Anti-CD38 antibodies and formulations

Abstract

The present invention relates to anti-CD38 antibodies and formulations, and specifically provides antibodies that specifically bind to human CD38, formulations and unit dosage forms comprising the antibodies, methods of making the antibodies, and methods of using the antibodies.

Description

[0001] This invention application is a divisional application based on a patent application with an application date of April 23, 2020, application number "202080030148.3" (international application number PCT / US2020 / 029531), and name "Anti-CD38 Antibodies and Preparations".

[0002] Related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 837,518, filed April 23, 2019; U.S. Provisional 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.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on April 17, 2020, is named 704023_SA9-289PC_ST25.txt and is 44,783 bytes in size. Technical Field

[0006] Provided herein are anti-CD38 antibodies with improved cytotoxic activity and stable formulations thereof. Background Art

[0007] CD38 is a type II glycosylated 45 kilodalton (kDa) membrane protein that is identified as a lymphocyte marker. CD38 plays a role in leukocyte homeostasis by regulating hematopoietic cell survival and differentiation (Richards JO et al., Mol Cancer Ther. 2008; 7 (8): 2517-27). CD38 acts as a receptor bound to CD31 and participates in cell adhesion and signal transduction. The function of CD38 in signal transduction appears to be diverse, depending on the cell lineage, differentiation stage, and possible association with different co-receptors (Richards JO et al., 2008). CD38 is also an extracellular enzyme that catalyzes the synthesis and hydrolysis of cyclic adenosine diphosphate ribose (cADPR) from nicotinamide adenine dinucleotide (NAD+) to ADP-ribose (DiLillo DJ, Ravetch JV., Cell. 2015; 161 (5): 1035-45). These reaction products are involved in calcium mobilization and intracellular signaling (Derer S et al., MAbs. 2014; 6(2): 409-21).

[0008] CD38 expression in healthy humans can be detected on NK cells, monocytes, dendritic cells, macrophages, granulocytes, activated T and B cells, and plasma cells. In contrast, expression has not been detected in hematopoietic stem cells, resting T and B cells, or tissue macrophages. In addition, several hematological malignancies express CD38, such as malignant plasma cell diseases (e.g., multiple myeloma (MM), amyloidosis) and other hematopoietic cancers (including, including, for example, Waldenstrom's disease, non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML)).

[0009] CD38 expression is particularly prominent in MM, as >98% of patients are positive for this protein (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). The strong and consistent expression of CD38 on malignant clones of MM cells contrasts with the limited expression pattern on normal cells, suggesting that this antigen can be used to specifically target tumor cells.

[0010] MM is a malignant plasma cell disorder characterized by expression of CD38 on the cell surface, clonal proliferation of plasma cells in the bone marrow (BM), and the production of excess monoclonal immunoglobulins (usually of the IgG or IgA type) or free urinary light chains (also known as paraprotein, M-protein, or M-component). It is a disease primarily associated with advancing age, with over 80% of patients aged 60 years and older.

[0011] The course of MM varies with 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 changes include del(17p), t(4;14) and t(14;16), and 1q gain. Over the past two decades, median survival has improved from 3 to 6 years; however, some patients can live for more than 10 years (Ocio EM et al., Expert Rev Hematol. 2014;7(1):127-41). Treatment options and survival depend on the patient's age, health, and disease status. Patients under approximately 65 years of age, in good health, and presenting with symptomatic active disease will usually receive autologous stem cell transplantation (ASCT) therapy first. Induction chemotherapy is given to achieve cytoreduction of the disease before stem cells are collected. Induction regimens include alkylating agents, dexamethasone alone, thalidomide plus dexamethasone, and vincristine, (doxorubicin) and dexamethasone (VAD; or modifications thereof); however, the latter two regimens are associated with higher toxicity (Richardson PG et al., Blood. 2010; 116(5): 679-86; Arnulf B et al., Haematologica. 2012; 97: 1925-8). (bortezomib), bortezomib combination with Treatment with lenalidomide plus dexamethasone as induction therapy has shown improved outcomes, and these agents have shown higher response rates and lower toxicity (Richardson PG 2010, 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). Other approved drugs include pomalidomide (which belongs to the same class as lenalidomide) and dapoxetine. ) and carfilzomib and ixazomib (belong to the same class of proteasome inhibitors as bortezomib). In addition to these new treatments, monoclonal antibodies (particularly anti-CD38 antibodies) have also begun to play an important role in the treatment of myeloma patients. Daratumumab is an anti-CD38 antibody that has been approved for the treatment of MM as a single agent or in combination with other MM therapies (Touzeau C, Moreau P., Expert Opin Biol Ther. 2017; 17(7): 887-93; Tzogani K. et al., Oncologist. 2018; 23: 1-11). It has been reported that the anti-CD38 antibody ixatuximab induces a response rate of 25%-29% as a single agent and a response rate of 60% in combination therapy in relapsed or refractory multiple myeloma (Martin T et al., Blood. 2017; 129(25): 3294-303); recent phase 3 study results indicate that the addition of ixatuximab can improve progression-free survival in relapsed and refractory multiple myeloma treated with pomalidomide-dexamethasone (J Clin Oncol 37, 2019 (Supplement; Abstract 8004)). In addition, the anti-Slam-F7 antibody elozumab has been approved for combination with lenalidomide and dexamethasone for the treatment of adult MM patients who have received one to three prior lines of therapy, and for combination with pomalidomide and dexamethasone for the treatment of adult MM patients who have received at least two prior therapies including lenalidomide and a proteasome inhibitor (Bristol-Myers Squibb Company. (eloizumab) [Package insert]. U.S. Food and Drug Administration website: www-dot-accessdata-dot-fda.gov / drugsatfda_docs / label / 2018 / 761035s008lbl.pdf. Revised November 2018.

[0012] Current therapies for these CD38-expressing diseases aim to effectively control the disease, maximize quality of life, and prolong survival. For example, the average MM patient undergoes four to eight different treatment regimens during their lifetime. Consequently, despite improved prognosis for patients with newer therapies, MM remains a fatal disease. Consequently, treating patients who have progressed on current therapies, including antibody therapies, remains a significant unmet medical need. Summary of the Invention

[0013] It is contemplated that the antibodies, uses, and methods of treatment provided herein can provide superior clinical responses or treatments for diseases that express CD38 compared to currently available treatments, including antibody treatments. 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.

[0014] In one aspect, antibodies that specifically bind to human CD38 are provided. In some embodiments, the antibodies provided herein comprise 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.

[0015] In some embodiments, the antibodies provided herein comprise an LC having an 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 an amino acid sequence of SEQ ID NO: 7 and an HC having an amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibodies provided herein comprise an LC having an amino acid sequence of SEQ ID NO: 7 and an HC having an amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibodies provided herein comprise an LC having an amino acid sequence of SEQ ID NO: 7 and an HC having an amino acid sequence of SEQ ID NO: 4.

[0016] In some embodiments, the antibodies provided herein comprise an LC having an 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 an amino acid sequence of SEQ ID NO: 8 and an HC having an amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibodies provided herein comprise an LC having an amino acid sequence of SEQ ID NO: 8 and an HC having an amino acid sequence of SEQ ID NO: 6.

[0017] In some embodiments, the antibodies provided herein comprise an LC having an 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 an amino acid sequence of SEQ ID NO: 9 and an HC having an amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibodies provided herein comprise an LC having an amino acid sequence of SEQ ID NO: 9 and an HC having an amino acid sequence of SEQ ID NO: 7.

[0018] On the other hand, provided herein is a pharmaceutical composition comprising the antibody prepared. In some embodiments of the pharmaceutical composition, the antibody comprises HC and LC with amino acid sequence as described above, combined with sucrose, L-histidine and polysorbate 80. In some embodiments, the antibody is present at a concentration of 50 mg / mL, the sucrose is present at a concentration of 8% (w / v), the L-histidine is present at a concentration of 10 mM, the 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.

[0019] On the other hand, provided herein are unit dosage forms comprising formulated antibodies. In some embodiments, the antibody comprises HC and LC having an amino acid sequence as described above, and the unit dosage form comprises 215 mg of the 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.

[0020] On the other hand, provided herein is a method for preparing a pharmaceutical composition comprising an antibody that specifically binds to human CD38. In some embodiments, the antibody comprises an HC and an LC having an amino acid sequence as described above, wherein the method comprises expressing the antibody in cell culture, subjecting the antibody to at least one of a chromatography 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 the purified antibody, sucrose, L-histidine, and polysorbate 80. In some embodiments, the 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 polysorbate 80 (PS80) at a concentration of 0.05% v / v. In some embodiments, the antibody formulation is prepared so that it has a pH of 6.2. In some embodiments of the method for preparing the antibody formulation, the antibody formulation is lyophilized.

[0021] On the other hand, a method for preparing a reconstructed 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 HC and LC having an amino acid sequence as described above. In some embodiments, the lyophilized antibody formulation is reconstructed in a diluent to prepare the reconstructed antibody formulation. In some embodiments, the reconstructed 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 reconstructed antibody formulation has a pH of 6.2.

[0022] 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, wherein the antibody comprises an HC and an LC having an amino acid sequence as described above.

[0023] In another aspect, an antibody for use in a method of treating multiple myeloma is provided. In some embodiments, the antibody specifically binds to human CD38, wherein the antibody comprises an HC and an LC having an amino acid sequence as described above.

