JP7602485B2 - Anti-CD38 Antibodies and Formulations - Google Patents

Description

RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62/837,518, filed April 23, 2019; U.S. Provisional Patent Application No. 62/859,699, filed June 10, 2019; European Patent Application No. 20305145.3, filed February 17, 2020; and European Patent Application No. 20305146.6, filed February 17, 2020, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on April 17, 2020, is named 704023_SA9-289PC_ST25.txt and is 44,783 bytes in size.

An anti-CD38 antibody with improved cytotoxic activity and a stable formulation thereof are provided herein.

CD38 is a type 2 glycosylated 45 kilodalton (kDa) membrane protein identified as a lymphocyte marker. CD38 has a role in leukocyte homeostasis through modulation of hematopoietic cell survival and differentiation (Non-Patent Document 1). CD38 functions as a receptor that binds to CD31 and is involved in cell adhesion and signal transduction. The function of CD38 in signal transduction appears to be versatile depending on the cell lineage, differentiation stage, and possibly association with different co-receptors (Richards JO, et al., 2008). CD38 is also an ectoenzyme that catalyzes the synthesis of cyclic adenosine diphosphate ribose (cADPR) from nicotinamide adenine dinucleotide (NAD+) and its hydrolysis to ADP-ribose (Non-Patent Document 2). These reaction products are involved in calcium mobilization and intracellular signal transduction (Non-Patent Document 3).

In healthy humans, CD38 expression is detected on NK cells, monocytes, dendritic cells, macrophages, granulocytes, activated T and B cells, and plasma cells. In contrast, no expression has been detected on hematopoietic stem cells, resting T and B cells, or tissue macrophages. In addition, several hematological malignancies, such as plasma cell dysplasias [e.g., multiple myeloma (MM), amyloidosis] and other cancers of hematopoietic origin, including, for example, Waldenstrom's disease, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), and acute myeloid leukemia (AML), express CD38.

CD38 expression is particularly prominent in MM, with >98% of patients testing positive for this protein (Non-Patent Document 4, Non-Patent Document 5). The strong and uniform expression of CD38 on malignant clonal MM cells contrasts with the restricted expression pattern on normal cells, suggesting that this antigen may be useful for specific targeting of tumor cells.

MM is a malignant plasma cell disorder characterized by cell surface expression of CD38, clonal proliferation of plasma cells in the bone marrow (BM), and production of excessive amounts of monoclonal immunoglobulins, usually of type IgG or IgA, or free light chains in urine (also called paraproteins, M proteins, or M components). MM is a predominantly age-related disease with more than 80% of patients over 60 years of age.

The disease course of MM varies depending on the aggressiveness of the disease and associated prognostic factors. Certain chromosomal abnormalities in multiple myeloma have been shown to be associated with poor clinical outcomes. High-risk cytogenetic alterations include del(17p), t(4;14), and t(14;16), as well as 1q gain, among others. In the past 20 years, median survival has improved from 3 to 6 years; however, some patients can live longer than 10 years (6). Treatment options and survival are based on the patient's age, health, and disease status. Patients younger than approximately 65 years who exhibit symptomatic active disease in good physical health will generally undergo initial therapy with autologous stem cell transplantation (ASCT). Induction chemotherapy is administered to achieve cytoreduction of the disease before stem cell harvest. Induction treatment regimens include alkylating agents, dexamethasone alone, thalidomide plus dexamethasone, and vincristine, Adriamycin® (doxorubicin) and dexamethasone (VAD; or modifications to this regimen); however, the latter two regimens are associated with higher toxicity (Non-Patent Document 7, Non-Patent Document 8). Treatment with Velcade® (bortezomib) alone, in combination with bortezomib, and Revlimid® (lenalidomide) plus dexamethasone have demonstrated improved outcomes as induction therapy, with these agents demonstrating higher response rates and lower toxicity (Richardson PG 2010, Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11). Additional approved drugs include pomalidomide (which belongs to the same IMiD® class as lenalidomide) and carfilzomib and ixazomib (which belong to the same proteasome inhibitor class as bortezomib). In addition to these new treatments, monoclonal antibodies, particularly anti-CD38 antibodies, have begun to play a major role in the treatment of myeloma patients. Daratumumab, an anti-CD38 antibody, has been approved for the treatment of MM as a single agent and in combination with other treatments for MM (Non-Patent Document 12; Non-Patent Document 13). Isatuximab, an anti-CD38 antibody, has been reported to induce response rates of 25-29% as a single agent and 60% with combination therapy in relapsed or refractory multiple myeloma (Non-Patent Document 14); recent results of a phase 3 study showed that the addition of isatuximab can improve progression-free survival with pomalidomide-dexamethasone in relapsed and refractory multiple myeloma (Non-Patent Document 15). In addition, elotuzumab, an anti-Slam-F7 antibody, has been approved in combination with lenalidomide and dexamethasone for the treatment of adult patients with MM who have received one to three prior lines of therapy, and in combination with pomalidomide and dexamethasone for the treatment of adult patients with MM who have received at least two prior lines of therapy, including lenalidomide and a proteasome inhibitor [Bristol-Myers Squibb Company, Empliciti (registered trademark) (elotuzumab) [package insert]]. U.S. Food and Drug Administration website. Non-Patent Document 16. Amended November 2018.

The current goal of therapy for these CD38-expressing diseases is to control the disease as effectively as possible, maximizing quality of life, and prolonging survival. For example, MM patients will receive an average of four to eight different regimens over their lifetime. Thus, despite improved patient outcomes with newer therapies, MM remains a fatal disease. Thus, treatment of patients who progress with current therapies, including antibody therapies, remains a challenging and significant unmet medical need.

Richards, J.O., et al., Mol Cancer Ther. 2008;7(8):2517-27 DiLillo DJ, Ravetch JV. Cell. 2015;161(5):1035-45 Derer S, et al., MAbs. 2014;6(2):409-21 Reinherz EL, et al., Proc Natl Acad Sci USA. 1980;77(3):1588-92 Lin P, et al., Am J Clin Pathol. 2004;121(4):482-8 Ocio EM, et al. Expert Rev Hematol. 2014;7(1):127-41 Richardson PG, et al., Blood. 2010;116(5):679-86 Arnulf B, et al. Haematologica. 2012;97:1925-8 Kumar S, et al., Blood. 2012;119(19):4375-82 Roussel M, et al., J Clin Oncol. 2014;32:2712-7 Durie BGM, et al., Lancet. 2017;389:519-27 Touzeau C, Moreau P. Expert Opin Biol Ther. 2017;17(7):887-93 Tzogani K. et al., Oncologist. 2018;23:1-11 Martin T, et al., Blood. 2017;129(25):3294-303 J Clin Oncol 37, 2019 (Addendum; Abstract 8004) www. accessdata. fda. gov/drugsatfda_docs/label/2018/761035s008lbl. pdf

Compared to currently available treatments for CD38-expressing diseases, including antibody treatments, it is believed that the antibodies, uses and methods of treatment provided herein may provide superior clinical responses or treatments of the diseases. Provided herein are antibodies that specifically bind to human CD38 and mediate superior killing of cells expressing CD38, formulations and unit dosage forms comprising the antibodies, methods of preparing the antibodies, and methods of using the antibodies.

In one aspect, an antibody that specifically binds to human CD38 is provided. In some embodiments, the antibody provided herein comprises a light chain (LC) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, and 9, and a heavy chain (HC) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6.

In some embodiments, the antibodies provided herein comprise an LC having the amino acid sequence of SEQ ID NO:7 and an HC having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the antibodies provided herein comprise an LC having the amino acid sequence of SEQ ID NO:7 and an HC having the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibodies provided herein comprise an LC having the amino acid sequence of SEQ ID NO:7 and an HC having the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibodies provided herein comprise an LC having the amino acid sequence of SEQ ID NO:7 and an HC having the amino acid sequence of SEQ ID NO:4.

In some embodiments, the antibodies provided herein comprise an LC having the amino acid sequence of SEQ ID NO:8 and an HC having an amino acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, the antibodies provided herein comprise an LC having the amino acid sequence of SEQ ID NO:8 and an HC having the amino acid sequence of SEQ ID NO:5. In some embodiments, the antibodies provided herein comprise an LC having the amino acid sequence of SEQ ID NO:8 and an HC having the amino acid sequence of SEQ ID NO:6.

In some embodiments, the antibodies provided herein comprise an LC having the amino acid sequence of SEQ ID NO:9 and an HC having an amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7. In some embodiments, the antibodies provided herein comprise an LC having the amino acid sequence of SEQ ID NO:9 and an HC having the amino acid sequence of SEQ ID NO:6. In some embodiments, the antibodies provided herein comprise an LC having the amino acid sequence of SEQ ID NO:9 and an HC having the amino acid sequence of SEQ ID NO:7.

In another aspect, provided herein is a pharmaceutical composition comprising a formulated antibody. In some embodiments of the pharmaceutical composition, the antibody comprises an HC and an LC having the amino acid sequence described above in combination with sucrose, L-histidine, and polysorbate 80. In some embodiments, the antibody is present at a concentration of 50 mg/mL, sucrose is present at a concentration of 8% (w/v), L-histidine is present at a concentration of 10 mM, polysorbate 80 (PS80) is present at a concentration of 0.05% (v/v), and the formulation has a pH of 6.2. In some embodiments, the pharmaceutical composition is lyophilized.

In another aspect, provided herein is a unit dosage form comprising a formulated antibody. In some embodiments, the antibody comprises an HC and an LC having the amino acid sequences described above, and the unit dosage form comprises 215 mg of antibody, 6.21 mg of L-histidine, 344 mg of sucrose, and 2.15 mg of polysorbate 80. In some embodiments, the unit dosage form of the antibody is lyophilized.

