TW202104269A - Anti-cd38 antibodies and formulations - Google Patents

Abstract

Provided herein are antibodies that specifically bind human CD38, formulations and unit dosage forms comprising the antibodies, methods of preparing the antibodies and methods of using the antibodies.

Description

Anti-CD38 antibodies and formulations

Provided herein are anti-CD38 antibodies with improved cytotoxic activity and stable formulations thereof.

CD38 is a type II glycosylated 45k canal ear swallow (kDa) membrane protein, which is identified as a lymphocyte marker. CD38 plays a role in white blood cell homeostasis by regulating the survival and differentiation of hematopoietic cells (Richards JO, et al., Mol Cancer Ther. 2008; 7(8): 2517-27). CD38 acts as a receptor that binds to CD31, and is involved in cell adhesion and signal transduction. The functions of CD38 in signal transduction appear 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 nicotine amide 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 signal transduction (Derer S, et al., MAbs. 2014; 6(2):409-21).

The expression of CD38 in healthy humans can be detected on NK cells, monocytes, dendritic cells, macrophages, granulocytes, activated T and B cells, and plasma cells. In contrast, no manifestations have 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 disease (for example, multiple myeloma (MM), amyloidosis) and other hematopoietic cancers (including, including, for example, Waldenstrom's disease). disease), non-Hodgkin’s lymphoma (NHL), acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML)).

The performance of CD38 is particularly evident in MM, because> 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 colonizing MM cells contrasts with the limited expression pattern on normal cells, which indicates that this antigen can be used to specifically target tumor cells.

MM is a malignant plasma cell disease characterized by CD38 expression on the cell surface, proliferation of plasma cells in the bone marrow (BM), and excess monoclonal immunoglobulin (usually IgG or IgA type or free urine light chain (also called It is the production of paraprotein, M protein or M component). This is a disease mainly related to aging, and more than 80% of patients are over 60 years old.

The course of MM varies with the aggressiveness of the disease and related prognostic factors. Certain chromosomal abnormalities in multiple myeloma have been shown to be related to poor clinical outcomes. High-risk cytogenetic changes include del(17p), t(4;14), t(14;16), and 1q increase. In the past two decades, the median survival has increased from 3 years to 6 years; however, some patients can live 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 younger than about 65 years old, in good health, and presenting with symptomatic active disease will usually receive autologous stem cell transplantation (ASCT) therapy first. In order to achieve cell reduction of the disease before collecting the stem cells, induction chemotherapy is administered. Induction treatment regimens include alkylating agents, dexamethasone alone, thalidomide plus dexamethasone, as well as vincristine, Adriamycin® (adriamycin) and dexamethasone (VAD; or a modification of this regimen); However, the latter two regimens are accompanied by higher toxicity (Richardson PG, et al., Blood. 2010; 116(5):679-86; Arnulf B, et al., Haematologica. 2012; 97:1925-8). The use of Velcade® (bortezomib), the combination of bortezomib and Revlimid® (lenalidomide) plus dexamethasone as induction therapy has shown improved results, 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. People, Lancet. 2017; 389:519-27). Other approved drugs include ^{pomalidomide (in the same class of IMiD ®} as lenalidomide) and carfilzomib and ixazomib (in the same class of proteasome inhibitors as bortezomib). In addition to these new treatments, monoclonal antibodies (especially anti-CD38 antibodies) have also begun to play an important role in the treatment of myeloma patients. Darimumab is an anti-CD38 antibody, which has been approved as a single agent or in combination with other MM treatments for the treatment of MM (Touzeau C, Moreau P., Expert Opin Biol Ther. 2017; 17(7):887-93 ; Tzogani K. et al., Oncologist. 2018; 23:1-11). According to reports, the anti-CD38 antibody ixatuximab induces a response rate of 25% to 29% as a single agent in relapsed or refractory multiple myeloma, and a 60% response rate in the form of combination therapy (Martin T, et al., Blood. 2017; 129(25):3294-303); the results of the recent phase 3 study pointed out that in relapsed and refractory multiple myeloma, the addition of ixatuximab can increase the use of po Progression-free survival of maturamide-dexamethasone treatment (J Clin Oncol 37, 2019 (Supplement; Abstract 8004)). In addition, the anti-Slam-F7 antibody ilolizumab has been approved in combination with lenalidomide and dexamethasone for the treatment of adult MM patients who have received one to three previous treatment lines (line), and is combined with pomalidomide and pomalidomide and dexamethasone. The dexamethasone combination is used to treat adult MM patients who have received at least two previous therapies including lenalidomide and proteasome inhibitors (Bristol-Myers Squibb Company. EMPLICITI® (Ilocizumab) [package insert]. The US Food and Drug Administration website: www-dot-accessdata-dot-fda.gov/drugsatfda_docs/label/2018/761035s008lbl.pdf, revised in November 2018.

The current goals of therapies for these CD38-expressing diseases are to control the disease as effectively as possible, maximize the quality of life, and prolong survival. For example, MM patients will receive an average of 4 to 8 different treatment options during their lifetime. Therefore, although refresher therapy improves the patient's prognosis, MM is still a fatal disease. Therefore, the treatment of patients who have received current therapies including antibody therapy and progressed remains a major challenge for unmet medical needs.

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

In one aspect, an antibody that specifically binds to human CD38 is provided. In some embodiments, the antibody provided herein comprises a light chain (LC) having an amino acid sequence selected from SEQ ID NO: 7, 8 and 9 and a light chain (LC) having an amino acid sequence selected from SEQ ID NO: 2, 3, 4, 5. And the heavy chain (HC) of the amino acid sequence of 6.

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

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

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

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

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

In another aspect, provided herein is a method for preparing a pharmaceutical composition comprising an antibody that specifically binds to human CD38. In some embodiments, the antibody comprises HC and LC having amino acid sequences as described above, wherein the method includes expressing the antibody in cell culture, subjecting the antibody to a chromatographic purification step and ultra Filtering at least one of the steps 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 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 at a concentration of 0.05% v/v. Ester 80 (PS80). In some embodiments, the antibody formulation is prepared such that it has a pH of 6.2. In some embodiments of the method for preparing the antibody formulation, the antibody formulation is lyophilized.

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

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

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 HC and LC having amino acid sequences as described above.

Provided herein are anti-CD38 antibodies that have superior antibody-dependent cellular cytotoxicity (ADCC) to cells that exhibit high, medium, and low amounts 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 a lower than expected isoelectric point (pi). 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 (especially in the commercial production process), the antibody system provided herein is suitable for administration to human patients. To provide it in a stable form. antibody

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 isolated and inserted into a replicable vector for further selection (DNA amplification) or performance. General procedures (for example, by using oligonucleotide probes that specifically bind to genes encoding antibody heavy and light chains) can be used to easily isolate and sequence antibody-encoding DNA. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. 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 lines (COS-7, ATCC CRL 1651) transformed by SV40; human embryonic kidney cell lines (293 or sub-selected for growth in suspension culture) 293 cells, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse testicular support 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 cancer 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 liver cells (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human liver cancer 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, Volume 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268 page. For example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography (wherein affinity chromatography is a generally preferred purification step) can be used to purify the anti-CD38 antibodies produced by the cells. Generally, various methods for preparing antibodies for research, testing, and clinical applications are well established in the industry, consistent with the methods described above, and/or deemed appropriate by those familiar with the technology.

In some embodiments, a 500 L disposable bioreactor operating in a feed batch mode was used to express antibodies from the CHO 8D6 host cell line (DXB11 derivative). The cell culture process starts with thawing the vial of the main cell bank, followed by a series of seed train cell expansion steps. The cells are then transferred to a bioreactor and cultured using a serum-free chemically defined cell culture medium. The culture was terminated after 10 to 14 days to collect the antibody. The cells and cell culture debris can be removed from the harvested cell culture by depth filtration.

The harvested material is then further processed through chromatography and filtration steps to produce purified antibodies. Prepare purified antibodies in liquid solutions and store them at ≤ -30ºC.

Before distributing the formulated antibody into suitable vials, the liquid solution is thawed and sterile filtered using a 0.2 μm filter device. Dispense the formulated antibody into a suitable container (eg USP Type 1 glass vial, 4.3 mL/vial). In some embodiments, the filled vial contains a 0.3 mL excess of formulated antibody. In some embodiments, the formulated antibody is lyophilized in a vial.