[0024] Specifically, the present invention includes but is not limited to the following:

[0025] 1. An antibody that specifically binds to human CD38, wherein the antibody comprises

[0026] 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

[0027] 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

[0028] 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

[0029] 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

[0030] 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

[0031] 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

[0032] 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.

[0033] 2. A pharmaceutical composition comprising a formulated antibody, wherein the formulated antibody comprises an antibody, sucrose, L-histidine, and polysorbate 80 (PS80), wherein the antibody specifically binds to human CD38 and is present at a concentration of 50 mg / mL, the sucrose is present at a concentration of 8% (w / v), the L-histidine is present at a concentration of 10 mM, and the PS80 is present at a concentration of 0.05% (v / v), wherein the formulation has a pH of 6.2, and wherein the antibody comprises

[0034] 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

[0035] 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

[0036] 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

[0037] 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

[0038] 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

[0039] 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

[0040] 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.

[0041] 3. A unit dosage form of a formulated antibody that specifically binds to human CD38, wherein the formulated antibody comprises 215 mg of the antibody, 6.21 mg of L-histidine, 344 mg of sucrose, and 2.15 mg of polysorbate 80, wherein the formulated antibody is lyophilized, and wherein the antibody comprises

[0042] 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

[0043] 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

[0044] 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

[0045] 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

[0046] 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

[0047] 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

[0048] 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.

[0049] 4. The pharmaceutical composition according to item 2 or the unit dosage form according to item 3, wherein the formulated antibody is lyophilized.

[0050] 5. The antibody, pharmaceutical composition or unit dosage form according to any one of items 1 to 4, wherein the amino acid at position 1 of the HC amino acid sequence is pyroglutamine.

[0051] 6. The antibody, pharmaceutical composition or unit dosage form according to any one of items 1 to 5, wherein said antibody has an isoelectric point (pi) of 5.8 to 9.0 when measured by capillary isoelectric focusing (cIEF).

[0052] 7. The antibody, pharmaceutical composition or unit dosage form according to any one of items 1 to 6, wherein the antibody comprises a major charged variant and at least one acidic charged variant and wherein the antibody has at least one of the following

[0053] a) the HC of the at least one charged variant comprises at least one deamidated asparagine selected from N289, N318, N387 and N392 as numbered according to SEQ ID NO: 1, or

[0054] b) the antibody comprises a predominantly charged variant and at least one acidic charged variant, wherein the predominantly charged variant comprises at least 71% of the antibody and the acidic charged variant comprises no more than 30% of the antibody, or

[0055] c) the major charged variant has an isoelectric point (pi) of 7.5 and wherein the one or more acidic charged variants have a pi of 5.8 when measured by capillary isoelectric focusing (cIEF).

[0056] 8. The antibody, pharmaceutical composition or unit dosage form according to any one of items 1 to 7, wherein the antibody comprises at least one basic charged variant, and wherein the antibody has at least one of the following

[0057] a) the basic charged variants comprise no more than 4% of the antibody, or

[0058] b) the one or more basic charged variants have a pi of 9.0 when measured by capillary isoelectric focusing (cIEF).

[0059] 9. The antibody, pharmaceutical composition or unit dosage form according to any one of items 1 to 8, wherein the antibody

[0060] a) is capable of killing cells expressing CD38 by antibody-dependent cellular cytotoxicity (ADCC) in the presence of natural killer cells expressing 158F, 158V, or both 158F and 158V variants of CD16a (FcγRIIIa), wherein the cells have a CD38 receptor density of ≤13,000 CD38 sites on the cell surface, and / or

[0061] b) with a K of 59 nM as measured by surface plasmon resonance (SPR) D Binds to CD16a (FcγRIIIa) (158F) with a phenylalanine at amino acid position 158, and wherein the antibody has a K of 75 nM as measured by SPR. D binds to CD16a with valine at amino acid position 158 (158V), and / or

[0062] c) with a K of 96 nM as measured by binding to HEK cells expressing FcγRIIIa 158F D Binds to CD16a (FcγRIIIa) with a phenylalanine at amino acid position 158 (158F), and wherein the antibody binds with a K of 40 nM as measured by binding to HEK cells expressing FcγRIIIa 158V D binds to FcγRIIIa with valine at amino acid position 158 (158V), and / or

[0063] d) with a K of 94 nM as measured by binding to HEK cells expressing FcγRIIa 131R D binds to CD32a (FcγRIIa) with an arginine at amino acid position 131 (131R), and wherein the antibody binds to CD32a with an arginine at amino acid position 131 (131R) with a K of 222 nM as measured by binding to HEK cells expressing FcγRIIa 131H D Binds to FcγRIIa with histidine at amino acid position 131 (131H).

[0064] 10. The antibody, pharmaceutical composition or unit dosage form according to any one of items 1 to 8, wherein

[0065] a) in the presence of natural killer cells expressing 158F, 158V, or both 158F and 158V variants of CD16a (FcγRIIIa), the antibody is capable of killing by ADCC cells expressing CD38, wherein the cells have a density of >400,000 CD38 receptors on the cell surface; and is capable of killing by ADCC cells expressing CD38, wherein the cells have a density of ≥100,000 CD38 receptors on the cell surface; and is capable of killing by ADCC cells expressing CD38, wherein the cells have a density of ≤13,000 CD38 receptors on the cell surface, and / or

[0066] b) the antibody is capable of killing cells expressing CD38 by antibody-dependent cellular phagocytosis (ADCP) in the presence of human peripheral blood mononuclear cells (PMBCs), wherein the cells have a density of >400,000 CD38 receptors on the cell surface.

[0067] 11. The antibody, pharmaceutical composition or unit dosage form according to item 9 or 10, wherein

[0068] a) the antibody is capable of killing KMS-12BM cells by ADCC with an EC50 of about 0.6 ng / mL in the presence of natural killer cells expressing a higher affinity variant (158V) of CD16a (FcγRIIIa), and / or

[0069] b) the antibody is capable of killing KMS-12BM cells by ADCC with an EC50 of about 0.65 ng / mL in the presence of natural killer cells expressing a lower affinity variant (158F) of CD16a (FcγRIIIa), and / or

[0070] c) the antibody is capable of killing RPMI-8226 cells by ADCC with an EC50 of about 0.09 ng / mL in the presence of natural killer cells expressing a higher affinity variant (158V) of CD16a (FcγRIIIa), and / or

[0071] d) the antibody is capable of killing RPMI-8226 cells by ADCC with an EC50 of about 0.09 ng / mL in the presence of natural killer cells expressing a lower affinity variant (158F) of CD16a (FcγRIIIa), and / or

[0072] e) the antibody is capable of killing MOLP-8 cells by ADCC with an EC50 of about 0.14 ng / mL in the presence of natural killer cells expressing a higher affinity variant (158V) of CD16a (FcγRIIIa), and / or

[0073] f) In the presence of natural killer cells expressing a lower affinity variant (158F) of CD16a (FcγRIIIa), the antibody was able to kill MOLP-8 cells by ADCC with an EC50 of approximately 0.28 ng / mL.

[0074] 12. The antibody, pharmaceutical composition or unit dosage form of any one of items 9-11, wherein the antibody is capable of killing MOLP-8 cells by ADCP with an EC50 of about 31.87 ng / mL in the presence of human PMBCs expressing the higher affinity variant (158V) of CD16a (FcγRIIIa).

[0075] 13. The antibody, pharmaceutical composition or unit dosage form of any one of items 1 to 12, wherein the antibody is capable of improving the survival of mice in a murine tumor model, wherein the mice in the model express human CD16a (158F) and wherein the mice have been injected with 500,000 EL4 cells expressing human CD38.

[0076] 14. A method for preparing a pharmaceutical composition comprising an antibody that specifically binds to human CD38, wherein the antibody comprises

[0077] 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

[0078] 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

[0079] 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

[0080] 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

[0081] 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

[0082] 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

[0083] 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,

[0084] The method comprises the following steps:

[0085] i) expressing the antibody in cell culture;

[0086] ii) subjecting the antibody to at least one purification step selected from chromatography and ultrafiltration to produce a purified antibody solution; and

[0087] iii) adjusting the purified antibody solution to produce a formulated antibody, the formulated antibody comprising: the purified antibody at a concentration of 50 mg / mL,

[0088] - sucrose at a concentration of 8% w / v,

[0089] - L-histidine at a concentration of 10 mM, and

[0090] - Polysorbate 80 at a concentration of 0.05% v / v,

[0091] wherein the formulated antibody has a pH of 6.2; and

[0092] iv) lyophilizing the formulated antibody;

[0093] Thus, the pharmaceutical composition is prepared.

[0094] 15. A method for preparing a remodeled formulated antibody comprising

[0095] a) providing a lyophilized antibody formulation comprising sucrose, L-histidine, polysorbate 80, and an antibody that specifically binds to human CD38; and

[0096] b) reconstitute the lyophilized antibody formulation in a diluent, wherein the reconstituted antibody formulation comprises

[0097] - said antibody at a concentration of 50 mg / mL,

[0098] - sucrose at a concentration of 8% w / v,

[0099] - L-histidine at a concentration of 10 mM, and

[0100] - Polysorbate 80 at a concentration of 0.05% v / v,

[0101] wherein the reconstituted antibody formulation has a pH of 6.2;

[0102] wherein the antibody comprises

[0103] 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

[0104] 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

[0105] 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

[0106] 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

[0107] 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

[0108] 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

[0109] 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.