In another aspect, provided herein is a process for preparing a pharmaceutical composition comprising an antibody that specifically binds to human CD38. In some embodiments, the antibody comprises a HC and a LC having the amino acid sequence described above, and the process comprises expressing the antibody in cell culture, subjecting the antibody to at least one chromatographic purification step and an ultrafiltration step to produce a purified antibody solution; and adjusting the purified antibody solution to produce an antibody formulation. In some embodiments, the antibody formulation comprises purified antibody, sucrose, L-histidine, and polysorbate 80. In some embodiments, the antibody formulation comprises antibody at a concentration of 50 mg/mL, sucrose at a concentration of 8% w/v, L-histidine at a concentration of 10 mM; and polysorbate 80 (PS80) at a concentration of 0.05% v/v. In some embodiments, the antibody formulation is prepared to have a pH of 6.2. In some embodiments of the process for preparing an antibody formulation, the antibody formulation is lyophilized.

In another aspect, a method of preparing a reconstituted antibody formulation is provided. In some embodiments, the lyophilized antibody formulation comprises sucrose, L-histidine, PS80, and an antibody that specifically binds to human CD38. In some embodiments, the antibody comprises an HC and an LC having the amino acid sequence described above. In some embodiments, the lyophilized antibody formulation is reconstituted in a diluent, thereby preparing a reconstituted antibody formulation. In some embodiments, the reconstituted antibody formulation comprises an antibody at a concentration of 50 mg/mL, sucrose at a concentration of 8% w/v, L-histidine at a concentration of 10 mM; and PS80 at a concentration of 0.05% v/v. In some embodiments, the reconstituted antibody formulation has a pH of 6.2.

In another aspect, a method of treating a patient with multiple myeloma is provided. In some embodiments, the method comprises administering to the patient one or more doses of an antibody that specifically binds to human CD38, the antibody comprising a HC and a LC having the amino acid sequence described above.

In another aspect, an antibody is provided for use in a method for treating multiple myeloma. In some embodiments, the antibody specifically binds to human CD38, and the antibody comprises a HC and a LC having the amino acid sequence described above.

Graph showing the onset of unfolding of mAbs 1, 2, 3, 5, 6, 7 and 8. 1 is a graph showing the isoelectric points (pI) of mAbs 1, 3, 5, 6, 7 and 8.

Provided herein are anti-CD38 antibodies that have excellent antibody-dependent cellular cytotoxicity (ADCC) against cells expressing high, medium and low levels of CD38 on the cell surface. In some embodiments, the anti-CD38 antibodies provided herein also have excellent antibody-dependent cellular phagocytosis (ADCP) activity against cells expressing CD38 on the cell surface. In some embodiments, the antibodies provided herein have a lower than expected isoelectric point (pI). Despite the lower than expected pI and the onset of protein unfolding at lower temperatures, which may negatively affect the stability of the antibody, especially in commercial production processes, the antibodies provided herein are provided in a suitable form that is stable for administration to human patients.
antibody

Provided herein is an anti-CD38 antibody that specifically binds to human CD38. The anti-CD38 antibody provided herein is made using recombinant methods. When producing an anti-antigen antibody recombinantly, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. The DNA encoding the antibody is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Many vectors are available. Vector components typically include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The vector is typically transformed into a host cell suitable for expression of the nucleic acid. In some embodiments, the host cell is a eukaryotic or prokaryotic cell. In some embodiments, the eukaryotic host cell is a mammalian cell. Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7, ATCC Accession No. CRL 1651); human embryonic kidney line [293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)]; baby hamster kidney cells (BHK, ATCC Accession No. CCL 10); mouse Sertoli cells [TM4, Mather, Biol. Reprod. 23:243-251 (1980)]; monkey kidney cells (CV1 ATCC Accession No. CCL 70); African green monkey kidney cells (VERO-76, ATCC Accession No. CRL-1587); human cervical carcinoma cells (HELA, ATCC Accession No. CCL 2); dog kidney cells (MDCK, ATCC Accession No. CCL 34); buffalo rat liver cells (BRL 3A, ATCC Accession No. CRL 1442); human lung cells (W138, ATCC Accession No. CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC Accession No. CCL51); TRI cells [Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)]; MRC 5 cells; FS4 cells; and the human hepatoma line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells [Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)]; and myeloma cell lines such as NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J. 2003), pp. 255-268. Anti-CD38 antibodies prepared from cells are purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being one of the generally preferred purification steps. In general, various methodologies for preparing antibodies for use in research, testing, and clinical applications are well established in the art and are consistent with the above methodologies and/or deemed appropriate by those skilled in the art.

In some embodiments, the antibody is expressed by a CHO 8D6 host cell line (DXB11 derivative) using a 500 L disposable bioreactor operated in a semi-batch culture mode. The cell culture process is initiated by thawing a vial of a master cell bank, followed by a series of seed train cell expansion steps. The cells are then transferred to the bioreactor and cultured using serum-free, chemically defined cell culture medium. The culture is stopped after 10-14 days for antibody harvest. Cells and cell culture debris are removed from the harvested cell culture by depth filtration.

The harvested material is then further processed by chromatography and filtration steps to produce purified antibody. The purified antibody is formulated into a liquid solution and stored at ≦-30°C.

Prior to dispensing the formulated antibody into suitable vials, the liquid solution is thawed and sterile filtered using a 0.2 μm filtration device. The formulated antibody is dispensed into suitable containers, such as USP Type 1 glass vials, 4.3 mL/vial. In some embodiments, the filled vials contain 0.3 mL excess of formulated antibody. In some embodiments, the formulated antibody is lyophilized within the vial.

The term "antibody" generally refers to a tetrameric protein containing two heavy chains (HC) and two light chains (LC). Each tetramer is generally composed of two pairs of identical polypeptide chains, each pair having one LC (generally having a molecular weight of about 25 kDa) and one HC (generally having a molecular weight of about 50-70 kDa). As used herein, the terms "HC" and "LC" refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain generally contains a variable domain of about 100-110 or more amino acids generally responsible for antigen recognition. The carboxy-terminal portion of each chain generally defines a constant domain responsible for effector function. Thus, in a typical antibody, a full-length HC immunoglobulin polypeptide comprises a variable domain (VH) and three constant domains (CH1, CH2 and CH3), with the VH domain at the amino terminus of the polypeptide and the CH3 domain at the carboxyl terminus, and a full-length LC immunoglobulin polypeptide comprises a variable domain (VL) and a constant domain (CL), with the VL domain at the amino terminus of the polypeptide and the CL domain at the carboxyl terminus.

The term "antibody" is used herein in the broadest sense and specifically encompasses the exemplary antibodies described above, including monoclonal antibodies and multispecific antibodies (e.g., bispecific and trispecific antibodies, so long as they exhibit the desired biological activity, including specific binding to the CD38 target and the ability to induce ADCC and ADCP).

As used herein, the term "Fc" refers to a molecule comprising a sequence of non-antigen-binding fragments resulting from the digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and may contain a hinge region. Fc molecules consist of monomeric polypeptides linked in dimeric or multimeric form by covalent (i.e., disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between the monomeric subunits of a typical Fc molecule ranges from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgG2, and IgG4). One example of an Fc is the disulfide-linked dimer resulting from papain digestion of IgG.

The antibodies described herein are isolated. The term "isolated protein," "isolated polypeptide," or "isolated antibody" refers to a protein, polypeptide, or antibody that, based on the origin or source of the derivative, (1) is not associated with naturally associated components with which it is naturally associated; (2) is substantially free of other proteins from the same species; (3) is expressed by cells from a different species; and/or (4) is not naturally occurring. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell of natural origin will be "isolated" from its naturally associated components. A protein is rendered substantially free of naturally associated components by isolation using protein purification techniques well known to those of skill in the art.

In some embodiments, the antibodies provided herein comprise the HC and LC amino acid sequences provided in Table 1.

Binding properties The term "affinity" refers to a measure of the affinity between two polypeptides, such as receptor/ligand or antigen/antibody. The intrinsic attractive effect between two polypeptides is expressed as the binding affinity equilibrium constant (KD) of a particular interaction. An antibody is said to specifically bind to an antigen when KD is ≦1 μM, preferably ≦100 nM. The KD binding affinity constant is measured, for example, by surface plasmon resonance (SPR) [BIAcore®] or biolayer interferometry.

The term "koff" refers to the dissociation rate constant of a particular antibody-antigen interaction. The koff dissociation rate constant is measured, for example, by biolayer interferometry.

Binding to CD38 In some embodiments, the antibodies provided herein comprise a tetrameric protein comprising two HCs and two LCs and bind to human CD38 with an affinity or KD of about 1.91×10 ⁻¹⁰ M to about 6.7×10 ⁻¹⁰ M as measured by surface plasmon resonance (SPR) as described below. In some embodiments, the HC and LC have the amino acid sequences of SEQ ID NOs: 2 and 7 (mAb2), 3 and 7 (mAb3), 4 and 7 (mAb4), 5 and 8 (mAb5), 5 and 9 (mAb6), 6 and 8 (mAb7), or 6 and 9 (mAb8), respectively.

