activity** 574 600 or 584? 609

* Percentage of control ** U/ml.

The agitation (shaking) stability of formulation 1 was also evaluated using the assays described above to evaluate the formulations and investigate the effect of PS concentrations by varying only the PS20 concentrations in this formulation (compositions 1, 3, 4, 5 and 6 in Table 8, where the content PS20 varied and was 0, 0.01, 0.02, 0.04, 0.06%). The data indicated that the combined formulation was stable under shaking conditions for both daratumumab and the enzyme as shown in various assays. The profile observed for particles, color, turbidity, sec, etc. was very similar to that of standard stable antibodies for all but 0% PS concentrations (PS200% formulation contained particles and was not stable) and the data were comparable with stability data for some commercial mAb formulations (data not shown).

The storage stability of formulation 2 (120 mg/ml daratumumab, 10 mM histidine, 300 mM sorbitol, 0.04% PS20, 1 mg/ml methionine, 2000 U/ml rhuPH20, pH 5.5) was assessed using the assays described. Samples were stored in 25R vials filled to a volume of 13.27 ml overflow (1500 mg dose) at various temperatures and analyzed using the various assays described below. The data collected showed that the combination product was stable under the above storage conditions for both daratumumab and rHuPH20. The profile observed for particles, color, turbidity, sec, etc. was very similar to that of standard stable antibodies, and the data were comparable to the stability data of some commercial mAb formulations. rhuPH20 is highly sensitive at high temperatures and loses activity very quickly when stored at 40°C. In table. 19 shows the characteristics of the composition.

- 40 042589

Table 19

Storage time, temperature and relative humidity (RH) Characteristics and/or analysis 0 months 1 month 1 month 5°C 40°C/ 75% ABOUT 25°C/ 60% ABOUT Average total number/ml of particles 2-10 µm in size 58.89 375.68 4285.81 Average total number/ml of particles with a size of 10-25 µm 3, 34 12.96 1.98 Mean total/mL particles >25 µm 1.23 0.49 0.12 PS20 (% w/v) 0.038 0.02 0.03 pH 5, 6 5, 6 5, 6 Turbidity (NTU) 5 eleven 6 % purity (cSDS, recovery) 98.4 98.6 98.1 % AGHC, aglycosylated heavy chains (cSDS, recovery) 0.4 0.5 0.4 % purity (cSDS, no recovery) 97.7 95.2 97.7 % monomers, exclusion HPLC 99.1 98.0 98.8 % Clusters, Exclusive HPLC 0.9 16 1.1 % fragments, exclusive HPLC < 0.10 0.4 < 0.10 Biological activity of daratumumab, CDC (% of control) Biological activity of daratumumab, ADCC (% of control) 105 99 88 73 99 103 rhuPH20 activity (U/ml) 2205 0 2258

Formulations 3-8 were tested for storage stability or shake stability using some or all of the assays described. The data showed that formulations 3-8 were stable under the conditions assessed for both daratumumab and HuPH20 (formulations 7 and 8 did not contain rHuPH20). Methionine has been included in Formulations 1-6 and 9-12 to provide additional oxidation stability. The profile observed for particles, color, turbidity, sec, etc. was very similar to that of standard stable antibodies, and the data were comparable to the stability data of some commercial mAb formulations (data not shown).

The agitation (shaking) stability of formulation 1 was also assessed using the above assays to evaluate the formulations and investigate the effect of PS20 concentrations by varying only the PS20 concentrations in this formulation (formulations 1, 3, 4, 5 and 6 in Table 8, where the PS20 content varied and amounted to 0, 0.01, 0.02, 0.04, 0.06%). The data showed that the combination formulation was stable under shaking conditions for both daratumumab and rHuPH20, as shown in fold.

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Claims (37)