[0110] 16. A method of treating a patient in need thereof, comprising administering to the patient one or more doses of an antibody that specifically binds to human CD38, wherein the patient has a disease comprising cells expressing CD38, wherein the antibody comprises

[0111] 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

[0112] 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

[0113] 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

[0114] 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

[0115] 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

[0116] 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

[0117] 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.

[0118] 17. An antibody for use in treating a disease comprising cells expressing CD38, wherein the antibody specifically binds to human CD38, wherein the antibody comprises

[0119] 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

[0120] 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

[0121] 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

[0122] 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

[0123] 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

[0124] 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

[0125] 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.

[0126] 18. The method of item 16 or the antibody for use of item 17, wherein the disease is of hematological origin.

[0127] 19. The method of claim 18 or the antibody for use thereof, wherein the disease of blood origin is selected from the group consisting of amyloidosis, non-Hodgkin's lymphoma (NHL), Waldenstrom's disease, multiple myeloma (MM), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

[0128] 20. The method or antibody for use of any one of items 16 to 19, wherein the one or more doses of the antibody are provided in the form of a lyophilized antibody formulation and wherein, prior to administration, the lyophilized antibody formulation is reconstituted to form a reconstituted antibody formulation such that the reconstituted antibody formulation has a pH of 6.2 and comprises the antibody at a concentration of 50 mg / mL in a liquid comprising 10 mM L-histidine, 8% (w / v) sucrose and 0.05% (v / v) polysorbate 80.

[0129] 21. The method or antibody for use according to any one of items 16 to 20, wherein 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.

[0130] 22. The method of any one of items 16 to 21 or the antibody for use thereof, wherein the pharmaceutical composition comprises a formulated antibody, wherein the formulated antibody comprises the 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), wherein the formulated antibody has a pH of 6.2.

[0131] 23. The method or antibody for use according to any one of items 16 to 22, wherein the antibody has an isoelectric point (pi) of 5.8 to 9.0 when measured by capillary isoelectric focusing (cIEF).

[0132] 24. The method according to any one of items 16 to 23 or the antibody for use, wherein the amino acid at position 1 of the HC amino acid sequence is pyroglutamine.

[0133] 25. The method of or antibody for use according to any one of items 16-25, wherein the antibody comprises a predominantly charged variant and at least one acidic charged variant and wherein the antibody has at least one of:

[0134] a) the HC of the at least one charged variant comprises at least one deamidated asparagine selected from N289, N318, N387, and N392 as numbered according to SEQ ID NO: 1, or

[0135] b) the antibody comprises a predominantly charged variant and at least one acidic charged variant, wherein the predominantly charged variant comprises at least 71% of the antibody and the acidic charged variant comprises no more than 30% of the antibody, or

[0136] c) the predominantly charged variant has an isoelectric point (pi) of 7.5 and wherein the one or more acidic charged variants have a pi of 5.8 when measured by capillary isoelectric focusing (cIEF).

[0137] 26. The method of or antibody for use according to any one of items 16-25, wherein the antibody comprises at least one basic charged variant, and wherein the antibody has at least one of

[0138] a) the basic charged variant comprises no more than 4% of the antibody, or

[0139] b) the one or more basic charged variants have a pi of 9.0 when measured by capillary isoelectric focusing (cIEF). BRIEF DESCRIPTION OF DRAWINGS

[0140] Figure 1 is a graph showing the unfolding of mAbs 1, 2, 3, 5, 6, 7, and 8.

[0141] Figure 2 is a graph showing the isoelectric points (PI) of mAbs 1, 3, 5, 6, 7, and 8. DETAILED DESCRIPTION

[0142] Provided herein are anti-CD38 antibodies that have superior 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 superior antibody-dependent cellular phagocytosis (ADCP) activity against cells expressing CD38 on the cell surface. In some embodiments, the antibodies provided herein have an isoelectric point (pi) lower than expected. Although the pi is lower than expected and the protein unfolds at lower temperatures, which may have a negative impact on the stability of the antibody (particularly in commercial production processes), the antibodies provided herein are provided in a stable form suitable for administration to human patients.

[0143] Antibody

[0144] Provided herein are anti-CD38 antibodies that specifically bind to human CD38. Recombinant methods can be used to produce the anti-CD38 antibodies provided herein. In order to recombinantly produce anti-antigen antibodies, the nucleic acid encoding the antibody is separated and inserted into a replicable vector for further cloning (DNA amplification) or expression. Conventional procedures can be used (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody) to easily separate the DNA encoding the antibody and sequence it. Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: 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 nucleic acid expression. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the eukaryotic host cell is a mammalian cell. Examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell 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 CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell 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 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268. Anti-CD38 antibodies produced by cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography (with affinity chromatography being a generally preferred purification step). In general, various methods for preparing antibodies for research, testing, and clinical use are well established in the art, consistent with the methods described above, and / or deemed appropriate by those skilled in the art.

[0145] In some embodiments, antibody is expressed by CHO8D6 host cell line (DXB11 derivative) using a 500L disposable bioreactor operated in fed-batch mode. The cell culture process begins with thawing the master cell bank bottle, followed by a series of seed culture (train) cell expansion steps. The cells are then transferred to a bioreactor and cultivated using a serum-free chemically determined cell culture medium. After 10-14 days, culture is terminated to harvest the antibody. Cells and cell culture debris can be removed from the cell culture harvested by deep filtration.

[0146] The harvested material is then further processed through chromatography and filtration steps to produce purified antibodies.The purified antibodies are formulated in liquid solutions and stored at ≤-30°C.

[0147] Before the antibody of preparation is distributed into suitable vials, the liquid solution is thawed and aseptically filtered using a 0.2 μm filter unit. The antibody of preparation is distributed into suitable containers (such as USP type 1 glass vials, 4.3 mL / vial). In some embodiments, the vial filled contains an excess of 0.3 mL of the antibody of preparation. In some embodiments, the antibody of preparation is lyophilized in a vial.

[0148] The term "antibody" generally refers to a tetrameric protein comprising two heavy chains (HC) and two light chains (LC). Each such tetramer is generally composed of two pairs of identical polypeptide chains, each pair of polypeptide chains 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 a variable domain sequence sufficient to confer specificity to the target antigen. The amino terminal portion of each light and heavy chain comprises a variable domain of about 100 to 110 or more amino acids, which is generally responsible for antigen recognition. The carboxyl 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 one variable domain (VH) and three constant domains (CH1, CH2, and CH3), wherein the VH domain is located at the amino-terminus of the polypeptide and the CH3 domain is located at the carboxyl-terminus, and a full-length LC immunoglobulin polypeptide comprises one variable domain (VL) and one constant domain (CL), wherein the VL domain is located at the amino-terminus of the polypeptide and the CL domain is located at the carboxyl-terminus.

[0149] The term "antibody" is used herein in the broadest sense and specifically includes typical antibodies as 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 trigger ADCC and ADCP).

[0150] As used herein, the term "Fc" refers to a molecule comprising a sequence of a non-antigen binding fragment obtained by antibody digestion or produced by other means, the molecule being in monomeric or multimeric form, and the "Fc" may contain a hinge region. The Fc molecule is composed of monomeric polypeptides that can be linked into dimer or multimeric form by covalent (i.e., disulfide bonds) and non-covalent binding. Depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG4), the number of intermolecular disulfide bonds between the monomer subunits of a typical Fc molecule is in the range of 1 to 4. An example of Fc is a disulfide-bonded dimer produced by papain digestion of IgG.

[0151] The antibodies described herein may be isolated. The terms "isolated protein," "isolated polypeptide," or "isolated antibody" refer to a protein, polypeptide, or antibody that, by virtue of its origin or source of derivation, is: (1) not associated with naturally associated components that accompany it in its native state, (2) substantially free of other proteins from the same species, (3) expressed by cells from a different species, and / or (4) not found in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0152] In some embodiments, the antibodies provided herein comprise HC and LC amino acid sequences as provided in Table 1.

[0153] Table 1

[0154]

[0155] Binding characteristics

[0156] The term "affinity" refers to the measure of the attraction between two polypeptides (such as, for example, receptor / ligand or antigen / antibody). The intrinsic attractive force between two polypeptides can be expressed as the binding affinity equilibrium constant (K) for a specific interaction. D ). When K D When the concentration is ≤1 μM, preferably ≤100 nM, the antibody is said to bind specifically to the antigen. D The binding affinity constant can be determined, for example, by surface plasmon resonance (SPR) Or bio-layer interferometry (Bio-Layer Interferometry) to measure.

[0157] The term "k dissociation" refers to the dissociation rate constant of a specific antibody-antigen interaction. The k dissociation rate constant can be measured, for example, by biofilm interferometry.

[0158] Binding to CD38

[0159] In some embodiments, the antibodies provided herein comprise a tetrameric protein comprising two HCs and two LCs and have a molecular weight of about 1.91 x 10 -10 M is about 6.7x10 -10 The affinity or K of M DBinds to human CD38. In some embodiments, the HC and LC have the following amino acid sequences, respectively: SEQ ID NO: 2 and 7 (mAb 2), 3 and 7 (mAb 3), 4 and 7 (mAb 4), 5 and 8 (mAb 5), 5 and 9 (mAb 6), 6 and 8 (mAb 7), or 6 and 9 (mAb 8).