In some embodiments, the antibody comprises a HC and LC having the amino acid sequences of SEQ ID NOs: 3 and 7, respectively, and the antibody binds to human CD38 with an affinity or KD of about 2×10 ⁻¹⁰ M. In some embodiments, the antibody comprises a HC and LC having the amino acid sequences of SEQ ID NOs: 5 and 8, respectively, and the antibody binds to human CD38 with an affinity or KD of about 6.7×10 ⁻¹⁰ M. In some embodiments, the antibody comprises a HC and LC having the amino acid sequences of SEQ ID NOs: 5 and 9, respectively, and the antibody binds to human CD38 with an affinity or KD of about 4.15×10 ⁻¹⁰ M. In some embodiments, the antibody comprises a HC and LC having the amino acid sequences of SEQ ID NOs: 6 and 8, respectively, and the antibody binds to human CD38 with an affinity or KD of about 3.85×10 ⁻¹⁰ M. In some embodiments, the antibody comprises a HC and LC having the amino acid sequences of SEQ ID NOs: 6 and 9, respectively, and the antibody binds to human CD38 with an affinity or KD of about 1.91×10 ⁻¹⁰ M.

Binding to FcγRIIIa (CD16a) and FcγRIIa (CD32a) The antibodies provided herein are also capable of binding to a low affinity FcγRIIIa (CD16a) variant (158F) having a phenylalanine (F) at amino acid position 158 with a KD within an order of magnitude, each KD less than 100 nM, as measured by SPR, and are capable of binding to a high affinity FcγRIIIa (CD16a) variant (158V) having a valine (V) at amino acid position 158. In some embodiments, the antibodies provided herein bind the 158F and 158V variants of FcγRIIIa (CD16a) with a KD of less than 100 nM as measured by SPR, and differ by less than 2-fold in binding to the 158F and 158V variants. In some embodiments, the antibodies provided herein bind to FcγRIIIa (CD16a) variant (158F) with a KD of about 59 nM or lower, e.g., as measured by SPR.

In some embodiments, the antibody comprises a HC having the amino acid sequence of SEQ ID NO:2 and a LC having the amino acid sequence of SEQ ID NO:7, and is capable of binding to FcγRIIIa(158F) with a KD of about 53 nM and to FcγRIIIa(158V) with a KD of about 47 nM, e.g., as measured by SPR. In some embodiments, the antibody comprises a HC having the amino acid sequence of SEQ ID NO:3 and a LC having the amino acid sequence of SEQ ID NO:7, and is capable of binding to FcγRIIIa(158F) with a KD of about 59 nM and to FcγRIIIa(158V) with a KD of about 75 nM, e.g., as measured by SPR. In some embodiments, the antibody comprises a HC having the amino acid sequence of SEQ ID NO:4 and a LC having the amino acid sequence of SEQ ID NO:7, and is capable of binding to FcγRIIIa(158F) with a KD of about 51 nM and to FcγRIIIa(158V) with a KD of about 47 nM, e.g., as measured by SPR.

The antibodies provided herein are also capable of binding to a low affinity FcγRIIa (CD32a) variant (131R) having an arginine at amino acid position 131 with a KD of ≦690 nM as measured by SPR, and to a high affinity FcγRIIa (CD32a) variant (131H) having a histidine at amino acid position 131 with a KD of less than about 270 nM.

In some embodiments, the antibody comprises a HC having the amino acid sequence of SEQ ID NO:2 and a LC having the amino acid sequence of SEQ ID NO:7, and is capable of binding to FcγRIIa (CD32a) (131R) with a KD of about 690 nM or to FcγRIIa (CD32a) (131H) with a KD of about 120 nM, e.g., as measured by SPR. In some embodiments, the antibody comprises a HC having the amino acid sequence of SEQ ID NO:3 and a LC having the amino acid sequence of SEQ ID NO:7, and is capable of binding to FcγRIIa (CD32a) (131R) with a KD of about 125 nM or ≦100 nM, e.g., as measured by SPR, and is capable of binding to FcγRIIa (CD32a) (131H) with a KD of about 220 nM. In some embodiments, the antibody comprises a HC having the amino acid sequence of SEQ ID NO:4 and a LC having the amino acid sequence of SEQ ID NO:7, and is capable of binding to FcγRIIa (CD32a) (131R) with a KD of about 510 nM and FcγRIIa (CD32a) (131H) with a KD of about 60 nM, e.g., as measured by SPR.

In some embodiments, the antibody is also capable of binding to FcγRIIIa (CD16a) 158F with an apparent KD of about 70 nM or less, and to FcγRIIIa (CD16a) 158V with an apparent KD of about 32 nM or less, as measured by binding to HEK cells expressing FcγRIIIa (CD16a) 158F or 158V, respectively. In some embodiments, an antibody comprising a HC having the amino acid sequence of SEQ ID NO:2 and a LC having the amino acid sequence of SEQ ID NO:7 is also capable of binding to FcγRIIIa (CD16a) 158F with an apparent KD of about 70 nM, and to FcγRIIIa (CD16a) 158V with an apparent KD of about 18 nM, as measured by binding to HEK cells expressing FcγRIIIa (CD16a) 158F or 158V, respectively. In some embodiments, an antibody comprising a HC having the amino acid sequence of SEQ ID NO:3 and a LC having the amino acid sequence of SEQ ID NO:7 is capable of binding to FcγRIIIa (CD16a) 158F with an apparent KD of about 60 nM and to FcγRIIIa (CD16a) 158V with an apparent KD of about 32 nM, as measured by binding to HEK cells expressing FcγRIIIa (CD16a) 158F or 158V, respectively. In some embodiments, an antibody comprising a HC having the amino acid sequence of SEQ ID NO:4 and a LC having the amino acid sequence of SEQ ID NO:7 is capable of binding to FcγRIIIa (CD16a) 158F with an apparent KD of about 44 nM and to FcγRIIIa (CD16a) 158V with an apparent KD of about 28 nM, as measured by binding to HEK cells expressing FcγRIIIa (CD16a) 158F or 158V, respectively.

In some embodiments, the antibody is also capable of binding to an FcγRIIa (CD32a) variant having an arginine at amino acid position 131 (131R) with an apparent KD of about 890 nM or lower, and to an FcγRIIa (CD32a) variant 131H with an apparent KD of about 840 nM or lower, as measured by binding to HEK cells expressing FcγRIIa (CD32a) 131R or 131H, respectively. In some embodiments, an antibody comprising a HC having the amino acid sequence of SEQ ID NO:2 and a LC having the amino acid sequence of SEQ ID NO:7 is also capable of binding to FcγRIIa (CD32a) 131R with an apparent KD of about 890 nM, and to FcγRIIa (CD32a) 131H with an apparent KD of about 840 nM, as measured by binding to HEK cells expressing FcγRIIa (CD32a) 131R or 131H, respectively. In some embodiments, an antibody comprising a HC having the amino acid sequence of SEQ ID NO:3 and a LC having the amino acid sequence of SEQ ID NO:7 is capable of binding to FcγRIIa (CD32a) 131R with an apparent KD of about 87 nM and FcγRIIa (CD32a) 131H with an apparent KD of about 222 nM, as measured by binding to HEK cells expressing FcγRIIa (CD32a) 131R or 131H, respectively. In some embodiments, an antibody comprising a HC having the amino acid sequence of SEQ ID NO:4 and a LC having the amino acid sequence of SEQ ID NO:7 is capable of binding to FcγRIIa (CD32a) 131R with an apparent KD of about 467 nM and FcγRIIa (CD32a) 131H with an apparent KD of about 544 nM, as measured by binding to HEK cells expressing FcγRIIa (CD32a) 131R or 131H, respectively.

Mechanism of Action The antibodies provided herein are capable of killing CD38-expressing cells by antibody-dependent cellular cytotoxicity (ADCC) of cells expressing high (approximately 400,000/cell), medium (approximately 100,000/cell) and low (approximately 13,000/cell) levels of CD38 molecules on the cell surface. The antibodies provided herein induce such ADCC in the presence of natural killer (NK) cells expressing the low affinity FcγIIIa (CD16a) receptor variant (158F) and/or the high affinity FcγIIIa (CD16a) receptor variant (158V). ADCC is measured using methods known to those of skill in the art, for example, by measuring target cell lysis in the presence of the antibody and effector cells in a cell-based potency assay. The target cells can be, for example, CD38-expressing cells such as KMS12-BM, RPMI-8226 or MOLP-8. In addition, effector cells can be any cell that can be induced to kill target cells, for example, by ADCC. In some embodiments, effector cells are primary cells isolated from human blood, such as peripheral blood mononuclear cells (PBMCs), or human NK cells purified from PBMCs. In another embodiment, the cells are a natural killer cell line, such as NK-92 cells, which express FcγRIIIa(158V) or FcγRIIIa(158F) on the cell surface (see WO 06/023148). In yet other embodiments, the NK cell line is a cell line, such as a Jurkat cell line, engineered to express FcγIIIa (CD16a) 158F or 158V (see, e.g., Promega, G701A).

The antibodies provided herein are capable of killing cells expressing high levels of CD38 on the cell surface by antibody-dependent cellular phagocytosis (ADCP). The antibodies provided herein induce such ADCP in the presence of human PBMCs. In one embodiment, the antibodies provided herein induce phagocytosis of about 41% of CD38-expressing cells by human PBMCs expressing FcγRIIIa(158V) with a relative EC50 of 212.6 pM.

Pharmaceutical Compositions and Formulated Antibodies The term "pharmaceutical formulation" refers to a preparation that is in such a form as to effect the biological activity of the active ingredient and does not contain any additional ingredients that are unacceptably toxic to the subject to whom the formulation is to be administered. Such formulations are generally sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that are reasonably administered to a target mammal to obtain an effective dose of the active ingredient utilized.

The pharmaceutical compositions and formulations of the antibodies provided herein are prepared by mixing the antibody having the desired purity with one or more optional pharma- ceutical acceptable carriers [Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)] and are provided in the form of a lyophilized formulation or an aqueous solution.