The term "antibody" generally refers to a tetrameric protein containing two heavy chains (HC) and two light chains (LC). Each such tetramer is usually composed of two pairs of identical polypeptide chains, each pair of polypeptide chains having an LC (usually with a molecular weight of about 25 kDa) and an HC (usually with 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 for the target antigen. The amino terminal portion of each light chain and heavy chain contains a variable domain of about 100 to 110 or more amino acids, which is generally responsible for antigen recognition. The carboxy terminal part of each chain usually defines the constant domain responsible for effector functions. Therefore, in a typical antibody, a full-length HC immunoglobulin polypeptide contains one variable domain (VH) and three constant domains (CH1, CH2, and CH3), where the VH domain is located at the amino terminal end of the polypeptide and is The CH3 domain is located at the carboxy terminus, and the full-length LC immunoglobulin polypeptide comprises a variable domain (VL) and a constant domain (CL), wherein the VL domain is located at the amino terminus of the polypeptide and the CL structure The domain is located at the carboxy terminus.

The term "antibody" is used in the broadest sense herein and specifically includes typical antibodies as described above, which include monoclonal antibodies and multispecific antibodies (eg, bispecific and trispecific antibodies, as long as they exhibit all The required biological activity (including the specific binding to the CD38 target and the ability to trigger ADCC and ADCP).

As used herein, the term "Fc" refers to a molecule containing a sequence of a non-antigen-binding fragment obtained by digestion of an antibody or produced by other means. The molecule is in the form of a monomer or a multimer, and the "Fc" may contain a hinge Area. Fc molecules are composed of monomeric polypeptides that can be linked into dimers or multimers by covalent (ie, disulfide bonds) and non-covalent bonds. Depending on the class (eg, IgG, IgA, and IgE) or subclass (eg, IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG4), the number of intermolecular disulfide bonds between monomer subunits of a typical Fc molecule is 1 To the range of 4. An example of Fc is a disulfide bond dimer produced by papain digestion of IgG.

The antibodies described herein can be isolated. The term "isolated protein", "isolated polypeptide" or "isolated antibody" refers to the following protein, polypeptide or antibody, said protein, polypeptide or antibody due to its origin or derived source: (1) and in its natural state Naturally associated components that accompany it are non-associative, (2) substantially free of other proteins from the same species, (3) expressed by cells from different species, and/or (4) not present in nature. Therefore, a polypeptide synthesized chemically or in a cell system different from the cell of its natural origin will be "separated" from its naturally associated components. It is also possible to use protein purification techniques well-known in the industry to make the protein substantially free of naturally associated components through separation.

In some embodiments, the antibodies provided herein comprise the HC and LC amino acid sequences as provided in Table 1. Table 1 mAb HC SEQ ID NO: LC SEQ ID NO: 2 2 7 3 3 7 4 4 7 5 5 8 6 5 9 7 6 8 8 6 9 9 10 7 Combination characteristics

The term "affinity" refers to the measure of attraction between two polypeptides (such as receptors/ligands or antigens/antibodies). The intrinsic attractive force between two polypeptides can be expressed as the binding affinity equilibrium constant (KD) for a specific interaction. When KD ≤ 1 μM, preferably ≤ 100 nM, the antibody is said to specifically bind to the antigen. The KD binding affinity constant can be measured, for example, by Surface Plasma Resonance (SPR) (BIAcore®) or Bio-Layer Interferometry.

The term "k _off "refers to the dissociation rate constant of a specific antibody-antigen interaction. The k _off dissociation rate constant can be measured, for example, by the biofilm layer interference technique. Combination with CD38

In some embodiments, the antibody provided herein comprises a tetrameric protein containing two HCs and two LCs, and when measured by surface plasmon resonance (SPR) as described below, it is approximately 1.91 x 10 ^-10 M to about 6.7 x 10 ^-10 M affinity or KD binds to human CD38. In some embodiments, 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).

In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 3 and 7, respectively, and the antibody can bind to human CD38 with an affinity or KD of ^{about 2×10 -10 M.} In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NO: 5 and 8, respectively, and the antibody can bind to human CD38 with an affinity or KD of ^{about 6.7 x 10 -10 M.} In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 5 and 9, respectively, and the antibody can bind to human CD38 with an affinity or KD of ^{about 4.15 x 10 -10 M.} In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NO: 6 and 8, respectively, and the antibody can bind to human CD38 with an affinity or KD of ^{about 3.85 x 10 -10 M.} In some embodiments, the antibody comprises HC and LC having the amino acid sequences of SEQ ID NOs: 6 and 9, respectively, and the antibody can bind to human CD38 with an affinity or KD of ^{about 1.91 x 10 -10 M.} Combination with FcγRIIIa ( CD16a ) and FcγRIIa ( CD32a)

The antibodies provided herein can also have a KD of less than one order of magnitude and a KD of less than 100 nM as measured by SPR, and a lower affinity FcγRIIIa with phenylalanine (F) at the amino acid position 158 ( CD16a) variant (158F) binds and can bind to the higher affinity FcγRIIIa (CD16a) variant (158V) with amino acid (V) at position 158 of the amino acid. In some embodiments, the antibodies provided herein bind to the 158F and 158V variants of FcγRIIIa (CD16a) with a KD of less than 100 nM as measured by SPR, and the binding to the 158F and 158V variants is different Less than 2 times. In some embodiments, the antibodies provided herein bind to the FcγRIIIa (CD16a) variant (158F) with a KD of about 59 nM or less when measured by, for example, SPR.

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

The antibodies provided herein can also bind with a lower affinity FcγRIIa (CD32a) variant (131R) with arginine at position 131 of the amino acid with a KD of ≤ 690 nM, and with a KD of less than about 270 nM with The higher affinity FcγRIIa (CD32a) variant (131H) binding with histidine at position 131, as measured by, for example, SPR.

In some embodiments, the antibody comprises HC with the amino acid sequence of SEQ ID NO: 2 and LC with the amino acid sequence of SEQ ID NO: 7, and is capable of interacting with FcγRIIa (CD32a) with a KD of about 690 nM ( 131R) binds and binds to FcγRIIa (CD32a) (131H) with a KD of about 120 nM, as measured by, for example, SPR. In some embodiments, the antibody comprises an HC having the amino acid sequence of SEQ ID NO: 3 and an LC having the amino acid sequence of SEQ ID NO: 7, and can be combined with a KD of ≤ about 125 nM or ≤ 100 nM. FcγRIIa (CD32a) (131R) binds and binds to FcγRIIa (CD32a) (131H) with a KD of about 220 nm, as measured by, for example, SPR. In some embodiments, the antibody comprises an HC having the amino acid sequence of SEQ ID NO: 4 and an LC having the amino acid sequence of SEQ ID NO: 7, and is capable of interacting with FcγRIIa (CD32a) with a KD of about 510 nM ( 131R) binds and binds to FcγRIIa (CD32a) (131H) with a KD of about 60 nM, as measured by, for example, SPR.

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

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

The antibodies provided herein are capable of expressing high amounts (about 400,000/cell), medium (about 100,000/cell) and low amounts (about 13,000/cell) of CD38 molecule antibody-dependent cells on the cell surface. Toxic to kill cells that express CD38. The antibodies provided herein trigger this ADCC in the presence of natural killer (NK) cells that exhibit a lower affinity FcγIIIa (CD16a) receptor variant (158F) and/or a higher affinity FcγIIIa (CD16a) receptor variant (158V). ADCC can be measured using methods known in the industry, for example, by measuring target cell lysis in the presence of antibodies and effector cells in cell-based potency analysis. The target cell may be, for example, a cell expressing CD38, such as KMS12-BM, RPMI-8226, or MOLP-8. In addition, the effector cell may be any cell that can be induced to kill the target cell by ADCC, for example. In some embodiments, the effector cells are primary cells isolated from human blood, such as peripheral blood mononuclear cells (PBMC) or human NK cells purified from PBMC. In another embodiment, the cell is a natural killer cell line (eg, NK-92 cell), which expresses 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 the Jurkat cell line, which has been engineered to express FcyIIIa (CD16a) 158F or 158V (see, for example, Promega, G701A).

The antibodies provided herein can kill cells that exhibit high amounts of CD38 on the cell surface through antibody-dependent cellular phagocytosis (ADCP). The antibodies provided herein trigger this ADCP in the presence of human PBMC. In one example, the antibody provided herein triggers about 41% phagocytosis of CD38-expressing cells by human PMBC expressing FcγRIIIa (158V) with a relative EC50 of 212.6 pM. Pharmaceutical composition and formulated antibody

The term "pharmaceutical formulation" refers to a preparation that is in a form that makes the biological activity of the active ingredient effective and does not contain additional components that have unacceptable toxicity to the subject receiving the preparation. Such formulations are usually sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those active ingredients that can be reasonably administered to the tested mammal to provide an effective dose.