[0160] In some embodiments, the antibody comprises a HC and a LC having the amino acid sequences of SEQ ID NOs: 3 and 7, respectively, and the antibody can be expressed at about 2 x 10 -10 The affinity or K of M D In some embodiments, the antibody comprises an HC and a LC having the amino acid sequences of SEQ ID NOs: 5 and 8, respectively, and the antibody can bind to human CD38 at a concentration of about 6.7 x 10 -10 The affinity or K of M D In some embodiments, the antibody comprises an HC and an LC having the amino acid sequences of SEQ ID NOs: 5 and 9, respectively, and the antibody can bind to human CD38 at a concentration of about 4.15 x 10 -10 The affinity or K of M D In some embodiments, the antibody comprises an HC and a LC having the amino acid sequences of SEQ ID NOs: 6 and 8, respectively, and the antibody can bind to human CD38 at a concentration of about 3.85 x 10 -10 The affinity or K of M D Binds to human CD38. In some embodiments, the antibody comprises a HC and a LC having the amino acid sequences of SEQ ID NOs: 6 and 9, respectively, and the antibody can be expressed at about 1.91 x 10 -10 The affinity or K of M D Binds to human CD38.

[0161] Binding to FcγRIIIa (CD16a) and FcγRIIa (CD32a)

[0162] The antibodies provided herein can also be expressed with a K D and with a K of less than 100 nM as measured by SPR D The antibodies provided herein bind to a lower affinity FcγRIIIa (CD16a) variant (158F) having a phenylalanine (F) at amino acid position 158 and are capable of binding to a higher affinity FcγRIIIa (CD16a) variant (158V) having a valine (V) at amino acid position 158. In some embodiments, the antibodies provided herein bind to FcγRIIIa (CD16a) with a K of less than 100 nM as measured by SPR. DBinds to the 158F variant and the 158V variant of FcγRIIIa (CD16a) and wherein the binding to the 158F and 158V variants differs by less than 2-fold. In some embodiments, the antibodies provided herein have a K of about 59 nM or less when measured by, for example, SPR. D Binds to FcγRIIIa (CD16a) variant (158F).

[0163] 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 activating the antibody with a K of about 53 nM. D Binds to FcγRIIIa(158F) with a K of approximately 47 nM D Binding to FcγRIIIa(158V) as measured by, for example, 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(158V) with a K of about 59 nM. D Binds to FcγRIIIa(158F) with a K of approximately 75 nM D Binding to FcγRIIIa(158V) as measured by, for example, 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(158V) with a K of about 51 nM. D Binds to FcγRIIIa(158F) with a K of approximately 47 nM D Binding to FcγRIIIa(158V) as measured by, e.g., SPR.

[0164] The antibodies provided herein can also be expressed with a K of ≤690 nM D Binds to a lower affinity FcγRIIa (CD32a) variant (131R) with an arginine at amino acid position 131 with a K of less than about 270 nM. D Binding to a higher affinity FcγRIIa(CD32a) variant (131H) with a histidine at position 131 as measured by, eg, SPR.

[0165] 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 activating the antibody with a K of about 690 nM. D Binds to FcγRIIa(CD32a)(131R) with a K of approximately 120 nM DBinding to FcγRIIa (CD32a) (131H) as measured by, for example, 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 with a K of ≤ about 125 nM or ≤ 100 nM. D Binds to FcγRIIa (CD32a) (131R) with a K of approximately 220 nm D Binds to FcγRIIa (CD32a) (131H) as measured by, for example, 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 FcγRIIa (CD32a) (131H) with a K of about 510 nM. D Binds to FcγRIIa(CD32a)(131R) with a K of approximately 60 nM D Binding to FcyRIIa(CD32a)(131H) as measured by, e.g., SPR.

[0166] In some embodiments, the antibody is further capable of expressing the antibody with an apparent K of about 70 nM or less. D Binds to FcγRIIIa (CD16a)158F with an apparent K of approximately 32 nM or less D Binding to FcγRIIIa(CD16a)158V as measured by binding to HEK cells expressing FcγRIIIa(CD16a)158F or 158V, respectively. In some embodiments, the 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 further capable of binding to FcγRIIIa(CD16a)158V with an apparent K of about 70 nM. D Binds to FcγRIIIa (CD16a)158F with an apparent K of approximately 18 nM D Binding to FcγRIIIa(CD16a)158V as measured by binding to HEK cells expressing FcγRIIIa(CD16a)158F or 158V, respectively. In some embodiments, the 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 further capable of binding to FcγRIIIa(CD16a)158V with an apparent K of about 60 nM. D Binds to FcγRIIIa (CD16a)158F with an apparent K of approximately 32 nM DBinding to FcγRIIIa(CD16a)158V as measured by binding to HEK cells expressing FcγRIIIa(CD16a)158F or 158V, respectively. In some embodiments, the 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 further capable of binding to FcγRIIIa(CD16a)158V with an apparent K of about 44 nM. D Binds to FcγRIIIa (CD16a)158F with an apparent K of approximately 28 nM D Binding to FcγRIIIa(CD16a)158V as measured by binding to HEK cells expressing FcγRIIIa(CD16a)158F or 158V, respectively.

[0167] In some embodiments, the antibody is further capable of expressing the antibody with an apparent K of about 890 nM or less. D Binds to an FcγRIIa (CD32a) variant (131R) with an arginine at amino acid position 131 with an apparent K of approximately 840 nM or less. D Binding to FcγRIIa(CD32a) variant 131H as measured by binding to HEK cells expressing FcγRIIa(CD32a) 131R or 131H, respectively. In some embodiments, the 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 further capable of binding to FcγRIIa(CD32a) variant 131H with an apparent K of about 890 nM. D Binds to FcγRIIa(CD32a)131R with an apparent K of approximately 840 nM D Binding to FcγRIIa(CD32a)131H as measured by binding to HEK cells expressing FcγRIIa(CD32a)131R or 131H, respectively. In some embodiments, the 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 further capable of binding to FcγRIIa(CD32a)131H with an apparent K of about 87 nM. D Binds to FcγRIIa (CD32a) 131R with an apparent K of approximately 222 nM D Binding to FcγRIIa(CD32a)131H as measured by binding to HEK cells expressing FcγRIIa(CD32a)131R or 131H, respectively. In some embodiments, the 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 further capable of binding to FcγRIIa(CD32a)131H with an apparent K of about 467 nM. D Binds to FcγRIIa (CD32a) 131R with an apparent K of approximately 544 nM. DBinding to FcyRIIa (CD32a) 131H, as measured by binding to HEK cells expressing FcyRIIa (CD32a) 131R or 131H, respectively.

[0168] Mechanism of action

[0169] The antibodies provided herein are capable of killing cells expressing CD38 through antibody-dependent cellular cytotoxicity of cells expressing high (about 400,000 / cell), medium (about 100,000 / cell), and low (about 13,000 / cell) levels of CD38 molecules on the cell surface. The antibodies provided herein trigger such ADCC in the presence of natural killer (NK) cells that express the lower affinity FcyIIIa (CD16a) receptor variant (158F) and / or express the higher affinity FcyIIIa (CD16a) receptor variant (158V). ADCC can be measured using methods known in the art, for example, by measuring target cell lysis in the presence of antibody and effector cells in a cell-based potency assay. The target cells can be, for example, cells expressing CD38, such as KMS12-BM, RPMI-8226, or MOLP-8. Additionally, the effector cells can be, for example, any cell that can be induced to kill target cells through ADCC. In some embodiments, the 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 natural killer cell lines, such as NK-92 cells, which express FcyRIIIa (158V) or FcyRIIIa (158F) on the cell surface (see WO 06 / 023148). In still other embodiments, the NK cell line is a cell line such as a Jurkat cell line that has been engineered to express FcyIIIa (CD16a) 158F or 158V (see, e.g., Promega, G701A).

[0170] The antibodies provided herein are capable of killing cells expressing high levels of CD38 through antibody-dependent cellular phagocytosis (ADCP). The antibodies provided herein trigger such ADCP in the presence of human PBMCs. In one embodiment, the antibodies provided herein trigger about 41% phagocytosis of cells expressing CD38 by human PMBCs expressing FcyRIIIa (158V) with a relative EC50 of 212.6 pM.

[0171] Pharmaceutical compositions and formulated antibodies

[0172] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such preparations are generally sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient used.

[0173] Pharmaceutical compositions and formulations of the antibodies provided herein can be prepared by mixing antibodies having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) and can be provided in the form of lyophilized formulations or aqueous solutions.

[0174] In some embodiments, the pharmaceutical compositions provided herein comprise a formulated antibody comprising 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 sequences of any 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 so that the pharmaceutical composition can be diluted for administration by infusion. For example, in some embodiments, sucrose is present at a concentration of 8% (w / v) to 10% (w / v). In some embodiments, sucrose is present at a concentration of 8% (w / v). In some embodiments, sucrose is present at a concentration of 10% (w / v). In some embodiments, L-histidine is present at a concentration of 10mM to 20mM. In some embodiments, L-histidine is present at a concentration of 10mM. In some embodiments, L-histidine is present at a concentration of 20mM. 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, for example, the stability of the antibody 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.