In some embodiments, the pharmaceutical compositions provided herein include an antibody formulated with the antibody and one or more of the following excipients: sucrose, L-histidine, and polysorbate 80 (PS80). In some embodiments, the antibody comprises the HC and LC amino acid sequence of any one of the monoclonal antibodies provided in Table 1. In some embodiments, the antibody is present in the pharmaceutical composition at a concentration of 5 mg/mL to 50 mg/mL. In some embodiments, the antibody is present in the pharmaceutical composition at a concentration of 5 mg/mL. In some embodiments, the antibody is present in the pharmaceutical composition at a concentration of 10 mg/mL. In some embodiments, the antibody is present in the pharmaceutical composition at a concentration of 20 mg/mL. In some embodiments, the antibody is present in the pharmaceutical composition at a concentration of 50 mg/mL. The concentration of each excipient is selected such that the pharmaceutical composition is diluted for administration by injection. For example, in some embodiments, sucrose is present at a concentration of 8% (weight/volume) to 10% (weight/volume). In some embodiments, sucrose is present at a concentration of 8% (weight/volume). In other embodiments, sucrose is present at a concentration of 10% (w/v). In some embodiments, L-histidine is present at a concentration of 10 mM to 20 mM. In some embodiments, L-histidine is present at a concentration of 10 mM. In some embodiments, L-histidine is present at a concentration of 20 mM. In some embodiments, PS80 is present at a concentration of 0.005% (v/v) to 0.05% (v/v). In some embodiments, PS80 is present at a concentration of 0.005% (v/v). In some embodiments, PS80 is present at a concentration of 0.02% (v/v). In some embodiments, PS80 is present at a concentration of 0.05% (v/v). In some embodiments, the pH of the pharmaceutical composition is optimized to maintain the stability of the antibody, for example when the pharmaceutical composition is in liquid form. In some embodiments, the pH of the formulated antibody has a pH of about 6.0 to about 6.5. In some embodiments, the formulated antibody has a pH of about 6.0. In some embodiments, the formulated antibody has a pH of about 6.2. In some embodiments, the formulated antibody has a pH of about 6.5. In some embodiments, the formulated antibody has a pH of 6.0. In some embodiments, the formulated antibody has a pH of 6.2. In some embodiments, the formulated antibody has a pH of 6.5.

In some embodiments, the antibodies of the pharmaceutical compositions and/or formulated antibodies provided herein comprise one or more charged isoforms. Charged isoforms are generally described as the main isoform, the acidic isoform, and the basic isoform. The main isoform refers to the most prevalent isoform within a given batch of antibody. The acidic isoform has a lower isoelectric point (pI) compared to the main isoform, and the basic isoform has a higher pI compared to the main isoform. Charged isoforms result from post-translational modifications to the amino acid sequences of the HC and/or LC. For example, increased deamidation, glycosylation, and sialylation can cause the pI of the antibody to decrease. The conversion of the N-terminal glutamic acid to pyroGlu (or pyroQ) results in the loss of one positive charge, which can cause the pI to decrease. In addition, the presence of a C-terminal lysine can cause the pI of the antibody to increase. Furthermore, amidation of the C-terminal proline can cause the pI of the antibody to increase.

In some embodiments of the antibodies provided herein (including antibodies in pharmaceutical compositions and formulated antibodies), the amino acid at position 1 of the HC amino acid sequence is pyroglutamine. In some embodiments of the antibodies provided herein, the HC has a C-terminal amino acid consisting of glycine.

The charged isoforms of the antibodies provided herein are separated and quantified using methods known to those skilled in the art for separating polypeptides based on charge. For example, in some embodiments, a weak cation exchange column is used in a high performance liquid chromatography (HPLC) system with a phosphate/sodium chloride gradient buffer. In this system, after loading the antibody onto the column, the acidic isoform of the antibody will elute first, followed by the main isoform, and then the basic isoform of the antibody. In another embodiment, capillary isoelectric focusing (cIEF) is used. Capillary isoelectric focusing is a method of separating proteins by their isoelectric point (pI) value. In cIEF, the antibody sample migrates to its isoelectric point on a pH gradient in a capillary, thereby separating the different charge isoforms along the length of the capillary. To identify and characterize the cIEF variants, the charge isoforms are isolated by strong cation exchange chromatography (SCX) and analyzed by cIEF. Images of the separated charged isoforms in the capillary are captured using a charge-coupled device camera that uses whole-column detection and measures absorbance at 280 nm. The charge isoform distribution of the test sample is compared to a reference standard using cIEF to identify the charge isoforms of the antibody. In some embodiments, cIEF is used as a quality control measure to confirm the identity of an antibody during commercial production by determining the charge isoform distribution of the antibody compared to a reference standard, e.g., a test antibody having a charge isoform distribution pattern within a predefined distribution pattern, or compared to the distribution pattern of the reference standard.

In some embodiments, the antibody (including antibodies in pharmaceutical compositions and formulated antibodies) comprises the HC and LC amino acid sequence of any one of the monoclonal antibodies provided in Table 1 and one or more charged isoform parameters selected from the group consisting of at least about 70% major isoform, no more than about 30% acidic isoform, and less than 4% basic isoform. In some embodiments, the antibody comprises 71% major isoform, 28% acidic isoform, and less than 4% basic isoform. In some embodiments, the antibody contains charge isoforms with isoelectric points ranging from about 5.8 to about 9.0. In some embodiments, the antibody contains charge isoforms with isoelectric points ranging from about 5.85 to about 8.97.

In some embodiments, the antibody (including the antibody in a pharmaceutical composition and the formulated antibody) is lyophilized. The antibody (including the antibody in a pharmaceutical composition and the formulated antibody) can be in a container (e.g., a vial) such that a defined dose of the antibody can be removed from the container and administered to a patient.

Provided herein are unit dosage forms of formulated antibodies that specifically bind to CD38. In some embodiments, the antibody comprises the HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1, and the unit dosage form comprises the antibody and one or more excipients selected from the group of sucrose, L-histidine, and polysorbate 80. In some embodiments, the unit dosage form comprises about 215 mg of antibody, about 6.21 mg of L-histidine, about 344 mg of sucrose, and about 2.15 mg of polysorbate 80. In some embodiments, the unit dosage form comprises 215 mg of antibody, 6.21 mg of L-histidine, 344 mg of sucrose, and 2.15 mg of polysorbate 80. In some embodiments, the unit dosage form is lyophilized. The unit dosage form can be in a container (e.g., a vial) such that a defined dosage of the antibody can be removed from the container and administered to a patient. In some embodiments, the antibody, pharmaceutical formulation and/or formulated antibody is in a glass vial fitted with an elastomeric seal stopper. In some embodiments, the vial contains about 215 mg of antibody. In some embodiments, the vial contains 215 mg of antibody. In some embodiments, the fill volume of the vial has been determined to allow for approximately 4 mL to be dispensed. In some embodiments, the fill volume of the vial has been determined to allow for 4 mL to be dispensed.
Preparation process

Provided herein are processes for preparing antibodies, pharmaceutical compositions, and unit dosage forms. In some embodiments, the antibody comprises the HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1. In one embodiment, the process includes expressing the antibody in a suitable cell culture. The antibody is subjected to at least one chromatography step and at least one ultrafiltration step, thereby producing a purified antibody solution. The purified antibody solution is adjusted to concentrate the antibody, add excipients, and adjust the pH. In one embodiment, the antibody concentration is adjusted to about 50 mg/mL; sucrose, L-histidine, and polysorbate 80 are added, and the pH is adjusted to at least about 6.0. In one embodiment, the antibody concentration is adjusted to 50 mg/mL; sucrose, L-histidine, and polysorbate 80 are added, and the pH is adjusted to at least 6.0. In some embodiments, the pH is about 6.2. In some embodiments, the pH is 6.2. In some embodiments, the sucrose has a concentration of about 8% w/v, the L-histidine has a concentration of about 10 mM, and the polysorbate 80 has a concentration of about 0.05% v/v. In some embodiments, the sucrose has a concentration of 8% w/v, the L-histidine has a concentration of 10 mM, and the polysorbate 80 has a concentration of 0.05% v/v. In some embodiments, the antibody, pharmaceutical composition and/or formulated antibody is lyophilized.

In some embodiments, a method for preparing a reconstituted and formulated antibody is provided, the formulated antibody being provided in lyophilized form and comprising the HC and LC amino acid sequence of any one of the monoclonal antibodies provided in Table 1, sucrose at a concentration of about 8% w/v, L-histidine at a concentration of about 10 mM, and polysorbate 80 at a concentration of about 0.05% v/v. In some embodiments, a method for preparing a reconstituted and formulated antibody is provided, the formulated antibody being provided in lyophilized form and comprising the HC and LC amino acid sequence of any one of the monoclonal antibodies provided in Table 1, sucrose at a concentration of 8% w/v, L-histidine at a concentration of 10 mM, and polysorbate 80 at a concentration of 0.05% v/v. In some embodiments, prior to use, the formulated antibody is reconstituted in a suitable volume of water for injection to produce a solution containing about 50 mg/mL antibody, about 8% sucrose, about 10 mM L-histidine, and about 0.05% polysorbate 80, and the pH of the reconstituted antibody is about 6.2. In some embodiments, prior to use, the formulated antibody is reconstituted in a suitable volume of water for injection to produce a solution containing 50 mg/mL antibody, 8% sucrose, about 10 mM L-histidine, and about 0.05% polysorbate 80, and the pH of the reconstituted antibody is about 6.2.