The pharmaceutical compositions and formulations of the antibodies provided herein can be prepared by combining an antibody of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Edited (1980). )) It is prepared by mixing, and can be provided in the form of a lyophilized formulation or an aqueous solution.

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 10 mM to 20 mM. In some embodiments, L-histidine is present at a concentration of 10 mM. In some embodiments, L-histidine is present at a concentration of 20 mM. In some embodiments, PS80 is present at a concentration of 0.005% (v/v) to 0.05% (v/v). In some embodiments, PS80 is present at a concentration of 0.005% (v/v). In some embodiments, PS80 is present at a concentration of 0.02% (v/v). In some embodiments, PS80 is present at a concentration of 0.05% (v/v). In some embodiments, the pH of the pharmaceutical composition is optimized to maintain the stability of the antibody when the pharmaceutical composition is in liquid form, for example. In some embodiments, the pH of the formulated antibody has a pH of about 6.0 to about 6.5. In some embodiments, the formulated antibody has a pH of about 6.0. In some embodiments, the formulated antibody has a pH of about 6.2. In some embodiments, the formulated antibody has a pH of about 6.5. In some embodiments, the formulated antibody has a pH of 6.0. In some embodiments, the formulated antibody has a pH of 6.2. In some embodiments, the formulated antibody has a pH of 6.5.

In some embodiments, the antibodies and/or formulated antibodies of the pharmaceutical compositions provided herein comprise one or more charged isotypes. Charged isoforms are usually described as major isoforms, acidic isoforms, and basic isoforms. 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) compared to the main isotype, and the basic isotype has a higher pH than the main isotype. Charged isoforms can be generated by post-translational modification of the amino acid sequence of HC and/or LC. For example, an increase in deamidation, glycation, and sialylation may lead to a decrease in antibody pI. The conversion of N-terminal glutamic acid to pyroglutamic acid (or pyroQ) results in the loss of a positive charge, which may lead to a decrease in pI. In addition, the presence of C-terminal lysine may lead to an increase in antibody pI. In addition, the amidation of C-terminal proline may lead to an increase in antibody pI.

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

The charged isotypes 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 may 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 isotype of the antibody will be extracted first, followed by the main isotype of the antibody, and then the acidic isotype. In another embodiment, capillary isoelectric focusing (cIEF) can be used. Capillary isoelectric focusing is a method of separating proteins by their isoelectric point (pI) value. In cIEF, the antibody sample migrates to its isoelectric point on the pH gradient in the capillary, thereby resolving different charged isotypes along the length of the capillary. In order to identify and characterize cIEF variants, the charged isoforms can be separated by strong cation exchange chromatography (SCX) and analyzed by cIEF. The entire column is used to detect and measure the absorbance at 280 nm, and a charge-coupled device camera is used to collect images of the charged isoforms resolved in the capillary. Use cIEF to compare the charged isotype distribution of the test sample with a reference standard to identify the charged isotype of the antibody. In some embodiments, cIEF is used as a quality control measure to confirm the identity of the antibody during commercial production and pass comparison with a reference standard (for example, a test antibody with a charged isotype distribution pattern within a predefined distribution pattern ) Or compared with the distribution pattern of the reference standard to determine the distribution of the charged isotype of the antibody.

In some embodiments, the antibody (including the antibody in the pharmaceutical composition and the formulated antibody) comprises the HC and LC amino acid sequences of any of the monoclonal antibodies provided in Table 1, and is selected from at least about 70% of the major isotypes , No more than about 30% acid isotype and less than 4% basic isotype one or more charged isotype parameters. In some embodiments, the antibody comprises 71% major isotype, 28% acidic isotype, and less than 4% basic isotype. In some embodiments, the antibody contains a charged isotype with an isoelectric point in the range of about 5.8 to about 9.0. In some embodiments, the antibody contains a charged isotype having an isoelectric point in the range of about 5.85 to about 8.97.

In some embodiments, antibodies (including antibodies and formulated antibodies in pharmaceutical compositions) are lyophilized. Antibodies (including antibodies and formulated antibodies in pharmaceutical compositions) can be in a container (eg, vial) so that a prescribed dose of antibody can be removed from the container for administration to a patient.

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 of the monoclonal antibodies provided in Table 1, and the unit dosage form comprises the antibody and one or more excipients, the excipient being selected from sucrose, L -Histidine and Polysorbate 80. In some embodiments, the unit dosage form contains about 215 mg of antibody, about 6.21 mg of L-histidine, about 344 mg of sucrose, and about 2.15 mg of polysorbate 80. In some embodiments, the unit dosage form contains 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 (eg, vial) such that a prescribed dose of antibody can be removed from the container for administration to a patient. In some embodiments, the antibodies, pharmaceutical formulations, and/or formulated antibodies are in glass vials equipped with elastic closures. In some embodiments, the vial contains about 215 mg of antibody. In some embodiments, the vial contains 215 mg of antibody. In some embodiments, the fill volume of the vial has been determined so that about 4 mL can be removed. In some embodiments, the fill volume of the vial has been determined so that 4 mL can be removed. Preparation

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 includes expressing the antibody in a suitable cell culture. The antibody is subjected to at least one chromatography step and at least one ultrafiltration step, thereby producing a purified antibody solution. The purified antibody solution is adjusted to concentrate the antibody and add excipients and adjust the pH. In one embodiment, the concentration of the antibody is adjusted to about 50 mg/ml; sucrose, L-histidine and polysorbate 80 are added, and the pH is adjusted to at least about 6.0. In one embodiment, the 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 about 6.2. In some embodiments, the pH is 6.2. In some embodiments, the concentration of sucrose is about 8% w/v, the concentration of L-histidine is about 10 mM; and the concentration of polysorbate 80 is about 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.

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

As used herein, the term "treatment" or "treating" refers to a clinical intervention designed to change the natural progression of an individual or cell being treated in the course of clinical pathology. The desired therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and remission or improvement in prognosis. For example, if one or more symptoms associated with cancer are alleviated or eliminated, the individual will be successfully "treated", including but not limited to reducing the proliferation of cancer cells, destroying cancer cells, reducing symptoms caused by the disease, and increasing the risk of the disease. The quality of life of those individuals, reducing the dosage of other drugs needed to treat the disease, and/or prolonging the individual’s survival.

Provided herein is a method for treating or delaying the progression of multiple bone marrow (such as relapsed multiple myeloma or relapsed and refractory multiple myeloma) in an individual, the method comprising administering to a subject in need An effective amount of the anti-CD38 antibody provided herein. 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 can improve the survival time of the mouse in the murine MM model, where the mouse in the model exhibits a low-affinity human CD16a variant (158F), and where 500,000 expressing human CD38 have been injected into the mouse EL4 cells. In some embodiments, one or more doses of anti-CD38 antibody are provided in the form of a lyophilized formulation, wherein the lyophilized formulation is reconstituted to form a reconstituted antibody formulation before administration. The antibody formulation has a pH of 6.2 and contains a concentration of about 10 mM L-histidine, about 8% w/v sucrose, and about 0.05% v/v polysorbate 80 in a liquid 50 mg/mL antibody. In some embodiments, one or more doses of anti-CD38 antibody are provided in the form of a lyophilized formulation, wherein the lyophilized formulation is reconstituted to form a reconstituted antibody formulation before administration. The structured antibody formulation has a pH of 6.2 and contains a concentration of 50 mg/mL in a liquid (the liquid contains 10 mM L-histidine, 8% w/v sucrose and 0.05% v/v polysorbate 80) Of antibodies.

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

Unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "one/one (a)", "one/one (an)" and "the" include plural indications Things. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules and the like.

The term "about" as used herein refers to the usual error range of the corresponding value that is easily known by those familiar with the art. References herein to "about" a certain value or parameter include (and describe) embodiments related to the value or parameter itself. In some embodiments, the term "about" means the specified value plus or minus 10% of the stated 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 stated value; for example, "about 20 mg/kg" can encompass 19 to 21 mg/kg.

"Subject" or "individual" for therapeutic purposes 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.

In order to illustrate and describe some specific embodiments of the present invention, examples are set forth below. However, the scope of the patent application should not be limited by the examples set forth herein in any way. Example 1. Antibody design:

An Fc engineered anti-CD38 antibody was generated with mutations in the Fc portion of the antibody to increase the affinity for activated FcγRIIIa and FcγRIIa receptors on effector cells.