[0175] In some embodiments, the antibodies and / or formulated antibodies of the pharmaceutical compositions provided herein comprise one or more charged isotypes. Charged isotypes are generally described as major isotypes, acidic isotypes, and basic isotypes. The major isotype refers to the most common isotype in a given batch of antibodies. One or more acidic isotypes have a lower isoelectric point (pI) than the major isotype, and the basic isotype has a higher pH than the major isotype. Charged isotypes can be produced by post-translational modification of the amino acid sequence of HC and / or LC. For example, an increase in deamidation, saccharification, and sialylation may result in a decrease in the pI of the antibody. The conversion of N-terminal glutamic acid to pyroglutamic acid (or pyroQ) results in a loss of a positive charge, which may result in a decrease in pI. In addition, the presence of a C-terminal lysine may result in an increase in the pI of the antibody. Furthermore, amidation of a C-terminal proline may result in an increase in the pI of the antibody.

[0176] 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.

[0177] The charged isoforms of the antibodies provided herein can be separated and quantified using methods known in the art for separating polypeptides based on charge. For example, in some embodiments, a weak cation exchange column can be used in a high performance liquid chromatography (HPLC) system with a phosphate / sodium chloride gradient buffer. In this system, after the antibody is loaded onto the column, the acidic isoform of the antibody is eluted first, followed by the major isoform of the antibody, and then the acidic isoform. In another embodiment, capillary isoelectric focusing (cIEF) can be used. Capillary isoelectric focusing is a method for separating proteins by their isoelectric point (pI) values. In cIEF, an antibody sample migrates to its isoelectric point on a pH gradient within a capillary, thereby resolving different charged isoforms along the length of the capillary. In order to identify and characterize cIEF variants, charged isoforms can be separated by strong cation exchange chromatography (SCX) and analyzed by cIEF. Using full column detection and measuring the absorbance at 280 nm, a charge-coupled device camera is used to collect images of the charged isoforms resolved in the capillary. The charged isotype distribution of a test sample is compared to a reference standard using cIEF to identify the charged isotype of the antibody. In some embodiments, cIEF is used as a quality control measure to confirm the identity of an antibody during commercial production, and the charged isotype distribution of an antibody is determined by comparison to a reference standard (e.g., a test antibody having a charged isotype distribution pattern within a predefined distribution pattern) or by comparison to the distribution pattern of a reference standard.

[0178] In some embodiments, the antibodies (including antibodies in pharmaceutical compositions and formulated antibodies) comprise the HC and LC amino acid sequences of any one of the monoclonal antibodies provided in Table 1, and one or more charged isotype parameters selected from at least about 70% major isotype, no more than about 30% acidic isotype, and less than 4% basic isotype. In some embodiments, the antibodies comprise 71% major isotype, 28% acidic isotype, and less than 4% basic isotype. In some embodiments, the antibodies contain charged isotypes having an isoelectric point in the range of about 5.8 to about 9.0. In some embodiments, the antibodies contain charged isotypes having an isoelectric point in the range of about 5.85 to about 8.97.

[0179] In some embodiments, the antibody (including the antibody in the pharmaceutical composition and the antibody formulated) is lyophilized. The antibody (including the antibody in the pharmaceutical composition and the antibody formulated) can be in a container (e.g., a vial) so that a prescribed dose of the antibody can be removed from the container for administration to a patient.

[0180] Provided herein are unit dosage forms of formulated antibodies that specifically bind 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 sucrose, L-histidine, and polysorbate 80. In some embodiments, the unit dosage form comprises approximately 215 mg of antibody, approximately 6.21 mg of L-histidine, approximately 344 mg of sucrose, and approximately 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 prescription dose of antibody can be removed from the container for administration to a patient. In some embodiments, the antibody, pharmaceutical formulation, and / or formulated antibody is in a glass vial equipped with an elastic closure. In some embodiments, the vial contains approximately 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 so that about 4 mL can be pipetted. In some embodiments, the fill volume of the vial has been determined so that 4 mL can be pipetted.

[0181] Preparation method

[0182] Provided herein are methods for preparing antibodies, pharmaceutical compositions, and unit dosage forms. In some embodiments, the antibody comprises the HC and LC amino acid sequences of any of the monoclonal antibodies provided in Table 1. In one embodiment, the method comprises expressing the antibody in a suitable cell culture. The antibody is subjected to at least one chromatography step and at least one ultrafiltration step to produce a purified antibody solution. The purified antibody solution is adjusted to concentrate the antibody, add excipients, and adjust the pH. In one embodiment, the concentration of the antibody is adjusted to approximately 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 concentration of the antibody 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 approximately 6.2. In some embodiments, the pH is 6.2. In some embodiments, the concentration of sucrose is approximately 8% w / v, the concentration of L-histidine is approximately 10 mM, and the concentration of polysorbate 80 is approximately 0.05% v / v. In some embodiments, the concentration of sucrose is 8% w / v, the concentration of L-histidine is 10 mM; and the concentration of polysorbate 80 is 0.05% v / v. In some embodiments, the antibody, pharmaceutical composition, and / or formulated antibody is lyophilized.

[0183] In some embodiments, methods are provided for preparing a reconstituted formulated antibody, wherein the formulated antibody is provided in a lyophilized form and comprises the HC and LC amino acid sequences 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, methods are provided for preparing a reconstituted formulated antibody, wherein the formulated antibody is provided in a lyophilized form and comprises the HC and LC amino acid sequences 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 comprising the antibody at a concentration of about 50 mg / mL, 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, wherein 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 comprising the 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 at a concentration of 0.05% v / v, wherein the pH of the reconstituted antibody is 6.2.

[0184] As used herein, the term "treatment" or "treating" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during a clinical pathological process. Desirable therapeutic effects include a reduction in the rate of disease progression, improvement or alleviation of the disease state, and regression or improved 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 cancer cells, destroying cancer cells, reducing the symptoms caused by the disease, improving the quality of life of those individuals suffering from the disease, reducing the dosage of other drugs required to treat the disease, and / or prolonging the survival of an individual.

[0185] Provided herein are methods for treating multiple myeloma (e.g., relapsed multiple myeloma or relapsed and refractory multiple myeloma) in an individual or delaying its progression, the methods 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 the survival of mice in a murine MM model, wherein the mice in the model express a low-affinity human CD16a variant (158F), and wherein 500,000 EL4 cells expressing human CD38 have been injected into the mice. In some embodiments, one or more doses of the anti-CD38 antibody are provided in the form of a lyophilized formulation, wherein prior to administration, the lyophilized formulation is reconstituted to form a reconstituted antibody formulation such that the reconstituted antibody formulation has a pH of 6.2 and comprises an antibody concentration of about 50 mg / mL in a liquid comprising about 10 mM L-histidine, about 8% w / v sucrose, and about 0.05% v / v polysorbate 80. In some embodiments, one or more doses of an anti-CD38 antibody are provided in the form of a lyophilized formulation, wherein prior to administration, the lyophilized formulation is reconstituted to form a reconstituted antibody formulation such that the reconstituted antibody formulation has a pH of 6.2 and comprises the antibody at a concentration of 50 mg / mL in a liquid comprising 10 mM L-histidine, 8% w / v sucrose, and 0.05% v / v polysorbate 80.

[0186] In some embodiments, one or more doses of an antibody are administered to a 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 the 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 the antibody. In some embodiments, the antibody is administered intravenously. In some embodiments, the antibody is administered subcutaneously.

[0187] definition

[0188] 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 a combination of two or more such molecules, and so forth.

[0189] As used herein, the term "about" refers to the usual error range for the corresponding value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments involving the value or parameter itself. In some embodiments, the term "about" means the specified value plus or minus 10% of the value; for example, "about 10 mg / kg" can encompass 9 to 11 mg / kg. In some embodiments, the term "about" means the specified value plus or minus 5% of the value; for example, "about 20 mg / kg" can encompass 19 to 21 mg / kg.

[0190] "Subject" or "individual" for purposes of treatment refers to any animal classified as a mammal, including humans, poultry and farm animals, as well as zoo animals, sports animals or pets such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

[0191] To illustrate and describe certain specific embodiments of the present invention, the following examples are set forth. However, the scope of the claims should not be limited in any way by the examples set forth herein.

[0192] Example

[0193] 1. Antibody design:

[0194] Fc-engineered anti-CD38 antibodies were generated that possess mutations in the Fc portion of the antibody to increase affinity for activating FcγRIIIa and FcγRIIa receptors on effector cells.

[0195] The two humanized heavy chains (HC) (SEQ ID NO: 5 and SEQ ID NO: 6) carry 14 and 10 mutations in the framework regions (compared to SEQ ID NO: 1), which resulted in an increase in the Homo sapiens germinality score from 74.49% to 88.78% and 84.69%, respectively. The two humanized light chains (LC) (SEQ ID NO: 8 and SEQ ID NO: 9) carry 17 and 15 mutations in the framework regions (compared to SEQ ID NO: 7), which resulted in an increase in the Homo sapiens germinality score from 64.36% to 81.19% and 79.21%, respectively. In addition, in LCs 2 and 3, the methionine at position 11 was substituted 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.