personal analyses. The profile observed for particles, color, turbidity, sec, etc. was very similar to that of standard stable antibodies for all PS concentrations except 0% and the data were comparable to the stability data of some commercial mAb formulations (data not shown). Formulations 9-12 were also evaluated for subcutaneous administration of daratumumab with varying enzyme concentrations in a porcine model as described in Example 2. These studies were performed to determine the rhPH20 concentration acceptable to deliver 16 ml of daratumumab. Parameters assessed were infusion pressure, swelling or blister area (if measurable), and site quality score. A dose-dependent increase in infusion pressure was observed. All test concentrations of rhPH20 (50, 500, 2000, 5000 U/ml) were acceptable for delivery of 16 ml of daratumumab. CLAIM 1. Pharmaceutical composition for the treatment of CD38-positive hematological malignant tumor and to reduce infusion-related reactions, containing about 1800 mg of anti-CD38 antibodies and about 30,000 units of hyaluronidase, where: a) the anti-CD38 antibody has an IgG1 isotype and contains a heavy chain variable region (VH) with the sequence of SEQ ID NO: 4 and a light chain variable region (LH) with the sequence of SEQ ID NO: 5; b) hyaluronidase is a recombinant hyaluronidase rHuPH20 and c) the total volume of the composition is about 15 ml. 2. A pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier. 3. The pharmaceutical composition according to claim 1 or 2, wherein the anti-CD38 antibody comprises a heavy chain of SEQ ID NO: 12 and a light chain of SEQ ID NO: 13. 4. Pharmaceutical composition according to any one of claims 1 to 3, further comprising: a) about 5 to about 50 mM histidine, and b) about 50 to about 400 mM sorbitol. 5. Pharmaceutical composition according to any one of claims 1 to 4, further comprising: a) about 10 mM histidine; about 300 mm sorbitol; about 0.04% w/v polysorbate-20 (PS-20) and about 1 mg/ml methionine at a pH of about 5.6; b) about 10 mM histidine; about 300 mm sorbitol; about 0.04% w/v PS-20 and about 2 mg/ml methionine at a pH of about 5.5; c) about 10 mM histidine; about 300 mm sorbitol; about 0.01% w/v PS-20 and about 2 mg/ml methionine at a pH of about 5.5; d) about 10 mM histidine; about 300 mm sorbitol; about 0.02% w/v PS-20 and about 2 mg/ml methionine at a pH of about 5.5; e) about 10 mM histidine; about 300 mm sorbitol; about 0.06% wt./about. PS-20 and about 2 mg/ml methionine at a pH of about 5.5; or f) about 10 mM histidine; about 300 mm sorbitol; about 0.04% w/v PS-20 and about 1 mg/ml methionine at a pH of about 5.5. 6. Pharmaceutical composition according to claim 4, additionally containing from about 0.01 to about 0.1% wt./about. polysorbate-20 (PS-20) and/or about 0.1 to about 2.5 mg/ml methionine. 7. The pharmaceutical composition according to claim 4, additionally containing from about 0.01 to about 0.04% wt./about. polysorbate-20 (PS-20); and about 1 to about 2 mg/ml methionine. 8. Pharmaceutical composition according to claim 4, additionally containing from about 0.01 to about 0.04% wt./about. polysorbate-20 (PS-20) and/or about 0.1 to about 2.5 mg/ml methionine. 9. A pharmaceutical composition according to any one of claims 4 to 8, containing about 10 mM histidine and about 100 to about 300 mM sorbitol. 10. Pharmaceutical composition according to any one of claims 1 to 9, which is a unit dosage form. 11. The pharmaceutical composition of claims 1 to 10, wherein the CD38-positive hematologic malignancy is CD38-positive multiple myeloma. 12. A single dosage form for the treatment of CD38-positive hematological malignant tumors, which allows to reduce infusion-related reactions, containing: a) an anti-CD38 antibody of the IgG1 isotype, comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5, in an amount of from about 1800 mg; b) hyaluronidase in an amount of about 30,000 U; c) histidine at a concentration of about 5 to about 15 mM; d) sorbitol at a concentration of about 100 to about 300 mM; e) polysorbate-20 (PS-20) at a concentration of about 0.01 to about 0.04% w/v. And - 42 042589 f) methionine at a concentration of about 1 to about 2 mg/ml at a pH of about 5.5; wherein the total volume of the unit dosage form is about 15 ml; and wherein the hyaluronidase is a recombinant rHuPH20 hyaluronidase. 13. A unit dosage form according to claim 12, where c) histidine is present at a concentration of about 10 mM. 14. A unit dosage form according to claim 12 or 13, wherein the sorbitol is present at a concentration of about 300 mM. 15. Single dosage form according to claims 12-14, where polysorbate-20 (PS-20) is present at a concentration of about 0.04% wt./about. 