The two humanized heavy chains (HC) (SEQ ID NO: 5 and SEQ ID NO: 6) carry 14 and 10 mutations in the framework region (compared to SEQ ID NO: 1), which leads to the germination of Homo sapiens (germinality) The score increased from 74.49% to 88.78% and 84.69% respectively. Two humanized light chains (LC) (SEQ ID NO: 8 and SEQ ID NO: 9) carry 17 and 15 mutations in the framework region (compared to SEQ ID NO: 7), which resulted in a reproduction score of 64.36 for Homo sapiens % Increased to 81.19% and 79.21% respectively. In addition, in LC 2 and 3, leucine was substituted for methionine at position 11 to avoid potentially problematic oxidation of methionine. The HC and LC sequences are provided in Table 2, and the mAb HC and LC pairs are provided in Table 3. Table 2 SEQ ID NO: chain Amino acid sequence 1 Heavy chain 1 QVQLVQSGAE VAKPGTSVKL SCKASGYTFT DYWMQWVKQR PGQGLEWIGT IYPGDGDTGY AQKFQGKATL TADKSSKTVY MHLSSLASED SAVYYCARGD YYGSNSLDYW GQGTSVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 2 Heavy chain 2 QVQLVQSGAE VAKPGTSVKL SCKASGYTFT DYWMQWVKQR PGQGLEWIGT IYPGDGDTGY AQKFQGKATL TADKSSKTVY MHLSSLASED SAVYYCARGD YYGSNSLDYW GQGTSVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PDVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPEEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 3 Heavy chain 3 QVQLVQSGAE VAKPGTSVKL SCKASGYTFT DYWMQWVKQR PGQGLEWIGT IYPGDGDTGY AQKFQGKATL TADKSSKTVY MHLSSLASED SAVYYCARGD YYGSNSLDYW GQGTSVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLAG PDVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPEEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 4 Heavy chain 4 QVQLVQSGAE VAKPGTSVKL SCKASGYTFT DYWMQWVKQR PGQGLEWIGT IYPGDGDTGY AQKFQGKATL TADKSSKTVY MHLSSLASED SAVYYCARGD YYGSNSLDYW GQGTSVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLAG PDVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPLPEEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 5 Heavy chain 5 QVQLVQSGAE VAKPGASVKV SCKASGYTFT DYWMQWVRQA PGQGLEWIGT IYPGDGDTSY AQKFQGRVTM TADTSTSTVY MELSSLRSED TAVYYCARGD YYGSNSLDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLAG PDVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPEEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 6 Heavy chain 6 QVQLVQSGAE VAKPGASVKV SCKASGYTFT DYWMQWVKQR PGQGLEWIGT IYPGDGDTGY AQKFQGRVTM TADKSTSTVY MELSSLRSED TAVYYCARGD YYGSNSLDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLAG PDVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPEEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 7 Light chain 1 DIVMTQSHLS MSTSLGDPVS ITCKASQDVS TVVAWYQQKP GQSPRRLIYS ASYRYIGVPD RFTGSGAGTD FTFTISSVQA EDLAVYYCQQ HYSPPYTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCYQQS VTEKLKSVKSA SVVCNSKVKV ESVQSTKs 8 Light chain 2 DIVMTQSPDS LAVSLGERAT INCKSSQDVS TVLAWYQQKP GQSPRRLIYS ASYRYIGVPD RFSGSGSGTD FTLTISSLQA EDVAVYYCQQ HYSPPYTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCYQQLS VTEKLQSVKSA SVVCSVKSA SVVQSKNSKVKV ESV 9 Light chain 3 DIVMTQSPDS LAVSLGERAT INCKSSQDVS TVVAWYQQKP GQSPRRLIYS ASYRYIGVPD RFSGSGSGTD FTFTISSLQA EDVAVYYCQQ HYSPPYTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCYQQLS VTSV HQLKSGTA SVVCYQQS VTEKSVQSTKNSKVKSA VTSKs 10 Heavy chain 7 QVQLVQSGAE VAKPGTSVKL SCKASGYTFT DYWMQWVKQR PGQGLEWIGT IYPGDGDTGY AQKFQGKATL TADKSSKTVY MHLSSLASED SAVYYCARGD YYGSNSLDYW GQGTSVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 11 huCD16a (FcγRIIIa) UniProtKB/Swiss-Prot P08637 "158V" variant bold MWQLLLPTAL LLLVSAGMRT EDLPKAVVFL EPQWYRVLEK DSVTLKCQGA YSPEDNSTQW FHNESLISSQ ASSYFIDAAT VDDSGEYRCQ TNLSTLSDPV QLEVHIGWLL LQAPRWVFKE EDPIHLRCHS WKNTALHKVT YLQNGKGRKY FHHNSDFYIP KATLKDSGSY FCRGL V GSKN VSSETVNITI TQGLAVSTIS SFFPPGYQVS FCLVMVLLFA VDTGLYFSVK TNIRSSTRDW KDHKFKWRKD PQDK 12 huFcγRIIa UniProtKB/Swiss-Prot P12318-1 "131H" variant bold MTMETQMSQN VCPRNLWLLQ PLTVLLLLAS ADSQAAAPPK AVLKLEPPWI NVLQEDSVTL TCQGARSPES DSIQWFHNGN LIPTHTQPSY RFKANNNDSG EYTCQT G QTS LSDPVHLTVL SEWLVLQTPH LEFQEGETIM LRCHSWKDKP LVKVTFFQNG KSQKFSHLDP TFSIPQANHS HSGDYHCTGN IGYTLFSSKP VTITVQVPSM GSSSPMGIIV AVVIATAVAA IVAAVVALIY CRKKRISANS TDPVKAAQFE PPGRQMIAIR KRQLEETNND YETADGGYMT LNPRAPTDDD KNIYLTLPPN DHVNSNN 13 Heavy chain 8 EVQLLESGGG LVQPGGSLRL SCAVSGFTFN SFAMSWVRQA PGKGLEWVSA ISGSGGGTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYFCAKDK ILWFGEPVFD YWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 14 Light chain 4 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPPTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCSGNNFY PRETEAKVQDSKSPTY VTEKLS VTEKS VTEKSV VTEKS VVCSGNSKV VTEKS Table 3 mAb Heavy chain SEQ ID NO: Light chain SEQ ID NO: 1 1 7 2 2 7 3 3 7 4 4 7 5 5 8 6 5 9 7 6 8 8 6 9 9 10 7 10 13 14 2. Biochemical characterization: A: Combination with CD38

In one experiment, the binding affinity and kinetic constant of mAb 1-8 for human CD38 were measured by SPR using a Biacore® instrument. In short, the anti-CD38 antibody is captured to the CM5 chip that has been covalently bound by the anti-Fc antibody. Various concentrations of huCD38 were injected on the capture antibody to be tested. Use Biacore evaluation software to generate binding curve (sensing map) and perform kinetic analysis.

As shown in Table 4, mAbs 3 and 8 have the highest affinity for human CD38 (T test for each variant: p value> 2). Table 4 Affinity for human CD38 (n = 3 ) mAb ka (1/Ms) CV ka (%) kd (1/s) CV kd (%) KD (M) CV Kd (%) 1 3.55E+06 11.6 6.55E-04 3.4 1.80E-10 5.0 3 3.89E+06 13.8 6.59E-04 2.2 1.71E-10 13.1 5 2.78E+06 11.0 1.88E-03 29.1 6.99E-10 22.1 6 3.10E+06 15.8 8.55E-03 76.2 3.85E-10 18.7 7 3.18E+06 15.4 1.30E-03 9.5 4.15E-10 14.5 8 3.51E+06 19.2 6.50E-04 4.7 1.91E-10 24.0

In another experiment, the Biacore® instrument used as described above measured the binding affinity of mAb 1 and 3 for human CD38 by SPR. mAb 1 has a Kd of 0.22 nM, and mAb 3 has a Kd of 0.20 nM.

The effect of thermal stress on the binding affinity of human CD38: SPR measurement using Biacore® instrument after mAb subjected to thermal stress (at 40ºC, in 10 mM histidine pH 6, 8% sucrose, 0.02% PS80 for 14 days) The binding affinity and kinetic constant of mAb 1-8 for human CD38. The same protocol described in the previous paragraph has been used.