[0196] Table 2

[0197]

[0198]

[0199]

[0200] Table 3

[0201]

[0202]

[0203] 2. Biochemical characterization:

[0204] A: Binding to CD38

[0205] In one experiment, using The instrument measured the binding affinity and kinetic constants of mAbs 1-8 for human CD38 by SPR. Briefly, anti-CD38 antibodies were captured onto a CM5 chip covalently bound to an anti-Fc antibody. Various concentrations of huCD38 were injected onto the captured antibody being tested. Binding curves (sensorgrams) were generated and kinetic analysis was performed using Biacore evaluation software.

[0206] As shown in Table 4, mAbs 3 and 8 had the highest affinity for human CD38 (T-test for each variant: p-value > 2).

[0207] Table 4 Affinity for human CD38 (n=3)

[0208]

[0209] In another experiment, the The instrument measures the binding affinity of mAbs 1 and 3 to human CD38 by SPR. mAb 1 has a Kd of 0.22 nM and mAb 3 has a Kd of 0.20 nM.

[0210] Effect of thermal stress on binding affinity to human CD38: Using The instrument measures the binding affinity and kinetic constants of mAbs 1-8 to human CD38 after the mAbs were subjected to thermal stress (14 days at 40°C in 10 mM histidine pH 6, 8% sucrose, 0.02% PS80). The same protocol described in the previous paragraph has been used.

[0211] As shown in Table 5, the samples subjected to thermal stress show very similar affinity and kinetic constants to the corresponding non-stressed samples CD38 (Table 4) (T-test for each variant: p-value > 2).

[0212] Table 5 Effect of thermal stress on affinity to human CD38 (n=3)

[0213]

[0214] B: Affinity to FcyRIIa, FcyRIIIa and FcyRIIb measured by computer

[0215] In one experiment, the affinity of mAbs to FcyRIIIa 158V receptor or FcyRIIIa 158F receptor 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). FcyRIIIa (CD16a) extracellular domain with V158 or F158 with a C-terminal histag were produced in HEK293 cells by transient expression according to the supplier (Ni-Sepharose GE, Healthcare) and purified by immobilized metal affinity chromatography (IMAC). The affinity was measured by SPR on a BIACore2000 (GE Healthcare) via a capture assay using anti-His IgG immobilized on a CM5 chip. FcyRIIIa-histag was then added to the running buffer followed by anti-CD38 mAbs at concentrations ranging from 8 to 256 nM. Analysis was performed using BIAevaluation Software 4_1 (GE Healthcare) with a two-state reaction, where acceptable χ2 2The value is less than 5 (meaning 2% of the highest tested concentration) and the percentage of the maximum theoretical resonance units is greater than 25 (meaning more than 1 / 4 of the interactions are productive).

[0216] As shown in Table 6, mAb 2-4 had a 9-fold higher affinity for FcγRIIIa 158V compared to mAb 1, and mAb 2-4 had a more than 27-fold higher affinity for the FcγRIIIa 158F receptor compared to mAb 1.

[0217] Table 6

[0218]

[0219] In another experiment, the affinity of mAbs 1 and 3 for the FcγRIIIa 158V receptor or the FcγRIIIa 158F receptor was measured using SPR as described above. mAb 3 had a K of 75 nM. D Binds to FcγRIIIa 158V, and mAb 3 binds with a K of 59 nM D Binds to FcγRIIIa 158F.

[0220] The affinity of the mAbs for the FcγRIIa receptor with either arginine at amino acid position 131 (FcγRIIa 131R) or histidine at amino acid position 131 (FcγRIIa 131H) was measured using the AlphaScreen proximity assay.

[0221] FcγRIIa extracellular domains with R131 or H131 were 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), which allows sorting of anti-CD38 Fc variants by competing with anti-CD38 with natural IgG1. Biotinylated anti-CD38 with natural IgG1 was captured on donor beads, and FcγRIIa histag protein was captured on acceptor beads. When anti-CD38 interacted with FcγRIIa, the beads became proximal. Excitation of the donor beads at 680nm caused the release of singlet oxygen, which, when in the proximal state, diffused and triggered the emission of light at 520-620nm from the acceptor beads. The amount of light was proportional to the degree of interaction and was measured using an EnVision multi-mode plate reader (Perkin Elmer). During the competition assay, unlabeled competing mAbs 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 for the interaction between biotinylated anti-CD38 and FcγRIIa-histag protein with native IgG1. The concentration of biotinylated anti-CD38 was set by a pre-performed calibration curve. Unlabeled competing mAbs were used at different levels so that increasing concentrations of unlabeled mAbs could displace biotinylated anti-CD38 and reduce the signal. Data were normalized to the maximum signal. Two experiments were run and IC50s were estimated using BIOST@T-SPEED-LTS2.1.0.

[0222] As shown in Table 7, mAb 3 showed over 14-fold and 18-fold higher affinity than mAb 1 for FcγRIIa 131R and FcγRIIa 131H, respectively.

[0223] Table 7 Affinity for FcγRIIa R131 and FcγRIIa H131

[0224]

[0225] C: Measurement of affinity for FcγRIIa, FcγRIIIa, and FcγRIIb in vitro

[0226] HEK293T-FcγRIIIa-158F, HEK293T-FcγRIIIa-158V, HEK293T-FcγRIIa-131H, HEK293T-FcγRIIa-131R, HEK293T-FcγRI or HEK293T-FcγRIIb were added at 10 5 Cells / well were seeded in 96-well microplates. These plates were centrifuged at 800 g for 1 minute and resuspended in 50 μL PBS containing different concentrations of antibodies at 4 ° C for 30 minutes. 200 μL PBS1% FBS was then added to wash the cells, followed by centrifugation at 800 g for 1 minute. The washing step was repeated three times in total, and then the cells were stained with a secondary antibody (fragment goat anti-human IgG (H+L) FITC, numbering 109-546-088 Jackson ImmunoResearch, final 10 μg / mL) conjugated to FITC for 30 minutes at 4 ° C. The cells were washed three times with 200 μL PBS1% FBS and resuspended in 100 μL PBS, and then acquired on MACSVYB (B1 channel). As shown in Table 8, mAbs 2, 3, and 4 had 32-fold, 38-fold, and 51-fold higher affinities for FcγRIIIa 158F expressed on HEK cells, respectively, compared to mAb 1, and mAbs 2, 3, and 4 had 9-fold, 5-fold, and 6-fold higher affinities for FcγRIIIa 158V expressed on HEK cells, respectively, compared to mAb 1.

[0227] Table 8

[0228]

[0229] As shown in Table 9, mAb 3 had approximately 5-fold higher affinity for FcγRIIa 131H expressed on HEK cells compared to mAb 1, and mAb 3 had approximately 16-fold higher affinity for FcγRIIa 131R expressed on HEK cells compared to mAb 1.

[0230] Table 9

[0231]

[0232] As shown in Table 10, mAbs 2, 3, and 4 had similar levels of binding affinity for FcγRI compared to mAb 1, and mAbs 2, 3, and 4 had 1.8- to 3.9-fold lower affinity than mAb 1 for FcγRIIb expressed on HEK cells.

[0233] Table 10

[0234]

[0235] D: Measurement of cytotoxic activity in vitro

[0236] Antibody-dependent cellular cytotoxicity (ADCC):

[0237] Calcein-acetoxymethyl (Calcein-AM; Invitrogen) release assay was used to examine antibody-dependent cellular cytotoxicity (ADCC) assays using an NK92 cell line engineered to overexpress FcγRIIIa receptors as effector cells. In living cells, non-fluorescent calcein AM was converted to green fluorescent calcein. Labeled target cells were incubated with SAR442085 antibody and NK-92 effector cells. Dissolution of target cells by ADCC resulted in the release of fluorescent calcein. Fluorescence intensity was proportional to the level of ADCC activity present.

[0238] Multiple myeloma target cells (MOLP-8, RPMI 8226, MM1R, or KMS12-BM) were incubated with Calcein-AM (50 μg diluted in 25 μL DMSO, followed by 10 μL of Calcein diluted in 4 mL RPMI 1640 + 1% FBS + 1% probenecid for 4 × 10 6 cells) were labeled for 30 min, then washed and washed with 2 × 10 4 Cells were plated at a density of 10 cells / well in 96-well round-bottom plates. Different concentrations of the indicated test mAbs or control isotype mAbs were added from 10 μg / mL to 0.01 pg / mL for 30 min to allow opsonization, after which natural killer (NK) cells transduced with the indicated FcγRIIIa variants were added. The cells were cultured at an E:T ratio of 5:1 (1×10 5 NK cells for 2×10 4 Target cells) were added with NK cells expressing 158F or 158V as effector cells. These plates were then incubated at 37°C in a humidified incubator with 5% CO2 for 1 hour, and 100 μL of supernatant was harvested and transferred to an opaque 96-well microplate for analysis using fluorescence determination on a Tecan Infinite M1000 to measure calcein release (excitation filter: 492 nm; emission: 515 nm). For maximum release, cells were dissolved with 2% TritonX-100. The fluorescence value of the culture medium background was subtracted from the fluorescence value of the experimental release (A), spontaneous release of target cells (B), and maximum release of target cells (C). The cytotoxicity and ADCC percentages of each plate (in duplicate) were calculated using the following formula:

[0239] Cytotoxicity (%) = (AB) / (CB) × 100%

[0240] Each experiment was repeated at least three times. Half-maximal effective concentration (EC50) values ​​were calculated by fitting the data points to a 4-parameter equation using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA).