As used herein, the term "treatment" or "treating" refers to a clinical intervention designed to alter the natural course of the individual or cells being treated during clinical pathology. Desired effects of treatment include slowing the rate of disease progression, ameliorating or alleviating the disease state, and achieving remission or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are alleviated or eliminated, including, but not limited to, reducing the proliferation of cancerous cells, destroying cancerous cells, reducing symptoms caused by the disease, increasing the quality of life of an individual suffering from the disease, reducing the dose of other pharmaceuticals required to treat the disease, and/or prolonging the survival of the individual.

Provided herein is a method of treating or delaying the progression of multiple myeloma (such as relapsed multiple myeloma or relapsed and refractory multiple myeloma) in an individual, comprising administering to a subject in need thereof an effective amount of an anti-CD38 antibody provided herein. In some embodiments, the antibody comprises the HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1. In some embodiments, the antibody is capable of improving survival of mice in a mouse MM model, where the mice in the model express a low affinity variant of human CD16a (158F), and the mice are injected with 500,000 EL4 cells expressing human CD38. In some embodiments, one or more doses of an anti-CD38 antibody are provided as a lyophilized formulation, and the lyophilized formulation is reconstituted prior to administration to form a reconstituted antibody formulation, such that the reconstituted antibody formulation has a pH of 6.2 and comprises an antibody at a concentration of about 50 mg/mL in a liquid comprising about 10 mM L-histidine, about 8% wt/vol sucrose, and about 0.05% v/vol polysorbate 80. In some embodiments, one or more doses of an anti-CD38 antibody are provided as a lyophilized formulation, and the lyophilized formulation is reconstituted prior to administration to form a reconstituted antibody formulation, such that the reconstituted antibody formulation has a pH of 6.2 and comprises an antibody at a concentration of 50 mg/mL in a liquid comprising about 10 mM L-histidine, 8% wt/vol sucrose, and 0.05% v/vol polysorbate 80.

In some embodiments, one or more doses of antibody are administered to the patient. In some embodiments, the dose can be about 0.1, about 0.2, about 0.3, about 0.5, about 1.0, about 2.5, about 5, about 10, or about 20 mg/kg of antibody. In some embodiments, the dose can be 0.1, 0.2, 0.3, 0.5, 1.0, 2.5, 5, 10, and 20 mg/kg of antibody. In some embodiments, the antibody is administered intravenously. In some embodiments, the antibody is administered subcutaneously.

DEFINITIONS As used in this specification and the appended claims, the singular forms "a,""an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "molecule" optionally includes combinations of two or more such molecules, and so forth.

As used herein, the term "about" refers to the normal error range for the respective value, which is readily known to one of ordinary skill in the art. Reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. In some embodiments, the term "about" refers to the stated value of said value plus or minus 10%; for example, "about 10 mg/kg" can encompass 9-11 mg/kg. In some embodiments, the term "about" refers to the stated value of said value plus or minus 5%; for example, "about 20 mg/kg" can encompass 19-21 mg/kg.

For purposes of treatment, a "subject" or "individual" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

Examples are set forth below for illustrative purposes and to describe some particular embodiments of the present invention. However, the scope of the claims should in no way be limited by the examples set forth herein.

Example 1. Antibody design:
To enhance affinity for the activating FcγRIIIa and FcγRIIa receptors on effector cells, Fc-engineered anti-CD38 antibodies were generated that had mutations in the Fc portion of the antibody.

The two humanized heavy chains (HC) (SEQ ID NO:5 and SEQ ID NO:6) carried 14 and 10 mutations in the framework regions (compared to SEQ ID NO:1) that resulted in an increase in germinality score from 74.49% to 88.78% and 84.69%, respectively, in Homo sapiens. The two humanized light chains (LC) (SEQ ID NO:8 and SEQ ID NO:9) carried 17 and 15 mutations in the framework regions (compared to SEQ ID NO:7) that resulted in an increase in germinality score from 64.36% to 81.19% and 79.21%, respectively, in Homo sapiens. In addition, in LC2 and 3, the methionine at position 11 was replaced with leucine to avoid potentially problematic methionine oxidation. The HC and LC sequences are provided in Table 2, and the mAb HC and LC pairings are provided in Table 3.

2. Biochemical characterization:
A: Binding to CD38 In one experiment, the binding affinity and kinetic constants of mAbs 1-8 to human CD38 were measured by SPR using a Biacore® instrument. Briefly, anti-CD38 antibodies were captured on a CM5 chip to which anti-Fc antibodies were covalently attached. Multiple concentrations of huCD38 were injected onto the captured antibodies to be tested. Binding curves (sensorgrams) were generated and kinetic analysis was performed using Biacore Evaluation software.

As shown in Table 4, mAbs 3 and 8 had the highest affinity for human CD38 (t-test for each variant: p-value > 2).

In a separate experiment, the binding affinity of mAbs 1 and 3 to human CD38 was measured by SPR using a Biacore® instrument as described above. mAb1 had a Kd of 0.22 nM and mAb3 had a Kd of 0.20 nM.

Effect of heat stress on binding affinity to human CD38: The binding affinity and kinetic constants of mAbs 1-8 to human CD38 were measured by SPR using a Biacore® instrument after the mAbs were subjected to heat stress (14 days at 40°C in 10 mM histidine pH 6, 8% sucrose, 0.02% PS80). The same protocol as described in the paragraph above was used.

As shown in Table 5, the heat-stressed samples showed very similar affinity and kinetic constants to the corresponding unstressed CD38 samples (Table 4) (t-test for each variant: p-value > 2).

B: In silico measurement of affinity for FcγRIIa, FcγRIIIa and FcγRIIb In one experiment, the affinity of mAbs for the FcγRIIIa 158V or FcγRIIIa 158F receptors was measured using surface plasmon resonance (SPR). The mAbs were produced in HEK293 cells by transient transfection according to the supplier (ThermoFisher Scientific) and purified by protein A affinity chromatography according to the supplier (GE Healthcare). FcγRIIIa (CD16a) extracellular domain containing V158 or F158 was produced with a C-terminal His tag in HEK293 cells by transient expression and purified by immobilized metal affinity chromatography (IMac) according to the supplier (Ni-Sepharose, GE Healthcare). Affinity by SPR on a BIACore 2000 (GE Healthcare) was performed by a capture assay using anti-His IgG immobilized on a CM5 chip. FcγRIIIa-His tag was then added to the running buffer followed by the addition of anti-CD38 mAb at concentrations ranging from 8 to 256 nM. Analyses were performed using BIAevaluation Software4_1 (GE Healthcare) using a two-state reaction with an acceptable ^χ2 value of less than 5 (meaning 2% of the highest concentration tested) and a percentage of maximum theoretical resonance units greater than 25 (meaning a productive interaction greater than ¼).

As shown in Table 6, mAbs 2-4 had 9-fold higher affinity for the FcγRIIIa 158V receptor than mAb 1 and 27-fold higher affinity for the FcγRIIIa 158F receptor than mAb 1.

In a separate experiment, the affinity of mAbs 1 and 3 for the FcγRIIIa 158V or FcγRIIIa 158F receptors was measured using SPR as described above. mAb 3 bound to FcγRIIIa 158V with a KD of 75 nM, and mAb 3 bound to FcγRIIIa 158F with a KD of 59 nM.

The affinity of the mAbs for the FcγRIIa receptor with arginine at amino acid position 131 (FcγRIIa 131R) or histidine at amino acid position 131 (FcγRIIa 131H) was measured using an AlphaScreen proximity assay.

FcγRIIa extracellular domain containing R131 or H131 was provided by R&D System (batch 1330-CD) or Biorbyt (batch ORB138408), respectively. The assay was a bead-based proximity assay described by the supplier (Perkin Elmer) that allowed ranking of anti-CD38 Fc variants by competition against anti-CD38 with native IgG1. Biotinylated anti-CD38 with native IgG1 was captured on donor beads and FcγRIIa His-tagged protein was captured on acceptor beads. Upon interaction of anti-CD38 and FcγRIIa, the beads are brought into close proximity. Excitation of the donor beads at 680 nm causes the release of singlet oxygen, which diffuses and triggers the emission of light at 520-620 nm from the acceptor beads when in close proximity. The amount of light is directly proportional to the degree of interaction, and the amount of light was measured with an EnVision multimode plate reader (Perkin Elmer). During the competition assay, untagged competitor mAb, anti-CD38 Fc-variants DE (S239D/I332E, Eu numbering), ADE (G236A/S239D/I332E, Eu numbering), or ADLE (G236A/S239D/F243L/I332E, Eu numbering) were used to compete the interaction between biotinylated anti-CD38 and native IgG1 and FcγRIIa-His tagged proteins. The concentration of biotinylated anti-CD38 was set from a previously performed calibration curve. Different levels of untagged competitor mAb were used so that increasing concentrations of untagged mAb could displace biotinylated anti-CD38 and reduce the signal. Data was normalized to the maximum signal. Two experiments were performed and IC50 was evaluated by BIOST@T-SPEED-LTS 2.1.0.

As shown in Table 7, mAb3 showed 14-fold and 18-fold higher affinity for FcγRIIa 131R and FcγRIIa 131H, respectively, compared to mAb1.