As shown in Table 5, the samples subjected to thermal stress showed very similar affinity and kinetic constants to the corresponding non-stressed sample CD38 (Table 4) (T test for each variant: p value> 2). Table 5 The influence of thermal stress on the affinity for human CD38 (n = 3 ) mAb ka (1/Ms) CV ka (%) kd (1/s) CV kd (%) KD (M) CV KD (%) 1 4.00E+06 21.9 7.14E-04 5.5 1.83E-10 15.7 3 3.71E+06 8.2 6.71E-04 3.0 1.81E-10 5.3 5 2.95E+06 21.2 2.36E-03 4.4 8.28E-10 25.7 6 3.86E+06 27.9 1.28E-03 3.7 3.46E-10 24.0 7 3.39E+06 6.8 1.33E-03 5.9 3.94E-10 10.5 8 3.53E+06 25.2 6.63E-04 11.7 1.98E-10 31.5 B: Measure the affinity to FcγRIIa , FcγRIIIa and FcγRIIb by computer ( in silico)

In one experiment, surface plasmon resonance (SPR) was used to measure the affinity of mAb for FcγRIIIa 158V receptor or FcγRIIIa 158F receptor. MAb was 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). According to the supplier (Ni-Sepharose GE, Healthcare), the extracellular domain of FcγRIIIa (CD16a) with V158 or F158 with C-terminal histag was produced in HEK293 cells through transient performance, and passed through immobilized metal affinity chromatography (IMAC) Purify it. Using anti-His IgG immobilized on a CM5 chip, the affinity was measured by SPR on BIACore 2000 (GE Healthcare) by capture analysis. FcyRIIIa-histag was then added to the running buffer, followed by anti-CD38 mAb in a concentration ranging from 8 to 256 nM. Using BIAevaluation Software 4_1 (GE Healthcare), the two-state reaction is used for analysis, where the acceptable χ ² value is less than 5 (meaning 2% of the highest test concentration) and the percentage of the maximum theoretical resonance unit is greater than 25 (meaning more than ¼ productive interaction effect).

As shown in Table 6, compared with mAb 1, mAb 2-4 has a 9-fold higher affinity for FcyRIIIa 158V, and compared with mAb 1, mAb 2-4 has more than 27-fold higher affinity for FcyRIIIa 158F receptor. Table 6 K _D (nM) mAb FcγRIIIa 158V FcγRIIIa 158F 1 425 ＞1500 2 47 53 3 50 55 4 47 51

In another experiment, the affinity of mAbs 1 and 3 for FcyRIIIa 158V receptor or FcyRIIIa 158F receptor was measured using SPR as described above. mAb 3 binds FcyRIIIa 158V with a KD of 75 nM, and mAb 3 binds FcyRIIIa 158F with a KD of 59 nM.

The AlphaScreen affinity analysis was used to measure the affinity of the mAb for the FcγRIIa receptor with arginine at amino acid position 131 (FcγRIIa 131R) or histidine at amino acid position 131 (FcγRIIa 131H).

The FcyRIIa extracellular domain with R131 or H131 is provided by R&D System (batch 1330-CD) or Biorbyt (batch ORB138408), respectively. The analysis is a bead-based proximity analysis described by the supplier (Perkin Elmer), which allows ranking by anti-CD38 Fc variants by competing with anti-CD38 with native IgG1. The biotinylated anti-CD38 with native IgG1 was captured on the donor bead, and the FcγRIIa histag protein was captured on the acceptor bead. When anti-CD38 interacts with FcγRIIa, the beads will approach. Excitation of the donor beads at 680 nm causes the release of singlet oxygen, which when in the close state diffuses and triggers light emission at 520 to 620 nm from the acceptor beads. The amount of light is proportional to the degree of interaction and is measured with EnVision multi-mode plate reader (Perkin Elmer). During the competition analysis, unlabeled competitive mAb anti-CD38 Fc variant DE (S239D/I332E, Eu numbering), ADE (G236A/S239D/I332E, Eu numbering) or ADLE (G236A/S239D/F243L/I332E, Eu numbering) ) Used to compete for the interaction between biotinylated anti-CD38 with natural IgG1 and FcγRIIa-histag protein. The concentration of biotinylated anti-CD38 is set by a pre-completed calibration curve. Unlabeled competitive mAb is used at different levels, so that increasing concentrations of unlabeled mAb can replace biotinylated anti-CD38 and reduce signal. Normalize the data with respect to the maximum signal. Two experiments were run and IC50 was evaluated through BIOST@T-SPEED-LTS 2.1.0.

As shown in Table 7, compared to mAb 1, mAb 3 showed more than 14-fold and 18-fold higher affinities for FcyRIIa 131R and FcyRIIa 131H, respectively. Table 7 for FcγRIIa R131 and affinity of FcγRIIa H131 Relative IC50 (nM) mAb FcγRIIa R131 FcγRIIa H131 1 1741 268 2 687 119 3 125 15 4 508 58 C : Measurement of affinity for FcγRIIa , FcγRIIIa and FcγRIIb in vitro

The HEK293T-FcγRIIIa-158F, HEK293T- FcγRIIIa-158V, HEK293T-FcγRIIa-131H, HEK293T-FcγRIIa-131R, HEK293T-FcγRI or HEK293T-FcγRIIb at ¹⁰⁵ cells / well in 96-well micro plate. The plates were centrifuged at 800 g for 1 minute and resuspended in 50 μL PBS containing different concentrations of antibodies at 4ºC for 30 minutes. Then add 200 μL of PBS 1% FBS to wash the cells, followed by centrifugation at 800 g for 1 minute. The washing step was repeated three times in total, after which the cells were stained with FITC-conjugated secondary antibody (fragment goat anti-human IgG (H+L) FITC, number 109-546-088 Jackson ImmunoResearch, final 10 μg/mL) at 4ºC for 30 minute. The cells were washed three times with 200 μL PBS 1% FBS, and resuspended in 100 μL PBS, and then harvested on MACSVYB (channel B1). As shown in Table 8, compared with mAb 1, mAb 2, 3, and 4 have 32-fold, 38-fold, and 51-fold higher affinity for FcγRIIIa 158F expressed on HEK cells, respectively, and compared with mAb 1, mAb 2 , 3 and 4 have 9-fold, 5-fold and 6-fold higher affinity for FcγRIIIa 158V expressed on HEK cells, respectively. Table 8 mAb Average apparent Kd (nM) SEM Affinity fold difference relative to mAb 1 FcγRIIIa 158F 1 2269.1 771.4 1 2 70.1 13.1 32.4 3 60.4 10.4 37.6 4 44.2 9.1 51.4 FcγRIIIa 158V 1 165.3 44.2 1 2 17.6 3.3 9.4 3 32.0 5.3 5.2 4 28.1 7.8 5.9

As shown in Table 9, compared with mAb 1, mAb 3 has about 5-fold higher affinity for FcγRIIa 131H expressed on HEK cells, and compared with mAb 1, mAb 3 has an affinity for FcγRIIa 131R expressed on HEK cells About 16 times higher affinity. Table 9 mAb Average Kd (nM) SEM Difference in affinity relative to mAb 1 FcγRIIa 131H 1 1180.8 406.1 1 2 839.8 438.7 1.4 3 221.9 74.9 5.3 4 543.8 277.7 2.2 FcγRIIa 131R 1 1,420.3 166.5 1 2 891.1 131.1 1.6 3 87.4 13.9 16.3 4 467.3 153.6 3.0

As shown in Table 10, compared with mAb 1, mAbs 2, 3, and 4 have similar binding affinity levels for FcγRI, and compared with mAb 1, mAbs 2, 3, and 4 have low levels of FcγRIIb expressed on HEK cells. 1.8 to 3.9 times the affinity. Table 10 mAb Average Kd (nM) SEM Difference in affinity relative to mAb 1 FcγRI 1 24.2 4.2 1 2 13.5 1.7 1.8 3 14.7 3.2 1.6 4 15.9 2.7 1.5 FcγRIIb 1 1299.1 406.1 1 2 839.8 438.7 3.9 3 221.9 74.9 3.3 4 543.8 277.7 1.8 D : Measurement of cytotoxic activity in vitro, antibody-dependent cytotoxicity ( ADCC ):

Calcein-acetoxymethyl (Calcein-AM; Invitrogen) release analysis was used to examine the antibody-dependent cytotoxicity (ADCC) analysis using the NK92 cell line engineered to overexpress the FcγRIIIa receptor as effector cells. In living cells, non-fluorescent calcein AM is converted into green fluorescent calcein. The labeled target cells were incubated with SAR442085 antibody and NK-92 effector cells. The lysis of target cells by ADCC results in the release of fluorescent calcein. The fluorescence intensity is proportional to the degree of ADCC activity present.