[0241] Target cells with high CD38 receptor density

[0242] ADCC of the multiple myeloma cell line MOLP-8 was assessed in vitro as described above. MOLP-8 cells exhibit a high density of CD38 receptors on the cell surface (approximately 400,000 per cell).

[0243] Table 11 shows the EC50 of each tested antibody against MOLP-8 cells in the presence of NK cells expressing 158V. As shown in Table 11, mAbs 2, 3, and 4 triggered ADCC of MOLP-8 cells with EC50s 24 to 33 fold lower than mAb 1 in the presence of NK cells expressing the high affinity FcγRIIIa variant (158V).

[0244] Table 11

[0245]

[0246] Table 12 shows the EC50 of each test antibody against MOLP-8 cells in the presence of NK cells expressing 158F. As shown in Table 12, in the presence of NK cells expressing low-affinity FcγRIIIa variants (158F), mAb 4 showed the strongest ADCC activity against MOLP-8 cells, with an EC50 approximately 97-fold lower than mAb 1. In the presence of NK cells expressing low-affinity FcγRIIIa variants (158F), mAb 2 and 3 triggered ADCC of MOLP-8 cells with an EC50 approximately 59 to 69-fold lower than mAb 1.

[0247] Table 12

[0248]

[0249] Medium CD38 receptor density target cells

[0250] ADCC of the multiple myeloma cell line RPMI-8226 was assessed in vitro as described above. RPMI-8226 cells exhibit a moderate density of CD38 receptors on the cell surface (approximately 70,000 per cell).

[0251] Table 13 shows the EC50 of each test antibody against RPMI-8226 cells in the presence of NK cells expressing 158V. As shown in Table 13, mAb 2-4 showed similar increased levels of ability to trigger ADCC against MM cells expressing intermediate CD38 receptor density in the presence of NK cells expressing the higher affinity FcyRIIIa variant (158V), with an EC50 that was 27-fold lower compared to mAb 1.

[0252] Table 13

[0253]

[0254] Table 14 shows the EC50 of each test antibody against RPMI-8226 cells in the presence of NK cells expressing 158F. In the presence of NK cells expressing the low affinity FcyRIIIa variant, mAb 4 showed about a 73-fold increased ability to trigger ADCC against MM cells expressing intermediate CD38 receptor density compared to mAb 1; while in the presence of NK cells expressing the low affinity FcyRIIIa variant, mAb 3 showed about a 65-fold increased ability to trigger ADCC against MM cells expressing intermediate CD38 receptor density compared to mAb 1 and mAb 2 showed about a 49-fold increased ability of said triggering.

[0255] Table 14

[0256]

[0257] Low CD38 receptor density target cells

[0258] ADCC of the multiple myeloma cell line KMS-12BM was assessed in vitro as described above. KMS-12BM cells exhibit low CD38 receptor density on the cell surface (about 13,000 per cell).

[0259] Table 15 shows the EC50 of each test antibody against KMS-12BM cells in the presence of NK cells expressing the higher affinity FcyRIIIa variant (158V). Interestingly, as shown in Table 15, mAb 2, 3 and 4 showed increased ability to trigger ADCC against MM cells expressing low CD38 receptor density in the presence of NK cells expressing the higher affinity FcyRIIIa variant (158V) compared to mAb 1. mAb 3 had an EC50 that was about 69-fold lower than mAb 1 and mAb 4 had an EC50 that was about 91-fold lower than mAb 1.

[0260] Table 15

[0261]

[0262] Table 16 shows the EC50 of each test antibody against KMS-12BM cells in the presence of NK cells expressing lower affinity FcγRIIIa variants (158F). Significantly, as shown in Table 16, in the presence of NK cells expressing lower affinity FcγRIIIa variants (158F), mAb 2, 3, and 4 showed improved ability to trigger ADCC against MM cells expressing low CD38 receptor density 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.

[0263] Table 16

[0264]

[0265] Antibody-dependent cellular phagocytosis (ADCP):

[0266] First, from the French Blood Research Institute (Etablissement Human PBMCs were isolated from buffy coats of seven healthy donors from the laboratory of Dr. du Sang. All of these donors exhibited the same FCGR3A and FCGR2A genotypes (FcγRIIIa-158V / V and FcγRIIa-131H / H). Blood was first collected in 50 mL Falcon tubes. 15 mL Ficoll-Plaque TMPLUS 96% gently added to the bottom of the Sepmate tube, then blood was slowly added to the tube, after which it was centrifuged at 1,300g for 10 minutes. The PBMC ring was then collected and washed with 50 mL of PBS and centrifuged at 300g for another round of 5 minutes. The washing process was repeated twice, after which the PBMCs were stained with anti-CD14 magnetic beads according to the manufacturer's instructions (Miltenyi; ref. 130-050-201): incubation time was essentially 15 minutes at 4°C, after which a positive selection was performed via AutoMACS pro (Posseld selection). Mononuclear cells were collected and then washed with 50 mL of PBS. Mononuclear cells were cultured in T75 flasks in RPMI1640, 10% FBS, 2% inactivated human serum, 50 ng / ml GM-CSF at 37°C, 5% CO2 for 5 days. After 5 days of culture, mononuclear cells differentiated into macrophages. To collect macrophages, a cell digest (ThermoFisher, ref. A1110501) was placed in the flask at 37°C for 15 minutes to detach the cells, then 20 mL of RPMI 10% FBS 1% glutamine was added to stop the cell digest activity. The collected cells were centrifuged at 350g for 10 minutes, then washed in 20 mL of PBS and centrifuged at 300g for another round of 10 minutes. For both experiments, the macrophages used were from a batch of macrophages frozen in liquid nitrogen.

[0267] Macrophages were resuspended at 20 x 10 6 cells / mL in diluent C (provided in the manufacturer's staining kit from Sigma Aldrich ref. PKH26GL-1KT). For 1 volume of macrophages, 1 volume of PKH26 (20 μΐ, diluted in 1 mL of diluent C) was added to the cells to incubate in the dark for 5 minutes. Then 5 mL of FBS was added to deactivate the staining process and for 1 minute, after which it was made up to 50 mL with RPMI1640 10% FBS. CD38-expressing MOLP-8 cells were stained with PKH67 fluorescent dye and macrophages obtained from purified mononuclear cells were stained with PKH26, so that they could be identified by flow cytometry. MOLP-8 cells and macrophages were then placed in culture in the presence of mAb 1 or mAb 3 overnight, after which a double positive population (macrophages phagocytosing MOLP-8 cells) analysis was performed by flow cytometry.

[0268] As demonstrated by the higher total phagocytosis percentage (%) and lower EC50 (relative) value (EC50rel) obtained in all experiments, mAb 3 shows the ADCP activity for the MOLP-8 cell line improved compared with mAb 1. The geometric mean of total phagocytosis % is 40.85% for mAb 3, and 18.57% for mAb 1, and the geometric mean of EC50rel is 31.87ng / mL (or 212.57pM) for mAb 3, and for mAb 1, described value exceeds 64.86ng / mL (or exceeds 432.62pM). Based on EC50rel value, mAb 3 is better than mAb 1 at least 2.035 times in ADCP activity.

[0269] In vivo effects

[0270] The in vivo efficacy of mAb 3 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 were deleted of all murine genes encoding FcγRs and human FcγRs encoded as transgenes were inserted into the mouse genome. Humanized FcγR C57BL / 6 mice recapitulate the huFcγR expression pattern and expression levels and are functional in multiple huIgG-mediated models of inflammation, cytotoxicity, and tumor clearance. For the activating FcγRIIIa and FcγRIIa receptors, the human variants inserted into the mouse model were huFcγRIIIa 158F and huFcγRIIa 131R (a low affinity variant of the receptor), respectively. The characteristics of human FcγR expression patterns and expression levels are recapitulated in mice.

[0271] Starting on day 1 after tumor cell injection, test or control mAbs were administered intraperitoneally on days 1, 4, 7, and 14. Isotype control mAbs were administered at 10 mg / kg. mAbs 1, 3, and 10 were administered at 10 and 1.25 mg / kg. In this survival model, the primary efficacy endpoints were median survival time (MST), percentage of extended lifespan (ILS%), and long-term survivors (defined as a survival duration greater than or equal to twice the MST of the control group). Cox regression was used to evaluate differences in survival between groups.

[0272] 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 10 mg / kg of mAb 1 had an MST of over 82.5 days and a lifespan extension of greater than 112%. Fifty percent of the group treated with 10 mg / kg of mAb 1 were long-term survivors. Mice treated with 1.25 mg / kg of mAb 1 had an MST of greater than 46.5 days and a lifespan extension of greater than 19%. Thirty percent of the group treated with 1.25 mg / kg of mAb 1 were long-term survivors.

[0273] The group treated with 10 mg / kg of mAb 10 had an MST greater than 70 days and a 70% extended lifespan. Fifty percent of the group treated with 10 mg / kg of mAb 10 were long-term survivors. Mice treated with 1.25 mg / kg of mAb 10 had an MST greater than 42 days and an 8% extended lifespan. Only 10% of the group treated with 1.25 mg / kg of mAb 10 were long-term survivors.