C: Measurement of affinity to FcγRIIa, FcγRIIIa and FcγRIIb in vitro. HEK293T-FcγRIIIa-158F, HEK293T-FcγRIIIa-158V, HEK293T-FcγRIIa-131H, HEK293T-FcγRIIa-131R, HEK293T-FcγRI or HEK293T-FcγRIIb were seeded at ¹⁰⁵ cells/well in 96-well microplates. Plates were centrifuged at 800g for 1 min and resuspended in 50 μL PBS containing antibodies at different concentrations for 30 min at 4°C. 200 μL PBS 1% FBS was then added to wash the cells, followed by centrifugation at 800g for 1 min. The washing steps were repeated a total of three times before staining the cells with a secondary antibody conjugated with FITC [goat anti-human IgG (H+L) FITC fragment, ref. 109-546-088 Jackson ImmunoResearch, final concentration 10 μg/mL] for 30 min at 4° C. The cells were washed three times with 200 μL PBS 1% FBS and resuspended in 100 μL PBS before acquisition with MACSVYB (B1 flow path). As shown in Table 8, mAbs 2, 3 and 4 had 32-, 38- and 51-fold higher affinity, respectively, for FcγRIIIa 158F expressed in HEK cells compared to mAb1, and mAbs 2, 3 and 4 had 9-, 5- and 6-fold higher affinity, respectively, for FcγRIIIa 158V expressed in HEK cells compared to mAb1.

As shown in Table 9, mAb3 had approximately 5-fold higher affinity for FcγRIIa 131H expressed in HEK cells compared to mAb1, and mAb3 had approximately 16-fold higher affinity for FcγRIIa 131R expressed in HEK cells compared to mAb1.

As shown in Table 10, mAbs 2, 3, and 4 had similar levels of binding affinity to FcγRI compared to mAb1, and 1.8-3.9-fold lower affinity to FcγRIIb expressed in HEK cells compared to mAb1.

D: Measurement of in vitro cytotoxic activity Antibody-dependent cellular cytotoxicity (ADCC):
Antibody-dependent cellular cytotoxicity (ADCC) assays using the NK92 cell line engineered to overexpress the FcγRIIIa receptor as effector cells were investigated using a calcein-acetoxymethyl (calcein-AM; Invitrogen) release assay. In live cells, non-fluorescent calcein AM is converted to green fluorescent calcein. Labeled target cells were incubated with SAR442085 antibody and NK92 effector cells. Lysis of target cells by ADCC resulted in the release of fluorescent calcein. Fluorescence intensity was proportional to the level of ADCC activity present.

Multiple myeloma target cells (MOLP-8, RPMI 8226, MM1R or KMS12-BM) were labeled with calcein AM (50 μg diluted in 25 μL DMSO, 10 μL of calcein was then diluted in 4 mL RPMI 1640 + 1% FBS + 1% probenecid to stain 4×10 ⁶ cells) for 30 minutes, then washed and plated at a density of 2×10 ⁴ cells/well in 96-well round bottom plates. Cells were opsonized with the indicated test mAb or control isotype mAb at various concentrations ranging from 10 μg/mL to 0.01 pg/mL for 30 minutes prior to the addition of natural killer (NK) cells transduced with the indicated FcγRIIIa variants. NK cells expressing 158F or 158V were added as effector cells at an E:T ratio of 5:1 ( ^1x105 NK cells to ^2x104 target cells). Plates were then incubated for 1 hour at 37°C in a humidified incubator with 5% _CO2 and 100 μL of supernatant was harvested and transferred to an opaque 96-well microplate and analyzed using fluorimetry on a Tecan Infinite M1000 (excitation filter: 492 nm; emission filter: 515 nm), thereby measuring calcein release. To maximize release, cells were lysed with 2% Triton X-100. Culture medium background fluorescence values were subtracted from experimental release (A), spontaneous target cell release (B) and maximum target cell release (C). Cytotoxicity and ADCC percentages for each plate (replicate) were calculated using the following formula:
Cytotoxicity (%) = (AB)/(CB) x 100%

Each experiment was repeated at least three times. Half-maximal effective concentration (EC50) values were calculated by fitting data points to a four-parameter equation using GraphPad Prism 5 (GraphPad Software, Inc. San Diego, CA).

Target Cells with High CD38 Receptor Density ADCC of the multiple myeloma cell line MOLP-8 was evaluated in vitro as described above. MOLP-8 cells display a high density of CD38 receptors on the cell surface (approximately 400,000/cell).

Table 11 shows the EC50 of each antibody tested against MOLP-8 cells in the presence of NK cells expressing 158V. As shown in Table 11, in the presence of NK cells expressing the high affinity variant of FcγRIIIa (158V), mAbs 2, 3, and 4 induced ADCC of MOLP-8 cells with EC50s 24-33-fold lower than that of mAb 1.

Table 12 shows the EC50 of each antibody tested against MOLP-8 cells in the presence of NK cells expressing 158F. As shown in Table 12, mAb4 showed the strongest ADCC activity against MOLP-8 cells in the presence of NK cells expressing the low affinity FcγRIIIa variant (158F), with an EC50 approximately 97-fold lower than that of mAb1. mAbs 2 and 3 induced ADCC of MOLP-8 cells in the presence of NK cells expressing the low affinity FcγRIIIa variant (158F), with an EC50 approximately 59-69-fold lower than that of mAb1.

Intermediate CD38 Receptor Density Target Cells ADCC of the multiple myeloma cell line RPMI-8226 was assessed in vitro as described above. RPMI-8226 cells display an intermediate density of CD38 receptors on the cell surface (approximately 70,000/cell).

Table 13 shows the EC50 of each antibody tested against RPMI-8226 cells in the presence of NK cells expressing 158V. As shown in Table 13, mAbs 2-4 demonstrated a similar level of enhanced ability with an EC50 approximately 27-fold lower than mAb 1 to induce ADCC against MM cells expressing intermediate CD38 receptor density in the presence of NK cells expressing the high affinity variant of FcγRIIIa (158V).

Table 14 shows the EC50 of each antibody tested against RPMI-8226 cells in the presence of NK cells expressing 158F. mAb4 demonstrated approximately 73-fold enhanced ability compared to mAb1 to induce ADCC against MM cells expressing intermediate CD38 receptor density in the presence of NK cells expressing a low affinity variant of FcγRIIIa, and mAb3 demonstrated approximately 65-fold and mAb2 approximately 49-fold enhanced ability compared to mAb1 to induce ADCC against MM cells expressing intermediate CD38 receptor density in the presence of NK cells expressing a low affinity variant of FcγRIIIa.

ADCC of target cells with low CD38 receptor density, the multiple myeloma cell line KMS-12BM, was assessed in vitro as described above. KMS-12BM cells exhibit a low density of CD38 receptors on the cell surface (approximately 13,000/cell).

Table 15 shows the EC50 of each antibody tested against KMS-12BM cells in the presence of NK cells expressing the high affinity variant of FcγRIIIa (158V). Interestingly, as shown in Table 15, mAbs 2, 3, and 4 demonstrated the ability to induce enhanced ADCC against MM cells expressing low CD38 receptor density in the presence of NK cells expressing the high affinity variant of FcγRIIIa (158V) compared to mAb 1. mAb 3 had an EC50 approximately 69-fold lower than mAb 1, and mAb 4 had an EC50 approximately 91-fold lower than mAb 1.

Table 16 shows the EC50 of each antibody tested against KMS-12BM cells in the presence of NK cells expressing a low affinity variant of FcγRIIIa (158F). Unexpectedly, as shown in Table 16, mAbs 2, 3 and 4 demonstrated an ability to induce enhanced ADCC against MM cells expressing low CD38 receptor density in the presence of NK cells expressing a low affinity variant of FcγRIIIa (158F) compared to mAb 1. mAb 3 had an EC50 approximately 145-fold lower than mAb 1, and mAb 4 had an EC50 approximately 269-fold lower than mAb 1.

Antibody-dependent cellular phagocytosis (ADCP):
Human PBMCs were initially isolated from the buffy coat of seven healthy donors from the Etables Français du Sang, all of whom had the same FCGR3A and FCGR2A genotypes (FcγRIIIa-158V/V and FcγRIIa-131H/H). Blood was first collected in a 50 mL Falcon tube. 15 mL of 96% Ficoll-Plaque™ Plus was added very gently to the bottom of the Sepmate tube, and then the blood was centrifuged at 1300 g for 10 minutes. was slowly added to the tube. The PBMC ring was then collected, washed with 50 mL of PBS, and centrifuged once more at 300 g for 5 min. The washing process was repeated twice before PBMC staining with anti-CD14 magnetic beads (Miltenyi; ref. 130-050-201) according to the manufacturer's instructions: essentially before positive selection with AutoMACS pro (Posseld selection). Incubation time 15 min at 4° C. Monocytes were harvested and subsequently washed with 50 mL of PBS. Monocytes were cultured in T75 flasks in RPMI 1640, 10% FBS, 2% human heat inactivated serum, 50 ng/mL GM-CSF. The cells were cultured at 37° C. and 5% CO ₂ for 5 days. After 5 days of culture, the monocytes were differentiated into macrophages. To harvest macrophages, Accutase (ThermoFisher, ref: A1110501) was added to the flask for 15 min at 37°C to detach the cells, and then 20 mL of RPMI, 10% FBS, 1% glutamine was added to increase the Accutase activity. The collected cells were centrifuged at 350 g for 10 min, then washed in 20 mL of PBS and centrifuged once more at 300 g for 10 min. For the two experiments, the macrophages used were: It was from a batch of macrophages frozen in liquid nitrogen.