Target multiple myeloma cells (MOLP-8, RPMI 8226, MM1R or KMS12-BM) with calcein-AM (50 µg diluted in 25 µL DMSO, and then 10 µL calcein diluted in 4 mL RPMI 1640 + 1 % FBS + 1% probenecid, used for ^{staining 4 × 10 6} cells) label for 30 min, then wash and spread on a 96-well circular dish at a density of ^{2 × 10 4 cells/well.} Different concentrations of the indicated test mAb or control isotype mAb from 10 μg/mL to 0.01 pg/mL were added for 30 min to allow opsonization, after which natural killer (NK) cells transduced with the indicated FcγRIIIa variant were added. With an E: T ratio of 5:1 (1 × 10 ⁵ NK cells to 2 × 10 ⁴ target cells), NK cells expressing 158F or 158V are added as effector cells. The plates were then _{incubated at 37ºC in a humidified incubator with 5% CO 2} for 1 h, and 100 µL of the supernatant was harvested and transferred to an opaque 96-well microplate for use on Tecan Infinite M1000 Analyze by fluorometry to measure calcein release (excitation filter: 492 nm; emission: 515 nm). For maximum release, use 2% Triton X-100 to lyse the cells. Subtract the fluorescence value of the medium background from the fluorescence values of the experimental release (A), spontaneous release of target cells (B), and maximum release of target cells (C). Use the following formula to calculate the cytotoxicity and ADCC percentage of each disc (in duplicate): Cytotoxicity (%) = (A-B)/(C-B)×100%

Each experiment is repeated at least 3 times. The half maximum effective concentration (EC50) value was calculated by fitting the data points to a 4-parameter equation using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, California). High CD38 receptor density target cells

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

Table 11 shows the EC50 of each test antibody against MOLP-8 cells in the presence of NK cells expressing 158V. As shown in Table 11, in the presence of NK cells exhibiting a high-affinity FcγRIIIa variant (158V), mAbs 2, 3, and 4 trigger ADCC of MOLP-8 cells with EC50 that is 24 to 33 times lower than that of mAb 1. Table 11 mAb EC50 (ng/ml) SEM (ng/ml) 1 3.3 0.91 2 0.1 0.03 3 0.14 0.02 4 0.12 0.02

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 exhibiting low-affinity FcγRIIIa variant (158F), mAb 4 showed the strongest ADCC activity against MOLP-8 cells, with EC50 about 97 times lower than mAb 1. In the presence of NK cells exhibiting a low-affinity FcγRIIIa variant (158F), mAbs 2 and 3 trigger ADCC in MOLP-8 cells with an EC50 that is approximately 59 to 69 times lower than that of mAb 1. Table 12 mAb EC50 (ng/ml) SEM (ng/ml) 1 16.5 8.77 2 0.24 0.11 3 0.28 0.09 4 0.17 0.05 Medium CD38 receptor density target cells

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

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, in the presence of NK cells exhibiting a higher affinity FcγRIIIa variant (158V), mAbs 2 to 4 showed similarly improved levels of ability to trigger ADCC against MM cells exhibiting moderate CD38 receptor density , Where compared with mAb 1, EC50 is 27 times lower. Table 13 mAb EC50 (ng/ml) SEM (ng/ml) 1 2.45 0.73 2 0.09 0.02 3 0.09 0.003 4 0.09 0.003

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 exhibiting low-affinity FcγRIIIa variants, compared with mAb 1, mAb 4 showed an approximately 73-fold improved ability to trigger ADCC against MM cells exhibiting moderate CD38 receptor density; In the case of low-affinity FcγRIIIa variants of NK cells, compared to mAb 1, mAb 3 showed approximately 65-fold improved ability to trigger ADCC against MM cells exhibiting moderate CD38 receptor density and mAb 2 showed approximately 49-fold increased Improved ability to trigger. Table 14 mAb EC50 (ng/ml) SEM (ng/ml) 1 5.83 1.67 2 0.12 0.04 3 0.09 0.01 4 0.08 0.01 Low CD38 receptor density target cells

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

Table 15 shows the EC50 of each test antibody against KMS-12BM cells in the presence of NK cells exhibiting a higher affinity FcyRIIIa variant (158V). Interestingly, as shown in Table 15, in the presence of NK cells that exhibited a higher affinity FcγRIIIa variant (158V), compared to mAb 1, mAbs 2, 3, and 4 showed improved triggering for low CD38 The ADCC capacity of MM cells for receptor density. mAb 3 has an EC50 that is about 69 times lower than mAb 1, and mAb 4 has an EC50 that is about 91 times lower than mAb 1. Table 15 mAb EC50 (ng/ml) SEM (ng/ml) 1 41 12 2 0.65 0.05 3 0.6 0.1 4 0.45 0.35

Table 16 shows the EC50 of each test antibody against KMS-12BM cells in the presence of NK cells exhibiting a lower affinity FcyRIIIa variant (158F). Significantly, as shown in Table 16, in the presence of NK cells exhibiting a lower affinity FcγRIIIa variant (158F), compared to mAb 1, mAbs 2, 3, and 4 showed increased triggering for low CD38 receptors. The ADCC capacity of MM cells at body density. mAb 3 has an EC50 that is about 145 times lower than that of mAb 1, and mAb 4 has an EC50 that is about 269 times lower than that of mAb 1. Table 16 mAb EC50 (ng/ml) SEM (ng/ml) 1 94 2.55 2 1.05 0.25 3 0.65 0.15 4 0.35 0.15 Antibody-dependent cellular phagocytosis ( ADCP ):

First, human PBMCs were isolated from the buffy coat of seven healthy donors from the French Institute of Blood Research (Etablissement Français du Sang). All these donors exhibited the same FCGR3A and FCGR2A genotypes (FcγRIIIa-158V/V and FcγRIIa-131H/H). First collect the blood in a 50 mL Falcon test tube. Gently add 15 mL of Ficoll-Plaque ^TM PLUS 96% to the bottom of the Sepmate test tube, then slowly add blood to the test tube, and then centrifuge at 1,300 g for 10 minutes. The PBMC loop was then collected, washed with 50 mL PBS, and subjected to another round of centrifugation at 300 g for 5 minutes. Repeat the washing process twice, then stain PBMC with anti-CD14 magnetic beads according to the manufacturer's instructions (Miltenyi; No. 130-050-201): incubate at 4ºC for basically 15 minutes, and then use AutoMACS pro (Posseld selection) Positive selection. Collect monocytes and wash them with 50 mL PBS. Monocytes were cultured in RPMI1640, 10% FBS, 2% inactivated human serum, 50 ng/ml GM-CSF in T75 flasks at 37ºC and 5% CO ₂ for 5 days. After 5 days of culture, monocytes differentiate into macrophages. To collect macrophages, place the cell digest (ThermoFisher, number: A1110501) in a flask at 37ºC for 15 minutes to separate the cells, and then add 20 mL of RPMI 10% FBS 1% glutamic acid to stop the cells Digestive juice activity. The collected cells were centrifuged at 350 g for 10 minutes, followed by washing in 20 mL of PBS, and another round of centrifugation at 300 g for 10 minutes. For both experiments, the macrophages used were from batches of macrophages frozen in liquid nitrogen.

The macrophages were 20 x 10 ⁶ cells / mL were resuspended in a diluent in C (provided in the kit manufacturer staining PKH26GL-1KT number of from Sigma Aldrich). For 1 volume of macrophages, add 1 volume of PKH26 (20 μL diluted in 1 mL of Diluent C) to the cells to incubate in the dark for 5 minutes. Then add 5 mL FBS to inactivate the staining process and continue for 1 minute, and then supplement to 50 mL with RPMI1640 10% FBS. MOLP-8 cells expressing CD38 were stained with PKH67 fluorescent dye, and macrophages obtained from purified monocytes 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 double-positive populations (macrophages that phagocytose MOLP-8 cells) were analyzed by flow cytometry.

As demonstrated by the higher percentage of total phagocytosis (%) and lower EC50 (relative) value (EC50rel) obtained in all experiments, compared to mAb 1, mAb 3 exhibited improved resistance against MOLP-8 cell lines. ADCP activity. 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 31.87 ng/mL (or 212.57 pM) for mAb 3, and for mAb 1, the The value exceeds 64.86 ng/mL (or exceeds 432.62 pM). Based on the EC50rel value, mAb 3 is at least 2.035 times better than mAb 1 in terms of ADCP activity. In vivo efficacy

Evaluate the efficacy of mAb 3 in vivo. 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 on day 0 One EL4-huCD38 tumor cell. All mouse genes encoding FcγR in the humanized FcγR C57BL/6 mice were deleted and the human FcγR encoded as a transgene was inserted into the mouse genome. The humanized FcγR C57BL/6 mouse recapitulates the expression pattern and level of huFcγR and is functional in multiple huIgG-mediated models of inflammation, cytotoxicity, and tumor clearance. Regarding the activated FcγRIIIa and FcγRIIa receptors, the human variants inserted into the mouse model are huFcγRIIIa 158F and huFcγRIIa 131R (low affinity variants of the receptor), respectively. The characteristics of human FcγR expression patterns and performance levels are summarized in mice.