16. A unit dosage form according to claims 12-15, in which methionine is present at a concentration of about 1 mg/ml. 17. A unit dosage form according to any one of claims 12-16, additionally containing sucrose at a concentration of from about 100 to about 200 mm. 18. A unit dosage form according to any one of claims 12-17, wherein the anti-CD38 antibody comprises a heavy chain of SEQ ID NO: 12 and a light chain of SEQ ID NO: 13. 19. A single dosage form for the treatment of CD38-positive hematological malignant tumors and to reduce infusion-related reactions, containing: a) an anti-CD38 antibody of the IgG1 isotype containing the heavy chain variable region of SEQ ID NO: 4 and the light chain variable region of SEQ ID NO: 5 in an amount of about 1800 mg; b) hyaluronidase in an amount of about 30,000 U; 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 at a pH of about 5.6; where the total volume of a single dosage form is about 15 ml and where hyaluronidase is a recombinant hyaluronidase rHuPH20. 20. The unit dosage form of claims 12-19, wherein the CD38-positive hematologic malignancy is CD38-positive multiple myeloma. 21. A method of treating a CD38-positive hematological malignant tumor and reducing infusion-related reactions in an individual, comprising administering a pharmaceutical composition according to any one of paragraphs.1-11 or a unit dosage form according to any one of paragraphs. 12-20 subcutaneously to an individual for a period of time sufficient to treat a CD38 positive hematologic malignancy. 22. The method of claim 21 further comprising administering a second therapeutic agent. 23. The method of claim 22, wherein the second therapeutic agent is a proteasome inhibitor, an alkylating agent, or a glutamic acid derivative, or combinations thereof. 24. The method according to claim 22, where: a) the proteasome inhibitor is bortezomib, carfilzomib or ixazomib; b) the alkylating agent is busulfan, cyclophosphamide, bendamustine, chlorambucil, carboplatin, cisplatin, temozolomide, melphalan, carmustine, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, thiotepa, trabectedin or streptozocin, and c) the glutamic acid derivative is lenalidomide, thalidomide or pomalidomide. 25. The method of claim 22 further comprising administering a corticosteroid. 26. The method of claim 25 wherein the corticosteroid is dexamethasone. 27. The method of claim 25 wherein the corticosteroid is prednisolone. 28. The method according to claim 21, where the pharmaceutical composition additionally contains from about 5 to about 50 mm histidine and from about 50 to about 400 mm sorbitol and optionally additionally contains from about 0.01 to about 0.1% wt./about. polysorbate-20 (PS-20) and/or about 0.1 to about 2.5 mg/ml methionine. 29. The method according to item 21, where the pharmaceutical composition additionally contains about 10 mm histidine and from about 100 to about 300 mm sorbitol and optionally additionally contains from about 0.01 to about 0.04% wt./about. polysorbate-20 (PS-20) and/or about 0.1 to about 2 mg/ml methionine. 30. The method according to item 21, where the pharmaceutical composition additionally contains about 25 mm acetic acid, about 60 mm sodium chloride, about 140 mm mannitol and about 0.04% wt./about. polysorbate-20 (PS-20), pH about 5.5. 31. The method according to claim 21, where the pharmaceutical composition additionally contains about 10 mm histidine, about 300 mm sorbitol, about 0.04% wt./about. polysorbate-20 (PS-20) and about 2 mg/ml methionine; pH around 5.5. 32. The method according to claim 21, where the pharmaceutical composition additionally contains about 10 mm histidine, about 300 mm sorbitol, about 0.04% wt./about. polysorbate-20 (PS-20) and about 1 mg/ml methionine; - 43 042589 pH approx. 5.6. 33. The method of claims 21-32, wherein the CD38-positive hematologic malignancy is CD38-positive multiple myeloma. 34. Pharmaceutical combination for the treatment of CD38-positive hematological malignant tumor, containing about 1800 mg of anti-CD38 antibodies and about 30,000 IU of hyaluronidase, where: a) the anti-CD38 antibody has an IgGl isotype and contains a heavy chain variable region (VH) with the sequence of SEQ ID NO: 4 and a light chain variable region (VL) with the sequence of SEQ ID NO: 5; b) where hyaluronidase is a recombinant hyaluronidase rHuPH20 and c) the total volume of the combination is about 15 ml. 35. The pharmaceutical combination of claim 34, wherein the anti-CD38 antibody and hyaluronidase are combined in a single unit dosage form. 36. The pharmaceutical combination of claim 34, wherein the anti-CD38 antibody and hyaluronidase are in different unit dosage forms. 37. The pharmaceutical combination of claim 34, wherein the CD38 positive hematologic malignancy is CO38 positive multiple myeloma. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2