Macrophages were resuspended in Diluent C (provided in the Sigma Aldrich manufacturer's staining kit #PKH26GL-1KT) at ^20x106 cells/mL. For one volume of macrophages, one volume of PKH26 (20 μL diluted in 1 mL of Diluent C) was added to the cells and incubated in the dark for 5 minutes. 5 mL of FBS was then added to inactivate the staining process for 1 minute before completing to 50 mL with RPMI 1640, 10% FBS. CD38 expressing MOLP-8 cells were stained with PKH67 fluorescent dye and macrophages obtained from purified monocytes were stained with PKH26, making them identifiable by flow cytometry. MOLP-8 cells and macrophages were then placed into culture overnight in the presence of either mAb1 or mAb3 before analyzing the double positive population (macrophages that had phagocytosed MOLP-8 cells) by flow cytometry.

mAb3 exhibited enhanced ADCP activity against the MOLP-8 cell line compared to mAb1, as demonstrated by the higher percentage (%) of total phagocytosis and lower EC50 (relative) values (EC50rel) achieved in all experiments. The geometric mean of the % of total phagocytosis was 40.85% for mAb3 versus 18.57% for mAb1, and the geometric mean of the EC50rel was 31.87 ng/mL (or 212.57 pM) for mAb3 versus mAb1, whose values were greater than 64.86 ng/mL (or greater than 432.62 pM). Based on the EC50rel values, mAb3 appeared to be at least 2.035-fold superior to mAb1 for ADCP activity.

In vivo efficacy The in vivo efficacy of mAb3 was evaluated. Humanized FcγR C57BL/6 mice (Smith P, DiLillo DJ, Bournazos S, Li F, Ravetch JV; Proc Natl Acad Sci USA. 2012;109(16):6181-6) were injected intravenously with 500,000 EL4-huCD38 tumor cells on day 0. Humanized FcγR C57BL/6 mice have had the entire mouse gene encoding FcγR deleted and a human FcγR encoded as a transgene inserted into the mouse genome. Humanized FcγR C57BL/6 mice recapitulated huFcγR expression patterns and levels and were functional in various huIgG-mediated models of inflammation, cytotoxicity, and tumor clearance. For activating FcγRIIIa and FcγRIIa receptors, the human variants inserted into the mouse model were huFcγRIIIa 158F and huFcγRIIa 131R (low affinity variants of the receptor), respectively. The human FcγR expression pattern and expression level profile was recapitulated in the mouse.

Beginning on day 1 after tumor cell injection, test or control mAbs were administered intraperitoneally on days 1, 4, 7, and 14. Isotype control mAb was administered at 10 mg/kg. mAbs 1, 3, and 10 were administered at 10 and 1.25 mg/kg. In this survival model, primary efficacy endpoints were median survival time (MST), percent increased lifespan (%ILS), and long-term survivors (defined as mice with a survival time better than or equal to twice the MST of the control group). Differences in survival between groups were assessed with the Cox model.

As shown in Table 17, the isotype control group had an MST of 39 days. Twenty percent of the isotype control group exhibited long-term survival in this model. The group treated with mAb1 10 mg/kg had an MST of greater than 82.5 days, an increase in lifespan of more than 112%. Fifty percent of the group treated with mAb1 10 mg/kg were long-term survivors. Mice treated with mAb1 1.25 mg/kg had an MST of greater than 46.5 days, and an increase in lifespan of more than 19%. Thirty percent of the group treated with mAb1 1.25 mg/kg were long-term survivors.

The group treated with mAb10 10 mg/kg had an MST greater than 70 days, and a 70% increase in lifespan. Fifty percent of the group treated with mAb10 10 mg/kg were long-term survivors. Mice treated with mAb10 1.25 mg/kg had an MST greater than 42 days, and an 8% increase in lifespan. Only 10% of the group treated with mAb10 1.25 mg/kg were long-term survivors.

The group treated with mAb3 10 mg/kg had a MST longer than 90 days, and an increased life span of over 131%. Surprisingly, 90% of the group treated with mAb3 10 mg/kg were long-term survivors. mAb3 at 10 mg/kg was statistically significantly more active compared to the isotype control group (p=0.0351). Even more surprisingly, mice treated with mAb3 1.25 mg/kg also had a MST longer than 90 days, and an increased life span of over 131%. Furthermore, 90% of the group treated with mAb3 1.25 mg/kg were long-term survivors. mAb3 at 1.25 mg/kg was statistically significantly more active compared to the isotype control group (p=0.0380) and statistically significantly superior to mAb10 at the same dose (p=0.0380).

Treatment of this tumor model with mAb1, mAb3 and mAb10 at a dose of 10 mg/kg in mice with low affinity huFcγRIIIa (F158/F158) resulted in a statistically significant improvement in lifespan and number of long-term survivors. Surprisingly, however, at a dose of 1.25 mg/kg, mAb3 still exhibited a statistically significant improvement in lifespan and number of long-term survivors, while mAb1 and mAb10 were inactive.

E. Stability Studies Thermal Stability The thermal stability of mAbs 1, 3, 5, 6, 7 and 8 was measured by differential scanning calorimetry (DSC) using a Malvern MicroCal VP calorimeter. Samples were tested in buffer containing 10 mM histidine HCl at pH 6.0. Prepared samples were loaded into wells of a Wheaton 96-well round bottom plate in triplicate with buffer as a reference. Thermal scans were collected using a temperature ramp rate of 1° C./min and a range of 20-120° C. and processed using proprietary software 2.0.

As shown in Table 18, mAbs 3, 5, 6, 7 and 8 showed an onset of protein conformational changes at approximately 40°C, approximately 17°C lower than the onset temperature of mAb1 and 15-20°C lower than typical for mAbs (Figure 1). Furthermore, unfolding of the CH2 domain of mAbs 3, 5, 6, 7 and 8 began much earlier than mAb1, at approximately 50°C compared to 70°C.

The isoelectric point (pI) of each of mAbs 1, 3, 5, 6, 7 and 8 was measured by capillary isoelectric focusing (cIEF). The theoretical pI was determined from the composition using the analytical platform SEDNTERP and assuming the following amino acid pKa values: Arg=12, Asp=4.5, Glu=4.6, His=6.2, Lys=10.4 and Tyr=9.7. All sulfhydryl side chains of Cys residues were assumed to be disulfide-bonded with no contribution to pKa (Laue TM, Shah BD, Ridgeway TM, and Pelletier SL. Computer-aided interpretation of analytical sedimentation data for proteins. In Analytical Ultracentrifugation in Biochemistry and Polymer Science. Eds. Harding SE, Rowe AJ, and Horton JC, pp. 90-124. Royal Society of Chemistry, Chemistry, 1991).

Surprisingly, as shown in Table 19, the measured pI was lower than the calculated pI based on the sequence for mAb3.

Formulation Development of mAb3 Based on the thermal stability results, a lyophilized formulation was developed to ensure reasonable stability of mAb3 suitable for storage of mAb at ≦−30° C. and lyophilized mAb at 2-8° C. mAb3 formulations using different buffer systems and different pH were tested in stability studies consisting of freeze-thaw cycles, agitation studies and short-term (12 weeks) incubation at stressed, accelerated and intended storage conditions of 40° C., 25° C., 5° C., −30° C. and −80° C. In these experiments, protein aggregation and chemical degradation were assessed in addition to particle formation (visual inspection, particle counting based on light obscuration method, microflow imaging).

Based on the results of the formulation development studies, a formulation containing 10 mM L-histidine-HCl, 8% wt/vol sucrose and 0.05% wt/vol polysorbate 80, pH 6.2 was selected.

Lyophilization Process Development A lyophilization process for mAb3 was developed to ensure that the formulated antibody had acceptable stability and cake attributes after lyophilization. The process parameters for the lyophilization cycle (drying temperature, chamber pressure, etc.) were determined based on numerous development runs using a lab-scale lyophilizer (Genesis EL35 manufactured by SP Scientific). The robustness of the cycle was established by running a series of lyophilization runs at controlled ranges of freezing rate, drying temperature and chamber vacuum pressure. Stability studies for the lyophilized formulated antibody consisted of short-term (12 weeks) incubation at stressed, accelerated and intended storage conditions of 40°C, 25°C and 5°C. In these experiments, the attributes of the lyophilized powder (cake appearance, reconstitution time, oxygen headspace, moisture content) were evaluated in addition to aggregation, chemical degradation and particle formation. Based on formulation and lyophilization development studies, the composition of mAb3 was selected as 50 mg/mL mAb3, 10 mM L-histidine-HCl, 8% wt/vol sucrose, 0.05% wt/vol polysorbate 80, and pH 6.2. Formulated antibody was stored at ≦−30° C. and lyophilized formulated antibody was stored at 2-8° C.