Starting from day 1 after tumor cell injection, test or control mAbs were administered intraperitoneally on days 1, 4, 7 and 14. Isotype control mAb was administered at 10 mg/kg. mAb 1, 3, and 10 were administered at 10 and 1.25 mg/kg. In this survival model, the main efficacy endpoints are median survival time (MST), extended life percentage (ILS%), and long-term survivors (defined as survival duration that is better than or equal to twice the MST of the control group). The Cox model was used to evaluate the difference in survival between each group.

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 under this model. The group treated with mAb 1 at 10 mg/kg had an MST of more than 82.5 days and an extended life span of greater than 112%. Fifty percent of the group treated with mAb 1 at 10 mg/kg were long-term survivors. Mice treated with 1.25 mg/kg of mAb 1 had an MST greater than 46.5 days and an extended life span of greater than 19%. Thirty percent of the group treated with 1.25 mg/kg mAb 1 were long-term survivors.

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

The group treated with mAb 3 at 10 mg/kg had an MST over 90 days and an extended life span of greater than 131%. Significantly, 90% of the group treated with mAb 3 at 10 mg/kg were long-term survivors. Compared with the isotype control group, mAb 3 at 10 mg/kg was statistically significantly more active (p = 0.0351). Even more surprisingly, mice treated with 1.25 mg/kg of mAb 3 also had an MST greater than 90 days and an extended life span of greater than 131%. In addition, 90% of the group treated with 1.25 mg/kg mAb 3 were long-term survivors. Compared with the isotype control group, mAb 3 at 1.25 mg/kg was statistically significantly more active (p = 0.0380), and statistically significantly better than mAb 10 at the same dose (p = 0.0380).

Treatment of this tumor model in mice carrying low affinity huFcγRIIIa (F158/F158) with mAb 1, mAb 3, and mAb 10 at a dose of 10 mg/kg resulted in a statistically significant improvement 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. Table 17 Pharmacy Dose ( mg/kg ) Long-term survivors ( % ) MST (days) ILS% Isotype control 10 20 39 - mAb 3 10 90 ＞ 90 131% 1.25 90 ＞ 90 131% mAb 1 10 50 82.5 112% 1.25 30 46.5 19% mAb 10 10 50 70 79% 1.25 10 42 8% E. Stability study thermal stability

Using a Malvern MicroCal VP calorimeter, the thermal stability of mAb 1, 3, 5, 6, 7 and 8 was measured by differential scanning calorimetry (DSC). The samples were tested in a buffer (pH 6.0) containing 10 mM histidine HCl. Load the prepared sample together with the reference buffer into each well of the Wheaton 96-well circular bottom plate in triplicate. Using a temperature slope of 1ºC/min and a temperature range of 20ºC to 120ºC, the thermal scan was collected and processed using Origin Software 2.0.

As shown in Table 18, mAbs 3, 5, 6, 7 and 8 show protein conformational changes at about 40ºC, which is about 17ºC lower than the appearance temperature of mAb 1, and about 15ºC-20ºC lower than the typical temperature of mAb (figure 1). In addition, the unfolding of the CH2 domains of mAbs 3, 5, 6, 7 and 8 started much earlier than mAb 1 at about 50ºC compared to 70ºC. Table 18 Thermal stability ( ºC ) mAb T _appears T _m 1 (CH2) T _m 2 (Fab1) T _m 3 (Fab2) T _m 4 (CH3) 1 56.8 70.0 75.7 83.1 3 39.9 50.4 71.0 83.3 5 37.8 49.9 79.4 81.9 6 40.7 49.8 81.7 84.1 7 37.2 50.0 78.1 84.3 8 38.8 50.5 80.2 84.8 Isoelectric point

The isoelectric point (pI) of each of mAb 1, 3, 5, 6, 7 and 8 was measured by capillary isoelectric focusing (cIEF). The theoretical pI is determined by 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. Assuming that all sulfhydryl side chains in Cys residues are disulfide bonded and do not contribute 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 edited pages 90-124. Royal Society of Chemistry, 1991).

Surprisingly, as shown in Table 19, based on the sequence, the measured pI of mAb 3 is lower than the calculated pI. Table 19 mAb pI calculated value pI measurement value "Acid" form% "Alkaline" form% 1 7.46 7.47 16.6 19.0 3 7.46 6.95 20.1 44.0 5 6.72 5.67 20.7 23.6 6 6.72 5.66 16.8 21.5 7 7.11 6.01 25.8 48.5 8 7.11 6.22 11.5 25.7 Development of mAb 3 formulations

Based on the thermal stability results, a lyophilized formulation was developed to ensure sufficient stability of mAb 3 to be suitable for storage of mAb at ≤ -30ºC and lyophilized mAb for storage at 2ºC-8ºC. Test mAb 3 formulations using different buffer systems and at different pHs in stability studies. The stability studies include freeze-thaw cycles, agitation studies, and stress and acceleration at 40ºC, 25ºC, 5ºC, -30ºC and -80ºC And short-term (12 weeks) cultivation under expected storage conditions. In these experiments, in addition to particle formation (visual inspection, particle counting based on light blur, and microfluidic imaging), protein aggregation and chemical degradation were also evaluated.

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

A lyophilization procedure for mAb 3 was developed to ensure that the formulated antibody has acceptable stability and cake properties after lyophilization. Using a laboratory-scale lyophilizer (Genesis EL35 manufactured by SP Scientific), the program parameters (drying temperature, chamber pressure, etc.) of the lyophilization cycle are determined based on multiple development runs. The robustness of the cycle is established by performing a series of freeze-drying runs under controlled deviations of the freezing rate, drying temperature, and chamber vacuum pressure. Stability studies of lyophilized formulated antibodies include short-term (12 weeks) incubation under stress, acceleration, and expected storage conditions at 40ºC, 25ºC, and 5ºC. In these experiments, in addition to aggregation, chemical degradation, and particle formation, freeze-dried powder properties (cake appearance, reconstitution time, oxygen headspace, moisture content) were also evaluated. Based on the formulation and lyophilization development research, the composition of mAb 3 was selected as 50 mg/mL mAb 3, 10 mM L-histidine-HCl, 8% w/v sucrose, 0.05% w/v polysorbate 80 And the pH is 6.2. Store the formulated antibody at ≤ -30ºC and store the lyophilized formulated antibody at 2ºC to 8ºC.

no

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

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

Claims (26)