[0274] The group treated with 10 mg / kg of mAb 3 had an MST of over 90 days and an extended lifespan of greater than 131%. Remarkably, 90% of the group treated with 10 mg / kg of mAb 3 were long-term survivors. 10 mg / kg of mAb 3 was statistically significantly more active than the isotype control group (p=0.0351). Even more strikingly, mice treated with 1.25 mg / kg of mAb 3 also had an MST of greater than 90 days and an extended lifespan of greater than 131%. Furthermore, 90% of the group treated with 1.25 mg / kg of mAb 3 were long-term survivors. 1.25 mg / kg of mAb 3 was statistically significantly more active than the isotype control group (p=0.0380) and statistically significantly superior to mAb 10 at the same dose (p=0.0380).

[0275] Treatment of this tumor model in mice bearing low-affinity huFcγRIIIa (F158 / F158) with mAb 1, mAb 3, and mAb 10 at a dose of 10 mg / kg resulted in statistically significant improvements in lifespan and the number of long-term survivors. However, surprisingly, at a dose of 1.25 mg / kg, mAb 3 still exhibited a statistically significant improvement in lifespan and the number of long-term survivors, while mAb 1 and mAb 10 were inactive.

[0276] Table 17

[0277]

[0278] E. Stability Study

[0279] Thermal stability

[0280] 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 a buffer solution containing 10 mM histidine HCl (pH 6.0). The prepared samples were loaded into each well of a Wheaton 96-well round-bottom plate in triplicate along with a buffer solution as a reference. Thermal scans were collected and processed using Origin software 2.0 using a temperature ramp of 1°C / min and a temperature range of 20°C-120°C.

[0281] As shown in Table 18, mAbs 3, 5, 6, 7, and 8 exhibited protein conformational changes at approximately 40°C, which is approximately 17°C lower than the temperature at which mAb 1 appears and approximately 15°C-20°C lower than the typical temperature for mAbs ( Figure 1 Furthermore, at approximately 50°C relative to 70°C, the unfolding of the CH2 domains of mAbs 3, 5, 6, 7, and 8 began much earlier than that of mAb 1.

[0282] Table 18 Thermal stability (℃)

[0283]

[0284] Isoelectric point

[0285] 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 in Cys residues were assumed to be disulfide-bonded and not contribute to the pKa (Laue TM, Shah BD, Ridgeway TM, and Pelletier SL. Computer-aided interpretation of analytical sedimentation data for protein. In Analytical Ultracentrifugation in Biochemistry and Polymer Science. Harding SE, Rowe AJ, and Horton JC, eds., pp. 90-124. Royal Society of Chemistry, 1991).

[0286] Surprisingly, as shown in Table 19, the measured pi for mAb 3 was lower than the calculated pi based on the sequence.

[0287] Table 19

[0288]

[0289]

[0290] Development of mAb 3 formulations

[0291] Based on the thermal stability results, lyophilized formulations were developed to ensure sufficient stability of mAb 3 to be suitable for storage of mAb at ≤-30°C and storage of lyophilized mAb at 2°C-8°C. mAb 3 formulations using different buffer systems and at different pH were tested in stability studies that included freeze-thaw cycles, agitation studies, and short-term (12 weeks) incubation under stress, accelerated, and expected storage conditions at 40°C, 25°C, 5°C, -30°C, and -80°C. In these experiments, in addition to particle formation (visual inspection, light blur-based particle counting, and microfluidic imaging), protein aggregation and chemical degradation were also evaluated.

[0292] Based on the results from formulation development studies, a formulation containing 10 mM L-histidine-HCl, 8% w / v sucrose, and 0.05% w / v polysorbate 80 (pH 6.2) was selected.

[0293] Freeze-drying process development

[0294] The lyophilization process of mAb 3 is developed to ensure that the antibody of preparation has acceptable stability and cake attribute after lyophilization.Use laboratory-scale freeze dryer (Genesis EL35 manufactured by SP Scientific), based on multiple development runs, determine the process parameters (drying temperature, chamber pressure etc.) of lyophilization cycle.The robustness of circulation is to set up by carrying out a series of lyophilization runs under the controlled offset of freezing rate, drying temperature and chamber vacuum pressure.The stability study of the antibody of lyophilized preparation is included in the short-term (12 weeks) hatching under the stress, acceleration and expected storage conditions of 40 ℃, 25 ℃ and 5 ℃.In these experiments, except gathering, chemical degradation and particle formation, also evaluated lyophilized powder attribute (cake appearance, reconstruction time, oxygen head space, moisture content).Based on formulation and lyophilized development research, the composition of mAb 3 is selected as 50mg / mLmAb 3, 10mM L-histidine-HCl, 8%w / v sucrose, 0.05%w / v polysorbate 80 and pH is 6.2. Store formulated antibodies at ≤ -30°C and store lyophilized formulated antibodies at 2-8°C.

Claims

1. An antibody that specifically binds to human CD38, wherein the antibody comprises 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.

2. A pharmaceutical composition comprising a formulated antibody, wherein the formulated antibody comprises an antibody, sucrose, L-histidine, and polysorbate 80 (PS80), wherein the antibody specifically binds to human CD38 and is present at a concentration of 50 mg / mL, the sucrose is present at a concentration of 8% (w / v), the L-histidine is present at a concentration of 10 mM, and the PS80 is present at a concentration of 0.05% (v / v), wherein the formulation has a pH of 6.2, and wherein the antibody comprises 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.

3. A unit dosage form of a formulated antibody that specifically binds to human CD38, wherein the formulated antibody comprises 215 mg of the antibody, 6.21 mg of L-histidine, 344 mg of sucrose, and 2.15 mg of polysorbate 80, wherein the formulated antibody is lyophilized, and wherein the antibody comprises 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.

4. The pharmaceutical composition according to claim 2 or the unit dosage form according to claim 3, wherein the formulated antibody is lyophilized.

5. The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 4, wherein the amino acid at position 1 of the HC amino acid sequence is pyroglutamine.

6. The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 5, wherein the antibody has an isoelectric point (pi) of 5.8 to 9.0 when measured by capillary isoelectric focusing (cIEF).

7. The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 6, wherein the antibody comprises a major charged variant and at least one acidic charged variant and wherein the antibody has at least one of the following a) the HC of the at least one charged variant comprises at least one deamidated asparagine selected from N289, N318, N387 and N392 as numbered according to SEQ ID NO: 1, or b) the antibody comprises a predominantly charged variant and at least one acidic charged variant, wherein the predominantly charged variant comprises at least 71% of the antibody and the acidic charged variant comprises no more than 30% of the antibody, or c) the major charged variant has an isoelectric point (pi) of 7.5 and wherein the one or more acidic charged variants have a pi of 5.8 when measured by capillary isoelectric focusing (cIEF).

8. The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 7, wherein the antibody comprises at least one basic charged variant, and wherein the antibody has at least one of the following a) the basic charged variants comprise no more than 4% of the antibody, or b) the one or more basic charged variants have a pi of 9.0 when measured by capillary isoelectric focusing (cIEF).

9. The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 8, wherein the antibody a) is capable of killing cells expressing CD38 by antibody-dependent cellular cytotoxicity (ADCC) in the presence of natural killer cells expressing 158F, 158V, or both 158F and 158V variants of CD16a (FcγRIIIa), wherein the cells have a CD38 receptor density of ≤13,000 CD38 sites on the cell surface, and / or b) with a K of 59 nM as measured by surface plasmon resonance (SPR) D Binds to CD16a (FcγRIIIa) (158F) with a phenylalanine at amino acid position 158, and wherein the antibody has a K of 75 nM as measured by SPR. D binds to CD16a with valine at amino acid position 158 (158V), and / or c) with a K of 96 nM as measured by binding to HEK cells expressing FcγRIIIa 158F D Binds to CD16a (FcγRIIIa) with a phenylalanine at amino acid position 158 (158F), and wherein the antibody binds with a K of 40 nM as measured by binding to HEK cells expressing FcγRIIIa 158V D binds to FcγRIIIa with valine at amino acid position 158 (158V), and / or d) with a K of 94 nM as measured by binding to HEK cells expressing FcγRIIa 131R D binds to CD32a (FcγRIIa) with an arginine at amino acid position 131 (131R), and wherein the antibody binds to CD32a with an arginine at amino acid position 131 (131R) with a K of 222 nM as measured by binding to HEK cells expressing FcγRIIa 131H D Binds to FcγRIIa with histidine at amino acid position 131 (131H).

10. The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 8, wherein a) in the presence of natural killer cells expressing 158F, 158V, or both 158F and 158V variants of CD16a (FcγRIIIa), the antibody is capable of killing by ADCC cells expressing CD38, wherein the cells have a density of >400,000 CD38 receptors on the cell surface; and is capable of killing by ADCC cells expressing CD38, wherein the cells have a density of ≥100,000 CD38 receptors on the cell surface; and is capable of killing by ADCC cells expressing CD38, wherein the cells have a density of ≤13,000 CD38 receptors on the cell surface, and / or b) the antibody is capable of killing cells expressing CD38 by antibody-dependent cellular phagocytosis (ADCP) in the presence of human peripheral blood mononuclear cells (PMBCs), wherein the cells have a density of >400,000 CD38 receptors on the cell surface.

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