Claims (16)

An antibody that specifically binds to human CD38,
a) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:2 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or b) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:3 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or c) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:4 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or d) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or e) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9, or f) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or g) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9.
The antibody comprising: 1. A pharmaceutical composition comprising a formulated antibody, the formulated antibody comprising the antibody, sucrose, L-histidine and polysorbate 80 (PS80), wherein the antibody specifically binds human CD38 and is present at a concentration of 50 mg/mL, the sucrose is present at a concentration of 8% (wt/vol), the L-histidine is present at a concentration of 10 mM, and the PS80 is present at a concentration of 0.05% (v/v), the formulation has a pH of 6.2, and the antibody is
a) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:2 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or b) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:3 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or c) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:4 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or d) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or e) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9, or f) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or g) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9.
The pharmaceutical composition comprising: 1. A unit dosage form of a formulated antibody that specifically binds to human CD38, the formulated antibody comprising 215 mg of antibody, 6.21 mg of L-histidine, 344 mg of sucrose, and 2.15 mg of polysorbate 80, the formulated antibody being lyophilized, and the antibody being
a) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:2 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or b) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:3 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or c) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:4 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or d) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or e) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9, or f) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or g) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9.
The unit dosage form comprises The pharmaceutical composition of claim 2 or the unit dosage form of claim 3, wherein the formulated antibody is lyophilized. (i) the amino acid at position 1 of the HC amino acid sequence is pyroglutamine; and/or (ii) the antibody has an isoelectric point (pI) of 5.8 to 9.0 as measured by capillary isoelectric focusing (cIEF).
The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 4. (i) the antibody comprises a main charge variant and at least one acidic charge variant, the antibody comprising:
a) the HC of at least one charge variant comprises at least one deamidated asparagine selected from the group consisting of N289, N318, N387 and N392 as numbered by SEQ ID NO:1, or b) the antibody comprises a major charge variant and at least one acidic charge variant, wherein the major charge variant comprises at least 71% of the antibody and the acidic charge variant comprises no more than 30% of the antibody, or c) the major charge variant has an isoelectric point (pI) of 7.5 and the one or more acidic charge variants have a pI of 5.8 as measured by capillary isoelectric focusing (cIEF);
and/or (ii) the antibody comprises at least one basic charge variant, the antibody having at least one of:
a) the basic charge variant comprises 4% or less of the antibody; or b) one or more basic charge variants have a pI of 9.0 as measured by capillary isoelectric focusing (cIEF);
having at least one of
The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 5. The antibody is
a) is capable of killing CD38 expressing cells having a CD38 receptor density of ≦13,000 CD38 sites on their surface by antibody-dependent cellular cytotoxicity (ADCC) in the presence of natural killer cells expressing 158F, 158V, or both 158F and 158V variant CD16a (FcγRIIIa); and/or b) binds to CD16a (FcγRIIIa) having a phenylalanine at amino acid position 158 (158F) with a KD of 59 nM as measured by surface plasmon resonance (SPR), and the antibody binds to CD16a (158V) having a valine at amino acid position 158 with a KD of 75 nM as measured by SPR; and/or c) is capable of killing CD38 expressing cells having a CD38 receptor density of ≦13,000 CD38 sites on their surface by antibody-dependent cellular cytotoxicity (ADCC) in the presence of natural killer cells expressing 158F, 158V, or both 158F and 158V variant CD16a (FcγRIIIa) and/or d) binds to CD32a (FcγRIIa) (131R) having an arginine at amino acid position 131 with a KD of 94 nM as measured by binding to HEK cells expressing FcγRIIa 131R, and the antibody binds to FcγRIIa (131H) having a histidine at amino acid position 131 with a KD of 222 nM as measured by binding to HEK cells expressing FcγRIIa 131H.
The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 6. a) the antibody is capable of killing CD38 expressing cells having a cell surface density of >400,000 CD38 receptors by ADCC in the presence of natural killer cells expressing 158F, 158V or both 158F and 158V variant CD16a (FcγRIIIa); is capable of killing CD38 expressing cells having a cell surface density of >100,000 CD38 receptors by ADCC; is capable of killing CD38 expressing cells having a cell surface density of <13,000 CD38 receptors by ADCC; and/or b) the antibody is capable of killing CD38 expressing cells having a cell surface density of >400,000 CD38 receptors by antibody-dependent cellular phagocytosis (ADCP) in the presence of human peripheral blood mononuclear cells (PMBCs),
The antibody, pharmaceutical composition or unit dosage form of any one of claims 1 to 7. a) in the presence of natural killer cells expressing a high affinity variant (158V) of CD16a (FcγRIIIa), the antibody is capable of killing KMS-12BM cells by ADCC with an EC50 of about 0.6 ng/mL; and/or b) in the presence of natural killer cells expressing a low affinity variant (158F) of CD16a (FcγRIIIa), the antibody is capable of killing KMS-12BM cells by ADCC with an EC50 of about 0.65 ng/mL; and/or c) in the presence of natural killer cells expressing a high affinity variant (158V) of CD16a (FcγRIIIa), the antibody is capable of killing RPMI-8226 cells by ADCC with an EC50 of about 0.09 ng/mL; and/or d) in the presence of natural killer cells expressing a low affinity variant of CD16a (FcγRIIIa) (158F), the antibody is capable of killing RPMI-8226 cells by ADCC with an EC50 of about 0.09 ng/mL; and/or e) in the presence of natural killer cells expressing a high affinity variant of CD16a (FcγRIIIa) (158V), the antibody is capable of killing MOLP-8 cells by ADCC with an EC50 of about 0.14 ng/mL; and/or f) in the presence of natural killer cells expressing a low affinity variant of CD16a (FcγRIIIa) (158F), the antibody is capable of killing MOLP-8 cells by ADCC with an EC50 of about 0.28 ng/mL; and/or g) in the presence of human PMBCs expressing the high affinity variant (158V) of CD16a (FcγRIIIa), the antibody is capable of killing MOLP-8 cells via ADCP with an EC50 of approximately 31.87 ng/mL;
9. The antibody, pharmaceutical composition or unit dosage form of claim 7 or 8. The antibody, pharmaceutical composition or unit dosage form of any one of claims 1 to 9, wherein the antibody is capable of improving survival of mice in a mouse tumor model, the mice in the model expressing human CD16a (158F), and the mice are injected with 500,000 EL4 cells expressing human CD38. A method for preparing a pharmaceutical composition comprising an antibody that specifically binds to human CD38, the antibody comprising:
a) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:2 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or b) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:3 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or c) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:4 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or d) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or e) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9, or f) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or g) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9.
The method includes:
i) expressing the antibody in cell culture;
ii) subjecting the antibody to at least one purification step selected from the group consisting of chromatography and ultrafiltration to produce a purified antibody solution;
iii) preparing a purified antibody solution - purified antibody at a concentration of 50 mg/mL;
- sucrose at a concentration of 8% weight/volume,
- L-histidine at a concentration of 10 mM, and - polysorbate 80 at a concentration of 0.05% v/v.
wherein the formulated antibody has a pH of 6.2; and
iv) lyophilizing the formulated antibody;
and thereby preparing a pharmaceutical composition. 1. A method for preparing a reconstituted formulated antibody, comprising:
a) providing a lyophilized antibody formulation comprising sucrose, L-histidine, polysorbate 80 and an antibody that specifically binds to human CD38;
b) reconstituting the lyophilized antibody formulation in a diluent, the reconstituted antibody formulation comprising: antibody at a concentration of 50 mg/mL;
- sucrose at a concentration of 8% weight/volume,
- L-histidine at a concentration of 10 mM, and - polysorbate 80 at a concentration of 0.05% v/v.
wherein the reconstituted antibody formulation has a pH of 6.2;
The antibody is
i) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:2 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or ii) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:3 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or iii) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:4 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or iv) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or v) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9, or vi) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or vii) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9.
and 1. An antibody for use in the treatment of a disease involving CD38 expressing cells, comprising administering one or more doses of an antibody that specifically binds human CD38, the antibody comprising:
a) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:2 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or b) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:3 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or c) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:4 and a light chain (LC) having the amino acid sequence of SEQ ID NO:7, or d) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or e) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:5 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9, or f) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:8, or g) a heavy chain (HC) having the amino acid sequence of SEQ ID NO:6 and a light chain (LC) having the amino acid sequence of SEQ ID NO:9.
The antibody comprising: (i) the disease is hematological in origin; and/or (ii) the one or more doses of antibody are provided as a lyophilized antibody formulation, and the lyophilized antibody formulation is reconstituted prior to administration to form a reconstituted antibody formulation such that the reconstituted antibody formulation has a pH of 6.2 and contains the antibody at a concentration of 50 mg/mL in a liquid comprising 10 mM L-histidine, 8% wt/vol sucrose and 0.05% v/vol polysorbate 80; and/or (iii) the antibody is administered at a dose of 20 mg/kg, 10 mg/kg, 5 mg/kg, 3 mg/kg or 1.5 mg/kg; and/or (iv) the pharmaceutical composition comprises a formulated antibody, the formulated antibody comprising an antibody present at a concentration of 50 mg/mL, sucrose at a concentration of 8% (w/v), L-histidine at a concentration of 10 mM and polysorbate 80 (PS80) at a concentration of 0.05% (v/v), the formulated antibody having a pH of 6.2; and/or (v) the antibody has an isoelectric point (pI) of 5.8-9.0 as measured by capillary isoelectric focusing (cIEF); and/or (vi) the amino acid at position 1 of the HC amino acid sequence is pyroglutamine.
An antibody for use as claimed in claim 13. (i) the antibody comprises a main charge variant and at least one acidic charge variant, the antibody comprising:
a) the HC of at least one charge variant comprises at least one deamidated asparagine selected from the group consisting of N289, N318, N387 and N392 as numbered by SEQ ID NO:1, or b) the antibody comprises a major charge variant and at least one acidic charge variant, wherein the major charge variant comprises at least 71% of the antibody and the acidic charge variant comprises no more than 30% of the antibody, or c) the major charge variant has an isoelectric point (pI) of 7.5 and the one or more acidic charge variants have a pI of 5.8 as measured by capillary isoelectric focusing (cIEF);
and/or (ii) the antibody comprises at least one basic charge variant, the antibody having at least one of:
a) the basic charge variant comprises 4% or less of the antibody; or b) one or more basic charge variants have a pI of 9.0 as measured by capillary isoelectric focusing (cIEF);
having at least one of
An antibody for use according to claim 13 or 14. 16. The antibody for use according to claim 14 or 15, wherein the disease of hematological origin is selected from the group consisting of amyloidosis, non-Hodgkin's lymphoma (NHL), Waldenstrom's disease, multiple myeloma (MM), acute lymphocytic leukemia (ALL), and acute myeloid leukemia (AML).

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