An antibody that specifically binds to human CD38, wherein the antibody comprises a) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 2 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or b) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 3 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or c) a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 4 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or d) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or e) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9, or f) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or g) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9. 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 at a concentration of 50 mg/ The concentration of mL is present, 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) with the amino acid sequence of SEQ ID NO: 2 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or b) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 3 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or c) a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 4 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or d) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or e) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9, or f) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or g) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9. 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) with the amino acid sequence of SEQ ID NO: 2 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or b) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 3 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or c) a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 4 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or d) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or e) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9, or f) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or g) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9. The pharmaceutical composition according to claim 2 or the unit dosage form according to claim 3, wherein the formulated antibody is lyophilized. 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 pyroglutamic acid. 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) . The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 6, wherein the antibody comprises a main charged variant and at least one acidic charged variant, and wherein the antibody has at least one of the following One item a) The HC of the at least one charged variant contains at least one asparagine with a deamidated group selected from the group consisting of N289, N318, N318, and N289, N318 numbered according to SEQ ID NO: 1. N387 and N392, or b) The antibody comprises a main charged variant and at least one acidic charged variant, wherein the main charged variant accounts for at least 71% of the antibody and the acidic charged variant accounts for no more than 30% of the antibody ,or c) When measured by capillary isoelectric focusing (cIEF), the main charged variant has an isoelectric point (pI) of 7.5 and the one or more acidic charged variants have a pI of 5.8. 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 alkaline charged variant accounts for no more than 4% of the antibody, or b) When measured by capillary isoelectric focusing (cIEF), the one or more alkaline charged variants have a pI of 9.0. The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 8, wherein the antibody a) is naturally present in the presence of 158F, 158V, or both 158F and 158V variants expressing CD16a (FcγRIIIa) Killer cells can kill CD38-expressing cells by antibody-dependent cytotoxicity (ADCC), wherein the cells have a CD38 receptor density of ≤ 13,000 CD38 sites on the cell surface, and/or b) Binding to CD16a (FcγRIIIa) with phenylalanine (158F) at amino acid position 158 with _{a K D} of 59 nM as measured by surface plasmon resonance (SPR), and wherein the antibody is as measured by SPR The measured K _D of 75 nM binds to CD16a with amino acid (158V) at position 158 of the amino acid, and/or c) to a K of 96 nM as measured by binding to HEK cells expressing FcγRIIIa 158F _D binds to CD16a (FcγRIIIa) with phenylalanine (158F) at position 158 of the amino acid, and wherein the antibody binds to the amino acid with a K _{D of 40 nM as measured by binding to HEK cells expressing FcγRIIIa 158V.} acid at position 158 FcγRIIIa having captures leucine (158V) and / or d) to the amount of K _D as by binding to HEK cells FcγRIIa 131R performance measurement has a binding of 94 nM at amino acid position 131 CD32a (FcγRIIa) of arginine (131R), and wherein the antibody binds with a K _D of 222 nM as measured by binding to HEK cells expressing FcγRIIa 131H with histidine at position 131 of the amino acid (131H) FcγRIIa. 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 CD16a (FcγRIIIa) of 158F, 158V, or both 158F and 158V variants, the antibody can kill CD38-expressing cells through ADCC, where the cells are in the Having a CD38 receptor density> 400,000 on the cell surface; and capable of killing CD38-expressing cells by ADCC, wherein the cell has a CD38 receptor density> 100,000 on the cell surface; and capable of being killed by ADCC A cell expressing CD38, wherein the cell has a CD38 receptor density ≤ 13,000 on the cell surface, and/or b) The antibody can kill CD38-expressing cells through antibody-dependent cellular phagocytosis (ADCP) in the presence of human peripheral blood mononuclear cells (PMBC), wherein the cells have> CD38 receptor density of 400,000. The antibody, pharmaceutical composition or unit dosage form according to claim 9 or 10, wherein a) In the presence of natural killer cells expressing a higher affinity variant (158V) of CD16a (FcγRIIIa), the antibody can kill KMS-12BM cells through ADCC with an EC 50 of about 0.6 ng/mL, and/ or b) In the presence of natural killer cells expressing a lower affinity variant (158F) of CD16a (FcγRIIIa), the antibody can kill KMS-12BM cells through ADCC with an EC 50 of about 0.65 ng/mL, and/ or c) In the presence of natural killer cells expressing a higher affinity variant (158V) of CD16a (FcγRIIIa), the antibody can kill RPMI-8226 cells through ADCC with an EC 50 of about 0.09 ng/mL, and/ or d) In the presence of natural killer cells expressing a lower affinity variant (158F) of CD16a (FcγRIIIa), the antibody can kill RPMI-8226 cells through ADCC with an EC 50 of about 0.09 ng/mL, and/ or e) In the presence of natural killer cells expressing a higher affinity variant (158V) of CD16a (FcγRIIIa), the antibody can kill MOLP-8 cells through ADCC with an EC 50 of about 0.14 ng/mL, and/ or f) In the presence of natural killer cells expressing a lower affinity variant (158F) of CD16a (FcγRIIIa), the antibody can kill MOLP-8 cells through ADCC with an EC 50 of about 0.28 ng/mL. The antibody, pharmaceutical composition or unit dosage form according to any one of claims 9 to 11, wherein in the presence of human PMBC expressing a higher affinity variant (158V) of CD16a (FcγRIIIa), the antibody is capable of The MOLP-8 cells were killed by ADCP with an EC 50 of about 31.87 ng/mL. The antibody, pharmaceutical composition or unit dosage form according to any one of claims 1 to 12, wherein the antibody is capable of improving the survival period of mice in a murine tumor model, wherein the mouse line in the model expresses human CD16a (158F), and 500,000 EL4 cells expressing human CD38 have been injected into mice. A method for preparing a pharmaceutical composition, the pharmaceutical composition comprising an antibody that specifically binds to human CD38, wherein the antibody comprises a) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 2 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or b) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 3 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or c) a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 4 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or d) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or e) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9, or f) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or g) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9, The method includes the following steps: i) Express the antibody in cell culture; ii) passing the antibody through at least one purification step to produce a purified antibody solution, wherein the purification step is selected from chromatography and ultrafiltration; and iii) Adjust the purified antibody solution to produce a formulated antibody, the formulated antibody comprising -The purified antibody at a concentration of 50 mg/mL, -Sucrose with a concentration of 8% w/v, -L-histidine at a concentration of 10 mM, and -Polysorbate 80 with a concentration of 0.05% v/v, Wherein the formulated antibody has a pH of 6.2; and iv) freeze-drying the formulated antibody; Thus, the pharmaceutical composition is prepared. A method of preparing a reconstituted formulated antibody, which comprises a) Provide a freeze-dried antibody formulation, which contains sucrose, L-histidine, polysorbate 80 and an antibody that specifically binds to human CD38; and b) Reconstitute the lyophilized antibody formulation in a diluent, wherein the reconstituted antibody formulation comprises -The antibody at a concentration of 50 mg/mL, -Sucrose with a concentration of 8% w/v, -L-histidine at a concentration of 10 mM, and -Polysorbate 80 with a concentration of 0.05% v/v, Wherein the reconstituted antibody formulation has a pH of 6.2; Wherein the antibody comprises i) a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 2 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or ii) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 3 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or iii) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 4 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or iv) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or v) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 9, or vi) a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or vii) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9. A method of treating a patient in need, comprising administering to the patient one or more doses of an antibody that specifically binds human CD38, wherein the patient has a disease comprising cells expressing CD38, wherein the antibody comprises a) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 2 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or b) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 3 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or c) a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 4 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or d) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or e) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9, or f) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or g) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9. An antibody for the treatment of diseases comprising cells expressing CD38, wherein said antibody specifically binds to human CD38, wherein said antibody comprises a) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 2 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or b) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 3 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or c) a heavy chain (HC) with the amino acid sequence of SEQ ID NO: 4 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 7, or d) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or e) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 5 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9, or f) A heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and a light chain (LC) with the amino acid sequence of SEQ ID NO: 8, or g) The heavy chain (HC) with the amino acid sequence of SEQ ID NO: 6 and the light chain (LC) with the amino acid sequence of SEQ ID NO: 9. The method according to claim 16 or the antibody for the use according to claim 17, wherein the disease is of blood origin. The method according to claim 18 or the antibody for the use, wherein the blood-derived disease is selected from: amyloidosis, non-Hodgkin’s lymphoma (NHL), Waldenstrom’s disease (Waldenstrom’s disease), multiple myeloma (MM), acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML). The method according to any one of claims 16 to 19 or the antibody for the use, wherein the one or more doses of the antibody are provided in the form of a lyophilized antibody formulation, and wherein the antibody is administered Previously, the lyophilized antibody formulation was reconstituted to form a reconstituted antibody formulation, so that the reconstituted antibody formulation had a pH of 6.2 and the concentration of the antibody in the liquid was 50 mg/mL The liquid contains 10 mM L-histidine, 8% (w/v) sucrose and 0.05% (v/v) polysorbate 80. The method according to any one of claims 16 to 20 or the antibody for the use, wherein the antibody is 20 mg/kg, 10 mg/kg, 5 mg/kg, 3 mg/kg or 1.5 It is administered at a dose of mg/kg. The method according to any one of claims 16 to 21 or the antibody for the use, wherein the pharmaceutical composition comprises a formulated antibody, wherein the formulated antibody comprises an antibody present at a concentration of 50 mg/mL The antibody, 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 The antibody has a pH of 6.2. The method according to any one of claims 16 to 22 or the antibody for the use, wherein when measured by capillary isoelectric focusing (cIEF), the antibody has an isoelectric point (pI) of 5.8 to 9.0 ). The method according to any one of claims 16 to 23 or the antibody for the use, wherein the amino acid at position 1 of the HC amino acid sequence is pyroglutamic acid. The method according to any one of claims 16 to 25 or the antibody for the use, wherein the antibody comprises a main charged variant and at least one acidic charged variant and wherein the antibody has any of the following At least one a) The HC of the at least one charged variant contains at least one asparagine with a deamidated group selected from N289, N318, N387 numbered according to SEQ ID NO: 1 And N392, or b) The antibody comprises a main charged variant and at least one acidic charged variant, wherein the main charged variant accounts for at least 71% of the antibody and the acidic charged variant accounts for no more than 30% of the antibody ,or c) When measured by capillary isoelectric focusing (cIEF), the main charged variant has an isoelectric point (pI) of 7.5 and the one or more acidic charged variants have a pI of 5.8. The method according to any one of claims 16 to 25 or the antibody for the use, wherein the antibody comprises at least one basic charged variant, and wherein the antibody has at least one of the following a) The alkaline charged variant accounts for no more than 4% of the antibody, or b) When measured by capillary isoelectric focusing (cIEF), the one or more alkaline charged variants have a pI of 9.0.

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